WO2024160864A1 - Modulation de transporteurs de sucre - Google Patents

Modulation de transporteurs de sucre Download PDF

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Publication number
WO2024160864A1
WO2024160864A1 PCT/EP2024/052307 EP2024052307W WO2024160864A1 WO 2024160864 A1 WO2024160864 A1 WO 2024160864A1 EP 2024052307 W EP2024052307 W EP 2024052307W WO 2024160864 A1 WO2024160864 A1 WO 2024160864A1
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Prior art keywords
plant
sweet15
sweet12
expression
polynucleotide
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PCT/EP2024/052307
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English (en)
Inventor
Lucien Bovet
Cecilia CHEVAL
Aurore Ginette Denise HILFIKER
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Philip Morris Products S.A.
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Publication of WO2024160864A1 publication Critical patent/WO2024160864A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8245—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis

Definitions

  • the present invention relates in general to plants having modulated expression or activity of SWEET (Sugars Will Eventually be Exported Transporters).
  • the present invention is based, at least in part, on the finding that modulating expression or activity of certain SWEET s can modify the chemical profile of plant parts - such as leaf - during curing. This may alter the aromatic or sensory profile of the plant product - such as cured or dried tobacco leaf. Modulating expression or activity of certain SWEETs can impact plant growth and development. Modulating the expression or activity of certain SWEETs may therefore further modulate leaf yield and/or plant maturity.
  • the SWEETs described herein may be key regulators of reproductive growth and development, in addition to being induced during leaf curing. The discovery of these functions is unexpected considering the low expression levels of these certain SWEETs in non-cured tissues.
  • a mutant, non-naturally occurring or transgenic (for example, genetically engineered) Nicotiana tabacum plant comprising at least one modification which is capable of modulating expression or activity of one or more of SWEET12-S or SWEET12-T or SWEET15-S or SWEET15-T comprising or consisting or consisting essentially of: (i) a SWEET12-S polynucleotide sequence comprising, consisting 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 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,
  • the Nicotiana tabacum plant comprises at least one genetic alteration in a regulatory region or in the coding sequence of one or more of SWEET12-S or SWEET12-T or SWEET 15- S or SWEET15-T.
  • the modification comprises one or more of exogenous DNA or exogenous RNA.
  • the modification comprises one or more of a vector or a viral vector or an Agrobacterium vector or a CRISPR vector.
  • the modification is capable of driving one or more of RNA interference or transcriptional gene silencing or virus induced gene silencing.
  • the modification is capable of expressing one or more of double stranded RNA (dsRNA) or hairpin RNA (hpRNA) or small interfering RNA.
  • dsRNA double stranded RNA
  • hpRNA hairpin RNA
  • small interfering RNA the modification is capable of constitutively expressing one or more of SWEET12-S or SWEET12-T or SWEET15-S or SWEET15-T.
  • development during the vegetative phase of the Nicotiana tabacum plant is modulated as compared to the control Nicotiana tabacum plant and/or flowering time of the plant is modulated as compared to the control Nicotiana tabacum plant.
  • SWEET12-S and SWEET12-T or SWEET15-S and SWEET15-T or SWEET12-S and SWEET12-T and SWEET15-S and SWEET15-T is decreased and wherein plant height is increased during the vegetive phase and/or wherein flowering time is accelerated as compared to the control Nicotiana tabacum plant.
  • SWEET15-T is increased and wherein flowering time is accelerated as compared to the control Nicotiana tabacum plant.
  • the part of the mutant, non-naturally occurring or transgenic Nicotiana tabacum plant is cured or dried leaf.
  • the chemical profile of the cured or dried leaf is modulated as compared to cured or dried leaf derived from the control Nicotiana tabacum plant, suitably wherein the chemical profile is the sugar profile and/or the amino acid profile.
  • SWEET12-S or SWEET15-T is increased and wherein at least the fructose, glucose and sucrose content is decreased as compared to cured or dried leaf derived from the control Nicotiana tabacum plant.
  • SWEET15-S and SWEET15-T are decreased and wherein at least the fructose, glucose and sucrose content is decreased as compared to cured or dried leaf derived from the control Nicotiana tabacum plant.
  • SWEET12-S is increased and wherein asparagine, tryptophan, phenylalanine, glycine and methionine content is increased and wherein glutamic acid and proline content is decreased as compared to cured or dried leaf derived from the control Nicotiana tabacum plant.
  • ammonia content is increased as compared to cured or dried leaf derived from the control Nicotiana tabacum plant.
  • SWEET15-T is increased and wherein asparagine and tryptophan content is increased and wherein proline acid content is decreased as compared to cured or dried leaf derived from the control Nicotiana tabacum plant.
  • the plant is a Nicotiana tabacum plant, more suitably, a Virginia or Burley type.
  • plant material cured or dried plant material, or homogenized plant material, derived or obtained from the Nicotiana tabacum plant or part thereof; suitably, wherein the plant material is selected from the group consisting of biomass, seed, stem, flower, or leaf or a combination of two or more thereof; suitably, wherein the plant material is leaf; suitably, wherein the leaf is cured or dried leaf; suitably, wherein the cured leaf is selected from the group consisting of flue-cured leaf, sun-cured leaf or air-cured leaf.
