CN120530195A - Regulation of sugar transporters - Google Patents

Abstract

Disclosed is a mutant, non-naturally occurring or transgenic tobacco 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 comprising, consisting of, or consisting essentially of: (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: 6. NO:7 having at least 70% sequence identity; or (v) a polypeptide encoded by the polynucleotide shown 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:7; NO:8 has at least 97% sequence identity; wherein the plant or part thereof comprises at least one modification that is capable of modulating: (a) the expression of the polynucleotide in the plant or part thereof; or (b) the activity of the polypeptide in the plant or part thereof, as compared to a control tobacco plant or part thereof in which the expression of the polynucleotide or the 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 is modulated, as compared to a control tobacco 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.

Description

Modulation of sugar transport proteins

Technical Field

The present invention relates generally to plants having modulated expression or activity of SWEET (sugar efflux transporter (Sugars Will Eventually be Exported Transporters)).

Background

To make a tobacco product, different types of tobacco are mixed in various ratios to produce a blend having certain flavor characteristics. Flue-dried tobacco, such as Virginia tobacco (Virginia), is the most widely grown tobacco and is characterized by a high ratio of sugar to nitrogen, but has limited flavor characteristics. Other tobacco types, such as air-dried (e.g., burley (Burley), maryland (Maryland), and Galpao) or baked (e.g., dark) tobacco types, provide alternative flavor profiles. These different flavor profiles are important in the production of blended tobacco products. The flavor profile is the result of specific flavor compounds or precursors of these compounds present at levels in the tobacco plant. Due to the limited variety of tobacco used in commercial production, this means that there is limited opportunity to develop tobacco products having different flavors and aromas. The same applies to reconstituted tobacco materials for manufacturing heated tobacco rods for use in reduced risk products.

There remains a need in the art for improved opportunities to produce tobacco, thereby providing new flavor and/or sensory experience to consumers, while still maintaining commercially acceptable yields and characteristics. The present invention seeks to address this need and others.

Disclosure of Invention

The present invention is based, at least in part, on the discovery that modulating the expression or activity of a particular SWEET can alter the chemical characteristics of a plant part (such as a leaf) during desiccation. This can alter the aroma or organoleptic characteristics of the plant product, such as dried or dried tobacco leaves. Modulation of the expression or activity of a particular SWEET can affect plant growth and development. Thus, modulation of the expression or activity of a particular SWEET may further modulate leaf yield and/or plant maturity. The SWEET described herein may be a key regulator of reproductive growth and development in addition to being induced during leaf desiccation. The discovery of these functions was unexpected in view of the low expression levels of these specific SWEET in non-desiccating tissues. Without being bound by any particular theory, the gene expression of these particular SWEET may be induced at developmental stages different from those examined herein, or its regulation may be uncoupled from the gene expression.

In one aspect, disclosed is a mutant, non-naturally occurring or transgenic (e.g., genetically engineered) tobacco 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 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 as 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, which has at least 97% sequence identity to SEQ ID NO.6, or (ix) a SWEET15-T polypeptide which has at least 97% sequence identity to SEQ ID NO.8, wherein said plant or part thereof comprises at least one modification capable of modulating (a) the expression of a polynucleotide in a plant or part thereof, or (b) the activity of a polypeptide in a plant or part thereof, and wherein 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 tobacco plant in which the expression or activity of one or more of SWEET12-S or SWEET15-T is not modulated.

Suitably, the tobacco plant comprises at least one genetic alteration in a regulatory region or coding sequence of one or more of SWEET12-S or SWEET12-T or SWEET15-S or SWEET 15-T.

Suitably, the modification comprises one or more exogenous DNA or exogenous RNA.

Suitably, the modification comprises one or more vectors or viral vectors or Agrobacterium (Agrobacterium) vectors or CRISPR vectors.

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

Suitably, the modification is capable of expressing one or more double stranded RNAs (dsRNA) or hairpin RNAs (hpRNA) or small interfering RNAs.

Suitably, the modification is capable of constitutive expression of one or more of SWEET12-S or SWEET12-T or SWEET15-S or SWEET 15-T.

Suitably, the development of the tobacco plant during the vegetative stage is modulated compared to a control tobacco plant and/or the flowering time of the plant is modulated compared to a control tobacco plant.

Suitably, 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, and wherein the plant height is increased during the vegetative stage and/or wherein flowering time is accelerated compared to control tobacco plants.

Suitably, expression and/or activity of SWEET15-T is increased, and wherein flowering time is accelerated compared to control tobacco plants.

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

Suitably, the chemical characteristics of the dried or dried leaf are modulated, suitably wherein the chemical characteristics are sugar characteristics and/or amino acid characteristics, as compared to dried or dried leaf derived from a control tobacco plant.

Suitably, the expression and/or activity of SWEET12-S or SWEET15-T is increased and wherein at least the fructose, glucose and sucrose content is reduced compared to dried or desiccated leaves derived from control tobacco plants.

Suitably, the expression and/or activity of SWEET15-S and SWEET15-T is reduced, and wherein at least the fructose, glucose and sucrose content is reduced compared to dried or desiccated leaves derived from control tobacco plants.

Suitably, expression and/or activity of SWEET12-S is increased, and wherein asparagine, tryptophan, phenylalanine, glycine and methionine levels are increased, and wherein glutamic acid and proline levels are reduced as compared to dried or dried leaves derived from control tobacco plants.

Suitably, the ammonia content is increased as compared to dried or dried leaves derived from a control tobacco plant.

Suitably, expression and/or activity of SWEET15-T is increased, and wherein asparagine and tryptophan levels are increased, and wherein proline levels are reduced compared to dried or dried leaves derived from control tobacco plants.

Suitably, the plant is a tobacco plant, more suitably of the Virginia type (Virginia) or Burley type (Burley).

Also disclosed is a plant material, a dried or desiccated plant material or a homogenized plant material derived or obtained from a tobacco plant or part thereof, suitably wherein the plant material is selected from the group consisting of biomass, seeds, stems, flowers or leaves or a combination of two or more thereof, suitably wherein the plant material is a leaf, suitably wherein the leaf is a dried or desiccated leaf, suitably wherein the dried leaf is selected from the group consisting of flue dried leaf, sun dried leaf or desiccated leaf.

Also disclosed is a method of making a tobacco plant having a modulated flowering time and/or a modulated amino acid level and/or a modulated sugar level, the method comprising (a) providing a tobacco 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 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 as 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, which has at least 97% sequence identity to SEQ ID NO. 6, or (ix) a SWEET15-T polypeptide which has at least 97% sequence identity to SEQ ID NO. 8, wherein the modification modulates expression or activity as compared to a control tobacco plant in which expression of the same SWEET12-S or SWEET12-T or SWEET15-S or SWEET15-T is not modified.

Also disclosed is a method of making a tobacco plant having a modulated flowering time and/or a modulated amino acid level and/or a modulated sugar level, the method comprising (a) providing a tobacco plant having modulated expression or activity comprising, consisting of, or consisting essentially of one or more of SWEET12-S or SWEET12-T or SWEET15-S or SWEET15-T (i) a SWEET12-S polynucleotide sequence comprising, consisting of, or consisting of a sequence having at least 70% sequence identity with 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 as 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, which has at least 97% sequence identity to SEQ ID NO. 6, or (ix) a SWEET15-T polypeptide which has at least 97% sequence identity to SEQ ID NO. 8, and (b) introducing at least one modification capable of modulating the activity of (i) one or more of SWEET12-S or SWEET12-T or SWEET15-S or SWEET15-T in a tobacco plant, or (ii) one or more of SWEET12-S or SWEET12-T or SWEET15-S or SWEET15-T in a tobacco plant, compared to a control in which the expression of the same one or more of SWEET12-S or SWEET15-T is not modified.

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

Also disclosed is a plant obtainable or obtainable by such a method.

Also disclosed is a method of producing a dried or dried tobacco plant material having a modulated amino acid level and/or a modulated sugar level, the method comprising (a) preparing a tobacco plant as described above, or providing a tobacco plant as described above, (b) harvesting plant material (e.g., leaves) from the plant, and (c) drying or drying the plant material.

Also disclosed is a dried or desiccated plant material (e.g., leaves) obtained or obtainable by the method.

Also disclosed is a plant product comprising plant material, dried or dried plant material or homogenized plant material, or comprising dried or dried plant material.

Suitably, the plant product is a tobacco product from tobacco plant material.

Suitably, the tobacco product is a tobacco blend, suitably wherein the tobacco blend comprises virginia-type tobacco and/or burley-type tobacco.

Some advantages are

Modulation of the expression and/or activity of a particular SWEET as described herein may result in modulation of amino acid levels, particularly in dried or dried plant material. This can result in tobacco having new flavor or sensory characteristics.

Modulation of the expression and/or activity of a particular SWEET as described herein may result in the regulation of sugar levels, particularly in dried or dried plant material. This can result in tobacco having new flavor or sensory characteristics.

Modulating the expression and/or activity of a particular SWEET as described herein can result in plants with accelerated flowering time, which can shorten the growing season and enable faster introduction of new traits by crossing. This can lead to cost savings resulting from commercial plants.

Advantageously, non-genetically modified plants may be produced that are more acceptable to consumers.

Advantageously, the present disclosure is not limited to the use of EMS mutant plants. EMS mutant plants may not have the potential to improve crop characteristics after breeding. Once breeding begins, the desirable characteristics of the EMS mutant plant may be lost for various reasons. For example, multiple mutations may be required, which may be dominant or recessive, and it may be difficult to identify point mutations in a gene target. In contrast, the present disclosure utilizes the use of SWEET that can be specifically manipulated to produce plants having a desired phenotype.

Down-regulation of the Asparagine Synthetase (ASN) gene (WO 2017042162; bovet et al (2019) Plants (Basel) 11;8 (11): 492) also significantly altered the chemical composition (chemistry) of tobacco without affecting biomass. Advantageously, the combination of ASN-modulating plants and NtSWEET-modulating plants may rearrange their chemical components, such as the aminoacid-based components of burley or Dark (Dark) tobacco or virginia tobacco, and may further alter their flavor and/or organoleptic properties. Other genes and enzymes also play a role in the recombination of amino acids and/or sugars during leaf yellowing, such as diaminopimelate aminotransferase (DAPAT), aspartate Aminotransferase (AAT) and chloroplast sulfate transporter (SULTR) (which may also alter the chemical composition of the leaf). Modification NtSWEET of expression or NtSWEET activity, expression or activity of one or more of these other targets selected from one or more of ASN, DAPAT, and AAT can be used to further alter the flavor and/or organoleptic properties of the dried or dried tobacco.

Drawings

FIG. 1 is a series of four graphs (a) - (d) showing RNA-seq gene expression profiles of the SWEET gene during the drying time course of flue-cured tobacco (Nicotiana tabacum L.). These figures depict the expression profiles of (a) SWEET12-S, (b) SWEET12-T, (c) SWEET15-S, and (d) SWEET15-T in the detached leaves of stalks X, C, B and T before drying (0 hours) and during the drying process (24 hours, 48 hours, and 72 hours). Stalk leaf locations X, C, B and T are from the bottom to top of the plant, respectively. The left y-axis represents FPKM (mapping read fragments per million of kilobase transcripts). The x-axis represents the drying time course. The legend indicates stalk positions X (processed line), C (processed line, gray), B (dotted line, gray), and T (dotted line, black). Data were collected from at least three biological replicates (n=3). Each biological repeat was a pool of four leaves (n=20 leaves) harvested from five different plants that were inserted at the same stalk position in the plant.

FIG. 2 is a series of four graphs (a) - (d) showing RNA-seq gene expression profiles of the SWEET gene in lamina and midrib tissue of flue-cured tobacco (tobacco). These figures depict the expression profiles of (a) SWEET12-S, (b) SWEET12-T, (c) SWEET15-S and (d) SWEET15-T in leaf (treated line, black) and midrib (treated line, gray) tissue before (0 hours) and during the drying process (6 hours, 24 hours, 48 hours, 55 hours and 72 hours). The left y-axis represents FPKM (mapping read fragments per million of kilobase transcripts). The x-axis represents the drying time course. Legends indicate harvested tissue in the pool, leaf positions B-C. Data were collected from at least three biological replicates (n=3). Each biological repeat is a pool of five plants.

FIG. 3 is a series of two graphs showing the relative expression of the SWEET gene in flue-cured tobacco (tobacco) leaves of independent T1 plants. (a) Quantification of the relative transcript levels of SWEET12-S in each individual T1 plant indicated that SWEET12-S expression was higher in p35S SWEET12-S and decreased in RNAi-SWEET12-S compared to the WT control plants. (b) Quantification of the relative transcript levels of SWEET15-T in each individual T1 plant indicated that SWEET15-T expression was higher in p35S SWEET15-T and decreased in RNAi-SWEET15-T/-S compared to the WT control plant. The results represent the relative mRNA expression in mature leaves detached from stalk position C and dried for 48 hours. Data were normalized using PQ14 expression for correction of variability. The data are summarized in bar graphs, where each bar graph represents the gene expression level of the T1 individual plants.

Fig. 4 is a series of photographs showing the difference in growth and development of individual SWEET T1 lines of flue-cured tobacco (tobacco). (a, b and c) representative images of 10 week old plants grown under standard flue-cured fertilization conditions. Differences in height of individual T1 plants were observed. Images (b) and (c) show that RNAi lines RNAi-SWEET12-S and RNAi-SWEET15-T/-S are higher than the control plant (WT), while image (a) shows p35S that the SWEET12-S line is smaller than its corresponding control plant. No difference in growth and development of the p35s SWEET15-T plants compared to WT was observed (not shown).

Fig. 5 is two graphs showing flowering time of flue-cured tobacco (tobacco) SWEET T1 line. Flowering time of 15 week old plants grown under standard flue-cured fertilization conditions was evaluated. Flowering time is quantified as the percentage of plants that either show floral primordia or developing flowers, or have no inflorescences at all. Data were collected from at least ten individual plants. These data are summarized in stacked bar graphs.

Fig. 6 is a series of graphs showing leaf biomass of flue-cured tobacco (tobacco) SWEET T1 line. Fresh leaf biomass from 18 week old plants grown under standard flue-dry fertilization conditions. The results represent the average fresh weight of leaves detached from the stalks (C position). Data were collected from at least three biological replicates. The data are summarized in bar graphs, where bars represent the average of fresh amounts, and error bars represent standard deviation in grams (g). (a) Leaves of P35S: SWEET12-S plants were significantly smaller compared to WT and RNAi-SWEET12-S plants (student t test, < 0.05). (b) Leaves of P35s: SWEET15-T plants were significantly smaller compared to WT and RNAi-SWEET15-T plants (student T test, × P < 0.01).

FIG. 7 is a series of graphs showing the difference in sugar content of flue-dried tobacco Virginia (tobacco) SWEET12-S T1 plants. Sugar content was measured in 18 week old plants grown under standard flue-dried fertilization conditions and flue-dried under standard agronomic conditions. The results represent the average content of the sum of (a) fructose, (b) glucose, (C) sucrose and (d) sugar in the leaves separated from the stalks (C position) and flue dried. Data were collected from at least three biological replicates. The data are summarized in bar graphs, where bars represent mean values in g/100g and error bars represent standard deviation. Statistics showed significant differences in saccharide accumulation in SWEET12-S and (b, d) P35S, SWEET15-T compared to WT controls (student T test, P <0.05, P <0.01, P < 0.001).

Fig. 8 is a series of graphs showing the difference in sugar content of flue-dried virginia tobacco (tobacco) of SWEET 15T 1 plants. Sugar content was measured in 18 week old plants grown under standard flue-dried fertilization conditions and flue-dried under standard agronomic conditions. The results represent the average content of the sum of (a) fructose, (b) glucose, (C) sucrose and (d) sugar in the leaves separated from the stalks (C position) and flue dried. Data were collected from at least three biological replicates. The data are summarized in bar graphs, where bars represent combined content in g/100 g.

Detailed Description

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

The terms "comprising," "including," "having," "can," "containing," and variations thereof are intended to be open-ended terms, or expressions that do not exclude the possibility of additional actions or structures.

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

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

To describe the numerical ranges herein, each intervening value, to the same degree of accuracy, is expressly contemplated. For example, for ranges 6-9, the values 7 and 8 are covered in addition to 6 and 9, and for ranges 6.0-7.0, the values 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are expressly covered.

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

"coding sequence" or "polynucleotide coding" refers to a nucleotide (RNA or DNA molecule) comprising a polynucleotide encoding a polypeptide. The coding sequence may also include initiation and termination signals operably linked to regulatory elements including promoters and polyadenylation signals capable of directing expression in the cells of the individual or mammal to which the polynucleotide is administered. The coding sequence may be codon optimized.

"Complementary" or "complementary" can refer to Watson-Crick (e.g., A-T/U and C-G) or Hoogsteen base pairing between nucleotides or nucleotide analogs. "complementarity" refers to the property shared between two polynucleotides such that when they are arranged antiparallel to each other, the nucleotide bases at each position will be complementary.

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

In the context of a control plant or control plant cell, the term "control" refers to a plant or plant cell in which the expression, function or activity of one or more genes or polypeptides is not modified (e.g., increased or decreased) and which, therefore, can be compared to a plant in which the expression, function or activity of the same one or more genes or polypeptides has been modified. A "control plant" is a plant in which all parameters except the test parameter are substantially equivalent to the test plant or modified plant. For example, when referring to a plant into which a polynucleotide has been introduced, a control plant is an equivalent plant into which such a polynucleotide has not been introduced. The control plant may be an equivalent plant into which the control polynucleotide has been introduced. In such cases, the control polynucleotide is a polynucleotide that is expected to produce little or no phenotypic effect on the plant. The control plant may comprise a blank vector. The control plant may correspond to a wild type plant. The control plant may be a null isolate where the T1 isolate no longer has a transgene.

The term "reduced" or "reduced" refers to a reduction in quantity or function, such as polypeptide function, transcriptional function, or polypeptide expression, of from about 10% to about 99%, or a reduction of at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100%, or at least 150%, or at least 200% or more. The term "reduced" or the phrase "reduced amount" may refer to an amount or function that is less than the amount or function found in an unmodified plant processed in the same manner or a product from the same variety of plants. Thus, in some cases, wild-type plants of the same variety that have been processed in the same manner are used as controls by which it is measured whether a reduction in number is obtained.

"Donor DNA" or "donor template" refers to a double-stranded DNA fragment or molecule comprising at least a portion of a gene of interest. The donor DNA may encode a functional polypeptide.

An "endogenous gene or polypeptide" refers to a gene or polypeptide that originates from the genome of an organism and that has not undergone an alteration, such as loss, acquisition, or exchange of genetic material. Endogenous genes undergo standard gene delivery and gene expression. Endogenous polypeptides undergo normal expression.

