Modulation of genes encoding lysine ketoglutarate reductases

Abstract

There is disclosed a mutant, non-naturally occurring or transgenic tobacco plant or part thereof having modulated expression or activity of lysine ketoglutarate reductase (LKR) comprising, consisting of, or consisting essentially of: (i) a polynucleotide, the present invention relates to a protein comprising, consisting or consisting essentially of a sequence having at least 88% sequence identity to SEQ ID NO: 1 (NtLKR-S) and / or a sequence having 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 or consisting essentially of a sequence having at least 89% sequence identity to SEQ ID NO: 2 (NtLKR-S) and / or a sequence having 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) wherein the plant or part thereof comprises at least one modification as compared to a control plant or part thereof wherein the expression of the polynucleotide or the activity of the polypeptide is not modified, the at least one modification is capable of modulating: (a) expression of the polynucleotide in the plant or part thereof; or (b) the activity of the polypeptide in the plant or part thereof.

Description

Technical Field

[0001] The present invention generally relates to tobacco (Nicotiana tabacum) plants in which the expression or activity of the enzyme lysine ketoglutarate reductase (LKR), also known as saccharopine dehydrogenase, is modulated. Background Art

[0002] In order to manufacture tobacco products, different types of tobacco are mixed with various ratios, to produce the blend with some flavor profile. Flue-cured tobacco (for example, Virginia tobacco (Virginia)) is the most widely grown tobacco, and is characterized in that the high ratio of sugar to nitrogen, but its flavor profile is limited. Other tobacco types-such as drying (for example, burley tobacco (Burley), Maryland tobacco (Maryland) and Galpao (Galpao)) or baking (for example, dark tobacco (Dark)) tobacco types-provide alternative flavor profile. These different flavor profiles are very important in the production of mixed tobacco products. Flavor profile is the result of the specific flavor compounds or the precursor of these compounds existing with a certain level in the tobacco plant. Due to the limited kind of tobacco for commercial production, this means that the chance of developing a tobacco product with different flavors and fragrance is also limited. This is equally applicable to the reconstituted tobacco material of the heated tobacco stick for making risk-reducing products.

[0003] There remains a need in the art for improved opportunities for producing tobacco that provides new flavor and / or sensory experiences to consumers while still maintaining commercially acceptable yields and properties. The present invention seeks to address this need and others. Summary of the Invention

[0004] The present invention is based, at least in part, on the surprising discovery that modulation of tobacco LKR polynucleotide expression or tobacco LKR polypeptide expression can alter the chemical profile of cured tobacco plant material, such as cured leaves. Advantageously, this can be achieved without producing an abnormal phenotype in the tobacco plant, thereby conferring commercially acceptable yields and traits. Without wishing to be bound by theory, it is believed that the alteration in chemical composition occurs through alterations in the senescence pathway (during early curing), which can modulate the levels of essential amino acids, such as lysine. Observations of additional sustained changes in other amino acids (as well as sugars, etc.) have made it possible to engineer tobacco materials to have different flavors and / or sensory properties. This is valuable for tobacco types that are widely grown commercially but have limited flavor profiles.

[0005] Zhu et al. (2001) Plant Physiol. 126(4): 1539-45 describe Arabidopsis knockout mutants of LKR. Under normal growth conditions, the knockout phenotype is indistinguishable from wild-type plants. The relative levels of free lysine 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 in leaves of wild-type and LKR knockout mutants. Surprisingly, modulation of LKR can alter the amino acid profile of dried tobacco plant material, compared to results observed in Arabidopsis. For example, unexpectedly, reduction of LKR expression or activity in tobacco can enrich dried leaves with certain essential amino acids, as described herein.

[0006] Disclosed is a mutant, non-naturally occurring or transgenic or genetically engineered tobacco plant or part thereof (such as leaves, suitably dried leaves) having modulated (e.g., increased or decreased, suitably, decreased) expression or activity of a LKR comprising, consisting of or consisting essentially of: (i) a polynucleotide comprising, consisting of or consisting essentially of: (i) a sequence having at least 88% sequence identity to SEQ ID NO: 1 (NtLKR-S) and / or a sequence having at least 86% sequence identity to SEQ ID NO: 3 (NtLKR-T); (ii) a polypeptide encoded by the polynucleotide shown 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 a sequence having at least 85% sequence identity to SEQ ID NO: 4 (NtLKR-T); NO:4(NtLKR-T) having at least 88% sequence identity, consisting of or consisting essentially of the sequence; or (iv) a construct, vector or expression vector comprising the isolated polynucleotide shown in (i), wherein the plant or part thereof comprises at least one modification capable of modulating: (a) expression of the polynucleotide in the plant or part thereof; or (b) activity of the polypeptide in the plant or part thereof, compared to a control plant or part thereof in which expression of the polynucleotide or activity of the polypeptide is not modified.

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

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

[0009] Suitably, the modification comprises one or more vectors or viral vectors or Agrobacterium vectors or CRISPR vectors.

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

[0011] Suitably, the modification enables expression of one or more double-stranded RNA (dsRNA) or hairpin RNA (hpRNA) or small interfering RNA.

[0012] Suitably, the modulated expression or activity of the LKR is capable of modulating the level of one or more amino acids in the plant or part thereof compared to the level of the one or more amino acids in a control plant, suitably wherein the modulated expression or activity of the LKR is capable of modulating the timing of leaf senescence.

[0013] Suitably, the amino acid is lysine.

[0014] Suitably, the part of the mutant, non-naturally occurring or transgenic tobacco plant is a dried or dried leaf.

[0015] Suitably, the levels of at least lysine, arginine, GABA, glutamine, alanine, tyrosine, isoleucine and threonine are modulated in the dried or desiccated leaves compared to dried or desiccated leaves from control plants.

[0016] Suitably, the tobacco plant or part thereof is of the Burley type.

[0017] Suitably, the levels of at least lysine, arginine, proline, GABA, glutamine, leucine, alanine, phenylalanine, tyrosine, isoleucine, methionine, threonine and glycine are modulated, and the levels of at least asparagine, aspartic acid, tryptophan, histidine, glutamate, serine and valine are not significantly altered in the dried or desiccated leaves compared to dried or desiccated leaves from control plants.

[0018] Suitably, expression of the LKR polynucleotide or activity of the LKR polypeptide is reduced or inhibited in the dried or dried leaves, compared to dried or dried leaves from a control plant, and wherein in the dried or dried leaves: (i) the levels of at least lysine, arginine, proline, GABA, glutamine, leucine, alanine, phenylalanine, tyrosine and isoleucine are increased; and (ii) the levels of at least methionine, threonine and glycine are reduced; and (iii) the levels of at least asparagine, aspartic acid, tryptophan, histidine, glutamate, serine and valine are not significantly changed.

[0019] Suitably, the tobacco plant or part thereof is of the Nicotiana virginiana type.

[0020] Suitably, the levels of at least lysine, arginine, glutamine, histidine, tyrosine, tryptophan, threonine, GABA, asparagine, alanine, isoleucine, valine and serine are modulated and the levels of at least proline, aspartic acid, leucine, phenylalanine, glutamate and methionine are not significantly changed in the dried or desiccated leaves compared to dried or desiccated leaves from control plants.

[0021] Suitably, expression of the LKR polynucleotide or activity of the LKR polypeptide is reduced or inhibited in the dried or dried leaves, compared to dried or desiccated leaves from a control plant, and wherein in the dried or desiccated leaves: (i) the levels of at least lysine, arginine, glutamine, histidine, tyrosine, tryptophan, threonine, GABA, asparagine and alanine are increased; and (ii) the levels of at least isoleucine, valine and serine are reduced; and (iii) the levels of at least proline, aspartic acid, leucine, phenylalanine, glutamate and methionine are not significantly changed.

[0022] Suitably, the total amount of sugar is adjusted, suitably reduced.

[0023] In another aspect, a tobacco plant material, a dried tobacco plant material or a homogenized tobacco plant material is disclosed, which is derived from or obtained from the tobacco plant or part thereof as described above; suitably, wherein the tobacco plant material is selected from the group consisting of: biomass, seeds, stems, flowers or leaves or a combination of two or more thereof; suitably, wherein the tobacco plant material is leaves; suitably, wherein the leaves are dried leaves; suitably, wherein the dried leaves are selected from the group consisting of: flue-dried leaves, sun-dried leaves or air-dried leaves.

[0024] In another aspect, a method for producing a tobacco plant in which the level of at least one amino acid is modulated is disclosed, the method comprising: (a) providing a tobacco 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 a sequence having at least 86% sequence identity to SEQ ID NO: 3 (NtLKR-T); (ii) a polypeptide encoded by the polynucleotide shown 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 a sequence having at least 81% sequence identity to SEQ ID NO: 4 (NtLKR-T); NO:4 (NtLKR-T) having at least 88% sequence identity, consisting of, or consisting essentially of a sequence thereof; or (iv) a construct, vector, or expression vector comprising the isolated polynucleotide set forth in (i); and (b) introducing at least one modification capable of modulating: (a) expression of the NtLKR polynucleotide in a tobacco plant; or (b) activity of a NtLKR polypeptide in a tobacco plant, as compared to a control in which expression of the NtLKR polynucleotide or activity of the NtLKR polypeptide is not modified.

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

[0026] In another aspect, there is disclosed a tobacco plant material obtained or obtainable by the method described above.

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

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

[0029] In another aspect, disclosed is a tobacco product comprising tobacco plant material, dried tobacco plant material, or the homogenized tobacco plant material described above or comprising the dried tobacco plant material described above.

[0030] Suitably, the tobacco product is a tobacco blend; suitably, wherein the tobacco blend comprises Virginia-type tobacco and / or Burley-type tobacco.

[0031] Some advantages

[0032] Advantageously, regulating the expression of NtLKR polynucleotides or the activity of NtLKR polypeptides can modulate amino acid levels, particularly in cured tobacco plant material, which can impart novel flavor and / or sensory properties to tobacco.

[0033] Advantageously, non-genetically modified plants can be produced that are more acceptable to consumers.

[0034] Advantageously, the present disclosure is not limited to the use of ethyl methanesulfonate (EMS) mutant plants. EMS mutant plants may not have the potential for improved crop properties after breeding. Once breeding begins, the desirable characteristics of EMS mutant plants may be lost for various reasons. For example, multiple mutations may be required, mutations may be dominant or recessive, and point mutations may be difficult to identify in the gene target. Instead, the present disclosure utilizes the use of NtLKRs, which can be specifically manipulated to produce plants with a desired phenotype.

[0035] Advantageously, no abnormal phenotypes were observed, making the plants suitable for commercial production.

[0036] 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, the combination of ASN-regulated plants and NtLKR-regulated plants can rearrange their chemical composition, such as the amino acid chemistry of burley or dark or Virginia tobacco, and can further alter their flavor and / or sensory properties. Other genes and enzymes also play a role in the reorganization of amino acids and / or sugars during leaf yellowing, such as diaminopimelate aminotransferase (DAPAT), aspartate aminotransferase (AAT), and chloroplast sulfate transporter (SULTR3) (which can also alter leaf chemistry). Modifying NtLKR expression or NtLKR activity, as well as the expression or activity of one or more of these other targets selected from one or more of ASN, DAPAT, and AAT, can be used to further alter the flavor and / or sensory properties of cured tobacco.

[0037] Advantageously, no significant differences in alkaloid content were observed, meaning that nicotine levels were unchanged, thereby delivering the same amount of nicotine to tobacco consumers. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Figures 2 show NtLKR-RNAi plants identified by qRT-PCR as exhibiting low NtLKR expression. Relative expression is shown for TN90 (CT1-E438-4, n=4; CT1-E438-5, n=5; CT1-E438-6); T1-E438-2, n=4; T1-E438-5, n=5; T1-E438-11, n=5). Relative expression is shown for Nicotiana virginiana K326 (CT1-E437-5, n=5; CT1-E437-6, n=4; T1-E437-11, n=4; T1-E437-12, n=5; T1-E437-15, n=4). Tissue used for RNA isolation was the green midrib / leaf.

[0039] Figure 2 It is a graph showing chlorophyll measurements at the C stem position (CCI, 3 measurements per plant) 21 days after nutrient solution switching.

[0040] Figure 3 are three graphs showing the contents of three free amino acids Lys, Arg and Pro in three independent NtLKR-RNAi lines (E438-2, -5-11) compared with WT (CT1-E438). Statistical analysis was performed using ANOVA and Tukey's HSD test.

[0041] Figure 4 are six graphs showing the contents of three free amino acids Lys, Arg, and Gln, reducing sugars glucose and fructose, and nitrate in three independent NtLKR-RNAi lines (E43711, -12-15) compared to WT (CT1-E437). Statistical analysis was performed using ANOVA and Tukey's HSD test.

[0042] Figure 5 is a graph showing the expression of NtLKR during early air curing and early flue curing. DETAILED DESCRIPTION

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art. In the event of a 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 to implement or test the present invention. The materials, methods, and examples disclosed herein are illustrative only and are not intended to be limiting.

[0044] The terms "include," "including," "having / has," "may," "contain," and variations thereof are intended to be open transitional phrases, terms, or words that do not exclude the possibility of additional actions or structures.

[0045] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0046] The present disclosure contemplates other embodiments "comprising," "consisting of," and "consisting essentially of" the embodiments or elements presented herein, whether or not explicitly stated.

[0047] For the purposes of reciting numerical ranges herein, each intervening value therebetween is expressly contemplated with equal precision. For example, for the range 6-9, the values 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the values 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.

[0048] As used throughout the specification and claims, the following terms have the following meanings:

[0049] "Coding sequence" or "polynucleotide code" refers to the nucleotides (RNA or DNA molecule) comprising a polynucleotide that encodes a polypeptide. The coding sequence may also include initiation and termination signals operably linked to regulatory elements, including a promoter and polyadenylation signal, capable of directing expression in the cells of an individual or mammal to which the polynucleotide is administered. The coding sequence may be codon-optimized.

[0050] "Complementary" or "complementary" can refer to Watson-Crick (e.g., AT / U and CG) or Hoogs teen base pairing between nucleotides or nucleotide analogs. "Complementarity" refers to a property shared between two polynucleotides such that when they are aligned antiparallel to each other, the nucleotide bases at every position will be complementary.

[0051] "Construct" refers to a double-stranded recombinant polynucleotide fragment comprising one or more polynucleotides. The construct includes a "template strand" that base pairs with a complementary "sense strand or coding strand." A given construct can be inserted into a vector in two possible orientations: the same (or sense) orientation or the opposite (or antisense) orientation with respect to the orientation of a promoter within the vector (e.g., an expression vector).

[0052] In the context of a control plant or control plant cell, the term "control" refers to a plant or plant cell in which the expression, function or activity of one or more genes or polypeptides is not modified (e.g., increased or decreased) and which can therefore be compared to a plant in which the expression, function or activity of the same one or more genes or polypeptides is modified. A "control plant" is a plant whose parameters are substantially equivalent to those of a test plant or modified plant except for the test parameters. For example, when referring to a plant into which a polynucleotide has been introduced, a control plant is an equivalent plant into which such polynucleotides have not been introduced. A control plant can be an equivalent plant into which a control polynucleotide has been introduced. In such cases, a control polynucleotide is a polynucleotide that is expected to produce little or no phenotypic effect on a plant. A control plant can comprise a blank vector. A control plant can correspond to a wild-type plant. A control plant can be an empty segregant in which a T1 segregant no longer has a transgenic.

[0053] The term "reduce" or "reduce" refers to a reduction of about 10% to about 99%, 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 quantity or function, such as polypeptide function, transcription function or polypeptide expression. The term "reduce" or phrase "amount reduced" can refer to an amount or function that is less than the amount or function found in the unmodified plant or the product from the plant of the same variety processed in the same manner. Therefore, in some cases, the wild-type plant of the same variety processed in the same manner is used as a control, by which the reduction in quantity is measured.

[0054] "Donor DNA" or "donor template" refers to a double-stranded DNA fragment or molecule that includes at least a portion of a gene of interest. The donor DNA can encode a functional polypeptide.

[0055] "Endogenous gene or polypeptide" refers to a gene or polypeptide that originates from the genome of an organism and has not undergone changes (such as loss, gain, or exchange of genetic material). Endogenous genes undergo standard gene delivery and gene expression. Endogenous polypeptides undergo normal expression.

[0056] "Enhancer sequences" refer to sequences that increase gene expression. These sequences can be located upstream, within introns, or downstream of the transcribed region. The transcribed region extends from the promoter to the transcription termination region, including exons and intervening introns. Enhancement of gene expression can occur through a variety of mechanisms, including increased transcription efficiency, stabilization of mature mRNA, and enhanced translation.

[0057] "Expression" refers to the production of a functional product. For example, expression of a polynucleotide fragment can refer to the transcription of the polynucleotide fragment (e.g., transcription to produce mRNA or functional RNA) or the translation of mRNA into a precursor or mature polypeptide, or a combination thereof.

[0058] "Overexpression" refers to the production of a gene product in a transgenic organism that exceeds the level produced in an empty isolate (or non-transgenic) organism from the same experiment.

