Regulation of the gene encoding lysine ketoglutarate reductase.

Abstract

Disclosed are mutant, non-native, or transgenic Nicotiana tabacum plants or parts thereof having regulated expression or activity of lysine-ketoglutarate reductase (LKR), the Nicotiana tabacum plants or parts thereof comprising at least one modification capable of modulating (a) the expression of a polynucleotide in the plant or part thereof, or (b) the activity of a polypeptide in the plant or part thereof, compared to a control plant or part thereof in which the expression of the polynucleotide or the activity of the polypeptide is not modified.

Description

[Technical Field]

[0001] The present invention generally relates to Nicotiana tabacum plants having regulated expression or activity of lysine-ketoglutarate reductase (LKR) (also known as saccharopine dehydrogenase). [Background technology]

[0002] To produce tobacco products, different types of tobacco are mixed in various ratios to create blends with specific flavor characteristics. Flue-cured tobacco (e.g., Virginia) is the most widely grown tobacco and is characterized by a high sugar-to-nitrogen ratio, but it has a limited flavor profile. Other tobacco varieties, such as air-cured (e.g., Burley, Maryland, and Galpao) or flue-cured (e.g., dark) varieties, offer alternative flavor profiles. These different flavor profiles are important in the production of blended tobacco products. These flavor characteristics are the result of specific flavor compounds or precursors of these compounds present at specific levels in tobacco plants. Because the number of tobacco varieties available for commercial production is limited, this also means that there are limited opportunities to develop tobacco products with different flavor and aroma profiles. This also applies to the production of reconstituted tobacco materials used in heat-not-burn tobacco sticks in reduced-risk products.

[0003] There remains a need in the art for improved opportunities to create tobaccos that offer consumers new flavor and / or sensory experiences while still maintaining commercially acceptable yields and traits. The present invention seeks to address this and other needs. Summary of the Invention

[0004] The present invention is based, at least in part, on the surprising discovery that modulating Nicotiana tabacum LKR polynucleotide expression or Nicotiana tabacum LKR polypeptide expression can alter the chemical profile of cured Nicotiana tabacum plant material, such as cured leaves. Advantageously, this can be achieved without producing abnormal phenotypes in the Nicotiana tabacum plant, thereby conferring commercially acceptable yields and traits. Without wishing to be bound by theory, it is believed that the chemical changes occur through alterations in the senescence pathway (during the initial curing process) that can modulate the levels of essential amino acids, such as lysine. Further consistent changes in other amino acids (and sugars, etc.) have been observed, allowing for the engineering of tobacco materials with different flavor and / or sensory characteristics. This would be valuable for tobacco varieties that are widely grown commercially but have limited flavor profiles.

[0005] Zhu et al. (2001) Plant Physiol. 126(4):1539-45 described an Arabidopsis knockout mutant of LKR. The phenotype of this knockout was indistinguishable from that of wild-type plants under normal growth conditions. The relative levels of free Lys in leaves were measured and found to be similar between wild-type and LKR knockout mutants. No significant differences were observed in the relative levels of other free amino acids between wild-type and LKR knockout mutant leaves. Surprisingly, in contrast to the results seen in Arabidopsis, modulation of LKR can alter the amino acid profile of drought-treated Nicotiana tabacum plant material. For example, as described herein, surprisingly, reducing LKR expression or activity in Nicotiana tabacum can enrich drought-treated leaves with certain essential amino acids.

[0006] Disclosed is a mutant, non-native, or transgenic or engineered Nicotiana tabacum plant or part thereof (e.g., leaf, suitably cured leaf, etc.) having modulated (e.g., increased or decreased, suitably decreased) expression or activity of LKR, wherein the LKR is selected from the group consisting of (i) a polynucleotide comprising, consisting of, or consisting essentially of a sequence having at least 88% sequence identity with SEQ ID NO: 1 (NtLKR-S) and / or at least 86% sequence identity with SEQ ID NO: 3 (NtLKR-T); (ii) a polypeptide encoded by the polynucleotide of (i); (iii) a polypeptide having at least 89% sequence identity with SEQ ID NO: 2 (NtLKR-S) and / or at least 88% sequence identity with SEQ ID NO: 4 (NtLKR-T); (iv) a polypeptide comprising, consisting of, or consisting essentially of a sequence having the same identity as or similar to the sequence of any of the isolated polynucleotides described in (i), or (iv) a construct, vector, or expression vector comprising the isolated polynucleotide described in (i), wherein the plant or part thereof comprises at least one modification capable of modulating (a) the expression of the polynucleotide in the plant or part thereof, or (b) the activity of the polypeptide in the plant or part thereof, compared to a control plant or part thereof in which the expression of the polynucleotide or the activity of the polypeptide is not modified.

[0007] Preferably, the modification comprises at least one genetic alteration in the coding sequence of the polynucleotide or in a regulatory region of the polynucleotide.

[0008] Preferably, the modification comprises one or more of exogenous DNA or exogenous RNA.

[0009] Preferably, the modification comprises one or more of a vector, or a viral vector, or an Agrobacterium vector, or a CRISPR vector.

[0010] Preferably, the modification is capable of driving one or more of RNA interference or transcriptional gene silencing, or virus-induced gene silencing.

[0011] Preferably, the modification is capable of expressing one or more of double-stranded RNA (dsRNA) or hairpin RNA (hpRNA) or small interfering RNA.

[0012] Preferably, the regulated expression or activity of the LKR confers regulation of the level of one or more amino acids in the plant or part thereof compared to the level of one or more amino acids in a control plant, and preferably, the regulated expression or activity of the LKR confers regulation of the timing of leaf senescence.

[0013] Preferably, the amino acid is lysine.

[0014] Preferably, the part of the mutant, non-native or transgenic Nicotiana tabacum plant is a cured or dried leaf.

[0015] Preferably, the levels of at least lysine, arginine, GABA, glutamine, alanine, tyrosine, isoleucine, and threonine are modulated in the desiccation-treated or dried leaves compared to desiccation-treated or dried leaves obtained from a control plant.

[0016] Preferably, the Nicotiana tabacum plant or part thereof is of the burley species.

[0017] Preferably, the levels of at least lysine, arginine, proline, GABA, glutamine, leucine, alanine, phenylalanine, tyrosine, isoleucine, methionine, threonine, and glycine are adjusted in the desiccation-treated or desiccation-treated leaves, and there is no significant change in the levels of at least asparagine, aspartic acid, tryptophan, histidine, glutamic acid, serine, and valine, compared to the desiccation-treated or desiccation-treated leaves obtained from a control plant.

[0018] Preferably, the expression of LKR polynucleotide or activity of LKR polypeptide is reduced or inhibited in the desiccation-treated or desiccation-treated leaves, and compared to the desiccation-treated or desiccation-treated leaves obtained from a control plant, the desiccation-treated or desiccation-treated leaves exhibit (i) increased levels of at least lysine, arginine, proline, GABA, glutamine, leucine, alanine, phenylalanine, tyrosine, and isoleucine, (ii) decreased levels of at least methionine, threonine, and glycine, and (iii) no significant changes in the levels of at least asparagine, aspartic acid, tryptophan, histidine, glutamic acid, serine, and valine.

[0019] Preferably, the Nicotiana tabacum plant or part thereof is of the Virginia species.

[0020] Preferably, the levels of at least lysine, arginine, glutamine, histidine, tyrosine, tryptophan, threonine, GABA, asparagine, alanine, isoleucine, valine, and serine are modulated in the desiccated or dried leaves compared to desiccated or dried leaves obtained from a control plant, and there is no significant change in the levels of at least proline, aspartic acid, leucine, phenylalanine, glutamic acid, and methionine.

[0021] Preferably, the expression of LKR polynucleotide or the activity of LKR polypeptide is reduced or inhibited in the desiccation-treated or desiccation-treated leaves, and compared to the desiccation-treated or desiccation-treated leaves obtained from a control plant, the desiccation-treated or desiccation-treated leaves exhibit (i) increased levels of at least lysine, arginine, glutamine, histidine, tyrosine, tryptophan, threonine, GABA, asparagine, and alanine, (ii) decreased levels of at least isoleucine, valine, and serine, and (iii) no significant changes in the levels of at least proline, aspartic acid, leucine, phenylalanine, glutamic acid, and methionine.

[0022] Preferably the total sugar content is adjusted, preferably reduced.

[0023] In yet another aspect, disclosed is Nicotiana tabacum plant material, dried Nicotiana tabacum plant material, or homogenized Nicotiana tabacum plant material derived from or obtained from the above-mentioned Nicotiana tabacum plant or part thereof, preferably said Nicotiana tabacum plant material is selected from the group consisting of biomass, seeds, stems, flowers, or leaves, or a combination of two or more thereof, preferably said Nicotiana tabacum plant material is a leaf, preferably said leaf is dried, preferably said dried leaf is selected from the group consisting of hot-tube-dried leaves, sun-dried leaves, or air-dried leaves.

[0024] In yet another aspect, a method for producing a Nicotiana tabacum plant having a modulated level of at least one amino acid is disclosed, the method comprising: (a) providing a Nicotiana tabacum plant comprising (i) a polynucleotide comprising, consisting of, or consisting essentially of a sequence having at least 88% sequence identity to SEQ ID NO:1 (NtLKR-S) and / or at least 86% sequence identity to SEQ ID NO:3 (NtLKR-T), (ii) a polypeptide encoded by the polynucleotide set forth in (i), (iii) a polypeptide comprising, consisting of, or consisting essentially of a sequence having at least 89% sequence identity to SEQ ID NO:2 (NtLKR-S) and / or at least 88% sequence identity to SEQ ID NO:4 (NtLKR-T), or (iv) a construct, vector, or expression vector comprising the isolated polynucleotide set forth in (i); and (b) expressing an NtLKR polynucleotide in (a) the Nicotiana tabacum plant, or (b) expressing an NtLKR polynucleotide in the Nicotiana tabacum plant. The present invention also includes introducing at least one modification that is capable of modulating the activity of an NtLKR polypeptide in a tabacum plant compared to a control in which the expression of the NtLKR polynucleotide or the activity of the NtLKR polypeptide is not modified.

[0025] Preferably, in step (b), the at least one modification is introduced by genome editing, preferably the genome editing is selected from CRISPR-mediated genome editing, zinc finger nuclease-mediated mutagenesis, chemical or radiation mutagenesis, homologous recombination, oligonucleotide-directed mutagenesis, and meganuclease-mediated mutagenesis, or in step (b), the at least one modification is introduced using an interfering polynucleotide.

[0026] In yet another aspect, Nicotiana tabacum plant material obtained or obtainable by the above-described method is disclosed.

[0027] In yet another aspect, a method for producing dried Nicotiana tabacum plant material having an altered level of at least one amino acid is disclosed, the method comprising: (a) producing the Nicotiana tabacum plant described above; (b) harvesting plant material (e.g., leaves) from the Nicotiana tabacum plant; and (c) drying the plant material.

[0028] In yet another aspect, dried Nicotiana tabacum plant material (eg, leaves) obtained or obtainable by the above-described method is disclosed.

[0029] In yet another aspect, tobacco products are disclosed that comprise the Nicotiana tabacum plant material, cured Nicotiana tabacum plant material, or homogenized Nicotiana tabacum plant material, or that comprise the cured Nicotiana tabacum plant material.

[0030] Preferably, the tobacco product is a tobacco blend, and preferably the tobacco blend comprises Virginia and / or Burley tobacco.

[0031] Some advantages Advantageously, modulating the expression of NtLKR polynucleotides or the activity of NtLKR polypeptides can result in modulating the levels of amino acids, particularly in flue-cured tobacco plant material, which can result in tobacco with novel flavor and / or organoleptic properties.

[0032] Advantageously, non-genetically modified plants can be produced that are more acceptable to consumers.

[0033] Advantageously, the present disclosure is not limited to the use of ethyl methanesulfonate (EMS) mutant plants. EMS mutant plants may be less likely to bring trait improvements to subsequent crops. Once breeding begins, desirable traits of EMS mutant plants may be lost for various reasons. For example, several mutations may be required, but these mutations may be dominant or recessive, and identifying point mutations in gene targets may be difficult to achieve. In contrast, the present disclosure utilizes the use of NtLKR, which can be specifically engineered to produce plants with desired phenotypes.

[0034] Advantageously, no abnormal phenotypes were observed, making the plants suitable for commercial production.

[0035] Downregulation of the asparagine synthetase (ASN) gene (WO2017042162; Bovet et al. (2019) Plants (Basel) 11;8(11):492) also significantly alters tobacco chemistry without affecting biomass. Advantageously, combining both ASN-regulated plants with NtLKR-regulated plants could potentially reconfigure the chemistry, such as the amino acid chemistry, of Burley, Dark, or Virginia tobacco, further altering flavor and / or sensory characteristics. Other genes and enzymes also play a role in amino acid and / or sugar reorganization during leaf yellowing, such as diaminopimelate aminotransferase (DAPAT), aspartate aminotransferase (AAT), and the chloroplast sulfate transporter (SULTR3), which may also alter leaf chemistry. Altering NtLKR expression or activity in conjunction with the expression or activity of one or more of these other targets selected from one or more of ASN, DAPAT, and AAT may be used to further modify the flavor and / or sensory properties of flue-cured tobacco.

[0036] Advantageously, no significant difference in alkaloid content was observed, which means that the nicotine levels remain unchanged and therefore the same amount of nicotine can be delivered to the tobacco consumer. [Brief explanation of the drawings]

[0037] [Figure 1] Figure 1 shows two graphs demonstrating the identification of NtLKR-RNAi plants with low NtLKR expression via qRT-PCR. The relative expression of TN90 (CT1-E438-4 n=4, CT1-E438-5 n=5, CT1-E438-6 n=5, T1-E438-2 n=4, T1-E438-5 n=5, and T1-E438-11 n=5) is shown. The relative expression of Virginia K326 (CT1-E437-5 n=5, CT1-E437-6 n=4, T1-E437-11 n=4, T1-E437-12 n=5, and T1-E437-15 n=4) is shown. The tissue used for RNA isolation was the green midrib / leaf blade. [Figure 2] FIG. 2 is a graph showing chlorophyll measurements (CCI, 3 measurements per plant) at stem position C 21 days after switching the nutrient solution. [Figure 3] Figure 3 shows three graphs showing the content of three free amino acids, Lys, Arg, and Pro, in three independent NtLKR-RNAi lines (E438-2, -5, -11) compared with the WT (CT1-E438). Statistical analysis was performed using ANOVA and Tukey's HSD test. [Figure 4] Figure 4 shows six graphs showing the contents of the three free amino acids Lys, Arg, and Gln, the reducing sugars glucose and fructose, and nitrate in three independent NtLKR-RNAi lines (E43711, -12, and -15) compared with the WT (CT1-E437). Statistical analysis was performed using ANOVA and Tukey's HSD test. [Figure 5] FIG. 5 is a graph showing the expression of NtLKR during early air-drying and early hot-tube drying treatments. DETAILED DESCRIPTION OF THE INVENTION

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. In case of conflict, the present document, including definitions, shall prevail. Preferred methods and materials are described below, but methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. The materials, methods, and examples disclosed herein are illustrative only and are not intended to be limiting.

[0039] The terms "comprise," "include," "having," "having," "can," "containing," and variations thereof are intended to be open-ended transitional phrases, terms, or words that do not exclude the possibility of additional acts or configurations.

[0040] The singular forms "a," "and," and "the" include plural references unless the context clearly dictates otherwise.

[0041] The present disclosure contemplates other embodiments that "comprise," "consist," and "consist essentially of" the embodiments or elements presented herein, whether or not explicitly stated.

[0042] For purposes of describing numerical ranges herein, each intervening number of the same degree of precision is expressly contemplated. For example, for the range 6 to 9, the numbers 7 and 8 are contemplated in addition to the numbers 6 and 9, and for the range 6.0 to 7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are expressly contemplated.

[0043] As used throughout the specification and claims, the following terms have the following meanings:

[0044] "Coding sequence" or "encoding polynucleotide" refers to a nucleotide (RNA or DNA molecule) comprising a polynucleotide that encodes a polypeptide. The coding sequence may further comprise initiation and termination signals operably linked to regulatory elements, including a promoter and polyadenylation signal, capable of directing expression in the cells of an individual or mammal to which the polynucleotide is administered. The coding sequence may be codon-optimized.

[0045] "Complement" or "complementary" can refer to Watson-Crick (e.g., AT / U and CG) or Hoogsteen base pairing between nucleotides or nucleotide analogs. "Complementarity" refers to the property shared between two polynucleotides such that when aligned antiparallel to each other, the nucleotide bases at each position are complementary.

[0046] A "construct" refers to a double-stranded recombinant polynucleotide fragment comprising one or more polynucleotides. A construct comprises a "template strand" base-paired with a complementary "sense or coding strand." A given construct can be inserted into a vector in either of two possible orientations: the same (or sense) orientation or the opposite (or antisense) orientation relative to the orientation of a promoter placed within a vector, such as an expression vector.

[0047] The term "control," in the context of a control plant or control plant cell, refers to a plant or plant cell in which the expression, function, or activity of one or more genes or polypeptides has not been altered (e.g., increased or decreased), thereby providing a comparison with a plant in which the expression, function, or activity of one or more genes or polypeptides has been altered. A "control plant" is a plant that is substantially equivalent to a test plant or modified plant in all parameters except the test parameter. For example, when referring to a plant into which a polynucleotide has been introduced, the control plant is an equivalent plant into which such polynucleotide has not been introduced. A control plant can be an equivalent plant into which a control polynucleotide has been introduced. In such cases, the control polynucleotide is one that is expected to result in little or no phenotypic effect on the plant. A control plant can contain an empty vector. A control plant can correspond to a wild-type plant. A control plant can be a null segregant, in which the T1 segregant no longer carries the transgene.

[0048] The term "reduced" or "reduced" refers to a reduction of about 10% to about 99% in amount or function, such as polypeptide function, transcription function, or polypeptide expression, or a reduction of at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100%, or at least 150%, or at least 200% or more. The term "reduced" or the phrase "reduced amount" can refer to an amount or function that is less than that found in an unmodified plant or a product derived from a plant of the same variety processed in the same manner. Thus, in some contexts, wild-type plants of the same variety processed in the same manner are used as a control to determine whether a reduction in amount is obtained.

[0049] "Donor DNA" or "donor template" refers to a double-stranded DNA fragment or molecule that contains at least a portion of a gene of interest. The donor DNA can encode a functional polypeptide.

[0050] An "endogenous gene or polypeptide" refers to a gene or polypeptide that originates from the genome of an organism and has not undergone alteration, such as deletion, gain, or replacement of genetic material. An endogenous gene undergoes normal gene transmission and gene expression. An endogenous polypeptide undergoes normal expression.

[0051] "Enhancer sequences" refer to sequences that can increase gene expression. These sequences can be located upstream, within introns, or downstream of the transcribed region. The transcribed region consists of exons and intervening introns from the promoter to the transcription termination region. Enhancement of gene expression can be through various mechanisms, including increasing transcription efficiency, stabilizing mature mRNA, and enhancing translation.

