Regulation of sugar transporters

Abstract

Disclosed is a mutant, non-naturally occurring, or transgenic Nicotiana tabacum plant comprising at least one modification capable of modulating the expression or activity of one or more of SWEET12-S or SWEET12-T or SWEET15-S or SWEET15-T, wherein the plant or portion thereof comprises at least one modification capable of modulating (a) the expression of a polynucleotide in the plant or portion thereof, or (b) the activity of a polypeptide in the plant or portion thereof, compared to a control Nicotiana tabacum plant or portion thereof, and wherein expression or activity of one or more of SWEET12-S or SWEET12-T or SWEET15-S or SWEET15-T is modulated compared to the control Nicotiana tabacum plant.

Description

[Technical Field]

[0001] The present invention generally relates to plants in which the expression or activity of SWEET (transporters that ultimately export sugars) is modulated. [Background technology]

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

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

[0004] The present invention is based, at least in part, on the discovery that modulating the expression or activity of certain SWEETs can modify the chemical profile of plant parts, such as leaves, during curing. This can alter the aroma or sensory profile of plant products, such as cured or cured tobacco leaves. Modulating the expression or activity of certain SWEETs can affect plant growth and development. Thus, modulating the expression or activity of certain SWEETs can further regulate leaf yield and / or plant maturation. In addition to being induced during leaf curing, the SWEETs described herein may also be important regulators of reproductive growth and development. The discovery of these functions is unexpected, given the low expression levels of these specific SWEETs in non-cured tissues. Without being bound by any particular theory, it is possible that gene expression of these specific SWEETs is induced at developmental stages different from those investigated herein, or that their regulation is uncoupled from gene expression.

[0005] In one embodiment, (i) a SWEET12-S polynucleotide sequence comprising, consisting of, or consisting essentially of a sequence having at least 70% sequence identity to SEQ ID NO: 1, or (ii) a SWEET12-T polynucleotide sequence comprising, consisting of, or consisting essentially of a sequence having at least 70% sequence identity to SEQ ID NO: 3, or (iii) a SWEET15-S polynucleotide sequence comprising, consisting of, or consisting essentially of a sequence having at least 70% sequence identity to SEQ ID NO: 5, or (iv) a SWEET15-T polynucleotide sequence comprising, consisting of, or consisting essentially of a sequence having at least 70% sequence identity to SEQ ID NO: 7, and or (v) a polypeptide encoded by a polynucleotide set forth in (i) or (ii) or (iii) or (iv), or (vi) a SWEET12-S polypeptide having at least 97% sequence identity to SEQ ID NO:2, or (vii) a SWEET12-T polypeptide having at least 93% sequence identity to SEQ ID NO:4, or (viii) a SWEET15-S polypeptide having at least 97% sequence identity to SEQ ID NO:6, or (ix) a SWEET15-T polypeptide having at least 97% sequence identity to SEQ ID NO:8, comprising at least one modification capable of modulating the expression or activity of one or more of SWEET12-S or SWEET12-T or SWEET15-S or SWEET15-T, comprising, consisting of, or consisting essentially ofand (b) an activity of a polypeptide in the plant or portion thereof, compared to a control plant or portion thereof in which the expression of the polynucleotide or activity of the polypeptide is not altered, wherein expression or activity of one or more of SWEET12-S or SWEET12-T or SWEET15-S or SWEET15-T is modulated compared to a control Nicotiana tabacum plant in which the expression or activity of one or more of SWEET12-S or SWEET12-T or SWEET15-S or SWEET15-T is not modulated.

[0006] Suitably, the Nicotiana tabacum plant comprises at least one genetic alteration in the regulatory region or in the coding sequence of one or more of SWEET12-S or SWEET12-T or SWEET15-S or SWEET15-T.

[0007] Preferably, the modification comprises one or more of exogenous DNA or exogenous RNA.

[0008] Preferably, the modification comprises one or more of a vector, or a viral vector, or an Agrobacterium vector, or a CRISPR vector.

[0009] Suitably, the modification is capable of driving one or more of RNA interference or transcriptional gene silencing, or virus-induced gene silencing.

[0010] Preferably, the modification is capable of expressing one or more of double-stranded RNA (dsRNA) or hairpin RNA (hpRNA) or small interfering RNA.

[0011] Preferably, the modification is capable of constitutively expressing one or more of SWEET12-S or SWEET12-T or SWEET15-S or SWEET15-T.

[0012] Suitably, the development of the vegetative stage of the Nicotiana tabacum plant is modulated compared to a control Nicotiana tabacum plant and / or the flowering time of the plant is modulated compared to a control Nicotiana tabacum plant.

[0013] Preferably, compared to a control Nicotiana tabacum plant, the expression and / or activity of SWEET12-S and SWEET12-T or SWEET15-S and SWEET15-T or SWEET12-S and SWEET12-T and SWEET15-S and SWEET15-T is reduced, the plant height is increased during the vegetative growth phase, and / or flowering time is earlier.

[0014] Preferably, the expression and / or activity of SWEET15-T is increased and flowering time is accelerated compared to control Nicotiana tabacum plants.

[0015] Preferably, the part of the mutant, non-native or transgenic Nicotiana tabacum plant is a cured or dried leaf.

[0016] Preferably, the chemical profile of the dried or desiccated leaves is modulated compared to dried or desiccated leaves from a control Nicotiana tabacum plant, and preferably the chemical profile is a sugar profile and / or an amino acid profile.

[0017] Suitably, the expression and / or activity of SWEET12-S or SWEET15-T is increased and at least the fructose, glucose and sucrose content is decreased compared to desiccation-treated or desiccant leaves from a control Nicotiana tabacum plant.

[0018] Suitably, the expression and / or activity of SWEET15-S and SWEET15-T is reduced and at least the fructose, glucose and sucrose content is reduced compared to desiccation-treated or desiccant leaves from a control Nicotiana tabacum plant.

[0019] Preferably, the expression and / or activity of SWEET12-S is increased, the asparagine, tryptophan, phenylalanine, glycine, and methionine content is increased, and the glutamic acid and proline content is decreased compared to desiccation-treated or desiccant leaves from a control Nicotiana tabacum plant.

[0020] Suitably, the ammonia content is increased compared to dried treated or dried leaves from a control Nicotiana tabacum plant.

[0021] Suitably, the expression and / or activity of SWEET15-T is increased, the asparagine and tryptophan content is increased, and the prophosphate content is decreased compared to desiccation-treated or desiccant leaves from a control Nicotiana tabacum plant.

[0022] Preferably, the plant is a Nicotiana tabacum plant, more preferably a Virginia or Burley species.

[0023] Also disclosed is plant material, dried or desiccated plant material, or homogenized plant material derived from or obtained from a Nicotiana tabacum plant or part thereof, preferably the plant material is selected from the group consisting of biomass, seeds, stems, flowers, or leaves, or a combination of two or more thereof, preferably the plant material is leaves, preferably the leaves are dried or desiccated leaves, preferably the dried leaves are selected from the group consisting of hot-tube dried leaves, sun dried leaves, or air dried leaves.

[0024] Also disclosed are methods for preparing Nicotiana tabacum plants with modulated flowering time and / or modulated amino acid levels and / or modulated sugar levels, the methods comprising: (a) producing a polynucleotide sequence comprising (i) a SWEET12-S polynucleotide sequence comprising, consisting of, or consisting essentially of a sequence having at least 70% sequence identity to SEQ ID NO: 1; or (ii) a SWEET12-T polynucleotide sequence comprising, consisting of, or consisting essentially of a sequence having at least 70% sequence identity to SEQ ID NO: 3; or (iii) a SWEET15-S polynucleotide sequence comprising, consisting of, or consisting essentially of a sequence having at least 70% sequence identity to SEQ ID NO: 5; or (iv) a SWEET15-T polynucleotide sequence comprising, consisting of, or consisting essentially of a sequence having at least 70% sequence identity to SEQ ID NO: 7; or (v) a polynucleotide sequence comprising, consisting of, or consisting essentially of a sequence having at least 70% sequence identity to SEQ ID NO: 8; (iii) or (iv), or (vi) a SWEET12-S polypeptide having at least 97% sequence identity to SEQ ID NO: 2, or (vii) a SWEET12-T polypeptide having at least 93% sequence identity to SEQ ID NO: 4, or (viii) a SWEET15-S polypeptide having at least 97% sequence identity to SEQ ID NO: 6, or (ix) a SWEET15-T polypeptide having at least 97% sequence identity to SEQ ID NO: 8, wherein the modification modulates expression or activity compared to a control Nicotiana tabacum plant in which expression of the same one or more of SWEET12-S, SWEET12-T, SWEET15-S, or SWEET15-T is not modified.

[0025] Also disclosed are methods for preparing Nicotiana tabacum plants with modulated flowering time and / or modulated amino acid levels and / or modulated sugar levels, the methods comprising producing (a) (i) a SWEET12-S polynucleotide sequence comprising, consisting of, or consisting essentially of a sequence having at least 70% sequence identity to SEQ ID NO:1, or (ii) a SWEET12-T polynucleotide sequence comprising, consisting of, or consisting essentially of a sequence having at least 70% sequence identity to SEQ ID NO:3, or (iii) a SWEET15-S polynucleotide sequence comprising, consisting of, or consisting essentially of a sequence having at least 70% sequence identity to SEQ ID NO:5, or (iv) a SWEET15-T polynucleotide sequence comprising, consisting of, or consisting essentially of a sequence having at least 70% sequence identity to SEQ ID NO:7, or (v) a polynucleotide sequence comprising, consisting of, or consisting essentially of a sequence having at least 70% sequence identity to SEQ ID NO:7, or or a polypeptide encoded by a polynucleotide described in (iii) or (iv), or (vi) a SWEET12-S polypeptide having at least 97% sequence identity to SEQ ID NO:2, or (vii) a SWEET12-T polypeptide having at least 93% sequence identity to SEQ ID NO:4, or (viii) a SWEET15-S polypeptide having at least 97% sequence identity to SEQ ID NO:6, or (ix) a SWEET15-T polypeptide having at least 97% sequence identity to SEQ ID NO:8, (b) (i) expression of one or more of SWEET12-S or SWEET12-T or SWEET15-S or SWEET15-T in a Nicotiana tabacum plant, or (ii) expression of the same one or more of SWEET12-S or SWEET12-T or SWEET15-S or SWEET15-T in a Nicotiana tabacum plant, compared to a control in which the expression of the same one or more of SWEET12-S or SWEET12-T or SWEET15-S or SWEET15-T is not altered.The method includes introducing at least one modification capable of modulating the activity of one or more of SWEET12-S or SWEET12-T or SWEET15-S or SWEET15-T in a tabacum plant.

[0026] Preferably, in step (b), the at least one modification is introduced by genome editing, preferably the genome editing is selected from CRISPR-mediated genome editing, mutagenesis, zinc finger nuclease-mediated mutagenesis, chemical or radiation mutagenesis, homologous recombination, oligonucleotide-directed mutagenesis, and meganuclease-mediated mutagenesis, or in step (b), the at least one modification is introduced using an interfering polynucleotide, or in step (b), the at least one modification is a promoter located 5' to the polynucleotide.

[0027] Also disclosed are plants obtained or obtainable by this method.

[0028] Also disclosed is a method for producing dried or dehydrated Nicotiana tabacum plant material having modulated amino acid levels and / or modulated sugar levels, the method comprising: (a) preparing or providing the Nicotiana tabacum plant described above; (b) harvesting plant material (e.g., leaves) from the plant; and (c) dehydrating or drying the plant material.

[0029] Dried or dried plant material (eg, leaves) obtained or obtainable by the method is also disclosed.

[0030] Also disclosed are plant products comprising plant material, dried or dehydrated plant material, or homogenized plant material, or plant products comprising dried or dehydrated plant material.

[0031] Preferably, the plant product is a tobacco product derived from Nicotiana tabacum plant material.

[0032] Preferably, the tobacco product is a tobacco blend, and preferably the tobacco blend comprises Virginia and / or Burley tobacco.

[0033] Some advantages

[0034] Modulating the expression and / or activity of certain SWEETs described herein can modulate the levels of amino acids, particularly in curing processes or dried plant materials, which can result in tobacco with novel aroma and / or sensory characteristics.

[0035] Modulating the expression and / or activity of certain SWEETs described herein can modulate sugar levels, particularly in curing processes or dried plant materials, which can result in tobacco with novel aroma and / or sensory characteristics.

[0036] Modulating the expression and / or activity of certain SWEETs described herein can result in plants that flower earlier, which can shorten the growing season and allow for more rapid introduction of new traits through breeding, which can result in reduced costs for commercial plant production.

[0037] Advantageously, non-genetically modified plants can be produced that may be more acceptable to consumers.

[0038] Advantageously, the present disclosure is not limited to the use of EMS mutant plants. EMS mutant plants may be less likely to bring improved characteristics to crops after breeding. Once breeding begins, the desirable characteristics of EMS mutant plants may be lost for various reasons. For example, several mutations may be required, the mutations may be dominant or recessive, and identifying point mutations in gene targets may be difficult to achieve. In contrast, the present disclosure utilizes the use of SWEET, which can be specifically engineered to produce plants with desired phenotypes.

[0039] Downregulation of the asparagine synthetase (ASN) gene (WO2017042162; Bovet et al. (2019) Plants (Basel) 11;8(11):492) also significantly alters tobacco chemistry without affecting biomass. Advantageously, combining both ASN-regulated plants with NtSWEET-regulated plants could potentially reconfigure the chemistry, such as the amino acid chemistry, of Burley, Dark, or Virginia tobacco, further altering flavor and / or sensory characteristics. Other genes and enzymes also play a role in amino acid and / or sugar reorganization during leaf yellowing, such as diaminopimelate aminotransferase (DAPAT), aspartate aminotransferase (AAT), and the chloroplast sulfate transporter (SULTR3), which may also alter leaf chemistry. Altering NtSWEET expression or activity in conjunction with the expression or activity of one or more of these other targets selected from one or more of ASN, DAPAT, and AAT may be used to further modify the flavor and / or sensory properties of flue-cured or cured tobacco. [Brief explanation of the drawings]

[0040] [Figure 1]Figure 1 is a series of four graphs (a)–(d) showing RNA-seq gene expression profiles of SWEET genes during the curing time course of tube-cured tobacco (Nicotiana tabacum L.). The graphs show the expression profiles of (a) SWEET12-S, (b) SWEET12-T, (c) SWEET15-S, and (d) SWEET15-T in leaves detached from petioles X, C, B, and T before (0 h) and during (24 h, 48 h, and 72 h) the curing treatment. Leaf positions X, C, B, and T in the petiole are from bottom to top of the plant, respectively. The left y-axis shows FPKM (fragments per kilobase of transcript per million mapped reads). The x-axis shows the curing time course. The legend indicates the petiole positions X (dashed, gray), C (dashed, black), B (dotted, gray), and T (dotted, black). Data are collected from at least three biological replicates (n = 3). Each biological replicate is a pool of four leaves harvested from five different plants and inserted at the same petiole position on the plant (n = 20 leaves). [Figure 2] Figure 2 is a series of four graphs (a)–(d) showing RNA-seq gene expression profiles of SWEET genes in the leaf blade and midrib tissues of tube-cured tobacco (Nicotiana tabacum L.). The graphs show the expression profiles of (a) SWEET12-S, (b) SWEET12-T, (c) SWEET15-S, and (d) SWEET15-T in the leaf blade (dashed black line) and midrib (dashed gray line) tissues before (0 h) and during (6 h, 24 h, 48 h, 55 h, and 72 h) the curing treatment. The left y-axis shows FPKM (fragments per kilobase of transcript per million mapped reads). The x-axis shows the curing time course. The legend indicates the harvested tissue, leaf position B–C within the pool. Data are collected from at least three biological replicates (n=3). Each biological replicate is a pool of five plants. [Figure 3]Figure 3 is a series of two graphs showing relative SWEET gene expression in flue-cured tobacco (Nicotiana tabacum L.) leaves from independent T1 plants. (a) Quantification of relative SWEET12-S transcript levels in each individual T1 plant shows that SWEET12-S expression is higher in p35s:SWEET12-S compared to WT control plants and lower in RNAi-SWEET12-S. (b) Quantification of relative SWEET15-T transcript levels in each individual T1 plant shows that SWEET15-T expression is higher in p35s:SWEET15-T compared to WT control plants and lower in RNAi-SWEET15-T / -S. Results represent relative mRNA expression in mature leaves isolated from petiole position C and cured for 48 hours. PQ14 expression was used to normalize data for variation. Data are summarized in bar graphs, with each bar representing the gene expression level of an individual T1 plant. [Figure 4] Figure 4 is a series of photographs showing differences in growth and development of individual SWEET T1 lines of flue-cured tobacco (Nicotiana tabacum L.). (a, b, and c) Representative images of 10-week-old plants grown under standard flue-cured and fertilized conditions. Differences in the height of individual T1 plants are observed. Images (b) and (c) show that the RNAi lines RNAi-SWEET12-S and RNAi-SWEET15-T / -S are taller than the control plants (WT), while image (a) shows that the p35s:SWEET12-S lines are shorter than their respective control plants. No differences were observed in the growth and development of p35s:SWEET15-T plants compared to WT plants (not shown). [Figure 5] Figure 5 shows two graphs showing the flowering time of flue-cured tobacco (Nicotiana tabacum L.) SWEET T1 lines. Flowering time is assessed on 15-week-old plants grown under standard flue-cured fertilization conditions. Flowering time is quantified as the percentage of plants with flower buds, open flowers, or no inflorescences at all. Data are collected from at least 10 individual plants. These data are summarized in stacked bar graphs. [Figure 6] Figure 6 is a series of graphs showing leaf biomass of tube-cured tobacco (Nicotiana tabacum L.) SWEET T1 lines. Fresh leaf biomass from 18-week-old plants grown under standard tube-cured fertilization conditions. Results represent the average wet weight (C position) of leaves separated from the petiole. Data are collected from at least three biological replicates. Data are summarized in bar graphs, with bars representing the mean wet weight in grams (g) and error bars representing the standard deviation. (a) p35s:SWEET12-S plants have significantly smaller leaves than WT and RNAi-SWEET12-S plants (Student t-test, *P<0.05). (b) p35s:SWEET15-T plants have significantly smaller leaves than WT and RNAi-SWEET15-T plants (Student t-test, **P<0.01). [Figure 7-1] Figure 7 is a series of graphs showing the difference in sugar content in tube-cured Virginia tobacco (Nicotiana tabacum L.) SWEET12-S T1 plants. Sugar content was measured in 18-week-old plants grown under standard tube-curing and fertilized conditions and tube-cured under standard agronomic conditions. Results represent the average content of (a) fructose, (b) glucose, (c) sucrose, and (d) total sugars in tube-cured leaves separated from the petiole (position C). Data are collected from at least three biological replicates. Data are summarized in a bar graph, with bars representing the mean in g / 100 g and error bars representing the standard deviation. Statistics show that there is a significant difference in sugar accumulation in (a, c) p35s:SWEET12-S and (b, d) p35s:SWEET15-T compared to the WT control (Student t test, **P<0.05, **P<0.01, ***P<0.001). [Figure 7-2]Figure 7 is a series of graphs showing the difference in sugar content in tube-cured Virginia tobacco (Nicotiana tabacum L.) SWEET12-S T1 plants. Sugar content was measured in 18-week-old plants grown under standard tube-curing and fertilized conditions and tube-cured under standard agronomic conditions. Results represent the average content of (a) fructose, (b) glucose, (c) sucrose, and (d) total sugars in tube-cured leaves separated from the petiole (position C). Data are collected from at least three biological replicates. Data are summarized in a bar graph, with bars representing the mean in g / 100 g and error bars representing the standard deviation. Statistics show that there is a significant difference in sugar accumulation in (a, c) p35s:SWEET12-S and (b, d) p35s:SWEET15-T compared to the WT control (Student t test, **P<0.05, **P<0.01, ***P<0.001). [Figure 8-1] Figure 8 is a series of graphs showing the difference in sugar content of tube-cured Virginia tobacco (Nicotiana tabacum L.) in SWEET15 T1 plants. Sugar content was measured in 18-week-old plants grown under standard tube-curing and fertilized conditions and tube-cured under standard agronomic conditions. Results represent the average content of (a) fructose, (b) glucose, (c) sucrose, and (d) total sugars in tube-cured leaves separated from the petiole (position C). Data are collected from at least three biological replicates. Data are summarized in a bar graph, with bars representing pooled content in g / 100 g. [Figure 8-2]Figure 8 is a series of graphs showing the difference in sugar content of tube-cured Virginia tobacco (Nicotiana tabacum L.) in SWEET15 T1 plants. Sugar content was measured in 18-week-old plants grown under standard tube-curing and fertilized conditions and tube-cured under standard agronomic conditions. Results represent the average content of (a) fructose, (b) glucose, (c) sucrose, and (d) total sugars in tube-cured leaves separated from the petiole (position C). Data are collected from at least three biological replicates. Data are summarized in a bar graph, with bars representing pooled content in g / 100 g. DETAILED DESCRIPTION OF THE INVENTION

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. In case of conflict, the present document, including definitions, governs. Preferred methods and materials are described below, but methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. The materials, methods, and examples disclosed herein are merely illustrative and are not intended to be limiting.

[0042] The terms "comprise," "include," "having," "having," "can," "containing," and variations thereof are intended to be open-ended transitional phrases, terms, or words that do not exclude the possibility of additional acts or configurations.

[0043] The singular forms "a," "and," and "the" include plural references unless the context clearly dictates otherwise.

[0044] The present disclosure contemplates other embodiments "comprising," "consisting of," and "consisting essentially of" the embodiments or elements presented herein, whether explicitly stated or not.

[0045] For purposes of reciting numerical ranges herein, each intermediate number therebetween of the same degree of precision is expressly contemplated. For example, for the range 6 to 9, the numbers 7 and 8 are contemplated in addition to the numbers 6 and 9, and for the range 6.0 to 7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are expressly contemplated.

[0046] As used throughout the specification and claims, the following terms have the following meanings:

[0047] "Coding sequence" or "encoding polynucleotide" refers to a nucleotide (RNA or DNA molecule) comprising a polynucleotide that encodes a polypeptide. The coding sequence may further comprise initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in the cells of an individual or mammal to which the polynucleotide is administered. The coding sequence may be codon-optimized.