[0059] "Function" describes a polypeptide that has a biological function or activity. "Functional gene" refers to a gene that is transcribed into mRNA, which is translated into a functional or active polypeptide.

[0060] "Gene construct" refers to a DNA or RNA molecule comprising a polynucleotide encoding 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.

[0061] "Genome editing" generally refers to a process by which the genomic nucleic acids in a cell are altered. For example, this can be done by removing, inserting, or replacing one or more nucleotides in the genomic nucleic acids. Endonucleases can be used to create specific breaks or nicks at defined locations in the genome and are further described herein.

[0062] The terms "homology" or "similarity" refer to the degree of sequence similarity between two polypeptides or between two polynucleotide molecules compared by sequence alignment. The degree of homology between two discrete polynucleotides being compared is a function of the number of identical or matching nucleotides at comparable positions. Homology or similarity can be determined over the entire length of the subject sequence.

[0063] In the context of two or more polynucleotides or polypeptides, "identical" or "identity" refers to sequences that have a specific percentage of identical residues over a specified region. The percentage can be calculated by optimally aligning the two sequences, comparing a specified region of the two sequences, determining the number of positions where identical residues are present in the two sequences to produce the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to produce the percentage of sequence identity. Where the two sequences are of different lengths or the alignment produces one or more staggered ends and the specified comparison region includes only a single sequence, the residues of the single sequence are included in the denominator, not the numerator, of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) are considered equivalent. Identity can be identified manually or by using a computer sequence algorithm such as ClustalW, ClustalX, BLAST, FASTA, or Smith-Waterman. Suitable parameters for ClustalW may be as follows: for polynucleotide alignments: gap open penalty = 15.0, gap extension penalty = 6.66, and matrix = identity. For polypeptide alignments: Gap Open Penalty = 10.o, Gap Extension Penalty = 0.2, and Matrix = Gonnet. For DNA and protein alignments: ENDGAP = -1, and GAPDIST = 4.

[0064] The term "increase" or "increased" refers to an increase of about 10% to about 99%, 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 in quantity or function or activity, such as, but not limited to, one or more of polypeptide function or activity, transcriptional function or activity, and polypeptide expression. The term "increased" or the phrase "increased amount" can refer to a quantity or function or activity in a plant or a product produced by a plant that is greater than the quantity or function or activity found in an unmodified plant or a product from a plant of the same variety that has been processed in the same manner. Thus, in some cases, a wild-type plant of the same variety that has been processed in the same manner is used as a control against which to measure whether an increase in quantity is achieved.

[0065] The term "inhibit" or "inhibited" refers to a reduction of about 98% to about 100%, or a reduction of at least 98%, at least 99%, but particularly 100% in quantity or function or activity, such as but not limited to one or more of polypeptide function or activity, transcription function or activity, and polypeptide expression.

[0066] The term "introducing" can refer to providing a polynucleotide (e.g., a construct) or a polypeptide into a cell. Introducing includes reference to the incorporation of a polynucleotide into a eukaryotic cell, wherein the polynucleotide can be incorporated into the genome of the cell, and includes reference to the transient provision of a polynucleotide or polypeptide to a cell. Introducing includes stable or transient transformation methods, as well as sex crossing. Thus, in the context of inserting a polynucleotide (e.g., a recombinant construct / expression construct) into a cell, "introducing" refers to "transfection" or "transformation" or "transduction", and includes reference to the incorporation of a polynucleotide into a eukaryotic cell, wherein the polynucleotide can be incorporated into the genome of the cell (e.g., a chromosome, plasmid, plastid or mitochondrial DNA), converted into an autonomous replicon or transiently expressed (e.g., transfected mRNA).

[0067] The term "isolated" or "purified" refers to a material that is substantially or essentially free of components that normally accompany it 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 produces essentially one band in an electrophoretic gel. In particular, it refers to a polynucleotide or polypeptide that has a purity of at least 85%, more suitably at least 95%, and most suitably at least 99%. An isolated polynucleotide can be purified from the host cell in which it naturally occurs. Conventional polynucleotide purification methods known to those skilled in the art can be used to obtain isolated polynucleotides. The term also encompasses recombinant polynucleotides and chemically synthesized polynucleotides.

[0068] "Liquid tobacco extract" describes the direct product of an extraction process performed on a tobacco starting material. The extraction process used to produce a liquid tobacco extract may include heating the tobacco starting material under specific heating conditions and collecting the resulting volatile compounds. The liquid tobacco extract may contain a mixture of compounds derived from the tobacco starting material and removed during the extraction process, typically in combination with a liquid carrier or solvent.

[0069] "Modulate" refers to a qualitative or quantitative change, alteration, or modification that causes or promotes a process, pathway, function, or activity of interest. Without limitation, such a change, alteration, or modification can be an increase or decrease in the process, pathway, function, or activity of interest. For example, gene expression, polypeptide expression, or polypeptide function or activity can be modulated. Typically, the relevant change, alteration, or modification will be determined by comparison with a control.

[0070] The term "non-naturally occurring" describes entities that are not formed in nature or that do not exist in nature, such as polynucleotides, genetic mutations, polypeptides, plants, plant cells, and plant materials. Such non-naturally occurring entities or artificial entities can be prepared, synthesized, initiated, modified, intervened, or manipulated by methods described herein or known in the art. Such non-naturally occurring entities or artificial entities can be prepared, synthesized, initiated, modified, intervened, or manipulated by humans. Therefore, non-naturally occurring plants cannot be produced using essentially biological methods. Therefore, for example, non-naturally occurring plants, non-naturally occurring plant cells, or non-naturally occurring plant materials can be prepared using traditional plant breeding techniques (e.g., backcrossing) or by genetic manipulation techniques (e.g., antisense RNA, interfering RNA, meganucleases, etc.). By way of further example, a non-naturally occurring plant, non-naturally occurring plant cell, or non-naturally occurring plant material can be prepared by introgressing a first plant or plant cell gene into a second plant or plant cell (which itself can be naturally occurring), or by 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, such that the resulting plant, plant cell, or plant material, or its progeny, includes a genetic makeup (e.g., a genome, chromosome, or segment thereof) that is not naturally occurring or does not exist in nature. The resulting plant, plant cell, or plant material is therefore artificial or non-naturally occurring. Accordingly, an artificial or non-naturally occurring plant or plant cell can be prepared by modifying a gene sequence in a first naturally occurring plant or plant cell, even if the resulting gene sequence is naturally occurring in a second plant or plant cell, the second plant or plant cell including a different genetic background than 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).

[0071] "Oligonucleotide" or "polynucleotide" refers to at least two nucleotides covalently linked together. The description of a single strand also defines the sequence of the complementary strand. Therefore, 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. Therefore, 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. Therefore, a polynucleotide also encompasses probes that hybridize under stringent hybridization conditions. A polynucleotide can be single-stranded or double-stranded, or can contain portions of double-stranded and single-stranded sequences. A polynucleotide can be DNA (both genomic DNA and cDNA), RNA, or a hybrid, wherein the polynucleotide can comprise a combination of deoxyribonucleotides and ribonucleotides, and a combination 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.

[0072] The specificity with which single-stranded DNA hybridizes to complementary fragments is determined by the "stringency" of the reaction conditions (Sambrook et al., Molecular Cloning and Laboratory Manual, 2nd ed., Cold Spring Harbor (1989)). Hybridization under "stringent conditions" describes a hybridization protocol in which polynucleotides that are at least 60% homologous to each other remain hybridized. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) of the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH, and polynucleotide concentration) at which 50% of the probes complementary to a given sequence hybridize to the given sequence at equilibrium. Since a given sequence is typically present in excess, at the Tm, 50% of the probes are at equilibrium.

[0073] Stringent conditions typically include: (1) low ionic strength and high temperature for washing, e.g., 15 mM sodium chloride, 1.5 mM sodium citrate, 0.1% sodium dodecyl sulfate at 50°C; (2) a denaturing agent during hybridization, 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) at 42°C; or (3) 50% formamide. Washes also typically include 5xSSC (0.75 M NaCl, 75 mM sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5x Denhardt's solution, sonicated salmon sperm DNA (50 μg / mL), 0.1% SDS, and 10% dextran sulfate at 42° C., and 0.2xSSC (sodium chloride / sodium citrate) at 42° C. and 50% formamide at 55° C., followed by a high stringency wash consisting of 0.1xSSC containing EDTA at 55° C. Suitably, the conditions are such that sequences at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous to each other typically remain hybridized to each other.

[0074] "Moderate stringency conditions" use a washing solution and less stringent hybridization conditions so that the polynucleotide will hybridize to the entirety, fragment, derivative or analog of the polynucleotide. An example includes hybridization in 6xSSC, 5x Denport 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 adapt to experimental factors, such as probe length. Other moderate stringency conditions have been described (see Ausubel et al., Current Protocols in Molecular Biology, Vol. 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)).

[0075] "Low stringency conditions" use a wash solution and hybridization conditions that are less stringent than moderate stringency, such that a polynucleotide will hybridize to the entirety, fragment, derivative, or analog of a polynucleotide. Non-limiting examples of low stringency hybridization conditions include hybridization in 35% formamide, 5xSSC, 50mM Tris HCl (pH 7.5), 5mM 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, 25mM Tris HCl (pH 7.4), 5mM EDTA, and 0.1% SDS at 50°C. Other conditions of low stringency, such as conditions for cross-species hybridization, have been fully described (see Ausubel et al., 1993; Kriegler, 1990).

[0076] "Operably linked" means that the expression of a gene is under the control of a promoter to which it is spatially linked. A promoter under its control can be located 5' (upstream) or 3' (downstream) of a gene. The distance between the promoter and the gene can be roughly the same as the distance between the promoter and the gene it controls in the gene that produces the promoter. As is known in the art, variations in this distance can be adjusted without losing promoter function. "Operably linked" refers to the association of polynucleotide fragments in a single fragment so that the function of one fragment is regulated by another fragment. For example, when a promoter is capable of regulating the transcription of a polynucleotide fragment, it is operably linked to the polynucleotide fragment.

[0077] Term " plant " refers to any plant and offspring thereof that are in any stage of its life cycle or growth.In one embodiment, plant is tobacco plant, and it refers to the plant that belongs to Nicotiana.This term comprises the complete plant, plant organ, plant tissue, plant propagule, plant seed, vegetable cell and offspring thereof of mentioning.Plant cell includes, but is not limited to the cell from seed, suspension culture, plumule, meristematic zone, callus, leaf, root, tender shoot, gametophyte, sporophyte, pollen and pollen grain.This paper has described suitable kind, cultivar, hybrid and the kind of tobacco plant.

[0078] "Plant material" includes leaves, roots, sepals, root tips, petals, flowers, buds, stems, seeds and stalks. Plant material may be living or non-viable plant material.

[0079] "Polynucleotide," "polynucleotide sequence," or "polynucleotide fragment" are used interchangeably herein and refer to a polymer of single-stranded or double-stranded RNA or DNA, optionally containing synthetic, non-natural, or altered nucleotide bases. The polynucleotides of the present disclosure are listed in the accompanying sequence listing.

[0080] "Polypeptide" or "polypeptide sequence" refers to polymers of amino acids in which one or more amino acid residues is an artificial chemical analog of a corresponding naturally occurring amino acid, as well as polymers of naturally occurring amino acids. The term also includes modifications including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation. The polypeptides of the present disclosure are listed in the accompanying sequence listing.

[0081] "Promoter" refers to a molecule of synthetic or natural origin that can confer, activate or enhance the expression of a polynucleotide in a cell. The term refers to a polynucleotide element / sequence that is generally located upstream of a double-stranded polynucleotide fragment and operably connected thereto. A promoter can be completely derived from a region adjacent to a natural gene of interest, or can be composed of different elements derived from different natural promoters or synthetic polynucleotide fragments. A promoter can include one or more specific transcriptional regulatory sequences to further enhance expression or change spatial expression or change temporal expression. A promoter can also include terminal enhancers or repressor elements, which can be located up to several thousand base pairs from the transcription start site. Promoters can be derived from sources including viruses, bacteria, fungi, plants, insects and animals. A promoter can constitutively or differentially regulate the expression of a gene component with respect to the cell, tissue or organ in which expression occurs or with respect to the developmental stage in which expression occurs, or in response to external stimuli (such as physiological stress, pathogens, metal ions or inducers).

[0082] "Tissue-specific promoter" and "tissue-preferred promoter," as used interchangeably herein, refer to promoters that are primarily, but not necessarily exclusively, expressed in one tissue or organ, but may also be expressed in one specific 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, such as by a chemical compound (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 regulated by light, heat, pressure, flooding or drought, pathogens, plant hormones, wounds, or chemicals such as ethanol, jasmonates, salicylic acid, or safeners.

[0083] "Recombination" refers to the artificial combination of two otherwise isolated sequence fragments, such as isolated polynucleotide fragments obtained by chemical synthesis or by genetic engineering techniques. The term also includes reference to cells or vectors that have been modified by the introduction of heterologous polynucleotides, or cells derived from such modified cells, but does not encompass alterations to cells or vectors due to naturally occurring events (e.g., spontaneous mutations, natural transformations, or transductions or transpositions), such as those that occur without human intervention.

[0084] A "recombinant construct" refers to a combination of polynucleotides not normally found together in nature. Thus, a recombinant construct may comprise regulatory sequences and coding sequences derived from different sources, or regulatory sequences and coding sequences derived from the same source but arranged in a manner different from that normally found in nature. A recombinant construct may be a recombinant DNA construct.

[0085] "Regulatory sequence" and "regulatory element" are used interchangeably herein and refer to polynucleotide sequences that are located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence and that influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences include promoters, translation leader sequences, introns, and polyadenylation recognition sequences. The terms "regulatory sequence" and "regulatory element" are used interchangeably herein.

[0086] The term "tobacco" is used to refer to tobacco crops (e.g., various tobacco plants grown in the field rather than the tobacco grown by hydroponics), tobacco plants and parts thereof in a general sense, including but not limited to roots, stems, leaves, flowers and seeds prepared or obtained as described herein. Should be understood that "tobacco" refers to plants and products thereof belonging to the genus Nicotiana, and includes tobacco plants and products thereof.

[0087] The term "tobacco product" refers to consumer tobacco products, including but not limited to smoking materials (e.g., cigarettes, cigars, and pipe tobacco), snuff, chewing tobacco, chewing gum, and lozenges, as well as components, materials, and ingredients used to make consumer tobacco products. Suitably, these tobacco products are made from the leaves and stems of tobacco harvested from tobacco, and are cut, dried, cured, or fermented according to conventional techniques in tobacco preparation.

[0088] "Transcription terminator," "termination sequence," or "terminator" refers to a DNA sequence located downstream of a coding sequence and includes a polyadenylation recognition sequence and other sequences encoding regulatory signals that can affect mRNA processing or gene expression. The polyadenylation signal is typically characterized by affecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor.

[0089] "Transgenic" refers to any cell, cell line, callus, plant part, or plant whose genome is altered by the presence of a heterologous polynucleotide, such as a recombinant construct, including those initial transgenic events and those generated from the initial transgenic event by sexual hybridization or asexual propagation. The term does not include alterations in the genome (chromosomal or extrachromosomal) by conventional plant breeding methods or by naturally occurring events such as random cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation. Thus, in embodiments, transgenic plants or parts thereof are not generated using essentially biological methods.

[0090] "Transgenic plants" refer to plants that contain one or more heterologous polynucleotides in their genome, that is, plants that contain recombinant genetic material not normally found therein and that has been introduced into the plant (or into the progenitor cells of the plant) through artificial (manual) manipulation. For example, heterologous polynucleotides can be stably integrated into the genome so that the polynucleotides are passed on to successive generations. Heterologous polynucleotides can be integrated into the genome alone or as part of a recombinant construct. The commercial development of genetically modified embryos has also developed to the stage of introducing multiple characteristics into crop plants, commonly known as gene stacking. In this method, multiple genes that confer different characteristics of interest can be introduced into plants. Gene stacking can be achieved in many ways, including (but not limited to) co-transformation, re-transformation, and crossing with different transgenic lines. Therefore, plants grown from plant cells into which recombinant DNA has been introduced through transformation are transgenic plants, all of which are progeny (sexually or asexually) of the plant into which the transgene was introduced. It should be understood that the term transgenic plant includes the entire plant or tree and parts of the plant or tree, such as grains, seeds, flowers, leaves, roots, fruits, pollen, stems, etc. Each heterologous polynucleotide can confer a different trait on the transgenic plant.

[0091] A "transgene" refers to a gene or genetic material containing a gene sequence that has been isolated from one organism and introduced into a different organism. This non-natural segment of DNA may retain the ability to produce RNA or polypeptides in the transgenic organism, or it may alter the normal function of the genetic code of the transgenic organism.