[0052] "Expression" refers to the production of a functional product. For example, expression of a polynucleotide fragment can refer to transcription of the polynucleotide fragment (e.g., transcription to produce mRNA or functional RNA), or translation of mRNA into a precursor or mature polypeptide, or a combination thereof.

[0053] "Overexpression" means the production of a gene product in transgenic organisms that exceeds production levels in null segregating (or non-transgenic) organisms of the same experiment.

[0054] "Functional" describes a polypeptide that has a biological function or activity. A "functional gene" refers to a gene that is transcribed into mRNA that is translated into a functional or active polypeptide.

[0055] A "genetic construct" refers to a DNA or RNA molecule comprising a polynucleotide that encodes a polypeptide. The coding sequence may include initiation and termination signals operably linked to regulatory elements, including a promoter and polyadenylation signal, capable of directing expression.

[0056] "Genome editing" generally refers to a process in which genomic nucleic acid is altered in a cell. This can be, for example, by removing, inserting, or substituting one or more nucleotides in the genomic nucleic acid. Endonucleases can be used to create specific cuts or nicks at defined locations within the genome, as further described herein.

[0057] The terms "homology" or "similarity" refer to the degree of sequence similarity between two polypeptide molecules or two polynucleotide molecules compared by sequence alignment. The degree of homology between two separate polynucleotides being compared is a function of the number of identical or matching nucleotides at comparable positions. Homology or similarity can be determined over the entire length of the subject sequence.

[0058] "Identical" or "identity" in the context of two or more polynucleotides or polypeptides means that the sequences have a specified percentage of residues that are the same over a specified region. This percentage is calculated by optimally aligning two sequences, comparing the two sequences over a specified region, determining the number of positions where identical residues occur in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the specified region, and multiplying the result by 100 to obtain the percentage of sequence identity. If the two sequences are of different lengths or the alignment results in one or more staggered ends and a specified comparison region contains only a single sequence, the residues of the single sequence are included in the denominator of the calculation but not in the numerator. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identity can be determined manually or by using a computer sequence algorithm such as ClustalW, ClustalX, BLAST, FASTA, or Smith-Waterman. Suitable parameters for ClustalW can be as follows: For polynucleotide alignments: Gap opening penalty = 15.0, gap extension penalty = 6.66, and matrix = Identity. For polypeptide alignments: Gap opening penalty = 10.0, gap extension penalty = 0.2, and matrix = Gonnet. For DNA and protein alignments: ENDGAP = -1, and GAPDIST = 4.

[0059] The term "increase" or "increased" refers to an increase of about 10% to about 99% in the amount or function or activity of one or more of, for example, but not limited to, polypeptide function or activity, transcriptional function or activity, and polypeptide expression, or an increase of at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, at least 100%, at least 150%, or at least 200% or more. The term "increase" or "increased amount" can refer to an amount or function or activity in a plant or a product produced from a plant that is greater than that which would be found in an unmodified plant or a product derived from a plant of the same variety processed in the same manner. Thus, in some contexts, wild-type plants of the same variety processed in the same manner are used as a control to determine whether an increase in amount is obtained.

[0060] The term "inhibit" or "inhibited" refers to about a 98% to about 100% decrease, or at least a 98%, at least a 99%, but particularly a 100% decrease, in the amount or function or activity of one or more of, for example, but not limited to, a polypeptide function or activity, a transcriptional function or activity, and polypeptide expression.

[0061] The term "introduced" means providing a polynucleotide (e.g., a construct) or polypeptide into a cell. "Introduced" includes reference to the uptake of a polynucleotide into a eukaryotic cell, where the polynucleotide may be incorporated into the genome of the cell, and includes reference to the transient provision of a polynucleotide or polypeptide to a cell. "Introduced" includes reference to stable or transient transformation methods, as well as sexual crossing. Thus, "introduced" in the context of inserting a polynucleotide (e.g., a recombinant / expression construct) into a cell means "transfection" or "transformation" or "transduction," and includes reference to the uptake of a polynucleotide into a eukaryotic cell, where the polynucleotide may be integrated into the genome of the cell (e.g., a chromosome, a plasmid, a plastid, or a mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).

[0062] The terms "isolated" or "purified" refer to a material that is substantially or essentially free from components that normally accompany it when found in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high-performance liquid chromatography. A polypeptide that is the predominant species present in a preparation is substantially purified. In particular, an isolated polynucleotide is separated from open reading frames that flank the desired gene and encode polypeptides other than the desired polypeptide. The term "purified" means that the polynucleotide or polypeptide gives rise to essentially one band in an electrophoretic gel. Specifically, this means that the polynucleotide or polypeptide is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure. An isolated polynucleotide can be purified from a naturally occurring host cell. Conventional polynucleotide purification methods known to those skilled in the art can be used to obtain an isolated polynucleotide. The term also encompasses recombinant and chemically synthesized polynucleotides.

[0063] "Liquid tobacco extract" describes the direct product of an extraction process performed on tobacco starting material. The extraction process to produce a liquid tobacco extract may involve heating the tobacco starting material under specific heating conditions and collecting the volatile compounds that are produced. A liquid tobacco extract may contain a mixture of compounds that originate from the tobacco starting material and that have been removed during the extraction process, typically in combination with a liquid carrier or solvent.

[0064] "Modulate" or "modulating" refers to causing or promoting a qualitative or quantitative change, alteration, or modification in a process, pathway, function, or activity of interest. Without limitation, such a change, alteration, or modification can be an increase or decrease in the relative process, pathway, function, or activity of interest. For example, gene expression or polypeptide expression, or polypeptide function or activity, can be modulated. Typically, the relative change, alteration, or modification will be determined by comparison with a control.

[0065] The term "non-natural" describes entities such as polynucleotides, genetic mutations, polypeptides, plants, plant cells, and plant materials that are not naturally occurring or do not exist in nature. Such non-natural or artificial entities can be produced, synthesized, initiated, modified, intervened, or manipulated by methods described herein or known in the art. Such non-natural or artificial entities can be produced, synthesized, initiated, modified, intervened, or manipulated by humans. Thus, non-natural plants may not be produced using essentially biological processes. Thus, for example, non-natural plants, non-natural plant cells, or non-natural plant materials can be produced using traditional plant breeding techniques such as backcrossing, or by genetic engineering techniques such as antisense RNA, interfering RNA, and meganucleases. As yet another example, a non-naturally occurring plant, plant cell, or plant material can be created by introgressing or transferring one or more genetic mutations (e.g., one or more polymorphisms) from a first plant or plant cell into a second plant or plant cell (which itself may be natural) so that the resulting plant, plant cell, or plant material, or its progeny, contains a genetic makeup (e.g., a genome, chromosome, or segment thereof) that is not naturally formed or occurs naturally. The resulting plant, plant cell, or plant material is therefore artificial or non-natural. Thus, an artificial or non-naturally occurring plant or plant cell can be created by modifying a genetic sequence in a first naturally occurring plant or plant cell, even if the resulting genetic sequence is naturally occurring in a second plant or plant cell that contains a different genetic background from the first plant or plant cell. In certain embodiments, the mutation is not a naturally occurring mutation that occurs naturally in a polynucleotide or polypeptide, such as a gene or polypeptide. Differences in genetic background can be detected by phenotypic differences or by molecular biology techniques known in the art, such as polynucleotide sequencing, the presence or absence of genetic markers (e.g., microsatellite RNA markers).

[0066] "Oligonucleotide" or "polynucleotide" refers to at least two nucleotides covalently linked together. A depiction of a single strand also defines the sequence of the complementary strand. Thus, a polynucleotide also encompasses the complementary strand of the depicted single strand. Many variants of a polynucleotide can be used for the same purpose as a given polynucleotide. Thus, a polynucleotide also encompasses substantially identical polynucleotides and their complements. A single strand provides a probe that can hybridize to a given sequence under stringent hybridization conditions. Thus, a polynucleotide also encompasses a probe that hybridizes under stringent hybridization conditions. A polynucleotide can be single-stranded or double-stranded, or can contain portions of both double-stranded and single-stranded sequences. The polynucleotides can be DNA, both genomic and cDNA, RNA, or hybrids, and can contain combinations of deoxyribonucleotides and ribonucleotides, as well as combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. Polynucleotides can be obtained by chemical synthesis or by recombinant methods.

[0067] The specificity of single-stranded DNA for hybridizing complementary fragments is determined by the "stringency" of the reaction conditions (Sambrook et al., Molecular Cloning and Laboratory Manual, Second Ed., Cold Spring Harbor (1989)). Hybridization under "stringent conditions" describes a hybridization protocol in which polynucleotides that share at least 60% homology with each other remain hybridized. Generally, stringent conditions are selected to be approximately 5°C lower than the thermal melting point (Tm) of a specific sequence at a defined ionic strength and pH. Tm is the temperature (under defined ionic strength, pH, and polynucleotide concentration) at which 50% of the probes complementary to a given sequence hybridize to the given sequence at equilibrium. Since a given sequence is generally present in excess at Tm, 50% of the probes are occupied at equilibrium.

[0068] Stringent conditions typically include (1) low ionic strength and high temperature washes, e.g., 15 mM sodium chloride, 1.5 mM sodium citrate, 0.1% sodium dodecyl sulfate at 50°C; (2) denaturing agents, e.g., 50% (v / v) formamide, 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer (750 mM sodium chloride, 75 mM sodium citrate; pH 6.5), during hybridization at 42°C; or (3) 50% formamide. Washing also typically includes a wash in 0.2×SSC (sodium chloride / sodium citrate) containing 5×SSC (0.75 M NaCl, 75 mM sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50 μg / mL), 0.1% SDS, and 10% dextran sulfate at 42° C., a wash in 50% formamide at 55° C., followed by a high stringency wash consisting of 0.1×SSC containing EDTA at 55° C. Preferably, the conditions are such that sequences having at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% homology to each other typically remain hybridized to each other.

[0069] "Moderately stringent conditions" use less stringent washing solutions and hybridization conditions so that a polynucleotide hybridizes to the entire polynucleotide, fragments, derivatives, or analogs thereof. One example includes hybridization in 6xSSC, 5xDenhardt's solution, 0.5% SDS, and 100 μg / mL denatured salmon sperm DNA at 55°C, followed by one or more washes in 1xSSC, 0.1% SDS at 37°C. Temperature, ionic strength, etc. can be adjusted to accommodate experimental factors such as probe length. Other conditions of moderate stringency have been described (see Ausubel et al., Current Protocols in Molecular Biology, Volumes 1-3, John Wiley & Sons, Inc., Hoboken, NJ (1993); Kriegler, Gene Transfer and Expression: A Laboratory Manual, Stockton Press, New York, NY (1990); Perbal, A Practical Guide to Molecular Cloning, 2nd edition, John Wiley & Sons, New York, NY (1988)).

[0070] "Low stringency conditions" use washing solutions and hybridization conditions that are less stringent than those used for moderate stringency, such that a polynucleotide hybridizes to the entire polynucleotide, a fragment, a derivative, or an analog thereof. Non-limiting examples of low stringency hybridization conditions include hybridization in 35% formamide, 5xSSC, 50 mM Tris HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg / mL denatured salmon sperm DNA, 10% (weight / volume) dextran sulfate at 40°C, followed by one or more washes in 2xSSC, 25 mM Tris HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS at 50°C. Other conditions of low stringency, such as those for cross-species hybridizations, have been well described (see Ausubel et al., 1993; Kriegler, 1990).

[0071] "Operably linked" means that the expression of a gene is under the control of a spatially linked promoter. The promoter can be located 5' (upstream) or 3' (downstream) of the gene under its control. The distance between the promoter and the gene can be approximately the same as the distance between the promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, variation in this distance can be adjusted without loss of promoter function. "Operably linked" refers to the association of polynucleotide fragments within a single fragment such that the function of one is controlled by the other. For example, a promoter is operably linked to a polynucleotide fragment when it is capable of controlling the transcription of the polynucleotide fragment.

[0072] The term "plant" refers to any plant at any stage of its life cycle or development, and its progeny. In one embodiment, the plant is a tobacco plant and refers to a plant belonging to the genus Nicotiana. The term includes reference to whole plants, plant organs, plant tissues, plant propagules, plant seeds, plant cells, and their progeny. Plant cells include, but are not limited to, cells obtained from seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and pollen grains. Suitable species, cultivars, hybrids, and varieties of tobacco plants are described herein.

[0073] "Plant material" includes leaves, roots, sepals, root tips, petals, flowers, shoots, stems, seeds, and stalks. Plant material can be viable or non-viable plant material.

[0074] "Polynucleotide," "polynucleotide sequence," or "polynucleotide fragment" are used interchangeably herein and refer to a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural, or altered nucleotide bases. The polynucleotides of this disclosure are set forth in the accompanying Sequence Listing.

[0075] "Polypeptide" or "polypeptide sequence" refers to a polymer of amino acids in which one or more amino acid residues are artificial chemical analogues of corresponding naturally occurring amino acids, as well as naturally occurring polymers of amino acids. These terms also include modifications, including, but not limited to, glycosylation, lipid addition, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation. The polypeptides of the present disclosure are set forth in the accompanying sequence listing.

[0076] "Promoter" refers to a synthetic or naturally occurring molecule capable of conferring, activating, or enhancing expression of a polynucleotide in a cell. The term refers to a polynucleotide element / sequence, typically located upstream and operably linked to a double-stranded polynucleotide fragment. A promoter can be derived entirely from the region adjacent to a native gene of interest or can be composed of different elements derived from different native promoters or synthetic polynucleotide segments. A promoter can contain one or more specific transcription control sequences to further enhance expression or to alter spatial or temporal expression. A promoter can also contain distal enhancer or repressor elements and can be located up to thousands of base pairs from the start site of transcription. Promoters can be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. A promoter can constitutively or differentially control expression of genetic components depending on the cell, tissue, or organ in which expression occurs, the developmental stage in which expression occurs, or in response to external stimuli such as physiological stress, pathogens, metal ions, or inducers.

[0077] As used interchangeably herein, "tissue-specific promoter" and "tissue-preferred promoter" refer to promoters that are preferentially expressed in one tissue or organ, although not necessarily exclusively, and that can also be expressed in one particular cell. A "developmentally regulated promoter" refers to a promoter whose function is determined by developmental events. A "constitutive promoter" refers to a promoter that causes a gene to be expressed in most cell types at most times. An "inducible promoter" selectively expresses an operably linked DNA sequence in response to the presence of an endogenous or exogenous stimulus, for example, a chemical compound (a chemical inducer), or in response to an environmental, hormonal, chemical, or developmental signal, or a combination of two or more thereof. Examples of inducible or regulated promoters include promoters that are regulated by light, heat, pressure, waterlogging or drought, pathogens, plant hormones, wounding, or chemicals such as ethanol, jasmonic acid, salicylic acid, or safeners.

[0078] "Recombinant" refers to the artificial combination of two otherwise separated segments of sequence, such as by chemical synthesis or by the manipulation of isolated segments of polynucleotides by genetic engineering techniques. The term also includes reference to a cell or vector that has been modified by the introduction of a heterologous polynucleotide, or a cell derived from a cell so modified, but does not encompass the alteration of a cell or vector by natural events (e.g., spontaneous mutation, natural transformation or transduction or transposition), such as those that occur without deliberate human intervention.

[0079] "Recombinant construct" refers to a combination of polynucleotides that are not normally found together in nature. Thus, a recombinant construct can contain control and coding sequences from different sources, or control and coding sequences from the same source but arranged in a manner different from that normally found in nature. A recombinant construct can be a recombinant DNA construct.

[0080] As used interchangeably herein, "control sequence" and "control element" refer to polynucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence that influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Control sequences include promoters, translation leader sequences, introns, and polyadenylation recognition sequences. The terms "control sequence" and "control element" are used interchangeably herein.

[0081] The term "tobacco" is used collectively to refer to tobacco crops (e.g., field-grown, non-hydroponic tobacco plants) prepared or obtained as described herein, tobacco plants and parts thereof, including, but not limited to, roots, stems, leaves, flowers, and seeds. "Tobacco" refers to plants belonging to the genus Nicotiana and their products, and is understood to include Nicotiana tabacum plants and their products.

[0082] The term "tobacco products" refers to consumer tobacco products, including, but not limited to, smoking materials (e.g., cigarettes, cigars, and pipe tobacco), snuff, chewing tobacco, gum, and lozenges, as well as components, materials, and ingredients for the manufacture of consumer tobacco products. Preferably, these tobacco products are made from tobacco leaves and stems harvested from tobacco plants and cut, dried, cured, or fermented according to conventional techniques for tobacco preparation.

[0083] "Transcription terminator," "termination sequence," or "terminator" refers to a DNA sequence located downstream of a coding sequence, including polyadenylation recognition sequences and other sequences encoding regulatory signals that can affect mRNA processing or gene expression. Polyadenylation signals are typically characterized by affecting the addition of polyadenylic acid moieties to the 3' end of a pre-mRNA.

[0084] "Transgenic" refers to any cell, cell line, callus, tissue, plant part, or plant whose genome has been altered by the presence of a heterologous polynucleotide, such as a recombinant construct, including the original transgenic event, as well as those produced from the original transgenic event by sexual crossing or asexual propagation. The term does not encompass alterations of the genome (chromosomal or extrachromosomal) by conventional plant breeding methods or by natural events, such as random cross-pollination, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation. Thus, in embodiments, a transgenic plant or part thereof is not produced using an essentially biological process.

[0085] A "transgenic plant" refers to a plant that contains one or more heterologous polynucleotides within its genome, i.e., a plant containing recombinant genetic material not normally found therein that has been introduced into the plant (or an ancestor of the plant) by human manipulation. For example, a heterologous polynucleotide may be stably integrated into the genome so that the polynucleotide is transmitted to successive generations. A heterologous polynucleotide may be integrated into the genome alone or as part of a recombinant construct. Commercial development of genetically improved germplasm has also progressed to the stage of introducing multiple traits into crop plants, often referred to as gene stacking. In this approach, multiple genes that confer different characteristics of interest may be introduced into a plant. Gene stacking can be achieved by many means, including, but not limited to, co-transformation, retransformation, and cross-breeding of lines harboring different transgenes. Thus, a plant grown from a plant cell into which recombinant DNA has been introduced by transformation is a transgenic plant, as are all progeny of the plant that contain the introduced transgenes (whether produced sexually or asexually). The term transgenic plant is understood to encompass whole plants or trees as well as parts of plants or trees, such as grains, seeds, flowers, leaves, roots, fruit, pollen, stems, etc. Each heterologous polynucleotide may confer a different trait to the transgenic plant.

[0086] "Transgene" refers to a gene or genetic material containing a genetic sequence that has been isolated from one organism and introduced into a different organism. This non-native segment of DNA may retain the ability to produce RNA or polypeptides in the transgenic organism, or may alter the normal function of the transgenic organism's genetic code.