  • a method of preparing a Nicotiana tabacum plant with modulated flowering time and/or modulated amino acid levels and/or modulated sugar levels comprising: (a) providing a Nicotiana tabacum plant comprising at least one modification which is capable of modulating expression or activity of one or more of SWEET 12-S or SWEET12- T or SWEET15-S or SWEET15-T comprising or consisting or consisting essentially of: (i) a SWEET12-S polynucleotide sequence comprising, consisting 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 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 or consisting essentially of a sequence having at least 70 % sequence identity
  • a method of preparing a Nicotiana tabacum plant with modulated flowering time and/or modulated amino acid levels and/or modulated sugar levels comprising: (a) providing a Nicotiana tabacum plant having modulated expression or activity of one or more of SWEET12-S or SWEET12-T or SWEET15-S or SWEET15-T comprising or consisting or consisting essentially of: (i) a SWEET12-S polynucleotide sequence comprising, consisting 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 or consisting essentially of a sequence having at least 70 % sequence identity to SEQ ID NO: 3; or (iii) a SWEET 15-S polynucleotide sequence comprising, consisting or consisting essentially of a sequence having at least 70 % sequence identity to SEQ ID NO: 5;
  • the at least one modification is introduced by genome editing; suitably, wherein the genome editing is selected from CRISPR-mediated genome editing, mutagenesis, zinc finger nuclease-mediated mutagenesis, chemical or radiation mutagenesis, homologous recombination, oligonucleotide-directed mutagenesis and meganuclease-mediated mutagenesis; or wherein in step (b) the at least one modification is introduced using an interference polynucleotide; or wherein in step (b) the at least one modification is a promoter located 5’ to the polynucleotide.
  • a method of producing cured or dried Nicotiana tabacum plant material having modulated amino acid levels and/or modulated sugar levels comprising: (a) preparing a Nicotiana tabacum plant as described above or providing the Nicotiana tabacum plant as described above; (b) harvesting plant material (for example, leaf) from the plant; and (c) curing or drying the plant material.
  • cured or dried plant material for example, leaf obtained or obtainable by the method.
  • a plant product comprising the plant material, the cured or dried plant material, or the homogenised plant material or comprising the cured or dried plant material.
  • the plant product is a tobacco product from Nicotiana tabacum plant material.
  • the tobacco product is a tobacco blend; suitably, wherein the tobacco blend comprises Virginia type tobacco and/or Burley type tobacco.
  • Modulating the expression and/or activity of certain SWEETs as described herein can result in modulated levels of amino acids, especially in cured or dried plant material. This can result in tobacco with novel aromatic or sensory properties.
  • SWEETs as described herein can result in modulated levels of sugars, especially in cured or dried plant material. This can result in tobacco with novel aromatic or sensory properties.
  • SWEETs Modulating the expression and/or activity of certain SWEETs as described herein can result in plants with accelerated flowering time which can shorten the growth season and enable the faster introduction of new traits by crossing. This can result in cost savings for commercial plant production.
  • non-genetically modified plants can be created which may be more acceptable to consumers.
  • the present disclosure is not restricted to the use of EMS mutant plants.
  • An EMS mutant plant can have less potential to bring improved properties to a crop after breeding. Once breeding is started, the desirable characteristic(s) of the EMS mutant plant can be lost for different reasons. For example, several mutations may be required, the mutation can be dominant or recessive, and the identification of a point mutation in a gene target can be difficult to reach.
  • the present disclosure exploits the use of SWEET that can be specifically manipulated to produce plants with a desirable phenotype.
  • ASN asparagine synthetase
  • genes and enzymes also play a role in the reorganization of amino acids and/or sugars during leaf yellowing - such as diaminopimelate aminotransferase (DAPAT), aspartate amino transferases (AAT) and chloroplast sulphate transporters (SLILTR3) which may also change leaf chemistry.
  • DAPAT diaminopimelate aminotransferase
  • AAT aspartate amino transferases
  • SLILTR3 chloroplast sulphate transporters
  • Modifying NtSWEET expression or NtSWEET activity together with the expression or activity of one or more of these other targets selected from one or more of ASN, DAPAT and AAT may be used to further modify the flavour and/or sensory properties of cured or dried tobacco.
  • Figure 1 is a series of four graphs (a)-(d) showing the RNA-seq gene expression profile of SWEET genes during the curing time-course of flue-cured tobacco (Nicotiana tabacum L.).
  • the graphs depict expression profiles of (a) SWEET12-S, (b) SWEET12-T, (c) SWEET15-S and (d) SWEET15-T before curing (Oh) and during the curing process (24h, 48h, and 72h) in detached leaves from stalks X, C, B and T.
  • the stalk leaf positions X, C, B and T are from bottom to top of the plants, respectively.
  • the left y-axis indicates the FPKM (Fragments Per
  • the x-axis indicates the curing time-course.
  • the legend indicates the stalk positions X (processed line, grey), C (processed line, black), B (dotted line, grey) and T (dotted line, black).
  • FIG. 2 is a series of four graphs (a)-(d) showing RNA-seq gene expression profile of SWEET genes in the lamina and midrib tissues of flue-cured tobacco (Nicotiana tabacum L.).
  • the graphs depict expression profiles of (a) SWEET12-S, (b) SWEET12-T, (c) SWEET15-S and (d) SWEET15-T before curing (Oh) and during the curing process (6h, 24h, 48h, 55h and 72h) in the lamina (processed line, black) and midrib (processed line, grey) tissues.
  • the left y-axis indicates the FPKM (Fragments Per Kilobase of transcript per Million mapped reads).
  • the x- axis indicates the curing time-course.
  • FIG. 3 is a series of two graphs in which SWEET genes relative expression in flue-cured tobacco (Nicotiana tabacum L.) leaves of independent T1 plants is shown, (a) The quantification of SWEET12-S relative transcript levels in each individual T1 plant shows that SWEET12-S expression is higher in p35s; SWEET12-S and reduced in RNAi-SWEET12-S compared to WT control plants, (b) The quantification of SWEET15-T relative transcript levels in each individual T1 plant shows that SWEET15-T expression is higher in p35s; SWEET15- T and reduced in RNAi-SWEET15-T/-S compared to WT control plants.
  • results represent the relative mRNA expression in mature leaves detached from the stalk position C and cured for 48 hours. Data are normalised for variation using PQ14 expression. Data are summarised in bar plots in which each bar plot represents the gene expression level of a T1 individual plant.
  • Figure 4 is a series of photographs showing the differences in growth and development of flue-cured tobacco (Nicotiana tabacum L.) individual SWEET T1 lines, (a, b, and c) Representative images of 10 week-old plants grown in standard flue-cured fertilization conditions. Differences are observed in the height of individual T1 plants. Images (b) and (c) show that RNAi lines - RNAi-SWEET12-S and RNAi-SWEET15-T/-S - are taller than control plants (WT) whereas the image (a) shows that p35s: SWEET12-S lines are smaller than their respective control plants. No differences are observed in the growth and development of p35s; SWEET15-T plants compared to WT (not shown).