[0092] "Variant" with respect to a polynucleotide refers to: (i) a portion or fragment of a polynucleotide; (ii) a complement of a polynucleotide or a portion thereof; (iii) a polynucleotide that is substantially identical to a polynucleotide of interest or its complement; or (iv) a polynucleotide that hybridizes under stringent conditions to a polynucleotide of interest, its complement, or a polynucleotide substantially identical thereto.

[0093] "Variant" with respect to a peptide or polypeptide refers to a peptide or polypeptide that differs in sequence by insertion, deletion, or conservative substitution of amino acids but retains at least one biological function or activity. A variant may also refer to a polypeptide that retains at least one biological function or activity. Conservative substitution of amino acids, i.e., replacing an amino acid with a different amino acid that has similar properties (e.g., hydrophilicity, degree and distribution of charged regions), is generally considered in the art to involve minor changes.

[0094] The term "variety" refers to a population of plants that share consistent characteristics that distinguish them from other plants of the same species. Varieties are further characterized by minimal overall variation among individuals within the variety, despite possessing one or more unique traits. Varieties are typically commercially available.

[0095] "Vector" refers to a polynucleotide vehicle comprising a combination of polynucleotide components, polynucleotide constructs, and polynucleotide conjugates, etc., for enabling the transport of polynucleotides. The vector may be a viral vector, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. The vector may be a DNA or RNA vector. Suitable vectors include episomes capable of extrachromosomal replication, such as circular double-stranded nucleotide plasmids; linearized double-stranded nucleotide plasmids; and other vectors of any origin. "Expression vector" is a polynucleotide vehicle comprising a combination of polynucleotide components, polynucleotide constructs, and polynucleotide conjugates, etc., for enabling the expression of polynucleotides. Suitable expression vectors include episomes capable of extrachromosomal replication, such as circular double-stranded nucleotide plasmids; linearized double-stranded nucleotide plasmids; and other functionally equivalent expression vectors of any origin. The expression vector comprises at least one promoter located upstream of and operably linked to the polynucleotide, polynucleotide construct, or polynucleotide conjugate, as defined below.

[0096] Unless otherwise defined herein, the scientific and technical terms used in conjunction with the present invention will have the meanings commonly understood by those of ordinary skill in the art. For example, any nomenclature and techniques used in connection with cell and tissue culture, molecular biology, plant biology, microbiology, genetics, and polypeptide and polynucleotide chemistry and hybridization described herein are those well known and commonly used in the art. The meaning and scope of the terms should be clear; however, in the event of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definition. In addition, unless the context otherwise requires, singular terms should include the plural and plural terms should include the singular.

[0097] Disclosed is an isolated polynucleotide comprising, consisting of, or consisting essentially of a sequence having at least 60% sequence identity to any sequence described herein, including any of the polynucleotides shown in the sequence listing. Suitably, 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. Suitably, 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. Suitably, 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.

[0098] Suitably, 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 shown in the sequence listing.

[0099] In another embodiment, an isolated LKR polynucleotide from tobacco (NtLKR) is provided, 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 a polynucleotide having at least 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).

[0100] In another embodiment, a polynucleotide is provided that comprises, consists of, or consists essentially of a polynucleotide having substantial homology (ie, sequence similarity) or substantial identity to SEQ ID NO: 1 or SEQ ID NO: 3.

[0101] In another embodiment, fragments having substantial homology (i.e., sequence similarity) or substantial identity to SEQ ID NO: 1 or SEQ ID NO: 3 are provided, wherein the fragments have 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 fragments of SEQ ID NO: 1 or SEQ ID NO: 3.

[0102] In another embodiment, fragments having substantial homology (i.e., sequence similarity) or substantial identity to SEQ ID NO: 1 are provided, wherein the fragments have 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.

[0103] In another embodiment, fragments having substantial homology (i.e., sequence similarity) or substantial identity to SEQ ID NO: 3 are provided, wherein the fragments have 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.

[0104] 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 is provided, which encodes a polypeptide that functions as an LKR.

[0105] In another embodiment, a polymer of polynucleotides is provided that comprises, consists of, or consists essentially of the polynucleotide designated herein as SEQ ID NO: 1 or SEQ ID NO: 3.

[0106] Suitably, the polynucleotides described herein encode a NtLKR polypeptide having LKR activity.

[0107] A polynucleotide may comprise a polymer of nucleotides, which may be unmodified or modified deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Thus, a polynucleotide may be, but is not limited to, genomic DNA, complementary DNA (cDNA), mRNA, or antisense RNA, or a fragment thereof. In addition, a polynucleotide may be single-stranded or double-stranded DNA, a mixture of single-stranded and double-stranded regions, a hybrid comprising DNA and RNA, or a hybrid comprising a mixture of single-stranded and double-stranded regions, or a fragment thereof. In addition, a polynucleotide may be composed of triple-stranded regions comprising DNA, RNA, or both, or a fragment thereof. A polynucleotide may contain one or more modified bases, such as phosphorothioates, and may be a peptide nucleic acid. In general, a polynucleotide may be assembled from isolated or cloned cDNA fragments, genomic DNA, oligonucleotides, or individual nucleotides, or a combination of the foregoing. Although the polynucleotides described herein are shown as DNA sequences, they include their corresponding RNA sequences and their complementary (e.g., fully complementary) DNA or RNA sequences, including their reverse complements.

[0108] Fragments of a polynucleotide 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, and up to a full-length polynucleotide encoding a polypeptide described herein.

[0109] Polynucleotides will typically contain phosphodiester bonds, although in some cases, polynucleotide analogs are included that may have alternative backbones, including, for example, phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphosphoramidite bonds; as well as peptide polynucleotide backbones and bonds. Other similar polynucleotides include polynucleotides with positive backbones; non-ionic backbones and non-ribose backbones. Modification of the ribose-phosphate backbone can be done for a variety of reasons, such as to increase the stability and half-life of such molecules in physiological environments, or as probes on biochips. Mixtures of naturally occurring polynucleotides and analogs can be prepared; alternatively, mixtures of different polynucleotide analogs can be prepared, as well as mixtures of naturally occurring polynucleotides and analogs.

[0110] Various polynucleotide analogs are known, including for example phosphoramidate, phosphorothioate, phosphorodithioate, O-methylphosphoramidite bond and peptide polynucleotide backbone and bond. Other similar polynucleotides include polynucleotides with positive backbone, nonionic backbone and non-ribose backbone. Also include polynucleotides containing one or more carbocyclic sugars.

[0111] Other analogs include peptide polynucleotides that are peptide polynucleotide analogs.

[0112] In the purposes of the disclosed polynucleotides and fragments thereof, there are fragments for use as probes in hybridization assays or as primers in amplification assays. Such fragments generally include at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more adjacent nucleotides of the dna sequence. In other embodiments, the dna fragment includes at least about 10, 15, 20, 30, 40, 50 or 60 or more adjacent nucleotides of the dna sequence. Therefore, in one aspect, a method for detecting polynucleotides is also provided, which method includes using probes or primers or both.

[0113] The basic parameters that influence the selection of hybridization conditions and guidance for designing appropriate 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). Using knowledge of the genetic code in combination with the polypeptide sequences described herein, degenerate oligonucleotide sets can be prepared. Such oligonucleotides can be used, for example, as primers in polymerase chain reaction (PCR) to isolate and amplify DNA fragments.

[0114] At least one modification (eg, mutation) may be included in one or more of SEQ ID NO: 1 or SEQ ID NO: 3.

[0115] Provided is an isolated LKR polypeptide encoded by a polynucleotide described herein.

[0116] 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 shown in the sequence listing. Suitably, 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.

[0117] Also provided is a LKR polypeptide 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).

[0118] Also provided is a LKR polypeptide 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).

[0119] Also provided is an LKR polypeptide 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.

[0120] Also provided is a polypeptide encoded by SEQ ID NO: 2 or SEQ ID NO: 4.

[0121] The polypeptide may comprise a sequence having a sufficient or substantial degree of identity or similarity to SEQ ID NO: 2 or SEQ ID NO: 4 to function as an LKR.

[0122] Fragments of the polypeptides described herein are also contemplated. Fragments of polypeptides generally retain some or all of the function or activity, such as LKR activity, of the full-length sequence. Fragments of polypeptides 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, about 500 amino acids, and up to the full-length polypeptides described herein.

[0123] Polypeptides also include mutants produced by introducing any type of change (e.g., insertion, deletion or substitution of amino acids; changes in glycosylation status; changes affecting refolding or isomerization, three-dimensional structure or self-association state), which can be intentionally engineered or isolated naturally, provided that they still possess some or all of their function or activity. Suitably, the function or activity is modulated.

[0124] 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 polypeptide at a predetermined site. Insertions can involve 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 (such as similar hydrophobicity, hydrophilicity, antigenicity, or propensity to form or disrupt α-helical or β-sheet structures). Amino acid substitutions are typically single residues but can be clustered, depending on the functional constraints imposed on the polypeptide, and can range from about 1 to about 10 amino acids. Amino acid substitutions are suitably conservative amino acid substitutions, as described below. Amino acid substitutions, deletions, or insertions can be prepared using peptide synthesis techniques such as solid-phase peptide synthesis or by recombinant DNA manipulation. Methods for manipulating DNA sequences to generate substitution, insertion, or deletion variants of polypeptides are well known in the art. Such variants can have alterations that produce silent changes and result in functionally equivalent polypeptides. Deliberate amino acid substitutions can be made based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and amphipathic properties of the residues, as long as the secondary binding properties of the substance are maintained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with similar hydrophilicity values containing uncharged polar head groups 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 column and, where appropriate, in the same row in the third column can be substituted for each other:

[0125]

[0126]

[0127] The polypeptide can be a mature polypeptide or an immature polypeptide or a polypeptide derived from an immature polypeptide. The polypeptide can be in linear form or cyclized using known methods. The polypeptide typically comprises at least 10, at least 20, at least 30 or at least 40 contiguous amino acids.

[0128] At least one modification (eg, mutation) may be included in one or more of SEQ ID NO:2 or SEQ ID NO:4.

[0129] Recombinant constructs can be used to transform plants or plant cells to regulate polypeptide expression, function or activity. Recombinant polynucleotide constructs can include polynucleotides encoding one or more polynucleotides as described herein, which are operably linked to regulatory regions suitable for expressing polypeptides. Therefore, polynucleotides can include coding sequences encoding polypeptides as described herein. Plants or plant cells that have regulated polypeptide expression, function or activity can include mutant, non-natural, transgenic, artificial or genetically engineered plants or plant cells. Suitably, transgenic plants or plant cells include genomes that have been altered by the stable integration of recombinant DNA. Recombinant DNA comprises DNA that has been genetically engineered and constructed outside the cell, and comprises DNA containing naturally occurring DNA or cDNA or synthetic DNA. Transgenic plants can include plants regenerated from initially transformed plant cells, as well as offspring transgenic plants from later generations or hybridization of transformed plants. Suitably, compared to control plants, transgenic modification has altered the expression, function or activity of polynucleotides or polypeptides as described herein.

[0130] The polypeptide encoded by the recombinant polynucleotide can be a native polypeptide, or can be heterologous to the cell. In some cases, the recombinant construct contains a polynucleotide operably linked to the regulatory expression of a regulatory region. Examples of suitable regulatory regions are described herein.

[0131] Also provided are vectors containing recombinant polynucleotide constructs, such as those described herein. Suitable vector backbones include, for example, those conventionally 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, bacteriophages, baculoviruses, and retroviruses. Numerous vectors and expression systems are commercially available.

[0132] The vector may comprise, for example, an origin of replication, a support attachment region, or a marker. A marker gene may confer a selectable phenotype to a plant cell. For example, a marker may 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, an expression vector may comprise a tag sequence designed to facilitate manipulation or detection (e.g., purification or positioning) 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 fusions with the encoded polypeptide. Such tags may be inserted anywhere within the polypeptide, including at the carboxyl or amino terminus.

[0133] Plants or plant cells can be transformed by integrating the recombinant polynucleotide into their genome to become stably transformed. The plants or plant cells described herein can be stably transformed. Stably transformed cells typically retain the introduced polynucleotides in each cell division. Plants or plant cells can be transiently transformed so that the recombinant polynucleotides are not integrated into their genome. Transiently transformed cells typically lose all or part of the introduced recombinant polynucleotides in each cell division so that after a sufficient number of cell divisions, the introduced recombinant polynucleotides cannot be detected in daughter cells.

[0134] Many methods are available in the art for transforming plant cells, including biolistics, gene gun technology, Agrobacterium-mediated transformation, viral vector-mediated transformation, freeze-thaw methods, microprojectile bombardment, direct DNA uptake, sonication, microinjection, plant virus-mediated transfer, and electroporation.

[0135] If cells or cultured tissue are used as the recipient tissue for transformation, plants can be regenerated from the transformed culture, if desired, by techniques known to those skilled in the art.

[0136] The selection of regulatory regions to be included in the recombinant construct depends on several factors, including but not limited to efficiency, selectivity, inducibility, desired expression level and cell or tissue preferential expression. By appropriately selecting the regulatory region and placing the regulatory region relative to the coding sequence, regulating the expression of the coding sequence is routine work for those skilled in the art. The transcription of the polynucleotide can be regulated in a similar manner. Some suitable regulatory regions only or predominantly initiate transcription in certain cell types. Methods for identifying and characterizing regulatory regions in plant genomic DNA are known in the art.

[0137] In one embodiment, the present invention relates to a promoter that is capable of expressing a polypeptide of the present invention. The promoter is a tissue-specific promoter that is recognized by a tissue-specific factor, and the tissue-specific promoter is present in different tissues or cell types (e.g., root-specific promoters, branch-specific promoters, xylem-specific promoters), or is present during different developmental stages, or exists in response to different environmental conditions. Suitable promoters include constitutive promoters, which can be activated in most cell types without the need for specific inducing agents. Examples of promoters for controlling polypeptide expression include cauliflower mosaic virus 35S (CaMV / 35S), SSU, OCS, lib4, usp, STLS1, B33, nos or ubiquitin promoters or phaseolin promoters. Those skilled in the art can produce a variety of variants of recombinant promoters.

[0138] Tissue-specific promoters are transcriptional control elements that are active only in specific cells or tissues (such as vegetative or reproductive tissues) at specific times during plant development. Examples of tissue-specific promoters under developmental control include promoters that can initiate transcription only (or primarily only) in certain tissues, such as vegetative tissues (e.g., roots or leaves) or reproductive tissues (such as fruit, ovules, seeds, pollen, pistils, flowers, 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.

[0139] Exemplary leaf-specific promoters include the pyruvate orthophosphate dikinase (PPDK) promoter from a C4 plant (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 expressed in leaves and light-grown seedlings, RBCS1 and RBCS2 expressed in developing tomato fruit, or the ribulose bisphosphate carboxylase promoter expressed almost exclusively at high levels in mesophyll cells of leaves and sheaths).

[0140] Exemplary senescence-specific promoters include tomato promoters active during fruit ripening, leaf wilting and abscission, maize promoters of genes encoding cysteine proteases, promoters of 82E4, and promoters of SAG genes. 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 seed-specific promoters (those active during seed development, such as promoters of seed storage polypeptides) and seed germination promoters (those active during seed germination).

[0141] Examples of inducible promoters include promoters that respond to pathogen attack, anaerobic conditions, high temperature, light, drought, cold temperature or high salt concentration. Pathogen-inducible promoters include promoters from polypeptides associated with pathogenesis (PR polypeptides) that are induced following infection by pathogens (e.g., PR polypeptides, SAR polypeptides, β-1,3-glucanase, chitinase).

[0142] In addition to plant promoters, other suitable promoters can be derived from bacterial sources, for example, the octopine synthase promoter, the nopaline synthase promoter, and others from Ti plasmids, or can be derived from viral promoters (e.g., the 35S and 19S RNA promoters of cauliflower mosaic virus (CaMV), the constitutive promoter of tobacco mosaic virus, the cauliflower mosaic virus (CaMV) 19S and 35S promoters, or the figwort mosaic virus 35S promoter).

[0143] Disclosed is a plant or plant cell comprising at least one mutation in one or more polynucleotides or polypeptides as described herein, wherein the mutation results in modulation of the function or activity of NtLKR or a polypeptide encoded thereby.

[0144] Provided is a method for modulating the level of an NtLKR polypeptide in a (dried) plant or (dried) plant material, the method comprising introducing one or more mutations into the genome of the plant that modulate the expression of at least one NtLKR, wherein the at least one NtLKR gene is selected from one or more of the NtLKR sequences according to the present disclosure.

[0145] Also provided is a method for identifying a plant having modulated levels of one or more amino acids in the plant or a part thereof as compared to the levels of the one or more amino acids in a control plant, the method comprising screening a polynucleotide sample from a 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 levels of the one or more amino acids.

[0146] Also disclosed is a plant or plant cell that is heterozygous or homozygous for one or more mutations in a NtLKR gene according to the disclosure, wherein the mutations result in modulated expression of the NtLKR gene or the function or activity of the NtLKR polypeptide encoded thereby.