[0048] "Complement" or "complementary" can refer to Watson-Crick (e.g., AT / U and CG) or Hoogsteen base pairing between nucleotides or nucleotide analogs. "Complementarity" refers to the property shared between two polynucleotides, e.g., when aligned antiparallel to each other, that the nucleotide bases at each position are complementary.

[0049] A "construct" refers to a double-stranded recombinant polynucleotide fragment containing one or more polynucleotides. A construct comprises a "template strand" base-paired with a complementary "sense or coding strand." A given construct can be inserted into a vector in either of two possible orientations: the same (or sense) orientation or the opposite (or antisense) orientation relative to the orientation of a promoter placed within the vector (e.g., an expression vector).

[0050] The term "control," in the context of a control plant or control plant cell, refers to a plant or plant cell in which the expression, function, or activity of one or more genes or polypeptides is not altered (e.g., increased or decreased), thereby providing a comparison with a plant in which the expression, function, or activity of one or more genes or polypeptides is altered. A "control plant" is a plant that is substantially equivalent to a test plant or modified plant in all parameters except the test parameter. For example, when referring to a plant into which a polynucleotide has been introduced, the control plant is an equivalent plant into which such polynucleotide has not been introduced. A control plant can also be an equivalent plant into which a control polynucleotide has been introduced. In such cases, the control polynucleotide is one that is predicted to have little or no phenotypic effect on the plant. A control plant can contain an empty vector. A control plant can correspond to a wild-type plant. A control plant can be a null segregant, in which the T1 segregant no longer carries the transgene.

[0051] The term "reduction" or "reduced" refers to a reduction of about 10% to about 99% in amount or function, such as polypeptide function, transcription function, or polypeptide expression, or a reduction of at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100%, or at least 150%, or at least 200% or more. The term "reduced" or the phrase "reduced amount" can refer to an amount or function that is less than that found in an unmodified plant or a product from the same variety of plant processed in the same manner. Thus, in some contexts, wild-type plants of the same variety processed in the same manner are used as a control to determine whether a reduction in amount is obtained.

[0052] "Donor DNA" or "donor template" refers to a double-stranded DNA fragment or molecule that contains at least a portion of a gene of interest. The donor DNA can encode a functional polypeptide.

[0053] An "endogenous gene or polypeptide" refers to a gene or polypeptide that originates in the genome of an organism and has not undergone alteration, such as deletion, gain, or replacement of genetic material. An endogenous gene is subject to normal gene transmission and gene expression. An endogenous polypeptide is subject to normal expression.

[0054] "Enhancer sequences" refer to sequences that can increase gene expression. These sequences can be located upstream, within introns, or downstream of the transcribed region. The transcribed region consists of exons and intervening introns from the promoter to the transcription termination region. Enhancement of gene expression can be through various mechanisms, including increasing transcription efficiency, stabilizing mature mRNA, and enhancing translation.

[0055] "Expression" refers to the production of a functional product. For example, expression of a polynucleotide fragment can refer to transcription of the polynucleotide fragment (e.g., transcription resulting in mRNA or functional RNA), or translation of mRNA into a precursor or mature polypeptide, or a combination thereof.

[0056] "Overexpression" means the production of a gene product in transgenic organisms that exceeds production levels in null segregating (or non-transgenic) organisms of the same experiment.

[0057] "Functional" describes a polypeptide that has a biological function or activity. A "functional gene" refers to a gene that is transcribed into mRNA that is translated into a functional or active polypeptide.

[0058] A "genetic construct" refers to a DNA or RNA molecule comprising a polynucleotide that encodes a polypeptide. The coding sequence may include initiation and termination signals operably linked to regulatory elements, including a promoter and polyadenylation signal, capable of directing expression.

[0059] "Genome editing" generally refers to a process in which genomic nucleic acid is altered in a cell. This can be, for example, by removing, inserting, or substituting one or more nucleotides in the genomic nucleic acid. Endonucleases can be used to create specific cuts or nicks at defined locations in the genome, as further described herein.

[0060] The terms "homology" or "similarity" refer to the degree of sequence similarity between two polypeptide molecules or two polynucleotide molecules compared by sequence alignment. The degree of homology between two separate polynucleotides being compared is a function of the number of identical or matching nucleotides at comparable positions. Homology or similarity can be determined over the entire length of the subject sequence.

[0061] "Identical" or "identity" in the context of two or more polynucleotides or polypeptides means that the sequences have a specified percentage of residues that are the same over a specified region. The percentage is calculated by optimally aligning the two sequences, comparing the two sequences over a specified region, determining the number of positions where identical residues occur in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the specified region, and multiplying the result by 100 to obtain the percentage of sequence identity. If the two sequences are of different lengths or the alignment results in one or more staggered ends and a specified region of comparison contains only a single sequence, the residues of the single sequence are included in the denominator of the calculation but not in the numerator. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identity can be determined manually or by using a computer sequence algorithm such as ClustalW, ClustalX, BLAST, FASTA, or Smith-Waterman. Suitable parameters for ClustalW can be as follows: For polynucleotide alignments: Gap opening penalty = 15.0, gap extension penalty = 6.66, and matrix = Identity. For polypeptide alignments: Gap opening penalty = 10.0, gap extension penalty = 0.2, and matrix = Gonnet. For DNA and protein alignments: ENDGAP = -1, and GAPDIST = 4.

[0062] The term "increase" or "increased" refers to an increase of about 10% to about 99% in the amount or function or activity, including, but not limited to, one or more polypeptide functions or activities, transcriptional functions or activities, and polypeptide expression, or an increase of at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, at least 100%, at least 150%, or at least 200% or more. The term "increased" or the phrase "increased amount" can refer to an amount or function or activity in a plant or a product produced from a plant that is greater than that found in an unmodified plant or a product derived from a plant of the same variety processed in the same manner. Thus, in some contexts, wild-type plants of the same variety processed in the same manner are used as a control to determine whether an increase in amount is obtained.

[0063] The term "inhibit" or "inhibited" refers to about a 98% to about 100% decrease, or at least a 98%, at least a 99%, but particularly a 100% decrease, in the amount or function or activity of one or more of, for example, but not limited to, a polypeptide function or activity, a transcriptional function or activity, and polypeptide expression.

[0064] The term "introduced" means providing a polynucleotide (e.g., a construct) or polypeptide into a cell. "Introduced" includes reference to the uptake of a polynucleotide into a eukaryotic cell, where the polynucleotide may be integrated into the genome of the cell, and includes reference to the transient provision of a polynucleotide or polypeptide to a cell. Introduced includes reference to stable or transient transformation methods, as well as sexual crossing. Thus, "introduced" in the context of inserting a polynucleotide (e.g., a recombinant / expression construct) into a cell means "transfection" or "transformation" or "transduction," and includes reference to the uptake of a polynucleotide into a eukaryotic cell, where the polynucleotide may be integrated into the genome of the cell (e.g., a chromosome, a plasmid, a plastid, or a mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).

[0065] The terms "isolated" or "purified" refer to a material that is substantially or essentially free from components that normally accompany it when found in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high-performance liquid chromatography. A polypeptide that is the predominant species present in a preparation is substantially purified. In particular, an isolated polynucleotide is separated from open reading frames that flank the desired gene and encode polypeptides other than the desired polypeptide. The term "purified" means that the polynucleotide or polypeptide gives rise to essentially one band in an electrophoretic gel. Specifically, this means that the polynucleotide or polypeptide is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure. An isolated polynucleotide can be purified from a naturally occurring host cell. Conventional polynucleotide purification methods known to those skilled in the art can be used to obtain an isolated polynucleotide. The term also encompasses recombinant and chemically synthesized polynucleotides.

[0066] "Liquid tobacco extract" describes the direct product of an extraction process performed on tobacco starting material. The extraction process to produce a liquid tobacco extract can involve heating the tobacco starting material under specific heating conditions and recovering the volatile compounds that are produced. A liquid tobacco extract can contain a mixture of compounds that originate from the tobacco starting material and are removed during the extraction process, typically in combination with a liquid carrier or solvent.

[0067] "Modulate" or "modulation" refers to causing or promoting a qualitative or quantitative change, alteration, or modification in a process, pathway, function, or activity of interest. Without limitation, such a change, alteration, or modification can be an increase or decrease in the relative process, pathway, function, or activity of interest. For example, gene expression or polypeptide expression, or polypeptide function or activity, can be modulated. Typically, the relative change, alteration, or modification will be determined by comparison with a control.

[0068] The term "non-natural" describes entities such as polynucleotides, genetic mutations, polypeptides, plants, plant cells, and plant materials that are not naturally occurring or do not exist in nature. Such non-natural or artificial entities can be created, synthesized, initiated, modified, intervened, or manipulated by methods described herein or known in the art. Such non-natural or artificial entities can be created, synthesized, initiated, modified, intervened, or manipulated by humans. Thus, non-natural plants may not be produced using essentially biological processes. Thus, for example, non-natural plants, non-natural plant cells, or non-natural plant materials can be produced using traditional plant breeding techniques such as backcrossing, or by genetic engineering techniques such as antisense RNA, interfering RNA, and meganucleases. As a further example, a non-naturally occurring plant, plant cell, or plant material can be created by introgressing or transferring one or more genetic mutations (e.g., one or more polymorphisms) from a first plant or plant cell into a second plant or plant cell (which itself may be natural) so that the resulting plant, plant cell, or plant material, or its progeny, contains a genetic makeup (e.g., a genome, chromosome, or segment thereof) that is not naturally formed or does not exist in nature. The resulting plant, plant cell, or plant material is therefore artificial or non-natural. Thus, an artificial or non-naturally occurring plant or plant cell can be created by modifying a genetic sequence in a first naturally occurring plant or plant cell, even if the resulting genetic sequence is naturally occurring in a second plant or plant cell that contains a different genetic background from the first plant or plant cell. In certain embodiments, the mutation is not a naturally occurring mutation that occurs 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).

[0069] "Oligonucleotide" or "polynucleotide" refers to at least two nucleotides covalently linked together. A depiction of a single strand also defines the sequence of the complementary strand. Thus, a polynucleotide also encompasses the complementary strand of a depicted single strand. Many variants of polynucleotides can be used for the same purpose as a given polynucleotide. Thus, a polynucleotide also encompasses substantially identical polynucleotides and their complements. A single strand provides a probe that can hybridize to a given sequence under stringent hybridization conditions. Thus, a polynucleotide also encompasses probes that hybridize under stringent hybridization conditions. A polynucleotide can be single-stranded or double-stranded, or can contain portions of both double-stranded and single-stranded sequence. A polynucleotide can be DNA, both genomic and cDNA, RNA, or a hybrid, and can contain combinations of deoxyribonucleotides and ribonucleotides, as well as combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. Polynucleotides can be obtained by chemical synthesis or by recombinant methods.

[0070] The specificity of single-stranded DNA for hybridizing complementary fragments is determined by the "stringency" of the reaction conditions (Sambrook et al., Molecular Cloning and Laboratory Manual, Second Ed., Cold Spring Harbor (1989)). Hybridization under "stringent conditions" describes a hybridization protocol in which polynucleotides at least 60% homologous to each other remain hybridized. Generally, stringent conditions are selected to be approximately 5°C lower than the thermal melting point (Tm) of a specific sequence at a defined ionic strength and pH. 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 generally present in excess at the Tm, 50% of the probes are occupied at equilibrium.

[0071] Stringent conditions typically include: (1) a low ionic strength and high temperature wash, 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 involve a wash in 0.2×SSC (sodium chloride / sodium citrate) at 42° C. containing 5×SSC (0.75 M NaCl, 75 mM sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50 μg / mL), 0.1% SDS, and 10% dextran sulfate, and a high stringency wash consisting of 50% formamide at 55° C., followed by 0.1×SSC containing EDTA at 55° C. Suitably, 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.

[0072] "Moderately stringent conditions" use wash solutions and hybridization conditions that are less stringent, such that polynucleotides hybridize to the entire polynucleotide, fragments, derivatives, or analogs. One example includes hybridization in 6xSSC, 5xDenhardt's solution, 0.5% SDS, and 100 μg / mL denatured salmon sperm DNA at 55°C, followed by one or more washes in 1xSSC, 0.1% SDS at 37°C. Temperature, ionic strength, etc., can be adjusted to accommodate experimental factors such as probe length. Other moderate stringency conditions have been described (see Ausubel et al., Current Protocols in Molecular Biology, Volumes 1-3, John Wiley & Sons, Inc., Hoboken, NJ (1993); Kriegler, Gene Transfer and Expression: A Laboratory Manual, Stockton Press, New York, NY (1990); Perbal, A Practical Guide to Molecular Cloning, 2nd edition, John Wiley & Sons, New York, NY (1988)).

[0073] "Low stringency conditions" use wash solutions and hybridization conditions that are less stringent than those for moderate stringency, such that polynucleotides hybridize to the entire polynucleotide, fragments, derivatives, or analogs. Non-limiting examples of low stringency hybridization conditions include hybridization in 35% formamide, 5xSSC, 50 mM Tris HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg / mL denatured salmon sperm DNA, 10% (wt / vol) dextran sulfate at 40°C, followed by one or more washes in 2xSSC, 25 mM Tris HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS at 50°C. Other conditions of low stringency, such as those for cross-species hybridization, have been well described (see Ausubel et al., 1993; Kriegler, 1990).

[0074] "Operably linked" means that the expression of a gene is under the control of a spatially linked promoter. The promoter can be located 5' (upstream) or 3' (downstream) of the gene under its control. The distance between the promoter and the gene can be approximately the same as the distance between the promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, variations in this distance can be adjusted without loss of promoter function. "Operably linked" refers to the association of polynucleotide fragments in a single fragment, such that one function is controlled by the other. For example, a promoter is operably linked to a polynucleotide fragment when it is capable of controlling the transcription of the polynucleotide fragment.

[0075] The term "plant" refers to any plant at any stage of its life cycle or development, and its progeny. In one embodiment, the plant is a tobacco plant and refers to a plant belonging to the genus Nicotiana. The term includes reference to whole plants, plant organs, plant tissues, plant propagules, plant seeds, plant cells, and their progeny. Plant cells include, but are not limited to, cells from seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and pollen grains. Suitable species, cultivars, hybrids, and varieties of tobacco plants are described herein.

[0076] "Plant material" includes leaves, roots, sepals, root tips, petals, flowers, shoots, stems, seeds, and stalks. Plant material can be viable or non-viable plant material.

[0077] "Polynucleotide," "polynucleotide sequence," or "polynucleotide fragment" are used interchangeably herein and refer to a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural, or altered nucleotide bases. The polynucleotides of this disclosure are set forth in the accompanying Sequence Listing.

[0078] "Polypeptide" or "polypeptide sequence" refers to a polymer of amino acids in which one or more amino acid residues are artificial chemical analogues of a corresponding naturally occurring amino acid, as well as to naturally occurring polymers of amino acids. These terms also include 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 set forth in the accompanying sequence listing.

[0079] "Promoter" refers to a synthetic or naturally occurring molecule capable of conferring, activating, or enhancing expression of a polynucleotide in a cell. The term typically refers to a polynucleotide element / sequence located upstream and operably linked to a double-stranded polynucleotide fragment. A promoter can be derived entirely from the region adjacent to a native gene of interest or can be composed of different elements derived from different native promoters or synthetic polynucleotide segments. A promoter can contain one or more specific transcription control sequences to further enhance expression or alter spatial or temporal expression. A promoter can also contain distal enhancer or repressor elements, located as many as several thousand base pairs from the start site of transcription. Promoters can be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. A promoter can constitutively or differentially control the expression of gene components depending on the cell, tissue, or organ in which expression occurs, the developmental stage in which expression occurs, or in response to external stimuli such as physiological stress, pathogens, metal ions, or inducers.

[0080] As used interchangeably herein, "tissue-specific promoter" and "tissue-preferred promoter" refer to a promoter that is expressed primarily, although not necessarily exclusively, in one tissue or organ, but can also be expressed in one particular cell. A "developmentally regulated promoter" refers to a promoter whose function is determined by developmental events. A "constitutive promoter" refers to a promoter that causes a gene to be expressed in most cell types at most times. An "inducible promoter" selectively expresses an operably linked DNA sequence in response to the presence of an endogenous or exogenous stimulus, for example, a chemical compound (a chemical inducer), or in response to an environmental, hormonal, chemical, or developmental signal, or a combination of two or more thereof. Examples of inducible or regulated promoters include promoters that are regulated by light, heat, pressure, waterlogging or drought, pathogens, plant hormones, wounding, or chemicals such as ethanol, jasmonic acid, salicylic acid, or safeners.

[0081] "Recombinant" refers to the artificial combination of two otherwise separate segments of sequence, either by chemical synthesis or by the manipulation of isolated segments of polynucleotides by genetic engineering techniques. The term also includes reference to a cell or vector that has been modified by the introduction of a heterologous polynucleotide, or to cells derived from such modified cells, but does not encompass the alteration of a cell or vector by natural events (e.g., spontaneous mutation, natural transformation or transduction or transposition), such as those that occur without deliberate human intervention.

[0082] "Recombinant construct" refers to a combination of polynucleotides that are not normally found together in nature. Thus, a recombinant construct can contain control sequences and coding sequences from different sources, or control sequences and coding sequences from the same source but arranged in a manner different from that normally found in nature. A recombinant construct can be a recombinant DNA construct.

[0083] As used interchangeably herein, "control sequence" and "control element" refer to polynucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence that influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Control sequences include promoters, translation leader sequences, introns, and polyadenylation recognition sequences. The terms "control sequence" and "control element" are used interchangeably herein.

[0084] The term "tobacco" is used collectively to refer to tobacco crops (e.g., tobacco plants that are field-grown and not hydroponically grown), tobacco plants, and parts thereof, including, but not limited to, roots, stems, leaves, flowers, and seeds, prepared or obtained as described herein. "Tobacco" refers to plants and products thereof belonging to the genus Nicotiana, and is understood to include Nicotiana tabacum plants and their products.

[0085] The term "tobacco products" means consumer tobacco products, including, but not limited to, smoking materials (e.g., cigarettes, cigars, and pipe tobacco), snuff, chewing tobacco, gum, and lozenges, as well as components, materials, and ingredients for the manufacture of consumer tobacco products. Preferably, these tobacco products are made from tobacco leaves and stems harvested from tobacco plants and cut, dried, cured, or fermented according to conventional techniques for tobacco preparation.

[0086] "Transcription terminator," "termination sequence," or "terminator" refers to a DNA sequence located downstream of a coding sequence and includes polyadenylation recognition sequences and other sequences encoding regulatory signals that can affect mRNA processing or gene expression. Polyadenylation signals are usually characterized by affecting the addition of polyadenylic acid moieties to the 3' end of a pre-mRNA.

[0087] "Transgenic" refers to any cell, cell line, callus, tissue, plant part, or plant whose genome has been altered by the presence of a heterologous polynucleotide, such as a recombinant construct, including those original transgenic events, as well as those produced from original transgenic events by sexual crossing or asexual propagation. The term does not encompass alterations of the genome (chromosomal or extrachromosomal) by conventional plant breeding methods or by natural events (e.g., 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 produced using essentially biological processes.

[0088] A "transgenic plant" refers to a plant that contains one or more heterologous polynucleotides within its genome, i.e., a plant containing recombinant genetic material not normally found therein and introduced into the plant (or an ancestor of the plant) by human manipulation. For example, a heterologous polynucleotide may be stably integrated into the genome so that the polynucleotide is transmitted to successive generations. A heterologous polynucleotide may be integrated into the genome alone or as part of a recombinant construct. Commercial development of genetically improved germplasm has also progressed to the introduction of multiple traits into crop plants, often referred to as gene stacking. In this approach, multiple genes that confer different characteristics of interest may be introduced into a plant. Gene stacking can be achieved by many means, including, but not limited to, co-transformation, retransformation, and cross-breeding lines carrying different transgenes. Thus, a plant grown from a plant cell into which recombinant DNA has been introduced by transformation is a transgenic plant, as are all progeny of the plant that contain the introduced transgenes (whether produced sexually or asexually). The term transgenic plant is understood to encompass whole plants or trees as well as parts of the plants or trees, such as grains, seeds, flowers, leaves, roots, fruit, pollen, stems, etc. Each heterologous polynucleotide may confer a different trait to the transgenic plant.

[0089] "Transgene" refers to a gene or genetic material containing a genetic sequence that has been isolated from one organism and introduced into a different organism. This non-native segment of DNA may retain the ability to produce RNA or polypeptides in the transgenic organism, or may alter the normal function of the transgenic organism's genetic code.

[0090] "Variant" with respect to a polynucleotide means (i) a portion or fragment of the polynucleotide, (ii) the complement of a polynucleotide or a portion thereof, (iii) a polynucleotide that is substantially identical to a reference polynucleotide or its complement, or (iv) a polynucleotide that hybridizes under stringent conditions to a reference polynucleotide, its complement, or a substantially identical polynucleotide.

[0091] A "variant" with respect to a peptide or polypeptide refers to a peptide or polypeptide that differs in sequence by amino acid insertion, deletion, or conservative substitution, but retains at least one biological function or activity. A variant can also refer to a polypeptide that retains at least one biological function or activity. Conservative amino acid substitutions, i.e., replacing one amino acid with a different amino acid with similar properties (e.g., hydrophilicity, degree and distribution of charged regions), are recognized in the art as typically resulting in minor changes.

[0092] The term "variety" refers to a group of plants that share certain characteristics that distinguish them from other plants of the same species. While possessing one or more distinctive traits, varieties are further characterized by very little overall variation among individuals within the variety. Varieties are often sold commercially.

[0093] A "vector" refers to a polynucleotide vehicle containing a combination of polynucleotide components to enable the delivery of polynucleotides, polynucleotide constructs, polynucleotide conjugates, and the like. A vector can be a viral vector, bacteriophage, bacterial artificial chromosome, or yeast artificial chromosome. A vector can be a DNA or RNA vector. Suitable vectors include episomes capable of extrachromosomal replication, such as circular double-stranded nucleotide plasmids, linear double-stranded nucleotide plasmids, and other vectors of any origin. An "expression vector" is a polynucleotide vehicle containing a combination of polynucleotide components to enable the expression of polynucleotides, polynucleotide constructs, polynucleotide conjugates, and the like. Suitable expression vectors include episomes capable of extrachromosomal replication, such as circular double-stranded nucleotide plasmids, linear double-stranded nucleotide plasmids, and other functionally equivalent expression vectors of any origin. An expression vector contains at least a promoter, as defined below, located upstream and operably linked to a polynucleotide, polynucleotide construct, or polynucleotide conjugate.