  • FIG. 5 is two graphs showing the flowering time of flue-cured tobacco (Nicotiana tabacum L.) SWEET T 1 lines. Flowering time is evaluated in 15-week-old plants grown in standard flue-cured tobacco (Nicotiana tabacum L.) SWEET T 1 lines. Flowering time is evaluated in 15-week-old plants grown in standard flue-cured tobacco (Nicotiana tabacum L.) SWEET T 1 lines. Flowering time is evaluated in 15-week-old plants grown in standard flue-cured tobacco (Nicotiana tabacum L.) SWEET T 1 lines. Flowering time is evaluated in 15-week-old plants grown in standard flue-
  • Figure 6 is a series of graphs showing leaf biomass of flue-cured tobacco (Nicotiana tabacum L.) SWEET T1 lines. Fresh leaf biomass of 18-week-old plants grown in standard flue-cured fertilization conditions. The results represent the average fresh weight of leaves detached from the stalk (C position). Data are collected from at least three biological replicates. Data are summarized in bar plots in which the bars represent the mean of the fresh weight in gram (g) and the error bars represent standard deviation, (a) Leaves are significantly smaller in p35s: SWEET12-S plants compared to WT and RNAi-SWEET12-S plants (Student t-test, * P ⁇ 0.05). (b) Leaves are significantly smaller in p35s: SWEET15-T plants compared to WT and RNAI-SWEET15-T plants (Student t-test, ** P ⁇ 0.01).
  • Figure 7 is a series of graphs showing the differences in sugar content of flue-cured Virginia tobacco (Nicotiana tabacum L.) SWEET12-S T1 plants.
  • Sugar content is measured in 18- week-old plants grown in standard flue-cured fertilization conditions and flue-cured under standard agronomic conditions.
  • the results represent the average content of (a) fructose, (b) glucose, (c) sucrose and (d) the sum of sugars in leaves detached from the stalk (C position) and flue cured.
  • Data are collected from at least three biological replicates. Data are summarized in bar plots in which the bars represent the mean in g/100g, and the error bars represent standard deviation.
  • Figure 8 is a series of graphs showing the differences in sugar content of flue-cured Virginia tobacco (Nicotiana tabacum L.) in SWEET15 T1 plants.
  • Sugar content is measured in 18- week-old plants grown in standard flue-cured fertilization conditions and flue-cured under standard agronomic conditions.
  • the results represent the average content of (a) fructose, (b) glucose, (c) sucrose and (d) the sum of sugars in leaves detached from the stalk (C position) and flue-cured.
  • Data are collected from at least three biological replicates. Data are summarized in bar plots in which the bars represent the pooled content in g/100g.
  • each intervening number there between with the same degree of precision is explicitly contemplated.
  • the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the numbers 6.0, 6.1 , 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 and 7.0 are explicitly contemplated.
  • Coding sequence or “polynucleotide encoding” means the nucleotides (RNA or DNA molecule) that comprise a polynucleotide which encodes a polypeptide.
  • the coding sequence can further include initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in the cells of an individual or mammal to which the polynucleotide is administered.
  • the coding sequence may be codon optimized.
  • “Complement” or “complementary” can mean Watson-Crick (for example, A-T/U and C-G) or Hoogsteen base pairing between nucleotides or nucleotide analogues. “Complementarity” refers to a property shared between two polynucleotides, such that when they are aligned 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.
  • the construct comprises a "template strand” base-paired with a complementary "sense or coding strand.”
  • a given construct can be inserted into a vector in two possible orientations, either in the same (or sense) orientation or in the reverse (or antisense) orientation with respect to the orientation of a promoter positioned within a vector - such as an expression vector.
  • control in the context of a control plant or control plant cells means a plant or plant cells in which the expression, function or activity of one or more genes or polypeptides has not been modified (for example, increased or decreased) and so it can provide a comparison
  • control plant is a plant that is substantially equivalent to a test plant or modified plant in all parameters with the exception of the test parameters.
  • a control plant is an equivalent plant into which no such polynucleotide has been introduced.
  • a control plant can be an equivalent plant into which a control polynucleotide has been introduced. In such instances, the control polynucleotide is one that is expected to result in little or no phenotypic effect on the plant.
  • the control plant may comprise an empty vector.
  • the control plant may correspond to a wild-type plant.
  • the control plant may be a null segregant wherein the T 1 segregant no longer possesses a transgene.
  • decrease refers to a decrease of from about 10% to about 99%, or a decrease 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, or at least 150%, or at least 200% more of a quantity or a function - such as polypeptide function, transcriptional function, or polypeptide expression.
  • a decreased amount can refer to a quantity or a function that is less than what would be found in a plant or a product from the same variety of plant processed in the same manner, which has not been modified.
  • a wild-type plant of the same variety that has been processed in the same manner is used as a control by which to measure whether a decrease in quantity is obtained.
  • Donor DNA or “donor template” refers to a double-stranded DNA fragment or molecule that includes at least a portion of the gene of interest.
  • the donor DNA may encode a functional polypeptide.
  • Endogenous gene or polypeptide refers to a gene or polypeptide that originates from the genome of an organism and has not undergone a change, such as a loss, gain, or exchange of genetic material. An endogenous gene undergoes normal gene transmission and gene expression. An endogenous polypeptide undergoes normal expression.
  • Enhancer sequences refer to the sequences that can increase gene expression. These sequences can be located upstream, within introns or downstream of the transcribed region. The transcribed region is comprised of the exons and the intervening introns, from the promoter to the transcription termination region. The enhancement of gene expression can be through various mechanisms including increasing transcriptional efficiency, stabilization of mature mRNA and translational enhancement.
  • “Expression” refers to the production of a functional product.