"Enhancer sequence" refers to a sequence that can increase gene expression. These sequences may be located upstream, within introns or downstream of the transcribed region. The transcribed region comprises an exon and an insert intron from the promoter to the transcription termination region. Enhancement of gene expression can be performed by a variety of mechanisms, including increased transcription efficiency, stable mature mRNA, and translational enhancement.

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

By "over-expression" is meant that the level of gene product produced in a transgenic organism exceeds that produced by an empty isolated (or non-transgenic) organism from the same experiment.

"Functional" describes a polypeptide having a biological function or activity. "functional gene" refers to a gene transcribed into mRNA that is translated into a functional or active polypeptide.

"Genetic construct" refers to a DNA or RNA molecule comprising a polynucleotide encoding a polypeptide. The coding sequence may include initiation and termination signals operably linked to regulatory elements including promoters capable of directing expression and polyadenylation signals.

"Genome editing" generally refers to a process by which genomic nucleic acid in a cell is altered. This may be done, for example, by removing, inserting or replacing one or more nucleotides in the genomic nucleic acid. Endonucleases can be used to create specific breaks or breaks at defined locations in the genome and are further described herein.

The term "homology" or "similarity" refers to the degree of sequence similarity between two polypeptides or between two polynucleotide molecules that are aligned by sequence. The degree of homology between two discrete polynucleotides being compared is a function of the number of identical or matched nucleotides at the comparable positions. Homology or similarity may be determined over the entire length of the subject sequence.

In the context of two or more polynucleotides or polypeptides, "identical" or "identity" refers to a sequence having a particular percentage of identical residues over a particular region. The percentage may be calculated by optimally aligning two sequences, comparing designated regions of the two sequences, determining the number of positions in the two sequences where the same residue is present to produce a number of matched positions, dividing the number of matched positions by the total number of positions in the designated region, and multiplying the result by 100 to produce a percentage of sequence identity. Where two sequences have different lengths or alignments yielding one or more staggered ends and the designated comparison region comprises only a single sequence, the residues of the single sequence are included in the denominator rather than the numerator of the calculation. Thymine (T) and uracil (U) are considered equivalent when comparing DNA and RNA. Identity may be identified manually or by using a computer sequence algorithm such as ClustalW, clustalX, BLAST, FASTA or Smith-Waterman assay. Suitable parameters for ClustalW may be gap open penalty = 15.0, gap extension penalty = 6.66, and matrix = identity for polynucleotide alignment. For polypeptide alignment, gap open penalty = 10.o, gap extension penalty = 0.2, and matrix = Gonnet. ENDGAP = -1 and GAPDIST =4 for DNA and protein alignment.

The term "increase" or "increased" refers to an increase in number or function or activity, such as, but not limited to, one or more of polypeptide function or activity, transcriptional function or activity, and polypeptide expression, of about 10% to about 99%, or an increase of at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, at least 100%, at least 150%, or at least 200% or more. The term "increased" or the phrase "increased amount" may refer to an amount or function or activity in a plant or a product produced by a plant that is greater than the amount or function or activity found in an unmodified plant processed in the same manner or a product from the same variety of plants. Thus, in some cases, wild-type plants of the same variety that have been processed in the same manner are used as controls by which it is measured whether an increase in number is obtained.

The term "inhibit" or "inhibited" refers to an amount or function or activity that is reduced by about 98% to about 100%, or by at least 98%, at least 99%, but particularly 100%, such as, but not limited to, one or more of polypeptide function or activity, transcriptional function or activity, and polypeptide expression.

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

The term "isolated" or "purified" refers to a material that is substantially or essentially free of components that normally accompany it in its natural state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The polypeptides that are the major species present in the formulation are substantially purified. In particular, the isolated polynucleotide is separated from the open reading frame flanking the desired gene and encoding a polypeptide other than the desired polypeptide. The term "purified" means that the polynucleotide or polypeptide produces substantially one band in an electrophoresis gel. In particular, it means that the polynucleotide or polypeptide has a purity of at least 85%, more suitably at least 95%, and most suitably at least 99%. The isolated polynucleotide may be purified from its naturally occurring host cell. Conventional polynucleotide purification methods known to the skilled artisan may be used to obtain isolated polynucleotides. The term also encompasses recombinant polynucleotides and chemically synthesized polynucleotides.

"Liquid tobacco extract" describes the direct product of an extraction process performed on tobacco starting materials. An extraction process for producing a liquid tobacco extract may include heating a tobacco starting material under specific heating conditions and collecting volatile compounds generated. The liquid tobacco extract may be composed of a mixture of compounds derived from the tobacco starting material and removed during the extraction process, typically in combination with a liquid carrier or solvent.

"Modulation" refers to a qualitative or quantitative change, change or modification that causes or facilitates a process, pathway, function or activity of interest. Such a change, change or modification may be, without limitation, an increase or decrease in a related process, pathway, function or activity of interest. For example, gene expression or polypeptide function or activity may be modulated. Typically, the relevant change, alteration or modification will be determined by comparison to a control.

The term "non-naturally occurring" describes entities that are not naturally occurring or do not exist in nature, such as polynucleotides, gene mutations, polypeptides, plants, plant cells, and plant material. Such non-naturally occurring entities or artificial entities may be prepared, synthesized, initiated, modified, interfered with or manipulated by methods described herein or known in the art. Such non-naturally occurring entities or artificial entities may be prepared, synthesized, initiated, modified, intervening or manipulated by humans. Thus, non-naturally occurring plants cannot be produced using substantial biological methods. Thus, for example, non-naturally occurring plants, non-naturally occurring plant cells, or non-naturally occurring plant material can be prepared using conventional plant breeding techniques (e.g., backcrossing) or by genetic manipulation techniques (e.g., antisense RNA, interfering RNA, meganucleases, etc.). Further by way of example, a non-naturally occurring plant, non-naturally occurring plant cell, or non-naturally occurring plant material may be prepared by introgression of a first plant or plant cell into a second plant or plant cell (which may itself be naturally occurring), or by transferring one or more genetic mutations (e.g., one or more polymorphisms) from the first plant or plant cell into the second plant or plant cell, such that the resulting plant, plant cell, or plant material, or progeny thereof, includes genetic makeup (e.g., genome, chromosome, or segment thereof) that is not naturally occurring or does not occur in nature. The resulting plant, plant cell or plant material is thus artificial or non-naturally occurring. Accordingly, an artificial or non-naturally occurring plant or plant cell may be prepared by modifying a gene sequence in a first naturally occurring plant or plant cell even if the resulting gene sequence naturally occurs in a second plant or plant cell comprising a different genetic background than the first plant or plant cell. In certain embodiments, the mutation is not a naturally occurring mutation naturally occurring in a polynucleotide or polypeptide (such as a gene or polypeptide). Differences in genetic background can be detected by phenotypic differences or by molecular biological techniques known in the art, such as polynucleotide sequencing, the presence or absence of genetic markers (e.g., microsatellite RNA markers).

An "oligonucleotide" or "polynucleotide" refers to at least two nucleotides that are covalently linked together. The description of single strand also defines the sequence of the complementary strand. Thus, polynucleotides also encompass the depicted single-stranded complementary strand. Many variants of a polynucleotide may be used for the same purpose as a given polynucleotide. Thus, polynucleotides also encompass substantially identical polynucleotides and complements thereof. Single strands provide probes that can hybridize to a given sequence under stringent hybridization conditions. Thus, polynucleotides also encompass probes that hybridize under stringent hybridization conditions. The polynucleotide may be single-stranded or double-stranded, or may comprise portions of double-stranded and single-stranded sequences. The polynucleotide may be DNA (both genomic DNA and cDNA), RNA, or a hybrid, wherein the polynucleotide may comprise a combination of deoxyribonucleotides and ribonucleotides, as well as combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. Polynucleotides may be obtained by chemical synthesis methods or by recombinant methods.

The specificity of the hybridized complementary fragment of single-stranded DNA is determined by the "stringency" of the reaction conditions (Sambrook et al, molecular Cloning and Laboratory Manual, second edition, cold Spring Harbor (1989)). Hybridization under "stringent conditions" describes a hybridization protocol in which polynucleotides that are at least 60% homologous to each other remain hybridized. In general, stringent conditions are selected to be about 5 ℃ lower than the thermal melting point (Tm) for specific sequences at the specified ionic strength and pH. Tm is the temperature (under defined ionic strength, pH and polynucleotide concentration) at which 50% of the probe complementary to a given sequence hybridizes to the given sequence at equilibrium. Since a given sequence is usually present in excess, 50% of the probes are in equilibrium at Tm.

Stringent conditions will typically include (1) low ionic strength and high temperature washes, e.g., 15mM sodium chloride, 1.5mM sodium citrate, 0.1% sodium dodecyl sulfate at 50 ℃, (2) denaturants during hybridization, e.g., 50% (v/v) formamide, 0.1% bovine serum albumin, 0.1% Ficoll,0.1% polyvinylpyrrolidone, 50mM sodium phosphate buffer (750 mM sodium chloride, 75mM sodium citrate, pH 6.5), at 42 ℃, or (3) 50% formamide. The wash typically also contained 5XSSC (0.75M NaCl, 75mM sodium citrate), 50mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5X Deng Bote solution (Denhardt' ssolution), sonicated salmon sperm DNA (50. Mu.g/mL), 0.1% SDS and 10% dextran sulfate at 42 ℃, and 0.2XSSC (sodium chloride/sodium citrate) at 42 ℃ and 50% formamide at 55 ℃, followed by a high stringency wash consisting of 0.1XSSC containing EDTA at 55 ℃. Suitably, the conditions are such that sequences at least about 65%, 70%, 75%, 85%, 90%, 95%, 98% or 99% homologous to each other generally remain hybridized to each other.

"Moderately stringent conditions" use wash solutions and less stringent hybridization conditions such that a polynucleotide will hybridize to an entire, fragment, derivative or analog of a polynucleotide. One example comprises hybridization in 6XSSC, 5x Deng Bote solution, 0.5% SDS, and 100. Mu.g/mL denatured salmon sperm DNA at 55 ℃, followed by one or more washes in 1XSSC, 0.1% SDS at 37 ℃. Temperature, ionic strength, etc. can be adjusted to suit experimental factors such as probe length. Other moderately stringent conditions have been described (see Ausubel et al Current Protocols in Molecular Biology, volume 1-3, ,John Wiley&Sons,Inc.,Hoboken,N.J.(1993);Kriegler,Gene Transfer and Expression:A Laboratory Manual,Stockton Press,New York,N.Y.(1990);Perbal,A Practical Guide to Molecular Cloning,, 2 nd edition, john Wiley & Sons, new York, N.Y. (1988)).

"Low stringency conditions" use wash solutions and less stringent hybridization conditions than medium stringency such that polynucleotides will hybridize to the whole, fragment, derivative, or analog of a polynucleotide. Non-limiting examples of low stringency hybridization conditions include hybridization at 40℃in 35% formamide, 5XSSC, 50mM Tris HCl (pH 7.5), 5mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100. Mu.g/mL denatured salmon sperm DNA, 10% (weight/volume) dextran sulfate followed by one or more washes at 50℃in 2XSSC, 25mM Tris HCl (pH 7.4), 5mM EDTA and 0.1% SDS. Other conditions of low stringency, such as those for cross-species hybridization, have been described fully (see Ausubel et al, 1993; kriegler, 1990).

"Operably linked" means that the expression of a gene is under the control of a promoter to which it is spatially linked. The promoter may be located 5 '(upstream) or 3' (downstream) of the gene under its control. The distance between the promoter and the gene may be about the same as the distance between the promoter and the gene which it controls in the gene which produces the promoter. As is known in the art, this change in distance can be accommodated without loss of promoter function. "operably linked" refers to the association of polynucleotide fragments in a single fragment such that the function of one fragment is modulated by the other fragment. For example, a promoter is operably linked to a polynucleotide fragment when it is capable of regulating the transcription of the polynucleotide fragment.

The term "plant" refers to any plant and its progeny at any stage of its life cycle or development. In one embodiment, the plant is a tobacco plant, which 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 progeny thereof. 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.

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

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

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

"Promoter" refers to a molecule of synthetic or natural origin that is capable of conferring, activating or enhancing expression of a polynucleotide in a cell. The term refers to polynucleotide elements/sequences that are typically located upstream of and operably linked to a double-stranded polynucleotide fragment. Promoters may be derived entirely from regions adjacent to the native gene of interest, or may be composed of different elements derived from different native promoters or synthetic polynucleotide fragments. Promoters may contain one or more specific transcriptional regulatory sequences to further enhance expression or alter spatial expression or alter temporal expression. Promoters may also contain terminal enhancer or repressor elements, which may be located up to several kilobase pairs from the transcription initiation site. Promoters may be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. Promoters may regulate expression of a genomic component constitutively or differentially with respect to the cell, tissue, or organ in which expression occurs or with respect to the developmental stage in which expression occurs, or in response to an external stimulus (e.g., physiological stress, pathogen, metal ion, or inducer).

"Tissue-specific promoter" and "tissue-preferred promoter" as used interchangeably herein refer to promoters that are expressed primarily, but not necessarily exclusively, in a tissue or organ, but may also be expressed in a specific cell. "developmentally regulated promoter" refers to a promoter whose function is determined by developmental events. "constitutive promoter" refers to a promoter that causes a gene to be expressed in most cell types most of the time. An "inducible promoter" selectively expresses an operably linked DNA sequence in response to the presence of an endogenous or exogenous stimulus, e.g., by a 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 regulated by light, heat, pressure, flooding or drought, pathogens, plant hormones, wounds or chemicals such as ethanol, jasmonate, salicylic acid or safeners.

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

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

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

The term "tobacco" is used in a generic sense to refer to tobacco crops (e.g., a variety of tobacco plants grown in the field, rather than hydroponically grown tobacco), tobacco plants, and parts thereof, including, but not limited to, roots, stems, leaves, flowers, and seeds prepared or obtained as described herein. It is understood that "tobacco" refers to plants belonging to the genus Nicotiana and products thereof, and includes tobacco plants and products thereof.

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

"Transcription terminator", "termination sequence" or "terminator" refers to a DNA sequence located downstream of a coding sequence, including polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression. Polyadenylation signals are generally characterized as affecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor.

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

"Transgenic plant" refers to a plant that comprises within its genome one or more heterologous polynucleotides, i.e., a plant that contains recombinant genetic material that is not normally found therein and that has been introduced into the plant (or into progenitor cells of the plant) by manual (or artificial) manipulation. For example, a heterologous polynucleotide may be stably integrated into the genome such that the polynucleotide is delivered to successive generations. The heterologous polynucleotide may be integrated into the genome alone or as part of a recombinant construct. Commercial development of genetically modified embryology has also progressed to the stage of introducing various characteristics into crop plants, commonly referred to as gene stacking. In this method, multiple genes can be introduced into the plant that confer different characteristics of interest. Gene stacking can be accomplished in a number of ways including, but not limited to, cotransformation, re-transformation, and crossing with different transgenic lines. Thus, plants grown from plant cells into which recombinant DNA has been introduced by transformation are transgenic plants, all containing progeny (sexual or asexual) of the plant into which the transgene has been introduced. It is understood that the term transgenic plant includes whole plants or trees as well as parts of such plants or trees, such as grains, seeds, flowers, leaves, roots, fruits, pollen, stems, etc. Each heterologous polynucleotide may confer a different trait to the transgenic plant.

"Transgene" refers to a gene or genetic material comprising a sequence of genes that has been isolated from one organism and introduced into a different organism. Such a non-natural fragment of DNA may retain the ability to produce RNA or polypeptides in a transgenic organism, or it may alter the normal function of the genetic code of a transgenic organism.

"Variant" of a polynucleotide refers to (i) a portion or fragment of a polynucleotide, (ii) a complement of a polynucleotide or portion thereof, (iii) a polynucleotide that is substantially identical to a polynucleotide of interest or complement thereof, or (iv) a polynucleotide that hybridizes under stringent conditions to a polynucleotide of interest, complement thereof, or substantially identical thereto.

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

The term "variety" refers to a population of plants that share a constant characteristic that separates them from other plants of the same species. Despite having one or more unique traits, a variety is further characterized by minimal overall variation between individuals within the variety. Varieties are generally marketed.

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

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

An isolated polynucleotide is disclosed comprising, consisting of, or consisting essentially of a sequence having at least 60% sequence identity to any of the sequences described herein, including any of the polynucleotides shown in the sequence listing. Suitably, the isolated polynucleotide comprises, consists of, or consists essentially of a sequence having at least 60％、61％、62％、63％、64％、65％、66％、67％、68％、69％、70％、75％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％ or 100% sequence identity thereto. Suitably, an 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. Suitably, an 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, the isolated polynucleotide comprises, consists of, or consists essentially of a sequence having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto.

Suitably, the polynucleotides described herein encode an active polypeptide having at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 100% or more of the SWEET function or activity of the polypeptides shown in the sequence listing.

In another embodiment, an isolated SWEET polynucleotide (NtSWEET) from tobacco is provided comprising 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 (NtSWEET-S), or

A polynucleotide having or consisting essentially of 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 (NtSWEET-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 (NtSWEET-S), 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 (NtSWEET-T).

In another embodiment, a polynucleotide is provided comprising, consisting of, or consisting 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.

In another embodiment, fragments 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 are provided that have at least about 60％、61％、62％、63％、64％、65％、66％、67％、68％、69％、70％、75％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、99.1％、99.2％、99.3％、99.4％、99.5％、99.6％、99.7％、99.8％、99.9％ or 100% sequence identity to the corresponding fragment of SEQ ID NO. 1 or SEQ ID NO. 3 or SEQ ID NO. 5 or SEQ ID NO. 7.

In another embodiment, fragments 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 are provided that have at least about 60％、61％、62％、63％、64％、65％、66％、67％、68％、69％、70％、75％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、99.1％、99.2％、99.3％、99.4％、99.5％、99.6％、99.7％、99.8％、99.9％ or 100% sequence identity to the corresponding fragment of SEQ ID NO. 1 or SEQ ID NO. 3 or SEQ ID NO. 5 or SEQ ID NO. 7.

In another embodiment, a polynucleotide comprising a sufficient degree 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 is provided that encodes a polypeptide that functions as a SWEET.

In another embodiment, a polymer of polynucleotides is provided 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.

Suitably, the polynucleotides described herein encode a SWEET polypeptide having SWEET activity.

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

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

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

A variety of polynucleotide analogs are known, including, for example, phosphoramidates, phosphorothioates, phosphorodithioates, O-methylphosphite linkages, and peptide polynucleotide backbones and linkages. Other similar polynucleotides include polynucleotides having a positive backbone, a non-ionic backbone, and a non-ribose backbone. Also included are polynucleotides comprising one or more carbocyclic sugars.

Other analogs include peptide polynucleotides that are analogs of the peptide polynucleotide.

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

Basic parameters influencing the choice of hybridization conditions and guidance for designing appropriate conditions are described by Sambrook, j. Degenerate oligonucleotide sets may be prepared using knowledge of the genetic code in combination with the polypeptide sequences described herein. Such oligonucleotides can be used, for example, as primers in the Polymerase Chain Reaction (PCR), thereby isolating and amplifying DNA fragments.