[0087] "Variant" with respect to a polynucleotide means (i) a portion or fragment of the polynucleotide, (ii) the complement of a polynucleotide or a portion thereof, (iii) a polynucleotide that is substantially identical to a subject polynucleotide or its complement, or (iv) a polynucleotide that hybridizes under stringent conditions to a subject polynucleotide, its complement, or a substantially identical polynucleotide.

[0088] "Variant" with respect to a peptide or polypeptide refers to a peptide or polypeptide that differs in sequence by amino acid insertion, deletion, or conservative substitution, but retains at least one biological function or activity. A variant can also refer to a polypeptide that retains at least one biological function or activity. Conservative amino acid substitutions, i.e., replacing one amino acid with a different amino acid with similar properties (e.g., hydrophilicity, degree and distribution of charged regions), are recognized in the art as typically resulting in minor changes.

[0089] The term "variety" refers to a group of plants that share certain characteristics that distinguish them from other plants of the same species. While possessing one or more distinctive traits, varieties are further characterized by very little overall variation among individuals within the variety. Varieties are often commercially available.

[0090] A "vector" refers to a polynucleotide vehicle containing a combination of polynucleotide components to enable the transport of polynucleotides, polynucleotide constructs, and polynucleotide conjugates. A vector can be a viral vector, bacteriophage, bacterial artificial chromosome, or yeast artificial chromosome. A vector can be a DNA vector or an RNA vector. Suitable vectors include episomes capable of extrachromosomal replication, such as circular double-stranded nucleotide plasmids, linear double-stranded nucleotide plasmids, and other vectors of any origin. An "expression vector" is a polynucleotide vehicle containing a combination of polynucleotide components to enable the expression of polynucleotides, polynucleotide constructs, and polynucleotide conjugates. Suitable expression vectors include episomes capable of extrachromosomal replication, such as circular double-stranded nucleotide plasmids, linear double-stranded nucleotide plasmids, and other functionally equivalent expression vectors of any origin. An expression vector at least includes a promoter, as defined below, located upstream and operably linked to a polynucleotide, polynucleotide construct, or polynucleotide conjugate.

[0091] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those skilled in the art. For example, any nomenclature used in connection with cell and tissue culture, molecular biology, plant biology, microbiology, genetics, and polypeptide and polynucleotide chemistry, and hybridization described herein, and the techniques thereof, are well known and commonly used in the art. The meaning and scope of terms should be clear, but in the event of any potential ambiguity, the definitions provided herein shall take precedence over any dictionary or external definitions. Furthermore, unless otherwise required by context, singular terms shall include plurals, and plural terms shall include the singular.

[0092] Disclosed are isolated polynucleotides comprising, consisting of, or consisting essentially of a sequence having at least 60% sequence identity to any of the sequences described herein, including any polynucleotide set forth in the Sequence Listing. Preferably, the isolated polynucleotide comprises, consists of, or consists essentially of a sequence having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto. Preferably, the isolated polynucleotide comprises, consists of, or consists essentially of a sequence having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto. Preferably, the isolated polynucleotide comprises, consists of, or consists essentially of a sequence having at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto.

[0093] Preferably, the polynucleotides described herein encode active polypeptides having at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 100% or more of the LKR function or activity of the polypeptides set forth in the sequence listing.

[0094] In another embodiment, an LKR polynucleotide from Nicotiana tabacum is disclosed, comprising, consisting of, or consisting essentially of a polynucleotide having at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1 (NtLKR-S), or 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 3 (NtLKR-T).

[0095] In another embodiment, a polynucleotide is provided that comprises, consists of, or consists essentially of a polynucleotide having substantial homology (i.e., sequence similarity) or substantial identity to SEQ ID NO:1 or SEQ ID NO:3.

[0096] In another embodiment, fragments of SEQ ID NO:1 or SEQ ID NO:3 are provided that have substantial homology (i.e., sequence similarity) or substantial identity thereto, having at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to the corresponding fragment of SEQ ID NO:1 or SEQ ID NO:3.

[0097] In another embodiment, fragments of SEQ ID NO:1 are provided that have substantial homology (i.e., sequence similarity) or substantial identity thereto, having at least about 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to the corresponding fragment of SEQ ID NO:1.

[0098] In another embodiment, fragments of SEQ ID NO: 3 are provided that have substantial homology (i.e., sequence similarity) or substantial identity thereto, having at least about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to the corresponding fragment of SEQ ID NO: 3.

[0099] In another embodiment, a polynucleotide comprising a sufficient or substantial degree of identity or similarity to SEQ ID NO: 1 or SEQ ID NO: 3 that encodes a polypeptide that functions as an LKR is provided.

[0100] In another embodiment, there is provided a polymer of polynucleotides comprising, consisting of, or consisting essentially of the polynucleotide referred to herein as SEQ ID NO:1 or SEQ ID NO:3.

[0101] Preferably, the polynucleotides described herein encode an NtLKR polypeptide having LKR activity.

[0102] A polynucleotide can comprise a polymer of nucleotides, which can be unmodified or modified deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Thus, a polynucleotide can be, but is not limited to, genomic DNA, complementary DNA (cDNA), mRNA, or antisense RNA, or fragments thereof. Moreover, a polynucleotide can be single-stranded or double-stranded DNA, a DNA that is a mixture of single-stranded and double-stranded regions, a hybrid molecule containing DNA and RNA, or a hybrid molecule with a mixture of single-stranded and double-stranded regions or fragments thereof. Additionally, a polynucleotide can be composed of triple-stranded regions containing DNA, RNA, or both, or fragments thereof. A polynucleotide can contain one or more modified bases, such as phosphothioates, and can be a peptide nucleic acid. Generally, a polynucleotide can be assembled from isolated or cloned fragments of cDNA, genomic DNA, oligonucleotides, or individual nucleotides, or combinations of the foregoing. The polynucleotides described herein are depicted as DNA sequences, but also include their corresponding RNA sequences and their complementary (e.g., perfectly complementary) DNA or RNA sequences, including their reverse complements.

[0103] Fragments of polynucleotides can range from at least about 25 nucleotides, about 50 nucleotides, about 75 nucleotides, about 100 nucleotides, about 150 nucleotides, about 200 nucleotides, about 250 nucleotides, about 300 nucleotides, about 400 nucleotides, about 500 nucleotides, about 600 nucleotides, about 700 nucleotides, about 800 nucleotides, about 900 nucleotides, about 1000 nucleotides, about 1100 nucleotides, about 1200 nucleotides, about 1300 nucleotides, or about 1400 nucleotides, up to a full-length polynucleotide encoding a polypeptide described herein.

[0104] Polynucleotides generally contain phosphodiester bonds, but in some cases, polynucleotide analogs may have alternative backbones, such as phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphosphoramidite bonds, as well as peptide polynucleotide backbones and their linkages. Other analog polynucleotides include those with positively charged backbones, non-ionic backbones, and non-ribose backbones. Modifications of the ribose-phosphate backbone may be made for various reasons, such as to increase the stability and half-life of such molecules in physiological environments or as probes on biochips. Mixtures of natural polynucleotides and analogs may be produced, or alternatively, mixtures of different polynucleotide analogs and mixtures of natural polynucleotides and analogs may be produced.

[0105] A variety of polynucleotide analogs are known, including, for example, phosphoramidate, phosphorothioate, phosphorodithioate, O-methylphosphoramidite, and peptide polynucleotide backbones and linkages. Other analog polynucleotides include those with positively charged backbones, non-ionic backbones, and non-ribose backbones. Polynucleotides containing one or more carbocyclic sugars are also included.

[0106] Other analogs include peptide polynucleotides, which are peptide polynucleotide analogs.

[0107] Among the uses of the disclosed polynucleotides and their fragments are the use of the fragments as probes in hybridization assays or as primers for use in amplification assays.Such fragments generally comprise at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more consecutive nucleotides of a DNA sequence.In other embodiments, the DNA fragments comprise at least about 10, 15, 20, 30, 40, 50, or 60 or more consecutive nucleotides of a DNA sequence.Therefore, in one aspect, a method for detecting a polynucleotide is also provided, comprising the use of a probe or a primer, or both.

[0108] The basic parameters that influence the selection of hybridization conditions, and guidance for devising suitable conditions, are described by Sambrook, J., E.F. Fritsch, and T. Maniatis (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). Knowledge of the genetic code, combined with the polypeptide sequences described herein, can be used to prepare sets of degenerate oligonucleotides. Such oligonucleotides are useful, for example, as primers in polymerase chain reactions (PCR), in which DNA fragments are isolated and amplified.

[0109] At least one modification (eg, mutation) may be included in one or more of SEQ ID NO:1 and SEQ ID NO:3.

[0110] Isolated LKR polypeptides encoded by the polynucleotides described herein are provided.

[0111] Also provided is an isolated LKR polypeptide comprising, consisting of, or consisting essentially of a polypeptide having at least 60% sequence identity to any of the polypeptides described herein, including any of the polypeptides set forth in the Sequence Listing. Preferably, the isolated polypeptide comprises, consists of, or consists essentially of a sequence having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, 80%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity thereto. Suitably, the isolated LKR polypeptide comprises, consists of, or consists essentially of a sequence having at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity thereto. Suitably, the isolated LKR polypeptide comprises, consists of, or consists essentially of a sequence having at least 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity thereto.

[0112] Also provided are LKR polypeptides comprising, consisting of, or consisting essentially of a sequence having at least 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity to SEQ ID NO: 2 (NtLKR-S).

[0113] Also provided are LKR polypeptides comprising, consisting of, or consisting essentially of a sequence having at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity to SEQ ID NO: 4 (NtLKR-T).

[0114] Also provided are LKR polypeptides comprising, consisting of, or consisting essentially of a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity to SEQ ID NO:2 or SEQ ID NO:4.

[0115] Polypeptides encoded by SEQ ID NO:2 or SEQ ID NO:4 are also provided.

[0116] The polypeptide comprises a sequence that contains a sufficient or substantial degree of identity or similarity to SEQ ID NO:2 or SEQ ID NO:4 and is capable of functioning as an LKR.

[0117] Fragments of the polypeptides described herein are also contemplated. Polypeptide fragments typically retain some or all of the function or activity of the full-length sequence, such as LKR activity. Polypeptide fragments can range from at least about 25 amino acids, about 50 amino acids, about 75 amino acids, about 100 amino acids, about 150 amino acids, about 200 amino acids, about 250 amino acids, about 300 amino acids, about 400 amino acids, or about 500 amino acids up to the full-length polypeptides described herein.

[0118] Polypeptides can also be intentionally engineered or naturally isolated, and include variants produced by introducing any type of alteration (e.g., amino acid insertion, deletion, or substitution; changes in glycosylation status; changes affecting refolding or isomerization, three-dimensional structure, or self-association), provided they still possess some or all of their function or activity. Preferably, this function or activity is modulated.

[0119] Deletion refers to the removal of one or more amino acids from a polypeptide. Insertion refers to the introduction of one or more amino acid residues into a predetermined site within a polypeptide. Insertion can include the intrasequence insertion of single or multiple amino acids. Substitution refers to the replacement of an amino acid in a polypeptide with another amino acid having similar properties (e.g., similar hydrophobicity, hydrophilicity, antigenicity, tendency to form or disrupt an α-helical or β-sheet structure). Amino acid substitutions are typically of single residues but may be clustered depending on the functional constraints placed on the polypeptide and may range from about 1 to about 10 amino acids. Amino acid substitutions are preferably conservative amino acid substitutions, as described below. Amino acid substitutions, deletions, or insertions can be performed using peptide synthesis techniques, such as solid-phase peptide synthesis, or by recombinant DNA engineering. Methods for manipulating DNA sequences to produce substitution, insertion, or deletion variants of polypeptides are well known in the art. Variants may have alterations that produce silent changes and result in functionally equivalent polypeptides. As long as the secondary binding of the substance is maintained, deliberate amino acid substitutions can be made based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and amphipathicity of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid, positively charged amino acids include lysine and arginine, and amino acids with uncharged polar head groups with similar hydrophilicity values ​​include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine. Conservative substitutions can be made, for example, according to the following table. Amino acids in the same block in the second row and preferably in the same family in the third row can be substituted for each other.

[0120] [Table 1]

[0121] The polypeptide may be a mature or immature polypeptide, or a polypeptide derived from an immature polypeptide. The polypeptide may be linear or may be cyclized using known methods. The polypeptide typically contains at least 10, at least 20, at least 30, or at least 40 consecutive amino acids.

[0122] At least one modification (eg, mutation) may be included in one or more of SEQ ID NO:2 and SEQ ID NO:4.

[0123] Recombinant constructs can be used to transform plants or plant cells to modulate polypeptide expression, function, or activity. Recombinant polynucleotide constructs can include polynucleotides encoding one or more polynucleotides described herein, operably linked to regulatory regions suitable for expressing the polypeptides. Thus, polynucleotides can include coding sequences encoding polypeptides described herein. Plants or plant cells in which polypeptide expression, function, or activity is modulated can include mutant, non-naturally occurring, transgenic, artificial, or genetically engineered plants or plant cells. Preferably, transgenic plants or plant cells contain genomes that have been altered by stable integration of recombinant DNA. Recombinant DNA includes DNA that has been genetically engineered and constructed extracellularly, including DNA containing natural DNA, cDNA, or synthetic DNA. Transgenic plants can include plants regenerated from initially transformed plant cells, as well as progeny or progeny transgenic plants obtained from crosses of transformed plants. Preferably, the transgenic modification alters the expression, function, or activity of the polynucleotides or polypeptides described herein compared to control plants.

[0124] The polypeptide encoded by the recombinant polynucleotide may be a native polypeptide or may be heterologous to the cell. In some cases, the recombinant construct contains a polynucleotide operably linked to a control region that regulates expression. Examples of suitable control regions are described herein.

[0125] Also provided is a vector containing recombinant polynucleotide constructs such as those described herein.Suitable vector backbones include those commonly used in the art, such as plasmids, viruses, artificial chromosomes, bacterial artificial chromosomes, yeast artificial chromosomes, or bacteriophage artificial chromosomes.Suitable expression vectors include, but are not limited to, plasmids and viral vectors derived from, for example, bacteriophage, baculovirus, and retrovirus.Many vectors and expression systems are commercially available.

[0126] A vector can include, for example, an origin of replication, a scaffold attachment region, or a marker. A marker gene can confer a selectable phenotype to plant cells. For example, a marker can confer biocide resistance, such as resistance to antibiotics (e.g., kanamycin, G418, bleomycin, or hygromycin) or herbicides (e.g., glyphosate, chlorsulfuron, or phosphinothricin). In addition, expression vectors can contain tag sequences designed to facilitate manipulation or detection (e.g., purification or localization) of the expressed polypeptide. Tag sequences, such as luciferase, beta-glucuronidase, green fluorescent polypeptide, glutathione S-transferase, polyhistidine, c-myc, or hemagglutinin sequences, are typically expressed as a fusion with the encoded polypeptide. Such tags can be inserted anywhere within the polypeptide, including at either the carboxyl or amino terminus.

[0127] A plant or plant cell can be transformed by having a recombinant polynucleotide integrated into its genome so that it is stably transformed. The plants or plant cells described herein can be stably transformed. Stably transformed cells typically retain the introduced polynucleotide with each cell division. A plant or plant cell can be transiently transformed so that the recombinant polynucleotide is not integrated into its genome. Transiently transformed cells typically lose all or part of the introduced recombinant polynucleotide with each cell division, and therefore, the introduced recombinant polynucleotide cannot be detected in daughter cells after a sufficient number of cell divisions.

[0128] Many methods for transforming plant cells are available in the art, including biolistics, gene gun technology, Agrobacterium-mediated transformation, viral vector-mediated transformation, freeze-thaw techniques, particle bombardment, direct DNA uptake, sonication, microinjection, plant virus-mediated transduction, and electroporation.

[0129] When cells or cultured tissues are used as recipient tissues for transformation, plants can be regenerated from the transformed cultures, if desired, by techniques known to those skilled in the art.

[0130] The selection of a regulatory region to be included in a recombinant construct depends on several factors, including, but not limited to, efficiency, selectability, inducibility, desired expression level, and cell- or tissue-preferential expression. It is routine for those skilled in the art to regulate the expression of a coding sequence by appropriately selecting and positioning a regulatory region relative to the coding sequence. Transcription of polynucleotides can be regulated in a similar manner. Some suitable regulatory regions initiate transcription only in certain cell types, or primarily in certain cell types. Methods for identifying and characterizing regulatory regions in plant genomic DNA are known in the art.

[0131] Exemplary promoters include tissue-specific promoters recognized by tissue-specific factors present in different tissues or cell types (e.g., root-specific promoters, shoot-specific promoters, xylem-specific promoters), or present during different developmental stages, or present in response to different environmental conditions. Suitable promoters include constitutive promoters that can be activated in most cell types without the need for specific inducers. Examples of promoters that can be used to control polypeptide expression include the cauliflower mosaic virus 35S (CaMV / 35S), SSU, OCS, lib4, usp, STLS1, B33, nos, or ubiquitin or phaseolin promoters. Those skilled in the art can generate multiple variations of recombinant promoters.

[0132] Tissue-specific promoters are transcriptional regulators that are only active in specific cells or tissues at specific times during plant development, such as vegetative tissues or reproductive tissues. Examples of tissue-specific promoters under developmental control include promoters that can initiate transcription only (or mainly only) in certain tissues, such as vegetative tissues, such as roots or leaves, or reproductive tissues, such as fruit, ovule, seed, pollen, pistil, flower, or any embryonic tissue. Reproductive tissue-specific promoters can be, for example, anther-specific, ovule-specific, embryo-specific, endosperm-specific, integument-specific, seed and seed coat-specific, pollen-specific, petal-specific, sepal-specific, or a combination thereof.

[0133] Exemplary leaf-specific promoters include the pyruvate phosphate dikinase (PPDK) promoter from C4 plants (maize), the cab-m1Ca+2 promoter from maize, the Arabidopsis thaliana myb-related gene promoter (Atmyb5), the ribulose bisphosphate carboxylase (RBCS) promoter (e.g., the tomato RBCS1, RBCS2, and RBCS3A genes, which are expressed in leaves and light-grown seedlings, RBCS1 and RBCS2, which are expressed in developing tomato fruit, or the ribulose bisphosphate carboxylase promoter, which is expressed at high levels and nearly exclusively in mesophyll cells in leaf blades and leaf sheaths).

[0134] Exemplary senescence-specific promoters include the tomato promoter active during fruit ripening, senescence, and leaf fall, the maize promoter of the gene encoding cysteine ​​protease, the promoter of 82E4, and the promoter of the SAG gene.Exemplary anther-specific promoters can be used.Exemplary root-preferred promoters known to those skilled in the art can be selected.Exemplary seed-preferred promoters include both seed-specific promoters (promoters active during seed development, such as promoters of seed storage polypeptides) and seed germination promoters (promoters active during seed germination).

[0135] Examples of inducible promoters include promoters that respond to pathogen attack, anaerobic conditions, high temperature, light, drought, low temperature, or high salinity. Pathogen-inducible promoters include those derived from pathogenesis-related polypeptides (PR polypeptides), which are induced following infection by a pathogen (e.g., PR polypeptides, SAR polypeptides, beta-1,3-glucanases, chitinases).