  • expression of a polynucleotide fragment may refer to transcription of the polynucleotide fragment (for example,
  • “Overexpression” refers to the production of a gene product in transgenic organisms that exceeds levels of production in a null segregating (or non-transgenic) organism from the same experiment.
  • “Functional” describes a polypeptide that has biological function or activity.
  • a “functional gene” refers to a gene transcribed to mRNA, which is translated to a functional or active polypeptide.
  • Genetic construct refers to DNA or RNA molecules that comprise a polynucleotide that encodes a polypeptide.
  • the coding sequence can include initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression.
  • Genome editing generally refers to the process by which genomic nucleic acid in a cell is altered. This can be by removing, inserting or replacing one or more nucleotides in the genomic nucleic acid, for example. Endonucleases can be used to create specific breaks or nicks at defined locations in the genome and are further described herein.
  • homology refers to the degree of sequence similarity between two polypeptides or between two polynucleotide molecules compared by sequence alignment.
  • the degree of homology between two discrete polynucleotides being compared is a function of the number of identical, or matching, nucleotides at comparable positions. Homology or similarity can be determined across the full length of a subject sequence.
  • Identity in the context of two or more polynucleotides or polypeptides means that the sequences have a specified percentage of residues that are the same over a specified region. The percentage may be calculated by optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which the identical residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to yield the percentage of sequence identity.
  • increase refers to an increase of from 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 or more of a quantity or a function or an activity, such as but not limited to one or more of polypeptide function or activity, transcriptional function or activity and polypeptide expression.
  • the term “increased,” or the phrase “an increased amount” can refer to a quantity or a function or an activity in a plant or a product generated from the plant that is more than what would be found in a plant or a product from the same variety of plant processed in the same manner, which has not been modified.
  • a wild-type plant of the same variety that has been processed in the same manner is used as a control by which to measure whether an increase in quantity is obtained.
  • inhibitor refers to a decrease of from about 98% to about 100%, or a decrease of at least 98%, at least 99%, but particularly of 100%, of a quantity or a function or an activity, such as but not limited to one or more of polypeptide function or activity, transcriptional function or activity and polypeptide expression.
  • the term “introduced” can mean providing a polynucleotide (for example, a construct) or polypeptide into a cell. Introduced includes reference to the incorporation of a polynucleotide into a eukaryotic cell where the polynucleotide may be incorporated into the genome of the cell, and includes reference to the transient provision of a polynucleotide or polypeptide to the cell. Introduced includes reference to stable or transient transformation methods, as well as sexually crossing.
  • "introduced” in the context of inserting a polynucleotide (for example, a recombinant construct/expression construct) into a cell means “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 (for example, chromosome, plasmid, plastid or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (for example, transfected mRNA).
  • a polynucleotide for example, a recombinant construct/expression construct
  • transduction includes reference to the incorporation of a polynucleotide into a eukaryotic cell where the polynucleotide may be incorporated into the genome of the cell (for example, chromosome, plasmid, plastid or mitochondrial DNA), converted into an autonomous replicon,
  • isolated or “purified” refer to material that is substantially or essentially free from components that normally accompany it as found in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A polypeptide that is the predominant species present in a preparation is substantially purified. In particular, an isolated
  • polynucleotide is separated from open reading frames that flank the desired gene and encode polypeptides other than the desired polypeptide.
  • purified denotes that a polynucleotide or polypeptide gives rise to essentially one band in an electrophoretic gel. Particularly, it means that the polynucleotide or polypeptide is at least 85% pure, more suitably at least 95% pure, and most suitably at least 99% pure.
  • Isolated polynucleotides may be purified from a host cell in which they naturally occur. Conventional polynucleotide purification methods known to skilled artisans may be used to obtain isolated polynucleotides. The term also embraces recombinant polynucleotides and chemically synthesized polynucleotides.
  • Liquid tobacco extract describes the direct product of an extraction process carried out on a tobacco starting material.
  • the extraction process for producing the liquid tobacco extract can comprise heating the tobacco starting material under specific heating conditions and collecting the volatile compounds generated.
  • the liquid tobacco extract can contain a mixture of compounds that have derived from the tobacco starting material and have been removed during the extraction process, typically in combination with a liquid carrier or solvent.
  • Modulate refers to causing or facilitating a qualitative or quantitative change, alteration, or modification in a process, pathway, function or activity of interest. Without limitation, such a change, alteration, or modification may be an increase or decrease in the relative process, pathway, function or activity of interest. For example, gene expression or polypeptide expression or polypeptide function or activity can be modulated. Typically, the relative change, alteration, or modification will be determined by comparison to a control.
  • non-naturally occurring describes an entity - such as a polynucleotide, a genetic mutation, a polypeptide, a plant, a plant cell and plant material - that is not formed by nature or that does not exist in nature.
  • entity - such as a polynucleotide, a genetic mutation, a polypeptide, a plant, a plant cell and plant material - that is not formed by nature or that does not exist in nature.
  • Such non-naturally occurring entities or artificial entities may be made, synthesized, initiated, modified, intervened, or manipulated by methods described herein or that are known in the art.
  • Such non-naturally occurring entities or artificial entities may be made, synthesized, initiated, modified, intervened, or manipulated by man.
  • a non-naturally occurring plant may not be produced using an essentially biological process.
  • a non-naturally occurring plant, a non-naturally occurring plant cell or non-naturally occurring plant material may be made using traditional plant breeding techniques - such as backcrossing - or by genetic manipulation technologies - such as antisense RNA, interfering RNA, meganuclease and the like.
  • a non-naturally occurring plant, a non-naturally occurring plant cell or non-naturally occurring plant material may be made by introgression of or by transferring one or more genetic mutations (for example one or more polymorphisms) from a first plant or plant cell into a second plant or plant cell (which may itself be naturally occurring), such that the resulting plant,
  • plant cell or plant material or the progeny thereof comprises a genetic constitution (for example, a genome, a chromosome or a segment thereof) that is not formed by nature or that does not exist in nature.
  • the resulting plant, plant cell or plant material is thus artificial or non- naturally occurring.