At least one modification (e.g., 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.

An isolated SWEET polypeptide encoded by a polynucleotide as described herein is provided.

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

Also provided is a SWEET polypeptide 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.

Also provided is a SWEET polypeptide 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.

The polypeptide may comprise a sequence having a sufficient degree 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 use as a SWEET.

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

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

Deletions refer to the removal of one or more amino acids from a polypeptide. Insertion refers to one or more amino acid residues introduced into a polypeptide at a predetermined site. Insertions may comprise single or multiple amino acid intra-sequence insertions. Substitution refers to the substitution of an amino acid of a polypeptide with another amino acid having similar properties, such as similar hydrophobicity, hydrophilicity, antigenicity, propensity to form or disrupt an a-helix structure or a β -sheet structure. Amino acid substitutions are typically single residues, but may be clustered, depending on the functional constraints imposed on the polypeptide, and may range from about 1 to about 10 amino acids. The amino acid substitutions are suitably conservative amino acid substitutions as described below. Amino acid substitutions, deletions or insertions may be made using peptide synthesis techniques such as solid phase peptide synthesis or by recombinant DNA manipulation. Methods for manipulating DNA sequences to produce substitution, insertion or deletion variants of polypeptides are well known in the art. The variant may have alterations that produce silent changes and produce functionally equivalent polypeptides. Deliberate amino acid substitutions may be made based on the similarity of the polarity, charge, solubility, hydrophobicity, hydrophilicity, and the amphipathic nature of the residues as long as secondary binding of the substance is maintained. For example, negatively charged amino acids include aspartic acid and glutamic acid, positively charged amino acids include lysine and arginine, and amino acids having similar hydrophilicity values that contain uncharged polar head groups include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine. Conservative substitutions may be made, for example, according to the following table. The same blocks in the second column and suitably the same rows in the third column may be substituted for each other:

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

At least one modification (e.g., 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.

Recombinant constructs can be used to transform plants or plant cells to modulate polypeptide expression, function or activity. A recombinant polynucleotide construct may comprise a polynucleotide encoding one or more polynucleotides as described herein operably linked to a regulatory region suitable for expression of the polypeptide. Thus, a polynucleotide may include a coding sequence that encodes a polypeptide as described herein. Plants or plant cells that modulate the expression, function or activity of a polypeptide may include mutant, non-naturally occurring, transgenic, artificial or genetically engineered plants or plant cells. Suitably, the transgenic plant or plant cell comprises a genome that has been altered by stable integration of the recombinant DNA. Recombinant DNA comprises DNA that has been genetically engineered and constructed outside of the cell and comprises DNA that contains naturally occurring DNA or cDNA or synthetic DNA. Transgenic plants can include plants regenerated from initially transformed plant cells, as well as progeny transgenic plants from later generations of transformed plants or crosses. Suitably, the transgenic modification alters the expression, function or activity of a polynucleotide or polypeptide described herein as compared to a control plant.

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 comprises a polynucleotide that modulates expression operably linked to a regulatory region. Examples of suitable regulatory regions are described herein.

Vectors containing recombinant polynucleotide constructs, such as those described herein, are also provided. Suitable vector backbones include, for example, those conventionally used in the art, such as plasmids, viruses, artificial chromosomes, bacterial artificial chromosomes, yeast artificial chromosomes, or phage artificial chromosomes. Suitable expression vectors include, but are not limited to, plasmids and viral vectors derived from, for example, phages, baculoviruses and retroviruses. Numerous vectors and expression systems are commercially available.

The vector may comprise, for example, an origin of replication, a scaffold attachment region or a marker. The marker gene may confer a selectable phenotype on the plant cell. For example, the marker may confer biocide resistance, such as resistance to antibiotics (e.g., kanamycin (kanamycin), G418, bleomycin (bleomycin), or hygromycin) or herbicides (e.g., glyphosate), chlorsulfuron (chlorsulfuron), or phosphinothricin). In addition, the expression vector may comprise a tag sequence designed to facilitate manipulation or detection (e.g., purification or localization) of the expressed polypeptide. Tag sequences, such as luciferase, β -glucuronidase, green fluorescent polypeptide, glutathione S-transferase, polyhistidine, c-myc or hemagglutinin sequences are typically expressed as fusions with the encoded polypeptide. Such tags may be inserted anywhere within the polypeptide, including at the carboxy or amino terminus.

Plants or plant cells can be transformed by integrating the recombinant polynucleotide into their genome to become stably transformed. The plants or plant cells described herein may be stably transformed. Stably transformed cells typically retain the introduced polynucleotide at each cell division. The plant or plant cell may be transiently transformed such that the recombinant polynucleotide is not integrated into its genome. Transiently transformed cells typically lose all or a portion of the introduced recombinant polynucleotide at each cell division, such that after a sufficient number of cell divisions, the introduced recombinant polynucleotide is not detected in the daughter cells.

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

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

The choice of regulatory region to be included in the recombinant construct depends on several factors including, but not limited to, efficiency, selectivity, inducibility, desired expression levels, and cell or tissue-preferred expression. By appropriate selection of regulatory regions and placement of the regulatory regions relative to the coding sequence, it is routine for one skilled in the art to regulate expression of the coding sequence. Transcription of polynucleotides may be regulated in a similar manner. Some suitable regulatory regions initiate transcription only or predominantly in certain cell types. Methods for identifying and characterizing regulatory regions in plant genomic DNA are known in the art.

Exemplary promoters include tissue-specific promoters recognized by tissue-specific factors, which are present in different tissues or cell types (e.g., root-specific promoters, shoot-specific promoters, xylem-specific promoters), or during different stages of development, or in response to different environmental conditions. Suitable promoters include constitutive promoters which can be activated in most cell types without the need for specific inducers. Examples of promoters for controlling the expression of the polypeptide include cauliflower mosaic virus 35S (CaMV/35S), SSU, OCS, lib4, usp, STLS1, B33, nos or ubiquitin promoters or phaseolin promoters. Those skilled in the art are capable of producing a variety of variants of the recombinant promoter.

Tissue-specific promoters are transcriptional control elements that are active in a particular cell or tissue (such as in vegetative or reproductive tissue) only at a particular time during plant development. Examples of tissue-specific promoters under developmental control include promoters that initiate transcription only (or predominantly only) in certain tissues, such as vegetative tissues (e.g., roots or leaves) or reproductive tissues (such as fruits, ovules, seeds, pollen, pistils, flowers, or any embryonic tissue). The reproductive tissue specific promoter may 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.

Exemplary leaf-specific promoters include the pyruvate orthophosphate dikinase (PPDK) promoter from C4 plants (maize), the cab-m1ca+2 promoter from maize, the arabidopsis thaliana (Arabidopsis thaliana) myb-related gene promoter (Atmyb 5), the rubisco carboxylase (RBCS) promoter (e.g., the tomato RBCS1, RBCS2, and RBCS3A genes expressed in leaf and light-grown seedlings, the RBCS1 and RBCS2 expressed in developing tomato fruits, or the rubisco carboxylase promoter expressed almost exclusively at high levels in mesophyll cells of leaf and leaf sheath).

Exemplary senescence-specific promoters include tomato promoters active during fruit ripening, leaf blight and abscission, maize promoters of genes encoding cysteine proteases, promoters of 82E4, and promoters of SAG genes. Exemplary anther-specific promoters may be used. Exemplary root-preferred promoters known to those skilled in the art may be selected. Exemplary seed preferred promoters include seed specific promoters (those promoters active during seed development, such as promoters of seed storage polypeptides) and seed germination promoters (those promoters active during seed germination).

Examples of inducible promoters include promoters that respond to pathogen attack, anaerobic conditions, high temperature, light, drought, cold temperature, or high salt concentrations. Pathogen inducible promoters include promoters from polypeptides associated with pathogenesis (PR polypeptides) that are induced upon infection by a pathogen (e.g., PR polypeptide, SAR polypeptide, beta-1, 3-glucanase, chitinase).

In addition to plant promoters, other suitable promoters may be derived from bacterial sources, e.g., octopine synthase promoter, nopaline synthase promoter, and other promoters derived from Ti plasmid, or may be derived from viral promoters (e.g., 35S and 19S RNA promoters of cauliflower mosaic virus (CaMV), constitutive promoters of tobacco mosaic virus, cauliflower mosaic virus (CaMV) 19S and 35S promoters, or figwort mosaic virus 35S promoter).

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

A method for modulating the level of NtSWEET polypeptide in a (dried or desiccated) plant or (dried or desiccated) plant material is provided, the method comprising introducing into the genome of the plant one or more mutations that modulate the expression of at least one NtSWEET, wherein the at least one NtSWEET gene is selected from one or more NtSWEET sequences according to the present disclosure.

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

Also disclosed is a plant or plant cell that is heterozygous or homozygous for one or more mutations in a NtSWEET gene according to the disclosure, wherein the mutation results in modulated expression of the NtSWEET gene or the function or activity of a NtSWEET polypeptide encoded thereby.

A number of methods are available for combining mutations in a plant, including sexual crosses. Plants having one or more favorable heterozygous or homozygous mutations in a gene according to the present disclosure that regulate expression of the gene or function or activity of a polypeptide encoded thereby can be crossed with plants having one or more favorable heterozygous or homozygous mutations in one or more other genes that regulate expression of the gene or function or activity of a polypeptide encoded thereby. In one embodiment, a crossing is performed to introduce one or more advantageous heterozygous or homozygous mutations within a gene according to the present disclosure within the same plant.

If the function or activity of one or more polypeptides of the present disclosure in a plant is lower or higher than the function or activity of the same polypeptide in a plant, the function or activity is increased or decreased, the plant is not modified to inhibit the function or activity of the polypeptide and has been cultivated, harvested and dried or dried using the same protocol.

In some embodiments, the mutation is introduced into the plant or plant cell using mutagenesis methods, and the introduced mutation is identified or selected using methods known to those of skill in the art, such as Southern blot analysis, DNA sequencing, PCR analysis, or phenotypic analysis. Mutations that affect gene expression or interfere with the function of the encoded polypeptide can be determined using methods well known in the art. Insertion mutations in gene exons typically result in empty mutations. Mutations in conserved residues may be particularly effective in inhibiting the metabolic function of the encoded polypeptide. For example, it will be appreciated that mutations in one or more highly conserved regions may alter polypeptide function, while mutations outside those highly conserved regions may have little or no effect on polypeptide function. Furthermore, mutations in a single nucleotide can create a stop codon, which will result in a truncated polypeptide and, depending on the degree of truncation, loss of function.

Methods for obtaining mutant polynucleotides and polypeptides are also disclosed. Any plant of interest, including plant cells or plant material, may be genetically modified by a variety of known methods of induced mutagenesis, including site-directed mutagenesis, oligonucleotide-directed mutagenesis, chemically induced mutagenesis, radiation-induced mutagenesis, mutagenesis with modified bases, mutagenesis with gapped duplex DNA, double-strand break mutagenesis, mutagenesis with repair-deficient host strains, mutagenesis by total gene synthesis, DNA shuffling, and other equivalent methods.

Mutations in the polynucleotides and polypeptides described herein may include artificial mutations or synthetic mutations or genetic engineering mutations. Mutations in the polynucleotides and polypeptides described herein may be mutations obtained or obtainable by a process comprising in vitro or in vivo procedures. Mutations in the polynucleotides and polypeptides described herein may be mutations obtained or obtainable by a process that includes human intervention. The function or activity of a mutant polypeptide variant may be higher, lower or about the same as the unmutated polypeptide.

Methods of randomly introducing mutations in polynucleotides may include chemical mutagenesis and radiomutagenesis. Chemical mutagenesis involves the use of exogenously added chemicals, such as mutagenic, teratogenic or carcinogenic organic compounds, to induce mutations. Mutagens (including chemical mutagens or radiation) that produce predominantly point mutations and shortfall, insertions, missense mutations, simple sequence repeats, transversions or transitions can be used to produce mutations. Mutagens include ethyl methanesulfonate, methyl methanesulfonate, N-ethyl-N-nitrosourea, triethylmelamine, N-methyl-N-nitrosourea, procarbazine, chlorambucil, cyclophosphamide, diethyl sulfate, acrylamide monomers, melphalan, nitrogen mustard, vincristine, dimethylnitrosamine, N-methyl-N' -nitro-nitrosoguanidine, 2-aminopurine, 7, 12-dimethyl-benzo (a) anthracene, ethylene oxide, hexamethylphosphoramide, busulfan (bisulfan), dioxane (bisepoxyoctane, bisepoxybutane, etc.), 2-methoxy-6-chloro-9- [3- (ethyl-2-chloro-ethyl) aminopropylamino ] acridine dihydrochloride, and formaldehyde.

Spontaneous mutations in loci that may not be directly caused by mutagens are also contemplated, provided they produce the desired phenotype. Suitable mutagenizing agents may also include, for example, ionizing radiation, such as X-rays, gamma rays, fast neutron irradiation, and UV radiation. For each type of plant tissue, the dose of mutagenic substance or radiation is determined experimentally so that a frequency of mutations below a threshold level characterized by lethality or reproductive sterility is obtained. Any plant polynucleotide preparation method known to those skilled in the art may be used to prepare plant polynucleotides for mutation screening.

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

After mutation, screening can be performed to identify mutations that produce premature stop codons or non-functional genes. Following mutation, screening can be performed to identify mutations that result in functional genes that can be expressed at increased or decreased levels. Screening of mutants may be performed by sequencing or by using one or more probes or primers specific for the gene or polypeptide. Specific mutations can also be made in polynucleotides, which can result in regulated gene expression, regulated mRNA stability, or regulated polypeptide stability. Such plants are referred to herein as "non-naturally occurring" or "mutant" plants. Typically, a mutant or non-naturally occurring plant will include at least a portion (e.g., DNA or RNA) of a foreign or synthetic or artificial nucleotide that was not present in the plant prior to being manipulated. The foreign nucleotide may be a single nucleotide, two or more nucleotides, two or more consecutive nucleotides, or two or more non-consecutive nucleotides, for example at least 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or 1500 or more consecutive or non-consecutive nucleotides.

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

Zinc finger polypeptides may be used to modulate the expression of 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).

Meganucleases such as I-CreI can be used to modulate the expression or function or activity of one or more 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.

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

CRISPR systems can be used to modulate the expression or function or activity of one or more NtSWEET polynucleotides described herein and are preferred methods. This technique is described, for example, in Plant Methods (2016) 12:8;Front Plant Sci (2016) 7:506;Biotechnology Advances (2015) 33,1, pages 41-52, acta Pharmaceutica Sinica B (2017) 7,3, p292-302;Curr.Op.in Plant Biol (2017) 36,1-8 and Int J Mol Sci (2019) 20 (16), 4045. As is well known in the art, CRISPR editing systems typically comprise two components, a CRISPR-associated endonuclease (Cas) (e.g., cas 9) and a guide RNA (gRNA). Cas forms a double-stranded DNA break at a site in the genome defined by the sequence of the gRNA molecule that binds to Cas. The location of Cas cleavage DNA is defined by the unique sequence of the gRNA to which it binds. gRNA is a specially designed RNA sequence that recognizes the target DNA region of interest and directs Cas nuclease for editing. It has two segments, (i) a tracr RNA that serves as a binding scaffold for Cas nuclease, and (ii) CRISPR RNA (crRNA), 17-20 nucleotide sequences that are complementary to the target DNA. The exact region of DNA to be targeted will depend on the particular application. For example, to activate or inhibit 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, including Chop Chop Harvard. Applications of Cas 9-based genome editing in arabidopsis and tobacco are described, for example, in Methods enzymol (2014) 546:459-72 and Plant Physiol biochem (2018) 131:37-46. CRISPR technology has been widely used in plants (see e.g. WO 2015/189693). In addition to Cas9, other RNA-guided nucleases for use in CRISPR systems are described, including Casl、CaslB、Cas2、Cas3、Cas4、Cas5、Cas6、Cas7、Cas8、CaslO、Cpfl、Csyl、Csy2、Csy3、Csel、Cse2、Cscl、Csc2、Csa5、Csn2、Csm2、Csm3、Csm4、Csm5、Csm6、Cmrl、Cmr3、Cmr4、Cmr5、Cmr6、Csbl、Csb2、Csb3、Csxl7、Csxl4、CsxlO、Csxl6、CsaX、Csx3、Csxl、Csxl5、Csfl、Csf2、Csf3 and Csf4. In certain embodiments, it is preferred to use Cas 9. The present disclosure also provides CRISPR-based genome editing systems comprising an RNA-guided nuclease and a gRNA, wherein the CRISPR-based genome editing system modulates the activity of one or more polynucleotides described herein. The present disclosure also provides methods of cleaving one or more polynucleotides in a plant cell, the method comprising introducing into the plant cell a gRNA and an RNA-guided nuclease, wherein the gRNA works in conjunction with the RNA-guided nuclease to produce strand breaks in the one or more polynucleotides described herein. Also disclosed are CRISPR constructs comprising (i) a polynucleotide encoding a CRISPR-associated endonuclease, and (ii) a gRNA comprising a polynucleotide sequence (typically about 17-20 nucleotides) complementary to DNA of a polynucleotide as described herein to be targeted.

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

The NtSWEET polynucleotide may be targeted for inactivation by introducing a transposon (e.g., an IS element or other mobile genetic element) into the genome of the plant of interest. See, e.g., cytology AND GENETICS (2006) 40 (4): 68-81.

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

A mutant or non-naturally occurring plant or plant cell may have any combination of one or more modifications (e.g., mutations) in one or more NtSWEET polynucleotides described herein that result in the modulation of expression or function or activity of those polynucleotides or their polynucleotide products. For example, a mutant or non-naturally occurring plant or plant cell may 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. By way of further example, a mutant or non-naturally occurring plant or plant cell may have one or more modifications in a particular portion of a NtSWEET polynucleotide or NtSWEET polypeptide (such as in the region of NtSWEET encoding the active site of a NtSWEET polypeptide or portion thereof). By way of further example, a mutant or non-naturally occurring plant or plant cell may have one or more modifications in a region other than one or more NtSWEET nucleotides or NtSWEET polypeptides (such as in the upstream or downstream regions of the NtSWEET polynucleotide) provided that it modulates the function or expression of NtSWEET. The upstream element may include a promoter, enhancer or transcription factor. Some elements, such as enhancers, may be placed upstream or downstream of the gene that it modulates. An element need not be located close to the gene it regulates, as some elements have been found to be located several hundred thousand base pairs upstream or downstream of the gene it regulates. The mutant or non-naturally occurring plant or plant cell may have one or more modifications located within the first 100 nucleotides of the gene, within the first 200 nucleotides of the gene, within the first 300 nucleotides of the gene, within the first 400 nucleotides of the gene, within the first 500 nucleotides of the gene, within the first 600 nucleotides of the gene, within the first 700 nucleotides of the gene, within the first 800 nucleotides of the gene, within the first 900 nucleotides of the gene, within the first 1000 nucleotides of the gene, within the first 1100 nucleotides of the gene, within the first 1200 nucleotides of the gene, within the first 1300 nucleotides of the gene, within the first 1400 nucleotides of the gene, or within the first 1500 nucleotides of the gene. The mutant or non-naturally occurring plant or plant cell may have one or more modifications 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-naturally occurring plants or plant cells (e.g., mutant, non-naturally occurring or transgenic plants or plant cells, etc., as described herein) comprising mutant polypeptide variants.