[0094] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those skilled in the art. For example, any nomenclature used in connection with cell and tissue culture, molecular biology, plant biology, microbiology, genetics, and polypeptide and polynucleotide chemistry, and hybridization described herein, and the techniques thereof, are well known and commonly used in the art. The meaning and scope of terms should be clear, but in the event of any potential ambiguity, the definitions provided herein shall take precedence over any dictionary or external definitions. Furthermore, unless otherwise required by context, singular terms shall include plurals, and plural terms shall include the singular.

[0095] Disclosed are isolated polynucleotides comprising, consisting of, or consisting essentially of a sequence having at least 60% sequence identity to any of the sequences described herein, including any polynucleotide set forth in the Sequence Listing. Preferably, the isolated polynucleotide comprises, consists of, or consists essentially of a sequence having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto. Preferably, the isolated polynucleotide comprises, consists of, or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto. Preferably, the isolated polynucleotide comprises, consists of, or consists essentially of a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto. Suitably, an isolated polynucleotide comprises, consists of, or consists essentially of a sequence having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto.

[0096] Preferably, the polynucleotides described herein encode active polypeptides having at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 100% or more of the SWEET function or activity of a polypeptide set forth in the Sequence Listing.

[0097] In another embodiment, at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 (NtSWEET12-S), or

[0098] at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 3 (NtSWEET12-T) or at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 5 (NtSWEET15-S); Provided are SWEET polynucleotides isolated from Nicotiana tabacum (NtSWEET) comprising, consisting of, or consisting essentially of a polynucleotide having 4%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, or at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 7 (NtSWEET15-T).

[0099] In another embodiment, a polynucleotide is provided that comprises, consists of, or consists essentially of a polynucleotide having substantial homology (i.e., sequence similarity) or substantial identity to SEQ ID NO:1, or SEQ ID NO:3, or SEQ ID NO:5, or SEQ ID NO:7.

[0100] In another embodiment, there is provided a fragment of SEQ ID NO:1, or SEQ ID NO:3, or SEQ ID NO:5, or SEQ ID NO:7 with substantial homology (i.e., sequence similarity) or substantial identity thereto having at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to the corresponding fragment of SEQ ID NO:1, or SEQ ID NO:3, or SEQ ID NO:5, or SEQ ID NO:7.

[0101] In another embodiment, there is provided a fragment of SEQ ID NO:1, or SEQ ID NO:3, or SEQ ID NO:5, or SEQ ID NO:7 with substantial homology (i.e., sequence similarity) or substantial identity thereto having at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to the corresponding fragment of SEQ ID NO:1, or SEQ ID NO:3, or SEQ ID NO:5, or SEQ ID NO:7.

[0102] 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 or SEQ ID NO:5 or SEQ ID NO:7 that encodes a polypeptide that functions as SWEET is provided.

[0103] In another embodiment, there is provided a polymer of polynucleotides comprising, consisting of, or consisting essentially of a polynucleotide designated herein as SEQ ID NO:1 or SEQ ID NO:3 or SEQ ID NO:5 or SEQ ID NO:7.

[0104] Preferably, the polynucleotides described herein encode a SWEET polypeptide that has SWEET activity.

[0105] A polynucleotide can comprise a polymer of nucleotides, which can be unmodified or modified deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Thus, a polynucleotide can be, but is not limited to, genomic DNA, complementary DNA (cDNA), mRNA, or antisense RNA, or fragments thereof. Moreover, a polynucleotide can be single-stranded or double-stranded DNA, a DNA that is a mixture of single-stranded and double-stranded regions, a hybrid molecule containing DNA and RNA, or a hybrid molecule with a mixture of single-stranded and double-stranded regions or fragments thereof. Additionally, a polynucleotide can be composed of triple-stranded regions containing DNA, RNA, or both, or fragments thereof. A polynucleotide can contain one or more modified bases, such as phosphothioates, and can be a peptide nucleic acid. Generally, a polynucleotide can be assembled from isolated or cloned fragments of cDNA, genomic DNA, oligonucleotides, or individual nucleotides, or combinations of the foregoing. Although the polynucleotides described herein are presented as DNA sequences, they include their corresponding RNA sequences and their complementary (e.g., perfectly complementary) DNA or RNA sequences, including their reverse complements.

[0106] Fragments of polynucleotides can range from at least about 25 nucleotides, about 50 nucleotides, about 75 nucleotides, about 100 nucleotides, about 150 nucleotides, about 200 nucleotides, about 250 nucleotides, about 300 nucleotides, about 400 nucleotides, about 500 nucleotides, about 600 nucleotides, about 700 nucleotides, about 800 nucleotides, about 900 nucleotides, about 1000 nucleotides, about 1100 nucleotides, about 1200 nucleotides, about 1300 nucleotides, or about 1400 nucleotides, up to a full-length polynucleotide encoding a polypeptide described herein.

[0107] Polynucleotides generally contain phosphodiester bonds, but in some cases, polynucleotide analogs may have alternative backbones, including, for example, phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphosphoramidite linkages, as well as peptide polynucleotide backbones and linkages. Other analog polynucleotides include those with cationic, non-ionic, and non-ribose backbones. Modifications of the ribose-phosphate backbone may be made 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 natural polynucleotides and analogs may be made; alternatively, mixtures of different polynucleotide analogs and mixtures of natural polynucleotides and analogs may be made.

[0108] A variety of polynucleotide analogs are known, including, for example, phosphoramidate, phosphorothioate, phosphorodithioate, O-methylphosphoramidite linkages, and peptide polynucleotide backbones and linkages. Other analog polynucleotides include those with normal backbones, non-ionic backbones, and non-ribose backbones. Polynucleotides containing one or more carbocyclic sugars are also included.

[0109] Other analogs include peptide polynucleotides, which are peptide polynucleotide analogs.

[0110] Among the uses of the disclosed polynucleotides and their fragments are the use of the fragments as probes in hybridization assays or primers for use in amplification assays. Such fragments generally contain at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more consecutive nucleotides of a DNA sequence. In other embodiments, the DNA fragments contain at least about 10, 15, 20, 30, 40, 50, or 60 or more consecutive nucleotides of a DNA sequence. Thus, in one aspect, methods for detecting polynucleotides are also provided, including the use of probes or primers, or both.

[0111] The basic parameters that influence the selection of hybridization conditions, and guidance for devising suitable conditions, are described by Sambrook, J., E.F. Fritsch, and T. Maniatis (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). Using knowledge of the genetic code combined with the polypeptide sequences described herein, a set of degenerate oligonucleotides can be prepared. Such oligonucleotides are useful, for example, as primers in polymerase chain reactions (PCR), in which DNA fragments are isolated and amplified.

[0112] At least one alteration (eg, mutation) may be included in one or more of SEQ ID NO:1 or SEQ ID NO:3 or SEQ ID NO:5 or SEQ ID NO:7.

[0113] Isolated SWEET polypeptides encoded by the polynucleotides described herein are provided.

[0114]

[0013] Provided are isolated SWEET polypeptides comprising, consisting of, or consisting essentially of a polypeptide having at least 60% sequence identity to any of the polypeptides described herein, including any of the polypeptides set forth in the Sequence Listing. Preferably, the isolated polypeptide comprises, consists of, or consists essentially of a sequence having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, 80%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity thereto. Suitably, the isolated polypeptide comprises, consists of, or consists essentially of a sequence having at least 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.

[0115] Also provided are SWEET polypeptides comprising, consisting of, or consisting essentially of a sequence having at least 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 to SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:8.

[0116] Also provided are SWEET polypeptides comprising, consisting of, or consisting essentially of a sequence having at least 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 or SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:8.

[0117] The polypeptide may comprise a sequence that comprises a sufficient or substantial degree of identity or similarity to SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:8 for function as a SWEET.

[0118] Fragments of the polypeptides described herein are also contemplated. Polypeptide fragments typically retain some or all of the function or activity of the full-length sequence, such as SWEET activity. Polypeptide fragments can range from at least about 25 amino acids, about 50 amino acids, about 75 amino acids, about 100 amino acids, about 150 amino acids, about 200 amino acids, about 250 amino acids, about 300 amino acids, about 400 amino acids, or about 500 amino acids, up to the full-length polypeptides described herein.

[0119] Polypeptides also include variants produced by the introduction of any type of alteration (e.g., amino acid insertion, deletion, or substitution; changes in glycosylation status; changes affecting refolding or isomerization, three-dimensional structure, or self-association status), whether intentionally engineered or naturally isolated, provided that they still possess some or all of their function or activity. Preferably, this function or activity is modulated.

[0120] Deletion refers to the removal of one or more amino acids from a polypeptide. Insertion refers to the introduction of one or more amino acid residues into a predetermined site within a polypeptide. Insertion can include the intrasequence insertion of single or multiple amino acids. Substitution refers to the replacement of an amino acid in a polypeptide with another amino acid having similar properties (e.g., similar hydrophobicity, hydrophilicity, antigenicity, tendency to form or disrupt α-helical or β-sheet structures). Amino acid substitutions are typically of 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 preferably conservative amino acid substitutions, as described below. Amino acid substitutions, deletions, or insertions can be made using peptide synthesis techniques, such as solid-phase peptide synthesis, or by recombinant DNA engineering. Methods for manipulating DNA sequences to produce substitution, insertion, or deletion variants of polypeptides are well known in the art. Variants can have alterations that produce silent changes, resulting in functionally equivalent polypeptides. As long as the secondary binding of the substance is maintained, deliberate amino acid substitutions can be made based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and amphipathic properties of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid, positively charged amino acids include lysine and arginine, and amino acids with uncharged polar head groups with similar hydrophilicity values ​​include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine. Conservative substitutions can be made, for example, according to the following table. Amino acids in the same block in the second row and preferably in the same family in the third row can be substituted for each other.

[0121] [Table 1]

[0122] The polypeptide may be a mature or immature polypeptide, or a polypeptide derived from an immature polypeptide. The polypeptide may be linear or cyclized using known methods. The polypeptide typically contains at least 10, at least 20, at least 30, or at least 40 consecutive amino acids.

[0123] At least one modification (eg, mutation) may be included in one or more of SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:8.

[0124] Recombinant constructs can be used to transform plants or plant cells to modulate polypeptide expression, function, or activity. Recombinant polynucleotide constructs can include a polynucleotide encoding one or more of the polynucleotides described herein, operably linked to a regulatory region suitable for expressing the polypeptide. Thus, the polynucleotide can include a coding sequence encoding a polypeptide described herein. Plants or plant cells in which polypeptide expression, function, or activity is modulated can include mutant, non-naturally occurring, transgenic, artificial, or genetically modified plants or plant cells. Preferably, transgenic plants or plant cells contain genomes that have been altered by stable integration of recombinant DNA. Recombinant DNA includes DNA that has been genetically engineered and constructed outside the cell, including DNA containing natural DNA, cDNA, or synthetic DNA. Transgenic plants can include plants regenerated from the originally transformed plant cell, as well as progeny transgenic plants from subsequent generations or crosses of the transformed plant. Preferably, the transgenic modification alters the expression, function, or activity of the polynucleotides or polypeptides described herein compared to a control plant.

[0125] The polypeptide encoded by the recombinant polynucleotide may be a native polypeptide or may be heterologous to the cell. In some cases, the recombinant construct contains a polynucleotide that regulates expression and is operably linked to a regulatory region. Examples of suitable regulatory regions are described herein.

[0126] Also provided is a vector containing a recombinant polynucleotide construct such as those described herein.Suitable vector backbones include those commonly used in the art, such as plasmids, viruses, artificial chromosomes, bacterial artificial chromosomes, yeast artificial chromosomes, or bacteriophage artificial chromosomes.Suitable expression vectors include, but are not limited to, plasmids and viral vectors derived from, for example, bacteriophages, baculoviruses, and retroviruses.Many vectors and expression systems are commercially available.

[0127] A vector may contain, for example, an origin of replication, a scaffold attachment region, or a marker. Marker genes can confer a selectable phenotype to plant cells. For example, markers can confer biocide resistance, such as resistance to antibiotics (e.g., kanamycin, G418, bleomycin, or hygromycin) or herbicides (e.g., glyphosate, chlorsulfuron, or phosphinothricin). In addition, expression vectors can contain tag sequences designed to facilitate manipulation or detection (e.g., purification or localization) of the expressed polypeptide. Tag sequences, such as luciferase, beta-glucuronidase, green fluorescent polypeptide, glutathione S-transferase, polyhistidine, c-myc, or hemagglutinin sequences, are typically expressed as a fusion with the encoded polypeptide. Such tags can be inserted anywhere within the polypeptide, including at either the carboxyl or amino terminus.

[0128] Plants or plant cells can be transformed by having a recombinant polynucleotide integrated into their genome so that they are stably transformed. The plants or plant cells described herein can be stably transformed. Stably transformed cells typically retain the introduced polynucleotide with each cell division. Plants or plant cells can be transiently transformed so that the recombinant polynucleotide is not integrated into their genome. Transiently transformed cells typically lose all or part of the introduced recombinant polynucleotide with each cell division, so that the introduced recombinant polynucleotide is not detected in daughter cells after a sufficient number of cell divisions.

[0129] Many methods for transforming plant cells are available in the art, including biolistics, gene gun techniques, Agrobacterium-mediated transformation, viral vector-mediated transformation, freeze-thaw methods, particle bombardment, direct DNA uptake, sonication, microinjection, plant virus-mediated transfer, and electroporation.

[0130] If cells or cultured tissues are used as recipient tissues for transformation, plants can be regenerated from the transformed cultures, if desired, by techniques known to those skilled in the art.

[0131] The selection of a regulatory region to be included in a recombinant construct depends on several factors, including, but not limited to, efficiency, selectability, inducibility, desired expression level, and cell- or tissue-preferential expression. It is a routine matter for those skilled in the art to regulate the expression of a coding sequence by appropriately selecting and positioning a regulatory region relative to the coding sequence. Transcription of polynucleotides can be regulated in a similar manner. Some suitable regulatory regions initiate transcription only in certain cell types, or primarily in certain cell types. Methods for identifying and characterizing regulatory regions in plant genomic DNA are known in the art.

[0132] Exemplary promoters include tissue-specific promoters recognized by tissue-specific factors present in different tissues or cell types (e.g., root-specific promoters, shoot-specific promoters, xylem-specific promoters), or present during different developmental stages, or present in response to different environmental conditions. Suitable promoters include constitutive promoters that can be activated in most cell types without the need for specific inducers. Examples of promoters that can be used to control polypeptide expression include the cauliflower mosaic virus 35S (CaMV / 35S), SSU, OCS, lib4, usp, STLS1, B33, nos, or ubiquitin or phaseolin promoters. Those skilled in the art can generate multiple variations of recombinant promoters.

[0133] Tissue-specific promoters are transcriptional control elements that are only active in specific cells or tissues at specific times during plant development, such as in vegetative tissues or reproductive tissues. Examples of tissue-specific promoters under developmental control include promoters that can initiate transcription only (or mainly only) in certain tissues, such as vegetative tissues, such as roots or leaves, or reproductive tissues, such as fruit, ovule, seed, pollen, pistil, flower, or any embryonic tissue. Reproductive tissue-specific promoters can be, for example, anther-specific, ovule-specific, embryo-specific, endosperm-specific, integument-specific, seed and seed coat-specific, pollen-specific, petal-specific, sepal-specific, or a combination thereof.

[0134] Exemplary leaf-specific promoters include the bilbert orthophosphate dikinase (PPDK) promoter from C4 plants (maize), the cab-m1Ca+2 promoter from maize, the Arabidopsis thaliana myb-related gene promoter (Atmyb5), the ribulose biphosphate 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 biphosphate carboxylase promoter expressed at high levels almost exclusively in mesophyll cells in leaf blades and sheaths).

[0135] Suitable senescence-specific promoters include tomato promoters active during fruit ripening, senescence and leaf abscission, maize promoters of genes encoding cysteine ​​proteases, 82E4 promoters and SAG gene promoters.Exemplary anther-specific promoters can be used.Exemplary root-preferential promoters known to those skilled in the art can be selected.Exemplary seed-preferential promoters include both seed-specific promoters (those promoters active during seed development, such as promoters of seed storage polypeptides) and seed germination promoters (promoters active during seed germination).

[0136] Examples of inducible promoters include promoters that respond to pathogen attack, anaerobic conditions, high temperature, light, drought, low temperature, or high salinity. Pathogen-inducible promoters include those from pathogenesis-related polypeptides (PR polypeptides) and are induced following infection by a pathogen (e.g., PR polypeptides, SAR polypeptides, beta-1,3-glucanases, chitinases).

[0137] In addition to plant promoters, other suitable promoters may be derived from bacterial origin, such as the octopine synthase promoter, the nopaline synthase promoter, and other promoters derived from Ti plasmids, or may be derived from viral promoters (e.g., the 35S and 19S RNA promoters of the cauliflower mosaic virus (CaMV), the constitutive promoters of the tobacco mosaic virus, the cauliflower mosaic virus (CaMV) 19S and 35S promoters, or the figwort mosaic virus 35S promoter).

[0138] Disclosed are plants or plant cells comprising at least one mutation in one or more of the polynucleotides or polypeptides described herein, which mutation results in modulated function or activity of NtSWEET or the polypeptide encoded thereby.

[0139] Methods are provided for modulating levels of NtSWEET polypeptides in a (cured or cured) plant or (cured or cured) tobacco plant material, the methods comprising introducing into the genome of the plant one or more mutations that modulate expression of at least one NtSWEET gene, wherein the at least one NtSWEET gene is selected from one or more NtSWEET sequences according to the present disclosure.

[0140] Also provided is a method for identifying a plant having a modulated level of one or more amino acids in the plant or part thereof relative to the level of the one or more amino acids in a control plant, the method comprising screening a polynucleotide sample obtained from the plant of interest for the presence of one or more mutations in an NtSWEET polynucleotide sequence according to the present disclosure, and optionally correlating the identified mutations to mutations known to modulate the level of one or more amino acids.

[0141] Also disclosed are plants or plant cells that are heterozygous or homozygous for one or more mutations in an NtSWEET gene according to the present disclosure, which mutations result in modulation of expression of the NtSWEET gene or the function or activity of the NtSWEET polypeptide encoded thereby.

[0142] Numerous techniques, including sexual crossing, can be used to combine mutations in one plant. A plant having one or more favorable heterozygous or homozygous mutations in a gene according to the present disclosure that modulates the expression of the gene or the function or activity of the polypeptide encoded thereby can be crossed with a plant having one or more favorable heterozygous or homozygous mutations in one or more other genes that modulate the expression or the function or activity of the polypeptide encoded thereby. In one embodiment, crossing is performed to introduce one or more favorable heterozygous or homozygous mutations in a gene according to the present disclosure within the same plant.

[0143] The function or activity of one or more polypeptides of the present disclosure in a plant is increased or decreased if the function or activity is 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 that has been grown, harvested, and dried or desiccated using the same protocols.

[0144] In some embodiments, mutations are introduced into plants or plant cells using mutagenesis techniques, and the introduced mutations are identified or selected using methods known to those skilled in the art (e.g., Southern blot analysis, DNA sequencing, PCR analysis, or phenotypic analysis). Mutations that affect gene expression or disrupt the function of the encoded polypeptide can be determined using methods well known in the art. Insertional mutations in gene exons usually result in null mutants. Mutations in conserved residues can be particularly effective in disrupting the metabolic function of the encoded polypeptide. For example, it will be understood that mutations in one or more highly conserved regions are likely to alter polypeptide function, while mutations outside those highly conserved regions are likely to have little or no effect on polypeptide function. In addition, mutations in a single nucleotide can create a stop codon, resulting in a truncated polypeptide and, depending on the extent of truncation, loss of function.

[0145] Also disclosed are methods for obtaining mutant polynucleotides and polypeptides.Any plant of interest, including plant cells or plant materials, can be genetically modified by various methods known to induce mutagenesis, 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.

[0146] The mutations in the polynucleotides and polypeptides described herein may include man-made mutations, synthetic mutations, or genetically engineered mutations. The mutations in the polynucleotides and polypeptides described herein may be mutations that have been obtained or can be obtained through a process involving in vitro or in vivo manipulation steps. The mutations in the polynucleotides and polypeptides described herein may be mutations that have been obtained or can be obtained through a process involving human intervention. The function or activity of the mutant polypeptide variant may be higher, lower, or approximately the same as that of the unmutated polypeptide.

[0147] Methods for randomly introducing mutations into polynucleotides can include chemical mutagenesis and radiation mutagenesis. Chemical mutagenesis involves the use of exogenously added chemicals, such as mutagenic, teratogenic, or carcinogenic organic compounds, to induce mutations. Mutagens that primarily cause point mutations, as well as short deletions, insertions, missense mutations, simple sequence repeats, transversions, or transitions, can be used to induce mutations, including chemical mutagens or radiation. Mutagens include ethyl methanesulfonate, methyl methanesulfonate, N-ethyl-N-nitrosourea, triethylmelamine, N-methyl-N-nitrosourea, procarbazine, chlorambucil, cyclophosphamide, diethyl sulfate, acrylamide monomer, melphalan, nitrogen mustard, vincristine, dimethylnitrosamine, N-methyl-N'-nitro-nitrosoguanidine, nitrosoguanidine, 2-aminopurine, 7,12 dimethyl-benz(a)anthracene, ethylene oxide, hexamethylphosphoramide, bisulfane, diepoxyalkanes (diepoxyoctane, diepoxybutane, etc.), 2-methoxy-6-chloro-9[3-(ethyl-2-chloro-ethyl)aminopropylamino]acridine dihydrochloride, and formaldehyde.

[0148] Spontaneous mutations at loci that may not be directly caused by mutagens are also contemplated, provided that they result in the desired phenotype. Suitable mutagens may also include, for example, ionizing radiation (e.g., X-rays, gamma rays, fast neutron irradiation, and UV radiation). The dosage of mutagenic chemicals or radiation is experimentally determined for each type of plant tissue to obtain a mutation frequency that is below the threshold level characterized by lethality or reproductive sterility. Any method of plant polynucleotide preparation known to those skilled in the art can be used to prepare plant polynucleotides for mutation screening.

[0149] The mutation process may involve one or more plant cross-breeding steps.