  • an artificial or non-naturally occurring plant or plant cell may be made by modifying a genetic sequence in a first naturally occurring plant or plant cell, even if the resulting genetic sequence occurs naturally in a second plant or plant cell that comprises a different genetic background from the first plant or plant cell.
  • a mutation is not a naturally occurring mutation that exists naturally in a polynucleotide or a polypeptide - such as a gene or a polypeptide.
  • Differences in genetic background can be detected by phenotypic differences or by molecular biology techniques known in the art - such as polynucleotide sequencing, presence or absence of genetic markers (for example, microsatellite RNA markers).
  • “Oligonucleotide” or “polynucleotide” means at least two nucleotides covalently linked together.
  • the depiction of a single strand also defines the sequence of the complementary strand.
  • a polynucleotide also encompasses the complementary strand of a depicted single strand.
  • Many variants of a polynucleotide may be used for the same purpose as a given polynucleotide.
  • a polynucleotide also encompasses substantially identical polynucleotides and complements thereof.
  • a single strand provides a probe that may hybridize to a given sequence under stringent hybridization conditions.
  • a polynucleotide also encompasses a probe that hybridizes under stringent hybridization conditions.
  • Polynucleotides may be single stranded or double stranded, or may contain portions of both double stranded and single stranded sequence.
  • the polynucleotide may be DNA, both genomic and cDNA, RNA, or a hybrid, where the polynucleotide may contain combinations of deoxyribo- and ribo-nucleotides, and 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 single-stranded DNA to hybridize complementary fragments is determined by the "stringency" of the reaction conditions (Sambrook et al., Molecular Cloning and Laboratory Manual, Second Ed., Cold Spring Harbor (1989)).
  • stringent conditions describes hybridization protocols in which polynucleotides at least 60% homologous to each other remain hybridized.
  • stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.
  • Tm is the temperature (under defined ionic strength, pH, and polynucleotide concentration) at which 50% of the probes complementary to the given
  • Stringent conditions typically comprise: (1) low ionic strength and high temperature washes, for example 15 mM sodium chloride, 1.5 mM sodium citrate, 0.1% sodium dodecyl sulfate, at 50°C; (2) a denaturing agent during hybridization, for example, 50% (v/v) formamide, 0.1 % bovine serum albumin, 0.1% Ficoll, 0.1 % polyvinylpyrrolidone, 50 mM sodium phosphate buffer (750 mM sodium chloride, 75 mM sodium citrate; pH 6.5), at 42°C; or (3) 50% formamide.
  • low ionic strength and high temperature washes for example 15 mM sodium chloride, 1.5 mM sodium citrate, 0.1% sodium dodecyl sulfate, at 50°C
  • a denaturing agent during hybridization for example, 50% (v/v) formamide, 0.1 % bovine serum albumin, 0.1% Ficoll, 0.1 % polyvinylpyrrolidone, 50
  • Washes typically also comprise 5xSSC (0.75 M NaCI, 75 mM sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5xDenhardt's solution, sonicated salmon sperm DNA (50 ⁇ g/mL), 0.1 % SDS, and 10% dextran sulfate at 42°C, with a wash at 42°C in 0.2xSSC (sodium chloride/sodium citrate) and 50% formamide at 55°C, followed by a high-stringency wash consisting of O.IxSSC containing EDTA at 55°C.
  • the conditions are such that sequences at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous to each other typically remain hybridized to each other.
  • Modely stringent conditions use washing solutions and hybridization conditions that are less stringent, such that a polynucleotide will hybridize to the entire, fragments, derivatives, or analogs of the polynucleotide.
  • One example comprises hybridization in 6xSSC, 5xDenhardt's solution, 0.5% SDS and 100 ⁇ g/mL denatured salmon sperm DNA at 55°C, followed by one or more washes in 1xSSC, 0.1 % SDS at 37°C.
  • the temperature, ionic strength, etc. can be adjusted to accommodate experimental factors such as probe length.
  • Low stringent conditions use washing solutions and hybridization conditions that are less stringent than those for moderate stringency, such that a polynucleotide will hybridize to the entire, fragments, derivatives, or analogs of the polynucleotide.
  • a non-limiting example of low stringency hybridization conditions includes hybridization in 35% formamide, 5xSSC, 50 mM Tris HCI (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 pg/mL denatured salmon sperm DNA, 10% (wt/vol) dextran sulfate at 40°C, followed by one or more washes in 2xSSC, 25 mM Tris HCI (pH 7.4), 5 mM EDTA, and 0.1 % SDS at 50°C.
  • Other conditions of low stringency such as those for cross-species hybridizations, are well-described (see Ausubel et al., 1993; Kriegler, 1990).
  • “Operably linked” means that expression of a gene is under the control of a promoter with which it is spatially connected.
  • a promoter may be positioned 5' (upstream) or 3' (downstream)
  • operably linked refers to the association of polynucleotide fragments in a single fragment so that the function of one is regulated by the other.
  • a promoter is operably linked with a polynucleotide fragment when it is capable of regulating the transcription of that polynucleotide fragment.
  • the term "plant” refers to any plant at any stage of its life cycle or development, and its progenies.
  • 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 of same.
  • Plant cells include, without limitation, cells from seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores. Suitable species, cultivars, hybrids and varieties of tobacco plant are described herein.
  • Plant material includes leaf, root, sepal, root tip, petal, flower, shoot, stem, seed and stalk. Plant material can be viable or non-viable plant material.
  • Polynucleotide refers to a polymer of RNA or DNA that is single- or doublestranded, optionally containing synthetic, non-natural or altered nucleotide bases.
  • the polynucleotides of the present disclosure are set forth in the accompanying sequence listing.
  • Polypeptide or “polypeptide sequence” refer to a polymer of amino acids in which one or more amino acid residues is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring polymers of amino acids.
  • Promoter means a synthetic or naturally-derived molecule which is capable of conferring, activating or enhancing expression of a polynucleotide in a cell.