[0136] In addition to plant promoters, other suitable promoters may be derived from bacterial origin, such as the octopine synthase promoter, the nopaline synthase promoter, and other promoters derived from Ti plasmids, or may be derived from viral promoters (e.g., the 35S and 19S RNA promoters of the cauliflower mosaic virus (CaMV), the constitutive promoters of the tobacco mosaic virus, the cauliflower mosaic virus (CaMV) 19S and 35S promoters, or the figwort mosaic virus 35S promoter).

[0137] Disclosed are plants or plant cells comprising at least one mutation in one or more of the polynucleotides or polypeptides described herein, which mutation results in modulated function or activity of NtLKR or the polypeptide encoded thereby.

[0138] A method is provided for modulating the level of NtLKR polypeptides in a (cured) plant or (cured) tobacco plant material, the method comprising introducing into the genome of the plant one or more mutations that modulate the expression of at least one NtLKR gene, wherein the at least one NtLKR gene is selected from one or more NtLKR sequences according to the present disclosure.

[0139] Also provided is a method for identifying a plant having a modulated level of one or more amino acids in the plant or a portion thereof compared to the level of the one or more amino acids in a control plant, the method comprising screening a polynucleotide sample obtained from the plant of interest for the presence of one or more mutations in an NtLKR polynucleotide sequence according to the present disclosure, and optionally correlating the identified mutations with mutations known to modulate the level of one or more amino acids.

[0140] Also disclosed are plants or plant cells that are heterozygous or homozygous for one or more mutations in an NtLKR gene according to the present disclosure, which mutations result in modulation of expression of the NtLKR gene or the function or activity of the NtLKR polypeptide encoded thereby.

[0141] Numerous techniques, including sexual crossing, can be used to combine mutations in one plant. A plant having one or more favorable heterozygous or homozygous mutations in a gene according to the present disclosure that modulates the expression of the gene or the function or activity of the polypeptide encoded thereby can be crossed with a plant having one or more favorable heterozygous or homozygous mutations in one or more other genes that modulate the expression or the function or activity of the polypeptide encoded thereby. In one embodiment, crossing is performed to introduce one or more favorable heterozygous or homozygous mutations in a gene according to the present disclosure within the same plant.

[0142] The function or activity of one or more polypeptides of the present disclosure in a plant is increased or decreased if the function or activity is less or more than the function or activity of the same polypeptide in a plant that has not been modified to inhibit the function or activity of the polypeptide and that has been grown, harvested, and dried using the same protocols.

[0143] In some embodiments, the mutations are introduced into plants or plant cells using mutagenesis techniques, and the introduced mutations are identified or selected using methods known to those skilled in the art, such as Southern blot analysis, DNA sequencing, PCR analysis, or phenotypic analysis. Mutations that affect gene expression or disrupt the function of the encoded polypeptide can be determined using methods well known in the art. Insertional mutations in gene exons usually result in null mutants. Mutations in conserved residues can be particularly effective in disrupting the metabolic function of the encoded polypeptide. For example, it will be understood that mutations in one or more highly conserved regions are likely to alter polypeptide function, while mutations outside these highly conserved regions are likely to have little or no effect on polypeptide function. In addition, mutations in a single nucleotide can create a stop codon, resulting in a truncated polypeptide and, depending on the extent of truncation, resulting in loss of function.

[0144] Also disclosed are methods for obtaining mutant polynucleotides and polypeptides.Any plant of interest, including plant cell or plant material, can be genetically modified by various known methods for inducing mutagenesis, such methods include site-directed mutagenesis, oligonucleotide-directed mutagenesis, chemically induced mutagenesis, radiation-induced mutagenesis, mutagenesis using modified bases, mutagenesis using gapped duplex DNA, double-strand break mutagenesis, mutagenesis using repair-deficient host strains, mutagenesis by total gene synthesis, DNA shuffling and other equivalent methods.

[0145] Mutations in the polynucleotides and polypeptides described herein may include man-made mutations, synthetic mutations, or genetically engineered mutations. Mutations in the polynucleotides and polypeptides described herein may be mutations obtained or obtainable through a process involving in vitro or in vivo manipulation steps. Mutations in the polynucleotides and polypeptides described herein may be mutations obtained or obtainable through a process involving human intervention. The function or activity of the mutant polypeptide variant may be greater, less, or substantially the same as that of the unmutated polypeptide.

[0146] Methods for randomly introducing mutations into polynucleotides include chemical mutagenesis and radiation mutagenesis.Chemical mutagenesis involves the use of exogenously added chemicals, such as mutagenic, teratogenic, or carcinogenic organic compounds, to induce mutations.Mutagens that mainly produce point mutations, as well as short deletions, insertions, missense mutations, simple sequence repeats, transversions, or transitions, can be used to create mutations, including chemical mutagens or radiation. Mutagens include ethyl methanesulfonate, methyl methanesulfonate, N-ethyl-N-nitrosourea, triethylmelamine, N-methyl-N-nitrosourea, procarbazine, chlorambucil, cyclophosphamide, diethyl sulfate, acrylamide monomer, melphalan, nitrogen mustard, vincristine, dimethylnitrosamine, N-methyl-N'-nitro-nitrosoguanidine, nitrosoguanidine, 2-aminopurine, 7,12 dimethyl-benz(a)anthracene, ethylene oxide, hexamethylphosphoramide, bisulfane, diepoxyalkanes (such as diepoxyoctane and diepoxybutane), 2-methoxy-6-chloro-9[3-(ethyl-2-chloro-ethyl)aminopropylamino]acridine dihydrochloride, and formaldehyde.

[0147] Natural mutations at loci that may not be directly caused by mutagens are also considered, provided that they result in the desired phenotype.Suitable mutagen-inducing agents can also include, for example, ionizing radiation, such as X-rays, gamma rays, fast neutron irradiation, and UV radiation.The dose of mutagenic chemicals or radiation is experimentally determined for each type of plant tissue to obtain a mutation frequency that is below the threshold level associated with lethality or reproductive sterility.Any method of preparing plant polynucleotides known to those skilled in the art can be used to prepare plant polynucleotides for mutation screening.

[0148] The mutation process may involve one or more plant cross-breeding steps.

[0149] After mutation, screening can be performed to identify mutations that create premature stop codons or other non-functional genes. After mutation, screening can be performed to identify mutations that create functional genes that can be expressed at increased or decreased levels. Screening for mutants can be performed by sequencing or by using one or more probes or primers specific to the gene or polypeptide. Specific mutations in polynucleotides that can result in regulated gene expression, regulated mRNA stability, or regulated polypeptide stability can also be created. Such plants are referred to herein as "non-natural" or "mutant" plants. Typically, mutant or non-natural plants will contain at least a portion of exogenous, synthetic, or man-made nucleotides (e.g., DNA or RNA) that were not present in the plant prior to manipulation. The exogenous nucleotide can be a single nucleotide, two or more nucleotides, two or more contiguous nucleotides, or two or more non-contiguous nucleotides, for example, at least 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or 1500 or more contiguous or non-contiguous nucleotides.

[0150] Sequence-specific polynucleotides capable of interfering with the transcription of one or more endogenous genes, sequence-specific polynucleotides capable of interfering with the translation of RNA transcripts (e.g., double-stranded RNA, siRNA, ribozymes), sequence-specific polypeptides capable of interfering with the stability of one or more polypeptides, sequence-specific polynucleotides capable of interfering with the enzymatic function of one or more polypeptides or the binding function of one or more polypeptides with respect to a regulatory polypeptide, antibodies that exhibit specificity for one or more polypeptides, small molecule compounds that can interfere with the stability of one or more polypeptides or the enzymatic function of one or more polypeptides or the binding function of one or more polypeptides, zinc finger polypeptides that bind to one or more polynucleotides, and meganucleases that function on one or more polynucleotides can be used to regulate the expression, function, or activity of one or more of the polynucleotides or polypeptides described herein. Genome editing techniques are well known in the art and are discussed further below.

[0151] Zinc finger polypeptides can be used to regulate the expression or function or activity of one or more NtLKR polynucleotides described herein. The use of zinc finger nucleases is described in Nature Rev. Genet. (2010) 11(9):636-646).

[0152] Meganucleases, such as I-CreI, can be used to regulate the expression or function or activity of one or more of the NtLKR polynucleotides described herein. The use of meganucleases is described in Curr Gene Ther. (2011) Feb;11(1):11-27 and Int J Mol Sci. (2019) 20(16), 4045.

[0153] Transcription activator-like effector nucleases (TALENs) can be used to regulate the expression, function, or activity of one or more of the NtLKR polynucleotides described herein. The use of TALENs is described in Nature Rev. Mol. Cell Biol. (2013) 14:49-55 and Int J Mol Sci. (2019) 20(16), 4045.

[0154] CRISPR system can be used to regulate the expression or function or activity of one or more of the NtLKR polynucleotides described herein, and is a preferred method.This technology is described, for example, in Plant Methods (2016) 12:8; Front Plant Sci. (2016) 7:506; Biotechnology Advances (2015) 33,1,p41-52; Acta Pharmaceutica Sinica B (2017) 7,3,p292-302; Curr.Op.in Plant Biol. (2017) 36,1-8 and Int J Mol Sci (2019) 20 (16), 4045.As is well known in the art, CRISPR editing system generally includes two components: CRISPR-associated endonuclease (Cas) (e.g., Cas9) and guide RNA (gRNA). Cas creates double-stranded DNA breaks at sites in the genome defined by the sequence of the gRNA molecule bound to it. The location at which Cas cleaves DNA is dictated by the unique sequence of the gRNA bound to it. The gRNA is a specifically designed RNA sequence that recognizes a target DNA region and directs Cas nuclease to it for editing. It contains two components: (i) a tracer RNA that serves as a binding scaffold for the Cas nuclease, and (ii) a 17-20 nucleotide sequence of Crisp RNA (crRNA) that is complementary to the target DNA. The exact region of DNA targeted will depend on the specific application. For example, to activate or repress a target polynucleotide, the gRNA can target a promoter that drives expression of the target polynucleotide. Methods for designing gRNAs are well known in the art and include those from Chop Chop Harvard. The application of Cas9-based genome editing in Arabidopsis and tobacco is described, for example, in Methods Enzymol. (2014) 546: 459-72 and Plant Physiol Biochem (2018) 131: 37-46. CRISPR technology has been widely introduced into plants (see, for example, WO2015 / 189693).In addition to Cas9, other RNA-guided nucleases have been described for use in CRISPR system, including Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, CaslO, Cpfl, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3 and Csf4.In certain embodiments, it is preferred to use Cas9. The present disclosure further provides a CRISPR-based genome editing system comprising an RNA-guided nuclease and a gRNA, which modulates the activity of one or more of the polynucleotides described herein. The present disclosure also provides a method for cleaving one or more polynucleotides in a plant cell, comprising introducing a gRNA and an RNA-guided nuclease into the plant cell, wherein the gRNA acts in conjunction with the RNA-guided nuclease to create a strand break in one or more of the polynucleotides described herein. Also disclosed is a CRISPR construct comprising (i) a polynucleotide encoding a CRISPR-associated endonuclease and (ii) a gRNA comprising a polynucleotide sequence (typically about 17-20 nucleotides) complementary to the DNA of a targeted polynucleotide described herein.

[0155] Antisense technology is another well-known method that can be used to modulate the expression or activity of one or more of the NtLKR polypeptides described herein. See, e.g., Gene (1988) 10;72(1-2):45-50.

[0156] The NtLKR polynucleotide can be targeted for inactivation by introducing a transposon (e.g., an IS element or other mobile genetic element) into the genome of the target plant. See, e.g., Cytology and Genetics (2006) 40(4):68-81.

[0157] The NtLKR polynucleotide can be targeted for inactivation by introducing into the plant a ribozyme derived from a number of small circular RNAs capable of self-cleavage and replication. See, e.g., FEMS Microbiology Reviews (1999) 23, 3, 257-275.

[0158] A mutant or non-naturally occurring plant or plant cell can have any combination of one or more modifications (e.g., mutations) in one or more of the NtLKR polynucleotides described herein, which modifications result in regulated expression, function, or activity of the polynucleotide or its polynucleotide product. For example, a mutant or non-naturally occurring plant or plant cell can have a single modification in a single NtLKR polynucleotide or polypeptide; multiple modifications in a single NtLKR polynucleotide or polypeptide; a single modification in two or more NtLKR polynucleotides or polypeptides; or multiple modifications in two or more NtLKR polynucleotides or polypeptides. As yet another example, a mutant or non-naturally occurring plant or plant cell can have one or more modifications in a particular portion of an NtLKR polynucleotide or NtLKR polypeptide, such as a region of the NtLKR that encodes the active site of an NtLKR polypeptide or portion thereof. As yet another example, a mutant or non-naturally occurring plant or plant cell may have one or more modifications in a region outside one or more NtLKR polynucleotides or NtLKR polypeptides, for example, in a region upstream or downstream of an NtLKR polynucleotide if it regulates the function or expression of NtLKR. Upstream elements can include promoters, enhancers, or transcription factors. Some elements, such as enhancers, can be located upstream or downstream of the gene they regulate. Because some elements have been found to be located hundreds of thousands of base pairs upstream or downstream of the gene they regulate, the element need not be located near the gene it regulates.A mutant or non-naturally occurring plant or plant cell can have one or more modifications located within the first 100 nucleotides of the gene, the first 200 nucleotides of the gene, the first 300 nucleotides of the gene, the first 400 nucleotides of the gene, the first 500 nucleotides of the gene, the first 600 nucleotides of the gene, the first 700 nucleotides of the gene, the first 800 nucleotides of the gene, the first 900 nucleotides of the gene, the first 1000 nucleotides of the gene, the first 1100 nucleotides of the gene, the first 1200 nucleotides of the gene, the first 1300 nucleotides of the gene, the first 1400 nucleotides of the gene, or the first 1500 nucleotides of the gene. A mutant or non-naturally occurring plant or plant cell can have one or more modifications located within the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, or fifteenth set of 100 nucleotides of the gene, or a combination thereof. Disclosed are mutant or non-native plants or plant cells (such as mutant, non-native, or transgenic plants or plant cells described herein) that comprise the mutant polypeptide variants.

[0159] In one embodiment, seeds obtained from the plant are mutagenized and then grown into first-generation mutant plants. The first-generation plants are then self-pollinated, and seeds obtained from the first-generation plants are grown into second-generation plants and then screened for mutations at those loci. While mutagenized plant material can be screened for mutations, the advantage of screening second-generation plants is that all somatic mutations correspond to germline mutations. Those skilled in the art will understand that a variety of plant materials, including but not limited to seeds, pollen, plant tissues, or plant cells, can be mutagenized to create mutant plants. However, the type of mutagenized plant material can affect when plant polynucleotides are screened for mutations. For example, when pollen is subjected to mutagenesis before pollination of a non-mutagenized plant, the seeds resulting from that pollination grow into first-generation plants. All cells of the first-generation plants will contain the mutations created in the pollen, and therefore these first-generation plants may then be screened for mutations without waiting until the second generation.

[0160] NtLKR polynucleotides prepared from individual plants, plant cells, or plant materials can optionally be pooled to facilitate screening for mutations within a population of plants originating from the mutagenized plant tissues, cells, or materials. One or more subsequent generations of plants, plant cells, or plant materials can be screened. The size of the optionally pooled group depends on the sensitivity of the screening method used. After the samples are optionally pooled, they can be subjected to a polynucleotide-specific amplification technique, such as PCR. Any one or more primers or probes specific to genes or sequences immediately adjacent to the gene can be used to amplify sequences within the optionally pooled sample. Preferably, one or more primers or probes are designed to amplify a region of the locus where beneficial mutations are most likely to occur. Most preferably, primers are designed to detect mutations within the region of the polynucleotide. Additionally, primers and probes preferably avoid known polymorphic sites to facilitate screening for point mutations. To facilitate detection of the amplification product, one or more primers or probes can be labeled using any conventional labeling method. Primer or probe can be designed based on the sequences described herein using methods well understood in the art.In order to facilitate the detection of amplification products, primer or probe can be labeled using any conventional labeling method.They can be designed based on the sequences described herein using methods well understood in the art.

[0161] Polymorphisms can be identified by means known in the art, several of which are described in the literature.

[0162] In some embodiments, plants may be regenerated or grown from plants, plant tissues, or plant cells. Any suitable method for regenerating or growing plants from plant cells or plant tissues may be used, including, but not limited to, tissue culture or regeneration from protoplasts. Preferably, plants may be regenerated by growing transformed plant cells on callus, shoot, or root induction media. See, for example, McCormick et al., Plant Cell Reports 5:81-84 (1986). These plants are then grown and pollinated with either the same transformed line or a different line, and the resulting hybrids with the desired phenotypic characteristics are identified. Two or more generations may be grown to ensure that the expression of the desired phenotypic characteristics is stably maintained and inherited, and then the seeds may be harvested to ensure that the desired phenotypic characteristics have been achieved. Thus, "transformed seeds" refer to seeds containing a nucleotide construct stably integrated into the plant genome.

[0163] Thus, in yet another aspect, a method for preparing a mutant plant is provided. The method comprises providing at least one cell of a plant containing one or more NtLKR genes encoding a functional NtLKR. The at least one cell of the plant is then treated under conditions effective to modulate the function of an NtLKR polynucleotide. The at least one mutant plant cell is then grown in a mutant plant, wherein the mutant plant has a modulated level of an NtLKR polypeptide described herein compared to a control plant. In one embodiment of this method of producing a mutant plant, the treating step comprises subjecting at least one cell to a chemical mutagen, as described above, and under conditions effective to produce at least one mutant plant cell. In another embodiment of this method, the treating step comprises subjecting at least one cell to a radiation source under conditions effective to produce at least one mutant plant cell. The term "mutant plant" includes mutant plants whose genotype is altered compared to a control plant, preferably by means other than genetic engineering or genetic modification.