[0150] After mutation, screening can be performed to identify mutations that create premature stop codons or otherwise non-functional genes. After mutation, screening can be performed to identify mutations that create functional genes that can be expressed at increased or decreased levels. Screening for mutants can be performed by sequencing or by using one or more probes or primers specific to the gene or polypeptide. Specific mutations in polynucleotides can also be created that can result in regulated gene expression, regulated mRNA stability, or regulated polypeptide stability. Such plants are referred to herein as "non-natural" or "mutant" plants. Typically, mutant or non-natural plants will contain at least a portion of exogenous, synthetic, or man-made nucleotides (e.g., DNA or RNA) that were not present in the plant before manipulation. The exogenous nucleotide can be a single nucleotide, two or more nucleotides, two or more contiguous nucleotides, or two or more non-contiguous nucleotides (e.g., at least 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or 1500 or more contiguous or non-contiguous nucleotides).

[0151] 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 with respect to substrates or regulatory polypeptides, antibodies that show specificity for one or more polypeptides, small molecule compounds that can interfere with the stability of one or more polypeptides or the enzymatic function of one or more polypeptides or the binding function of one or more polypeptides, zinc finger polypeptides that bind to one or more polynucleotides, and meganucleases that function on one or more polynucleotides can be used to regulate the expression, function, or activity of one or more of the polynucleotides or polypeptides described herein. Genome editing techniques are well known in the art and will be further discussed below.

[0152] Zinc finger polypeptides can be used to modulate the expression or function or activity of one or more NtSWEET polynucleotides described herein. The use of zinc finger nucleases is described in Nature Rev. Genet. (2010) 11(9):636-646).

[0153] Meganucleases, such as I-CreI, can be used to regulate the expression or function or activity of one or more of the NtSWEET 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.

[0154] Transcription activator-like effector nucleases (TALENs) can be used to regulate the expression, function, or activity of one or more of the NtSWEET 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.

[0155] CRISPR system can be used to regulate the expression or function or activity of one or more of the NtSWEET polynucleotides described herein, and is a preferred method.This technology is described, for example, in Plant Methods (2016) 12:8; Front Plant Sci. (2016) 7:506, Biotechnology Advances (2015) 33,1,p41-52, Acta Pharmaceutica Sinica B (2017) 7,3,p292-302, Curr.Op.in Plant Biol. (2017) 36,1-8 and Int J Mol Sci (2019) 20 (16), 4045.As is well known in the art, CRISPR editing system generally includes two components: CRISPR-associated endonuclease (Cas) (for example, Cas9) and guide RNA (gRNA). Cas creates double-stranded DNA breaks at sites in the genome defined by the sequence of the gRNA molecule bound to it. The location at which Cas cleaves DNA is defined by the unique sequence of the gRNA bound to it. The gRNA is a specifically designed RNA sequence that recognizes a target DNA region and directs Cas nuclease to it for editing. It has two sections: (i) a tracer RNA that serves as a binding scaffold for the Cas nuclease, and (ii) a 17-20 nucleotide sequence of Crisp RNA (crRNA) that is complementary to the target DNA. The exact region of DNA targeted will depend on the specific application. For example, to activate or repress a target polynucleotide, the gRNA can be targeted to a promoter that drives expression of the target polynucleotide. Methods for designing gRNAs are well known in the art and include those from Chop Chop Harvard. The application of Cas9-based genome editing in Arabidopsis and tobacco is described, for example, in Methods Enzymol. (2014) 546: 459-72 and Plant Physiol Biochem (2018) 131: 37-46. CRISPR technology has been widely introduced into plants (see, for example, WO2015 / 189693).In addition to Cas9, other RNA-guided nucleases have been described for use in CRISPR systems, including Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, CaslO, Cpfl, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3 and Csf4. In certain embodiments, the use of Cas9 is preferred. The present disclosure 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 of the polynucleotides described herein. The present disclosure also provides a method for cleaving one or more polynucleotides in a plant cell, comprising introducing a gRNA and an RNA-guided nuclease into the plant cell, wherein the gRNA acts in conjunction with the RNA-guided nuclease to create a strand break in one or more of the polynucleotides described herein. A CRISPR construct is also disclosed, comprising (i) a polynucleotide encoding a CRISPR-associated endonuclease, and (ii) a gRNA comprising a polynucleotide sequence (typically about 17-20 nucleotides) complementary to the DNA of the targeted polynucleotide described herein.

[0156] Antisense technology is another well-known method that can be used to modulate the expression or activity of one or more of the NtSWEET polypeptides described herein. See, e.g., Gene (1988) 10;72(1-2):45-50.

[0157] NtSWEET polynucleotides can be targeted for inactivation by introducing a transposon (e.g., an IS element or other mobile genetic element) into the genome of a plant of interest. See, e.g., Cytology and Genetics (2006) 40(4):68-81.

[0158] NtSWEET polynucleotides can be targeted for inactivation by introducing into plants ribozymes derived from a number of small circular RNAs capable of self-cleavage and replication. See, e.g., FEMS Microbiology Reviews (1999) 23, 3, 257-275.

[0159] A mutant or non-naturally occurring plant or plant cell can have any combination of one or more modifications (e.g., mutations) in one or more of the NtSWEET polynucleotides described herein, which modifications result in regulated expression, function, or activity of the polynucleotide or its polynucleotide product. For example, a mutant or non-naturally occurring plant or plant cell can have a single modification in a single NtSWEET polynucleotide or polypeptide; multiple modifications in a single NtSWEET polynucleotide or polypeptide; a single modification in two or more NtSWEET polynucleotides or polypeptides; or multiple modifications in two or more NtSWEET polynucleotides or polypeptides. As yet another example, a mutant or non-naturally occurring plant or plant cell can have one or more modifications in a particular portion of an NtSWEET polynucleotide or NtSWEET polypeptide, such as a region of NtSWEET that encodes the active site of an NtSWEET polypeptide or portion thereof. As yet another example, a mutant or non-naturally occurring plant or plant cell may have one or more modifications in a region outside one or more NtSWEET polynucleotides or NtSWEET polypeptides, for example, in a region upstream or downstream of an NtSWEET polynucleotide if it regulates the function or expression of NtSWEET. Upstream elements may include promoters, enhancers, or transcription factors. Some elements, such as enhancers, may be located upstream or downstream of the gene they regulate. Elements do not need to be located near the gene they regulate, as some elements have been found to be located hundreds of thousands of base pairs upstream or downstream of the gene they regulate.A mutant or non-naturally occurring plant or plant cell can have one or more modifications located within the first 100 nucleotides of the gene, the first 200 nucleotides of the gene, the first 300 nucleotides of the gene, the first 400 nucleotides of the gene, the first 500 nucleotides of the gene, the first 600 nucleotides of the gene, the first 700 nucleotides of the gene, the first 800 nucleotides of the gene, the first 900 nucleotides of the gene, the first 1000 nucleotides of the gene, the first 1100 nucleotides of the gene, the first 1200 nucleotides of the gene, the first 1300 nucleotides of the gene, the first 1400 nucleotides of the gene, or the first 1500 nucleotides of the gene. A mutant or non-naturally occurring plant or plant cell can have one or more modifications located within the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, or fifteenth set of 100 nucleotides of the gene, or a combination thereof. Disclosed are mutant or non-native plants or plant cells (such as the mutant, non-native or transgenic plants or plant cells described herein) that comprise the mutant polypeptide variants.

[0160] In one embodiment, seeds from the plant are mutagenized and then grown into first-generation mutant plants. The first-generation plants are then self-pollinated, and seeds from the first-generation plants are grown into second-generation plants and then screened for mutations at those loci. While mutagenized plant material can be screened for mutations, the advantage of screening second-generation plants is that all somatic mutations correspond to germline mutations. Those skilled in the art will understand that a variety of plant materials, including but not limited to seeds, pollen, plant tissues, or plant cells, can be mutagenized to create mutant plants. However, the type of mutagenized plant material can affect when plant polynucleotides are screened for mutations. For example, if pollen is subjected to mutagenesis before pollination of a non-mutagenized plant, the seeds resulting from that pollination will grow into first-generation plants. All cells of the first-generation plants will contain the mutations created in the pollen, and therefore these first-generation plants can then be screened for mutations without waiting until the second generation.

[0161] NtSWEET polynucleotides prepared from individual plants, plant cells, or plant materials can optionally be pooled to facilitate screening for mutations within a population of plants originating from the mutagenized plant 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 a polynucleotide-specific amplification technique, such as PCR. Any one or more primers or probes specific to genes or sequences immediately adjacent to the gene can be used to amplify sequences within the optionally pooled sample. Preferably, the one or more primers or probes are designed to amplify a region of the locus where beneficial mutations are most likely to occur. Most preferably, the primers are designed to detect mutations within the region of the polynucleotide. Additionally, primers and probes preferably avoid known polymorphic sites to facilitate screening for point mutations. To facilitate detection of the amplification product, the 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 amplification products, primers or probes can be labeled using any conventional labeling method. These can be designed based on the sequences described herein using methods well understood in the art.

[0162] Polymorphisms can be identified by means known in the art, several of which are described in the literature.

[0163] In some embodiments, plants may be regenerated or grown from plants, plant tissues, or plant cells. Any suitable method for regenerating or growing plants from plant cells or plant tissues may be used, including, but not limited to, tissue culture or regeneration from protoplasts. Preferably, plants may be regenerated by growing transformed plant cells on callus, shoot, or root induction media. See, for example, McCormick et al., Plant Cell Reports 5:81-84 (1986). These plants are then grown and pollinated with either the same transformed line or a different line, and the resulting hybrids with the desired phenotypic characteristics are identified. Two or more generations may be grown to ensure that the expression of the desired phenotypic characteristics is stably maintained and inherited, and the seeds may then be harvested to ensure that the desired phenotypic characteristics have been achieved. Thus, "transformed seeds" refer to seeds containing a nucleotide construct stably integrated into the plant genome.

[0164] Thus, in a further aspect, a method for preparing a mutant plant is provided. The method includes providing at least one cell of a plant containing one or more NtSWEET genes encoding a functional NtSWEET. The at least one cell of the plant is then treated under conditions effective to modulate the function of an NtSWEET polynucleotide. The at least one mutant plant cell is then grown in a mutant plant, where the mutant plant has a modulated level of an NtSWEET polypeptide described herein compared to a control plant. In one embodiment of this method for producing a mutant plant, the treating step involves subjecting at least one cell to a chemical mutagen, as described above, and under conditions effective to obtain at least one mutant plant cell. In another embodiment of this method, the treating step involves subjecting at least one cell to a radiation source under conditions effective to obtain at least one mutant plant cell. The term "mutant plant" includes mutant plants whose genotype is altered compared to a control plant, preferably by means other than genetic engineering or genetic modification.

[0165] In certain embodiments, a mutant plant, mutant plant cell, or mutant plant material may contain one or more mutations that naturally occur in another plant, plant cell, or plant material and confer a desired trait. This mutation may be incorporated (e.g., introgressed) into another plant, plant cell, or plant material (e.g., a plant, plant cell, or plant material having a different genetic background than the plant from which the mutation originated) to confer the trait thereto. Thus, by way of example, a naturally occurring mutation in a first plant may be introduced into a second plant (e.g., a second plant having a different genetic background than the first plant). Thus, one skilled in the art can search for and identify plants that naturally harbor one or more mutant alleles of the genes described herein that confer a desired trait in their genome. The naturally occurring mutant alleles may be transferred into a second plant by various methods, including breeding, backcrossing, and introgression, to produce a line, variety, or hybrid having one or more mutations in the genes described herein. The same techniques may also be applied to the introgression of one or more non-naturally occurring mutations from a first plant into a second plant. Plants exhibiting desired traits can be screened out from a pool of mutant plants. Preferably, selection is performed using knowledge of the polynucleotides described herein. Consequently, it is possible to screen for genetic traits compared to controls. Such screening procedures may involve the application of conventional amplification or hybridization techniques as discussed herein. Accordingly, a further aspect of the present disclosure relates to a method for identifying mutant plants, comprising: (a) providing a sample containing one or more NtSWEET polynucleotides from a plant; and (b) sequencing the polynucleotides, wherein a difference in the sequence of the polynucleotides compared to the polynucleotides of a control plant indicates that the plant is a mutant plant.In another aspect, a method is provided for identifying a mutant plant that accumulates increased or decreased levels of an amino acid compared to a control plant, the method comprising: (a) providing a sample from the plant to be screened; (b) determining whether the sample contains one or more mutations in one or more NtSWEET polynucleotides described herein; and (c) determining the level of at least one amino acid in the plant. Preferably, the level of the at least one amino acid is determined in desiccated or dried leaves. In another aspect, a method is provided for preparing a mutant plant having increased or decreased levels of at least one amino acid compared to a control plant, the method comprising: (a) providing a sample from a first plant; (b) determining whether the sample contains one or more mutations in one or more NtSWEET polynucleotides described herein that result in a modulated level of at least one amino acid; and (c) introgressing the one or more mutations into a second plant. Preferably, the level of the at least one amino acid is determined in desiccated or dried leaves. The mutations can be transferred into a second plant using various methods known in the art (e.g., by genetic engineering, genetic manipulation, introgression, plant breeding, backcrossing, etc.). In one embodiment, the first plant is a wild-type plant. In one embodiment, the second plant has a different genetic background from the first plant. In another aspect, a method is provided for preparing a mutant plant having an increased or decreased level of at least one amino acid compared to a control plant, the method comprising: (a) providing a sample from a first plant; (b) determining whether the sample contains one or more mutations in one or more of the NtSWEET polynucleotides described herein that result in a modulated level of at least one amino acid; and (c) introgressing the one or more mutations into a second plant. Preferably, the level of the at least one amino acid is determined in desiccated or dried leaves. In one embodiment, the introgressing step comprises plant breeding, optionally including backcrossing, etc. In one embodiment, the first plant is a wild-type plant.In one embodiment, the second plant has a different genetic background than the first plant. In one embodiment, the first plant is not a cultivar or elite cultivar. In one embodiment, the second plant is a cultivar or elite cultivar. A further aspect relates to mutant plants (including mutant plants of cultivars or elite cultivars) obtained or obtainable by the methods described herein. In certain embodiments, the mutant plant may have one or more mutations localized only to a specific region of the plant, such as within the sequence of one or more NtSWEET polynucleotides described herein. According to this embodiment, the remaining genomic sequence of the mutant plant will be the same or substantially the same as that of the plant before mutagenesis.

[0166] In certain embodiments, the mutant plant may have one or more mutations localized to multiple genomic regions of the plant, such as within the sequence of one or more NtSWEET polynucleotides described herein and one or more additional regions of the genome, such that the remaining genomic sequence of the mutant plant will no longer be the same or substantially the same as the plant prior to mutagenesis. In certain embodiments, the mutant plant may not have one or more mutations in one or more, two or more, three or more, four or more, or five or more exons of an NtSWEET polynucleotide described herein; or may not have one or more mutations in one or more, two or more, three or more, four or more, or five or more introns of an NtSWEET polynucleotide described herein; or may not have one or more mutations in a promoter of an NtSWEET polynucleotide described herein; or may not have one or more mutations in a 3' untranslated region of an NtSWEET polynucleotide described herein; or may not have one or more mutations in a 5' untranslated region of an NtSWEET polynucleotide described herein; or may not have one or more mutations in a coding region of an NtSWEET polynucleotide described herein; or may not have one or more mutations in a non-coding region of an NtSWEET polynucleotide described herein.

[0167] In a further aspect, there is provided a method for identifying a plant, plant cell, or plant material containing a mutation in a gene encoding an NtSWEET polynucleotide described herein, the method comprising: (a) subjecting the plant, plant cell, or plant material to mutagenesis; (b) obtaining a sample from the plant, plant cell, or plant material, or a progeny thereof; and (c) determining the polynucleotide sequence of the NtSWEET 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 occurring in genomic regions that affect expression of the NtSWEET gene in plant cells, such as transcription start sites, start codons, intronic regions, exon-intron boundaries, terminators, or other regions.

[0168] Plants suitable for use in the present disclosure include monocotyledonous and dicotyledonous plants and plant cell lines, including members of the genus Nicotiana.

[0169] Various embodiments are directed to mutant, non-native, or transgenic tobacco plants or tobacco plant cells and may be applied to any species of the genus Nicotiana, including N. rustica and N. tabacum (e.g., LA B21, LN KY171, TI 1406, Basma, Galpao, Perique, Beinhart 1000-1, and Petico).Other species include N. acaulis, N. acuminata, N. africana, N. alata, N. ameghinoi, N. amplexicaulis, N. arentsii, N. attenuata, N. azambujae, and N. benavide sii, N. benthamiana, N. bigelovii, N. bonariensis, N. cavicola, N. clevelandii, N. cordifolia, N. corymbosa, N. debneyi, N. excelsior, and N. forget iana, N. fragrans, N. glauca, N. glutinosa, N. goodspeedii, N. gossei, N. hybrid, N. ingulba, N. kawakamii, N. knightiana, N. langsdorffii, N. lin earis、N.longiflora、N.maritima、N.megalosiphon、N.miersii、N.noctiflora、N.nudicaulis、N.obtusifolia、N.occidentalis、N.occidentalis subsp.hesperis、N.otophora、N.paniculata、N.pauciflora、N.petunioides、N.plumbaginifolia、N.quadrivalvis、N.raimondii、N.repanda、N.rosulata subsp.ingulba、N.rotundifolia、N.setchellii、N.simulans、N.solanifolia、N.spegazzinii、N.stocktonii、N.suaveolens、N.sylvestr is, N. thyrsiflora, N. tomentosa, N. tomentosiformis, N. trigonophylla, N. umbratica, N. undulata, N. velutina, N. wigandioides, Nx sanderae has a slightly different variety than N.tabacum.

[0170] The use of tobacco cultivars and elite tobacco cultivars is also contemplated herein. Thus, a transgenic, non-naturally occurring, or mutant plant may be a tobacco cultivar or elite tobacco cultivar that contains one or more introduced genes, or one or more genetic mutations, or a combination thereof. The genetic mutations (e.g., one or more polymorphisms) may be mutations that do not naturally occur in a particular tobacco cultivar or cultivar (e.g., elite tobacco cultivar), or may be genetic mutations that occur naturally, provided that the mutations do not naturally occur in a particular tobacco cultivar or cultivar (e.g., elite tobacco cultivar).

[0171] Particularly useful Nicotiana tabacum varieties include burley-type, dark-type, flue-cured-type, and Orient-type tobaccos. Non-limiting examples of varieties or cultivars include BD64, CC101, CC200, CC27, CC301, CC400, CC500, CC600, CC700, CC800, CC900, Coker 176, Coker 319, Coker 371 Gold, Coker 48, CD263, DF911, DT538 LC Galpao tobacco, GL26H, GL350, GL600, GL737, GL939, GL973, HB04P, HB04P LC, HB3307PLC, Hybrid 403LC, Hybrid 404LC, Hybrid 501 LC, K149, K326, K346, K358, K394, K399, K730, KDH959, KT200, KT204LC, KY10, KY14, KY160, KY17, KY171, KY907, KY907LC, KY14xL8 LC, Little Crittenden, McNair 373, McNair 944, msKY 14xL8, Narrow Leaf Madole, Narrow Leaf Madole LC, NBH98, N-126, N-777LC, N-7371LC, NC100, NC102, NC2000, NC291, NC297, NC299, NC3, NC4, NC5, NC6, NC7, NC606, NC71, NC72, NC810, NC BH 129, NC2002, Neal Smith Madole, OXFORD207, PD7302 LC, PD7309 LC, PD7312 LC, "Perique" cigarettes, PVH03, PVH09, PVH19, PVH50, PVH51, R610, R630, R7-11, R7-12, RG17, RG81, RGH51, RGH4, RGH51, RS1 410, Speight168, Speight172, Speight179, Speight210, Speight220, Speight225, Speight227, Speight234, Speight G-28, Speight G-70, Speight H-6, Speight H20, Speight NF3, TI1406, TI1269, TN86, TN86LC, TN90, TN97, TN97LC, TN D94, TN D950, TR (Tom Rosson) Madole, VA309, VA359, AA37-1, B13P, Xanthi (Mitchell-Mor), Bel-W3, 79-615, Samsun Holmes NN, KTRDC No. 2 Hybrid 49, Burley 21, KY8959, KY9, MD609, PG01, PG04, PO1, PO2, PO3, RG11, RG8, VA509, AS44, Banket A1, Basma Drama B84 / 31, Basma I Zichna ZP4 / B, Basma Xanthi BX 2A, Batek, Besuki Jember, C104, Coker 347, Criollo Misionero, Delcrest, Djebel 81, DVH405, Galpao Comum, HB04P, Hicks Broadleaf, Kabakulak Elassona, Kutsage E1, LA BU 21, NC2326, NC297, PVH2110, Red Russian, Samsun, Saplak, Simmaba, Talgar28, Wislica, Yayaldag, Prilep HC-72, Prilep P23, Prilep PB 156 / 1, Prilep P12-2 / 1, Yaka JK-48, Yaka JB 125 / 3, TI-1068, KDH-960, TI-1070, TW136, Basma, TKF4028, L8, TKF2002, GR141, Basma xanthi, GR149, GR153, Petit Havana. The above low converter sub-varieties are also contemplated, even if not specifically identified herein.

[0172] Embodiments are also directed to compositions and methods for producing mutant, non-naturally occurring, hybrid, or transgenic plants that have been modified to regulate the expression or function of one or more NtSWEET polynucleotides described herein (or any combination thereof described herein). Advantageously, the resulting mutant, non-naturally occurring, hybrid, or transgenic plants can be similar or substantially identical to control plants in overall appearance. Various phenotypic characteristics, such as degree of maturity, number of leaves per plant, petiole height, leaf insertion angle, leaf size (width and length), internode distance, and blade-to-midrib ratio, can be assessed by field observation.

[0173] One aspect relates to seeds of the mutant plants, non-natural plants, hybrid plants, or transgenic plants described herein. Preferably, the seeds are tobacco seeds. A further aspect relates to pollen or ovules of the mutant plants, non-natural plants, hybrid plants, or transgenic plants described herein. Additionally, provided are mutant plants, non-natural plants, hybrid plants, or transgenic plants described herein, further comprising a polynucleotide that confers male sterility.