In one embodiment, seeds from the plant are mutagenized and subsequently 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, which are then screened for mutations in their loci. Although mutagenized plant material may be screened for mutations, the advantage of screening second generation plants is that all somatic mutations correspond to germline mutations. Those skilled in the art will appreciate that a variety of plant materials, including but not limited to seeds, pollen, plant tissue or plant cells, may be subjected to mutagenesis in order to produce mutant plants. However, when screening for mutations in plant polynucleotides, the type of plant material that is mutagenized may have an effect. For example, when pollen is subjected to mutagenesis prior to pollination of non-mutagenized plants, the seeds obtained from pollination are grown into first generation plants. Each cell of the first generation plants will contain a mutation that is generated in pollen, so these first generation plants can then be screened for the mutation rather than waiting until the second generation.

NtSWEET polynucleotides prepared from individual plants, plant cells, or plant material may optionally be combined to facilitate selection of mutations in a plant population derived from mutagenized plant tissue, cells, or material. One or more subsequent generations of plants, plant cells, or plant material may be screened. The size of the optionally pooled groups depends on the sensitivity of the screening method used. After the samples are optionally pooled, they may be subjected to polynucleotide specific amplification techniques, such as PCR. Any one or more primers or probes specific for the gene or sequences immediately adjacent to the gene may be used to amplify sequences within the optionally pooled sample. Suitably, one or more primers or probes are designed to amplify the region of the locus where the useful mutation is most likely to occur. Most suitably, the primers are designed to detect mutations within the polynucleotide region. In addition, primers and probes preferably avoid known polymorphic sites to facilitate screening for point mutations. To facilitate detection of the amplified product, any conventional labeling method may be used to label one or more primers or probes. Primers or probes can be designed based on the sequences described herein using methods well understood in the art. To facilitate detection of the amplified product, any conventional labeling method may be used to label the primer or probe. These primers or probes can be designed based on the sequences described herein using methods well understood in the art.

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

In some embodiments, the plant may be regenerated or grown from a plant, plant tissue, or plant cell. Any suitable method of regenerating or growing a plant from plant cells or plant tissue may be used, such as, but not limited to, culture or regeneration from protoplast tissue. Suitably, the plant may be regenerated by growing transformed plant cells on callus induction medium, shoot induction medium or root induction medium. See, e.g., mcCormick et al PLANT CELL Reports 5:81-84 (1986). These plants can then be grown and pollinated either by the same transformed line or by a different line, and the resulting hybrids with the desired phenotypic characteristic expression identified. Two or more generations may be grown to ensure stable maintenance and inheritance of expression of the desired phenotypic characteristic, and seeds picked to ensure expression of the desired phenotypic characteristic is obtained. Thus, a "transformed seed" refers to a seed that contains a nucleotide construct that is stably integrated into the plant genome.

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

In certain embodiments, a mutant plant, mutant plant cell, or mutant plant material may comprise one or more mutations that occur naturally in another plant, plant cell, or plant material and confer a desired trait. The mutation may be introduced (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 impart the trait thereto. Thus, for example, a mutation that occurs naturally in a first plant can be introduced into a second plant, such as a second plant having a different genetic background than the first plant. The skilled artisan is thus able to search for and identify plants that naturally carry in the genome one or more mutant alleles of a gene described herein, which confers a desired trait. Naturally occurring mutant alleles can be transferred to a second plant by a variety of methods, including breeding, backcrossing, and introgression, to produce lines, varieties, or hybrids having one or more mutations in the genes described herein. The same technique may also be applied to the introgression of one or more non-natural mutations from a first plant into a second plant. Plants exhibiting the desired trait can be screened from a pool of mutant plants. Suitably, the selection is made using knowledge of the polynucleotides as described herein. Thus, the genetic trait can be screened as compared to the control. Such screening methods may involve the use of conventional amplification or hybridization techniques as discussed herein. Accordingly, another aspect of the present disclosure relates to a method for identifying a mutant plant, comprising the steps of (a) providing a sample comprising one or more NtSWEET polynucleotides from a plant, and (b) determining the sequence of the polynucleotide, wherein a difference in the sequence of the polynucleotide compared to the polynucleotide of a control plant indicates that the plant is a mutant plant. In another aspect, a method is provided for identifying a mutant plant which accumulates an increased or decreased level of amino acids as compared to a control plant, comprising (a) providing a sample from a plant to be screened, (b) determining whether the sample comprises one or more mutations in one or more NtSWEET polynucleotides described herein, and (c) determining the level of at least one amino acid in the plant. Suitably, the level of at least one amino acid in the dried or dried leaves is determined. 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, comprising (a) providing a sample from a first plant, (b) determining whether the sample comprises 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) transferring the one or more mutations into a second plant. Suitably, the level of at least one amino acid in the dried or dried leaves is determined. The mutation may be transferred into the second plant using a variety of methods known in the art, such as by genetic engineering, genetic manipulation, introgression, plant breeding, backcrossing, and the like. In one embodiment, the first plant is a naturally occurring plant. In one embodiment, the second plant has a different genetic background than the first plant. In another aspect, a method 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 comprises 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 from the first plant into a second plant. Suitably, the level of at least one amino acid in the dried or dried leaves is determined. In one embodiment, the introgression step comprises plant breeding, optionally comprising backcrossing, and the like. In one embodiment, the first plant is a naturally occurring plant. In one embodiment, the second plant has a different genetic background than the first plant. In one embodiment, the first plant is not a cultivar or elite cultivar. In one embodiment, the second plant is a cultivar or a elite cultivar. Another aspect relates to a mutant plant (including cultivar or elite cultivar mutant plants) obtained or obtainable by the method described herein. In certain embodiments, a "mutant plant" may have one or more mutations that are located only in a specific region of the plant, e.g., within the sequences of one or more NtSWEET polynucleotides described herein. According to this embodiment, the remaining genomic sequence of the mutant plant will be identical or substantially identical to the plant prior to mutagenesis.

In certain embodiments, a mutant plant may have one or more mutations located in more than one genomic region of the plant, such as within the sequences of one or more NtSWEET polynucleotides described herein, and within one or more other regions of the genome. According to this embodiment, the remaining genomic sequence of the mutant plant will be different or substantially different from the plant prior to mutagenesis. In certain embodiments, the mutant plants may not have one or more, two or more, three or more, four or more, or one or more mutations in five or more exons of the NtSWEET polynucleotide described herein, or may not have one or more, two or more, three or more, four or more, or one or more mutations in five or more introns of the NtSWEET polynucleotide described herein, or may not have one or more mutations in the promoter of the NtSWEET polynucleotide described herein, or may not have one or more mutations in the 3 'untranslated region of the NtSWEET polynucleotide described herein, or may not have one or more mutations in the 5' untranslated region of the NtSWEET polynucleotide described herein, or may not have one or more mutations in the coding region of NtSWEET polynucleotide described herein, or may not have one or more mutations in the coding region of the NtSWEET polynucleotide described herein.

In another aspect, a method is provided for identifying a plant, plant cell or plant material comprising a mutation in a gene encoding a NtSWEET polynucleotide described herein, the method comprising (a) mutagenizing the plant, plant cell or plant material, (b) obtaining a sample from the plant, plant cell or plant material or progeny thereof, and (c) determining the polynucleotide sequence of the NtSWEET gene or variant or fragment thereof, wherein a difference in the sequence is indicative of one or more mutations therein. The method also allows selection of plants with mutations that occur in genomic regions that affect NtSWEET gene expression in plant cells, such as transcription initiation sites, start codons, intronic regions, exon-intron boundaries, terminators.

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

Various embodiments relate to mutant tobacco, non-naturally occurring tobacco or transgenic tobacco plants or tobacco plant cells, and may be applied to any species of nicotiana, including nicotiana tabacum (n.rusica) and nicotiana tabacum (e.g., LA B21, LN KY171, TI 1406, basma, galpao, perique, beinhart 1000-1 and Petico). other species include smokeless tobacco (N.acaulis), acuminate tobacco (N.acuminata), african tobacco (N.africana), floral tobacco (N.ala), A Mi Jinuo's tobacco (N.ameghinoi), hubbed tobacco (N.amplificalis), arrentsii tobacco (N.arentii), progressive tobacco (N.attenuata), armbuji tobacco (N.azambujae), bei Namo's tobacco (N.benavidesii), saccharum sinensis (N.benthamiana), Indian tobacco (N.bigelovii), boehringer tobacco (N.bonariensis), dongsheng tobacco (N.cappuccino), kevlar tobacco (N.cleveland dii), portal tobacco (N.cordifolia), umbrella bed tobacco (N.corymbosa), diberna tobacco (N.debneyi), murraya (N.excelsior), ful Ji Teshi tobacco (N.forgetiana), cigarette grass (N.fragrans), pink blue tobacco (N.glauca), nicotiana tabacum, Nicotiana tabacum (N.glutinosa), gu Tesi Nicotiana tabacum (N.goodpspeed ii), nicotiana griseus (N.gossei), nicotiana tabacum (N.hybrid), nicotiana tabacum (N.ingulba), nicotiana tabacum (N.kawakami), nicotiana tabacum (N.knaghtiana), nicotiana tabacum (N.Iangsdoffii), nicotiana tabacum (N.lineris), nicotiana longifolia (N.Iongifolia), nicotiana maritima (N.maritima), nicotiana tabacum (N.kanagana), Tobacco of king pipe (N.megalophos), nicotiana moelleriensis (N.miersii), nicotiana nocturna (N.noctiflora), nicotiana nudiflora (N.nudiflora), nicotiana tabacum (N.obtusifolia), nicotiana tabacum (N.occidentalis subsp. Heperis), nicotiana otophylla (N.otophora), nicotiana circulans (N.paniculata), nicotiana parvifolia (N.pa, Petunia (n. Petunia), jasmine leaf tobacco (n. Plumbeginfolia), quarry tobacco (n. Quadrivalvis), raymond tobacco (n. Raimondii), tabacco (n. Renda), rosette tobacco (n. Rossulata), rosette tobacco Gu Erba (n. Rossulata subsp. Inguba), round leaf tobacco (n. Rotundifolia), saururi tobacco (n. Setchelli), Tobacco mimetics (N.simulas), front leaf tobacco (N.solanifolia), tobacco of the tobacco Sa Peg (N.spegazzinii), tobacco of the tobacco Stokes (N.stononii), tobacco of the tobacco Suaviens (N.suaveolens), tobacco of the tobacco Melastoma (N.sylvestris), tobacco of the tobacco pseudoscion (N.thyrsislope), tobacco of the tobacco villus (N.tomtosa), tobacco of the tobacco villus (N.tomotosiformis), tobacco of the tobacco Trigonophila (N.trigofila), shadow tobacco (n.umbral), boswellia (n.undurata), scirpus (n.velutina), scirpus (n.wigandioides) and nicotiana tabacum (N.x sanderae). In one embodiment, the plant is tobacco.

Also contemplated herein are the use of tobacco cultivars and elite tobacco cultivars. Thus, the transgenic, non-naturally occurring or mutant plant may be a tobacco variety or elite tobacco cultivar comprising one or more transgenes, or one or more genetic mutations, or a combination thereof. The genetic mutation (e.g., one or more polymorphisms) may be a mutation that is not naturally occurring in the individual tobacco variety or tobacco cultivar (e.g., elite tobacco cultivar), or may be a genetic mutation that does naturally occur, provided that the mutation is not naturally occurring in the individual tobacco variety or tobacco cultivar (e.g., elite tobacco cultivar).

Particularly useful tobacco varieties include burley-, black-, flue-and oriental-type tobacco. Non-limiting examples of varieties or cultivars are ：BD 64、CC 101、CC 200、CC 27、CC 301、CC 400、CC 500、CC 600、CC 700、CC 800、CC 900、Coker 176、Coker 319、Coker 371Gold、Coker 48、CD 263、DF911、DT 538LC Galpao tobacco, GL 26H, GL, GL 600, GL 737, GL 939, GL 973, HB 04P, HB P LC, HB3307PLC, hybrid 403LC, hybrid 404LC, hybrid 501LC、K 149、K326、K 346、K 358、K394、K 399、K 730、KDH 959、KT 200、KT204LC、KY10、KY14、KY 160、KY 17、KY 171、KY 907、KY907LC、KY14xL8 LC、Little Crittenden、McNair 373、McNair944、msKY 14×L8、 narrow leaf Madole, narrow leaf Madole LC、NBH 98、N-126、N-777LC、N-7371LC、NC 100、NC 102、NC 2000、NC 291、NC 297、NC 299、NC 3、NC 4、NC 5、NC 6、NC7、NC 606、NC 71、NC 72、NC 810、NC BH 129、NC 2002、Neal Smith Madole、OXFORD 207、PD 7302LC、PD 7309LC、PD 7312LC、'Perique' tobacco 、PVH03、PVH09、PVH19、PVH50、PVH51、R 610、R 630、R 7-11、R 7-12、RG 17、RG 81、RG H51、RGH 4、RGH 51、RS1410、Speight 168、Speight 172、Speight 179、Speight 210、Speight 220、Speight 225、Speight 227、Speight 234、Speight G-28、Speight G-70、Speight H-6、Speight H20、Speight NF3、TI 1406、TI 1269、TN 86、TN86LC、TN 90、TN 97、TN97LC、TN D94、TN D950、TR(Tom Rosson)Madole、VA 309、VA359、AA 37-1、B13P、Xanthi(Mitchell-Mor)、Bel-W3、79-615、Samsun HolmesNN、KTRDC 2, hybrid 49, burley 21, KY8959, KY9, MD 609, PG01, PG04, PO1, PO2, PO3, RG11, RG 8, VA509, AS44, banket A, basma Drama B84/31, basma I Zichna ZP/B, basma Xanthi BX 2A, batek, besuki Jember, C104, coker 347, criollo Misionero, delcrest, djebel 81, DVH 405,Comum, HB04P, hicks broadleaf 、Kabakulak Elassona、Kutsage E1、LA BU 21、NC 2326、NC 297、PVH 2110、Red Russian、Samsun、Saplak、Simmaba、Talgar 28、Wislica、Yayaldag、Prilep HC-72、Prilep P23、Prilep PB 156/1、Prilep P12-2/1、Yaka JK-48、Yaka JB 125/3、TI-1068、KDH-960、TI-1070、TW136、 basma, TKF 4028, L8, TKF 2002, GR141, basma xanthi, GR149, GR153, PETIT HAVANA. Even if not specifically indicated herein, the low-transformation subvariants described above are contemplated.

Embodiments also relate to compositions and methods for producing mutant plants, non-naturally occurring plants, hybrid plants, or transgenic plants that have been modified to modulate expression of or function of one or more NtSWEET polynucleotides described herein (or any combination thereof as described herein). Advantageously, the obtained mutant, non-naturally occurring, hybrid or transgenic plant may be similar or substantially identical in overall appearance to the control plant. Various phenotypic characteristics, such as maturity, leaf number per plant, stalk height, she Charu angle, leaf size (width and length), internode distance, and leaf-to-midvein ratio can be assessed by field observations.

One aspect relates to seeds of a mutant plant, non-naturally occurring plant, hybrid plant, or transgenic plant described herein. Suitably, the seed is tobacco seed. Another aspect relates to pollen or ovules of a mutant, non-naturally occurring, hybrid or transgenic plant described herein. In addition, provided are mutant plants, non-naturally occurring plants, hybrid plants, or transgenic plants as described herein, further comprising a polynucleotide that confers male sterility.

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

The plant material described herein may be a dried or dried tobacco material. The CORESTA recommendation for tobacco drying is described in CORESTA guide No. 17, month 4 of 2016, sustainabilityin Leaf Tobacco Production.

According to the present disclosure, expression of one or more of SWEET12-S or SWEET12-T or SWEET15-S or SWEET15-T or activity of one or more of SWEET12-S or SWEET12-T or SWEET15-S or SWEET15-T is modulated as compared to control plants.

In one embodiment, the expression of SWEET12-S or the activity of SWEET12-S is modulated as compared to a control plant. In another embodiment, the expression of SWEET12-T or the activity of SWEET12-T is modulated as compared to a control plant. In another embodiment, the expression of SWEET12-S and SWEET12-T or the activity of SWEET12-S and SWEET12-T is modulated as compared to control plants.

In another embodiment, the expression of SWEET15-S or the activity of SWEET15-S is modulated as compared to a control plant. In another embodiment, the expression of SWEET15-T or the activity of SWEET15-T is modulated as compared to a control plant. In another embodiment, the expression of SWEET15-S and SWEET15-T or the activity of SWEET15-S and SWEET15-T is modulated as compared to control plants.

In another embodiment, the expression of SWEET12-S and SWEET15-S or the activity of SWEET12-S and SWEET15-S is modulated as compared to control plants.

In another embodiment, the expression of SWEET12-S and SWEET15-T or the activity of SWEET12-S and SWEET15-T is modulated as compared to control plants.

In another embodiment, the expression of SWEET12-T and SWEET15-S or the activity of SWEET12-T and SWEET15-S is modulated as compared to control plants.

In another embodiment, the expression of SWEET12-T and SWEET15-T or the activity of SWEET12-T and SWEET15-T is modulated as compared to control plants.

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 as compared to control plants.

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 as compared to control plants.

In another embodiment, the expression of SWEET12-S and SWEET15-T or the activity of SWEET12-S and SWEET15-T is modulated as compared to control plants.

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 as compared to control plants.

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 as compared to control plants.

Modulating expression of one or more of SWEET12-S or SWEET12-T or SWEET15-S or SWEET15-T or activity of one or more of SWEET12-S or SWEET12-T or SWEET15-S or SWEET15-T compared to a control plant may modulate plant development during the vegetative stage compared to a control plant and/or it may modulate plant flowering time compared to a control plant. For example, modulating (suitably, reducing) the expression of SWEET12-S and/or SWEET12-T or the activity of SWEET12-S and/or SWEET12-T may modulate (suitably, increase) plant height during the vegetative phase of growth and may result in an acceleration of flowering time. By way of further example, modulating (suitably reducing) the expression of SWEET15-S and/or SWEET15-T or the activity of SWEET15-S and/or SWEET15-T may modulate (suitably increase) plant height during the vegetative phase of growth and may result in an acceleration of flowering time. By way of further example, modulating (suitably increasing) the expression of SWEET15-T and/or SWEET15-S or the activity of SWEET15-T and/or SWEET15-S may modulate (e.g., accelerate or increase) flowering time.