  • the term refers to a polynucleotide element/sequence, typically positioned upstream and operably-linked to a double-stranded polynucleotide fragment.
  • Promoters can be derived entirely from regions proximate to a native gene of interest, or can be composed of different elements derived from different native promoters or synthetic polynucleotide segments.
  • a promoter may comprise one or more specific transcriptional regulatory sequences to further enhance expression, or to alter spatial expression or to alter temporal expression.
  • a promoter may also comprise distal enhancer or repressor elements, which may be located as much as several thousand base
  • a promoter may be derived from sources including viral, bacterial, fungal, plants, insects, and animals.
  • a promoter may regulate the expression of a gene component constitutively or differentially with respect to cell, the tissue or organ in which expression occurs or, with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, pathogens, metal ions, or inducing agents.
  • tissue-specific promoter and “tissue-preferred promoter” as used interchangeably herein refer to a promoter that is expressed predominantly but not necessarily exclusively in one tissue or organ, but that may also be expressed in one specific cell.
  • a “developmentally regulated promoter” refers to a promoter whose function is determined by developmental events.
  • a “constitutive promoter” refers to a promoter that causes a gene to be expressed in most cell types at most times.
  • An “inducible promoter” selectively express an operably linked DNA sequence in response to the presence of an endogenous or exogenous stimulus, for example by chemical compounds (chemical inducers) or in response to environmental, hormonal, chemical or developmental signals or a combination of two or more thereof. Examples of inducible or regulated promoters include promoters regulated by light, heat, stress, flooding or drought, pathogens, phytohormones, wounding, or chemicals such as ethanol, jasmonate, salicylic acid, or safeners.
  • “Recombinant” refers to an artificial combination of two otherwise separated segments of sequence - such as by chemical synthesis or by the manipulation of isolated segments of polynucleotides by genetic engineering techniques.
  • the term also includes reference to a cell or vector, that has been modified by the introduction of a heterologous polynucleotide or a cell derived from a cell so modified, but does not encompass the alteration of the cell or vector by naturally occurring events (for example, spontaneous mutation, natural transformation or transduction or transposition) such as those occurring without deliberate human intervention.
  • “Recombinant construct” refers to a combination of polynucleotides that are not normally found together in nature.
  • a recombinant construct may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that normally found in nature.
  • the recombinant construct can be a recombinant DNA construct.
  • regulatory sequences and “regulatory elements” as used interchangeably herein refer to polynucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences include promoters, translation leader sequences, introns, and
  • tobacco is used in a collective sense to refer to tobacco crops (for example, a plurality of tobacco plants grown in the field and not 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 that belong to the Nicotiana genus and products thereof and includes Nicotiana tabacum plants and products thereof.
  • tobacco products refers to consumer tobacco products, including but not limited to, smoking materials (for example, cigarettes, cigars, and pipe tobacco), snuff, chewing tobacco, gum, and lozenges, as well as components, materials and ingredients for manufacture of consumer tobacco products.
  • smoking materials for example, cigarettes, cigars, and pipe tobacco
  • snuff for example, cigarettes, cigars, and pipe tobacco
  • chewing tobacco for example, tobacco, cigars, and pipe tobacco
  • lozenges as well as components, materials and ingredients for manufacture of consumer tobacco products.
  • these tobacco products are manufactured from tobacco leaves and stems harvested from tobacco and cut, dried, cured, or fermented according to conventional techniques in tobacco preparation.
  • Transcription terminator refers to DNA sequences located downstream of a coding sequence, including polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression.
  • the polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor.
  • Transgenic refers to any cell, cell line, callus, tissue, plant part or plant, the genome of which has been altered by the presence of a heterologous polynucleotide, such as a recombinant construct, including those initial transgenic events as well as those created by sexual crosses or asexual propagation from the initial transgenic event.
  • the term does not encompass the alteration of the genome (chromosomal or extra-chromosomal) by conventional plant breeding methods or by naturally occurring events - such as random cross-fertilization, nonrecombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation.
  • the transgenic plant or part thereof is not produced using an essentially biological process.
  • Transgenic plant refers to a plant which comprises within its genome one or more heterologous polynucleotides, that is, a plant that contains recombinant genetic material not normally found therein and which has been introduced into the plant in question (or into progenitors of the plant) by human (artificial) manipulation.
  • the heterologous polynucleotide can be stably integrated within the genome such that the polynucleotide is passed on to successive generations.
  • the heterologous polynucleotide can be integrated into the genome alone or as part of a recombinant construct.
  • Gene stacking can be accomplished by many means including but not limited to co-transformation, retransformation, and crossing lines with different transgenes.
  • a plant that is grown from a plant cell into which recombinant DNA is introduced by transformation is a transgenic plant, as are all offspring of that plant that contain the introduced transgene (whether produced sexually or asexually).
  • transgenic plant encompasses the entire plant or tree and parts of the plant or tree, for instance grains, seeds, flowers, leaves, roots, fruit, pollen, stems and the like. Each heterologous polynucleotide may confer a different trait to the transgenic plant.
  • Transgene refers to a gene or genetic material containing a gene sequence that has been isolated from one organism and is introduced into a different organism. This non-native segment of DNA may retain the ability to produce RNA or polypeptide in the transgenic organism, or it may alter the normal function of the transgenic organism's genetic code.
  • “Variant” with respect to a polynucleotide means: (i) a portion or fragment of a polynucleotide; (ii) the complement of a polynucleotide or portion thereof; (iii) a polynucleotide that is substantially identical to a polynucleotide of interest or the complement thereof; or (iv) a polynucleotide that hybridizes under stringent conditions to the polynucleotide of interest, complement thereof, or a polynucleotide substantially identical thereto.
  • “Variant” with respect to a peptide or polypeptide means a peptide or polypeptide that differs in sequence by the insertion, deletion, or conservative substitution of amino acids, but retain at least one biological function or activity. Variant may also mean a polypeptide that retains at least one biological function or activity.