[0174] Also provided are tissue cultures of regenerable cells of the mutant, non-naturally occurring, hybrid, or transgenic plants described herein, or portions thereof, which cultures regenerate plants capable of expressing all the morphological and physiological characteristics of the parent. Regenerable cells include cells from leaves, pollen, embryos, cotyledons, hypocotyls, roots, root tips, anthers, flowers and parts thereof, ovules, shoots, stems, petioles, pith, and capsules, or callus or protoplasts derived therefrom.

[0175] The plant material described herein can be cured or cured tobacco material. CORESTA recommendations for tobacco curing are set forth in CORESTA Guide No. 17, April 2016, Sustainability in Leaf Tobacco Production.

[0176] According to the present disclosure, the expression of one or more of SWEET12-S or SWEET12-T or SWEET15-S or SWEET15-T, or the activity of one or more of SWEET12-S or SWEET12-T or SWEET15-S or SWEET15-T, is regulated compared to a control plant.

[0177] In one embodiment, the expression or activity of SWEET12-S is regulated compared to a control plant. In another embodiment, the expression or activity of SWEET12-T is regulated compared to a control plant. In another embodiment, the expression or activity of SWEET12-S and SWEET12-T is regulated compared to a control plant.

[0178] In another embodiment, the expression or activity of SWEET15-S is regulated compared to a control plant. In another embodiment, the expression or activity of SWEET15-T is regulated compared to a control plant. In another embodiment, the expression or activity of SWEET15-S and SWEET15-T is regulated compared to a control plant. In another embodiment, the expression or activity of SWEET15-S and SWEET15-T is regulated compared to a control plant.

[0179] In another embodiment, the expression of SWEET12-S and SWEET15-S, or the activity of SWEET12-S and SWEET15-S, is modulated compared to a control plant.

[0180] In another embodiment, the expression of SWEET12-S and SWEET15-T, or the activity of SWEET12-S and SWEET15-T, is modulated compared to a control plant.

[0181] In another embodiment, the expression of SWEET12-T and SWEET15-S, or the activity of SWEET12-T and SWEET15-S, is modulated compared to a control plant.

[0182] In another embodiment, the expression of SWEET12-T and SWEET15-T, or the activity of SWEET12-T and SWEET15-T, is modulated compared to a control plant.

[0183] In another embodiment, the expression of SWEET12-S and SWEET12-T and SWEET15-S, or the activity of SWEET12-S and SWEET12-T and SWEET15-S, is modulated compared to a control plant.

[0184] In another embodiment, the expression of SWEET12-S and SWEET12-T and SWEET15-T, or the activity of SWEET12-S and SWEET12-T and SWEET15-T, is modulated compared to a control plant.

[0185] In another embodiment, the expression of SWEET12-S and SWEET15-S and SWEET15-T, or the activity of SWEET12-S and SWEET15-S and SWEET15-T, is modulated compared to a control plant.

[0186] In another embodiment, the expression of SWEET12-T and SWEET15-S and SWEET15-T, or the activity of SWEET12-T and SWEET15-S and SWEET15-T, is modulated compared to a control plant.

[0187] In another embodiment, the expression of SWEET12-S and SWEET12-T and SWEET15-S and SWEET15-T, or the activity of SWEET12-S and SWEET12-T and SWEET15-S and SWEET15-T, is modulated compared to a control plant.

[0188] Regulating the expression of one or more of SWEET12-S, SWEET12-T, SWEET15-S, or SWEET15-T, or regulating the activity of one or more of SWEET12-S, SWEET12-T, SWEET15-S, or SWEET15-T, compared to a control plant, can regulate the development of the vegetative growth phase of a plant compared to a control plant and / or regulate the flowering time of a plant compared to a control plant. For example, regulating (preferably decreasing) the expression of SWEET12-S and / or SWEET12-T, or the activity of SWEET12-S and / or SWEET12-T can regulate (preferably increase) the height of a plant during the vegetative growth phase, which can lead to an accelerated flowering time. As a further example, modulating (preferably decreasing) the expression of SWEET15-S and / or SWEET15-T or the activity of SWEET15-S and / or SWEET15-T can modulate (preferably increase) the height of a plant during the vegetative growth phase, which can lead to an accelerated flowering time. As a further example, modulating (preferably increasing) the expression of SWEET15-T and / or SWEET15-S or the activity of SWEET15-T and / or SWEET15-S can modulate (e.g., accelerate or increase) the flowering time.

[0189] The chemical profile of dried leaves from a plant can be adjusted by regulating the expression of one or more of SWEET12-S, SWEET12-T, SWEET15-S, or SWEET15-T, or the activity of one or more of SWEET12-S, SWEET12-T, SWEET15-S, or SWEET15-T, compared to a control plant. The chemical profile can include a sugar profile and / or an amino acid profile. As an example, the fructose, glucose, and sucrose content of dried leaves can be adjusted (preferably decreased) by regulating (preferably increasing) the expression of SWEET12-S and / or SWEET12-T, or the activity of SWEET12-S and / or SWEET12-T. As a further example, the fructose, glucose, and sucrose content of dried leaves can be adjusted (preferably decreased) by regulating (preferably increasing) the expression of SWEET15-T or the activity of SWEET15-T. As a further example, the expression of SWEET15-S and SWEET15-T or the activity of SWEET15-S and SWEET15-T can be modulated (preferably increased) to modulate (preferably decrease) the fructose, glucose, and sucrose content of the drying process or dried leaves.

[0190] The amino acid profile of dried leaves from a plant can be adjusted by regulating the expression of one or more of SWEET12-S, SWEET12-T, SWEET15-S, or SWEET15-T, or the activity of one or more of SWEET12-S, SWEET12-T, SWEET15-S, or SWEET15-T, compared to a control plant. The amino acid profile can include asparagine, tryptophan, and proline, or asparagine, tryptophan, proline, phenylalanine, glycine, methionine, and glutamic acid. The amino acid profile can be adjusted to increase or decrease the levels of one or more amino acids. In one embodiment, the levels of asparagine and tryptophan are increased, and the levels of proline are decreased. In another embodiment, the levels of asparagine, tryptophan, phenylalanine, glycine, and methionine are increased, and the levels of glutamic acid and proline are decreased. For example, modulating (preferably increasing) the expression of SWEET12-S and / or SWEET12-T or the activity of SWEET12-S and / or SWEET12-T can modulate the levels of asparagine, tryptophan, proline, phenylalanine, glycine, methionine, and glutamic acid, preferably increasing the levels of asparagine, tryptophan, phenylalanine, glycine, and methionine and decreasing the levels of glutamic acid and proline. As a further example, modulating (preferably increasing) the expression of SWEET15-T and / or SWEET15-S or the activity of SWEET15-T and / or SWEET15-S can modulate the levels of asparagine, tryptophan, and proline, preferably increasing the levels of asparagine and tryptophan and decreasing the level of proline.When the expression of SWEET12-S and / or SWEET12-T, or the activity of SWEET12-S and / or SWEET12-T, is reduced, no significant changes in sugar or amino acid levels are observed, but the sensory profile of the flue-cured or cured tobacco still changes (see Table 6).

[0191] The amount of leaf biomass can be adjusted by regulating the expression of one or more of SWEET12-S, SWEET12-T, SWEET15-S, or SWEET15-T, or the activity of one or more of SWEET12-S, SWEET12-T, SWEET15-S, or SWEET15-T, compared to a control plant. As an example, the amount of leaf biomass can be adjusted (preferably decreased) by regulating (preferably increasing) the expression of SWEET12-S and / or SWEET12-T, or the activity of SWEET12-S and / or SWEET12-T. As a further example, the amount of leaf biomass can be adjusted (preferably decreased) by regulating (preferably increasing) the expression of SWEET15-S and / or SWEET15-T, or the activity of SWEET15-S and / or SWEET15-T. Under some conditions, such as when the expression of SWEET12-S and / or SWEET12-T or the activity of SWEET12-S and / or SWEET12-T is reduced, or when the expression of SWEET15-S and / or SWEET15-T or the activity of SWEET15-S and / or SWEET15-T is reduced, there is no significant change in the amount of leaf biomass. In certain embodiments, it is preferable that the amount of leaf biomass is not reduced, so as to maintain the yield that can be obtained during commercial tobacco production.

[0192] The ammonia level can be adjusted by modulating the expression of SWEET12-S and / or SWEET12-T or the activity of SWEET12-S or SWEET12-T compared to a control plant. As an example, the ammonia level can be adjusted (preferably increased) by modulating (preferably increasing) the expression of SWEET12-S and / or SWEET12-T or the activity of SWEET12-S and / or SWEET12-T. Ammonia compounds are known to react with sugars during tobacco processing and smoking to form flavor compounds that alter the taste of tobacco smoke. Therefore, modulating the ammonia level is expected to alter the flavor of tobacco.

[0193] Preferably, the adjusted level is observed in at least cured or dried leaves, preferably in fully cured or dried leaves. Tobacco is considered fully cured when there is no moisture in the midvein of the leaf, resulting in leaves that are light yellow-brown to reddish-brown to dark brown in color. Preferably, the cured leaves are taken from leaves at the middle position of the plant.

[0194] Further embodiments relate to mutant, non-native, or transgenic plants or cells in which the expression of one or more NtSWEET polynucleotides or the activity of one or more NtSWEET polypeptides is modulated compared to a control plant or part thereof in which NtSWEET expression or NtSWEET activity is not modulated.

[0195] Still further aspects relate to cured or dried plant material, such as cured or dried leaves or cured or dried tobacco, derived or derivable from a mutant, non-native, or transgenic plant or cell in which expression of one or more of the NtSWEET polynucleotides described herein, or the function of the NtSWEET polypeptide encoded thereby, is modulated compared to a control plant or portion thereof.

[0196] Embodiments also relate to compositions and methods for generating mutant, non-native, or transgenic plants or plant cells that have been modified to modulate the expression or activity of one or more of the NtSWEET polynucleotides or NtSWEET polypeptides described herein.

[0197] In one embodiment, the phenotype of the mutant, non-native, or transgenic plant is substantially the same as that of a control plant or portion thereof. In one embodiment, the leaf weight of the mutant, non-native, or transgenic plant is substantially the same as that of a control plant or portion thereof. In one embodiment, the leaf number of the mutant, non-native, or transgenic plant is substantially the same as that of a control plant or portion thereof. In one embodiment, the leaf weight and leaf number of the mutant, non-native, or transgenic plant are substantially the same as that of a control plant. In one embodiment, the petiole height of the mutant, non-native, or transgenic plant is substantially the same as that of a control plant or portion thereof, for example, one month, two months, or three months or more after field transplantation, or 10 days, 20 days, 30 days, or 36 days or more after topping. For example, the petiole height of the mutant, non-native, or transgenic plant does not exceed that of the control plant or portion thereof.

[0198] In another aspect, there is provided a method for modulating the amount of at least one amino acid and / or at least one sugar in at least a portion of a plant (e.g., leaves, such as cured or dried leaves), the method comprising: (i) modulating the expression or function of one or more NtSWEET polypeptides described herein, suitably wherein the NtSWEET polypeptide is encoded by a corresponding NtSWEET polynucleotide described herein; (ii) measuring the level of at least one amino acid and / or at least one sugar in at least a portion of a mutant, non-native, or transgenic plant obtained in step (i) (e.g., leaves, such as cured or dried leaves, or tobacco or smoke); and (iii) identifying a mutant, non-native, or transgenic plant or portion thereof in which the level of at least one amino acid and / or sugar is modulated compared to a control plant or portion thereof.

[0199] In another aspect, there is provided a method for modulating the amount of at least one amino acid and / or at least one sugar in dried leaves or dried plant material, such as dried leaves, comprising: (i) modulating the expression or function of one or more NtSWEET polypeptides (or any combination thereof as described herein), suitably wherein the NtSWEET polypeptides are encoded by corresponding NtSWEET polynucleotides as described herein; (ii) harvesting and drying one or more plant materials, such as leaves, for a period of time; (iii) measuring the level of at least one amino acid and / or at least one sugar in the dried plant material or dried plant material obtained in or during step (ii); and (iv) identifying the dried plant material or dried plant material in which the level of at least one amino acid and / or at least one sugar is modulated compared to a control plant or portion thereof.

[0200] The increase in expression compared to a control can be from about 5% to about 100%, or at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, or an increase of 100% or more, such as a 200%, 300%, 500%, 1000% or more increase, including transcriptional function, or NtSWEET polynucleotide expression, or NtSWEET polypeptide expression.

[0201] The increase in function or activity compared to a control can be from about 5% to about 100%, or at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, or an increase of 100% or more, such as 200%, 300%, 500%, 1000% or more, including transcriptional function, or NtSWEET polynucleotide expression, or NtSWEET polypeptide expression, or a combination thereof.

[0202] The reduction in expression compared to a control can be from about 5% to about 100%, or at least a 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, or 100% reduction, including transcriptional function, or NtSWEET polynucleotide expression, or NtSWEET polypeptide expression, or a combination thereof.

[0203] The decrease in function or activity compared to a control can be from about 5% to about 100%, or at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, or 100%, including a decrease in transcription function, or NtSWEET polynucleotide or polypeptide expression, or a combination thereof.

[0204] The polynucleotides and recombinant constructs described herein can be used to modulate the expression or function or activity of an NtSWEET polynucleotide or NtSWEET polypeptide described herein in a plant species of interest, preferably tobacco.

[0205] Numerous polynucleotide-based methods can be used to increase gene expression in plants and plant cells. As an example, a construct, vector, or expression vector compatible with the plant to be transformed can be prepared, containing a gene of interest along with an upstream promoter capable of overexpressing the gene in the plant or plant cell. Exemplary promoters are described herein. After transformation and when grown under suitable conditions, the promoter can drive expression to regulate the level of NtSWEET in the plant, or specific tissues thereof. In one exemplary embodiment, a vector carrying one or more of the NtSWEET 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, driving its constitutive expression in all tissues of the plant. The vector also carries an antibiotic resistance gene to allow for selection of transformed callus and cell lines.

[0206] Expression of the sequence from the promoter can be enhanced by including expression control sequences known in the art. Signals associated with aging and signals active during the desiccation process are specifically shown.

[0207] Accordingly, various embodiments are directed to methods for modulating the expression levels of one or more NtSWEET polynucleotides described herein (or any combination thereof as described herein) by integrating multiple copies of the NtSWEET polynucleotides into a plant genome, comprising transforming a plant cell host with an expression vector comprising a promoter operably linked to one or more NtSWEET polynucleotides described herein. The polypeptide encoded by the recombinant polynucleotide can be a native polypeptide or can be heterologous to the cell.

[0208] In one embodiment, a plant for use in the present disclosure is a hot-tube-dried mutant, non-native, or transgenic plant.

[0209] In one embodiment, a plant for use in the present disclosure is a sun-drought treated mutant, non-native, or transgenic plant.

[0210] In one embodiment, the plants used in the present disclosure are air-dried mutant, non-native, or transgenic plants.

[0211] In one embodiment, a plant for use in the present disclosure is a mutant, non-native, or transgenic Virginia tobacco plant that has been cured, for example, tube-cured.

[0212] In one embodiment, a plant for use in the present disclosure is a cured, e.g., air-cured, mutant, non-native, or transgenic burley tobacco plant.

[0213] In one embodiment, a plant for use in the present disclosure is a cured, e.g., flue-cured, mutant, non-native, or transgenic dark tobacco plant.

[0214] By modulating NtSWEET expression and / or NtSWEET activity, the sensory profile of tobacco can be advantageously modified, as can be seen from Table 6.

[0215] Plants carrying one or more mutant alleles of the NtSWEET polynucleotides described herein can be used in plant breeding programs to create useful lines, varieties, and hybrids. In particular, mutant alleles can be introgressed into commercially important varieties described herein. Accordingly, provided are methods for plant breeding, including crossing a mutant, non-naturally occurring, or transgenic plant described herein with a plant containing a different genetic identity. The method can further include crossing the progeny plant with another plant, and optionally repeating the crossing until progeny possessing desirable genetic traits or genetic backgrounds are obtained. One goal achieved by such breeding methods is to introduce desirable genetic traits into other varieties, breeding lines, hybrids, or cultivars, particularly those of commercial interest. Another goal is to facilitate stacking of genetic modifications of different genes in a single plant variety, line, hybrid, or cultivar. Both interspecific and intraspecific crosses are contemplated. The progeny plants resulting from such crosses are also referred to as breeding lines and are examples of the disclosed non-naturally occurring plants.

[0216] In one embodiment, a method for producing a non-naturally occurring plant is provided, comprising: (a) crossing a mutant or transgenic plant with a second plant to obtain progeny tobacco seeds; and (b) growing the progeny tobacco seeds under plant growth conditions to obtain a non-naturally occurring plant. The method may further comprise: (c) crossing the previous generation non-naturally occurring plant with itself or another plant to obtain progeny tobacco seeds; (d) growing the progeny tobacco seeds of step (c) under plant growth conditions to obtain additional non-naturally occurring plants; and (e) repeating the crossing and growing steps of (c) and (d) multiple times to generate additional generations of non-naturally occurring plants. The method may optionally comprise, prior to step (a), providing a parent plant that comprises a characterized genetic identity and is not identical to the mutant or transgenic plant. In some embodiments, depending on the breeding program, the crossing and growing steps are repeated 0-2, 0-3, 0-4, 0-5, 0-6, 0-7, 0-8, 0-9, or 0-10 times to generate generations of the non-naturally occurring plant. Backcrossing is an example of such a method in which the progeny is crossed with one of the parents or another plant genetically similar to that parent to obtain a next-generation progeny plant with a genetic identity closer to that of one of the parents. Plant breeding, particularly plant breeding techniques, are well known and can be used in the disclosed methods. The present disclosure further provides non-naturally occurring plants produced by these methods. Certain embodiments exclude the step of selecting the plants.

[0217] In some embodiments of the methods described herein, lines resulting from breeding and screening for variant genes are evaluated in the field using standard field procedures. Control genotypes, including the original, unmutagenized parents, are included, and entries are arranged in the field in a randomized complete block design or other suitable field design. For tobacco, standard agronomic methods are used, e.g., the tobacco is harvested, weighed, and sampled for chemical and other common tests before and during curing. Statistical analysis of the data is performed to confirm the similarity of the selected lines to the parent lines. Cytogenetic analysis of the selected plants is optionally performed to confirm chromosome complement and chromosome pairing relationships.

[0218] DNA fingerprinting, single nucleotide polymorphism, microsatellite markers, or similar techniques can be used in marker-assisted selection (MAS) breeding programs to introgress or breed mutant alleles of genes into other tobacco plants, as described herein. For example, breeders can create segregating populations from hybridization of genotypes containing mutant alleles with agronomically desirable genotypes. F2 or backcross generation plants can be screened using markers developed from the genomic sequence or a fragment thereof using one of the techniques listed herein. Plants identified as carrying the mutant allele can be backcrossed or self-pollinated to create a second population to be screened. Depending on the expected inheritance pattern or MAS technique used, it may be necessary to self-pollinated selected plants before each backcross cycle to help identify the desired individual plants. Backcrossing or other breeding procedures can be repeated until the desired phenotype of the recurrent parent is restored.

[0219] According to the present disclosure, in a breeding program, successful crosses produce fertile F1 plants. The selected F1 plants can be crossed with one of the parents, and the first backcross generation plants are self-pollinated to produce a population that is again screened for variant gene expression (e.g., a null version of the gene). The backcross, self-pollination, and screening process is repeated, for example, at least four times, until the final screening produces a plant that is fertile and reasonably similar to the recurrent parent. If desired, the plant is self-pollinated, and then the progeny are again screened to confirm that the plant exhibits variant gene expression. In some embodiments, the F2 generation plant population is screened for variant gene expression, for example, by using PCR methods with primers based on polynucleotide sequence information for the polynucleotides described herein (or any combination thereof described herein) according to standard methods, to identify plants that do not express the polypeptide due to a gene defect.

[0220] Hybrid tobacco varieties can be produced by preventing self-pollination of a female parent plant (i.e., seed parent) of a first variety, allowing pollen from a male parent plant of a second variety to pollinate the female parent plant, resulting in the formation of F1 hybrid seeds in the female plant. Self-pollination of female plants can be prevented by removing the stamens of flowers at an early stage of flower development. Alternatively, pollen formation can be prevented in female parent plants using male sterility formation. For example, male sterility can be produced by cytoplasmic male sterility (CMS) or transgenic male sterility, where the introduced gene inhibits microspore production and / or pollen formation, or self-incompatibility. Female parent plants containing CMS are particularly useful. In embodiments where the female parent plant is CMS, pollen is harvested from a male fertile plant and manually applied to the stigma of a CMS female parent plant, and the resulting F1 seed is harvested.

[0221] The varieties and lines described herein can be used to form single-cross tobacco F1 hybrids. In such embodiments, plants of the parent varieties can be grown as a substantially homogeneous, adjacent population to promote natural cross-pollination from the male parent plant to the female parent plant. The F1 seeds formed in the female parent plant are selectively harvested by conventional means. Two parent plant varieties can also be grown in large quantities, and a blend of F1 hybrid seeds formed in the female parent and seeds formed in the male parent as a result of self-pollination can be harvested. Alternatively, a three-way cross can be performed, in which a single-cross F1 hybrid is used as the female parent and crossed with a different male parent. As another alternative, a double-cross hybrid can be created, in which the F1 progeny of two different single crosses are crossed with themselves.

[0222] A population of mutant, non-native, or transgenic plants can be screened or selected for those members of the population that have 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 include Southern analysis or PCR amplification for detecting polynucleotides; Northern blots, S1 RNase protection, primer extension, or RT-PCR amplification for detecting RNA transcripts; enzymatic assays for detecting enzyme or ribozyme activity of polypeptides and polynucleotides; and polypeptide gel electrophoresis, Western blots, immunoprecipitation, and enzyme immunoassays for detecting polypeptides. Other techniques, such as in situ hybridization, enzyme staining, and immunostaining and enzyme assays, can also be used to detect the presence or expression, function, or activity of NtSWEET polypeptides or NtSWEET polynucleotides.

[0223] Described herein are mutant, non-native or transgenic plant cells and plants comprising 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.

[0224] Without limitation, the plants and parts thereof described herein can be modified either before or after the expression, function, or activity of one or more NtSWEET polynucleotides and / or NtSWEET polypeptides according to the present disclosure is modulated.