Modulating 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 as compared to a control plant can modulate the chemical characteristics of dried or dried leaves derived from the plant. Chemical features may include sugar features and/or amino acid features. For example, modulating (suitably increasing) the expression of SWEET12-S and/or SWEET12-T or the activity of SWEET12-S and/or SWEET12-T may modulate (suitably reduce) the fructose, glucose and sucrose content of the dried or dried leaves. By way of further example, modulating (suitably increasing) the expression of SWEET15-T or the activity of SWEET15-T may modulate (suitably decreasing) the fructose, glucose and sucrose content of the dried or dried leaves. By way of further example, modulating (suitably increasing) the expression of SWEET15-S and SWEET15-T or the activity of SWEET15-S and SWEET15-T may modulate (suitably reduce) the fructose, glucose and sucrose content of the dried or dried leaves.

Modulating expression of one or more of SWEET12-S or SWEET12-T or SWEET15-S or SWEET15-T or activity of one or more of SWEET12-S or SWEET12-T or SWEET15-S or SWEET15-T as compared to control plants can modulate amino acid characteristics of dried or dried leaves derived from the plant. Amino acid characteristics may include asparagine and tryptophan and proline or asparagine, tryptophan, proline, phenylalanine, glycine, methionine and glutamic acid. When amino acid characteristics are modulated, the level of one or more amino acids may be increased or decreased. 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 content are decreased. For example, modulating (suitably increasing) the expression of SWEET12-S and/or SWEET12-T or the activity of SWEET12-S and/or SWEET12-T may modulate the levels of asparagine and tryptophan and proline and phenylalanine and glycine and methionine and glutamic acid, suitably increasing the levels of asparagine, tryptophan, phenylalanine, glycine and methionine, and decreasing the levels of glutamic acid and proline. By way of further example, modulating (suitably increasing) the expression of SWEET15-T and/or SWEET15-S or the activity of SWEET15-T and/or SWEET15-S may modulate the levels of asparagine and tryptophan and proline, suitably increasing the levels of asparagine and tryptophan, and decreasing the levels of proline. When the expression of SWEET12-S and/or SWEET12-T or the activity of SWEET12-S and/or SWEET12-T was reduced, no significant change in the levels of sugar or amino acids was observed, however the organoleptic properties of the dried or dried tobacco were still altered (see Table 6).

Modulating 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 as compared to control plants can modulate the amount of leaf biomass. For example, modulating (suitably increasing) the expression of SWEET12-S and/or SWEET12-T or the activity of SWEET12-S and/or SWEET12-T may modulate (suitably reduce) the amount of leaf biomass. By way of further example, modulating (suitably increasing) the expression of SWEET15-S and/or SWEET15-T or the activity of SWEET15-S and/or SWEET15-T may modulate (suitably reduce) the amount of leaf biomass. 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, the amount of leaf biomass does not change significantly. In certain embodiments, it is preferred not to reduce the amount of leaf biomass, as this will preserve the yield that can be obtained during commercial production of tobacco.

Modulating the expression of SWEET12-S and/or SWEET12-T or the activity of SWEET12-S or SWEET12-T may modulate ammonia levels as compared to control plants. For example, modulating (suitably increasing) the expression of SWEET12-S and/or SWEET12-T or the activity of SWEET12-S and/or SWEET12-T may modulate (suitably increase) the level of ammonia. Ammonium compounds are known to react with sugars during tobacco processing and smoking to form flavor components that alter the taste of tobacco smoke. Thus, it is contemplated to adjust the ammonia level to alter tobacco flavor.

Suitably, the level of modulation is observed in at least dried or dried leaves (suitably, substantially dried or dried leaves). Tobacco is considered to be sufficiently dry when the ribbing in the leaf is free of moisture, resulting in a light tan to reddish brown to dark brown color of the leaf. Suitably, the dried leaves are taken from intermediate position leaves on the plant.

Another aspect relates to a mutant, non-naturally occurring or transgenic plant or cell in which expression of one or more NtSWEET polynucleotides or activity of one or more NtSWEET polypeptides has been modulated as compared to a control plant or portion thereof in which expression of NtSWEET or activity of NtSWEET has not been modulated.

Another aspect relates to a dried or desiccated plant material, such as dried or desiccated leaves or dried or desiccated tobacco, derived or derivable from a mutant, non-naturally occurring or transgenic plant or cell, wherein expression of or function of a NtSWEET polypeptide encoded by one or more of the NtSWEET polynucleotides described herein is modulated as compared to a control plant or portion thereof.

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

In one embodiment, the phenotype of the mutant, non-naturally occurring or transgenic plant is substantially the same as a control plant or part thereof. In one embodiment, the leaf weight of the mutant, non-naturally occurring or transgenic plant is substantially the same as the control plant or part thereof. In one embodiment, the number of leaves of the mutant, non-naturally occurring or transgenic plant is substantially the same as the control plant or portion thereof. In one embodiment, the leaf weight and leaf number of the mutant, non-naturally occurring or transgenic plant is substantially the same as the control plant. In one embodiment, the stalk height of the mutant, non-naturally occurring or transgenic plant is substantially the same as the control plant or part thereof, for example, one, two or three or more months after field transplantation or 10, 20, 30 or 36 or more days after topping. For example, the stalk height of a mutant, non-naturally occurring or transgenic plant is not lower than the stalk height of a control plant or portion thereof.

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., a leaf, such as a dried or desiccated leaf), the method comprising (i) modulating the expression or function of one or more NtSWEET polypeptides described herein, suitably wherein 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-naturally occurring or transgenic plant obtained in step (i) (e.g., a leaf (such as a dried or desiccated leaf) or tobacco or smoke), and (iii) identifying a mutant, non-naturally occurring or transgenic plant or portion thereof in which the level of at least one amino acid and/or sugar has been modulated as compared to a control plant or portion thereof.

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 a dried or desiccated plant material, such as dried or desiccated leaves, the method comprising (i) modulating the expression or function of one or more NtSWEET polypeptides (or any combination thereof as described herein), suitably wherein NtSWEET polypeptide is encoded by a corresponding NtSWEET polynucleotide as described herein, (ii) harvesting the plant material, such as one or more leaves, and desiccating for a period of time, (iii) measuring the level of at least one amino acid and/or at least one sugar in the dried or desiccated plant material obtained in or during step (ii), and (iv) identifying a dried or desiccated plant material in which the level of at least one amino acid and/or at least one sugar has been modulated compared to a control plant or part thereof.

The increase in expression may 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% or more, such as 200%, 300%, 500%, 1000% or more, as compared to a control, which includes an increase in transcription function or NtSWEET polynucleotide expression or NtSWEET polypeptide expression.

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

The reduction in expression may 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% as compared to a control, comprising a reduction in transcription function or NtSWEET polynucleotide expression or NtSWEET polypeptide expression, or a combination thereof.

The decrease in function or activity as compared to a control may 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% less, which includes a decrease in transcription function or NtSWEET polynucleotide expression or NtSWEET polypeptide expression, or a combination thereof.

The polynucleotides and recombinant constructs described herein may be used to modulate the expression of or function or activity of the NtSWEET polynucleotide or NtSWEET polypeptide described herein in a plant species of interest (suitably tobacco).

Many polynucleotide-based methods are available for increasing gene expression in plants and plant cells. As an example, a construct, vector or expression vector may be prepared that is compatible with the plant to be transformed, including the gene of interest together with an upstream promoter capable of overexpressing the gene in the plant or plant cell. Exemplary promoters are described herein. After transformation, and when grown under appropriate conditions, the promoter may drive expression to regulate NtSWEET levels in the plant or its particular tissue. In an exemplary embodiment, a vector carrying one or more 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 constitutive expression of the transgene in all tissues of the plant. The vector also carries an antibiotic resistance gene to confer selection on transformed calli and cell lines.

Expression of sequences from promoters may be enhanced by including expression control sequences, which are well known in the art. Signals associated with aging and signals active during the drying procedure are specifically indicated.

Thus, various embodiments relate to methods of modulating the expression levels of one or more NtSWEET polynucleotides described herein (or any combination thereof as described herein) by integrating multiple copies of NtSWEET polynucleotides into the genome of a plant, 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 may be a native polypeptide, or may be heterologous to the cell.

In one embodiment, the plant used in the present disclosure is a flue-dried mutant, non-naturally occurring or transgenic plant.

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

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

In one embodiment, the plant used in the present disclosure is a mutant, non-naturally occurring or transgenic tobacco virginia plant that is desiccated (e.g., flue-desiccated).

In one embodiment, the plants used in the present disclosure are mutant, non-naturally occurring or transgenic burley tobacco plants that are desiccated (e.g., air dried).

In one embodiment, the plants used in the present disclosure are mutant, non-naturally occurring or transgenic dark tobacco plants that are desiccated (e.g., roasted).

By modulating NtSWEET expression and/or NtSWEET activity, the organoleptic characteristics of the tobacco can be advantageously altered, as can be appreciated from table 6.

Plants carrying mutant alleles of one or more NtSWEET polynucleotides described herein can be used in plant breeding programs to produce useful lines, varieties, and hybrids. For example, mutant alleles can be introgressed into commercially important varieties as described herein. Thus, there is provided a method for plant breeding comprising crossing a mutant plant, non-naturally occurring plant or transgenic plant as described herein with a plant containing different genetic identity. The method may further comprise crossing the progeny plant with another plant, and optionally repeating the crossing until a progeny having the desired genetic trait or genetic background is obtained. One goal that such breeding methods exert is to introduce desirable genetic traits into other varieties, breeding lines, hybrids or cultivars, especially those of commercial interest. Another object is to facilitate the superposition of genetic modifications of different genes in a single plant variety, line, hybrid or cultivar. Intra-species and inter-species mating are considered. Progeny plants derived from such crosses, also known as breeding lines, are examples of non-naturally occurring plants of the present disclosure.

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 produce a progeny tobacco seed, and (b) growing the progeny tobacco seed under plant growth conditions to produce the non-naturally occurring plant. The method may further comprise (c) crossing the previous generation non-naturally occurring plant with itself or another plant to produce a progeny tobacco seed, (d) growing the progeny tobacco seed of step (c) under plant growth conditions to produce an additional non-naturally occurring plant, and (e) repeating the crossing and growing steps of (c) and (d) a plurality of times to produce further progeny of the non-naturally occurring plant. The method may optionally comprise the step of providing a parent plant prior to step (a), said parent plant comprising genetic identity which is characterized and which differs from the mutant or transgenic plant. In some embodiments, the crossing and growing steps are repeated 0 to 2 times, 0 to 3 times, 0 to 4 times, 0 to 5 times, 0 to 6 times, 0 to 7 times, 0 to 8 times, 0 to 9 times, or 0 to 10 times depending on the breeding program, in order to produce generations of non-naturally occurring plants. Backcrossing is an example of such a method in which progeny is crossed to one of its parents or another plant that is genetically similar to its parents, in order to obtain progeny plants that have a genetic identity in the next generation that is closer to that of one of the parents. Techniques for plant breeding, particularly plant breeding, are well known and may be used in the methods of the present disclosure. The present disclosure also provides non-naturally occurring plants produced by these methods. Certain embodiments do not include the step of selecting plants.

In some embodiments of the methods described herein, lines derived from breeding and screening variant genes are evaluated in the field using standard field procedures. Control genotypes containing original non-mutagenized parents are included and the entries are arranged in the field in a randomized complete block design or other suitable field design. For tobacco, standard agronomic practices are used, such as harvesting, weighing and sampling tobacco for chemical and other common testing prior to and during drying. Statistical analysis of the data was performed to confirm similarity between selected lines and parental lines. Cytogenomic analysis of the selected plants is optionally performed to confirm the genomic and chromosome pairing relationship.

DNA fingerprinting, single nucleotide polymorphisms, microsatellite markers, or similar techniques may be used in a breeding program for Marker Assisted Selection (MAS) to transfer or cultivate mutant alleles of genes into other tobacco as described herein. For example, a breeder may produce an isolated population by crossing a genotype containing mutant alleles with an agronomically desirable genotype. Plants in F2 or backcross generations can be screened using markers developed from genomic sequences or fragments thereof using one of the techniques listed herein. Plants identified as having mutant alleles can be backcrossed or self-pollinated to produce a second population to be screened. Depending on the desired genetic pattern or MAS technique used, it may be necessary to self-pollinate selected plants prior to each round of crossing to aid in the identification of the individual plants of interest. Backcrossing or other breeding operations may be repeated until the desired phenotype of the recurrent parent is restored.

In accordance with the present disclosure, successful crosses result in a fertile F1 plant in a breeding program. The selected F1 plant can be crossed with one of the parents and the first backcrossed generation plant self-pollinated to produce a population of rescreened variant gene expression (e.g., an inactive version of the gene). The process of backcrossing, self-pollination and screening is repeated, e.g., at least 4 times, until the final screening produces a plant that is fertile and quite similar to the recurrent parent. If desired, such plants are self-pollinated and the progeny are then screened again to confirm that the plants exhibit variant gene expression. In some embodiments, variant gene expression of a population of plants in the F2 generation is screened, e.g., plants that are not capable of expressing a polypeptide due to the lack of a gene are identified according to standard methods, e.g., by using PCR methods, wherein the primers are based on polynucleotide sequence information of the polynucleotides described herein (or any combination thereof as described herein).

Hybrid tobacco varieties may be produced by preventing self-pollination of the female parent plant of the first variety (i.e., the seed parent), allowing pollen from the male parent plant of the second variety to fertilize the female parent plant, and allowing F1 hybrid seed to form on the female plant. Self-pollination of female plants can be prevented by emasculating the flowers at an early stage of flower development. Alternatively, a form of male sterility may be used to prevent pollen formation on female parent plants. For example, male sterility may be produced by Cytoplasmic Male Sterility (CMS) or transgenic male sterility, wherein the transgene inhibits microspore or pollen formation, or is self-incompatible. Female parent plants containing CMS are particularly useful. In embodiments where the female parent plant is CMS, pollen is harvested from the male-fertile plant and applied artificially to the stigma of the CMS female parent plant, and the resulting F1 seed is harvested.

The varieties and lines described herein can be used to form single hybrid tobacco F1 hybrids. In such embodiments, the plants of the parent variety may be grown as a substantially homogeneous adjacent population to facilitate natural cross-pollination of the male parent plant with the female parent plant. F1 seeds formed on female parent plants are selectively harvested by conventional means. It is also possible to grow two parent plant varieties in bulk and harvest a blend of F1 hybrid seed formed on the female parent and seed formed on the male parent as a result of self-pollination. Alternatively, a three line cross may be made, wherein a single hybrid F1 hybrid is used as the female parent and is crossed with a different male parent. As another alternative, a two-hybrid can be produced in which two different singly crossed F1 progeny undergo self-crossing.

Those population members having the desired trait or phenotype may be screened or selected among mutant, non-naturally occurring or transgenic plant populations. For example, those plants in the progeny population of a single transformation event that have the desired expression level or function of the polypeptide encoded thereby can be screened. Physical and biochemical methods can be used to identify expression or activity levels. These methods include Southern analysis or PCR amplification for detecting polynucleotides, northern blotting, S1 RNase protection, primer extension or RT-PCR amplification for detecting RNA transcripts, enzyme analysis for detecting the enzyme or ribozyme function of polypeptides and polynucleotides, and polypeptide gel electrophoresis, western blotting, immunoprecipitation and enzyme-linked immunoassay for detecting polypeptides. Other techniques such as in situ hybridization, enzyme staining, immunostaining, and enzyme assays may also be used to detect the presence or expression, function, or activity of NtSWEET polypeptides or NtSWEET polynucleotides.

Mutant, non-naturally occurring or transgenic plant cells and plants as described herein include one or more recombinant polynucleotides, one or more polynucleotide constructs, one or more double stranded RNAs, one or more conjugates, or one or more vectors/expression vectors.

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

One or more of the following further genetic modifications may be present in mutant, non-naturally occurring or transgenic plants and parts thereof. One or more genes involved in nitrogen metabolism intermediate conversion may be modified to reduce the level of at least one Tobacco Specific Nitrosamine (TSNA). Non-limiting examples of such genes include those encoding nicotine demethylases (such as CYP82E4, CYP82E5 and CYP82E10 described in WO2006/091194, WO2008/070274, WO2009/064771 and WO 2011/088180), and nitrate reductase enzymes as described in WO 2016/046288. One or more genes involved in heavy metal uptake or heavy metal transport may be modified to reduce heavy metal content. Non-limiting examples include genes in the multi-drug resistance related polypeptide family, the Cation Diffusion Facilitator (CDF) family, the Zrt-Irt-like polypeptide (ZIP) family, the cation exchanger (CAX) family, the copper transporter (COPT) family, the heavy metal ATPase family (e.g., HMA as described in WO2009/074325 and WO 2017/129739), the homolog family of the natural resistance related macrophage polypeptide (NRAMP) and other members of the ATP Binding Cassette (ABC) transporter family (e.g., MRP) as described in WO2012/028309, which are involved in the transport of heavy metals such as cadmium.