[0164] In certain embodiments, a mutant plant, mutant plant cell, or mutant plant material may contain one or more mutations that naturally occur in another plant, plant cell, or plant material and confer a desired trait. This mutation may be incorporated (e.g., introgressed) into another plant, plant cell, or plant material (e.g., a plant, plant cell, or plant material having a different genetic background than the plant from which the mutation originated) to confer the trait thereto. Thus, by way of example, a naturally occurring mutation in a first plant may be introduced into a second plant, such as a second plant having a different genetic background than the first plant. Thus, one skilled in the art can search for and identify plants that naturally harbor one or more mutant alleles of the genes described herein that confer a desired trait within their genome. The naturally occurring mutant alleles can be transferred into a second plant by various methods, including breeding, backcrossing, and introgression, to produce a line, variety, or hybrid having one or more mutations in the genes described herein. The same techniques can be applied to the introgression of one or more non-naturally occurring mutations from a first plant into a second plant. Plants exhibiting desired traits can be found by screening from a pool of mutant plants. Preferably, this selection is carried out using knowledge of the polynucleotides described herein. Consequently, it is possible to screen for genetic traits compared to controls. Such screening procedures may involve the application of conventional amplification or hybridization techniques as discussed herein. Accordingly, a further aspect of the present disclosure relates to a method for identifying mutant plants, comprising: (a) providing a sample containing one or more NtLKR polynucleotides from a plant; and (b) determining the sequence of the polynucleotides, wherein a difference in the sequence of the polynucleotides compared to the polynucleotides of a control plant indicates that the plant is a mutant plant.In another aspect, a method is provided for identifying a mutant plant that accumulates an increased or decreased level of an amino acid compared to a control plant, the method comprising: (a) providing a sample from the plant to be screened; (b) determining whether the sample contains one or more mutations in one or more NtLKR polynucleotides described herein; and (c) determining the level of at least one amino acid in the plant. Preferably, the level of the at least one amino acid is determined in desiccation-treated leaves. In another aspect, a method is provided for preparing a mutant plant having an increased or decreased level of at least one amino acid compared to a control plant, the method comprising: (a) providing a sample from a first plant; (b) determining whether the sample contains one or more mutations in one or more NtLKR polynucleotides described herein that result in a modulated level of at least one amino acid; and (c) introgressing the one or more mutations into a second plant. Preferably, the level of the at least one amino acid is determined in desiccation-treated leaves. The mutation can be transferred into a second plant using various methods known in the art, such as genetic engineering, genetic manipulation, introgression, plant breeding, backcrossing, etc. In one embodiment, the first plant is a wild-type plant. In one embodiment, the second plant has a different genetic background from the first plant. In another aspect, a method is provided for preparing a mutant plant having an increased or decreased level of at least one amino acid compared to a control plant, the method comprising: (a) providing a sample from a first plant; (b) determining whether the sample contains one or more mutations in one or more of the NtLKR polynucleotides described herein that result in a modulated level of at least one amino acid; and (c) introgressing the one or more mutations into a second plant. Preferably, the level of the at least one amino acid is determined in desiccated leaves. In one embodiment, the introgression step comprises plant breeding, optionally including backcrossing, etc. In one embodiment, the first plant is a wild-type plant. In one embodiment, the second plant has a different genetic background from the first plant.In one embodiment, the first plant is not a cultivar or elite cultivar. In one embodiment, the second plant is a cultivar or elite cultivar. Yet another aspect relates to mutant plants (including mutant plants of cultivars or elite cultivars) obtained or obtainable by the methods described herein. In certain embodiments, the mutant plant may have one or more mutations localized only to a specific region of the plant, such as within the sequence of one or more NtLKR polynucleotides described herein. In accordance with this embodiment, the remaining genomic sequence of the mutant plant will be the same or substantially the same as that of the plant before mutagenesis.

[0165] In certain embodiments, the mutant plant may have one or more mutations localized in multiple genomic regions of the plant, such as within one or more of the NtLKR polynucleotide sequences described herein and in one or more additional regions of the genome, such that the remaining genomic sequences of the mutant plant are not the same or substantially the same as those of the plant prior to mutagenesis. In certain embodiments, the mutant plant may not have one or more mutations in one or more, two or more, three or more, four or more, or five or more exons of an NtLKR polynucleotide described herein; or may not have one or more mutations in one or more, two or more, three or more, four or more, or five or more introns of an NtLKR polynucleotide described herein; or may not have one or more mutations in the promoter of an NtLKR polynucleotide described herein; or may not have one or more mutations in the 3' untranslated region of an NtLKR polynucleotide described herein; or may not have one or more mutations in the 5' untranslated region of an NtLKR polynucleotide described herein; or may not have one or more mutations in a coding region of an NtLKR polynucleotide described herein; or may not have one or more mutations in a non-coding region of an NtLKR polynucleotide described herein.

[0166] In yet another aspect, there is provided a method for identifying a plant, plant cell, or plant material containing a mutation in a gene encoding an NtLKR polynucleotide described herein, comprising: (a) subjecting a plant, plant cell, or plant material to mutagenesis; (b) obtaining a sample from the plant, plant cell, or plant material, or its progeny; and (c) determining the polynucleotide sequence of the NtLKR gene or a variant or fragment thereof, wherein differences in the sequence indicate one or more mutations therein. This method also allows for the selection of plants having mutations occurring in genomic regions that affect expression of the NtLKR gene in plant cells, such as transcription start sites, start codons, intronic regions, exon-intron boundaries, and terminators.

[0167] Plants suitable for use in the present disclosure include monocotyledonous and dicotyledonous plants and plant cell lines, including members of the genus Nicotiana.

[0168] Various embodiments are directed to mutant, non-native, or transgenic tobacco plants or tobacco plant cells and may be applied to any species of the genus Nicotiana, including N. rustica and N. tabacum (e.g., LA B21, LN KY171, TI 1406, Basma, Galpao, Perique, Beinhart 1000-1, and Petico).Other species include N. acaulis, N. acuminata, N. africana, N. alata, N. ameghinoi, N. amplexicaulis, N. arentsii, N. attenuata, N. azambujae, and N. benavide sii, N. benthamiana, N. bigelovii, N. bonariensis, N. cavicola, N. clevelandii, N. cordifolia, N. corymbosa, N. debneyi, N. excelsior, and N. forget iana, N. fragrans, N. glauca, N. glutinosa, N. goodspeedii, N. gossei, N. hybrid, N. ingulba, N. kawakamii, N. knightiana, N. langsdorffii, N. lin earis、N.longiflora、N.maritima、N.megalosiphon、N.miersii、N.noctiflora、N.nudicaulis、N.obtusifolia、N.occidentalis、N.occidentalis subsp.hesperis、N.otophora、N.paniculata、N.pauciflora、N.petunioides、N.plumbaginifolia、N.quadrivalvis、N.raimondii、N.repanda、N.rosulata subsp.ingulba、N.rotundifolia、N.setchellii、N.simulans、N.solanifolia、N.spegazzinii、N.stocktonii、N.suaveolens、N.sylvestr is, N. thyrsiflora, N. tomentosa, N. tomentosiformis, N. trigonophylla, N. umbratica, N. undulata, N. velutina, N. wigandioides, Nx sanderae has a strong selection of N.tabacum.

[0169] The use of tobacco cultivars and elite tobacco cultivars is also contemplated herein. Thus, a transgenic, non-naturally occurring, or mutant plant can be a tobacco cultivar or elite tobacco cultivar that includes one or more introduced genes, or one or more genetic mutations, or a combination thereof. The genetic mutations (e.g., one or more polymorphisms) can be mutations that do not naturally occur in a particular tobacco cultivar or cultivar (e.g., an elite tobacco cultivar), or can be genetic mutations that occur naturally when the mutation does not naturally occur in a particular tobacco cultivar or cultivar (e.g., an elite tobacco cultivar).

[0170] Particularly useful Nicotiana tabacum species include burley, dark, flue-cured, and oriental tobacco. Non-limiting examples of varieties or cultivars include BD64, CC101, CC200, CC27, CC301, CC400, CC500, CC600, CC700, CC800, CC900, Coker 176, Coker 319, Coker 371 Gold, Coker 48, CD263, DF911, DT538 LC Galpao tobacco, GL26H, GL350, GL600, GL737, GL939, GL973, HB04P, and HB04P. LC, HB3307PLC, Hybrid 403LC, Hybrid 404LC, Hybrid 501LC, K149, K326, K346, K358, K394, K399, K730, KDH959, KT200, KT204LC, KY10, KY14, KY160, KY17, KY171, KY907, KY907LC, KY14xL8 LC, Little Crittenden, McNair 373, McNair 944, msKY 14xL8, Narrow Leaf Madole, Narrow Leaf Madole LC, NBH98, N-126, N-777LC, N-7371LC, NC100, NC102, NC2000, NC291, NC297, NC299, NC3, NC4, NC5, NC6, NC7, NC606, NC71, NC72, NC810, NC BH129, NC2002, Neal Smith Madole, OXFORD207, PD7302 LC, PD7309 LC, PD7312 LC, "Perique" cigarettes, PVH03, PVH09, PVH19, PVH50, PVH51, R610, R630, R7-11, R7-12, RG17, RG81, RG H51, RGH4, RGH51, RS1410, Speight168, Speight172, Speight179, Speight210, Speight220, Speight225, Speight227, Speight234, Speight G-28, Speight G-70, Speight H-6, Speight H20, SpeightNF3, TI1406, TI1269, TN86, TN86LC, TN90, TN97, TN97LC, TN D94, TN D950, TR (Tom Rosson) Madole, VA309, VA359, AA37-1, B13P, Xanthi (Mitchell-Mor), Bel-W3, 79-615, Samsun Holmes NN, KTRDC No. 2 Hybrid 49, Burley 21, KY8959, KY9, MD609, PG01, PG04, PO1, PO2, PO3, RG11, RG8, VA509, AS44, Banket A1, Basma Drama B84 / 31, Basma I Zichna ZP4 / B, Basma Xanthi BX 2A, Batek, Besuki Jember, C104, Coker347, Criollo Misionero, Delcrest, Djebel 81, DVH405, Galpao Comum, HB04P, Hicks Broadleaf, Kabakulak Elassona, Kutsage E1, LA BU 21, NC2326, NC297, PVH2110, Red Russian, Samsun, Saplak, Simmaba, Talgar28, Wislica, Yayaldag, Prilep HC-72, Prilep P23, Prilep PB 156 / 1, Prilep P12-2 / 1, Yaka JK-48, Yaka JB 125 / 3, TI-1068, KDH-960, TI-1070, TW136, Basma, TKF4028, L8, TKF2002, GR141, Basma xanthi, GR149, GR153, Petit Havana. Low converter sub-varieties of the above are also contemplated even if not specifically identified herein.

[0171] Embodiments are also directed to compositions and methods for producing mutant, non-naturally occurring, hybrid, or transgenic plants that have been modified to regulate the expression or function of one or more NtLKR polynucleotides described herein (or any combination thereof described herein). Advantageously, the resulting mutant, non-naturally occurring, hybrid, or transgenic plants can be similar or substantially identical to control plants in overall appearance. Various phenotypic characteristics can be evaluated by field observation, such as the degree of maturity, number of leaves per plant, stalk height, leaf insertion angle, leaf size (width and length), internode distance, and leaf blade-to-midrib ratio.

[0172] One aspect relates to seeds of the mutant plants, non-naturally occurring plants, hybrid plants, or transgenic plants described herein. Preferably, the seeds are tobacco seeds. Yet another aspect relates to pollen or ovules of the mutant plants, non-naturally occurring plants, hybrid plants, or transgenic plants described herein. Additionally, provided are mutant plants, non-naturally occurring plants, hybrid plants, or transgenic plants described herein that further comprise a polynucleotide that confers male sterility.

[0173] Also provided are tissue cultures of regenerable cells of the mutant, non-naturally occurring, hybrid, or transgenic plants described herein, or portions thereof, which culture will regenerate plants capable of expressing all the morphological and physiological characteristics of the parent. Regenerable cells include cells obtained from leaves, pollen, embryos, cotyledons, hypocotyls, roots, root tips, anthers, flowers and parts thereof, ovules, shoots, stems, stalks, pith, and capsules, or callus or protoplasts derived therefrom.

[0174] The plant material described herein may be cured tobacco material. CORESTA's recommendations for the curing of tobacco are set out in CORESTA Guide No. 17, April 2016, Sustainability in Leaf Tobacco Production.

[0175] The mutant, transgenic, or non-naturally occurring plants or parts thereof of the present disclosure exhibit modulated levels of at least one amino acid in plant material, for example, in desiccated leaves.

[0176] Preferably, the modulated level of at least one amino acid is observed in at least cured leaves, preferably in fully cured leaves. Tobacco is considered fully cured when the midrib of the leaf is devoid of moisture, resulting in leaves that are light yellow-brown to reddish-brown to dark brown in color.

[0177] Preferably, the dried leaves are taken from leaves at the middle position of the plant, and preferably there is no effect on the phenotype of the leaves having a modulated level of at least one amino acid compared to leaves of a control plant.

[0178] In one embodiment, the level of lysine is increased compared to a control plant or part thereof.

[0179] In one embodiment, the levels of lysine, arginine, glutamine, tyrosine, gamma aminobutyric acid (GABA), and alanine are increased compared to a control plant or portion thereof.

[0180] In one embodiment, the levels of lysine, arginine, glutamine, histidine, tyrosine, tryptophan, threonine, GABA, asparagine, and alanine are increased compared to a control plant or portion thereof.

[0181] In one embodiment, the levels of lysine, arginine, proline, GABA, glutamine, leucine, alanine, phenylalanine, tyrosine, and isoleucine are increased compared to a control plant or portion thereof.

[0182] In one embodiment, the levels of isoleucine, valine, and serine are reduced compared to a control plant or portion thereof.

[0183] In one embodiment, the levels of methionine, threonine, and glycine are reduced compared to a control plant or part thereof.

[0184] In one embodiment, the levels of aspartic acid and glutamic acid are not significantly altered compared to a control plant or portion thereof.

[0185] In one embodiment, the levels of proline, aspartic acid, leucine, phenylalanine, glutamic acid, and methionine are not significantly altered compared to a control plant or portion thereof.

[0186] In one embodiment, the levels of asparagine, aspartic acid, tryptophan, histidine, glutamic acid, serine, and valine are not significantly altered compared to a control plant or portion thereof.

[0187] In one embodiment, the level of total free amino acids is not significantly altered compared to a control plant or part thereof.

[0188] In one embodiment, the plant is a Virginia tobacco plant having increased levels of lysine, arginine, glutamine, histidine, tyrosine, tryptophan, threonine, GABA, asparagine, and alanine compared to a control plant or portion thereof, preferably wherein the increase is about a 4.6-fold increase in lysine, about a 2.81-fold increase in arginine, about a 2.06-fold increase in glutamine, about a 1.68-fold increase in histidine, about a 1.62-fold increase in tyrosine, about a 1.6-fold increase in tryptophan, about a 1.53-fold increase in threonine, about a 1.45-fold increase in GABA, about a 1.38-fold increase in asparagine, and about a 1.3-fold increase in alanine compared to a control plant or portion thereof.

[0189] The Virginia tobacco plant may also have reduced levels of isoleucine, valine, and serine compared to a control plant or portion thereof, but preferably the reduction in isoleucine is about 39%, the reduction in valine is about 17%, and the reduction in serine is about 15% compared to a control plant or portion thereof.

[0190] The Virginia tobacco plants may also have no significant changes in the levels of proline, aspartic acid, leucine, phenylalanine, glutamic acid, and methionine compared to a control plant or portion thereof.

[0191] The Virginia tobacco plants may have about a 15% reduction in total sugars compared to a control plant or portion thereof, including a reduction in glucose, preferably about a 23% reduction, a reduction in fructose, preferably about an 18% reduction, and no significant difference in sucrose content.

[0192] The Virginia tobacco plants may not have a significant difference in ammonia content compared to the control plants or portions thereof.

[0193] The Virginia tobacco plants may have a reduced nitrate content compared to the control plants or portions thereof, preferably reduced by about 34%.

[0194] In another embodiment, the plant is a burley tobacco plant having increased levels of lysine, arginine, proline, GABA, glutamine, leucine, alanine, phenylalanine, tyrosine, and isoleucine compared to a control plant or portion thereof, preferably the increase is about 11.2-fold for lysine, about 1.85-fold for arginine, about 1.41-fold for proline, about 1.26-fold for GABA, about 1.25-fold for glutamine, about 1.24-fold for leucine, about 1.20-fold for alanine, about 1.15-fold for phenylalanine, about 1.09-fold for tyrosine, and about 1.06-fold for isoleucine.

[0195] The burley tobacco plant may have reduced levels of methionine, threonine, and glycine compared to a control plant or portion thereof, but preferably the reduction in methionine is about 29%, the reduction in threonine is about 10%, and the reduction in glycine is about 8% compared to a control plant or portion thereof.

[0196] The burley tobacco plants may not have significant changes in the levels of asparagine, aspartic acid, tryptophan, histidine, glutamic acid, serine, and valine compared to a control plant or portion thereof.

[0197] Yet another embodiment relates to a mutant, non-naturally occurring, or transgenic plant or cell in which expression of one or more NtLKR polynucleotides or activity of one or more NtLKR polypeptides is modulated, preferably increased, and the plant or plant cell has at least a 4-fold or at least an 11-fold increased level of at least one amino acid, preferably lysine, compared to a control plant or part thereof in which expression of NtLKR or activity of NtLKR is not modulated, preferably increased.

[0198] Yet another embodiment relates to cured plant material, such as cured leaves or cured tobacco, derived or derivable from a mutant, non-native, or transgenic plant or cell, in which the expression of one or more of the NtLKR polynucleotides described herein or the function of the NtLKR polypeptide encoded thereby is modulated, and the level of one or more amino acids, preferably lysine, is modulated, preferably reduced, compared to a control plant or portion thereof.

[0199] Embodiments are also directed to compositions and methods for producing mutant, non-naturally occurring, or transgenic plants or plant cells that have been modified to modulate, preferably decrease, the expression or activity of one or more of the NtLKR polynucleotides or NtLKR polypeptides described herein, which can result in plants or plant parts (e.g., leaves, such as dried leaves) with modulated, preferably increased, amino acid (preferably lysine) content.

[0200] In one embodiment, the phenotype of the mutant, non-native, or transgenic plant is substantially the same as that of a control plant or portion thereof. In one embodiment, the leaf weight of the mutant, non-native, or transgenic plant is substantially the same as that of a control plant or portion thereof. In one embodiment, the leaf number of the mutant, non-native, or transgenic plant is substantially the same as that of a control plant or portion thereof. In one embodiment, the leaf weight and leaf number of the mutant, non-native, or transgenic plant are substantially the same as that of a control plant or portion thereof. In one embodiment, the stem height of the mutant, non-native, or transgenic plant is substantially the same as that of a control plant or portion thereof, for example, 1 month, 2 months, or 3 months or more after field transplantation, or 10 days, 20 days, 30 days, or 36 days or more after topping. For example, the stem height of the mutant, non-native, or transgenic plant does not exceed that of the control plant or portion thereof. In another embodiment, the chlorophyll content of the mutant, non-native, or transgenic plant is decreased relative to a control plant or portion thereof, hi another embodiment, the rate of leaf senescence is increased relative to a control plant or portion thereof.

[0201] In another aspect, a method is provided for modulating the amount of at least one amino acid in at least a portion of a plant (e.g., a leaf, such as a cured leaf), the method comprising: (i) modulating the expression or function of one or more NtLKR polypeptides described herein, preferably wherein the NtLKR polypeptide is encoded by a corresponding NtLKR polynucleotide described herein; (ii) measuring the level of at least one amino acid in at least a portion of a mutant, non-native, or transgenic plant obtained in step (i) (e.g., a leaf, such as a cured leaf, or tobacco or smoke); and (iii) identifying a mutant, non-native, or transgenic plant in which the level of at least one amino acid is modulated compared to a control plant or portion thereof.