[0225] One or more of the following additional genetic modifications may be present in mutant, non-native, or transgenic plants and their parts: One or more genes involved in the conversion of nitrogenous metabolic intermediates may be modified to result in lower levels of at least one tobacco-specific nitrosamine (TSNA). Non-limiting examples of such genes include those encoding nicotine demethylases, such as CYP82E4, CYP82E5, and CYP82E10, as described in WO2006 / 091194, WO2008 / 070274, WO2009 / 064771, and WO2011 / 088180, and nitrate reductase, as described in WO2016 / 046288. One or more genes involved in heavy metal uptake or heavy metal transport may be modified to result in lower heavy metal content. Non-limiting examples include genes in the family of polypeptides associated with multidrug resistance, the family of cation diffusion facilitators (CDFs), the family of Zrt-Irt-like polypeptides (ZIPs), the family of cation exchangers (CAXs), the family of copper transporters (COPTs), the family of heavy metal ATPases (e.g., HMAs described in WO2009 / 074325 and WO2017 / 129739), the family of homologs of natural resistance-associated macrophage polypeptide (NRAMP), and other members of the ATP-binding cassette (ABC) transporter family (e.g., MRPs) described in WO2012 / 028309, which are involved in the transport of heavy metals such as cadmium.

[0226] Another exemplary modification could result in a plant with regulated expression or function of isopropylmalate synthase, resulting in changes in sucrose ester composition that can be used to alter the beneficial profile (see WO 2013 / 029799). Another exemplary modification could result in a plant with regulated expression or function of threonine synthase, which can regulate methionine levels (see WO 2013 / 029800). Another exemplary modification could result in a plant with regulated expression or function of one or more of neoxanthin synthase, lycopene beta cyclase, and 9-cis-epoxycarotenoid dioxygenase, which regulates beta-damascenone content to alter the flavor profile (see WO 2013 / 064499). Another exemplary modification could result in a plant with regulated expression or function of a member of the CLC family of chloride channels, which regulates nitrate levels therein (see WO 2014 / 096283 and WO 2015 / 197727). Another exemplary modification can result in plants with regulated expression or function of one or more asparagine synthetases that regulate asparagine levels in leaves and regulated levels of acrylamide in the aerosol produced upon heating or burning of leaves (see WO2017 / 129739). Another exemplary modification can result in plants with regulated protease activity during desiccation (see WO2016 / 009006). Another exemplary modification can result in plants with reduced nitrate levels by altering the gene expression of nitrate reductase (e.g., Nia2) or the activity of the protein encoded thereby (see WO2016 / 046288).Another exemplary modification could result in plants with altered alkaloid levels by altering the gene expression of the putative ABC-2 transporters NtABCGl-T and NtABCGl-S or the activity of the protein encoded thereby (see WO 2019 / 086609). Another exemplary modification could result in plants that regulate the time to flowering by altering the gene expression of the gene encoding Terminal Flower 1 (TFL1) or the activity of the protein encoded thereby (see WO 2018 / 114641). Another exemplary modification could result in plants with regulated expression or function of one or more asparagine synthases to regulate the level of asparagine in leaves, and plants that regulate the level of acrylamide in the aerosol produced when leaves are heated or burned (see WO 2017 / 042162). Other exemplary modifications include herbicide tolerance, e.g., glyphosate is the active ingredient in numerous broad-spectrum herbicides. Glyphosate-tolerant transgenic plants have been developed by transferring the aroA gene (glyphosate EPSP synthase from Salmonella typhimurium and E. coli). Sulfonylurea-tolerant plants have been produced by transforming mutant ALS (acetolactate synthase) genes from Arabidopsis. Mutant photosystem II OB polypeptides from Amaranthus hybridus have been transferred to plants to produce atrazine-tolerant transgenic plants, and bromoxynil-tolerant transgenic plants have been produced by incorporating the bxn gene from Klebsiella pneumoniae bacteria. Another exemplary modification results in plants that are resistant to insects. Bacillus thuringiensis (Bt) toxins can provide an effective way to delay the emergence of Bt-resistant pests, as recently demonstrated in broccoli, where the cry1Ac and cry1C Bt genes in cones controlled diamondback moths resistant to either single polypeptide, significantly slowing the evolution of resistant insects.Another exemplary modification results in plants that are resistant to diseases caused by pathogens (e.g., viruses, bacteria, fungi). Plants expressing the Xa21 gene (resistance to bacterial leaf spot) have been engineered with plants expressing both a Bt fusion gene and a chitinase gene (resistance to yellow stem borer and pod disease). Another exemplary modification results in altered fertility, such as male sterility. Another exemplary modification results in plants that are tolerant to abiotic stress (e.g., drought, temperature, salinity); tolerant transgenic plants have been produced by transferring acylglycerol phosphate enzymes from Arabidopsis; genes encoding mannitol dehydrogenase and sorbitol dehydrogenase, involved in the synthesis of mannitol and sorbitol, improve drought tolerance. Another exemplary modification results in a plant in which the activity of one or more nicotine N-demethylases is regulated so that the levels of nornicotine and nornicotine metabolites formed during drying can be controlled (see WO2015169927). Other exemplary modifications can result in plants with improved polypeptide and oil storage capacity, increased photosynthetic efficiency, extended shelf life, increased carbohydrate content, and fungal resistance. Transgenic plants with regulated expression of S-adenosyl-L-methionine (SAM) or cystathionine gamma-synthase (CGS), or a combination thereof, are also contemplated. One or more genes involved in the nicotine synthesis pathway can be modified to control the nicotine level when a plant or plant part is subjected to a drying process or drying. The nicotine synthesis gene may be selected from the group consisting of A622, BBLa, BBLb, JRE5L1, JRE5L2, MATE1, MATE2, MPO1, MPO2, MYC2a, MYC2b, NBBl, nic1, nic2, NUP1, NUP2, PMT1, PMT2, PMT3, PMT4 and QPT or one or more combinations thereof.One or more genes involved in controlling the amount of one or more alkaloids can be modified to result in a plant or plant part that produces regulated levels of alkaloids. The alkaloid level control gene can be selected from the group consisting of BBLa, BBLb, JRE5L1, JRE5L2, MATE1, MATE2, MYC2a, MYC2b, nic1, nic2, NUP1 and NUP2, or a combination of two or more thereof.

[0227] Other exemplary modifications can result in plants with modulated amino acid content (see WO2019 / 185703 and WO2021 / 063863), or plants with modulated sugar content (see WO2019 / 185699 and WO2021 / 063860 and WO2021 / 063863), or plants with modulated nitrate levels (see WO2020 / 141062), or plants with modulated sugar and amino acid content (see WO2021 / 063863).

[0228] In a preferred embodiment, yet another genetic modification relates to the asparagine synthetase (ASN) gene 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 described in WO2017042162) or the activity of ASN (e.g., NtASN1-S, NtASN1-T, NtASN5-S, and NtASN5-T described in WO2017042162) significantly alters the chemical properties of cured or cured tobacco leaves without affecting biomass. Thus, modulating the expression and / or activity of a combination of ASN and NtSWEET may have the potential to reconfigure the chemical properties of cured or cured tobacco leaves (particularly the amino acid chemistry of burley or dark tobacco), thereby altering their sensory characteristics.

[0229] In addition to ASN, other genes and enzymes play a role in the reorganization of amino acids and / or sugars during leaf yellowing, such as diaminopimelate aminotransferase (DAPAT), which is involved in both lysine catabolism and assimilation, and aspartate aminotransferase (AAT), which is expressed during senescence and may alter leaf chemistry after drought treatment (WO2019 / 185703). The chloroplast sulfate transporter SULTR3, such as NtSULTR3;1A-S, NtSULTR3;1A-T, and NtSULTR3;3-T, plays a role in sugar and amino acid metabolism during drought treatment (see WO2021 / 063863). Thus, additional genetic modifications may involve DAPAT and / or AAT (e.g., one or more of NtAATI-S, NtAAT1-T, NtAAT2-S, NtAAT2-T, NtAAT3-S, NtAAT3-T, NtAAT4-S, or NtAAT4-T as described in WO2017042162), and / or one or more of NtSULTR3;1A-S, NtSULTR3;1A-T, and NtSULTR3;3-T as described in WO2021 / 063863. Modulating the expression and / or activity of a combination of DAPAT and / or AAT and / or SULTR3 and NtSWEET may have the potential to reconfigure the chemistry of flue-cured or cured tobacco leaves, thereby altering their organoleptic properties.Modifications to combinations of NtSWEET with one or more, or two or more, or three or more, or four or more of ASN and DAPAT and AAT and SULTR3 are disclosed, including NtSWEET and ASN, NtSWEET and DAPAT, NtSWEET and AAT, NtSWEET and ASN and DAPAT, NtSWEET and ASN and AAT, NtSWEET and ASN and DAPAT and AAT, NtSWEET and SULTR3, NtSWEET and ASN and SULTR3, NtSWEET and AAT and SULTR3, NtSWEET and ASN and DAPAT and SULTR3, NtSWEET and ASN ... AAT and SULTR3.

[0230] One or more traits can be introgressed from another cultivar into, or directly transformed into, a mutant, non-native, or transgenic plant.

[0231] Various embodiments provide mutant plants, non-naturally occurring plants, or transgenic plants, as well as 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.

[0232] The plant parts described herein, particularly the leaf blades and / or petioles and / or midribs of such plants, can be incorporated into or used in various consumable products, including, but not limited to, aerosol-forming materials, aerosol-forming devices, smoking articles, smokable articles, smokeless products, medical or cosmetic products, intravenous preparations, tablets, powders, and tobacco products. Examples of aerosol-forming materials include tobacco compositions, tobacco, tobacco extracts, cut tobacco, cut fillers, flue-cured or cured tobacco, expanded tobacco, homogenized tobacco, reconstituted tobacco, and pipe tobacco. Smoking articles and smokable articles are types of aerosol-forming devices. Examples of smoking articles or smokable articles include cigarettes, cigarillos, and cigars. Examples of smokeless products include chewing tobacco and snuff. In certain aerosol-forming devices, rather than combustion, the tobacco composition or another aerosol-forming material is heated by one or more electric heating elements to produce an aerosol. In another type of heated aerosol-forming device, the aerosol is produced by the transfer of heat from a combustible fuel element or heat source to a physically separated aerosol-forming material that may be located within, around, or downstream of the heat source. Smokeless tobacco products and various tobacco-containing aerosol-forming materials may contain tobacco in any form, including as dry particles, pieces, granules, powders, or slurries, or deposited on, mixed with, surrounded by, or combined with any form of other component, such as flakes, films, tabs, foams, or beads. The term "smoke" is used to describe the type of aerosol produced by a smoking article, such as a cigarette, or by burning an aerosol-forming material.

[0233] In one embodiment, cured or dried plant material derived from the mutant, transgenic, and non-naturally occurring plants described herein is also provided. Processes for curing green tobacco leaves are known to those skilled in the art and include, but are not limited to, the air-curing, flame-curing, hot-tube-curing, and sun-curing processes described herein.

[0234] In another embodiment, a tobacco product is described that includes a tobacco-containing aerosol-forming material, including plant material (e.g., leaves, preferably cured or dried leaves) from a mutant tobacco plant, a transgenic tobacco plant, or a non-naturally occurring tobacco plant described herein. The tobacco product described herein can be a blended tobacco product, which can further include unmodified tobacco.

[0235] Mutant, non-native or transgenic plants may have other uses, for example in agriculture.

[0236] The present disclosure also provides a method for producing seeds, including cultivating a mutant plant, a non-natural plant, or a transgenic plant described herein and collecting seeds from the cultivated plant.Seeds from the plants described herein can be prepared by means known in the art and packaged in packaging materials to form manufactured articles.Packaging materials such as paper and cloth are well known in the art.The seed package has a label, for example, a tag or label fixed to the packaging material, and a label describing the nature of the seeds therein is printed on the package.

[0237] Compositions, methods, and kits for genotyping plants for identification, selection, or breeding can include means for detecting the presence of NtSWEET polynucleotides in a sample of polynucleotides. Accordingly, compositions are described that include one or more primers for specifically amplifying at least a portion of one or more NtSWEET polynucleotides, and optionally one or more probes and optionally one or more reagents for carrying out the amplification or detection.

[0238] Thus, gene-specific oligonucleotide primers or probes are disclosed that comprise about 10 or more contiguous polynucleotides corresponding to the NtSWEET polynucleotides described herein. Such primers or probes can comprise or consist of more than about 15, 20, 25, 30, 40, 45, or 50 contiguous polynucleotides that hybridize (e.g., specifically hybridize) to the NtSWEET polynucleotides described herein. In some embodiments, the primers or probes can comprise or consist of about 10-50 contiguous nucleotides, about 10-40 contiguous nucleotides, about 10-30 contiguous nucleotides, or about 15-30 contiguous nucleotides, which can be used in sequence-dependent methods of gene identification (e.g., Southern hybridization) or isolation (e.g., in situ hybridization of bacterial colonies or bacteriophage plaques) or gene detection (e.g., as one or more amplification primers in amplification or detection). One or more specific primers or probes can be designed and used to amplify or detect part or all of the polynucleotide. As a specific example, two primers can be used in a PCR protocol to amplify a polynucleotide fragment. PCR can also be performed using one primer derived from the polynucleotide sequence and a second primer hybridizing to the upstream or downstream sequence of the polynucleotide sequence (for example, a promoter sequence, the 3' end of a pre-mRNA, or a sequence derived from a vector). Examples of temperature and isothermal techniques useful for in vitro amplification of polynucleotides are well known in the art. The sample can be or can be derived from a plant, a plant cell or plant material produced from a plant or derived from a plant, or a tobacco product, the plant cell or plant material described herein.

[0239] In a further aspect, a method for detecting an NtSWEET polynucleotide described herein (or any combination thereof as described herein) in a sample is also provided, the method comprising the steps of: (a) providing a sample containing or suspected of containing a polynucleotide; (b) contacting the sample with one or more primers or one or more probes to specifically detect at least a portion of the NtSWEET polynucleotide; and (c) detecting the presence of an amplification product, wherein the presence of the amplification product indicates the presence of the NtSWEET polynucleotide in the sample. In a further aspect, the use of one or more primers or probes to specifically detect at least a portion of an NtSWEET polynucleotide is also provided. Kits for detecting at least a portion of an NtSWEET polynucleotide are also provided, comprising one or more primers or probes for specifically detecting at least a portion of an NtSWEET polynucleotide. The kit may include reagents for polynucleotide amplification (e.g., PCR) or reagents for probe hybridization detection techniques (e.g., Southern blot, Northern blot, in situ hybridization, or microarray). The kit may include reagents for antibody binding detection techniques such as Western blot, ELISA, SELDI mass spectrometry, or test strips. The kit may include reagents for DNA sequencing. The kit may include the reagents and instructions for using the kit.

[0240] In some embodiments, the kits may include instructions for one or more of the described methods. The described kits may be useful for genetic identity determination using co-dominant scoring, phylogenetic studies, genotyping, haplotyping, genealogy analysis, or plant breeding, among other things.

[0241] The present disclosure also provides methods for genotyping plants, plant cells, or plant material containing the NtSWEET polynucleotides described herein. Genotyping provides a means of distinguishing between chromosomal pair homologs and can be used to identify segregants in plant populations. Molecular marker methods can be used for phylogenetic studies, characterizing genetic relationships between crop varieties, identifying crosses or somatic hybrids, localizing chromosomal segments affecting single genetic traits, map-based cloning, and quantitative inheritance studies. Specific methods of genotyping can employ any number of molecular marker analysis techniques, including amplified fragment length polymorphism (AFLP). AFLP is the product of allelic differences between amplified fragments caused by polynucleotide variability. Thus, the present disclosure further provides a means for tracking the segregation of one or more genes or polynucleotides, as well as chromosomal sequences genetically related to these genes or polynucleotides, using techniques such as AFLP analysis.

[0242] Also disclosed herein are methods for producing liquid tobacco extracts, and the liquid tobacco extracts produced by the methods.

[0243] A specific extraction temperature is selected for the tobacco starting material. The extraction temperature is typically selected within the range of about 100°C to about 160°C. The duration of the heating step may optionally be controlled to provide some control over the composition of the extract derived from the tobacco starting material. Preferably, the tobacco starting material is heated at the extraction temperature for at least about 90 minutes, more preferably at least about 120 minutes. The heating step is typically carried out in an inert atmosphere. Preferably, a stream of inert gas, such as nitrogen, is passed through the tobacco starting material during the heating step. Volatile tobacco compounds are released into the inert gas stream during the heating step, so that the inert gas acts as a carrier for the volatile components. The inert gas stream may have a flow rate of at least about 25 liters / minute, more preferably at least about 30 liters / minute. A relatively high flow rate of the inert gas may advantageously improve the efficiency of extraction from the tobacco starting material. Optionally, the heating step may be carried out under vacuum. Suitable heating methods for carrying out the heating of the tobacco starting material are known to those skilled in the art and include dry distillation, steam distillation, vacuum distillation, flash distillation, and thin film steam distillation.

[0244] If the volatile compounds are recovered by absorption in a liquid solvent, the step of forming a liquid tobacco extract may include drying the solution of the volatile compounds in the liquid solvent to concentrate the solution. Drying may be carried out using any suitable means, including, but not limited to, desiccation, molecular sieving, freeze-drying, phase separation, distillation, membrane permeation, controlled water crystallization and filtration, reverse hygroscopicity, ultracentrifugation, liquid chromatography, reverse osmosis, or chemical drying.

[0245] Liquid tobacco extracts are particularly suitable for producing compositions, formulations, or gel compositions for use in aerosol generating systems. Aerosol generating systems are disclosed that include the compositions, formulations, or gel compositions. In such aerosol generating systems, the compositions, formulations, or gels are typically heated in an aerosol generating device (e.g., a device with a heater element that interacts with the composition, formulation, or gel incorporating the liquid tobacco extract to produce an aerosol). During use, volatile compounds are released by heat transfer and are entrained in the air drawn through the aerosol generating device. As the released compounds cool, they condense to form an aerosol, which is inhaled by the consumer.

[0246] The present invention is further described in the following examples, which are provided to describe the invention in more detail. These examples describe preferred modes presently contemplated for carrying out the invention and are intended to illustrate, but not limit, the invention. [Example]

[0247] Example 1 - Materials and Methods Plant materials and cultivation conditions Before germination, seeds are sterilized with chlorine gas vapor. A 5% final chlorine solution is placed in a bell jar along with a glass tube containing the seeds. Hydrochloric acid (37%) is then added to the solution, and the seeds are incubated for 2 hours. The seeds are then placed on Murashige and Skoog (1962) growth medium and transferred to a plant growth chamber (24°C, 16 hours of light / 20°C, 8 hours of darkness) for 4 weeks. Well-developed plantlets are transferred to a greenhouse and grown in 10 L pots until fully grown. Artificial light is applied for 16 hours daily. All plants are grown at the same spacing at each stage of cultivation to ensure that spacing does not affect the leaves.

[0248] Flowering period Flowering time is assessed on 15-week-old plants grown under standard hot-tube drying and fertilization conditions and is quantified as the percentage of plants with flower buds, open flowers, or no inflorescence at all.

[0249] Methods for determining free amino acid, sugar, ammonia and nitrate levels Amino acid content is measured using Method MP 1471 rev 5 2011, Resana, Italy: Chelab Silliker Srl, Merieux NutriSciences Company. To determine amino acids in cured plant leaves, the cured leaf blades are dried at 40°C for 2-3 days after midrib removal, if necessary. The tobacco material is then ground to a fine powder (-100µM) before analysis of amino acid content. Alternatively, the amino acid content in plant material is measured as described in UNI EN ISO 13903:2005.

[0250] Reducing sugar content is measured using a segmented flow colorimetric method developed for the analysis of tobacco samples, as adapted by Skalar Instrument Co. (West Chester, PA) and described in Tobacco Science 20:139-144 (1976). Measurement of reducing sugar content is also described in Coresta Recommended Method 38, CRM38, CRM, and ISO 15154:2003. To determine reducing sugars in cured leaves, the cured leaf blades are dried at 40°C for 2-3 days after midrib removal, if necessary. The tobacco material is then ground to a fine powder (-100 μM) before analysis of reducing sugars. Alternatively, reducing sugar content is measured according to ISO 15154:2003.

[0251] Nitrate content is measured using a Lachet QuikChem 8500 instrument according to the manufacturer's protocol (Lachet QuikChem Method 12-107-04-1-J, Lachet Instruments, Loveland, CO, USA). Alternatively, nitrate content is measured according to ISO 15517:2003.

[0252] The ammonia content is determined using ion chromatography according to ISO 21045:2018.

[0253] Gene expression analysis The generated sequencing data was demultiplexed using Illumina BaseSpace® Clarity LIMS (Illumina, Inc.) and then imported into Qiagen CLC Genomics Workbench version 12.0.1 (CLC bio, QIAGEN). Transcriptome reads were mapped to an updated version of the N. tabacum reference genome (Sierro et al., (2014) Nat Commun 5, 3833) using the "RNA-Seq Analysis" 2.16 tool with a similarity of 0.8 (S = 0.8) and a fractional length of 0.8 (L = 0.8) as mapping criteria. The mismatch cost was set to 2, the insertion cost was set to 3, and the deletion cost was set to 3. No global alignment was performed; instead, paired distances were automatically detected. The maximum number of read hits was set to 10, and paired reads were counted as 1. Gene expression—FPKM values ​​were obtained for each gene in the reference genome and for genes without a transcription model.

[0254] RNAi procedure The DNA fragment of SEQ ID NO:9 was selected to suppress the expression of both copies of SWEET12 (SWEET12-S and SWEET12-T) and was cloned between the strong constitutive MMV promoter and the 3'nos terminator sequence of the nopaline synthase gene of Agrobacterium tumefaciens (Cheng et al. (1997) Plant Physiol. 115(3):971-980). Flue-cured Virginia tobacco (Nicotiana tabacum L.) cultivar K326 was transformed using standard Agrobacterium-mediated transformation protocols (Horsch et al. (1985) Science, 227, 1229-1232). Seeds were harvested from independent TO lines showing the strongest SWEET12 silencing. T1 plants derived from these T0 lines were grown in a greenhouse under standard agricultural practices and selected for RT-qPCR experiments to assess SWEET12 gene expression levels using the following primers (5' to 3'): SWEET12-S CLUSTER 3-F1 (SEQ ID NO: 11), SWEET12-S-R1 (SEQ ID NO: 12), SWEET12-T-F1 (SEQ ID NO: 13), and SWEET12-T-R1 (SEQ ID NO: 14).