Other exemplary modifications may result in plants with modulated expression or function of isopropyl malate synthase, which results in an alteration of sucrose ester composition, which may be used to alter the preference profile (see WO 2013/029799). Other exemplary modifications may produce plants with modulated expression or function of threonine synthase, wherein the level of methionine may be modulated (see WO 2013/029800). Other exemplary modifications may produce plants with one or more of neoxanthin synthase, lycopene beta cyclase and 9-cis-epoxide carotenoid dioxygenase with modulated expression or function to modulate beta-damascenone content to alter flavor profile (see WO 2013/064499). Other exemplary modifications may produce plants with modulated expression or function of CLC family members placed in the chloride channel to modulate nitrate levels therein (see WO2014/096283 and WO 2015/197727). Other exemplary modifications may produce plants with modulated expression or function of one or more asparagine synthetases to modulate levels of asparagine in leaves and to modulate levels of acrylamide in aerosols produced upon heating or burning leaves (see WO 2017/129739). Other exemplary modifications may result in plants having modulated protease activity during desiccation (see WO 2016/009006). Other exemplary modifications may result in plants having reduced nitrate levels by altering the gene expression of a nitrate reductase (e.g., nia 2) or the activity of the protein encoded thereby (see WO 2016/046288). Other exemplary modifications may result in plants having modified alkaloid levels by altering the gene expression of putative ABC-2 transporter NtABCGl-T and NtABCGl-S or the activity of the protein encoded thereby (see WO 2019/086609) other exemplary modifications may result in plants having modulated flowering-time by altering the gene expression of the gene encoding final flower 1 (TFL 1) or the activity of the protein encoded thereby (see WO 2018/114641). Other exemplary modifications may produce plants with modulated expression or function of one or more asparagine synthetases to modulate levels of asparagine in leaves and to modulate levels of acrylamide in aerosols produced upon heating or burning leaves (see WO 2017/042162). Examples of other modifications include modulation of herbicide tolerance, for example, glyphosate is an active ingredient of many broad-spectrum herbicides. Glyphosate resistant transgenic plants have been developed by transferring aroA genes (glyphosate EPSP synthase from salmonella typhimurium (Salmonella typhimurium) and escherichia coli (e.coli)). Sulfonylurea resistant plants have been produced by transformation of mutated ALS (acetolactate synthase) genes from arabidopsis thaliana. The OB polypeptide from optical system II of mutant amaranthus viridis (Amaranthus hybridus) has been transferred into plants to produce an atrazine-resistant transgenic plant, and the bromoxynil-resistant transgenic plant has been produced by incorporating a bxn gene from the bacterium Klebsiella pneumoniae (Klebsiella pneumoniae). Another exemplary modification results in plants that are resistant to insects. Bacillus thuringiensis (Bacillus thuringiensis, bt) toxins can provide an effective way to delay the emergence of anti-Bt pests, as recently described in broccoli, where the pyramidal cry1Ac and cry1C Bt genes control plutella xylostella that is resistant to either individual polypeptide, and significantly delay the evolution of resistant insects. Another exemplary modification results in plants that are resistant to disease caused by pathogens (e.g., viruses, bacteria, fungi). Plants expressing the Xa21 gene (bacterial leaf blight resistance) and plants expressing the Bt fusion gene and the chitinase gene (tryporyza incertulas and sheath resistance) have been designed. Another exemplary modification results in altered reproductive ability, e.g., male sterility. Another exemplary modification results in plants that are tolerant to abiotic stress (e.g., drought, temperature, salinity) and have been made tolerant by transferring acylglycerophosphatase from Arabidopsis, genes encoding mannitol dehydrogenase and sorbitol dehydrogenase, which are involved in mannitol and sorbitol synthesis, improve drought resistance. Another exemplary modification results in plants in which the activity of one or more nicotine N-demethylases is modulated, such that the level of nornicotine and nornicotine metabolites formed during drying can be modulated (see WO 2015169927). Other exemplary modifications may result in plants with improved storage polypeptides and oils, plants with enhanced photosynthetic efficiency, plants with extended shelf life, plants with enhanced carbohydrate content, and antifungal plants. Transgenic plants in which the expression of S-adenosyl-L-methionine (SAM) or cystathionine gamma-synthase (CGS) or a combination thereof has been modulated are also contemplated. One or more genes involved in the nicotine synthesis pathway may be modified to produce plants or plant parts that produce regulated levels of nicotine when dried or desiccated. The nicotine synthesis gene may be selected from the group ：A622、BBLa、BBLb、JRE5L1、JRE5L2、MATE1、MATE 2、MPO1、MPO2、MYC2a、MYC2b、NBB1、nic1、nic2、NUP1、NUP2、PMT1、PMT2、PMT3、PMT4 and QPT consisting of or a combination of one or more of them. One or more genes involved in controlling the amount of one or more alkaloids may be modified to yield plants or plant parts that produce regulated levels of alkaloids. The alkaloid level control gene may be selected from the group consisting of BBLa, BBLb, JRE L1, JRE5L2, MATE1, MATE 2, MYC2a, MYC2b, nic1, nic2, NUP1 and NUP2, or a combination of one or more thereof.

Other exemplary modifications may result in plants with a modulated amino acid content (see WO2019/185703 and WO 2021/063863) or with a modulated sugar content (see WO2019/185699 and WO 2021/063263) or with a modulated nitrate level (see WO 2020/141062) or with a modulated sugar and amino acid content (see WO 2021/063863).

In a preferred embodiment, the further genetic modification involves an Asparagine Synthetase (ASN) gene as described in WO 2017042162. Modulating the expression of an ASN gene (e.g., one or more of NtASN1-S, ntASN1-T, ntASN5-S and NtASN5-T as described in WO 2017042162) or the activity of an ASN (e.g., ntASN1-S, ntASN1-T, ntASN5-S and NtASN5-T as described in WO 2017042162) significantly alters the chemical composition of tobacco dried or dried leaves without affecting biomass. Thus, modulating the expression and/or activity of the combination of ASN and NtSWEET may have the potential to rearrange the chemical components of the dried or dried tobacco leaves (particularly the amino acid chemical components of burley or dark tobacco) and thereby alter the organoleptic properties.

In addition to ASN, other genes and enzymes also play a role in the recombination of amino acids and/or sugars during leaf yellowing, such as diaminopimelate aminotransferase (DAPAT), which is involved in catabolism and anabolism of lysine, and Aspartate Aminotransferase (AAT), which is expressed during senescence and has the potential to alter the chemical composition of the leaf after drying (WO 2019/185703). Chloroplast sulfate transporters SULTR3 (such as NtSULTR; 1A-S, ntSULTR; 1A-T and NtSULTR; 3-T) play a role in sugar and amino acid metabolism during desiccation (see WO 2021/063863). Thus, additional genetic modifications may involve DAPAT and/or AAT (e.g., one or more of NtAATI-S, ntAAT-T, ntAA T2-S, ntAAT2-T, ntAA T3-S, ntAAT-T, ntAA T4-S or NtAAT-T as described in WO 2017042162) and/or NtSULTR3, 1A-S, ntSULTR3, 1A-T and NtSULTR3, one or more of 3-T as described in WO 2021/063863. Modulation of DAPAT and/or AAT and/or expression and/or activity of combinations of SULTR and NtSWEET may have the potential to rearrange the chemical composition of the dried or dried tobacco leaves and thereby alter the organoleptic properties. Modifications to combinations of NtSWEET and one or more, or two or more, or three or more, or four or more ASNs and DAPAT and AAT and SULTR3 are also disclosed, including NtSWEET and ASNs, ntSWEET and DAPAT, ntSWEET and AAT, ntSWEET and ASNs and DAPAT, ntSWEET and ASNs and AAT, ntSWEET and ASNs and DAPAT and AAT, ntSWEET and SULTR3, ntSWEET and ASNs and SULTR3, ntSWEET and DAPAT and SULTR3, ntSWEET and AAT and SULTR3, ntSWEET and ASNs and DAPAT and SULTR3, ntSWEET and ASNs and AAT and SULTR3, ntSWEET and ASNs and DAPAT and AAT and SULTR3.

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

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

The parts of the plants described herein, particularly the leaves and/or stems and/or midribs of these plants, may be incorporated into or used to prepare various consumables, including but not limited to aerosol-forming materials, aerosol-forming devices, smoking articles, smokable articles, smokeless products, medicinal or cosmetic products, intravenous formulations, tablets, powders, and tobacco products. Examples of aerosol-forming materials include tobacco compositions, tobacco extracts, cut filler, dry or desiccated tobacco, expanded tobacco, homogenized tobacco, reconstituted tobacco, and pipe tobacco. Smoking articles and smokable articles are of the type of aerosol-forming devices. Examples of smoking or smokable articles include cigarettes, cigarillos and cigars. Examples of smokeless products include chewing tobacco and snuff. In some aerosol-forming devices, rather than combustion, the tobacco composition or another aerosol-forming material is heated by one or more electrical heating elements to produce an aerosol. In another type of heated aerosol-forming device, an aerosol is generated by transferring heat from a combustible fuel element or heat source to a physically separate aerosol-forming material, which may be located within, around or downstream of the heat source. The smokeless tobacco product and the plurality of tobacco-containing aerosol-forming materials can comprise tobacco in any form, including dry particles, flakes, small particles, powders, or slurries deposited on, mixed in, surrounded by, or otherwise combined with other ingredients in any form, such as flakes, films, cards (tabs), foams, or beads. The term "smoke" is used to describe a type of aerosol produced by a smoking article, such as a cigarette, or by burning an aerosol-forming material.

In one embodiment, roasted or dried plant material from the mutant, transgenic, and non-naturally occurring plants described herein is also provided. Processes for drying green tobacco leaves are known to those skilled in the art and include, but are not limited to, air drying, fire drying, flue drying, and sun drying as described herein.

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

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

The present disclosure also provides methods for producing seeds comprising culturing a mutant plant, non-naturally occurring plant, or transgenic plant described herein, and collecting the seeds from the cultivated plant. Seeds from the plants described herein may be conditioned by means known in the art and packaged in packaging materials to form articles of manufacture. Packaging materials such as paper and cloth are well known in the art. The package of seeds may be provided with indicia describing the nature of the seeds therein, such as labels or indicia affixed to the packaging material, indicia printed on the package.

Compositions, methods, and kits for genotyping plants to identify, select, or breed may include detecting the manner in which NtSWEET polynucleotides are present in a polynucleotide sample. Thus, a composition is described comprising 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 performing amplification or detection.

Accordingly, disclosed are gene-specific oligonucleotide primers or probes comprising about 10 or more contiguous polynucleotides corresponding to NtSWEET polynucleotides described herein. The primer or probe may comprise or consist of about 15, 20, 25, 30, 40, 45, or 50 or more contiguous polynucleotides that hybridizes (e.g., specifically hybridizes) to one or more NtSWEET polynucleotides described herein. In some embodiments, the primer or probe may comprise or consist of about 10 to 50 consecutive nucleotides, about 10 to 40 consecutive nucleotides, about 10 to 30 consecutive nucleotides, or about 15 to 30 consecutive nucleotides, which may be used in a sequence-dependent method of genetic identification (e.g., southern hybridization) or isolation (e.g., in situ hybridization of bacterial colonies or plaques) or genetic detection (e.g., as one or more amplification primers in amplification or detection). One or more specific primers or probes may be designed and used to amplify or detect some or all of the polynucleotide. As a specific example, two primers may be used in a PCR protocol to amplify a polynucleotide fragment. PCR can also be performed using one primer derived from a polynucleotide sequence, such as a promoter sequence, the 3' end of a pre-mRNA, or a sequence derived from a vector, and a second primer that hybridizes to a sequence upstream or downstream of the polynucleotide sequence. Examples of thermal and isothermal techniques for amplifying polynucleotides in vitro are well known in the art. The sample may be or may be derived from a plant, plant cell or plant material, or a tobacco product prepared or derived from a plant, plant cell or plant material as described herein.

In another aspect, there is also provided a method of detecting a NtSWEET polynucleotide described herein (or any combination thereof as described herein) in a sample, the method comprising the steps of (a) providing a sample comprising or suspected of comprising a polynucleotide, (b) contacting the sample with one or more primers or one or more probes to specifically detect at least a portion of a NtSWEET polynucleotide, and (c) detecting the presence of an amplification product, wherein the presence of an amplification product is indicative of the presence of a NtSWEET polynucleotide in the sample. In another aspect, there is also provided the use of one or more primers or probes for specifically detecting at least a portion of a NtSWEET polynucleotide. Also provided are kits for detecting at least a portion NtSWEET of the polynucleotides, comprising one or more primers or probes for specifically detecting at least a portion NtSWEET of the polynucleotides. The kit may comprise reagents for polynucleotide amplification (such as PCR) or for probe hybridization detection techniques (such as southern blotting, northern blotting, in situ hybridization or microarrays). The kit may include reagents for antibody binding detection techniques such as western blotting, ELISA, SELDI mass spectrometry, or test strips. The kit may include reagents for DNA sequencing. The kit may include reagents and instructions for use.

In some embodiments, the kit may include instructions for one or more of the methods. The kit can be used for genetic identity determination, phylogenetic studies, genotyping, haplotyping, pedigree analysis or plant breeding, in particular co-dominant scoring.

The present disclosure also provides methods of genotyping plants, plant cells, or plant material comprising NtSWEET polynucleotides as described herein. Genotyping provides a means of distinguishing homologs of chromosome pairs and can be used to distinguish isolates in a plant population. The molecular marker method can be used for phylogenetic research, characterization of genetic relationships between crop varieties, identification of hybrid or somatic hybrids, localization of chromosome segments affecting monogenic traits, map-based cloning and quantitative genetic research. Specific methods of genotyping may 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 methods of using techniques such as AFLP analysis to track the isolation of one or more genes or polynucleotides and chromosomal sequences linked to those genes or polynucleotide genes.

Also disclosed herein are methods of producing the liquid tobacco extract and liquid tobacco extracts produced by one or more methods.

A specific extraction temperature is selected for the tobacco starting material. The extraction temperature is typically selected in the range of about 100 degrees celsius to about 160 degrees celsius. The duration of the heating step may optionally be controlled to provide a degree of control over the composition of the extract derived from the tobacco starting material. Suitably, the tobacco starting material is heated at the extraction temperature for at least about 90 minutes, more suitably at least about 120 minutes. The heating step is typically performed in an inert atmosphere. Suitably, during the heating step, a stream of inert gas, such as nitrogen, is passed through the starting tobacco material. The volatile tobacco compounds are released into the inert gas stream during the heating step such that the inert gas acts as a carrier for the volatile components. The flow rate of the inert gas stream may be at least about 25 liters/minute, more suitably at least about 30 liters/minute. The relatively high inert gas flow rate may advantageously increase the efficiency of extraction from the tobacco starting material. Optionally, the heating step may be performed under vacuum. Suitable heating methods for carrying out heating of the tobacco starting material are known to the skilled person and include dry distillation, water distillation, vacuum distillation, flash distillation and thin film water distillation.

When the volatile compounds are collected by absorption in a liquid solvent, the step of forming the liquid tobacco extract may include drying a solution of the volatile compounds in the liquid solvent to concentrate the solution. Drying may be performed using any suitable means including, but not limited to, dehydration, molecular sieves, freeze drying, phase separation, distillation, membrane permeation, controlled crystallization and filtration of water, reverse osmosis, ultracentrifugation, liquid chromatography, reverse osmosis, or chemical drying.

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

The invention is also described in the following examples, which are provided to describe the invention in more detail. These examples illustrate the presently preferred modes of carrying out the invention that are presently contemplated, and are intended to illustrate and not to limit the invention.

Examples

Example 1 materials and methods

Plant material and culture conditions

The seeds were sterilized with chlorine steam prior to germination. The 5% final chlorine solution was placed in a bell jar with a glass tube containing seeds. Hydrochloric acid (37%) was then added to the solution and the seeds were incubated for 2 hours. The seeds were then placed on Murashige & Skoog (Murashige and Skoog, 1962) growth medium and transferred to a plant growth chamber (24 ℃,16 hours light/20 ℃ and 8 hours dark) for 4 weeks. Well-developed seedlings were transferred to a greenhouse and grown in 10L pots until adequate growth was achieved. Artificial light was used for 16 hours per day. All plants were grown at the same pitch at each step of the cultivation to avoid any effect of pitch on the branches and leaves.

Flowering time

Flowering time of 15 week old plants grown under standard flue-cured fertilization conditions was evaluated. Flowering time is quantified as the percentage of plants that either show floral primordia or developing flowers, or have no inflorescences at all.

Method for determining free amino acid, sugar, ammonia and nitrate levels

Amino acid content was measured using Method MP 1471rev 5 2011,Resana,Italy:Chelab Silliker S.r.l,Merieux NutriSciences Company. To determine the amino acids in the leaves of the desiccated plant, the desiccated leaves were dried at 40℃for 2-3 days after removal of the middle rib, if desired. The tobacco material was then ground to a fine powder (-100 uM) prior to analysis of amino acid content. Or the amino acid content in the plant material is measured as described in UNI EN ISO 13903:2005.

The reducing sugar content was measured using a split-flow colorimetry developed by Skalar Instrument Co (WEST CHESTER, PA) and described in Tobacco Science 20:139-144 (1976) for analysis of tobacco samples. Measurement of the reducing sugar content is also described in Coresta recommendations 38, CRM and ISO 15154:2003 to determine the reducing sugar in dried leaves, the dried leaves were dried at 40℃for 2-3 days after removal of the middle ribs if desired. The tobacco material was then ground to a fine powder (-100 uM) prior to analysis of the reducing sugars. Or the reducing sugar content is measured according to ISO 15154:2003.

Nitrate content was measured according to the manufacturer's protocol using Lachet QuikChem 8500 instrument (Lachat QuikChem method-107-04-1-J, lachet Instruments, loveland, CO, USA). Or nitrate content is measured according to ISO 15517:2003.

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

Gene expression analysis

Using IlluminaClarity LIMS(Illumina, inc.) the generated sequencing data was split and subsequently imported QIAGEN CLC Genomics Workbench version 12.0.1 (CLC bio, QIAGEN Company). Transcriptome reads were mapped to an updated version of the tobacco reference genome by the "RNA-SEQ ANALYSIS"2.16 tool (Sierro et al, (2014) Nat com 5,3833), using a similarity of 0.8 (s=0.8) and a length fraction of 0.8 (l=0.8) as mapping criteria. The mismatch cost is set to 2, the insertion cost is set to 3, and the deletion cost is set to 3. Global alignment is not performed, but pairing distances are automatically detected. The maximum read match (read hit) number is set to 10 and paired reads are counted as one. Gene expression FPKM values for each gene in the reference genome and those genes without transcript models are retrieved.

RNAi procedure

The DNA fragment SEQ ID NO 9 was selected for inhibiting the expression of both copies of SWEET12 (SWEET 12-S and SWEET 12-T) and cloned between the strong constitutive MMV promoter and the 3' NOs terminator sequence of the nopaline synthase gene of Agrobacterium tumefaciens (Agrobacterium tumefaciens) (Cheng et al (1997) Plant physiol.115 (3): 971-980). Standard Agrobacterium-mediated transformation protocols were used to transform flue-dried tobacco variety K326 (Horsch et al, (1985) Science,227, 1229-1232). Seeds were harvested from independent T0 lines exhibiting the strongest SWEET12 silencing. T1 plants from those T0 lines were grown in the greenhouse under standard agronomic practices and selected by RT-qPCR experiments to assess the 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).

The same strategy was used for silencing SWEET15 using the DNA fragment SEQ ID NO 10. The selection was performed by RT-qPCR experiments to evaluate the SWEET15 gene expression level 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).

Constitutive expression program

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 the 3' NOs terminator sequence of the nopaline synthase gene of Agrobacterium tumefaciens (Cheng et al 1997Plant Physiol.115 (3): 971-980). A standard Agrobacterium-mediated transformation protocol was used to transform Virginia tobacco (tobacco) variety K326 (Horsch et al, (1985) Science,227, 1229-1232). Seeds were harvested from independent T0 lines exhibiting the strongest SWEET12-S and SWEET 15-T. T1 plants from those T0 lines were grown in the greenhouse under standard agronomic practices and selected by RT-qPCR experiments to assess SWEET12 gene expression levels using the primers described in the preceding section.