  • a conservative substitution of an amino acid that is, replacing an amino acid with a different amino acid of similar properties (for example, hydrophilicity, degree and distribution of charged regions) is recognized in the art as typically involving a minor change.
  • variable refers to a population of plants that share constant characteristics which separate them from other plants of the same species. While possessing one or more distinctive traits, a variety is further characterized by a very small overall variation between individuals within that variety. A variety is often sold commercially.
  • Vector refers to a polynucleotide vehicle that comprises a combination of polynucleotide components for enabling the transport of polynucleotides, polynucleotide constructs and polynucleotide conjugates and the like.
  • a vector may be a viral vector, bacteriophage, bacterial artificial chromosome or yeast artificial chromosome.
  • a vector may be a DNA or
  • RNA vector 19 RNA vector.
  • Suitable vectors include episomes capable of extra-chromosomal replication such as circular, double-stranded nucleotide plasmids; linearized double-stranded nucleotide plasmids; and other vectors of any origin.
  • An "expression vector” is a polynucleotide vehicle that comprises a combination of polynucleotide components for enabling the expression of polynucleotide(s), polynucleotide constructs and polynucleotide conjugates and the like.
  • Suitable expression vectors include episomes capable of extra-chromosomal replication such as circular, double-stranded nucleotide plasmids; linearized double-stranded nucleotide plasmids; and other functionally equivalent expression vectors of any origin.
  • An expression vector comprises at least a promoter positioned upstream and operably-linked to a polynucleotide, polynucleotide constructs or polynucleotide conjugate, as defined below.
  • an isolated polynucleotide comprising, consisting or consisting essentially of a sequence having at least 60% sequence identity to any of the sequences described herein, including any of polynucleotides shown in the sequence listing.
  • the isolated polynucleotide comprises, consists 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.
  • the isolated polynucleotide comprises, consists 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.
  • the isolated polynucleotide comprises, consists 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.
  • the isolated polynucleotide comprises, consists or consists essentially of a sequence having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto.
  • the polynucleotide(s) described herein encode an active polypeptide that has at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 100% or more of the SWEET function or activity of the polypeptide(s) shown in the sequence listing.
  • an isolated SWEET polynucleotide from Nicotiana tabacum comprising, consisting or consisting essentially of a polynucleotide 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 to SEQ ID NO: 1 (NtSWEET12-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: 3 (NtSWEET12-T), or at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
  • polynucleotides comprising a sufficient or substantial degree of identity or similarity to SEQ ID NO: 1 or SEQ ID NO: 3 or SEQ ID NO: 5 or SEQ ID NO: 7 that encode a polypeptide that functions as a SWEET.
  • polymer of polynucleotides which comprises, consists or consists 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.
  • the polynucleotides described herein encode SWEET polypeptides that have SWEET activity.
  • a polynucleotide can include a polymer of nucleotides, which may be unmodified or modified deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Accordingly, a polynucleotide can be, without limitation, a genomic DNA, complementary DNA (cDNA), mRNA, or antisense RNA or a fragment(s) thereof. Moreover, a polynucleotide can be single-stranded or double-stranded DNA, DNA that is a mixture of single-stranded and double-stranded regions, a hybrid molecule comprising DNA and RNA, or a hybrid molecule with a mixture of single-stranded and doublestranded regions or a fragment(s) thereof.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • polynucleotide can be composed of triple-stranded regions comprising DNA, RNA, or both or a fragment(s) thereof.
  • a polynucleotide can contain one or more modified bases, such as phosphothioates, and can be a peptide nucleic acid.
  • polynucleotides can be assembled from isolated or cloned fragments of cDNA, genomic DNA, oligonucleotides, or individual nucleotides, or a combination of the foregoing.
  • the polynucleotides described herein are shown as DNA sequences, they include their corresponding RNA sequences, and their complementary (for example, completely complementary) DNA or RNA sequences, including the reverse complements thereof.
  • 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 the full-length polynucleotide encoding the polypeptides described herein.
  • a polynucleotide will generally contain phosphodiester bonds, although in some cases, polynucleotide analogues are included that may have alternate backbones, comprising, for example, phosphoramidate, phosphorothioate, phosphorodithioate, or O- methylphophoroamidite linkages; and peptide polynucleotide backbones and linkages.
  • Other analogue polynucleotides include those with positive backbones; non-ionic backbones, and non-ribose backbones. Modifications of the ribose-phosphate backbone may be done for a variety of reasons, for example, to increase the stability and half-life of such molecules in physiological environments or as probes on a biochip. Mixtures of naturally occurring
  • polynucleotides and analogues can be made; alternatively, mixtures of different polynucleotide analogues, and mixtures of naturally occurring polynucleotides and analogues may be made.
  • a variety of polynucleotide analogues are known, including, for example, phosphoramidate, phosphorothioate, phosphorodithioate, O-methylphophoroamidite linkages and peptide polynucleotide backbones and linkages.
  • Other analogue polynucleotides include those with positive backbones, non-ionic backbones and non-ribose backbones.
  • Polynucleotides containing one or more carbocyclic sugars are also included.
  • analogues include peptide polynucleotides which are peptide polynucleotide analogues.
  • fragments generally comprise at least about 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more contiguous nucleotides of a DNA sequence.
  • a DNA fragment comprises at least about 10, 15, 20, 30, 40, 50 or 60 or more contiguous nucleotides of a DNA sequence.
  • oligonucleotides are useful as primers, for example, in polymerase chain reactions (PCR), whereby DNA fragments are isolated and amplified.
  • At least one modification can 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.
  • SWEET polypeptide encoded by the polynucleotide(s) described herein.
  • an isolated SWEET polypeptide comprising, consisting 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.
  • the isolated polypeptide comprises, consists 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.
  • the isolated polypeptide comprises, consists 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%,
  • SWEET polypeptide comprising, consisting 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.
  • SWEET polypeptide comprising, consisting 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 can include sequences comprising a sufficient or substantial degree of identity or similarity to SEQ ID NO: 2 or SEQ ID NO: 4 or SEQ ID NO: 6 or SEQ ID NO: 8 to function as a SWEET.