[0202] In another aspect, there is provided a method for modulating the amount of at least one amino acid in dry-processed plant material, such as dry-processed leaves, comprising: (i) modulating the expression or function of one or more NtLKR polypeptides (or any combination thereof as described herein), preferably wherein the NtLKR polypeptides are encoded by corresponding NtLKR polynucleotides as described herein; (ii) harvesting and dry-processing the plant material, such as one or more leaves, for a period of time; (iii) measuring the level of at least one amino acid in the dry-processed plant material obtained in or during step (ii); and (iv) identifying dry-processed plant material in which the level of at least one amino acid is modulated compared to a control plant or portion thereof.

[0203] The increase in expression compared to a control can be from about 5% to about 100%, or at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, or an increase of 100% or more, such as 200%, 300%, 500%, 1000% or more, including transcriptional function, or NtLKR polynucleotide expression, or NtLKR polypeptide expression.

[0204] The increase in function or activity compared to a control can be from about 5% to about 100%, or at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, or an increase of 100% or more, such as 200%, 300%, 500%, 1000% or more, including transcriptional function, or NtLKR polynucleotide expression, or NtLKR polypeptide expression, or a combination thereof.

[0205] The reduction in expression compared to a control can be from about 5% to about 100%, or at least a 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, or 100% reduction, including transcriptional function, or NtLKR polynucleotide expression, or NtLKR polypeptide expression, or a combination thereof.

[0206] The reduction in expression compared to a control can be from about 5% to about 100%, or at least a 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, or 100% reduction, including transcriptional function, or NtLKR polynucleotide expression or NtLKR polypeptide expression, or a combination thereof.

[0207] The polynucleotides and recombinant constructs described herein can be used to modulate the expression or function or activity of an NtLKR polynucleotide or NtLKR polypeptide described herein in a plant species of interest, preferably tobacco.

[0208] Numerous polynucleotide-based methods can be used to increase gene expression in plants and plant cells. As an example, a construct, vector, or expression vector compatible with the plant to be transformed can be prepared, containing a gene of interest along with an upstream promoter capable of overexpressing the gene in the plant or plant cell. Exemplary promoters are described herein. After transformation and when grown under suitable conditions, the promoter can drive expression to regulate the level of NtLKR in the plant or specific tissues thereof. In an exemplary embodiment, a vector carrying one or more NtLKR polynucleotides described herein (or any combination thereof described herein) is generated to overexpress the gene in a plant or plant cell. The vector carries a suitable promoter, such as the cauliflower mosaic virus (CaMV) 35S promoter, upstream of the transgene, which drives its constitutive expression in all tissues of the plant. The vector also carries an antibiotic resistance gene to allow for selection of transformed calli and cell lines.

[0209] Expression of the sequence from the promoter can be enhanced by including expression control sequences known in the art. Signals associated with aging and signals active during the desiccation process are specifically labeled.

[0210] Accordingly, various embodiments are directed to methods for modulating the expression level of one or more NtLKR polynucleotides described herein (or any combination thereof described herein) by integrating multiple copies of the NtLKR polynucleotides into a plant genome, the method comprising transforming a plant cell host with an expression vector comprising a promoter operably linked to one or more NtLKR polynucleotides described herein. The polypeptide encoded by the recombinant polynucleotide may be a native polypeptide or may be heterologous to the cell.

[0211] In one embodiment, a plant for use in the present disclosure is a hot-tube-dried mutant, non-native, or transgenic plant.

[0212] In one embodiment, a plant for use in the present disclosure is a sun-drought treated mutant, non-native, or transgenic plant.

[0213] In one embodiment, the plants used in the present disclosure are air-dried mutant, non-native, or transgenic plants.

[0214] In one embodiment, a plant for use in the present disclosure is a mutant, non-native, or transgenic Virginia tobacco plant that has been cured, for example, hot-tube cured.

[0215] In one embodiment, a plant for use in the present disclosure is a cured, e.g., air-cured, mutant, non-native, or transgenic burley tobacco plant.

[0216] In one embodiment, a plant for use in the present disclosure is a cured, e.g., flue-cured, mutant, non-native, or transgenic dark tobacco plant.

[0217] Modulation of NtLKR expression and / or activity can advantageously alter the sensory profile of tobacco. For example, as can be seen from Table 5, RNAi-modified Burley TN90 tobacco exhibits reduced harshness, a smoother, more mellow aerosol, and less of the typical dark notes, but more animalic and nutty notes (less trigeminal impact). As yet another example, as can be seen from Table 6, RNAi-modified Virginia K326 tobacco exhibits an increased hay note and a smoother, darker flavor profile. Plants carrying mutant alleles of one or more NtLKR polynucleotides described herein can be used in plant breeding programs to create useful lines, varieties, and hybrids. In particular, mutant alleles can be introgressed into commercially important varieties described herein. Accordingly, provided are methods of plant breeding, including crossing a mutant, non-native, or transgenic plant described herein with a plant containing a different genetic identity. The method may further include crossing the progeny plant with another plant, and optionally repeating the crossing until progeny with the desired genetic traits or genetic background are obtained. One goal achieved by such breeding methods is to introduce desirable genetic traits into other varieties, breeding lines, hybrids, or cultivars, particularly those of commercial interest. Another goal is to facilitate stacking of genetic modifications of different genes in a single plant variety, line, hybrid, or cultivar. Intraspecific and interspecific crosses are contemplated. The progeny plants resulting from such crosses are also referred to as breeding lines and are examples of non-natural plants of the present disclosure.

[0218] In one embodiment, a method for producing a non-naturally occurring plant is provided, comprising: (a) crossing a mutant or transgenic plant with a second plant to obtain a progeny tobacco seed; (b) growing the progeny tobacco seed under plant growth conditions to produce a non-naturally occurring plant. The method may further comprise: (c) crossing the previous generation non-naturally occurring plant with itself or another plant to obtain a progeny tobacco seed; (d) growing the progeny tobacco seed of step (c) under plant growth conditions to obtain an additional non-naturally occurring plant; and (e) repeating the crossing and growing steps of (c) and (d) multiple times to generate additional generations of the non-naturally occurring plant. The method may optionally comprise, prior to step (a), providing a parent plant that has been characterized and contains a genetic identity that is not identical to that of the mutant or transgenic plant. In some embodiments, depending on the breeding program, the crossing and growing steps are repeated 0-2, 0-3, 0-4, 0-5, 0-6, 0-7, 0-8, 0-9, or 0-10 times to generate a generation of non-naturally occurring plants. Backcrossing is an example of such a method, in which the progeny is crossed with one of the parents or another plant genetically similar to the parent to obtain a next-generation progeny plant with a genetic identity closer to that of the parent. Plant breeding, particularly plant breeding techniques, are well known and can be used in the methods of the present disclosure. The present disclosure further provides non-naturally occurring plants produced by these methods. Certain embodiments exclude the step of selecting the plants.

[0219] In some embodiments of the methods described herein, lines resulting from breeding and screening for variant genes are evaluated in the field using standard field procedures. Control genotypes, including the original, unmutagenized parent, are included and entries are sequenced in the field in a randomized complete block design or other suitable field design. For tobacco, standard agronomic practices are used. For example, tobacco is harvested, weighed, and sampled for chemical and other common tests before and during curing. Statistical analysis of the data is performed to confirm the similarity of the selected lines to the parent lines. Cytogenetic analysis of the selected plants is optionally performed to confirm chromosome complement and chromosome pairing relationships.

[0220] DNA fingerprinting, single nucleotide polymorphism, microsatellite markers, or similar techniques may be used in marker-assisted selection (MAS) breeding programs to introgress or breed mutant alleles of genes into other tobacco plants, as described herein. For example, breeders can create segregating populations by hybridizing genotypes containing mutant alleles with agronomically desirable genotypes. F2 or backcross generation plants can be screened using markers developed from the genome sequence or a fragment thereof using one of the techniques listed herein. Plants identified as carrying the mutant allele can be backcrossed or self-pollinated to create a second population to be screened. Depending on the expected inheritance pattern or MAS technique used, it may be necessary to self-pollinated selected plants before each backcross cycle to help identify the desired individual plants. Backcrossing or other breeding procedures can be repeated until the desired phenotype of the recurrent parent is restored.

[0221] According to the present disclosure, in a breeding program, successful crosses produce fertile F1 plants. The selected F1 plants can be crossed with one of the parents, and the backcross first-generation plants are self-pollinated to produce a population that is again screened for variant gene expression (e.g., a null version of the gene). The backcross, self-pollination, and screening process is repeated, for example, at least four times, until a final screen produces a plant that is fertile and reasonably similar to the recurrent parent. The plant is self-pollinated if desired, and then the progeny are again screened to confirm that the plant exhibits variant gene expression. In some embodiments, the F2 generation plant population is screened for variant gene expression, for example, by using PCR methods with primers based on polynucleotide sequence information for the polynucleotides described herein (or any combination thereof described herein) according to standard methods to identify plants that are unable to express the polypeptide due to the absence of the gene.

[0222] Hybrid tobacco varieties can be produced by preventing self-pollination of a first variety of female parent plant (i.e., seed parent) and allowing pollen from a second variety of male parent plant to pollinate the female parent plant, resulting in the formation of F1 hybrid seeds in the female plant. Self-pollination of female plants can be prevented by emasculating the flowers at an early stage of flower development. Alternatively, pollen formation can be prevented in the female parent plant by forming male sterility. For example, male sterility can be caused by cytoplasmic male sterility (CMS) or transgenic male sterility, where the introduced gene inhibits microspore production or pollen formation, or self-incompatibility. Female parent plants containing CMS are particularly useful. In embodiments where the female parent plant is CMS, pollen is harvested from a male fertile plant and manually applied to the stigma of a CMS female parent plant, and the resulting F1 seeds are harvested.

[0223] The varieties and lines described herein can be used to form single-cross tobacco F1 hybrids. In such embodiments, plants of the parent varieties can be grown as a substantially homogeneous, adjacent population to promote natural cross-pollination from the male parent plants to the female parent plants. The F1 seeds formed in the female parent plants can be selectively harvested by conventional means. Two parent plant varieties can also be grown in large quantities, and a blend of F1 hybrid seeds formed in the female parent and seeds formed in the male parent as a result of self-pollination can be harvested. Alternatively, a three-way cross can be performed, in which a single-cross F1 hybrid is used as the female parent and crossed with a different male parent. As another alternative, a double-cross hybrid can be produced, in which the F1 progeny of two different single crosses are crossed with themselves.

[0224] A population of mutant, non-native, or transgenic plants can be screened or selected for those members of the population that have the desired trait or phenotype. For example, a population of progeny from a single transformation event can be screened for those plants that have the desired level of expression or function of the polypeptide encoded thereby. Physical and biochemical methods can be used to identify expression or activity levels. These include Southern analysis or PCR amplification for detecting polynucleotides; Northern blots, S1 RNase protection, primer extension, or RT-PCR amplification for detecting RNA transcripts; enzymatic assays for detecting enzyme or ribozyme activity of polypeptides and polynucleotides; and polypeptide gel electrophoresis, Western blots, immunoprecipitation, and enzyme immunoassays for detecting polypeptides. Other techniques, such as in situ hybridization, enzyme staining, and immunostaining and enzyme assays, can also be used to detect the presence or expression, function, or activity of polypeptides or polynucleotides.

[0225] Described herein are mutant, non-native, or transgenic plant cells and plants that comprise one or more recombinant polynucleotides, one or more polynucleotide constructs, one or more double-stranded RNAs, one or more conjugates, or one or more vectors / expression vectors.

[0226] Without limitation, the plants and parts thereof described herein can be modified either before or after the expression, function, or activity of one or more NtLKR polynucleotides and / or NtLKR polypeptides according to the present disclosure is modulated.

[0227] One or more of the following additional genetic modifications may be present in mutant, non-native, or transgenic plants and parts thereof: One or more genes involved in the conversion of nitrogenous metabolic intermediates may be modified to result in lower levels of at least one tobacco-specific nitrosamine (TSNA). Non-limiting examples of such genes include those encoding nicotine demethylases, such as CYP82E4, CYP82E5, and CYP82E10, as described in WO2006 / 091194, WO2008 / 070274, WO2009 / 064771, and WO2011 / 088180, and those encoding nitrate reductases, as described in WO2016 / 046288. One or more genes involved in heavy metal uptake or heavy metal transport may be modified to result in lower heavy metal content. Non-limiting examples include genes in the family of polypeptides associated with multidrug resistance, the family of cation diffusion facilitators (CDFs), the family of Zrt-Irt-like polypeptides (ZIPs), the family of cation exchangers (CAXs), the family of copper transporters (COPTs), the family of heavy metal ATPases (e.g., HMAs described in WO2009 / 074325 and WO2017 / 129739), the family of homologs of natural resistance-associated macrophage polypeptide (NRAMP), and other members of the ATP-binding cassette (ABC) transporter family (e.g., MRPs) described in WO2012 / 028309 involved in the transport of heavy metals such as cadmium.

[0228] Another exemplary modification can result in a plant with regulated expression or function of isopropylmalate synthase, which results in changes in sucrose ester composition that can be used to alter the beneficial profile (see WO 2013 / 029799). Another exemplary modification can result in a plant with regulated expression or function of threonine synthase, which can regulate methionine levels (see WO 2013 / 029800). Another exemplary modification can result in a plant with regulated expression or function of one or more of neoxanthin synthase, lycopene beta cyclase, and 9-cis-epoxycarotenoid dioxygenase, which regulate beta-damascenone content to alter the flavor profile (see WO 2013 / 064499). Another exemplary modification can result in a plant with regulated expression or function of a member of the CLC family of chloride channels to regulate nitrate levels in the plant (see WO 2014 / 096283 and WO 2015 / 197727). Another exemplary modification can result in plants with regulated expression or function of one or more asparagine synthetases that regulate asparagine levels in leaves and regulated levels of acrylamide in the aerosol produced upon heating or burning of leaves (see WO2017 / 129739). Another exemplary modification can result in plants with regulated protease activity during desiccation treatments (see WO2016 / 009006). Another exemplary modification can result in plants with reduced nitrate levels by altering the gene expression of nitrate reductase (e.g., Nia2) or the activity of the protein encoded thereby (see WO2016 / 046288).Another exemplary modification can result in plants with altered alkaloid levels by altering the gene expression of the putative ABC-2 transporters NtABCGl-T and NtABCGl-S or the activity of the protein encoded thereby (see WO 2019 / 086609). Another exemplary modification can result in plants that regulate the time to flowering by altering the gene expression of the gene encoding Terminal Flower 1 (TFL1) or the activity of the protein encoded thereby (see WO 2018 / 114641). Another exemplary modification can result in plants with regulated expression or function of one or more asparagine synthases to regulate asparagine levels in leaves, and plants with regulated levels of acrylamide in the aerosol produced upon heating or burning leaves (see WO 2017 / 042162). Other exemplary modifications include herbicide tolerance; for example, glyphosate is the active ingredient in numerous broad-spectrum herbicides. Glyphosate-tolerant transgenic plants have been developed by transferring the aroA gene (glyphosate EPSP synthase from Salmonella typhimurium and E. coli). Sulfonylurea-tolerant plants have been produced by transforming mutant ALS (acetolactate synthase) genes from Arabidopsis. Mutant Amaranthus hybridus photosystem II OB polypeptides have been transferred to plants to produce atrazine-tolerant transgenic plants, and bromoxynil-tolerant transgenic plants have been produced by incorporating the bxn gene from Klebsiella pneumoniae bacteria. Another exemplary modification results in insect-resistant plants. Bacillus thuringiensis (Bt) toxins can provide an effective method for delaying the emergence of Bt-resistant pests, as recently demonstrated in broccoli, where pyramidalized cry1Ac and cry1C Bt genes controlled diamondback moths resistant to either single polypeptide, significantly delaying the evolution of resistant insects.Another exemplary modification results in plants resistant to diseases caused by pathogens (e.g., viruses, bacteria, fungi). Plants expressing the Xa21 gene (resistance to bacterial leaf spot) have been engineered along with plants expressing both a Bt fusion gene and a chitinase gene (resistance to yellow stem borer and elytra). Another exemplary modification results in altered fertility, such as male sterility. Another exemplary modification results in plants tolerant to abiotic stresses (e.g., drought, temperature, salinity); resistant transgenic plants have been produced by transferring acylglycerol phosphate enzymes from Arabidopsis; genes encoding mannitol dehydrogenase and sorbitol dehydrogenase, involved in the synthesis of mannitol and sorbitol, improve drought tolerance. Another exemplary modification results in a plant in which the activity of one or more nicotine N-demethylases is regulated so that the levels of nornicotine and nornicotine metabolites formed during the drying process can be controlled (see WO2015169927). Other exemplary modifications can result in plants with improved polypeptide and oil storage capacity, increased photosynthetic efficiency, extended shelf life, enhanced carbohydrate content, and fungal resistance. Transgenic plants in which the expression of S-adenosyl-L-methionine (SAM) or cystathionine gamma-synthase (CGS), or a combination thereof, is regulated are also contemplated. One or more genes involved in the nicotine synthesis pathway can be modified to result in a plant or plant part that produces controlled levels of nicotine when dried. The nicotine synthesis gene may be selected from the group consisting of A622, BBLa, BBLb, JRE5L1, JRE5L2, MATE1, MATE2, MPO1, MPO2, MYC2a, MYC2b, NBBl, nic1, nic2, NUP1, NUP2, PMT1, PMT2, PMT3, PMT4, and QPT, or one or more combinations thereof.One or more genes involved in regulating the amount of one or more alkaloids can be modified to result in a plant or part of a plant producing regulated levels of alkaloids. The alkaloid level regulating gene can be selected from the group consisting of BBLa, BBLb, JRE5L1, JRE5L2, MATE1, MATE2, MYC2a, MYC2b, nic1, nic2, NUP1, and NUP2, or a combination of two or more thereof.

[0229] Other exemplary modifications can result in plants with modulated amino acid content (see WO2019 / 185703 and WO2021 / 063863), or plants with modulated sugar content (see WO2019 / 185699 and WO2021 / 063860 and WO2021 / 063863), or plants with modulated nitrate levels (see WO2020 / 141062), or plants with modulated sugar and amino acid content (see WO2021 / 063863).

[0230] In a preferred embodiment, yet another genetic modification relates to the asparagine synthetase (ASN) gene described in WO2017042162. Modulating the expression of the ASN gene (e.g., one or more of NtASN1-S, NtASN1-T, NtASN5-S, and NtASN5-T described in WO2017042162) or the activity of ASN (e.g., NtASN1-S, NtASN1-T, NtASN5-S, and NtASN5-T described in WO2017042162) significantly alters the chemical properties of cured tobacco leaves without affecting biomass. Thus, modulating the expression and / or activity of a combination of ASN and NtLKR may have the potential to reconfigure the chemical properties of cured tobacco leaves (particularly the amino acid chemistry of burley or dark tobacco), thereby altering their sensory characteristics.