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 sugar accumulation changes that occur when tobacco leaves are dried. Based on identity with the corresponding arabidopsis and tomato orthologs, 25 genes and related gene products associated with putative SWEET functions were identified in tobacco. In FIG. 1, transcriptomic analysis showed that among these 25 genes and related gene products, SWEET12-S (SEQ ID NO: 1) and SWEET12-T (SEQ ID NO: 3) gene expression was strongly induced in the detached leaves of Virginia tobacco plants, which were dried in the flue-drying bin for 72 hours. The expression of those genes rose rapidly 24 hours after desiccation, remained increased in leaves detached from the lower stalk positions (X and C) on the plants 48 hours and 72 hours after desiccation, while slightly decreased in leaves at the higher stalk positions (B and T), and the transcript levels remained very high compared to non-desiccated tissue (0 hour time point). This suggests that SWEET12-S and SWEET12-T have specific functions in sugar transport in the early stages of drying (the so-called yellowing stage where chemical changes occur) (Bovet et al, (2020).Leaf Curing Practices Alter Gene Expression and the Chemical Constituents of Tobacco Leaves.In:Ivanov,N.V.,Sierro,N.,Peitsch,M.C.( edition) The Tobacco Plant genome of Plant genome Springer. Since many senescence-associated genes (SAG) were induced during the yellowing stage of drying (Bovet et al (2019) Plants (Basel) 11;8 (11): 492), the expression of putative senescence-associated target genes SWEET15-S (SEQ ID NO: 5) and SWEET15-T (SEQ ID NO: 7) was studied. The transcript levels of those genes were not significant during the time course of flue drying, transcript levels were below 5FPKM.

EXAMPLE 3 analysis of Gene expression of SWEET in different plant organs

SWEET12-S and SWEET12-T are highly expressed in both leaf and midrib tissues of plant leaves, with higher expression in leaf than in midrib tissue, as shown in FIG. 2. Similar trends were observed for SWEET15-S and SWEET15-T gene expression, however transcript expression levels can be considered below significant levels. Some SWEETs are not only regulated by transcription, but also by phosphorylation, which is critical for their biological function (Anjali et al (2020) Plant Physiol biochem. 156:1-6). Thus, small changes in the low expression levels of SWEET15-S and SWEET15-T do not rule out the possibility that they will play a positive role during drying. In Table 1, the expression of SWEET12-S and SWEET12-T and SWEET15-S and SWEET15-T in plant organs is shown. These data indicate that these four transcripts are mainly present in immature flowers and petals, demonstrating the active sugar requirements of energy supply and seed storage in such tissues. SWEET15-S showed very low expression in all organs, SWEET12-S was also expressed in the stem, possibly indicating sugar transport in phloem.

Example 4 functional characterization of SWEET 12-S and SWEET 12-T

To characterize the function of SWEET 12-S and SWEET 12-T as potential mediators of sugar accumulation in dried leaves, flue-cured virginia tobacco variety K326 plants were transformed with a fusion construct that placed the coding region of the SWEET 12-S cDNA under the control of a CaMV 35S promoter (p 35S) that directs constitutive high-level expression in plants (Tzfira and Citovsky (2006) Curr Opin biotechnol.17 (2): 147-54). In addition, the insert sequence SEQ ID NO 9 was used as an RNAi construct for silencing SWEET 12-S and SWEET 12-T to generate RNAi plants. Similarly, flue-cured tobacco (tobacco) variety K326 plants were transformed with constructs that placed the coding region of the SWEET 15-T cDNA under the control of p35S and used the insert sequence SEQ ID NO:10 to generate RNAi plants for down-regulation of SWEET 15-S and SWEET 15-T.

Selection of T0 putative transformants was accomplished by RT-qPCR screening on mature leaves. Seeds were collected for each individual p35s: SWEET15-T and p35: SWEET12-S T0 progeny exhibiting up-regulation of expression, and conversely, seeds exhibiting expression of silenced RNAi-SWEET12 and RNAi-SWEET 15T 0 progeny, to produce T1 plant lines. T1 independent lines were again selected by RT-qPCR screening on leaves detached from the plants and flue dried for 48 hours. Three p35s were selected for this screening, the SWEET12-S T1 line, three RNAi-SWEET 12T 1 lines, four p35s, the SWEET15-T T1 line and five RNAi-SWEET 15T 1 lines (FIG. 3). The growth and development phenotypes of these T1 lines were evaluated for chemical characteristics after flue drying.

Example 5-effect of SWEET12 cluster 3 and SWEET15 on the growth and development of flue-dried virginia tobacco (tobacco) variety K326 plants.

To characterize the effect of SWEET12 and SWEET15 on growth and development, detailed physiological and developmental analyses were performed on T1 lines. The T1 line was compared to its corresponding wild-type (WT) control line. The transformed lines were similar to the germination of the seeds of the control and appearance of the first true leaves of the seedlings, indicating that SWEET12 and SWEET15 did not play an important role in germination, growth and development during the early young stage. Although all lines exhibited similar overall structure and branches and leaves, some growth defects were observed. FIG. 4 shows a significant delay and decrease in growth of the p35S SWEET12-S line compared to the control line. In contrast, RNAi-SWEET12 line was higher than the control line. This suggests that SWEET12-S contributes to growth and development during the nutritional phase. Although no difference in growth and development of the SWEET15-T line was observed compared to the control plants, the RNAi-SWEET15 line was higher than its corresponding control, indicating that SWEET15 could also contribute to vegetative growth and development.

The transition from vegetative to reproductive development is an important stage of tobacco leaf maturation and leaf chemical composition change, with the purpose of harvesting the leaves for desiccation before and after such transition. Thus, the flowering-time of T1 transformants was studied. Tobacco produces leaves from the top inflorescence of a single upstanding stem (Smith and McDaniel (1992) Dev biol.153 (1): 176-84). The number of 15 week old plants that appeared in the floral primordia or developed flowers, or were completely devoid of inflorescences was counted for evaluation of flowering time. The data presented in fig. 5 shows that 12% of WT control plants and 100% of RNAi-SWEET12 flowering when plants reach 15 weeks of age. In contrast, no inflorescence was observed on the same age p35S: SWEET12-S plants. This comparative phenotype of RNAi-SWEET12 with p35S SWEET12-S strongly suggests that SWEET12 may act as a negative regulator affecting the function of the sugar transporter and thus the flowering-time. Interestingly, the opposite phenotype was observed in the SWEET15 line. 75% of p35s, SWEET15 week old plants show floral primordia or developmental flowers, while only 20% of RNAi-SWEET15 lines bloom. This suggests that, unlike SWEET12, SWEET15 acts as a positive regulator of flowering-time.

Overall, this data demonstrates that two glycotransporters from the SWEET polygene family are key regulators of reproductive growth and development in addition to being induced during leaf desiccation. In view of the low expression levels of these transporters in non-desiccating tissues, the discovery of this function was unexpected and it was postulated that gene expression of these transporters was induced at developmental stages different from those examined so far (see table 1), or that its regulation was uncoupled from gene expression as observed for many of the plasma membrane located transporters.

Auxotrophs often alter leaf biomass yield, particularly when those growth deficits are caused by the deregulation of genes involved in carbohydrate metabolism and transport (LASTDRAGER et al (2014) J Exp Bot.65 (3): 799-807). Thus, biomass from mature leaves prepared for drying was evaluated. Although no significant difference in leaf biomass was observed for RNAi-SWEET12 and RNAi-SWEET15 compared to the control, the data in FIG. 6 indicate that the two transgenic lines p35S SWEET12-S and p35S SWEET15-T exhibited a smaller but significant reduction in leaf biomass compared to the WT plant. This suggests that SWEET12 and SWEET15 can affect leaf biomass yield. It is worth mentioning that there was no difference in leaf biomass of RNAi lines compared to WT plants, possibly due to functional redundancy, or because we did not see any effect under the conditions. Other functional SWEET present in those plants may compensate for the effects of SWEET12 and SWEET15 downregulation. This also shows that the effect on flowering under greenhouse conditions is not transferred to leaf biomass.

Example 6-effect of SWEET12 cluster 3 and SWEET15 on chemical composition of flue-dried virginia tobacco (tobacco) variety K326 plants.

Since SWEET is known to fine tune the sugar balance between cells and plant tissue (see above), we hypothesize that SWEET may alter the chemical composition of tobacco leaves during desiccation. Therefore, leaf chemistry characteristics of T1 transformants were studied. Mature leaves ready for drying were removed and flue dried and evaluated for sugar, free amino acids, ammonia and nitrate content. The fructose, glucose and sucrose content in the SWEET12-S line was significantly and strongly reduced (fig. 7). This suggests that SWEET12 down regulates sugar accumulation in dried leaves. This also demonstrates the biological function of SWEET as a sugar transporter in flue-dried virginia tobacco (tobacco) variety K326 plants. No statistical difference in sugar accumulation was observed for RNAi-SWEET12 lines compared to WT plants. Again, this may be due to functional redundancy that may occur in such large multigene families.

Previous results demonstrated that the expression of the SWEET15-S and SWEET15-T genes was not or weakly induced during the yellowing stage of flue drying (fig. 2). Thus, after desiccation of detached leaves from p35s, SWEET15-T or RNAi-SWEET15 lines, no change in leaf chemistry was expected and metabolite content determinations were made on pools of T1 lines rather than individual plants. The data in FIG. 8 shows that sucrose is reduced 3-fold and fructose and glucose accumulation is reduced about 2-fold in the SWEET15-T line for p35s compared to WT plants. The combined leaf tissue material of the RNAi-SWEET15 line had slightly reduced sugar content compared to the control line. The data also indicate that SWEET15 plays a role in sugar accumulation during drying. The inconsistency between the absence of SWEET15-S and SWEET15-T gene expression during the early stages of drying and the phenotypic change in sugar accumulation in SWEET15 transformants may be due to (i) late induction of SWEET15 gene after 72 hours, which was not evaluated in our experimental design, (ii) regulation of sugar accumulation by SWEET15 prior to drying, sustained changes during drying being independent of its expression, (iii) observed phenotypic changes independent of transcriptional regulation, triggered by posttranscriptional regulation of SWEET transporter. The fact that the SWEET15 gene was induced in the late stages (after 4 days of drying) during air drying of burley tobacco (low sugar tobacco) supports in particular the views (i, ii).

In plants, the carbon stream is directed to sucrose, starch or amino acid synthesis (Yadav et al, (2015) Front Plant Sci.22; 6:275). Thus, changes in sugar accumulation may affect amino acid accumulation in dried leaves (Bovet et al (2019) Plants (Basel) 11;8 (11): 492). Table 2 shows the amino acid content in flue-dried tobacco Virginia (tobacco) SWEET12T1 line. The free amino acid content in 18 week old plants grown under standard flue-dried fertilization conditions and flue-dried under standard agronomic conditions was determined. The results represent the average content of leaves detached from stalks (C position) and flue dried in mg/kg (3 biological replicates = leaves from three individual lines). Data were collected from at least three biological replicates. Statistics showed significant differences in accumulation of glutamate, asparagine, tryptophan, phenylalanine, glycine, proline and methionine in P35S compared to WT controls (student t test, ns: not significant, < P0.05, < P0.01, < P0.001). The data in table 2 show that SWEET12-S line exhibits significantly higher levels of asparagine, tryptophan, phenylalanine, glycine and methionine, and reduced levels of glutamic acid and proline as compared to the control line. In other words, the sugar reduction in P35S-SWEET is accompanied by accumulation of more amino acids, demonstrating more aggressive consumption of carbon resources by amino acid synthesis. Similarly, as shown in table 3, asparagine and tryptophan content were higher and proline content was lower in the SWEET15-T line compared to the WT control. The free amino acid content in 18 week old plants grown under standard flue-dried fertilization conditions and flue-dried under standard agronomic conditions was measured. The results represent the content of the pool of T1 leaves detached from the stalks (C position) and flue dried. The data are summarized in mg/kg. No strong differences were observed in RNAi lines (see tables 2 and 3).

The reduction in proline correlates with the reduction in sugar in p35s, SWEET 15-T. Since proline is known to be an osmoprotectant, this suggests that the water stress during desiccation (senescence) of transgenic lines is different compared to CT 1. Regarding the effect on flavor, proline is known to form amadori compounds, which when heated by the production of acetyl-pyrroline produce a "popcorn taste (popcorn)" (Wei et al (2017) Food chem 232:531-544).

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), typically variations in sugar and amino acid content can affect ammonia, nitrate and alkaloid content. They were quantified in SWEET lines (tables 4 and 5). In table 4, the free amino acid content in 18 week old plants grown under standard flue-dried fertilization conditions and flue-dried under standard agronomic conditions was determined. The results represent the average content in leaves detached from the stalks (C position) and dried in the flue, ammonia expressed in mg/kg, nitrate expressed in mg/kg (calculated as NO 3) and alkaloids expressed in g/100 g. Data were collected from at least three biological replicates. Statistics show that there is a significant difference in ammonia accumulation in P35S: SWEET12-S compared to WT control (student t test, ns: not significant, < 0.05). In table 5, the free amino acid content in 18 week old plants grown under standard flue-dried fertilization conditions and flue-dried under standard agronomic conditions was measured. The results represent the content of the pool of T1 leaves detached from the stalks (C position) and flue dried. The ammonia data are summarized in mg/kg, nitrate (in NO 3) in mg/kg and alkaloid in g/100 g. The SWEET12-S line only showed a significant increase in ammonia content compared to WT plants.

Functional characterization of SWEET was well documented in arabidopsis, but not in crops. SWEET is described as a key target useful for improving yield or responding to environmental stress. This example demonstrates the importance of the SWEET gene, altering the chemical composition of tobacco leaves during drying, and thus potentially altering the aroma profile of tobacco. SWEET is also a key target that can be studied to increase leaf yield or to regulate plant maturity by affecting leaf biomass and flowering time (growth and development).

EXAMPLE 7 influence on the sense organs

The results are presented in table 6. The observed flowering-time acceleration in SWEET-12RNAi compared to control tobacco was independent of any significant change in sugar and free amino acids. However, at the sensory level of the P1 bars, tobacco SWEET12-RNAi had more properties (less bright and flat, more deep (dark), cigar taste (cigar)) and maturity than the control. On the other hand, P35S SWEET-12 is organoleptically closer to the control overall (although exhibiting more chemical composition differences in sugar and free amino acid content). However, P35S SWEET12 exhibited more dark smoke (ashy-smoky) than the control (probably caused by the different balance between sugar and amino acids).

Other aspects of the invention are set forth in the following numbered paragraphs:

1. A mutant, non-naturally occurring or transgenic plant comprising at least one modification capable of modulating expression or activity of one or more of a SWEET12-S or SWEET15-T comprising, consisting or consisting essentially of, or consisting of a sequence comprising, consisting of or consisting of at least 70% sequence identity to SEQ ID NO:1, or (ii) a SWEET12-T polynucleotide sequence comprising, consisting of or consisting of at least 70% sequence identity to SEQ ID NO:3, or (iii) a SWEET15-S polynucleotide sequence comprising, consisting of or consisting of at least 70% sequence identity to SEQ ID NO:5, or (iv) a SWEET15-S polynucleotide sequence comprising, consisting of or consisting of at least 70% sequence identity to SEQ ID NO:7, or consisting of at least 70% sequence identity to SEQ ID NO: 12-6) a polypeptide (iii) comprising, consisting of at least 70% sequence identity to SEQ ID NO:3, or consisting of at least 70% sequence identity to SEQ ID NO: 15-S or consisting of at least 70% to SEQ ID NO:5, or consisting of at least 70% to SEQ ID NO: 6) a polypeptide (iii) comprising, or consisting of at least 70% to SEQ ID NO:6, which has at least 70% sequence identity with SEQ ID NO. 8, wherein the plant or part thereof comprises at least one modification capable of modulating (a) expression of the polynucleotide in the plant or part thereof, or (b) activity of the polypeptide in the plant or part thereof, or wherein expression or activity of one or more of SWEET12-S or SWEET15-T is modulated, as compared to a control plant or part thereof, wherein expression or activity of one or more of SWEET12-S or SWEET15-T is not modulated.

2. The mutant, non-naturally occurring or transgenic plant or part thereof according to paragraph 1, wherein the plant comprises at least one genetic alteration in a regulatory region or coding sequence of one or more of SWEET12-S or SWEET12-T or SWEET15-S or SWEET15-T, and/or wherein the at least one genetic alteration comprises one or more exogenous DNA or exogenous RNA, and/or wherein the at least one genetic alteration comprises one or more vectors or viral vectors or Agrobacterium vectors or CRISPR vectors, and/or wherein the at least one genetic alteration is capable of driving one or more RNA interference or transcriptional gene silencing or virus induced gene silencing, and/or wherein the at least one genetic alteration is capable of expressing one or more double stranded RNA (dsRNA) or RNA (hpRNA) or small interfering RNA, and/or wherein the at least one genetic alteration is capable of constitutively expressing SWEET12-S or SWEET12-T or one or SWEET15-S or one or more of SWEET 15-S.

3. The mutant, non-naturally occurring or transgenic plant of paragraph 1 or paragraph 2, wherein the plant's development during the vegetative stage is modulated as compared to the control plant and/or the flowering time of the plant is modulated as compared to the control plant.

4. The mutant, non-naturally occurring or transgenic plant of 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, and wherein plant height is increased during the vegetative stage and/or wherein flowering time is accelerated compared to the control plant.

5. The mutant, non-naturally occurring or transgenic plant of paragraph 3, wherein expression and/or activity of SWEET15-T is increased, and wherein flowering time is accelerated compared to the control plant.

6. The mutant, non-naturally occurring or transgenic plant or part thereof of paragraph 1 or paragraph 2, wherein the part of the mutant, non-naturally occurring or transgenic plant is a dried or desiccated leaf.

7. The mutant, non-naturally occurring or transgenic plant or part thereof of paragraph 6, wherein the chemical profile of the dried or desiccated leaf is modulated as compared to a dried or desiccated leaf derived from the control plant, suitably wherein the chemical profile is the sugar profile and/or the amino acid profile, suitably wherein the expression and/or activity of SWEET12-S or SWEET15-T is increased, and wherein at least the fructose, glucose and sucrose content is reduced as compared to a dried or desiccated leaf derived from the control plant, or wherein the expression and/or activity of SWEET12-S is increased, and wherein the level of asparagine, tryptophan, phenylalanine, glycine and methionine is increased, and wherein the level of dried or desiccated leaf and proline is reduced as compared to a dried leaf derived from the control plant, wherein the level of proline and/or proline is increased as compared to a dried leaf derived from the control plant, wherein the level of dry proline and/or proline is increased as compared to a dried leaf derived from the control plant, and wherein the dry level of at least the fructose, glucose and sucrose content is reduced as compared to a dried leaf derived from the control plant.

8. A mutant, non-naturally occurring or transgenic plant or part thereof according to any one of the preceding paragraphs, wherein the plant is a tobacco plant, more suitably of the virginia type or burley type.

9. Plant material, dried plant material or homogenized plant material derived or obtained from a plant or part thereof according to any of paragraphs 1 to 8, suitably wherein said plant material is selected from the group consisting of biomass, seeds, stems, flowers or leaves or a combination of two or more thereof, suitably wherein said plant material is a leaf, suitably wherein said leaf is a dried leaf, suitably wherein said dried leaf is selected from the group consisting of flue dried leaf, sun dried leaf or air dried leaf.