[0255] The same strategy is adopted to silence SWEET15 using DNA fragment SEQ ID NO: 10. Selection is performed by RT-qPCR experiments to evaluate SWEET15 gene expression levels using the following primers (5' to 3'): SWEET15-T-F1 (SEQ ID NO: 15), SWEET15-T-R1 (SEQ ID NO: 16), SWEET15-S-F1 (SEQ ID NO: 17), and SWEET15-S-R1 (SEQ ID NO: 18).

[0256] Constitutive expression procedure The polynucleotide sequences of SWEET12-S (SEQ ID NO: 1) and SWEET15-T (SEQ ID NO: 7) were cloned between the strong constitutive 35S promoter and 3'nos terminator sequences of the nopaline synthase gene of Agrobacterium tumefaciens (Cheng et al., 1997 Plant Physiol. 115(3):971-980). Virginia tobacco (Nicotiana tabacum L.) cultivar K326 was transformed using standard Agrobacterium-mediated transformation protocols (Horsch et al. (1985) Science, 227, 1229-1232). Seeds were harvested from independent TO lines that exhibited the strongest SWEET12-S and SWEET15-T. T1 plants from these TO lines were grown in a greenhouse under standard agricultural practices and selected for RT-qPCR experiments to evaluate SWEET12 gene expression levels using the primers described in the previous section.

[0257] Example 2 - Gene expression analysis of SWEET12-S (SEQ ID NO: 1) and SWEET12-T (SEQ ID NO: 3) It is hypothesized that SWEET may contribute to the changes in sugar accumulation that occur during the curing process of tobacco leaves. Twenty-five genes and associated gene products were identified in Nicotiana tabacum L. associated with putative SWEET functions based on their identity with corresponding Arabidopsis and tomato orthologs. Figure 1 shows that, among these 25 genes and associated gene products, SWEET12-S (SEQ ID NO: 1) and SWEET12-T (SEQ ID NO: 3) gene expression was strongly induced in detached leaves of Virginia tobacco plants cured for 72 hours in a hot-air tube curing chamber. Expression of these genes rapidly increased 24 hours after curing in leaves detached from the lower petiole position of the plant (X and C) and continued to increase at 48 and 72 hours after curing, whereas it slightly decreased in leaves detached from the higher petiole position (B and T), although transcript levels were still significantly higher compared to uncured tissue (0 hour time point). This suggests that SWEET12-S and -T have specialized functions in sugar transport during the early stages of the desiccation process, the so-called yellowing stage, when chemical changes occur (Bovet et al. (2020). Leaf Curing Practices: Alter Gene Expression and the Chemical Constituents of Tobacco Leaves. In: Ivanov, NV, Sierro, N., Peitsch, MC (eds). The Tobacco Plant Genome. Compendium of Plant Genomes. Springer). Because many senescence-associated genes (SAGs) are induced during the yellowing stage of desiccation (Bovet et al. (2019). Plants (Basel) 11;8(11):492), we investigated the expression of putative senescence-associated target genes, SWEET15-S (SEQ ID NO: 5) and -T (SEQ ID NO: 7). The transcription levels of these genes were not significant during the hot air tube drying process, being lower than 5 FPKM.

[0258] Example 3 - Gene expression analysis of SWEET in different plant organs SWEET12-S and -T are highly expressed in both the lamina and midrib tissues of plant leaves, with higher expression in the lamina tissue than in the midrib tissue, as shown in Figure 2. A similar trend was observed for SWEET15-S and -T gene expression, but the transcript expression levels were considered to be below significant levels. Some SWEETs are not only transcriptionally regulated but can also be regulated by phosphorylation, and this post-transcriptional regulation is essential for biological function (Anjali et al. (2020) Plant Physiol Biochem. 156:1-6). Therefore, the slight changes in the low expression levels of SWEET15-S and -T do not preclude their possible active function during drought treatment. Table 1 shows the expression of SWEET12-S and -T, as well as SWEET15-S and -T, in plant organs. These data indicate that the four transcripts are primarily present in immature flowers and petals, demonstrating the need for active sugars in such tissues for energy supply and seed storage. SWEET15-S showed very low expression in all organs, and SWEET12-S was also expressed in the stem, which may indicate sugar transport in the phloem.

[0259] Example 4 - Functional Characterization of SWEET 12-S and -T To characterize the function of SWEET 12-S and -T as potential regulators of sugar accumulation in drought-treated leaves, flue-cured Virginia tobacco (Nicotiana tabacum L.) cultivar K326 plants were transformed with a fusion construct that placed the coding region of SWEET 12-S cDNA under the control of the CaMV 35S promoter (p35s), inducing constitutive high-level expression in plants (Tzfira and Citovsky (2006) Curr Opin Biotechnol. 17(2):147-54). Furthermore, RNAi plants were generated using insert SEQ ID NO: 9 as an RNAi construct for silencing SWEET 12-S and -T. Similarly, flue-cured Virginia tobacco (Nicotiana tabacum L.) cultivar K326 plants were transformed with a construct placing the coding region of SWEET 15-T cDNA under the control of p35s, and RNAi plants were generated using insert SEQ ID NO: 10 for downregulation of SWEET 15-S and -T.

[0260] Putative T0 transformants were selected by RT-qPCR screening of mature leaves. Seeds from each independent p35s:SWEET15-T and p35:SWEET12-S T0 progeny showing upregulated expression, and conversely, seeds from RNAi-SWEET12 and RNAi-SWEET15 T0 progeny showing silencing of expression, were collected to generate T1 plant lines. The T1 independent lines were then isolated from the plants and reselected by RT-qPCR screening of leaves after 48 hours of tube drying. From this screening, three p35s:SWEET12-S T1 lines, three RNAi-SWEET12 T1 lines, four p35s:SWEET15-T T1 lines, and five RNAi-SWEET15 T1 lines were selected (Figure 3). The growth and development phenotypes of these T1 lines, as well as their chemical profiles after tube drying, were evaluated.

[0261] Example 5 - Effect of SWEET12 Cluster 3 and SWEET15 on the growth and development of flue-cured Virginia tobacco (Nicotiana tabacum L.) cultivar K326 plants To characterize the effects of SWEET12 and SWEET15 on growth and development, we conducted detailed physiological and developmental analyses of T1 lines. T1 lines were compared with the corresponding wild-type (WT) control lines. Seed germination and the emergence of the first true leaf from the seeds were similar between the transformed lines and the control, suggesting that SWEET12 and SWEET15 do not play a major role in germination, growth, and development at the early seedling stage. Although the overall structure and leaves displayed by all lines were similar, some growth defects were observed. Figure 4 shows that the p35s:SWEET12-S lines exhibited significant growth retardation and reduction compared to the control lines. In contrast, the RNAi-SWEET12 lines were taller than the control lines. This suggests that SWEET12-S contributes to growth and development during the vegetative stage. The growth and development of the p35s:SWEET15-T lines showed no differences when compared with control plants, but the RNAi-SWEET15 lines were taller than their respective controls, suggesting that SWEET15 may also contribute to vegetative growth and development.

[0262] The transition from vegetative to reproductive development is a critical stage in tobacco leaf maturation, during which chemical properties change. It is common to harvest and desiccate leaves during this transition. Therefore, the flowering time of T1 transformants is investigated. Nicotiana tabacum produces leaves from a single upright stem, with an inflorescence at its apex (Smith and McDaniel (1992) Dev Biol. 153(1):176-84). The number of 15-week-old plants exhibiting flower buds, developed flowers, or no inflorescence at all is counted to assess flowering time. Data presented in Figure 5 show that, by the time the plants reached 15 weeks of age, 12% of the WT control plants had flowered, while 100% of the RNAi-SWEET12 plants had flowered. In contrast, no inflorescences were observed yet in p35s:SWEET12-S plants of the same age. This contrasting phenotype between RNAi-SWEET12 and p35s:SWEET12-S strongly suggests that SWEET12 may act as a negative regulator, affecting sugar transporter function and, therefore, flowering time. Interestingly, the opposite phenotype is observed in the SWEET15 line. 75% of 15-week-old p35s:SWEET15 plants exhibited flower buds or opened flowers, whereas only 20% of the RNAi-SWEET15 line flowered. This suggests that, unlike SWEET12, SWEET15 functions as a positive regulator of flowering time.

[0263] Overall, these data indicate that two sugar transporters of the SWEET multigene family, in addition to being induced during leaf drought treatment, are important regulators of reproductive growth and development. This functional finding was unexpected given their low expression levels in non-drought-treated tissues, suggesting that gene expression of these transporters may be induced at a different developmental stage than previously examined (see Table 1), or that their regulation is uncoupled from gene expression, as observed for many transporters located on the plasma membrane.

[0264] Vegetative growth defects often alter leaf biomass yield, especially when the defects result from dysregulation of genes involved in sugar metabolism and transport (Lastdrager et al. (2014) J Exp Bot. 65(3):799-807). Therefore, the biomass of mature leaves prepared for drought treatment is evaluated. While no significant differences in leaf biomass were observed between RNAi-SWEET12 and RNAi-SWEET15 plants compared to their controls, the data in Figure 6 show that both transgenic lines, p35s:SWEET12-S and p35s:SWEET15-T, showed a slight but significant decrease in leaf biomass compared to WT plants. This suggests that SWEET12 and SWEET15 may affect leaf biomass yield. It is important to note that the lack of difference in leaf biomass between the RNAi lines and WT plants is likely due to functional redundancy or a lack of conditions to confirm any effects. Other functional SWEETs present in these plants may compensate for the effects of downregulation of SWEET12 and SWEET15, again indicating that the effects on flowers are not translated into leaf biomass under greenhouse conditions.

[0265] Example 6 - Effects of SWEET12 Cluster 3 and SWEET15 on the Chemistry of Flue-Cured Virginia Tobacco (Nicotiana tabacum L.) Cultivar K326 Plants Because SWEET is known to fine-tune the sugar balance between cells and plant tissues (see above), we hypothesized that SWEET might alter tobacco leaf chemistry during curing. Therefore, we investigated the chemical profile of T1 transformant leaves. Mature leaves ready for curing were isolated, subjected to hot-air tube curing, and evaluated for sugar, free amino acid, ammonia, and nitrate content. Fructose, glucose, and sucrose contents were significantly and drastically reduced in the p35s:SWEET12-S line (Figure 7). This suggests that SWEET12 negatively regulates sugar accumulation in cured leaves. This also validates the biological function of SWEET as a sugar transporter in hot-air tube-cured Virginia tobacco (Nicotiana tabacum L.) cultivar K326 plants. No statistical difference in sugar accumulation was observed in the RNAi-SWEET12 line compared to WT plants. Again, this is likely due to functional redundancy that can occur in such a large multigene family.

[0266] Previous results indicate that SWEET15-S and -T gene expression is not induced or is weakly induced during the yellowing stage of the hot air tube drying treatment (Figure 2). Therefore, no changes in leaf chemistry are expected after the drying treatment of leaves isolated from the p35s:SWEET15-T or RNAi-SWEET15 lines, and metabolite content measurements are performed on pools of T1 lines instead of individual plants. The data in Figure 8 show that sucrose accumulation is reduced threefold and fructose and glucose accumulation is reduced approximately twofold in the p35s:SWEET15-T lines compared to WT plants. Sugar content in pooled leaf tissue material from the RNAi-SWEET15 lines is slightly reduced compared to the control lines. This data also suggests a role for SWEET15 in sugar accumulation during the drying treatment. The discrepancy between the lack of SWEET15-S and -T gene expression and the phenotypic changes in sugar accumulation in SWEET15 transformants at the early stage of the drying process could be due to one of the following reasons: (i) a delayed induction of the SWEET15 gene after 72 h, which was not assessed in the experimental design; (ii) SWEET15 regulates sugar accumulation before the drying process, and the changes persist during the drying process independently of its expression; or (iii) the observed phenotypic changes are not dependent on transcriptional regulation but are induced by post-transcriptional regulation of SWEET transporters. The fact that the SWEET15 gene is induced during air-curing in burley tobacco (low-sugar tobacco) (4 days after drying) particularly supports points (i) and (ii).

[0267] In plants, carbon flux is directed toward sucrose, starch, or amino acid synthesis (Yadav et al., (2015) Front Plant Sci. 22;6:275). Therefore, changes in sugar accumulation can affect amino acid accumulation in cured leaves (Bovet et al. (2019) Plants (Basel) 11;8(11):492). Table 2 shows the amino acid content in tube-cured Virginia tobacco (Nicotiana tabacum L.) SWEET12 T1. Free amino acid content was determined in 18-week-old plants grown under standard tube-curing and fertilized conditions and tube-cured under standard agronomic conditions. Results represent the average content in mg / kg of tube-cured leaves (position C) separated from the petiole (three biological replicates = leaves from three separate lines). Data are collected from at least three biological replicates. Statistics show significant differences in the accumulation of glutamate, asparagine, tryptophan, phenylalanine, glycine, proline, and methionine in p35s:SWEET12-S compared to the WT control (Student t-test, ns: not significant, **P<0.05, **P<0.01, ***P<0.001). The data in Table 2 show that the SWEET12-S line exhibits significantly higher asparagine, tryptophan, phenylalanine, glycine, and methionine contents and lower glutamate and proline contents compared to the control line. In other words, the sugar reduction in P35S-SWEET is accompanied by the accumulation of more amino acids, demonstrating a more active consumption of carbon resources for amino acid synthesis. Similarly, as shown in Table 3, the asparagine and tryptophan contents are higher, and the proline content is lower, in the SWEET12-S line compared to the WT control. Free amino acid content was measured in 18-week-old plants grown under standard tube-dried fertilization and tube-dried under standard agricultural conditions. Results represent the content in pools of T1 leaves separated from the petiole (position C) and tube-dried. Data are summarized in mg / kg. No significant differences were observed in the RNAi lines (see Tables 2 and 3).

[0268] The reduction in proline correlates with the reduction in sugars in p35s:SWEET15-T. Because proline is a known osmoprotectant, this suggests that water stress during the desiccation process (senescence) is different in the transgenic lines compared to CT1. Regarding its effect on flavor, proline is known to form Amadori compounds, and prolinofructose produces a "popcorn" flavor when heated by generating acetyl-pyrroline (Wei et al. (2017) Food Chem. 232:531-544).

[0269] Carbohydrate and nitrogen metabolism are closely related (Osuna et al., (2015) Front Plant Sci. 18;6:1023; Huarancca Reyes et al. (2018) Plant Cell Physiol. 59(6):1248-1254), and changes in sugar and amino acid content can generally affect ammonia, nitrate, and alkaloid content. Their quantification was performed in the SWEET line (Tables 4 and 5). In Table 4, free amino acid content was determined in 18-week-old plants grown under standard tube-dried and fertilized conditions and tube-dried under standard agronomic conditions. Results represent the average contents, expressed as mg / kg for ammonia, mg / kg for nitrate as NO3, and g / 100g for alkaloids, in tube-dried leaves isolated from petioles (position C). Data were collected from at least three biological replicates. Statistics show a significant difference in ammonia accumulation in p35s:SWEET12-S compared with the WT control (Student t-test, ns: not significant, *P<0.05). In Table 5, free amino acid content is measured in 18-week-old plants grown under standard tube-dried fertilization and tube-dried under standard agronomic conditions. Results represent the content in pools of T1 leaves separated from the petiole (position C) and tube-dried. Data are summarized in mg / kg for ammonia, mg / kg for nitrate as NO3, and g / 100g for alkaloids. Only the p35s:SWEET12-S line showed a significant increase in ammonia content compared with WT plants.

[0270] The functional properties of SWEET are well documented in Arabidopsis but not in other crops. SWEET is described as a key target that can be used to improve yield or in response to environmental stress. This example demonstrates the importance of the SWEET gene, altering tobacco leaf chemistry during curing and thus the aroma profile of tobacco. SWEET also influences leaf biomass and flowering time (growth and development), making it an important target that can be investigated to improve leaf yield or regulate plant maturity.

[0271] Example 7 - Sensory Effects The results are shown in Table 6 below. The accelerated flowering time observed with SWEET-12 RNAi tobacco does not correlate with any significant changes in sugars and free amino acids compared to control tobacco. However, at the sensory level of P1 sticks, SWEET12-RNAi tobacco is more distinctive (less bright and flat, more dark and cigar-like) and mature compared to the control. On the other hand, P35S SWEET-12 is sensorily closer to the control overall (but exhibits more chemical differences in sugar and free amino acid content). However, P35S SWEET-12 exhibits a more ashy, smoky character than the control (which may result from a different balance between sugars and amino acids).

[0272] Further aspects of the invention are described in the following numbered paragraphs.

[0273] Aspect 1. (i) a SWEET12-S polynucleotide sequence comprising, consisting of, or consisting essentially of a sequence having at least 70% sequence identity to SEQ ID NO: 1, or (ii) a SWEET12-T polynucleotide sequence comprising, consisting of, or consisting essentially of a sequence having at least 70% sequence identity to SEQ ID NO: 3, or (iii) a SWEET15-S polynucleotide sequence comprising, consisting of, or consisting essentially of a sequence having at least 70% sequence identity to SEQ ID NO: 5, or (iv) a SWEET15-T polynucleotide sequence comprising, consisting of, or consisting essentially of a sequence having at least 70% sequence identity to SEQ ID NO: 7, or (v) a polypeptide encoded by the polynucleotide set forth in (i) or (ii) or (iii) or (iv), or (iv) a SWEET12-S polypeptide having at least 70% sequence identity to SEQ ID NO: 2, or (v) a SWEET15-T polynucleotide sequence having at least 70% sequence identity to SEQ ID NO: 4. 12-T polypeptide, or (vi) a SWEET15-S polypeptide having at least 70% sequence identity to SEQ ID NO:6, or (vii) a SWEET15-T polypeptide having at least 70% sequence identity to SEQ ID NO:8, wherein the plant or portion thereof comprises at least one modification capable of modulating expression or activity of one or more of SWEET12-S or SWEET12-T or SWEET15-S or SWEET15-T, as compared to a control plant or portion thereof in which the expression of the polynucleotide or activity of the polypeptide is not modified, and wherein the expression or activity of one or more of SWEET12-S or SWEET12-T or SWEET15-S or SWEET15-T isA mutant, non-native, or transgenic plant in which the expression or activity of one or more of SWEET12-S, SWEET12-T, SWEET15-S, or SWEET15-T is modulated compared to an unmodulated control plant. Aspect 2. The plant comprises at least one genetic modification in a regulatory region or in the coding sequence of one or more of SWEET12-S or SWEET12-T or SWEET15-S or SWEET15-T, and / or the at least one genetic modification comprises one or more of exogenous DNA or exogenous RNA, and / or the at least one genetic modification comprises one or more of a vector or a viral vector or an Agrobacterium vector or a CRISPR vector, and / or the at least one genetic modification comprises an RNA interference or transcriptional gene. 10. A mutant, non-naturally occurring, or transgenic plant or part thereof described in paragraph 1, wherein the plant or part is capable of driving one or more of gene silencing or virus-induced gene silencing, and / or wherein at least one genetic modification is capable of expressing one or more of double-stranded RNA (dsRNA) or hairpin RNA (hpRNA) or small interfering RNA, and / or wherein at least one genetic modification is capable of constitutively expressing one or more of SWEET12-S or SWEET12-T or SWEET15-S or SWEET15-T. Aspect 3. 3. A mutant, non-native or transgenic plant according to paragraph 1 or paragraph 2, wherein the development of the vegetative growth phase of the plant is modulated compared to a control plant and / or the flowering time of the plant is modulated compared to a control plant. Aspect 4. A mutant, non-native, or transgenic plant according to paragraph 3, wherein the expression and / or activity of SWEET12-S and SWEET12-T or SWEET15-S and SWEET15-T or SWEET12-S and SWEET12-T and SWEET15-S and SWEET15-T is reduced, the plant height is increased during vegetative growth, and / or the flowering time is earlier compared to a control plant. Aspect 5. 4. A mutant, non-native or transgenic plant according to paragraph 3, which has increased expression and / or activity of SWEET15-T and an earlier flowering time compared to a control plant. Aspect 6. 3. The mutant, non-naturally occurring, or transgenic plant or part thereof of paragraph 1 or paragraph 2, wherein the mutant, non-naturally occurring, or transgenic plant is a dried leaf or a dried leaf. Aspect 7. The chemical profile of the dried leaves or dried leaves is modulated compared to dried leaves or dried leaves derived from a control plant, preferably the chemical profile is a sugar profile and / or an amino acid profile, preferably the expression and / or activity of SWEET12-S or SWEET15-T is increased and at least the fructose, glucose and sucrose contents are decreased compared to dried leaves or dried leaves derived from a control plant, or the expression and / or activity of SWEET15-S and SWEET15-T is decreased and at least the fructose, glucose and sucrose contents are decreased compared to dried leaves or dried leaves derived from a control plant. or the expression and / or activity of SWEET12-S is increased, and asparagine, tryptophan, phenylalanine, glycine and methionine contents are increased, and glutamic acid and proline contents are decreased, compared to desiccation-treated leaves or dried leaves derived from a control plant, and preferably the expression and / or activity of SWEET15-T is increased, and asparagine and tryptophan contents are increased, and prophosphate content is decreased, compared to desiccation-treated leaves or dried leaves derived from a control plant. Aspect 8. 8. A mutant, non-naturally occurring, or transgenic plant or part thereof according to any of paragraphs 1 to 7, wherein the plant is a Nicotiana tabacum plant, more preferably a Virginia or Burley species. Aspect 9. 9. The plant material, dried plant material, or homogenized plant material derived from or obtained from a plant or part thereof according to any of paragraphs 1 to 8, wherein the plant material is preferably selected from the group consisting of biomass, seeds, stems, flowers, or leaves, or a combination of two or more thereof, preferably the plant material is leaves, preferably the leaves are dried leaves, preferably the dried leaves are selected from the group consisting of hot-tube dried leaves, sun dried leaves, or air dried leaves. Aspect 10.1. A method for preparing a plant having a modulated flowering time and / or modulated amino acid levels and / or modulated sugar levels, the method comprising: (a) producing a polynucleotide sequence comprising (i) a SWEET12-S polynucleotide sequence comprising, consisting of, consisting essentially of a sequence having at least 70% sequence identity to SEQ ID NO: 1; or (ii) a SWEET12-T polynucleotide sequence comprising, consisting of, consisting essentially of a sequence having at least 70% sequence identity to SEQ ID NO: 3; or (iii) a SWEET15-S polynucleotide sequence comprising, consisting of, consisting essentially of a sequence having at least 70% sequence identity to SEQ ID NO: 5; or (iv) a SWEET15-T polynucleotide sequence comprising, consisting of, consisting essentially of a sequence having at least 70% sequence identity to SEQ ID NO: 7; or (v) a polypeptide encoded by a polynucleotide set forth in (i) or (ii) or (iii) or (iv); or (iv) a polypeptide encoded by a polynucleotide set forth in SEQ ID NO: 2. (v) a SWEET12-T polypeptide having at least 70% sequence identity to SEQ ID NO: 4; or (v) a SWEET12-T polypeptide having at least 70% sequence identity to SEQ ID NO: 4; or (vi) a SWEET15-S polypeptide having at least 70% sequence identity to SEQ ID NO: 6; or (vii) a SWEET15-T polypeptide having at least 70% sequence identity to SEQ ID NO: 8; (b) (i) the expression of one or more of SWEET12-S or SWEET12-T or SWEET15-S or SWEET15-T in the plant, or (ii) the expression of the same one or more of SWEET12-S or SWEET12-T or SWEET15-S or SWEET15-T in the plant, compared to a control in which the expression of the same one or more of SWEET12-S or SWEET12-T or SWEET15-S or SWEET15-T is not altered;A method comprising introducing at least one modification capable of regulating the activity of one or more of SWEET12-S or SWEET12-T or SWEET15-S or SWEET15-T in a plant. Aspect 11. 11. The method of paragraph 10, wherein in step (b), the at least one modification is introduced by genome editing, preferably wherein the genome editing is selected from CRISPR-mediated genome editing, mutagenesis, zinc finger nuclease-mediated mutagenesis, chemical or radiation mutagenesis, homologous recombination, oligonucleotide-directed mutagenesis, and meganuclease-mediated mutagenesis, or wherein in step (b), the at least one modification is introduced using an interfering polynucleotide, or wherein in step (b), the at least one modification is a promoter located 5' to the polynucleotide. Aspect 12. Plants obtained or obtainable by the methods of paragraph 10 or 11. Aspect 13. 10. A method for producing dried plant material having regulated amino acid levels and / or regulated sugar levels, the method comprising: (a) preparing a plant according to paragraph 10 or paragraph 11, or providing a plant according to paragraph 12; (b) harvesting plant material (e.g., leaves) from the plant; and (c) drying the plant material. Aspect 14. Dried plant material (e.g. leaves) obtained or obtainable by the method of paragraph 13. Aspect 15. Preferably, the plant product is a tobacco product derived from Nicotiana tabacum plant material, and / or the tobacco product is a tobacco blend, preferably the tobacco blend comprising Virginia tobacco and / or Burley tobacco, the plant material, the cured plant material, or the homogenized plant material described in paragraph 9, or the cured plant material described in paragraph 14. Any publications cited or described herein provide relevant information disclosed prior to the filing date of this application. Nothing herein should be construed as an admission that the inventors are not entitled to antedate such disclosure. All publications mentioned in the above specification are incorporated herein by reference. Various modifications and variations of the present invention will become apparent to those skilled in the art without departing from the scope and spirit of the present invention. While the present invention has been described in connection with certain preferred embodiments, it should be understood that the present invention, as claimed, should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in cell biology, molecular biology, and plant biology or related fields are intended to be within the scope of the following claims.