[0147] A variety of methods can be used to combine mutations in a plant, including sexual crossing. Plants having one or more favorable heterozygous or homozygous mutations in a gene according to the present disclosure that modulate the expression of the gene or the function or activity of a polypeptide encoded thereby can be crossed with plants having one or more favorable heterozygous or homozygous mutations in one or more other genes that modulate the expression of the gene or the function or activity of a 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.

[0148] A function or activity is increased or decreased if the function or activity of one or more polypeptides of the present disclosure in a plant is lower or higher 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 has been grown, harvested and dried using the same protocol.

[0149] In some embodiments, a mutation is introduced into a plant or plant cell using a mutagenesis method, and the mutation introduced is identified or selected using methods well known to those skilled in the art such as Southern blot analysis, DNA sequencing, PCR analysis or phenotypic analysis. Methods well known in the art can be used to determine mutations that affect gene expression or interfere with the function of the encoded polypeptide. Insertion mutations in gene exons typically result in null mutations. Mutations in conserved residues can be particularly effective in inhibiting the metabolic function of the encoded polypeptide. For example, it should be understood that mutations in one or more highly conserved regions may change polypeptide function, while mutations outside those highly conserved regions may have little or no effect on polypeptide function. In addition, mutations in single nucleotides can produce stop codons, which will result in truncated polypeptides and, depending on the degree of truncation, loss of function.

[0150] Also disclosed are methods for obtaining mutant polynucleotides and polypeptides. Any plant of interest, including plant cells or plant material, can be genetically modified by a variety of known mutagenesis-inducing methods, including 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.

[0151] Mutations in the polynucleotides and polypeptides described herein may include artificial mutations, synthetic mutations, or genetically engineered mutations. Mutations in the polynucleotides and polypeptides described herein may be mutations obtained or obtainable through a process comprising in vitro or in vivo manipulation steps. Mutations in the polynucleotides and polypeptides described herein may be mutations obtained or obtainable through a process comprising human intervention. The function or activity of a mutant polypeptide variant may be higher, lower, or about the same as that of the unmutated polypeptide.

[0152] Methods for randomly introducing mutations in polynucleotides may include chemical mutagenesis and radiomutagenesis. Chemical mutagenesis involves the use of exogenously added chemicals (such as mutagenic, teratogenic, or carcinogenic organic compounds) to induce mutations. Mutagens (including chemical mutagens or radiation) that primarily produce point mutations and short deletions, insertions, missense mutations, simple sequence duplications, transversions, or transitions can be used to generate mutations. 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, busulfan, diepoxyalkylenes (diepoxyoctane, diepoxybutane, etc.), 2-methoxy-6-chloro-9-[3-(ethyl-2-chloro-ethyl)aminopropylamino]acridine dihydrochloride and formaldehyde.

[0153] It is also envisioned that spontaneous mutations in the locus may not be directly caused by a mutagen, as long as they produce the desired phenotype. Suitable mutagenic agents can also comprise, for example, ionizing radiation, such as X-rays, gamma rays, fast neutron irradiation, and UV radiation. For each type of plant tissue, the dosage of the mutagenic chemical or radiation is determined experimentally so as to obtain a mutation frequency below a threshold level characterized by lethality or reproductive sterility. Any plant polynucleotide preparation method known to those skilled in the art can be used to prepare plant polynucleotides for mutation screening.

[0154] The mutation process may include one or more plant crossing steps.

[0155] After sudden change, screening can be performed to identify the sudden change that produces premature termination codon or non-functional gene.After sudden change, screening can be performed to identify the sudden change that produces the functional gene that can express with the level that increases or decreases.The screening of mutant can be carried out by order-checking or by using one or more probes or primers that are special to this gene or polypeptide. Specific mutation can also be produced in polynucleotide, and it can cause the gene expression of regulation, the mRNA stability of regulation or the polypeptide stability of regulation. This type of plant is referred to as " non-naturally occurring " or " mutant " plant in this article. Usually, mutant or non-naturally occurring plant will include at least a portion (for example, DNA or RNA) of external or synthetic or artificial nucleotide that did not exist in plant before being operated. The foreign nucleotides can be a single nucleotide, two or more nucleotides, two or more consecutive 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 consecutive or non-contiguous nucleotides.

[0156] Sequence-specific polynucleotides that can interfere with the transcription of one or more endogenous genes; sequence-specific polynucleotides that can interfere with the translation of RNA transcripts (e.g., double-stranded RNA, siRNA, ribozymes); sequence-specific polypeptides that can interfere with the stability of one or more polypeptides; sequence-specific polynucleotides that can interfere with the enzymatic function of one or more polypeptides or the binding function of one or more polypeptides relative to a substrate or 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 large-range nucleases that have a function against one or more polynucleotides can be used to regulate the expression or function or activity of one or more polynucleotides or polypeptides described herein. Genome editing technologies are well known in the art and are further discussed below.

[0157] Zinc finger polypeptides can be used to modulate the expression, 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).

[0158] Meganucleases (such as I-Crel) can be used to modulate 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.

[0159] Transcription activator-like effector nucleases (TALENs) can be used to modulate the expression or function or activity of one or more 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.

[0160] The CRISPR system can be used to regulate the expression or function or activity of one or more NtLKR polynucleotides described herein and is a preferred method. The technology is described in, for example, Plant Methods (2016) 12:8; Front Plant Sci. (2016) 7:506; Biotechnology Advances (2015) 33, 1, pages 41-52; 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 systems generally include two components: a CRISPR-associated nuclease (Cas) (e.g., Cas9) and a guide RNA (gRNA). Cas forms a double-stranded DNA break at a site in the genome that is defined by the sequence of the gRNA molecule to which Cas binds. The location of Cas DNA breaks is defined by the unique sequence of the gRNA bound to it. gRNA is a specially designed RNA sequence that recognizes the target DNA region of interest and guides the Cas nuclease for editing. It has two segments: (i) tracr RNA, which acts as a binding scaffold for the Cas nuclease; and (ii) crispr RNA (crRNA), a 17-20 nucleotide sequence complementary to the target DNA. The exact region of DNA to be targeted will depend on the specific application. For example, in order to activate or inhibit a target polynucleotide, the gRNA can be targeted to a promoter that drives the expression of the target polynucleotide. Methods for designing gRNA are well known in the art, including Chop Chop Harvard. The application of Cas9-based genome editing in Arabidopsis and tobacco is described in, for example, Methods Enzymol. (2014) 546: 459-72 and Plant Physiol Biochem. (2018) 131: 37-46. CRISPR technology has been widely used in plants (see, for example, WO2015 / 189693).In addition to Cas9, other RNA-guided nucleases for use in CRISPR systems have been described, including Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas10, Cpf1, Csy1, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, and Csf4. In certain embodiments, the use of Cas9 is preferred. The present disclosure also provides a CRISPR-based genome editing system comprising an RNA-guided nuclease and a gRNA, wherein the CRISPR-based genome editing system regulates the activity of one or more polynucleotides described herein. The present disclosure also provides a method for cutting one or more polynucleotides in a plant cell, comprising introducing a gRNA and an RNA-guided nuclease into a plant cell, wherein the gRNA and the RNA-guided nuclease act in conjunction to produce a chain break in one or more polynucleotides described herein. A CRISPR construct is also disclosed, comprising: (i) a polynucleotide encoding a CRISPR-associated nuclease; and (ii) a gRNA comprising a polynucleotide sequence (typically about 17-20 nucleotides) complementary to the DNA of a polynucleotide as described herein to be targeted.

[0161] Antisense technology is another well-known method that can be used to modulate the expression or activity of one or more NtLKR polypeptides. See, for example, Gene (1988) 10; 72(1-2): 45-50.

[0162] NtLKR polynucleotides can be targeted for inactivation by introducing a transposon (e.g., an IS element or other mobile genetic element) into the genome of the plant of interest. See, for example, Cytology and Genetics (2006) 40(4):68-81.

[0163] NtLKR polynucleotides can be targeted for inactivation by introducing into plants ribozymes derived from a number of small circular RNAs that are capable of self-cleavage and replication. See, for example, FEMS Microbiology Reviews (1999) 23, 3, 257-275.

[0164] 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 NtLKR polynucleotides described herein that result in modulated expression, function, or activity of those polynucleotides or their polynucleotide products. 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. By way of further example, a mutant or non-naturally occurring plant or plant cell can have one or more modifications in a specific portion of an NtLKR polynucleotide or NtLKR polypeptide, such as in a region of the NtLKR that encodes the active site of an NtLKR polypeptide or portion thereof. By way of further example, a mutant or non-naturally occurring plant or plant cell can have one or more modifications in a region other than one or more NtLKR polynucleotides or NtLKR polypeptides, such as in a region upstream or downstream of the NtLKR polynucleotide, provided that it modulates the function or expression of the NtLKR. Upstream elements can include promoters, enhancers, or transcription factors. Some elements, such as enhancers, can be placed upstream or downstream of the gene they regulate. An element need not be located close to the gene it regulates, as some elements have been found hundreds of thousands of base pairs upstream or downstream of 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 a gene, within the first 200 nucleotides of a gene, within the first 300 nucleotides of a gene, within the first 400 nucleotides of a gene, within the first 500 nucleotides of a gene, within the first 600 nucleotides of a gene, within the first 700 nucleotides of a gene, within the first 800 nucleotides of a gene, within the first 900 nucleotides of a gene, within the first 1000 nucleotides of a gene, within the first 1100 nucleotides of a gene, within the first 1200 nucleotides of a gene, within the first 1300 nucleotides of a gene, within the first 1400 nucleotides of a gene, or within the first 1500 nucleotides of a 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 a gene, or a combination thereof. Disclosed are mutant or non-naturally occurring plants or plant cells (eg, mutant, non-naturally occurring, or transgenic plants or plant cells as described herein, etc.) comprising mutant polypeptide variants.

[0165] In one embodiment, seeds from a plant are mutagenized and subsequently grown into first-generation mutant plants. The first-generation plants are then self-pollinated, and the seeds from the first-generation plants are grown into second-generation plants, which are then screened for mutations in their loci. Although mutagenized plant materials 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 appreciate that a variety of plant materials, including but not limited to seeds, pollen, plant tissues, or plant cells, can be mutagenized to produce mutant plants. However, when screening for mutations in plant polynucleotides, the type of mutagenized plant material may have an impact. For example, when pollen is mutagenized before pollination of a non-mutagenized plant, the seeds obtained from the pollination are grown into first-generation plants. Each cell of the first-generation plant will contain the mutations produced in the pollen; therefore, these first-generation plants can be screened for mutations subsequently, rather than waiting until the second generation.

[0166] NtLKR polynucleotides prepared from individual plants, plant cells, or plant materials can optionally be pooled to accelerate screening for mutations in a plant population derived from mutagenized plant tissue, cells, or material. 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 polynucleotide-specific amplification techniques, such as PCR. Any one or more primers or probes specific for the gene or sequences immediately adjacent to the gene can be used to amplify sequences within the optionally pooled samples. Suitably, one or more primers or probes are designed to amplify regions of the locus where useful mutations are most likely to occur. Most suitably, primers are designed to detect mutations within the polynucleotide region. In addition, primers and probes preferably avoid known polymorphic sites to facilitate screening for point mutations. To facilitate detection of the amplified product, one or more primers or probes can be labeled using any conventional labeling method. Primers or probes can be designed based on the sequences described herein using methods well understood in the art. To facilitate detection of the amplified product, any conventional labeling method can be used to label the primers or probes. These primers or probes can be designed based on the sequences described herein using methods well understood in the art.

[0167] Polymorphisms can be identified by methods known in the art, and some have been described in the literature.

[0168] In some embodiments, plants can 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 can be used, such as, but not limited to, tissue culture or regeneration from protoplasts. Suitably, plants can be regenerated by growing transformed plant cells on callus induction medium, shoot induction medium, or root induction medium. See, for example, McCormick et al., Plant Cell Reports 5:81-84 (1986). These plants can then be grown and pollinated with the same transformed strain or different strains, and the resulting hybrids with the desired phenotypic characteristic expression can be identified. Two or more generations can be grown to ensure that the expression of the desired phenotypic characteristic is stably maintained and inherited, and seeds are harvested to ensure that the desired phenotypic characteristic expression is obtained. Therefore, "transformed seeds" refer to seeds containing a nucleotide construct stably integrated into the plant genome.

[0169] Thus, in another aspect, a method for preparing a mutant plant is provided. The method involves providing at least one cell of a plant comprising one or more NtLKR genes encoding a functional NtLKR. Next, the at least one cell of the plant is treated under conditions effective to modulate the function of the NtLKR polynucleotide. The at least one mutant plant cell is then propagated into a mutant plant, wherein the level of an NtLKR polypeptide as described herein is modulated in the mutant plant compared to a control plant. In one embodiment of this method for preparing a mutant plant, the treating step involves subjecting the at least one cell to a chemical mutagen as described above under conditions effective to obtain the at least one mutant plant cell. In another embodiment of this method, the treating step involves subjecting the at least one cell to a radiation source under conditions effective to obtain the at least one mutant plant cell. The term "mutant plant" includes mutant plants in which the genotype has been modified (suitably, by means other than genetic engineering or genetic modification) compared to a control plant.

[0170] In certain embodiments, mutant plants, mutant plant cells or mutant plant materials can include one or more mutations, and the one or more mutations are naturally present in another plant, plant cell or plant material, and confer desired proterties. This mutation can be introduced into (e.g., gene infiltration) another plant, plant cell or plant material (e.g., plant, plant cell or plant material with a genetic background different from that of the plant from which the mutation originates) to confer this proterties. Therefore, for example, a mutation naturally occurring in a first plant can be introduced into a second plant, such as a second plant with a genetic background different from that of the first plant. Therefore, technicians can search for and identify plants that naturally carry one or more mutant alleles of a gene described herein in the genome, and the gene confer desired proterties. Naturally occurring mutant alleles can be transferred to a second plant by a variety of methods (comprising breeding, backcrossing and gene infiltration) to produce strains, varieties or hybrids with one or more mutations in a gene described herein. The same technology can also be applied to the infiltration of one or more non-natural mutations from a first plant to a second plant. Plants displaying desired proterties can be screened in the library of mutant plants. Suitably, selection is performed using knowledge of the polynucleotides as described herein. Thus, genetic traits can be screened compared to a control. Such screening methods may involve the application of conventional amplification or hybridization techniques as discussed herein. Accordingly, another aspect of the present disclosure relates to a method for identifying a mutant plant, the method comprising the steps of: (a) providing a sample comprising one or more NtLKR polynucleotides from a plant; and (b) determining the sequence of the polynucleotide, wherein a difference in the sequence of the polynucleotide compared to a polynucleotide 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 increased or decreased levels of an amino acid compared to a control plant, the method comprising: (a) providing a sample from a plant to be screened; (b) determining whether the sample comprises one or more mutations in one or more NtLKR polynucleotides as described herein; and (c) determining the level of at least one amino acid in the plant. Suitably, the level of at least one amino acid is determined in dried leaves. In another aspect, a method for producing a mutant plant having increased or decreased levels of at least one amino acid compared to a control plant is provided, 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 modulated levels of at least one amino acid; and (c) transferring the one or more mutations to a second plant. Suitably, the level of the at least one amino acid is determined in dried leaves. The mutations can be transferred to the second plant using various methods known in the art, such as genetic engineering, genetic manipulation, introgression, plant breeding, backcrossing, and the like.In one embodiment, the first plant is a naturally occurring plant. In one embodiment, the second plant has a different genetic background than the first plant. In another aspect, a method for producing a mutant plant having increased or decreased levels of at least one amino acid compared to a control plant is provided, the method comprising: (a) providing a sample from the first plant; (b) determining whether the sample comprises one or more mutations in one or more NtLKR polynucleotides described herein that result in modulated levels of at least one amino acid; and (c) introgressing the one or more mutations from the first plant into the second plant. Suitably, the level of at least one amino acid is determined in dried leaves. In one embodiment, the introgression step comprises plant breeding, optionally including backcrossing, etc. In one embodiment, the first plant is a naturally occurring plant. In one embodiment, the second plant has a different genetic background than the first plant. In one embodiment, the first plant is not a cultivar or an elite cultivar. In one embodiment, the second plant is a cultivar or an elite cultivar. Another aspect relates to mutant plants (including cultivar or elite cultivar mutant plants) obtained or obtainable by the methods described herein. In certain embodiments, a "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. According to this embodiment, the remaining genomic sequence of the mutant plant will be identical or substantially identical to that of the plant prior to mutagenesis.

[0171] In certain embodiments, the mutant plant may have one or more mutations located in more than one genomic region of the plant, such as within the sequence of one or more NtLKR polynucleotides described herein and within one or more other regions of the genome. According to this embodiment, the remaining genomic sequence of the mutant plant will be different or substantially different from that of the plant before 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 the NtLKR polynucleotides 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 the NtLKR polynucleotides described herein; or may not have one or more mutations in the promoter of the NtLKR polynucleotides described herein; or may not have one or more mutations in the 3' untranslated region of the NtLKR polynucleotides described herein; or may not have one or more mutations in the 5' untranslated region of the NtLKR polynucleotides described herein; or may not have one or more mutations in the coding region of the NtLKR polynucleotides described herein; or may not have one or more mutations in the non-coding regions of the NtLKR polynucleotides described herein.