10. A method of producing a plant having modulated flowering-time and/or modulated amino acid levels and/or modulated sugar levels, the method comprising (a) providing a plant having modulated expression or activity of one or more of a SWEET12-S or SWEET15-T 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 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 of, a sequence having at least 70% sequence identity to SEQ ID NO:7, or at least 70% sequence identity to SEQ ID NO: 12-6-v) a polypeptide (ii) having at least 70% sequence identity to SEQ ID NO:3, or consisting of, or consisting of, a polypeptide (iii) a sequence having at least 70% sequence identity to SEQ ID NO:3, or consisting of, a sequence having at least 70% sequence identity to SEQ ID NO:5, which has at least 70% sequence identity to SEQ ID NO. 6, or (vii) a SWEET15-T polypeptide which has at least 70% sequence identity to SEQ ID NO. 8, and (b) introducing at least one modification capable of modulating the activity of (i) one or more of SWEET12-S or SWEET12-T or SWEET15-S or SWEET15-T in said plant, or (ii) one or more of SWEET12-S or SWEET12-T or SWEET15-S or SWEET15-T in said plant, compared to a control in which the expression of the same one or more of SWEET12-S or SWEET15-T is not modified.

11. The method of paragraph 10, wherein in step (b) the at least one modification is introduced by genomic editing, suitably wherein the genomic editing is selected from CRISPR-mediated genomic editing, mutagenesis, zinc finger nuclease-mediated mutagenesis, chemical or radiological 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.

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

13. A method of producing a dried plant material having a modulated amino acid level and/or a modulated sugar level, 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.

14. A dried plant material (e.g. leaf) obtained or obtainable by a method according to paragraph 13.

15. A plant product comprising the plant material, the dried plant material or the homogenized plant material of paragraph 9, or the dried plant material of paragraph 14, suitably wherein the plant product is a tobacco product from a tobacco plant material, and/or wherein the tobacco product is a tobacco blend, suitably wherein the tobacco blend comprises virginia-type tobacco and/or burley-type tobacco. Any publications cited or described herein are provided for their disclosure prior to the filing date of the present application. The statements herein should not be construed as an admission that the inventors are not entitled to antedate such disclosure. All publications mentioned in the above specification are herein incorporated by reference. Various modifications and alterations of this application will become apparent to those skilled in the art without departing from the scope and spirit of this application. Although the application has been described in connection with specific preferred embodiments, the application as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the application which are obvious to those skilled in cellular biology, molecular biology and plant biology or related fields are intended to be within the scope of the following claims.

TABLE 1

RNA-seq gene expression profile of the SWEET gene in plant organs of flue dried tobacco Virginia (tobacco) collected in the field. Data are expressed in FPKM.

TABLE 2

Amino acid content in flue-dried Virginia tobacco (tobacco) SWEET 12T 1 line, SWEET12-S/-TRNAi line and SWEET12-S p S line

TABLE 3 Table 3

Amino acid content in flue-dried Virginia tobacco (tobacco) SWEET 15T 1 line, SWEET15-S/-TRNAi line and SWEET15-T p S line

TABLE 4 Table 4

Ammonia, nitrate and alkaloid content in flue dried virginia tobacco (tobacco) SWEET 12T 1 line.

TABLE 5

Ammonia, nitrate and alkaloid content in flue dried virginia tobacco (tobacco) SWEET 15T 1 line.

TABLE 6

Results of sensory analysis of tobacco in which the expression of SWEET-12 or the activity of SWEET-12 has been reduced ("T1 SWEET-RNAi") or increased ("T1 SWEET 35S (OE)") compared to a control in which the expression of SWEET-12 or the activity of SWEET-12 has not been modulated ("control CT"). "55% SWEET (K326)/45% FC BR" refers to 55% SWEET modified tobacco from a greenhouse blended with 45% flue dried tobacco (FC) from Brazil (BR), which is the base tobacco added to reconstituted tobacco (cast leaf).

SEQ ID NO. 1 Polynucleotide coding sequence of SWEET12-S from Nicotiana tabacum atggccatatttgatctccaccatccatggctatttgtgtttggagccttaggaaacattatttccatattcgtcttcttagctccagtgccaacatttcgccgaatctacaaagaaaaatcaaccatgggctttcaatcagtcccttacgtggtagcactgttttcatctatgctctggatgtattatgcatttatcaagaaaaatgctattctcctcatctccatcaactccttcggttgcattgtcgagacaatttacatctccattttccttctctacgcatccaaggaggctaggaggcagacggtgaaacttttggtatcattgattggaggattgtacacactgatatttcttgtaactttgttccctttgaatggagcccttcgagtacaagtagtgggttggatttgtgtagccgtagcagtggctgtctttgctgcacctcttagcattgtgtttcaagtggttcgaacgaagagtgtggagtttctgcccttcaccctgtctttctttcttacattaagtgctatcatgtggtttggttatggtctccttcaaaaggacctgtgtattgcactgccgaatgtattgggtttcttcctgggaatgattcagatgctgttgtatgggctataccgtaaggtaaagccagcagcagaattagagaaaaaggtgccggagcatatagtaaacatcgtcgtcgtaggaaactcagaacagatacatcctgtcaaatccgagaaaaatgaggatatgatcaagaagctggatgaagaagaaaacagggagagcagcgtaattagcccaccggtgccagctctgctgccggtggcgaacgaccatgaaaatgaagaacgtgcgggtggcgagctggcgcaagtgaacttgcagccgcagcagcagtttgaaaccccggtgcttgtggtgtgtgctgcagcttga

SEQ ID NO. 2A polypeptide sequence MAIFDLHHPWLFVFGALGNIISIFVFLAPVPTFRRIYKEKSTMGFQSVPYVVALFSSMLWMYYAFIKKNAILLISINSFGCIVETIYISIFLLYASKEARRQTVKLLVSLIGGLYTLIFLVTLFPLNGALRVQVVGWICVAVAVAVFAAPLSIVFQVVRTKSVEFLPFTLSFFLTLSAIMWFGYGLLQKDLCIALPNVLGFFLGMIQMLLYGLYRKVKPAAELEKKVPEHIVNIVVVGNSEQIHPVKSEKNEDMIKKLDEEENRESSVISPPVPALLPVANDHENEERAGGELAQVNLQPQQQFETPVLVVCAAASEQ ID NO:3： related to SEQ ID NO. 1a polynucleotide coding sequence of SWEET12-T from tobacco atggccatatttgacctccaccatccatggctatttgtgttcggagtcttaggaaacattatttccatattcgtcttcttagctccagtgccaacctttcgccgaatctacaaagaaaaatcaaccatgggttttcaatcagtcccctacgtggtagcactgttttcatccatgctctggatgtattatgcatttatcaagaaaaatgccactctcctcatctctatcaactccttcggttgcattgtcgagaccatttacatctccattttccttctctacgcatccaaggaggctaggaggcagacggtgaaacttttggtatcattgattggaggattgtacacactgatatttctcgtcactttgttccctttgaatggagcccttcgagtacaagtagtgggttggatttgtgtagccgtagcagtggctgtctttgctgcacctcttagcattgtatgtcaagtggttcggacgaagagtgtggagttcctgcccttcaccctgtctttctttcttacattgagtgctatcatgtggtttggttatggtctccttcaaaaggacctgtgtattgcactgccaaatgtattgggtttcttcctgggaatgattcagatgctgttgtatgggctataccgtaacgtaaagccagcagcagaattagagaaaaaggtgccggagcatgtagtaaacatcgtcgtccttggaaactcagaacagatacatcctgtcaaatccgagaaaaatgaggatatgatcaagaagctggatgaagaagtattagctgcagaagaaaacagggagagcagcgtaattagcccaccggtggcaaacgaccatgagaatgaagaacgtgcgggtggcgagctggcgcaagttaacttgcagccgcagcagcagtttgaaacaccggtgcttgtggtgtgtgctgcagcttga

SEQ ID NO. 4A polypeptide sequence MAIFDLHHPWLFVFGVLGNIISIFVFLAPVPTFRRIYKEKSTMGFQSVPYVVALFSSMLWMYYAFIKKNATLLISINSFGCIVETIYISIFLLYASKEARRQTVKLLVSLIGGLYTLIFLVTLFPLNGALRVQVVGWICVAVAVAVFAAPLSIVCQVVRTKSVEFLPFTLSFFLTLSAIMWFGYGLLQKDLCIALPNVLGFFLGMIQMLLYGLYRNVKPAAELEKKVPEHVVNIVVLGNSEQIHPVKSEKNEDMIKKLDEEVLAAEENRESSVISPPVANDHENEERAGGELAQVNLQPQQQFETPVLVVCAAASEQ ID NO:5： related to SEQ ID NO. 3a polynucleotide coding sequence from tobacco SWEET15-S atggctatcttcactgcttctcaattggcttttgtttttggcgttcttggaaatggggtgtcgttcttggtgtacttgtctccaataccgactttctataggatttataagagaaaatcaacggaaggattccagtctataccctattcggttgcactattcagtgccatgctctacttgtactatgcttatctcaaggagaagaatgggattttgctcgttactattaacagcttcgggactgccatcgaattgatatatctcacaatcttcttgatatatgctacccgagaggccaagatttacactacaaagctggttcttctgttaaatataggatcatatggagcaattgtggccttgacatatatattcgccaaagatgagacgcgagtcactattgtcggatggatctgtgctgtcttttctgtctgcgtcttcgctgctcctctaagcattatgagacgtgttataagaacaaggagcgttgagttcatgccattccctctttcattcttcctcacaatctgcgccgtcatgtggtttttctatggtctcttgataaaggacatgtacattgccacgccaaacattctagggtttacatttggaattgctcagatgatactgtacgcgatcttcagaaacagaaagcaacaaatccaaccggcggacagtaatctaaaagatttgacacaagtcgtcatagacatgaaagcaatggtattggagatgcaagaaaattctgatccgaataaggaagctgaagttgatgatactgatgaaaaaaagactaagcaagaagttgttgcacaaacaacttccaacgtatga

SEQ ID NO. 6 polypeptide sequence related to SEQ ID NO. 5 MAIFTASQLAFVFGVLGNGVSFLVYLSPIPTFYRIYKRKSTEGFQSIPYSVALFSAMLYLYYAYLKEKNGILLVTINSFGTAIELIYLTIFLIYATREAKIYTTKLVLLLNIGSYGAIVALTYIFAKDETRVTIVGWICAVFSVCVFAAPLSIMRRVIRTRSVEFMPFPLSFFLTICAVMWFFYGLLIKDMYIATPNILGFTFGIAQMILYAIFRNRKQQIQPADSNLKDLTQVVIDMKAMVLEMQENSDPNKEAEVDDTDEKKTKQEVVAQTTSNV

SEQ ID NO. 7 Polynucleotide coding sequence of SWEET15-T from Nicotiana tabacum atggctatcttcactgcttctcatttggcttttgtttttggcgttcttggaaatggggtgtcgttcttggtgtacttgtctccaataccgactttctataggatatataagagaaaatcaacggaaggattccagtctataccctattcggttgcactattcagtgccatgctctacttgtactatgcttatctcaaggagaagaatgggattttgctcattactattaacagcttcggaactgccatcgaattcatatatctcacaatcttcttgatgtatgctacccgagaggccaagatttacactacgaagctggttcttctgttaaatataggatcatttggagcaatcgtcgccttgacatatatattcgccaaagataagacgcgagtcactattgtcggatggatttgtgctgtcttttctgtctgcgtcttcgctgctcctcttagcattatgagacgcgttataaaaacaaggagcgttgagtttatgccattccctctttctttcttcctcacaatctgcgccgtcatgtggtttttctatggtctcttgataaaggacatgtacattgccacgccaaacattctagggtttacatttggaattgctcagatgatactgtacgcaatcttcagaaacagaaagcaacaaatccaaccggcagacagtaatctgaaagatttgacacaagtcgtcatagacatgaaagcaatggtattggagatgcaagaaaattctgatccaaataaggaagctgaagttgatgatactgatgaaaaaaagactaataagcaggaagttgttgcacaaacaacttctaacgtatga

SEQ ID NO. 8 polypeptide sequence related to SEQ ID NO. 7 MAIFTASHLAFVFGVLGNGVSFLVYLSPIPTFYRIYKRKSTEGFQSIPYSVALFSAMLYLYYAYLKEKNGILLITINSFGTAIEFIYLTIFLMYATREAKIYTTKLVLLLNIGSFGAIVALTYIFAKDKTRVTIVGWICAVFSVCVFAAPLSIMRRVIKTRSVEFMPFPLSFFLTICAVMWFFYGLLIKDMYIATPNILGFTFGIAQMILYAIFRNRKQQIQPADSNLKDLTQVVIDMKAMVLEMQENSDPNKEAEVDDTDEKKTNKQEVVAQTTSNV

SEQ ID NO. 9 selected Polynucleotide sequences (RNAi) to silence both SWEET 12-S and SWEET12-T

aatgtattgggtttcttcctgggaatgattcagatgctgttgtatgggctataccgtaa

SEQ ID NO. 10 selected Polynucleotide sequences (RNAi) to silence both SWEET 15-S and SWEET15-T

TTCTTCCTCACAATCTGCGCCGTCATGTGGTTTTTCTATGGTCTCTTGATAAAGGACATGTACATTGCC 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 (15)

1. A mutant, non-naturally occurring or transgenic tobacco 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 comprising, consisting of, or consisting essentially of: (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 shown 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; wherein the plant or part thereof comprises at least one modification capable of modulating: (a) expression of the polynucleotide in the plant or part thereof; or (b) activity of the polypeptide in the plant or part thereof, as compared to a control tobacco plant or part thereof in which expression of the polynucleotide or activity of the polypeptide is not modified, 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 a control tobacco plant in which expression or activity of one or more of SWEET12-S or SWEET12-T or SWEET15-S or SWEET15-T is not modulated. 2. The mutant, non-naturally occurring or transgenic tobacco plant or part thereof according to claim 1, wherein the plant comprises at least one genetic alteration in the regulatory region or coding sequence of one or more of SWEET12-S or SWEET12-T or SWEET15-S or SWEET15-T; and/or wherein the at least one genetic alteration comprises one or more exogenous DNA or exogenous RNA; and/or wherein the at least one genetic alteration comprises one or more vectors or viral vectors or Agrobacterium vectors or CRISPR vectors; and/or wherein the at least one genetic alteration is capable of driving one or more of RNA interference or transcriptional gene silencing or viral-induced gene silencing; and/or wherein the at least one genetic alteration is capable of expressing one or more double-stranded RNA (dsRNA) or hairpin RNA (hpRNA) or small interfering RNA; and/or 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. The mutant, non-naturally occurring or transgenic tobacco plant of claim 1 or claim 2, wherein the development of the plant during the vegetative stage is modulated compared to the control tobacco plant, and/or the flowering time of the plant is modulated compared to the control tobacco plant. 4. The mutant, non-naturally occurring or transgenic tobacco 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, and wherein plant height is increased during the vegetative stage and/or wherein flowering time is accelerated compared to the control tobacco plant. 5. The mutant, non-naturally occurring or transgenic tobacco plant of claim 3, wherein expression and/or activity of SWEET15-T is increased, and wherein flowering time is accelerated compared to the control tobacco plant. 6. The mutant, non-naturally occurring or transgenic tobacco plant or part thereof according to claim 1 or claim 2, wherein the part of the mutant, non-naturally occurring or transgenic plant is a cured or dried leaf. 7. The mutant, non-naturally occurring or transgenic tobacco plant or part thereof according to claim 6, wherein said chemical profile of said dried or dried leaves is modulated compared to dried or dried leaves derived from said control tobacco plant, suitably wherein said chemical profile is said sugar profile and/or said amino acid profile; suitably, wherein the expression and/or activity of SWEET12-S or SWEET15-T is increased, and wherein at least the fructose, glucose and sucrose contents are reduced compared to cured or dried leaves from said control tobacco plant; or wherein the expression and/or activity of SWEET15-S and SWEET15-T is reduced, and wherein at least the fructose, glucose and sucrose content is reduced compared to cured or dried leaves from said control tobacco plant; or wherein the expression and/or activity of SWEET12-S is increased, and wherein the asparagine, tryptophan, phenylalanine, glycine and methionine contents are increased, and wherein the glutamate and proline contents are reduced compared to cured or dried leaves derived from said control tobacco plant; suitably, wherein the ammonia content is increased compared to cured or dried leaves from said control tobacco plant; and/or wherein the expression and/or activity of SWEET15-T is increased, and wherein the asparagine and tryptophan contents are increased, and wherein the proline content is decreased compared to cured or dried leaves from said control tobacco plant. 8. The mutant, non-naturally occurring or transgenic tobacco plant or part thereof according to any one of the preceding claims, wherein the plant is of the Virginia or Burley type. 9. A plant material, dried or dried plant material or homogenised plant material, derived from or obtained from a tobacco plant or part thereof according to any one of claims 1 to 8; suitably, wherein the plant material is selected from the group consisting of: biomass, seeds, stems, flowers or leaves or a combination of two or more thereof; suitably, wherein said plant material is leaves; suitably, wherein said leaves are dried or dried leaves; suitably The dried leaves are selected from the group consisting of flue-dried leaves, sun-dried leaves or air-dried leaves. 10. A method of producing a tobacco plant having modulated flowering time and/or modulated amino acid levels and/or modulated sugar levels, the method comprising: (a) providing a tobacco 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 comprising, consisting of, or consisting essentially of: (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 shown 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, wherein the modification modulates expression or activity compared to a control tobacco plant in which expression of the same one or more of SWEET12-S or SWEET12-T or SWEET15-S or SWEET15-T is not modified. 11. The method according to claim 10, wherein in step (b), the at least one modification is introduced by genome editing; suitably, wherein the genome editing is selected from the group consisting of CRISPR-mediated genome editing, mutagenesis, zinc finger nuclease-mediated mutagenesis, chemical or radioactive mutagenesis, homologous recombination, oligonucleotide-directed mutagenesis, and meganuclease-mediated mutagenesis; or wherein in step (b), the at least one modification is introduced using an interfering polynucleotide; or wherein in step (b), the at least one modification is a promoter located 5' of the polynucleotide. 12. A tobacco plant obtained or obtainable by the method according to claim 10 or 11. 13. A method of producing cured tobacco plant material having modulated amino acid levels and/or modulated sugar levels, the method comprising: (a) preparing a tobacco plant according to claim 10 or claim 11, or providing a tobacco plant according to claim 12; (b) harvesting plant material (e.g., leaves) from the plant; and (c) drying the plant material. 14. Dried plant material (e.g. leaves) obtained or obtainable by the method according to claim 13. 15. A plant product comprising the plant material, dried plant material or homogenised plant material according to claim 9, or comprising the dried plant material according to claim 14; suitably, wherein the tobacco product is a tobacco blend; suitably, The tobacco blend comprises Virginia-type tobacco and/or Burley-type tobacco.