[0274] [Table 2]

[0275] [Table 3]

[0276] [Table 4]

[0277] [Table 5]

[0278] [Table 6]

[0279] [Table 7]

[0280] array SEQ ID NO: 1: Polynucleotide coding sequence of SWEET12-S from Nicotiana tabacum atggccatatttgatctccaccatccatggctatttgtgtttggagccttaggaaacattattccatattcgtcttcttagctccagtgccaacatttcgccgaatctacaaagaaaaatcaacacatgggctttcaatcagtcccttacgtggtagcactgttttcatctatgctctggatgtattatgcatttatcaagaaaaatgctattctcctcatctccatcaactcctt ggttgcattgtcgagacaatttacatctccattttccttctctacgcatccaaggaggctaggaggcagacggtgaaactttggtatcattgattggaggattgtacacactgatatttcttgtaactttgttccctttgaatggagcccttcgagtacaagtagtgggttggatttgtgtagccgtagcagtggctgtctttgctgcacctcttagcattgtgtttcaagtggtt cgaacgaagagtgtggagtttctgcccttcaccctgtctttctttcttacattaagtgctatcatgtggtttggttatggtctcttcaaaaggacctgtgtattgcactgccgaatgtattgggtttcttcctgggaatgattcagatgctgttgtatgggctataccgtaaggtaaagccagcagcagaattagagaaaaaggtgccggagcatatagtaaaaacatcgtcgtcgta ggaaactcagaacagatacatcctgtcaaatccgagaaaaatgaggatatgatcaagaagctggatgaagaagaaaacagggagagcagcgtaattagcccaccggtgccagctctgctgccggtggcgaacgaccatgaaaatgaagaacgtgcgggtggcgagctggcgcaagtgaacttgcagccgcagcagcagtttgaaaccccggtgcttgtggtgtgtgctgcagcttga sequence number2: polypeptide sequence related to sequence number1 MAIFDLHHPWLFVFGALGNIISIFVFLAPVPTFRRIYKEKSTMGFQSVPYVVALFSSMLWMYYAFIKKNAILLISINSFGCIVETIYISIFLLYASKEARRQTVKLLVSLIGGLYTLIFLVTLFPLNGALRVQVVGWICVAVAVAVFAAPLSIVFQV VRTKSVEFLPFTLSFFLTLSAIMWFGYGLLQKDLCIALPNVLGFFLGMIQMLLYGLYRKVKPAAELEKKVPEHIVNIVVVGNSEQIHPVKSEKNEDMIKKLDEEENRESSVISPPVPALLPVANDHENEERAGGELAQVNLQPQQQFETPVLVVCAAA SEQ ID NO: 3: Polynucleotide coding sequence of SWEET12-T from Nicotiana tabacum atggccatatttgacctccaccatccatggctatttgtgttcggagatcttaggaaacatatttccatattcgtcttcttagctccagtgccaacctttcgccgaatctacaaagaaaaatcaacatgggttttcaatcagtccctacgtggtagcactgttttcatccatgctctggatgtattatgcatttatcaagaaaaatgccactctcctcatctctatcaactcctt cggttgcattgtcgagaccatttacatctccattttccttctctacgcatccaaggaggctaggaggcagacggtgaaacttttggtatcattgattgaggattgtacacactgatattctcgtcactttgttccctttgaatggagcccttcgagtacaagtagtgggttggatttgtgtagccgtagcagtggctgtctttgctgcacctcttagcattgttgtcaagtgg ttcggacgaagagtgtggagttcctgcccttcaccctgtctttctttcttacattgagtgctatcatgtggtttggttatggtctccttcaaaggacctgtgtattgcactgccaaatgtattgggtttcttcctgggaatgattcagatgctgttgtatgggctataccgtaacgtaaagccagcagcagaattagagaaaaaggtgccggagcatgtagtaaaacatcgtcgtc cttggaaactcagaacagatacatcctcaaatccgagaaaaatgaggatatgatcaagaagctggatgaagaagtattagctgcagaagaaaacagggagagcagcgtaattagcccaccggtggcaaacgaccatgagaatgaagaacgtgcgggtggcgagctggcgcaagttaacttgcagccgcagcagcagtttgaaacaccggtgcttgtggtgtgtgctgcagcttga sequence number 4: polypeptide sequence related to sequence number 3 MAIFDLHHPWLFVFGVLGNIISIFVFLAPVPTFRRIYKEKSTMGFQSVPYVVALFSSMLWMYYAFIKKNATLLISINSFGCIVETIYISIFLLYASKEARRQTVKLLVSLIGGLYTLIFLVTLFPLNGALRVQVVGWICVAVAVAVFAAPLSIVCQV VRTKSVEFLPFTLSFFLTLSAIMWFGYGLLQKDLCIALPNVLGFFLGMIQMLLYGLYRNVKPAAELEKKVPEHVVNIVVLGNSEQIHPVKSEKNEDMIKKLDEEVLAAEENRESSVISPPVANDHENEERAGGELAQVNLQPQQQFETPVLVVCAAA SEQ ID NO: 5: Polynucleotide coding sequence of SWEET15-S from Nicotiana tabacum atggctatcttcactgcttctcaattggcttttgtttttggcgttcttggaaatggggtgtcgttcttggtgtacttgtctccaataccgactttctataggattttaataagaaaaatcaacggaaggattccagtctataccctattcggttgcactattcagtgccatgctctacttgtactatgcttatctcaaggaagaatg ggattttgctcgtttactattaacagcttcgggactgccatcgaattgatatatctcacaatcttcttgatatatgctacccgagaggccaagatttacactacaaagctggttcttctgttaaataggatcatatggagcaattgtggccttgacatatatattcgccaaagatgagacggagtcactattgtcggatggatctgt gctgtcttttctgtctgcgtcttcgctcctctctaagcattatgagacgtgttataagaacaaggagcgttgagttcatgccattccctctttcattcttcctcacaatctgcgccgtcatgtggttttctatggtctcttgataaaggacatgtacattgccacgccaaacattctagggtttacatttggaattgctcagatga tactgtacgcgatcttcagaaacagaaagcaacaaatccaaccggcggacagtaatctaaaagatttgacacaagtcgtcatagacatgaaagcaatggtattggagatgcaagaaaattctgatccgaataaggaagctgaagttgatgatactgatgaaaaaaagactaagcaagaagttgttgcacaaacaacttccaacgtatga sequence number 6: polypeptide sequence related to sequence number 5 MAIFTASQLAFVFGVLGNGVSFLVYLSPIPTFYRIYKRKSTEGFQSIPYSVALFSAMLYYAYLKEKNGILLVTINSFGTAIELIYLTIFLIYATREAKIYTTKLVLLLNIGSYGAIVALTYIFAKDETRVTIVGWI CAVFSVCVFAAPLSIMRRVIRTRSVEFMPFPLSFFLTICAVMWFFYGLLIKDMYIATPNILGFTFGIAQMILYAIFRNRKQQIQPADSNLKDLTQVVIDMKAMVLEMQENSDPNKEAEVDDTDEKKTKQEVVAQTTSNV SEQ ID NO: 7: Polynucleotide coding sequence of SWEET15-T from Nicotiana tabacum atggctatcttcactgcttctcatttggcttttgtttttggcgttcttggaaatggggtgtcgttcttggtgtacttgtctccaataccgactttctataggatatataagaaaaatcaacggaaggattccagtctataccctattcggttgcactattcagtgccatgctctacttgtactatgcttatctcaaggaagaatgg gattttgctcattactattaacagcttcggaactgccatcgaattcatatatctcacaatcttcttgatgtatgctacccgagaggccaagatttacactacgaagctggttcttctgttaaatataggatcatttggagcaatcgtcgccttgacatatatattcgccaaagataagacgcgagtcactattgtcggatggatttgtt ctgtcttttctgtctgcgtcttcgctctctcttagcattatgagacgcgttataaaaaacaaggagcgttgagtttatgccattccctctttctttcttcctcacaatctgcgccgtcatgtggttttctatggtctcttgataaaggacatgtacattgccacgccaaacattctagggtttacatttggaattgctcagatgata ctgtacgcaatcttcagaaacagaaagcaacaaatccaaccggcagacagtaatctgaaagatttgacacaagtcgtcatagacatgaaagcaatggtattggagatgcaagaaaattctgatccaaataaggaaggctgaagttgatgatactgatgaaaaaaagactaataagcaggaagttgttgcacaaacaactttaacgtatga sequence number 8: polypeptide sequence related to sequence number 7 MAIFTASHLAFVFGVLGNGVSFLVYLSPIPTFYRIYKRKSTEGFQSIPYSVALFSAMLYYAYLKEKNGILLITINSFGTAIEFIYLTIFLMYATREAKIYTTKLVLLLNIGSFGAIVALTYIFAKDKTRVTIVGWIC AVFSVCVFAAPLSIMRRVIKTRSVEFMPFPLSFFLTICAVMWFFYGLLIKDMYIATPNILGFTFGIAQMILYAIFRNRKQQIQPADSNLKDLTQVVIDMKAMVLEMQENSDPNKEAEVDDTDEKKTNKQEVVAQTTSNV SEQ ID NO: 9: Polynucleotide sequence selected to silence (RNAi) both SWEET 12-S and -T aatgtattgggtttcttcctgggaatgattcagatgctgttgtatgggctataccgtaa SEQ ID NO: 10: Polynucleotide sequence selected to silence (RNAi) both SWEET 15-S and -T ttcttcctcacaatctgcgccgtcatgtggttttctatggtctcttgataaaggacatgtacattgcc SEQ ID NO: 11: SWEET12-S CLUSTER 3-F1 amplification primer cagggagagcagcgtaattagc SEQ ID NO: 12: SWEET12-S-R1 amplification primer cccgcacgttcttcattttc SEQ ID NO: 13: SWEET12-T-F1 amplification primer gagcagcagcctacaacagttaatt SEQ ID NO: 14: SWEET12-T-R1 amplification primer ccatctttagcatcttcattttcagt SEQ ID NO: 15: SWEET15-T-F1 amplification primer acgcgatcttcagaaacagaaag SEQ ID NO: 16: SWEET15-T-R1 amplification primer catgtctatgacgacttgtgtcaaat SEQ ID NO: 17: SWEET15-S-F1 amplification primer aatcttcttgatgtatgctacc SEQ ID NO: 18: SWEET15-S-R1 amplification primer aatccatccgacaatagtga

Claims

1. A mutant, non-native, or transgenic Nicotiana tabacum plant, comprising: (i) a SWEET12-S polynucleotide sequence comprising, consisting of, or consisting essentially of a sequence having at least 70% sequence identity to SEQ ID NO: 1; or (ii) a SWEET12-T polynucleotide sequence comprising, consisting of, or consisting essentially of a sequence having at least 70% sequence identity to SEQ ID NO:3; or (iii) a SWEET15-S polynucleotide sequence comprising, consisting of, or consisting essentially of a sequence having at least 70% sequence identity to SEQ ID NO:5; or (iv) a SWEET15-T polynucleotide sequence comprising, consisting of, or consisting essentially of a sequence having at least 70% sequence identity to SEQ ID NO: 7; or (v) a polypeptide encoded by a polynucleotide according to (i) or (ii) or (iii) or (iv); or (vi) a SWEET12-S polypeptide having at least 97% sequence identity to SEQ ID NO: 2; or (vii) a SWEET12-T polypeptide having at least 93% sequence identity to SEQ ID NO: 4; or (viii) a SWEET15-S polypeptide having at least 97% sequence identity to SEQ ID NO: 6; or (ix) a SWEET15-T polypeptide having at least 97% sequence identity to SEQ ID NO: 8; comprising, consisting of, or consisting essentially of, at least one modification capable of regulating the expression or activity of one or more of SWEET12-S or SWEET12-T or SWEET15-S or SWEET15-T, the plant or part thereof comprises at least one modification that is capable of modulating (a) the expression of the polynucleotide in the plant or part thereof, or (b) the activity of the polypeptide in the plant or part thereof, compared to a control Nicotiana tabacum plant or part thereof in which the expression of the polynucleotide or the activity of the polypeptide is not modified; A mutant, non-native, or transgenic Nicotiana tabacum plant in which the expression or activity of one or more of SWEET12-S or SWEET12-T or SWEET15-S or SWEET15-T is modulated compared to a control Nicotiana tabacum plant in which the expression or activity of one or more of SWEET12-S or SWEET12-T or SWEET15-S or SWEET15-T is not modulated.

2. the plant comprises at least one genetic alteration in a regulatory region or in the coding sequence of one or more of SWEET12-S or SWEET12-T or SWEET15-S or SWEET15-T; and / or the at least one genetic alteration comprises one or more of exogenous DNA or exogenous RNA; and / or the at least one genetic modification comprises one or more of a vector, a viral vector, an Agrobacterium vector, or a CRISPR vector; and / or the at least one genetic alteration is capable of driving one or more of RNA interference or transcriptional gene silencing or virus-induced gene silencing; and / or the at least one genetic alteration is capable of expressing one or more of double-stranded RNA (dsRNA) or hairpin RNA (hpRNA) or small interfering RNA; and / or 2. The mutant, non-native, or transgenic Nicotiana tabacum plant or part thereof of claim 1, wherein the at least one genetic alteration is capable of constitutively expressing one or more of SWEET12-S or SWEET12-T or SWEET15-S or SWEET15-T.

3. 3. The mutant, non-native, or transgenic Nicotiana tabacum plant of claim 1 or claim 2, wherein the vegetative development of the plant is modulated compared to the control Nicotiana tabacum plant, and / or the flowering time of the plant is modulated compared to the control Nicotiana tabacum plant.

4. 4. The mutant, non-naturally occurring, or transgenic Nicotiana tabacum plant of claim 3, wherein the expression and / or activity of SWEET12-S and SWEET12-T, or SWEET15-S and SWEET15-T, or SWEET12-S and SWEET12-T and SWEET15-S and SWEET15-T is reduced, the plant grows taller during the vegetative growth phase, and / or the flowering time is earlier, compared to the control Nicotiana tabacum plant.

5. 4. The mutant, non-native, or transgenic Nicotiana tabacum plant of claim 3, wherein the expression and / or activity of SWEET15-T is increased and flowering time is advanced compared to a control Nicotiana tabacum plant.

6. 3. The mutant, non-natural, or transgenic Nicotiana tabacum plant or part thereof of claim 1 or claim 2, wherein the part of the mutant, non-natural, or transgenic plant is a dried leaf or a dried leaf.

7. The chemical profile of the dried or dehydrated leaves is modulated compared to the dried or dehydrated leaves from the control Nicotiana tabacum plant, preferably the chemical profile is a sugar profile and / or an amino acid profile, and preferably the expression and / or activity of SWEET12-S or SWEET15-T is increased and at least fructose, glucose, and sucrose content is decreased compared to desiccation-treated or desiccation-treated leaves from the control Nicotiana tabacum plant; or the expression and / or activity of SWEET15-S and SWEET15-T is reduced and at least the fructose, glucose, and sucrose content is reduced compared to desiccation-treated or desiccation-treated leaves from the control Nicotiana tabacum plant; or the expression and / or activity of SWEET12-S is increased, the asparagine, tryptophan, phenylalanine, glycine, and methionine content is increased, and the glutamic acid and proline content is decreased compared to the desiccation-treated or desiccant-treated leaves from the control Nicotiana tabacum plant, and preferably the ammonia content is increased compared to dried treated or dried leaves from said control Nicotiana tabacum plant, and / or 7. The mutant, non-naturally occurring, or transgenic Nicotiana tabacum plant or part thereof of claim 6, wherein the expression and / or activity of SWEET15-T is increased, the asparagine and tryptophan content is increased, and the prophosphate content is decreased compared to desiccation-treated or desiccant leaves derived from the control Nicotiana tabacum plant.

8. 8. The mutant, non-naturally occurring, or transgenic Nicotiana tabacum plant or part thereof according to any one of claims 1 to 7, wherein the plant is a Virginia or Burley species.

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

10. 1. A method for preparing a Nicotiana tabacum plant having a modified flowering time and / or a modified amino acid level and / or a modified sugar level, comprising: (a) providing a Nicotiana tabacum plant, (i) a SWEET12-S polynucleotide sequence comprising, consisting of, or consisting essentially of a sequence having at least 70% sequence identity to SEQ ID NO: 1; or (ii) a SWEET12-T polynucleotide sequence comprising, consisting of, or consisting essentially of a sequence having at least 70% sequence identity to SEQ ID NO:3; or (iii) a SWEET15-S polynucleotide sequence comprising, consisting of, or consisting essentially of a sequence having at least 70% sequence identity to SEQ ID NO:5; or (iv) a SWEET15-T polynucleotide sequence comprising, consisting of, or consisting essentially of a sequence having at least 70% sequence identity to SEQ ID NO: 7; or (v) a polypeptide encoded by a polynucleotide according to (i) or (ii) or (iii) or (iv); or (vi) a SWEET12-S polypeptide having at least 97% sequence identity to SEQ ID NO: 2; or (vii) a SWEET12-T polypeptide having at least 93% sequence identity to SEQ ID NO: 4; or (viii) a SWEET15-S polypeptide having at least 97% sequence identity to SEQ ID NO: 6; or (ix) providing a Nicotiana tabacum plant comprising at least one modification capable of modulating the expression or activity of one or more of SWEET12-S or SWEET12-T or SWEET15-S or SWEET15-T, wherein the SWEET15-T polypeptide comprises, consists of, or consists essentially of a SWEET15-T polypeptide having at least 97% sequence identity to SEQ ID NO:8; wherein said modification modulates said expression or activity compared to a control Nicotiana tabacum plant in which said expression of the same one or more of SWEET12-S or SWEET12-T or SWEET15-S or SWEET15-T is not modified.

11. In step (b), the at least one modification is introduced by genome editing, preferably the genome editing is selected from CRISPR-mediated genome editing, mutagenesis, zinc finger nuclease-mediated mutagenesis, chemical or radiation mutagenesis, homologous recombination, oligonucleotide-directed mutagenesis, and meganuclease-mediated mutagenesis; or In step (b), the at least one modification is introduced using an interfering polynucleotide, or 11. The method of claim 10, wherein in step (b), the at least one modification is a promoter located 5' to the polynucleotide.

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

13. 1. A method for producing dried processed Nicotiana tabacum plant material having modulated amino acid levels and / or modulated sugar levels, comprising: (a) preparing a Nicotiana tabacum plant according to claim 10 or claim 11, or providing a Nicotiana tabacum plant according to claim 12; (b) harvesting plant material (e.g., leaves) from the plant; (c) drying the plant material.

14. 14. Dried processed plant material (e.g. leaves) obtained or obtainable by the method of claim 13.

15. 15. A plant product comprising the plant material, dried plant material or homogenized plant material according to claim 9, or comprising the dried plant material according to claim 14, preferably The tobacco product is a tobacco blend, preferably A plant product wherein said tobacco blend comprises Virginia tobacco and / or Burley tobacco.

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