[0172] In another aspect, a method for identifying a plant, plant cell, or plant material comprising a mutation in a gene encoding an NtLKR polynucleotide as described herein is provided, the method comprising: (a) mutagenizing the plant, plant cell, or plant material; (b) obtaining a sample from the plant, plant cell, or plant material, or a progeny thereof; 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. The method also allows for the selection of plants having mutations in genomic regions that affect expression of the NtLKR gene in plant cells, such as the transcription start site, the start codon, intronic regions, exon-intron boundaries, or terminators.

[0173] Plants suitable for use in the present disclosure include monocotyledonous and dicotyledonous plants and plant cell systems, and include members of the genus Nicotiana.

[0174] Various embodiments relate to mutant tobacco, non-naturally occurring tobacco, or transgenic tobacco plants or tobacco plant cells and can be applied to any species of the Nicotiana genus, including N. rustica and Nicotiana tabacum (e.g., LAB21, 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, N. benavidesii, N. benthamiana, N. bigelovii, N. bonariensis, N. cavicola, N. clevelandii, N. cordifolia, N. corymbosa, N. debneyi, N. excelsior, N. forget iana), N. fragrans, N. glauca, N. glutinosa, N. goodspeedii, N. gossei, N. hybrid, N. ingulba, N. kawakamii, N. knightiana, N. langsdorffii, N. linearis, N. longiflora, N. maritima, N. megalosiphon, N. miersii, N. noctiflora, N. nudicaulis, N. obtusifolia, N. occidentalis, N. occidentalis subsp. hesperis), ear-shaped tobacco (N.otophora), round tobacco (N. paniculata), few-flowered tobacco (N. pauciflora), petunia-shaped tobacco (N.petunioides), blue jasmine tobacco (N. plumbaginifolia), Quadrivalvis tobacco (N. quadrivalvis), Raymond tobacco (N. raimondii), wave-edged tobacco (N. repanda), rosette tobacco (N. rosulata), rosette subspecies Ingulba tobacco (N. rosulata In one embodiment, the plant is tobacco.

[0175] This paper also contains and uses tobacco cultivar and fine tobacco cultivar.Therefore, transgenic, non-natural existence or mutant plant can be tobacco cultivar or fine tobacco cultivar, and it comprises one or more transgenic or one or more genetic mutations or its combination.Gene mutation (for example, one or more polymorphisms) can be that non-natural is present in the sudden change in individual tobacco cultivar or tobacco cultivar (for example, fine tobacco cultivar), or can be really naturally occurring genetic mutation, condition is that described sudden change is not naturally present in individual tobacco cultivar or tobacco cultivar (for example, fine tobacco cultivar).

[0176] Particularly useful tobacco varieties include Burley, Black, Flue-cured, and Oriental types. Non-limiting examples of varieties or cultivars are: BD 64, CC 101, CC 200, CC 27, CC 301, CC 400, CC 500, CC 600, CC 700, CC 800, CC 900, Coker 176, Coker 319, Coker 371 Gold, Coker 48, CD 263, DF911, DT538LC Galpao tobacco, GL 26H, GL 350, GL 600, GL 737, GL 939, GL 973, HB 04P, HB 04P LC, HB3307PLC, Hybrid 403LC, Hybrid 404LC, Hybrid 501LC, K 149, K 326, K 346, K 358, K394, K 399, K730, KDH 959, KT 200, KT204LC, KY10, KY14, KY 160, KY 17, KY 171, KY 907, KY907LC, KY14xL8 LC, Little Crittenden, McNair 373, McNair 944, msKY 14×L8, narrow leaf Madole, narrow leaf Madole LC, NBH 98, N-126, N-777LC, N-7371LC, NC 100, NC 102, NC 2000, NC 291, NC297, NC 299, NC 3, NC 4, NC 5, NC 6, NC7, NC 606, NC 71, NC 72, NC 810, NC BH 129, NC2002, Neal Smith Madole,OXFORD 207,PD 7302LC, PD 7309LC, PD 7312LC, 'Perique' tobacco, PVH03, PVH09, PVH19, PVH50, PVH51, R 610, R 630, R 7-11, R 7-12, RG 17, RG 81, RGH51, RGH 4, RGH 51, RS1410, Speight 168, Speight 172, Speight 179, Speight 210, Speight 220, Speight 225, Speight 227, Speight 234, Speight G-28, Speight G-70, Speight H-6, Speight H20, Speight NF3, TI 1406, TI 1269, TN 86, TN86LC, TN 90, TN 97、TN97LC、TN D94、TND950, TR (Tom Rosson) Madole, VA 309, VA359, AA 37-1, B13P, Xanthi (Mitchell-Mor), Bel-W3, 79-615, Samsun Holmes NN, KTRDC No. 2 Hybrid 49, Burley 21, KY8959, KY9, MD 609, PG01, PG04, PO1, PO2, PO3, RG11, RG 8, VA509, AS44, Banket A1, Basma Drama B84 / 31, Basma I Zichna ZP4 / B, Basma Xanthi BX 2A, Batek, Besuki Jember, C104, Coker347, Criollo Misionero, Delcrest, Djebel 81, DVH 405, Comum, HB04P, Hicks Broadleaf, Kabakulak Elassona, Kutsage E1, LA BU 21, NC 2326, NC 297, PVH 2110, RedRussian, Samsun, Saplak, Simmaba, Talgar 28, Wislica, Yayaldag, Prilep HC-72, Prilep P23, Prilep PB 156 / 1, Prilep P12-2 / 1, Yaka JK-48, Yaka JB 125 / 3, TI-1068, KDH-960, TI-1070, TW136, Basma, TKF 4028, L8, TKF 2002, GR141, Basma xanthi, GR149, GR153, Petit Havana. Even if not specifically stated herein, the low-conversion subvariants of the above are also contemplated.

[0177] Embodiments also relate to compositions and methods for producing mutant plants, non-naturally occurring plants, hybrid plants, or transgenic plants that have been modified to modulate the expression or function of one or more NtLKR polynucleotides described herein (or any combination thereof as described herein). Advantageously, the resulting mutant plants, non-naturally occurring plants, hybrid plants, or transgenic plants can be similar or substantially identical in overall appearance to control plants. Various phenotypic characteristics, such as degree of maturity, number of leaves per plant, culm height, leaf insertion angle, leaf size (width and length), internode distance, and leaf-to-midrib ratio, can be evaluated by field observation.

[0178] One aspect relates to the seed of mutant plant as described herein, non-natural plant, hybrid plant or transgenic plant. Suitably, the seed is a tobacco seed. Another aspect relates to the pollen or ovule of mutant plant as described herein, non-natural plant, hybrid plant or transgenic plant. In addition, mutant plant as described herein, non-natural plant, hybrid plant or transgenic plant is provided, which also comprises a polynucleotide that imparts male sterility.

[0179] Also provided are tissue cultures of regenerable cells of a mutant plant, non-naturally occurring plant, hybrid plant, or transgenic plant as described herein, or a portion thereof, wherein the culture regenerates a plant capable of expressing all the morphological and physiological characteristics of the parent plant. Regenerable cells include cells from leaves, pollen, embryos, cotyledons, hypocotyls, roots, root tips, anthers, flowers and parts thereof, ovules, buds, stems, stalks, pith, and sacs, or callus or protoplasts derived therefrom.

[0180] The plant material described herein may be a cured tobacco material. CORESTA recommendations for tobacco curing are described in: CORESTA Guideline No. 17, April 2016, Sustainability in Leaf Tobacco Production.

[0181] 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, such as in dried leaves.

[0182] Suitably, modulation of the level of at least one amino acid is observed in at least dried leaves, suitably fully dried leaves. Tobacco is considered to be fully cured when the leaf midribs are free of moisture resulting in a leaf colour ranging from light tan to reddish brown to dark brown.

[0183] Suitably, the dried leaves are taken from mid-level leaves on the plant. Suitably, the phenotype of the leaves in which the level of at least one amino acid is modulated is unaffected compared to leaves from a control plant.

[0184] In one embodiment, the level of lysine is increased compared to a control plant or part thereof.

[0185] In one embodiment, the levels of lysine, arginine, glutamine, tyrosine, gamma aminobutyric acid (GABA), and alanine are increased compared to a control plant or part thereof.

[0186] In one embodiment, the levels of lysine, arginine, glutamine, histidine, tyrosine, tryptophan, threonine, GABA, asparagine and alanine are increased compared to control plants or parts thereof.

[0187] In one embodiment, the levels of lysine, arginine, proline, GABA, glutamine, leucine, alanine, phenylalanine, tyrosine and isoleucine are increased compared to control plants or parts thereof.

[0188] In one embodiment, the levels of isoleucine, valine and serine are reduced compared to control plants or parts thereof.

[0189] In one embodiment, the levels of methionine, threonine and glycine are reduced compared to control plants or parts thereof.

[0190] In one embodiment, the levels of aspartate and glutamate are not significantly altered compared to control plants or parts thereof.

[0191] In one embodiment, the levels of proline, aspartic acid, leucine, phenylalanine, glutamate and methionine are not significantly altered compared to control plants or parts thereof.

[0192] In one embodiment, the levels of asparagine, aspartic acid, tryptophan, histidine, glutamate, serine and valine are not significantly altered compared to control plants or parts thereof.

[0193] In one embodiment, the levels of total free amino acids are not significantly altered compared to a control plant or part thereof.

[0194] In one embodiment, the plant is a Nicotiana tabacum plant, wherein the levels of lysine, arginine, glutamine, histidine, tyrosine, tryptophan, threonine, GABA, asparagine and alanine are increased compared to a control plant or part thereof, suitably wherein the increase in lysine is about 4.6 fold, the increase in arginine is about 2.81 fold, the increase in glutamine is about 2.06 fold, the increase in histidine is about 1.68 fold, the increase in tyrosine is about 1.62 fold, the increase in tryptophan is about 1.6 fold, the increase in threonine is about 1.53 fold, the increase in GABA is about 1.45 fold, the increase in asparagine is about 1.38 fold and the increase in alanine is about 1.3 fold compared to a control plant or part thereof.

[0195] The Virginia tobacco plant may also have reduced levels of isoleucine, valine and serine compared to control plants or parts thereof, suitably wherein the reduction in isoleucine is about 39%, the reduction in valine is about 17%, and the reduction in serine is about 15% compared to control plants or parts thereof.

[0196] The levels of proline, aspartic acid, leucine, phenylalanine, glutamic acid, and methionine were also not significantly changed in N. virginiana plants compared to control plants or parts thereof.

[0197] The total amount of sugars in the Nicotiana virginiana plant may be reduced by about 15% (including a reduction in glucose, suitably by about 23%; a reduction in fructose, suitably by about 18%; and no significant difference in sucrose content) compared to control plants or parts thereof.

[0198] The ammonia content of the Nicotiana virginiana plants may not be significantly different compared to control plants or parts thereof.

[0199] The nitrate content of the Nicotiana virginiana plants may be reduced, suitably by about 34%, compared to control plants or parts thereof.

[0200] In another embodiment, the plant is a Burley tobacco plant, wherein the levels of lysine, arginine, proline, GABA, glutamine, leucine, alanine, phenylalanine, tyrosine and isoleucine are increased compared to a control plant or part thereof, suitably wherein the increase in lysine is about 11.2 fold, the increase in arginine is about 1.85 fold, the increase in proline is about 1.41 fold, the increase in GABA is about 1.26 fold, the increase in glutamine is about 1.25 fold, the increase in leucine is about 1.24 fold, the increase in alanine is about 1.20 fold, the increase in phenylalanine is about 1.15 fold, the increase in tyrosine is about 1.09 fold, and the increase in isoleucine is about 1.06 fold.

[0201] The Burley tobacco plant may have reduced levels of methionine, threonine and glycine compared to control plants or parts thereof, suitably wherein the reduction in methionine is about 29%, the reduction in threonine is about 10%, and the reduction in glycine is about 8% compared to control plants or parts thereof.

[0202] The levels of asparagine, aspartic acid, tryptophan, histidine, glutamate, serine, and valine may not be significantly changed in the Burley tobacco plants compared to control plants or parts thereof.

[0203] Another aspect relates to a mutant, non-naturally occurring or transgenic plant or cell, wherein the expression of one or more NtLKR polynucleotides or the activity of one or more NtLKR polypeptides is modulated, suitably, reduced, and the level of at least one amino acid, suitably lysine, is increased at least 4-fold or at least 11-fold compared to a control plant or part thereof, wherein the expression of NtLKR or the activity of NtLKR is not modulated, suitably, reduced.

[0204] A further aspect relates to a mutant, non-naturally occurring or transgenic plant or cell derived from or derivable from dried plant material, such as dried leaves or dried tobacco, in which the expression of one or more of the NtLKR polynucleotides described herein or the function of the encoded NtLKR polypeptide is modulated, suitably reduced, and in which the level of one or more amino acids, suitably lysine, is modulated, suitably reduced, compared to a control plant or part thereof.

[0205] Embodiments also relate to compositions and methods for producing mutant, non-naturally occurring or transgenic plants or plant cells that have been modified to modulate (suitably reduce) the expression or activity of one or more of the NtLKR polynucleotides or NtLKR polypeptides described herein, which can result in modulated, suitably increased, amino acid (suitably lysine) content of the plant or plant part (e.g., leaves - such as dried leaves) or plant cell.

[0206] In one embodiment, the phenotype of mutant, non-natural existence or transgenic plant is basically the same as control plant or its part.In one embodiment, the leaf weight of mutant, non-natural existence or transgenic plant is basically the same as control plant or its part.In one embodiment, the leaf number of mutant, non-natural existence or transgenic plant is basically the same as control plant or its part.In one embodiment, the leaf weight and leaf number of mutant, non-natural existence or transgenic plant are basically the same as control plant or its part.In one embodiment, for example after field transplantation one, two or three or more months or after topping 10,20,30 or 36 or more days, the stalk of mutant, non-natural existence or transgenic plant is basically the same as control plant or its part.For example, the stalk of mutant, non-natural existence or transgenic plant is not less than the stalk of control plant or its part is high.In another embodiment, compared with control plant or its part, the chlorophyll content of mutant, non-natural existence or transgenic plant reduces.In another embodiment, compared with control plant or its part, the speed of leaf aging increases.

[0207] In another aspect, a method for modulating the amount of at least one amino acid in at least a part of a plant (e.g., leaves, such as dried leaves) is provided, the method comprising: (i) modulating the expression or function of one or more of the NtLKR polypeptides described herein, suitably 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 part of the mutant, non-naturally occurring or transgenic plant obtained in step (i) (e.g., leaves, such as dried leaves, or tobacco or smoke); and (iii) identifying the mutant, non-naturally occurring or transgenic plant or part thereof in which the level of the at least one amino acid is modulated compared to a control plant or part thereof.

[0208] In another aspect, there is provided a method for modulating the amount of at least one amino acid in dried plant material, such as dried leaves, the method comprising: (i) modulating the expression or function of one or more NtLKR polypeptides (or any combination thereof as described herein), suitably wherein the NtLKR polypeptides are encoded by the corresponding NtLKR polynucleotides described herein; (ii) harvesting the plant material, such as one or more leaves, and drying for a period of time; (iii) measuring the level of at least one amino acid in the dried plant material obtained in or during step (ii); and (iv) identifying dried plant material in which the level of at least one amino acid has been modulated compared to a control plant or part thereof.

[0209] The increase in expression can be from about 5% to about 100%, or an increase of at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, or 100% or more, such as 200%, 300%, 500%, 1000% or more, compared to a control, including an increase in transcriptional function or NtLKR polynucleotide expression or NtLKR polypeptide expression.

[0210] The increase in function or activity can be from about 5% to about 100%, or an increase of at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, or 100% or more, such as 200%, 300%, 500%, 1000% or more, compared to a control, including an increase in transcriptional function or NtLKR polynucleotide expression or NtLKR polypeptide expression, or a combination thereof.

[0211] The reduction in expression 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 100%, compared to a control, including a reduction in transcriptional function or NtLKR polynucleotide expression or NtLKR polypeptide expression, or a combination thereof.

[0212] The reduction in function or activity 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 100%, compared to a control, including a reduction in transcriptional function or NtLKR polynucleotide expression or NtLKR polypeptide expression, or a combination thereof.

[0213] The polynucleotides and recombinant constructs described herein can be used to modulate the expression or function or activity of a NtLKR polynucleotide or NtLKR polypeptide described herein in a plant species of interest, suitably tobacco.

[0214] Many 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 that includes the 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 appropriate conditions, the promoter can drive expression to modulate 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 as 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 to drive constitutive expression of the transgene in all tissues of the plant. The vector also carries an antibiotic resistance gene to confer selection for transformed callus and cell lines.

[0215] Expression of sequences from the promoter can be enhanced by including expression control sequences, which are well known in the art. Signals associated with aging and those active during the drying process are specifically indicated.