[0231] In addition to ASN, other genes and enzymes play a role in the reorganization of amino acids and / or sugars during leaf yellowing, such as diaminopimelate aminotransferase (DAPAT), which is involved in both lysine catabolism and assimilation, and aspartate aminotransferase (AAT), which is expressed during senescence and may alter leaf chemistry after drought treatment (WO2019 / 185703). The chloroplast sulfate transporter SULTR3, such as NtSULTR3;1A-S, NtSULTR3;1A-T, and NtSULTR3;3-T, plays a role in sugar and amino acid metabolism during drought treatment (see WO2021 / 063863). Thus, additional genetic modifications may involve DAPAT and / or AAT (e.g., one or more of NtAATI-S, NtAAT1-T, NtAAT2-S, NtAAT2-T, NtAAT3-S, NtAAT3-T, NtAAT4-S, or NtAAT4-T as described in WO2017042162), and / or one or more of NtSULTR3;1A-S, NtSULTR3;1A-T, and NtSULTR3;3-T as described in WO2021 / 063863. Modulating the expression and / or activity of DAPAT and / or AAT and / or SULTR3 and NtLKR in combination may have the potential to reconfigure the chemistry of flue-cured tobacco leaves, thereby altering their organoleptic properties. Modifications to one or more, or two or more, or three or more, or four or more combinations of NtLKR and ASN, NtLKR and DAPAT, NtLKR and AAT, NtLKR and ASN and DAPAT, NtLKR and ASN and AAT, NtLKR and ASN and DAPAT and AAT, NtLKR and SULTR3, NtLKR and ASN and SULTR3, NtLKR and AAT and SULTR3, NtLKR and ASN and DAPAT and SULTR3, NtLKR and ASN and AAT and SULTR3, NtLKR and ASN and DAPAT and SULTR3, NtLKR and ASN and AAT and SULTR3, NtLKR and ASN and DAPAT and SULTR3, NtLKR and ASN and AAT and SULTR3, NtLKR and ASN and DAPAT and SULTR3, NtLKR and ASN and AAT and SULTR3, NtLKR and ASN and DAPAT and AAT and SULTR3.

[0232] One or more traits can be introgressed from another cultivar into, or directly transformed into, a mutant, non-native, or transgenic plant.

[0233] Various embodiments provide mutant, non-naturally occurring, or transgenic plants, as well as biomass, in which the expression level of one or more polynucleotides according to the present disclosure is modulated to thereby modulate the level of the encoded polypeptide.

[0234] The plant parts described herein, particularly the leaf blades and / or petioles and / or midribs of such plants, can be incorporated into or used in various consumable products, including, but not limited to, aerosol-forming materials, aerosol-forming devices, smoking articles, smokable articles, smokeless products, medical or cosmetic products, intravenous preparations, tablets, powders, and tobacco products. Examples of aerosol-forming materials include tobacco compositions, tobacco, tobacco extracts, cut tobacco, cut fillers, flue-cured tobacco, expanded tobacco, homogenized tobacco, reconstituted tobacco, and pipe tobacco. Smoking articles and smokable articles are types of aerosol-forming devices. Examples of smoking articles or smokable articles include cigarettes, cigarillos, and cigars. Examples of smokeless products include chewing tobacco and snuff. In certain aerosol-forming devices, rather than combustion, the tobacco composition or another aerosol-forming material is heated by one or more electric heating elements to produce an aerosol. In another type of heated aerosol-forming device, aerosol is produced by the transfer of heat from a combustible fuel element or heat source to a physically separated aerosol-forming material that may be located within, around, or downstream of the heat source. Smokeless tobacco products and various tobacco-containing aerosol-forming materials may contain tobacco in any form, including as dry particles, pieces, granules, powders, or slurries, or deposited on, mixed with, surrounded by, or combined with any form of other component, such as flakes, films, tabs, foams, or beads. The term "smoke" is used to describe the type of aerosol produced by a smoking article, such as a cigarette, or by burning an aerosol-forming material.

[0235] In one embodiment, cured plant material derived from the mutant, transgenic, and non-naturally occurring plants described herein is also provided. Processes for curing green tobacco leaves are known to those skilled in the art and include, but are not limited to, the air-curing, flame-curing, hot-air-curing, and sun-curing processes described herein.

[0236] In another embodiment, a tobacco product is described that includes a tobacco-containing aerosol-forming material, including plant material, such as leaves, preferably cured leaves, obtained from a mutant tobacco plant, a transgenic tobacco plant, or a non-naturally occurring tobacco plant described herein. The tobacco product described herein can be a blended tobacco product, which can further include unmodified tobacco.

[0237] Mutant, non-native, or transgenic plants may have other uses, for example, in agriculture.

[0238] The present disclosure also provides a method for producing seeds, including cultivating a mutant plant, a non-natural plant, or a transgenic plant described herein and collecting seeds from the cultivated plant. The seeds obtained from the plants described herein can be prepared by means known in the art and packaged in packaging material to form an article of manufacture. Packaging materials such as paper and cloth are well known in the art. The seed package has a label, for example, a tag or label fixed to the packaging material, and a label describing the nature of the seeds therein is printed on the package.

[0239] Compositions, methods, and kits for genotyping plants for identification, selection, or breeding can include means for detecting the presence of NtLKR polynucleotides in a sample of polynucleotides. Accordingly, compositions are described that include one or more primers for specifically amplifying at least a portion of one or more NtLKR polynucleotides, and optionally one or more probes and optionally one or more reagents for carrying out the amplification or detection.

[0240] Thus, gene-specific oligonucleotide primers or probes are disclosed that comprise about 10 or more contiguous polynucleotides corresponding to the NtLKR polynucleotides described herein. Such primers or probes can comprise or consist of more than about 15, 20, 25, 30, 40, 45, or 50 contiguous polynucleotides that hybridize (e.g., specifically hybridize) to the NtLKR polynucleotides described herein. In some embodiments, the primers or probes can comprise or consist of about 10-50 contiguous nucleotides, about 10-40 contiguous nucleotides, about 10-30 contiguous nucleotides, or about 15-30 contiguous nucleotides, which can be used in sequence-dependent methods of gene identification (e.g., Southern hybridization) or isolation (e.g., in situ hybridization of bacterial colonies or bacteriophage plaques) or gene detection (e.g., as one or more amplification primers in amplification or detection). One or more specific primers or probes can be designed and used to amplify or detect part or all of a polynucleotide. As a specific example, two primers can be used in a PCR protocol to amplify a polynucleotide fragment. PCR can also be performed using one primer derived from a polynucleotide sequence and a second primer hybridizing to a sequence upstream or downstream of the polynucleotide sequence, such as a promoter sequence, the 3' end of a pre-mRNA, or a sequence derived from a vector. Examples of temperature and isothermal techniques useful for in vitro amplification of polynucleotides are well known in the art. The sample may or may not be obtained from a plant, plant cell, or plant material, or a tobacco product made or obtained from a plant, plant cell, or plant material described herein.

[0241] In yet another aspect, a method for detecting an NtLKR polynucleotide described herein (or any combination thereof described herein) in a sample is also provided, the method comprising: (a) providing a sample containing or suspected of containing a polynucleotide; (b) contacting the sample with one or more primers or one or more probes to specifically detect at least a portion of the NtLKR polynucleotide; and (c) detecting the presence of an amplification product, the presence of which indicates the presence of the NtLKR polynucleotide in the sample. In yet another aspect, the use of one or more primers or probes to specifically detect at least a portion of the NtLKR polynucleotide is also provided. A kit for detecting at least a portion of an NtLKR polynucleotide is also provided, the kit comprising one or more primers or probes to specifically detect at least a portion of the NtLKR polynucleotide. The kit may include reagents for polynucleotide amplification, such as PCR, or reagents for probe hybridization detection techniques, such as Southern blot, Northern blot, in situ hybridization, or microarray. The kit may include reagents for antibody binding detection techniques, such as Western blot, ELISA, SELDI mass spectrometry, or test strips. The kit may include reagents for DNA sequencing. The kit may include reagents and instructions for using the kit.

[0242] In some embodiments, the kits may include instructions for one or more of the described methods. The described kits may be useful for genetic identity determination using co-dominant scoring, phylogenetic studies, genotyping, haplotyping, genealogy analysis, or plant breeding, among other things.

[0243] The present disclosure also provides methods for genotyping plants, plant cells, or plant materials containing the NtLKR polynucleotides described herein. Genotyping provides a means of distinguishing between chromosomal pair homologs and can be used to identify segregants in plant populations. Molecular marker methods can be used for phylogenetic studies, characterizing genetic relationships between crop varieties, identifying crosses or somatic hybrids, localizing chromosomal segments affecting single inherited traits, map-based cloning, and quantitative genetic studies. Specific methods of genotyping can employ any number of molecular marker analysis techniques, including amplified fragment length polymorphism (AFLP). AFLP is the product of allelic differences between amplified fragments caused by polynucleotide variability. Thus, the present disclosure further provides a means for tracking the segregation of one or more genes or polynucleotides, as well as chromosomal sequences genetically related to these genes or polynucleotides, using techniques such as AFLP analysis.

[0244] Also disclosed herein are methods for producing liquid tobacco extracts, and the liquid tobacco extracts produced by the methods.

[0245] A specific extraction temperature is selected for the tobacco starting material. The extraction temperature is typically selected within the range of about 100°C to about 160°C. The duration of the heating step may optionally be controlled to provide some control over the composition of the extract derived from the tobacco starting material. Preferably, the tobacco starting material is heated at the extraction temperature for at least about 90 minutes, more preferably at least about 120 minutes. The heating step is typically carried out in an inert atmosphere. Preferably, a stream of inert gas, such as nitrogen, is passed through the tobacco starting material during the heating step. Volatile tobacco compounds are released into the inert gas stream during the heating step, so that the inert gas acts as a carrier for the volatile components. The inert gas stream may have a flow rate of at least about 25 liters / minute, more preferably at least about 30 liters / minute. A relatively high flow rate of the inert gas may advantageously improve the efficiency of extraction from the tobacco starting material. Optionally, the heating step may be carried out under vacuum. Suitable heating methods for carrying out the heating of the tobacco starting material are known to those skilled in the art and include dry distillation, steam distillation, vacuum distillation, flash distillation, and thin film steam distillation.

[0246] If the volatile compounds are recovered by absorption in a liquid solvent, the step of forming a liquid tobacco extract may include drying the solution of the volatile compounds in the liquid solvent to concentrate the solution. Drying may be carried out using any suitable means, including, but not limited to, desiccation, molecular sieving, freeze-drying, phase separation, distillation, membrane permeation, controlled water crystallization and filtration, reverse hygroscopicity, ultracentrifugation, liquid chromatography, reverse osmosis, or chemical drying.

[0247] Liquid tobacco extracts are particularly suitable for producing compositions, formulations, or gel compositions for use in aerosol generating systems. Aerosol generating systems are disclosed that include the compositions, formulations, or gel compositions. In such aerosol generating systems, the compositions, formulations, or gels are typically heated in an aerosol generating device, e.g., a device that includes a heater element that interacts with the composition, formulation, or gel incorporating the liquid tobacco extract to produce an aerosol. During use, volatile compounds are released by heat transfer and entrained in the air drawn through the aerosol generating device. As the released compounds cool, they condense to form an aerosol, which is inhaled by the consumer.

[0248] The present invention is further described in the following examples, which are provided to describe the invention in more detail. These examples describe preferred modes presently contemplated for carrying out the invention and are intended to illustrate, but not limit, the invention. [Example]

[0249] Example 1 - Materials and Methods plant material Before germination, seeds were sterilized by the vapor chlorine gas method. A 5% final chlorine solution was placed in a bell jar with the seed tube. Hydrochloric acid (37%) was then added to the solution, and the seeds were incubated for 2 hours. Then, under a laminar flow hood, the seeds were placed on Murashige and Skoog (Int J Mol Sci. (2020) 21(10):3441) growth medium and transferred to a plant growth room (24°C, 16 hours of light / 20°C, 8 hours of darkness) for 4 weeks. Well-developed plantlets were transferred to a greenhouse and grown in 10 L pots (2-6 replicates) until fully grown. Artificial light was applied for 16 hours daily. For each plant, a representative fully grown leaf was sampled at the flowering stage. The collected samples were then freeze-dried and disrupted by shaking at 400 rpm in a container containing glass beads for 8 hours for leaves and 24 hours for roots. Any roots that remain unbroken are now ground to the finest possible level in a mortar.

[0250] Methods for determining free amino acid, sugar, ammonia, and nitrate levels Amino acid content is measured using Method MP 1471 rev 5 2011, Resana, Italy: Chelab Silliker Srl, Merieux NutriSciences Company. To determine amino acids in cured plant leaves, the cured leaf pieces are dried at 40°C for 2-3 days after midrib removal, if necessary. The tobacco material is then ground to a fine powder (approximately 100 µM) before analysis of amino acid content. Alternatively, amino acid content in plant material is measured as described in UNI EN ISO 13903:2005.

[0251] Reducing sugar content is measured using a segmented flow colorimetric method developed for the analysis of tobacco samples, as adapted by Skalar Instrument Co. (West Chester, PA) and described in Tobacco Science 20:139-144 (1976). Measurement of reducing sugar content is also described in Coresta Recommended Method 38, CRM38, CRM, and ISO 15154:2003. To determine reducing sugars in cured leaves, the cured leaf sections are dried at 40°C for 2-3 days after midrib removal, if necessary. The tobacco material is then ground to a fine powder (approximately 100 μM) before analysis of reducing sugars. Alternatively, reducing sugar content is measured according to ISO 15154:2003.

[0252] Nitrate content is measured using a Lachet QuikChem 8500 instrument according to the manufacturer's protocol (Lachet QuikChem Method 12-107-04-1-J, Lachet Instruments, Loveland, CO, USA). Alternatively, nitrate content is measured according to ISO 15517:2003.

[0253] The ammonia content is determined using ion chromatography according to ISO 21045:2018.

[0254] Gene expression analysis The generated sequencing data was demultiplexed using Illumina BaseSpace® Clarity LIMS (Illumina, Inc.) and then imported into Qiagen CLC Genomics Workbench version 12.0.1 (CLC bio, QIAGEN). Transcriptome reads were mapped to an updated version of the N. tabacum reference genome (BMC Genomics (2017) 18(1):448) using the "RNA-Seq Analysis" 2.16 tool with a similarity of 0.8 (S = 0.8) and a fractional length of 0.8 (L = 0.8) as mapping criteria. The mismatch cost was set to 2, the insertion cost was set to 3, and the deletion cost was set to 3. No global alignment was performed; instead, paired distances were automatically detected. The maximum number of read hits was set to 10, and paired reads were counted as 1. Gene expression—FPKM values ​​were obtained for each gene in the reference genome and for genes without a transcription model.

[0255] RNAi procedure A specific DNA fragment (SEQ ID NO: 8) was selected to suppress the expression of both NtLKR-S and NtLKR-T. It was cloned between the strong constitutive MMV promoter and the 3'nos terminator sequence of the nopaline synthase gene from Agrobacterium tumefaciens (Plant Mol Biol (1999) 40(5):771-82). Burley tobacco cultivar TN90 and Virginia tobacco cultivar K326 were transformed using standard Agrobacterium-mediated transformation protocols (Methods Mol Biol. (2006) 343:143-54 and Transgenic Res. (2013) 22(3):643-9). Seeds were harvested from three independent TO lines that showed the strongest NtLKR silencing. T1 plants from 10 lines were grown in a greenhouse and selected for the presence of the construct insertion in genomic DNA by PCR using the following primers (5'-3'): MMV-F (SEQ ID NO: 9) and IPMS2-R (SEQ ID NO: 10). To verify that the progeny exhibited efficient transcriptional suppression, RNA was isolated from transgenic plants of each independent transformation event and their corresponding control plants, and qPCR experiments were performed using primers NtLKR-F1 (SEQ ID NO: 6) and NtLKR-R1 (SEQ ID NO: 7) to evaluate LKR gene expression levels.

[0256] Example 2 - Metabolic Analysis Metabolic analysis revealed that Lys levels increased in senescent tobacco leaves (48 hours after burley etiolated leaves during the air-curing process) and catabolic products resulting from endogenous LKR activity, such as saccharopine, 2-aminoadipate and 2-ketoadipate, and pipecholate. Furthermore, transcriptome analysis revealed that LKR genes involved in Lys degradation were also strongly upregulated during the etiolated stage. These data suggested that Lys accumulates early during the curing process.

[0257] As shown in Figure 5, NtLKR is strongly induced during the early drought treatment stage, so-called etiolation (senescence gene program, 0–192 h BU, and 0–60 h FC).

[0258] Example 3 - Downregulation of NtLKR via RNAi silencing approach NtLKR is downregulated via an RNAi silencing approach to determine the chemical effects on cured leaves of two tobacco species, burley and Virginia. Plants are grown in a greenhouse. Classical agricultural practices are used for plant cultivation and curing, such as air-curing for burley and hot-air tube curing for Virginia after harvesting mature leaves (Industrial Crops and Products, 167, 2021, 113534, ISSN 0926-6690 and Leaf Curing Practices: Alter Gene Expression and the Chemical Constituents of Tobacco Leaves. In: Ivanov, NV, Sierro, N., Peitsch, MC (eds) The Tobacco Plant Genome. Compendium of Plant Genomes. Springer, Cham).

[0259] To investigate the function of the NtLKR gene and the possible accumulation of Lys in drought-treated leaves when NtLKR (LKR / SDH) is downregulated, NtLKR-RNAi plants were generated using the insert of SEQ ID NO: 8 as an RNAi construct. Compared to wild-type plants, no distinctive phenotypes were observed when anti-NtLKR T1 plants were grown. Several transformed lines were grown at TO, screened, and selected via RT-qPCR (using intact green midrib / leaf blade as tissue for RNA isolation). Seeds from plants showing significant downregulation of NtLKR were re-grown (T1 plants) and rescreened by RT-qPCR to confirm NtLKR silencing (see Figure 1). Based on gene expression levels, three NtLKR-T1 lines were selected in both Burley TN90 and Virginia K326 tobacco varieties for further analysis of free amino acid, sugar, ammonia, and nitrate contents.

[0260] Example 4 - Effect of NtLKR silenced lines on senescence in the Burley TN90 background Because NtLKR-RNAi burley TN90 plants are highly efficient at accumulating more Lys and several other amino acids (Arg), their effect on etiolation (assuming Lys is the energy source for etiolated leaves) will be tested in leaves at the mid-stem.

[0261] Control and T2-NtLKR-RNAi plants on the burley TN90 background are grown in a greenhouse in a randomized fashion. After two months of growth, all plants are pinched and half of the cultures are switched from the burley nutrient solution (EC = 2.4) to water (EC = 0.8). The other half of the cultures remain in the burley nutrient solution.

[0262] The data show chlorophyll measurements (CCI, 3 measurements per plant) at stem position C 21 days after switching the nutrient solution (see Figure 2).

[0263] Chlorophyll (CCI) measurements at the mid-stem leaf position in control (CT2-E438) and LRK-RNAi (T2-E438-5) plants are shown in Table 1. Plants were pinched after two months of growth. At that time, half of the cultures were switched to water (H2O, EC = 0.8) instead of nutrient solution (EC = 2.4). Chlorophyll (CCI) was recorded after 21 days. Numbers indicate p-values ​​from a t-test.