[0216] Thus, various embodiments relate to methods for modulating the expression level of one or more NtLKR polynucleotides described herein (or any combination thereof as described herein) by integrating multiple copies of the NtLKR polynucleotides into the plant genome, 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 can be a native polypeptide or can be heterologous to the cell.

[0217] In one embodiment, the plants used in the present disclosure are flue-cured mutant, non-naturally occurring or transgenic plants.

[0218] In one embodiment, the plants used in the present disclosure are sun-dried mutant, non-naturally occurring or transgenic plants.

[0219] In one embodiment, the plants used in the present disclosure are dried mutant, non-naturally occurring or transgenic plants.

[0220] In one embodiment, the plant used in the present disclosure is a cured (eg, flue-cured) mutant, non-naturally occurring, or transgenic Nicotiana Virginiana plant.

[0221] In one embodiment, the plant used in the present disclosure is a cured (eg, air-cured) mutant, non-naturally occurring, or transgenic Burley tobacco plant.

[0222] In one embodiment, the plant used in the present disclosure is a cured (eg, cured) mutant, non-naturally occurring, or transgenic dark tobacco plant.

[0223] By regulating NtLKR expression and / or activity, the sensory characteristics of tobacco can be advantageously altered. For example, as understood from Table 5, RNAi-modified Burley tobacco TN90 tobacco has reduced irritation, making the aerosol more rounded and smooth, with less typical deep flavor, but more animal and nutty flavor (with less trigeminal influence). By way of further example, as understood from Table 6, RNAi-modified Virginia tobacco K326 tobacco has increased hay flavor, with a rounder, deeper flavor profile. Plants carrying mutant alleles of one or more NtLKR polynucleotides described herein can be used in plant breeding programs to produce useful strains, varieties, and hybrids. For example, mutant alleles can be introgressed into commercially important varieties described herein. Thus, methods for plant breeding are provided, comprising hybridizing a mutant plant, a non-naturally occurring plant, or a transgenic plant as described herein with a plant containing a different genetic identity. The method can further comprise hybridizing the offspring plant with another plant, and optionally repeating the hybridization until offspring with the desired genetic trait or genetic background are obtained. One purpose of such breeding methods is to introduce desired genetic traits into other varieties, breeding lines, hybrids, or cultivars, particularly those of commercial interest. Another purpose is to facilitate the genetic modification of different genes in a single plant variety, line, hybrid, or cultivar. Both intraspecific and interspecific mating are contemplated. Progeny plants derived from such hybridizations, also referred to as breeding lines, are examples of non-naturally occurring plants of the present disclosure.

[0224] In one embodiment, there is provided a method for producing a non-natural plant, the method comprising: (a) hybridizing a mutant or transgenic plant with a second plant to produce offspring tobacco seeds; (b) growing offspring tobacco seeds under plant growth conditions to produce non-natural plants. The method may also include: (c) hybridizing the non-natural plant of the previous generation with itself or another plant to produce offspring tobacco seeds; (d) growing the offspring tobacco seeds of step (c) under plant growth conditions, to produce other non-natural plants; and (e) repeating the hybridization and growth steps of (c) and (d) for many times to produce the further offspring of the non-natural plant. The method may optionally include providing a step of a parental plant before step (a), the parental plant comprising the genetic consistency characterized and different from the mutant or transgenic plant. In some embodiments, depending on a breeding plan, hybridization and growth steps are repeated 0 to 2 times, 0 to 3 times, 0 to 4 times, 0 to 5 times, 0 to 6 times, 0 to 7 times, 0 to 8 times, 0 to 9 times or 0 to 10 times, to produce the generations of non-natural plants. Backcrossing is an example of such a method, in which a progeny is crossed with one of its parents or another plant that is genetically similar to its parent in order to obtain a progeny plant that has a genetic identity closer to that of one of the parents in the next generation. Techniques for plant breeding, particularly plant breeding, are well known and can be used in the methods of the present disclosure. The present disclosure also provides non-naturally occurring plants produced by these methods. Certain embodiments do not include the step of selecting a plant.

[0225] In some embodiments of the method described herein, standard field program is used to evaluate the strain derived from breeding and screening variant genes in the field. Comprise the control genotype of original non-mutagenized parent and include, and by randomized complete block design or other suitable field design, the selected person (entry) is arranged in the field. For tobacco, the agronomy practice of standard is used, for example, tobacco is gathered in the crops, weighed and sampled, for chemical and other common tests before and during drying. The statistical analysis of the execution data is to confirm the similarity between the selected strain and the parental strain. Optionally, the cytogenetics analysis of selected plant is performed to confirm chromosome group and chromosome pairing relationship.

[0226] DNA fingerprinting, single nucleotide polymorphism, microsatellite markers or similar techniques can be used in the breeding program of marker assisted selection (MAS), with as described herein, the mutant allele of gene is transferred or cultivated in other tobacco.For example, the breeder can produce the colony of separation by the hybridization of the genotype containing the mutant allele and the genotype of agronomy expectation.Can use one of technology listed herein, use the mark developed from genomic sequence or its fragment to screen the plant or backcross generation among the F2.The plant that is accredited as having the mutant allele can backcross or self-pollinate, to produce the second colony to be screened.Depend on expected inheritance pattern or used MAS technology, be necessary before each round backcrossing, selected plant is carried out self-pollination, to help identify required individual plant.Can repeat backcrossing or other breeding operations, until recover the required phenotype of recurrent parent.

[0227] In some embodiments, the F1 plant of plant colony is screened for variant gene expression in the F2 generation.In some embodiments, the F1 plant of plant colony is screened for variant gene expression in the F2 generation.In some embodiments, the F1 plant of plant colony is screened for variant gene expression in the F2 generation.In some embodiments, the F1 plant of plant colony is screened for variant gene expression in the F2 generation.In some embodiments, the F1 plant of plant colony is screened for variant gene expression in the F2 generation.In some embodiments, the F1 plant of plant colony is screened for variant gene expression in the F2 generation.In some embodiments, the F1 plant of plant colony is screened for variant gene expression in the F2 generation.In some embodiments, the F1 plant of plant colony is screened for variant gene expression in the F2 generation.In some embodiments, the F1 plant of plant colony is screened for variant gene expression in the F2 generation.

[0228] Hybrid tobacco varieties can be produced in the following way: stop the self-pollination of the female parent plant (that is, seed parent) of the first kind, allow the pollen from the male parent plant of the second kind to fertilize the female parent plant, and allow F1 hybrid seed to form on female plant.Can be by flower emasculation to stop the self-pollination of female plant in the early stage of flower development.Or, can use male sterile form to stop forming pollen on female parent plant.For example, can produce male sterility by cytoplasm male sterility (CMS) or transgenic male sterility, wherein transgenic suppresses microspore or pollen formation or self-incompatibility.The female parent plant containing CMS is particularly useful.In the embodiment that female parent plant is CMS, from male fertile plant results pollen and artificially apply to the column cap of CMS female parent plant, and gather in the crops the F1 seed obtained.

[0229] Kind described herein and strain can be used for forming single cross tobacco F1 hybrid.In this class embodiment, the plant of parental kind can be grown as the adjacent colony of homogeneity basically, so that male parent plant and the natural cross pollination of female parent plant.Selectively gather in the crops the F1 seed that forms on female parent plant by conventional means.Also can plant two parental plant kinds in large quantities, and gather in the crops the F1 hybrid seed that forms on female parent and the admixture of the seed that forms on male parent owing to self-pollination.Perhaps, can carry out three-line hybridization, wherein single cross F1 hybrid is as female parent, and hybridizes with different male parents.As another alternative, can produce double cross hybrid, wherein the F1 filial generation of two different single crosses carries out self hybridization.

[0230] A population of mutant, non-naturally occurring, or transgenic plants can be screened or selected for those members of the population that possess a desired trait or phenotype. For example, a population of progeny from a single transformation event can be screened for those plants that have a 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 methods include Southern analysis or PCR amplification for detecting polynucleotides; Northern blotting, S1 RNase protection, primer extension, or RT-PCR amplification for detecting RNA transcripts; enzymatic assays for detecting enzyme or ribozyme function of polypeptides and polynucleotides; and polypeptide gel electrophoresis, Western blotting, immunoprecipitation, and enzyme-linked immunosorbent assays for detecting polypeptides. Other techniques such as in situ hybridization, enzyme staining, immunostaining, and enzyme assays can also be used to detect the presence or expression, function, or activity of NtLKR polypeptides or polynucleotides.

[0231] Mutant, non-naturally occurring or transgenic plant cells and plants as described herein include 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.

[0232] Without limitation, the plants and parts thereof described herein can be modified before or after the expression, function or activity of one or more NtLKR polynucleotides or NtLKR polypeptides according to the present disclosure has been modulated.

[0233] In mutant, non-natural or transgenic plants and parts thereof, one or more of the following further genetic modifications may be present. One or more genes related to the conversion of nitrogen metabolism intermediates may be modified to reduce the level of at least one tobacco-specific nitrosamine (TSNA). Non-limiting examples of such genes include those encoding nicotine demethylases (CYP82E4, CYP82E5, and CYP82E10, such as those described in WO2006 / 091194, WO2008 / 070274, WO2009 / 064771, and WO2011 / 088180), and nitrate reductases, such as those described in WO2016 / 046288. One or more genes involved in heavy metal absorption or heavy metal transport may be modified to reduce heavy metal content. Non-limiting examples include genes in the multidrug resistance-associated polypeptide family, the cation diffusion facilitating factor (CDF) family, the Zrt-Irt-like polypeptide (ZIP) family, the cation exchanger (CAX) family, the copper transporter (COPT) family, the heavy metal ATPase family (e.g., HMA, as described in WO2009 / 074325 and WO2017 / 129739), the family of homologs of the natural resistance-associated macrophage polypeptide (NRAMP), and other members of the ATP-binding cassette (ABC) transporter family (e.g., MRP), as described in WO2012 / 028309, which are involved in the transport of heavy metals such as cadmium.

[0234] Other exemplary modifications can produce plants with regulated expression or function of isopropylmalate synthase, which results in changes in sucrose ester composition, which can be used to change the preference profile (see WO2013 / 029799). Other exemplary modifications can produce plants with regulated expression or function of threonine synthase, in which methionine levels can be regulated (see WO2013 / 029800). Other exemplary modifications can produce plants with regulated expression or function of one or more of neoxanthin synthase, lycopene beta cyclase, and 9-cis-epoxycarotenoid dioxygenase to regulate beta-damascenone content to change flavor characteristics (see WO2013 / 064499). Other exemplary modifications can produce plants with regulated expression or function of CLC family members placed in chloride channels to regulate nitrate levels therein (see WO2014 / 096283 and WO2015 / 197727). Other exemplary modifications can produce plants with modulated expression or function of one or more asparagine synthetases to modulate the level of asparagine in the leaves and to modulate the level of acrylamide in the aerosols produced when the leaves are heated or burned (see WO2017 / 129739). Other exemplary modifications can result in plants with modulated protease activity during curing (see WO2016 / 009006). Other exemplary modifications 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). Other exemplary modifications can result in plants with modified alkaloid levels by altering the gene expression of the putative ABC-2 transporters NtABCG1-T and NtABCG1-S or the activity of the proteins encoded thereby (see WO2019 / 086609). Other exemplary modifications can result in plants with regulated flowering time by altering the gene expression of the gene encoding terminal flower 1 (TFL1) or the activity of the protein encoded thereby (see WO2018 / 114641). Other exemplary modifications can result in plants with regulated expression or function of one or more asparagine synthetases to regulate asparagine levels in leaves and to regulate acrylamide levels in aerosols generated when leaves are heated or burned (see WO2017 / 042162). Other examples of modifications include regulating herbicide tolerance, for example, glyphosate is the active ingredient in many broad-spectrum herbicides. Glyphosate-resistant transgenic plants have been developed by transferring the aroA gene (glyphosate EPSP synthase from Salmonella typhimurium and E. coli).Sulfonylurea-resistant plants have been generated by transforming a mutant ALS (acetolactate synthase) gene from Arabidopsis thaliana.The OB polypeptide from photosystem II of mutant Amaranthus hybridus has been transferred into plants to produce atrazine-resistant transgenic plants; and bromoxynil-resistant transgenic plants have been produced by incorporating the bxn gene from the bacterium Klebsiella pneumoniae. Another exemplary modification results in plants resistant to insects. Bacillus thuringiensis (Bt) toxins can provide an effective way to delay the emergence of Bt-resistant pests, as recently demonstrated in cauliflower, where the pyramidal cry1Ac and cry1C Bt genes control diamondback moths resistant to either individual polypeptide and significantly delay 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 (resistant to bacterial blight) and plants expressing a Bt fusion gene and a chitinase gene (resistant to yellow stem borer and sheath resistance) have been engineered. Another exemplary modification produces altered reproductive capacity, such as male sterility. Another exemplary modification produces plants that tolerate abiotic stress (e.g., drought, temperature, salinity), and by transferring acylglycerol phosphatase from Arabidopsis, tolerant transgenic plants have been produced; genes encoding mannitol dehydrogenase and sorbitol dehydrogenase improve drought resistance, and the mannitol dehydrogenase and sorbitol dehydrogenase are involved in the synthesis of mannitol and sorbitol. Another exemplary modification produces plants in which the activity of one or more nicotine N-demethylases is regulated, so that the levels of nornicotine and nornicotine metabolites formed during drying can be regulated (see WO2015169927). Other exemplary modifications can produce plants with improved storage polypeptides and oils, plants with enhanced photosynthetic efficiency, plants with extended shelf life, plants with enhanced carbohydrate content, and plants that are antifungal. It is also conceivable that transgenic plants in which the expression of S-adenosyl-L-methionine (SAM) or cystathionine γ-synthase (CGS) or a combination thereof has been regulated. One or more genes involved in the nicotine synthesis pathway can be modified to produce plants or plant parts that produce regulated levels of nicotine when dried. Nicotine synthesis genes can be selected from the group consisting of: A622, BBLa, BBLb, JRE5L1, JRE5L2, MATE1, MATE 2, MPO1, MPO2, MYC2a, MYC2b, NBB1, nic1, nic2, NUP1, NUP2, PMT1, PMT2, PMT3, PMT4 and QPT or one or more combinations thereof. One or more genes involved in controlling the amount of one or more alkaloids can be modified to produce plants or plant parts that produce regulated levels of alkaloids.The alkaloid level-controlling gene may be selected from the group consisting of BBLa, BBLb, JRE5L1, JRE5L2, MATE1, MATE 2, MYC2a, MYC2b, nic1, nic2, NUP1 and NUP2, or a combination of one or more thereof.

[0235] Other exemplary modifications can result in plants with modulated amino acid content (see WO2019 / 185703 and WO2021 / 063863) or with modulated sugar content (see WO2019 / 185699 and WO2021 / 063860 and WO2021 / 063863) or with modulated nitrate levels (see WO2020 / 141062) or with modulated sugar and amino acid content (see WO2021 / 063863).

[0236] In a preferred embodiment, the additional genetic modification involves an asparagine synthetase (ASN) gene as described in WO2017042162. Modulating the expression of an ASN gene (e.g., one or more of NtASN1-S, NtASN1-T, NtASN5-S, and NtASN5-T as described in WO2017042162) or the activity of an ASN (e.g., NtASN1-S, NtASN1-T, NtASN5-S, and NtASN5-T as described in WO2017042162) significantly alters the chemical composition of cured tobacco leaves without affecting biomass. Thus, modulating the expression and / or activity of ASNs and NtLKRs in combination has the potential to rearrange the chemical composition of cured tobacco leaves (particularly the amino acid chemical composition of burley or dark tobacco), thereby altering the sensory properties.

[0237] In addition to ASN, other genes and enzymes also play a role in the reorganization of amino acids and / or sugars during leaf yellowing, such as diaminopimelate aminotransferase (DAPAT), which is involved in the catabolism and anabolism of lysine, and aspartate aminotransferase (AAT), which is expressed during senescence and has the potential to change the chemical composition of leaves after drying (WO2019 / 185703). Chloroplast sulfate transporters SULTR3 (such as NtSULTR3; 1A-S, NtSULTR3; 1A-T and NtSULTR3; 3-T) play a role in sugar and amino acid metabolism during drying (see WO2021 / 063863). Thus, additional genetic modifications may involve DAPAT and / or AAT (e.g., one or more of NtAATI-S, NtAAT1-T, NtAA T2-S, NtAAT2-T, NtAA T3-S, NtAAT3-T, NtAA T4-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 a combination of DAPAT and / or AAT and / or SULTR3 and NtLKR may have the potential to reorder the chemical composition of cured tobacco leaves, thereby altering sensory properties. Also disclosed are modifications of NtLKR and one or more, or two or more, or three or more, or four or more combinations of ASN and DAPAT and AAT and SULTR3, including 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 DAPAT 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 AAT and SULTR3.

[0238] One or more traits can be introgressed into a mutant, non-naturally occurring, or transgenic plant from another cultivar, or can be directly transformed therein.

[0239] Various embodiments provide mutant plants, non-naturally occurring plants or transgenic plants, and biomass, in which the expression level of one or more polynucleotides according to the present disclosure is modulated, thereby modulating the level of the polypeptide encoded thereby.

[0240] The parts of plants described herein, particularly the leaves and / or stalks and / or midribs of these plants, can be incorporated into or used to prepare various consumables, including but not limited to aerosol-forming materials, aerosol-forming devices, smoking articles, smokeable products, smokeless products, medicine or beauty products, intravenous preparations, tablets, powders and tobacco products. The example of aerosol-forming material comprises tobacco composition, tobacco, tobacco extract, shredded tobacco, cut filler, dried tobacco, expanded tobacco, homogenized tobacco, reconstituted tobacco and pipe tobacco. Smoking articles and smokeable products are types of aerosol-forming devices. Examples of smoking articles or smokeable products include cigarettes, cigarillos and cigars. The example of smokeless products includes chewing tobacco and snuff. In some aerosol-forming devices, rather than burning, tobacco composition or another aerosol-forming material is heated by one or more electric heating elements to produce aerosol. In another type of heated aerosol-forming device, an aerosol is generated by transferring heat from a combustible fuel element or heat source to a physically separate aerosol-forming material, which may be located within, around, or downstream of the heat source. Smokeless tobacco products and various tobacco-containing aerosol-forming materials may contain tobacco in any form, including dry particles, fragments, small particles, powders, or slurries deposited on, mixed in, surrounded by, or otherwise combined with other ingredients, which may take any form, such as flakes, films, tabs, foams, or beads. The term "smoke" is used to describe a type of aerosol produced by smoking articles such as cigarettes or by burning aerosol-forming materials.

[0241] In one embodiment, the present invention also provides dried plant material from mutant, transgenic and non-naturally occurring plants as described herein. Processes for drying green tobacco leaves are known to those skilled in the art and include, but are not limited to, air-curing, fire-curing, flue-curing and sun-curing as described herein.

[0242] In another embodiment, the present invention describes a tobacco product comprising an aerosol-forming material comprising tobacco, the aerosol-forming material comprising plant material, such as leaves, suitably dried leaves, from a mutant tobacco plant, a transgenic tobacco plant, or a non-naturally occurring tobacco plant as described herein. The tobacco product described herein can be a blended tobacco product that can also include unmodified tobacco.

[0243] Mutant, non-naturally occurring, or transgenic plants may have other uses, for example, in agriculture.

[0244] The disclosure also provides the method for producing seed, it comprises cultivating mutant plant as herein described, non-naturally occurring plant or transgenic plant, and collecting seed from the plant of cultivation.Seed from plant as herein described can be conditioned by mode as known in the art, and packaged in packaging material, to form manufacturing article.Packaging materials such as paper and cloth are well known in the art.The packaging of seed can carry the mark of describing the character of seed wherein, for example, be fixed to the label or mark of packaging material, be printed on the mark on the packaging.

[0245] Compositions, methods, and kits for genotyping plants for identification, selection, or breeding can include a means for detecting the presence of NtLKR polynucleotides in a sample of polynucleotides. Thus, a composition comprising 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 performing amplification or detection is described.

[0246] Accordingly, gene-specific oligonucleotide primers or probes are disclosed that comprise about 10 or more contiguous polynucleotides corresponding to the NtLKR polynucleotides described herein. The primers or probes may comprise or consist of about 15, 20, 25, 30, 40, 45, or 50 or more contiguous polynucleotides that hybridize (e.g., specifically hybridize) to one or more NtLKR polynucleotides described herein. In some embodiments, the primers or probes may comprise or consist of about 10 to 50 contiguous nucleotides, about 10 to 40 contiguous nucleotides, about 10 to 30 contiguous nucleotides, or about 15 to 30 contiguous nucleotides, which can be used in sequence-dependent methods for gene identification (e.g., Southern hybridization) or isolation (e.g., in situ hybridization of bacterial colonies or 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 specific examples, can use two kinds of primers to increase polynucleotide fragments in the PCR scheme.PCR also can use a kind of primer that derives from polynucleotide sequence and carry out with the second primer of polynucleotide sequence upstream or downstream sequence hybridization, and described polynucleotide sequence is such as 3 ' end of promoter sequence, mRNA precursor or the sequence that derives from carrier.The example that is used for the heat of in vitro amplification polynucleotide and isothermal technology is well known in the art.Sample can be or can be derived from plant, vegetable cell or vegetable material, or by plant as described herein, vegetable cell or vegetable material preparation or derivative tobacco product.

[0247] In another aspect, a method for detecting an NtLKR polynucleotide as described herein (or any combination thereof as described herein) in a sample is provided, the method comprising the steps of: (a) providing a sample comprising or suspected of comprising 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, wherein the presence of the amplification product indicates the presence of the NtLKR polynucleotide in the sample. In another aspect, use of one or more primers or probes for specifically detecting at least a portion of the NtLKR polynucleotide is provided. A kit for detecting at least a portion of the NtLKR polynucleotide is also provided, comprising one or more primers or probes for specifically detecting at least a portion of the NtLKR polynucleotide. The kit may comprise 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. Kits may include reagents and instructions for use.

[0248] In some embodiments, the kit may include instructions for use in one or more of the methods. The kit may be used for genetic identity determination, phylogenetic studies, genotyping, haplotype typing, pedigree analysis, or plant breeding, particularly co-dominance scoring.

[0249] The present disclosure also provides a method for genotyping plants, plant cells or plant materials comprising the NtLKR polynucleotides described herein. Genotyping provides a means of distinguishing homologs of chromosome pairs and can be used to distinguish isolates in a plant population. Molecular marker methods can be used for phylogenetic studies, characterizing genetic relationships between crop varieties, identifying hybrids or somatic hybrids, locating chromosome segments that affect single gene traits, map-based cloning and quantitative genetic studies. Specific methods for 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. Therefore, the present disclosure further provides methods for tracking the separation of one or more genes or polynucleotides and chromosomal sequences genetically linked to these genes or polynucleotides using techniques such as AFLP analysis.

[0250] Also disclosed herein are methods of producing liquid tobacco extracts and liquid tobacco extracts produced by one or more methods.

[0251] For tobacco starting material, select specific extraction temperature. Extraction temperature is selected in the range of about 100 degrees Celsius to about 160 degrees Celsius usually. Can optionally control the duration of heating step to provide certain control degree to the composition of the extract derived from tobacco starting material. Suitably, tobacco starting material is heated at least about 90 minutes under extraction temperature, more suitably at least about 120 minutes. Heating step is carried out in an inert atmosphere usually. Suitably, during heating step, make inert gas stream such as nitrogen flow through starting tobacco material. Volatile tobacco compound is released into inert gas stream during heating step, makes inert gas serve as the carrier of volatile component. The flow rate of inert gas stream can be at least about 25 liters / minute, more suitably at least about 30 liters / minute. Relatively high inert gas flow rate can advantageously improve the efficiency of extracting from tobacco starting material. Optionally, heating step can be carried out under vacuum. Suitable heating method for carrying out the heating of tobacco starting material is known to technicians and comprises: dry distillation, water distillation, vacuum distillation, flash distillation and thin film water distillation.

[0252] When collecting volatile compounds by absorption in a liquid solvent, the step of forming the liquid tobacco extract may comprise a solution of dry volatile compounds in a liquid solvent to concentrate the solution. Drying may be carried out using any suitable measure, including but not limited to controlled crystallization and filtration of dehydration, molecular sieves, freeze drying, phase separation, distillation, membrane osmosis, water, reverse moisture absorption, ultracentrifugation, liquid chromatography, reverse osmosis or chemical drying.

[0253] Liquid tobacco extract is particularly suitable for producing compositions or formulations or gel compositions for use in aerosol generating systems. An aerosol generating system is disclosed, comprising the composition or formulation or gel composition. In such aerosol generating system, the composition or formulation or gel is typically heated in an aerosol generating device to produce an aerosol, such as a device comprising a heater element that interacts with the composition or formulation or gel incorporating the liquid tobacco extract. During use, volatile compounds are released by heat transfer and entrained in the air drawn in by the aerosol generating device. When the released compounds cool, the compounds condense to form an aerosol that is inhaled by the consumer.

[0254] The invention is further described in the following examples, which are provided to describe the invention in more detail. These examples set forth preferred modes presently contemplated for carrying out the invention and are intended to illustrate but not to limit the invention.

[0255] Example

[0256] Example 1 - Materials and Methods

[0257] Plant materials

[0258] Before germination, the seeds were sterilized using a chlorine vapor method. A chlorine solution with a final concentration of 5% was placed in a bell-shaped container together with the seed glass tube. Hydrochloric acid (37%) was then added to the solution and the seeds were incubated for 2 hours. The seeds were then placed on Murashige & Skoog (Int J Mol Sci. (2020) 21 (10): 3441) growth medium under a laminar flow hood and transferred to a plant growth chamber (24°C, 16 hours of light / 20°C, 8 hours of darkness) for 4 weeks. Well-developed seedlings were transferred to a greenhouse and cultivated in 10L pots until fully grown (2 to 6 replicates). Artificial light was used for 16 hours per day. For each plant, a representative, fully grown leaf sample was collected at flowering. The collected samples were then freeze-dried and crushed in a container filled with glass beads at 400 rpm: leaves for 8 hours and roots for 24 hours. At this point, the roots that were still not broken were placed in a mortar and ground as finely as possible.

[0259] Methods for determining free amino acid, sugar, ammonia and nitrate levels

[0260] Amino acid content was determined using Method MP 1471 rev 5 2011, Resana, Italy: Chelab Silliker S.rl, Merieux NutriSciences Company. To determine amino acids in dried plant leaves, the dried leaves were dried at 40°C for 2-3 days after removing the midrib, if necessary. The tobacco material was then ground into a fine powder (-100 μM) before analysis for amino acid content. Alternatively, the amino acid content of plant material was measured as described in UNI EN ISO 13903:2005.

[0261] Reducing sugar content was measured using a segmented flow colorimetric method for analyzing tobacco samples developed by Skalar Instrument Co (West Chester, PA) and described in Tobacco Science 20:139-144 (1976). Reducing sugar content was also measured in accordance with Coresta Recommended Method 38, CRM38, CRM, and ISO 15154:2003. To determine reducing sugars in dried leaves, the dried leaves were dried at 40°C for 2-3 days after removing the midrib, if necessary. The tobacco material was then ground into a fine powder (-100 μM) before analyzing for reducing sugars. Alternatively, reducing sugar content was measured according to ISO 15154:2003.

[0262] Nitrate content was measured according to the manufacturer's protocol using a Lachet QuikChem 8500 instrument (Lachat QuikChem method 12-107-04-1-J, Lachet Instruments, Loveland, CO, USA). Alternatively, nitrate content was measured according to ISO 15517:2003.

[0263] Ammonia content was measured using ion chromatography according to ISO 21045:2018.

[0264] Gene expression analysis

[0265] By using Illumina Clarity LIMS( The sequencing data generated by Illumina, Inc. were split and subsequently imported into Qiagen CLC Genomics Workbench version 12.0.1 (CLC bio, QIAGEN Company). The transcriptome reads were mapped to the latest version of the tobacco reference genome (BMC Genomics (2017) 18 (1): 448) using the "RNA-Seq Analysis" 2.16 tool, using 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. Global alignment was not performed, and the pairing distance was automatically detected. The maximum number of read hits was set to 10, and the paired reads were counted as one. The gene expression FPKM values for each gene in the reference genome and those genes without transcript models were retrieved.

[0266] RNAi procedures

[0267] A specific DNA fragment (SEQ ID NO: 8) was selected to suppress the expression of both NtLKR-S and NtLKR-T. This fragment 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 variety TN90 and Virginia tobacco variety K326 were transformed using a standard Agrobacterium-mediated transformation protocol (Methods Mol Biol. (2006) 343:143-54 and Transgenic Res. (2013) 22(3):643-9). Seeds were harvested from three independent TO lines that exhibited the strongest NtLKR silencing. T1 plants from 10 lines were grown in the greenhouse, and lines harboring the construct insertion in their genomic DNA were selected 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 effective transcriptional repression, RNA was isolated from transgenic plants from 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 assess LKR gene expression levels.

[0268] Example 2 - Metabolomics Analysis

[0269] Metabolomic analysis revealed that levels of lysine, as well as catabolites produced by endogenous LKR activity, such as saccharoine, 2-aminoadipic acid, 2-ketoadipic acid, and pipecolic acid, increased in senescent tobacco leaves (48 hours after burley tobacco leaves yellowed during the drying process). Furthermore, transcriptomic analysis revealed that LKR genes involved in lysine degradation were strongly upregulated during the yellowing period. These data suggest that lysine accumulates early in the curing process.

[0270] like Figure 5 As shown, NtLKR is strongly induced during the early curing period, the so-called etiolation (a genetic program of aging, 0-192 h BU and 0-60 h FC).

[0271] Example 3 - Downregulation of NtLKR by RNAi silencing approach

[0272] NtLKR was downregulated by RNAi silencing to determine the chemical effects on dried leaves of two tobacco types, Burley and Virginia tobacco. Plants were grown in a greenhouse. Plants were cultivated and dried using traditional agronomic practices (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 (ed.) The Tobacco Plant Genome. Compendium of Plant Genomes. Springer, Cham), such as after harvesting mature leaves, Burley tobacco was air-dried and Virginia tobacco was flue-dried.

[0273] To investigate the function of the NtLKR gene and the possible accumulation of lysine in dried leaves when NtLKR (LKR / SDH) is downregulated, NtLKR-RNAi plants were generated using the insert of SEQ ID NO: 8 as an RNAi construct. No specific phenotype was observed when NtLKRT1-resistant plants were grown compared to wild-type plants. Several transformed lines were grown in T0, screened, and selected by RT-qPCR (using whole green midribs / leaves as tissue for RNA isolation). Seeds from plants showing significant downregulation of NtLKR were replanted (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 from two tobacco types, Burley TN90 and Virginia K326, for further analysis of free amino acids, sugars, ammonia, and nitrate contents.

[0274] Example 4 - Effect of NtLKR-Silenced Lines on Senescence in Burley Tobacco TN90 Background

[0275] NtLKR-RNAi Burley tobacco TN90 plants were very efficient in accumulating more lysine and some other amino acids (Arg), so the effect on etiolation in mid-stem leaves was tested (assuming that lysine is the energy source for etiolated leaves).

[0276] Control and T2-NtLKR-RNAi plants in the Burley tobacco TN90 background were randomly planted in the greenhouse. After 2 months of cultivation, all plants were topped and half of the culture was switched from Burley tobacco nutrient solution (EC = 2.4) to water (EC = 0.8). The other half of the culture remained in Burley tobacco nutrient solution.

[0277] The data show (see Figure 2 ) Chlorophyll measurements at the C stem position 21 days after nutrient solution change (CCI, 3 measurements per plant).

[0278] Chlorophyll measurements (CCI) at the mid-stem leaf position for the control (CT2-E438) and LRK-RNAi (T2-E438-5) are shown in Table 1. Plants were topped after 2 months of cultivation. At this time, half of the culture was switched to water (H2O, EC = 0.8) instead of nutrient solution (EC = 2.4). Chlorophyll (CCI) was recorded after 21 days. Figures show t-test p values.

[0279] Interestingly, the absence of both the S and T copies of the NtLKR gene induced a more rapid decrease in chlorophyll. This suggests that these plants senesced earlier than the control. Therefore, the accumulation of lysine contributes to accelerated leaf senescence.

[0280] Example 5 - Effect of NtLKR Downregulation (T1 Plants) on Chemical Components of Burley Tobacco TN90

[0281] Free amino acids, sugars, ammonia and nitrates were determined for dried leaves in control (WT, n=14) and NtLKR-RNAi (n=13) plants. No differences were found for sugars, ammonia and nitrates for the burley tobacco types. Table 2 depicts the mean values for individual amino acids in control and NtLKR-RNAi plants for fully dried tobacco, as well as the percentage / fold change and statistical correlation (p-value ANOVA). As expected, blocking the activity of NtLKR / SDH significantly increased lysine in the dried leaves compared to control leaves, by about 11-fold, thereby changing the chemical composition of the final material. The content of other amino acids changed significantly, but to a lesser extent than lysine, namely: Arg, Pro, GABA, Gln, Leu, Ala, Phe, Tyr, Ileu, Met, Thr and Gly (see Table 2). Interestingly, Met decreased threefold. This suggests that a strong rearrangement of free amino acids occurs in dried leaves compared to green leaves, which is mainly produced by the activity of aminotransferases during leaf etiolation (early drying), as shown in Bovet et al. (2019) Plants (Basel) 11;8(11):492. In addition to asparagine synthetase, which is actually an aminotransferase (WO2017042162), other enzymes also play a role in the rearrangement of amino acids during leaf etiolation, such as diaminopimelate aminotransferase (DAPAT), which is involved in both the catabolism and anabolism of lysine, and aspartate aminotransferase (AAT), which is also expressed during senescence and has the potential to change the chemical composition of leaves after drying (WO2019185703). Since photosynthesis is no longer active during leaf etiolation, the only way for early senescent leaves to remobilize nitrogen resources is to transfer amino groups into the energy machinery (such as mitochondria) to produce reducing equivalents (NADPH or NADH) or to produce cytoplasmic lysine (see Figure 2 ) and asparagine (Bovet et al. (2019) Plants (Basel) 11; 8(11): 492) (for nitrogen storage in seeds). The balance of free amino acids in dried leaves was altered by blocking NtLKR activity and accumulating lysine.