[0264] Interestingly, the absence of the NtLKR gene (copies S and T) induced a faster chlorophyll loss, indicating that these plants senesce earlier compared to controls. Thus, Lys accumulation contributes to faster leaf senescence.

[0265] Example 5 - Effect of NtLKR downregulation (T1 plants) on the chemical properties of Burley tobacco TN90 Free amino acids, sugars, ammonia, and nitrate were determined in desiccation-treated leaves for control (WT, n = 14) and NtLKR-RNAi (n = 13) plants. For burley, no differences were observed in sugars, ammonia, and nitrate. Table 2 illustrates the averages of single amino acids in control and NtLKR-RNAi plants, as well as the percent / fold changes and statistical associations (p-value ANOVA) for fully desiccation-treated tobacco. As suspected by blocking NtLKR / SDH activity, Lys significantly increased by approximately 11-fold in the leaf blades of desiccation-treated leaves, thus altering the chemical properties of the final material compared to control leaves. The contents of other amino acids, namely, Arg, Pro, GABA, Gln, Leu, Ala, Phe, Tyr, Ileu, Met, Thr, and Gly, also changed significantly, but to a lesser extent than Lys (see Table 2). Interestingly, Met decreased threefold. This confirms a stronger reorganization of free amino acids in desiccation-treated leaves compared to green leaves, which is mostly driven by the activity of aminotransferases during leaf yellowing (early desiccation), as shown by Bovet et al. (2019) Plants (Basel) 11;8(11):492. In addition to asparagine synthetase, which is in fact an aminotransferase (WO2017042162), others play a role in amino acid reorganization during leaf yellowing, such as diaminopimelate aminotransferase (DAPAT), which is involved in both lysine catabolism and assimilation, and aspartate aminotransferase (AAT), which is also expressed during senescence and may alter leaf chemistry after desiccation (WO2019185703). Because photosynthesis is no longer active during leaf yellowing, the only way for early senescing leaves is to remobilize N sources for seed N stores by transferring amino groups to enter energy machinery such as mitochondria to produce reducing equivalents (NADPH or NADH) or by producing cytoplasmic Lys (see Figure 2) and Asn (Bovet et al. (2019) Plants (Basel) 11;8(11):492).By blocking NtLKR activity and accumulating Lys, this alters the balance of free amino acids in drought-treated leaves.

[0266] The increase in Lys (>11x) shown in Figure 3 is fully consistent with the silencing of NtLKR in three independent RNAi lines (see Figure 1). This also confirms the activity of Nitab09g012060.1.1 and Nitab18g020540.1.1 (LKR-T and LKR-S, respectively) as LKR enzymes. Although the Lys content in green leaves of NtLKR-RNAi lines was not measured, the expression profiles of NtLKR-S and NtLKR-T, which function as senescence-activating genes, suggest that the accumulation of Lys in drought-treated leaves is likely associated with the senescence process. If the yellowing stage occurs naturally in the stem, it can be assumed that Lys and Asn are then remobilized and transported to the seeds via the phloem. Increases in Arg and Pro, although mild, were also very evident in the three independent lines and resulted from NtLKR inactivation.

[0267] Example 6 - Effect of NtLKR downregulation (T1 plants) on the chemical properties of Virginia tobacco K326 Free amino acids, sugars, ammonia, and nitrate were analyzed in desiccation-treated leaf blades of control (WT, n = 8) and NtLKR-RNAi (n = 13) Virginia tobacco plants. In contrast to burley, Virginia species require less N fertilization and accumulate less free nitrate, ammonia, and free amino acids in desiccation-treated leaves, but more reducing sugars. Tables 3 and 4 show the means of free amino acids, sugars, ammonia, and nitrate in both control and NtLKR-RNAi plants, as well as the percent / fold change and statistical associations (p-values, ANOVA) for fully desiccation-treated tobacco. As suspected, blocking NtLKR / SDH activity significantly increased Lys in Virginia, as in desiccation-treated burley leaves, but to a lesser extent (approximately 4x).

[0268] This accumulation of Lys in virginia (4x) was accompanied by significant changes in other free amino acids, reducing sugars, and nitrate. The contents of other amino acids that were altered were Arg, Gln, His, Tyr, Trp, Thr, GABA, Asn, Ala, Ileu, Val, and Ser (see Table 3). Considering the fact that the total amino acid content of virginia is approximately 10-fold lower than that of burley (compare Tables 2 and 3), the effect of NtLKR gene silencing on the amino acids in desiccated virginia leaves was weaker. However, a significant 20% decrease in glucose and fructose, and a 34% decrease in nitrate, compared to WT, was observed. Regarding nitrate, the values ​​were so low that it is difficult to conclude whether this correlates with the lack of active NtLKR / SDH. On the other hand, the 20% reduction in each major reducing sugar is more consistent. Indeed, when evaluating both glucose and fructose, the sum of reducing sugars corresponds to 17.04 in WT and 13.49 in NtLKR-RNAi, resulting in a reduction of reducing sugars of approximately 3.5% in the transgenic lines. As in burley, no change in ammonia content is observed (where this gene was initially targeted).

[0269] The data presented in Figure 4 demonstrate a significant increase (>4x) in Lys levels, fully aligned with NtLKR silencing in three different, independent RNAi lines (see Figure 2). This also confirms the activity of Nitab09g012060.1.1 and Nitab18g020540.1.1 (LKR-T and LKR-S, respectively) as LKR enzymes. Increases in Arg and Gln levels, although modest, were also significant in three independent lines and result from NtLKR inactivation. An overall 20% decrease in glucose and fructose levels in Virginia NtLKR-RNAi plants reached as much as 30% in line E437-12. Such decreases could result from either improved carbon respiration, assimilation of aa already present in green leaves, or reduced starch accumulation in chloroplasts. It is evident that the reduction of NtLKR activity has low impact on the biological functions of vegetative plants and does not affect the fitness and growth of transgenic plants, as demonstrated when they enter the senescence program.

[0270] Our observations showed that when Nt LKR was downregulated, Lys increased, whereas methionine and threonine decreased (particularly in the N-accumulating tobacco species Burley). However, a decrease in glucose as a mitochondrial energy source was observed in the highly reducing sugar-accumulating tobacco species Virginia.

[0271] Example 7 - Sensory Effects in RNAi Modified Burley TN90 Plants The modified sensory profile is shown in Table 5. The accumulation of Lys in the modified Burley TN90 contributes to a reduction in harshness, making the aerosol smoother and more mellow, with less typical dark notes but more animalic and nutty notes (less trigeminal impact).

[0272] Example 8 - Sensory Effects in RNAi Modified Virginia K326 Plants The altered sensory profile is shown in Table 6 and contributes to an increase in hay notes along with a rounder and darker flavor profile.

[0273] Any publications cited or described herein provide relevant information disclosed prior to the filing date of the present application. Nothing herein should be construed as an admission that the inventors are not entitled to antedate such disclosure. All publications mentioned in the above specification are incorporated herein by reference. Various modifications and variations of the present invention will become apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described methods for carrying out the invention that are obvious to those skilled in the art of cell biology, molecular biology, plant biology, or related fields are intended to be within the scope of the following claims.

[0274] array SEQ ID NO: 1 Polynucleotide coding sequence of LKR-S from Nicotiana tabacum SEQ ID NO: 2 Polypeptide sequence of LKR-S from Nicotiana tabacum MTDLLKFGREILGPVIMFGNGVVGILSEATNKWERRAPLTPSHCARLLHGGGKTGVSRIIMQPSTKRVHHDALYEDVGCEISEDLSDCGLILGIKQPKLEMILPDRAYAFFSHTHKAQKENMPLLDKILAERASLFDYELIVGDTGKRLLAFGSFAGRAGMIDFLRGGLWYLNHGYSTPFLSLGSSY MYSSLAAAKAAVISVGEEIATMGLPAGICPLVFVFTGSGNVSRGAQEIFKLLPHTFVDPRKLSELHETARDLTQSKHPSKRIFQVYGCVTTCQDMVEHLNPSKSFDKIDYYAHPEQYRPAFHEKIAPYVSVIVNCMYWEKRFPRLLTTKQIQDLMRNGCPLVGICDITCDVGGSIEFINQTSSIDSPFFR YEPSNDSYHYDIEGKGVMCSAVDILPTEFAKEASQHFGDILSHFTGSLASFRNLEELPAHLKRACIAHGALTQLYEYIPRMRKSDLEDPSTVLSSSNANGRKYTVLVSLSGHLFDKFLINEALDIIEAAGGSHFHLVKCQVGQITSALSYSELEVGAEDKAVLDKIVDSLTSLANSRNSLGSQNKENNMI SLKVGEFQQSIIDEKSDAKKVLILGAGRVCRPAAELLASIGSMSSGQWLSSITADFEEQHCVQVIVASLYLKDAEEVTEGIPNAKAVQLDIMNHESLSSCISQVDVVISLLPPSCHGIVAKSCIELKKHLVTASYVNDSMLKLDEDAKCAGITILGEMGLDPGIVTLIRVSMNLLENSSWYEISSECDCA SEQ ID NO: 3 Polynucleotide coding sequence of LKR-T from Nicotiana tabacum SEQ ID NO: 4 Polypeptide sequence of LKR-T from Nicotiana tabacum SEQ ID NO: 5 NtLKR-RNAi insert: caggttgatgttgtcatcagcttactgcctcctagttgccatggtattgtagcaaaatcatgcattgagctgaagaaacatcttgtcacagctagctacgttaatgattc SEQ ID NO: 6 NtLKR-F1 primer for qPCR atattattgaagcagcaggtggc SEQ ID NO: 7 NtLKR-R1 primer for qPCR tgctttatcttcagctccaacct SEQ ID NO: 8 DNA fragment used to suppress the expression of NtLKR-S and NtLKR-T Caggttgatgttgtcatcagcttactgcctcctagttgccatggtattgtagcaaaatcatgcattgagctgaagaaacatcttgtcacagctagctacgttaatgattc SEQ ID NO: 9 MMV-F primer gacgtctaatcccaacttcgtc SEQ ID NO: 10 IPMS2-R primer gacgtctaatcccaacttcgtc

[0275] [Table 2]

[0276] [Table 3]

[0277] [Table 4]

[0278] Table 5

[0279] Table 6

[0280] Table 7

Claims

1. 1. A mutant, non-native, or transgenic Nicotiana tabacum plant or part thereof having regulated expression or activity of lysine-ketoglutarate reductase (LKR), wherein the LKR is (i) a polynucleotide comprising, consisting of, or consisting essentially of a sequence having at least 88% sequence identity to SEQ ID NO: 1 (NtLKR-S) and / or at least 86% sequence identity to SEQ ID NO: 3 (NtLKR-T); (ii) a polypeptide encoded by the polynucleotide according to (i); (iii) a polypeptide comprising, consisting of, or consisting essentially of a sequence having at least 89% sequence identity to SEQ ID NO: 2 (NtLKR-S) and / or at least 88% sequence identity to SEQ ID NO: 4 (NtLKR-T); or (iv) a construct, vector, or expression vector comprising the isolated polynucleotide of (i); comprising, consisting of, or consisting essentially of, A mutant, non-native, or transgenic Nicotiana tabacum plant or part thereof, wherein the plant or part thereof comprises at least one modification that is capable of modulating (a) the expression of the polynucleotide in the plant or part thereof, or (b) the activity of the polypeptide in the plant or part thereof, compared to a control plant or part thereof in which the expression of the polynucleotide or the activity of the polypeptide is not modified.

2. the modification comprises at least one genetic alteration in the coding sequence of the polynucleotide or in a regulatory region of the polynucleotide; and / or the modification comprises one or more of exogenous DNA or exogenous RNA; and / or the modification comprises one or more of a vector, or a viral vector, or an Agrobacterium vector, or a CRISPR vector; and / or the modification is capable of driving one or more of RNA interference or transcriptional gene silencing, or virus-induced gene silencing; and / or 2. The mutant, non-naturally occurring, or transgenic Nicotiana tabacum plant or part thereof of claim 1, wherein the modification is capable of expressing one or more of double-stranded RNA (dsRNA) or hairpin RNA (hpRNA) or small interfering RNA.

3. The modulated expression or activity of said LKR confers modulation of the level of said one or more amino acids in said plant or part thereof compared to the level of said one or more amino acids in said control plant, preferably the modulated expression or activity of said LKR confers modulation of the timing of leaf senescence, preferably 3. The mutant, non-native, or transgenic Nicotiana tabacum plant or part thereof of claim 1 or claim 2, wherein the amino acid is lysine.

4. The part of the mutant, non-naturally occurring, or transgenic Nicotiana tabacum plant is a dried leaf or a dried leaf, preferably 4. The mutant, non-naturally occurring, or transgenic Nicotiana tabacum plant or part thereof according to any one of claims 1 to 3, wherein the levels of at least lysine, arginine, GABA, glutamine, alanine, tyrosine, isoleucine, and threonine are modulated in the dried-treated or dried leaves compared to the dried-treated or dried leaves obtained from the control plant.

5. The Nicotiana tabacum plant or part thereof is a burley species, preferably the levels of at least lysine, arginine, proline, GABA, glutamine, leucine, alanine, phenylalanine, tyrosine, isoleucine, methionine, threonine, and glycine are modulated in the desiccation-treated or dried leaves compared to desiccation-treated or dried leaves obtained from a control plant, and / or the levels of at least asparagine, aspartic acid, tryptophan, histidine, glutamic acid, serine, and valine are not significantly changed; and / or 5. The mutant, non-naturally occurring, or transgenic Nicotiana tabacum plant or part thereof according to any one of claims 1 to 4, wherein the expression of the LKR polynucleotide or the activity of the LKR polypeptide is reduced or inhibited in the desiccation-treated leaves or desiccation-treated leaves, and wherein the desiccation-treated leaves or desiccation-treated leaves have (i) increased levels of at least lysine, arginine, proline, GABA, glutamine, leucine, alanine, phenylalanine, tyrosine, and isoleucine, (ii) decreased levels of at least methionine, threonine, and glycine, and (iii) no significant changes in the levels of at least asparagine, aspartic acid, tryptophan, histidine, glutamic acid, serine, and valine, compared to the desiccation-treated leaves or desiccation-treated leaves obtained from the control plant.

6. The Nicotiana tabacum plant or part thereof is of the species Virginia, and preferably 5. The mutant, non-naturally occurring, or transgenic Nicotiana tabacum plant or part thereof according to any one of claims 1 to 4, wherein the levels of at least lysine, arginine, glutamine, histidine, tyrosine, tryptophan, threonine, GABA, asparagine, alanine, isoleucine, valine, and serine are modulated, and there is no significant change in the levels of at least proline, aspartic acid, leucine, phenylalanine, glutamic acid, and methionine in the dried-treated or dried leaves, compared to the dried-treated or dried leaves obtained from the control plant.

7. 7. The mutant, non-naturally occurring, or transgenic Nicotiana tabacum plant or part thereof of claim 6, wherein the expression of the LKR polynucleotide or the activity of the LKR polypeptide is reduced or inhibited in the desiccation-treated leaves or desiccation-treated leaves, and the desiccation-treated leaves or desiccation-treated leaves have (i) increased levels of at least lysine, arginine, glutamine, histidine, tyrosine, tryptophan, threonine, GABA, asparagine, and alanine, (ii) decreased levels of at least isoleucine, valine, and serine, and (iii) no significant changes in the levels of at least proline, aspartic acid, leucine, phenylalanine, glutamic acid, and methionine, compared to desiccation-treated leaves or desiccation-treated leaves obtained from the control plant.

8. The total sugar content is adjusted, preferably 8. The mutant, non-native, or transgenic Nicotiana tabacum plant or part thereof of claim 6 or 7, wherein the total amount of sugars is reduced.

9. 9. A Nicotiana tabacum plant material, dried Nicotiana tabacum plant material, or homogenized Nicotiana tabacum plant material derived from or obtained from a Nicotiana tabacum plant or part thereof according to any one of claims 1 to 8, preferably comprising: The Nicotiana tabacum plant material is selected from the group consisting of biomass, seeds, stems, flowers, or leaves, or a combination of two or more thereof, and is preferably The Nicotiana tabacum plant material is a leaf, preferably The leaves are dried leaves, preferably 10. The Nicotiana tabacum plant material, dried Nicotiana tabacum plant material, or homogenized Nicotiana tabacum plant material, wherein the dried leaves are selected from the group consisting of hot-tube dried leaves, sun dried leaves, or air dried leaves.

10. 1. A method for producing a Nicotiana tabacum plant having a modulated level of at least one amino acid, comprising: (a) (i) a polynucleotide comprising, consisting of, or consisting essentially of a sequence having at least 88% sequence identity to SEQ ID NO: 1 (NtLKR-S) and / or at least 86% sequence identity to SEQ ID NO: 3 (NtLKR-T); (ii) a polypeptide encoded by the polynucleotide according to (i); (iii) a polypeptide comprising, consisting of, or consisting essentially of a sequence having at least 89% sequence identity to SEQ ID NO: 2 (NtLKR-S) and / or at least 88% sequence identity to SEQ ID NO: 4 (NtLKR-T); or (iv) providing a Nicotiana tabacum plant comprising, consisting of, or consisting essentially of a construct, vector, or expression vector comprising the isolated polynucleotide of (i); (b) introducing at least one modification capable of modulating (a) the expression of the LKR polynucleotide in the Nicotiana tabacum plant, or (b) the activity of the LKR polypeptide in the Nicotiana tabacum plant, compared to a control in which the expression of the LKR polynucleotide or the activity of the LKR polypeptide is not modified.

11. In step (b), the at least one modification is introduced by genome editing, preferably the genome editing is selected from CRISPR-mediated genome editing, mutagenesis, zinc finger nuclease-mediated mutagenesis, chemical or radiation mutagenesis, homologous recombination, oligonucleotide-directed mutagenesis, and meganuclease-mediated mutagenesis; or 11. The method of claim 10, wherein in step (b), the at least one modification is introduced using an interfering polynucleotide.

12. 12. A Nicotiana tabacum plant obtained or obtainable by the method of claim 10 or claim 11.

13. 1. A method for producing desiccated Nicotiana tabacum plant material having an altered level of at least one amino acid, comprising: (a) producing a Nicotiana tabacum plant according to claim 10 or claim 11; (b) harvesting plant material (e.g., leaves) from said Nicotiana tabacum plant; (c) drying the plant material.

14. 14. Dry-processed Nicotiana tabacum plant material (e.g. leaves) obtained or obtainable by the method of claim 13.

15. 15. A tobacco product comprising the Nicotiana tabacum plant material, cured Nicotiana tabacum plant material, or homogenized Nicotiana tabacum plant material of claim 9, or the cured Nicotiana tabacum plant material of claim 14, preferably comprising: The tobacco product is a tobacco blend, preferably A tobacco product, wherein the tobacco blend comprises Virginia tobacco and / or Burley tobacco.

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