[0282] like Figure 3 As shown, in three different independent RNAi lines, the increase in lysine (>11-fold) was completely consistent with the silencing of NtLKR (see Figure 1). This also confirms the activity of Nitab09g012060.1.1 and Nitab18g020540.1.1 as LKR enzymes (LKR-T and LKR-S, respectively). Although the content of Lys in the green leaves of the NtLKR-RNAi strains was not measured, it is likely that the accumulation of lysine in the dried leaves is related to the aging process within the framework of the expression characteristics of NtLKR-S and NtLKR-T, which act as aging activation genes. When the stems naturally turn yellow, we can assume that lysine and asparagine are then remobilized and transported to the seeds through the phloem. Although the increase in arginine and proline is small, it is also quite obvious in three independent strains, which is a result of NtLKR inactivation.

[0283] Example 6 - Effect of NtLKR downregulation (T1 plants) on the chemical composition of Nicotiana virginiana K326

[0284] Free amino acids, sugars, ammonia, and nitrates were analyzed in dried leaves of control (WT, n=8) and NtLKR-RNAi (n=13) tobacco plants. Compared to burley tobacco, the virginiana type requires less nitrogen fertilizer and accumulates less free nitrate, ammonia, and free amino acids in the dried leaves, but more reducing sugars. Tables 3 and 4 depict the mean values of free amino acids, sugars, ammonia, and nitrates in both control and NtLKR-RNAi plants for fully cured tobacco, as well as the percentage / fold change and statistical correlation (p-value, analysis of variance). As expected, lysine was also significantly increased in virginiana by blocking NtLKR / SDH activity, similar to the dried leaves of burley tobacco, but to a lesser extent, with a fold increase of approximately 4-fold.

[0285] This 4-fold increase in lysine in Virginia tobacco was accompanied by significant changes in other free amino acids, reducing sugars, and nitrate. The other amino acids whose contents changed were: Arg, Gln, His, Tyr, Trp, Thr, GABA, Asn, Ala, Ileu, Val, and Ser (see Table 3). Considering that the total amino acid content in Virginia tobacco is approximately one-tenth that of Burley tobacco (compare Tables 2 and 3), silencing the NtLKR gene had a relatively weak effect on the amino acid content of Virginia tobacco leaves. However, compared to the WT, glucose and fructose were significantly reduced by 20%, and nitrate was reduced by 34%. Regarding nitrate, this value is so low that it is difficult to conclude that it is related to the lack of active NtLKR / SDH. On the other hand, the 20% reduction in each of the major reducing sugars is quite consistent. In fact, when evaluating both glucose and fructose, the total amount of reducing sugars was equivalent to 17.04 in the WT and 13.49 in the NtLKR-RNAi line, resulting in a reduction of reducing sugars of approximately 3.5% in the transgenic line. No changes in ammonia levels were observed, nor in burley tobacco (where the gene was originally targeted).

[0286] Figure 4 The data presented show that in three different independent RNAi lines, a significant increase in lysine (>4-fold) is fully consistent with silencing of NtLKR (see Figure 2 ). This also confirms the activity of Nitab09g012060.1.1 and Nitab18g020540.1.1 as LKR enzymes (LKR-T and LKR-S, respectively). Although the increase in arginine and glutamine was small, it was also significant in three independent lines, which was the result of NtLKR inactivation. The overall reduction in glucose and fructose in Virginia tobacco NtLKR-RNAi plants was 20%, and even reached 30% in the E437-12 line. This reduction may be due to increased carbon respiration or assimilation of original amino acids in green leaves, or reduced accumulation of starch in chloroplasts. Obviously, the reduction in NtLKR activity has little effect on the biological functions of nutritional plants and will not affect the adaptability and growth of transgenic plants, but it will become apparent once the aging process is entered.

[0287] We observed that when NtLKR was downregulated, lysine increased, while methionine and threonine decreased (especially in the nitrogen-accumulating tobacco type Burley), while glucose, a mitochondrial energy source, decreased in the high-reducing sugar-accumulating Virginia tobacco.

[0288] Example 7 - Sensory Effects in RNAi-Modified Burley Tobacco TN90 Plants

[0289] The altered sensory characteristics are provided in Table 5. The accumulation of lysine in the modified Burley TN90 contributes to reduced harshness, making the aerosol rounder and smoother, with less of the typical dark flavor, but more animalic and nutty (less trigeminal impact).

[0290] Example 8 - Effects on sensory organs in RNAi-modified Nicotiana virginiana K326 plants

[0291] The altered sensory profile is provided in Table 6 and contributes to an increased hay note with a rounder, darker flavor profile.

[0292] Any publication cited or described in this article provides relevant information disclosed before the filing date of this application. Statements in this article should not be interpreted as admitting that the inventor loses the qualification prior to such disclosure. All publications mentioned in the above specification are incorporated herein by reference. Without departing from the scope and spirit of the present invention, various modifications and variations of the present invention will be apparent to those skilled in the art. Although the present invention has been described in conjunction with specific preferred embodiments, it should be understood that the present invention as required should not be unduly limited to such specific embodiments. In fact, different improvements to the described modes for realizing the present invention that are apparent to those skilled in the art of cell biology, molecular biology and plant biology or related fields are intended to be within the scope of the following claims.

[0293] sequence

[0294] SEQ ID NO: 1 Polynucleotide coding sequence of LKR-S from tobacco

[0295]

[0296] The polypeptide sequence of LKR-S from tobacco, SEQ ID NO:2

[0297] MTDLLKFGREILGPVIMFGNGVVGILSEATNKWERRAPLTPSHCARLLHGGRGKTGVSRIIMQPSTKRVHHDALYEDVGCEISEDLSDCGLILGIKQPKLEMILPDRAYAFFSHTHKAQKENMPLLDKILAERASLFDYELIVGDTGKRLLAFGSFAGRAGMIDFLRGLGLWYLNHGYSTPFLSLGSSYMYSSLAAAKAAVISVGEEIATMGLPAGICPLVFVFTGSGNVSRGAQEIFKLLPHTFVDPRKLSELHETARDLTQSKHPSKRIFQVYGCVTTCQDMVEHLNPSKSFDKIDYYAHPEQYRPAFHEKIAPYVSVIVNCMYWEKRFPRLLTTKQIQDLMRNGCPLVGICDITCDVGGSIEFINQTSSIDSPFFRYEPSNDSYHYDIEGKGVMCSAVDILPTEFAKEASQHFGDILSHFTGSLASFRNLEELPAHLKRACIAHHGALTQLYEYIPRMRKSDLEDPSTVLSSSNANGRKYTVLVSLSGHLFDKFLINEALDIIEAAGGSFHLVKCQVGQITSALSYSELEVGAEDKAVLDKIVDSLTSLANSRNSLGSQNKENNMISLKVGEFQQSIIDEKSDAKKVLILGAGRVCRPAAELLASIGSMSSGQWLSSITADFEEQHCVQVIVASLYLKDAEEVTEGIPNAKAVQLDIMNHESLSSCISQVDVVISLLPPSCHGIVAKSCIELKKHLVTASYVNDSMLKLDEDAKCAGITILGEMGLDPGIVTLIRVSMNLLENSSWYEISSECDCA

[0298] The polynucleotide coding sequence of LKR-T from tobacco, SEQ ID NO:3

[0299]

[0300] SEQ ID NO:4 polypeptide sequence of LKR-T from tobacco

[0301]

[0302] SEQ ID NO:5 NtLKR-RNAi insert:

[0303] caggttgatgttgtcatcagcttactgcctcctagttgccatggtattgtagcaaaatcatgcattgagctgaagaaacatcttgtcacagctagctacgttaatgattc

[0304] SEQ ID NO:6 NtLKR-F1 primer for qPCR

[0305] atattattgaagcagcaggtggc

[0306] SEQ ID NO:7 NtLKR-R1 primer for qPCR

[0307] tgctttatcttcagctccaacct

[0308] SEQ ID NO: 8 DNA fragment for inhibiting the expression of NtLKR-S and NtLKR-T

[0309] Caggttgatgttgtcatcagcttactgcctcctagttgccatggtattgtagcaaaatcatgcattgagctgaagaaacatcttgtcacagctagctacgttaatgattc

[0310] SEQ ID NO:9MMV-F primer

[0311] gacgtctaatcccaacttcgtc

[0312] SEQ ID NO: 10IPMS2-R primer

[0313] Gacgtctaatcccaacttcgtc

[0314] Table 1

[0315] Chlorophyll measurements (CCI) at the mid-stem leaf position of the control (CT2-E438) and LRK-RNAi (T2-E438-5)

[0316] Plant ID Plant n= Average (CCI) SD CT2-E438 10 31.54 ±4.32 CT2-E438-H20 10 32.23 ±2.80 T2-E438-5 7 37.79 ±3.81 T2-E438-5-H20 7 28 40 ±4.15

[0317] Table 2

[0318] Free amino acid content in control and LKR-RNAi Burley tobacco plants (mean of data from individual lines (WT n=14 and LKR-RNAi, n=13)) as well as percentage / fold change and statistical correlation (p-value). Statistical analysis was performed using ANOVA and Tukey's HSD test. 'LKR-RNAi' results are for fully cured Burley tobacco leaves.

[0319]

[0320] Table 3

[0321] Free amino acid content in control and LKR-RNAi N. virginiana plants (mean of data from individual lines (WT n=8 and LKR-RNAi, n=13)) as well as percentage / fold change and statistical correlation (p-value, ANOVA Tukey HSD test). 'LKR-RNAi' results are for whole-cured N. virginiana leaves.

[0322]

[0323] Table 4

[0324] Sugar, ammonia, and nitrate contents in control and LKR-RNAi N. virginiana plants (mean of data from individual lines (WT n=8 and LKR-RNAi, n=13)) as well as percentage / fold change and statistical correlation (p-value, ANOVA Tukey HSD test). 'LKR-RNAi' results are for fully dried N. virginiana leaves.

[0325]

[0326] Table 5

[0327] Sensory Analysis of RNAi-Modified Burley Tobacco Variety TN90 Plants

[0328]

[0329] Table 6

[0330] Sensory analysis of RNAi-modified Nicotiana virginiana K326 plants

[0331]

Claims

1. A mutant, non-naturally occurring or transgenic tobacco plant or part thereof having modulated expression or activity of a lysine ketoglutarate reductase (LKR), said LKR comprising, consisting of or consisting essentially of: (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 a sequence having at least 86% sequence identity to SEQ ID NO: 3 (NtLKR-T); (ii) a polypeptide encoded by the polynucleotide shown 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 a sequence having at least 88% sequence identity to SEQ ID NO: 4 (NtLKR-T); or (iv) a construct, vector or expression vector comprising the isolated polynucleotide shown 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, as 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 mutant, non-naturally occurring or transgenic tobacco plant or part thereof according to claim 1, wherein the modification comprises at least one genetic alteration in the coding sequence of the polynucleotide or in the regulatory region of the polynucleotide; and / or wherein the modification comprises one or more of exogenous DNA or exogenous RNA; and / or wherein the modification comprises one or more of a vector or a viral vector or an Agrobacterium vector or a CRISPR vector; and / or wherein the modification is capable of driving one or more of RNA interference or transcriptional gene silencing or viral-induced gene silencing; and / or wherein the modification is capable of expressing one or more of double-stranded RNA (dsRNA) or hairpin RNA (hpRNA) or small interfering RNA.

3. A mutant, non-naturally occurring or transgenic tobacco plant or part thereof according to claim 1 or claim 2, wherein the modulated expression or activity of LKR is capable of modulating 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 the control plant, suitably wherein the modulated expression or activity of LKR is capable of modulating the timing of leaf senescence; suitably, wherein the amino acid is lysine.

4. The mutant, non-naturally occurring or transgenic tobacco plant or part thereof according to any one of the preceding claims, wherein the part of the mutant, non-naturally occurring or transgenic tobacco plant is a cured or dried leaf; suitably, wherein the levels of at least lysine, arginine, GABA, glutamine, alanine, tyrosine, isoleucine and threonine are modulated in said dried or desiccated leaves compared to dried or desiccated leaves from said control plants.

5. A mutant, non-naturally occurring or transgenic tobacco plant or part thereof according to any one of the preceding claims, wherein the tobacco plant or part thereof is of the Burley type; suitably, wherein the levels of at least lysine, arginine, proline, GABA, glutamine, leucine, alanine, phenylalanine, tyrosine, isoleucine, methionine, threonine and glycine are modulated, and the levels of at least asparagine, aspartic acid, tryptophan, histidine, glutamate, serine and valine are not significantly changed in the dried or desiccated leaves compared to the dried or desiccated leaves from the control plant; and / or wherein the expression of an LKR polynucleotide or the activity of an LKR polypeptide is reduced or inhibited in the dried or dried leaves as compared to dried or desiccated leaves from the control plant, and wherein in the dried or desiccated leaves: (i) the levels of at least lysine, arginine, proline, GABA, glutamine, leucine, alanine, phenylalanine, tyrosine and isoleucine are increased; and (ii) the levels of at least methionine, threonine and glycine are reduced; and (iii) the levels of at least asparagine, aspartic acid, tryptophan, histidine, glutamate, serine and valine are not significantly changed.

6. A mutant, non-naturally occurring or transgenic tobacco plant or part thereof according to any one of claims 1 to 4, wherein the tobacco plant or part thereof is of the Nicotiana virginiana type; suitably, wherein the levels of at least lysine, arginine, glutamine, histidine, tyrosine, tryptophan, threonine, GABA, asparagine, alanine, isoleucine, valine and serine are modulated, and the levels of at least proline, aspartic acid, leucine, phenylalanine, glutamate and methionine are not significantly changed in the dried or dried leaves compared to the dried or dried leaves from the control plants.

7. The mutant, non-naturally occurring or transgenic tobacco plant or part thereof of claim 6, wherein expression of the LKR polynucleotide or activity of the LKR polypeptide is reduced or inhibited in the dried or dried leaves compared to dried or dried leaves from the control plant, and wherein in the dried or dried leaves: (i) the levels of at least lysine, arginine, glutamine, histidine, tyrosine, tryptophan, threonine, GABA, asparagine and alanine are increased; and (ii) the levels of at least isoleucine, valine and serine are reduced; and (iii) the levels of at least proline, aspartic acid, leucine, phenylalanine, glutamate and methionine are not significantly changed.

8. A mutant, non-naturally occurring or transgenic tobacco plant or part thereof according to claim 6 or claim 7, wherein the total amount of sugars is modulated; suitably, wherein the total amount of sugars is reduced.

9. Tobacco plant material, dried tobacco plant material or homogenised tobacco plant material, derived from or obtained from a tobacco plant or part thereof according to any one of claims 1 to 8; suitably, wherein the tobacco plant material is selected from the group consisting of: biomass, seeds, stems, flowers or leaves or a combination of two or more thereof; suitably, wherein said tobacco plant material is leaves; suitably, wherein said leaves are dried leaves; suitably, The dried leaves are selected from the group consisting of flue-dried leaves, sun-dried leaves or air-dried leaves.

10. A method for producing a tobacco plant in which the level of at least one amino acid is modulated, comprising: (a) providing a tobacco 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 a sequence having at least 86% sequence identity to SEQ ID NO: 3 (NtLKR-T); (ii) a polypeptide encoded by the polynucleotide shown 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 a sequence having at least 88% sequence identity to SEQ ID NO: 4 (NtLKR-T); or (iv) a construct, vector or expression vector comprising the isolated polynucleotide shown in (i); as well as (b) introducing at least one modification that modulates: (a) the expression of the NtLKR polynucleotide in the tobacco plant; or (b) the activity of the NtLKR polypeptide in the tobacco plant, as compared to a control in which the expression of the NtLKR polynucleotide or the activity of the NtLKR polypeptide is not modified.

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

12. A tobacco plant obtained or obtainable by the method according to claim 10 or claim 11.

13. A method of producing cured tobacco plant material having altered levels of at least one amino acid, comprising: (a) producing a tobacco plant according to claim 10 or claim 11; (b) harvesting plant material (e.g., leaves) from the tobacco plant; and (c) drying the plant material.

14. Dried tobacco plant material (e.g. leaves) obtained or obtainable by the method according to claim 13.

15. A tobacco product comprising tobacco plant material, dried tobacco plant material or homogenised tobacco plant material according to claim 9, or comprising dried tobacco plant material according to claim 14; suitably, wherein the tobacco product is a tobacco blend; suitably, The tobacco blend comprises Virginia-type tobacco and / or Burley-type tobacco.

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