  • Fragment of the polypeptides described herein are also contemplated.
  • the fragments of the polypeptide(s) typically retain some or all of the function or activity of the full-length sequence - such as SWEET activity.
  • Fragments of a polypeptide may 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 polypeptide described herein.
  • polypeptides also include mutants produced by introducing any type of alterations (for example, insertions, deletions, or substitutions of amino acids; changes in glycosylation states; changes that affect refolding or isomerization, three-dimensional structures, or selfassociation states), which can be deliberately engineered or isolated naturally provided that they still have some or all of their function or activity. Suitably, this function or activity is modulated.
  • any type of alterations for example, insertions, deletions, or substitutions of amino acids; changes in glycosylation states; changes that affect refolding or isomerization, three-dimensional structures, or selfassociation states
  • a deletion refers to removal of one or more amino acids from a polypeptide.
  • An insertion refers to one or more amino acid residues being introduced into a predetermined site in a polypeptide. Insertions may comprise intra-sequence insertions of single or multiple amino acids.
  • a substitution refers to the replacement of amino acids of the polypeptide with other amino acids having similar properties (such as similar hydrophobicity, hydrophilicity, antigenicity, propensity to form or break a-helical structures or p-sheet structures). Amino acid substitutions are typically of single residues, but may be clustered depending upon functional constraints placed upon the polypeptide and may range from about 1 to about 10 amino acids.
  • amino acid substitutions are suitably conservative amino acid substitutions as described below.
  • Amino acid substitutions, deletions or insertions can be made using peptide synthetic techniques - such as solid phase peptide synthesis or by recombinant DNA manipulation. Methods for the manipulation of DNA sequences to produce substitution, insertion or deletion variants of a polypeptide are well known in the art.
  • the variant may have alterations which produce a silent change and result in a functionally equivalent polypeptide.
  • Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and the amphipathic nature of the residues as long as the secondary binding of the substance is retained.
  • negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine. Conservative substitutions may be made, for example according to the Table below. Amino acids in the same block in the second column and suitably in the same line 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.
  • Polypeptides may be in linear form or cyclized using known methods. Polypeptides typically comprise at least 10, at least 20, at least 30, or at least 40 contiguous amino acids.
  • At least one modification can 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 in order to modulate polypeptide expression, function or activity.
  • a recombinant polynucleotide construct can comprise a polynucleotide encoding one or more polynucleotides as described herein, operably linked to a regulatory region suitable for expressing the polypeptide.
  • polynucleotide can comprise a coding sequence that encodes the polypeptide as described herein.
  • Plants or plant cells in which polypeptide expression, function or activity are modulated can include mutant, non-naturally occurring, transgenic, man-made or genetically engineered plants or plant cells.
  • the transgenic plant or plant cell comprises a genome that has been altered by the stable integration of recombinant DNA.
  • Recombinant DNA includes DNA which has been genetically engineered and constructed outside of a cell and includes DNA containing naturally occurring DNA or cDNA or synthetic DNA.
  • a transgenic plant can include a plant regenerated from an originally-transformed plant cell and progeny transgenic plants from later generations or crosses of a transformed plant.
  • the transgenic modification alters the expression or function or activity of the polynucleotide or the polypeptide described herein as compared to a control plant.
  • the polypeptide encoded by a recombinant polynucleotide can be a native polypeptide, or can be heterologous to the cell.
  • the recombinant construct contains 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 routinely used in the art such as plasmids, viruses, artificial chromosomes, bacterial artificial chromosomes, yeast artificial chromosomes, or bacteriophage artificial chromosomes.
  • Suitable expression vectors include, without limitation, plasmids and viral vectors derived from, for example, bacteriophage, baculoviruses, and retroviruses. Numerous vectors and expression systems are commercially available.
  • the vectors can include, for example, origins of replication, scaffold attachment regions or markers.
  • a marker gene can confer a selectable phenotype on a plant cell.
  • a marker can confer biocide resistance, such as resistance to an antibiotic (for example, kanamycin, G418, bleomycin, or hygromycin), or an herbicide (for example, glyphosate, chlorsulfuron or phosphinothricin).
  • an expression vector can include a tag sequence designed to facilitate manipulation or detection (for example, purification or localization) of the expressed polypeptide.
  • Tag sequences such as luciferase, betaglucuronidase, green fluorescent polypeptide, glutathione S-transferase, polyhistidine, c-myc or hemagglutinin sequences typically are expressed as a fusion with the encoded polypeptide.
  • Such tags can be inserted anywhere within the polypeptide, including at either the carboxyl or amino terminus.
  • a plant or plant cell can be transformed by having the recombinant polynucleotide integrated into its genome to become stably transformed.
  • the plant or plant cell described herein can be
  • Stably transformed cells typically retain the introduced polynucleotide with each cell division.
  • a plant or plant cell can be transiently transformed such that the recombinant polynucleotide is not integrated into its genome.
  • Transiently transformed cells typically lose all or some portion of the introduced recombinant polynucleotide with each cell division such that the introduced recombinant polynucleotide cannot be detected in daughter cells after a sufficient number of cell divisions.
  • a number of methods are available in the art for transforming a plant cell including biolistics, gene gun techniques, Agrobacterium-mediated transformation, viral vector-mediated transformation, freeze-thaw method, microparticle bombardment, direct DNA uptake, sonication, microinjection, plant virus-mediated transfer, and electroporation.
  • plants can be regenerated from transformed cultures if desired, by techniques known to those skilled in the art.
  • regulatory regions to be included in a recombinant construct depends upon several factors, including, but not limited to, efficiency, selectability, inducibility, desired expression level, and cell- or tissue-preferential expression. It is a routine matter for one of skill in the art to modulate the expression of a coding sequence by appropriately selecting and positioning regulatory regions relative to the coding sequence. Transcription of a polynucleotide can be modulated 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 present in different tissues or cell types (for example, root-specific promoters, shoot-specific promoters, xylem-specific promoters), or present during different developmental stages, or present in response to different environmental conditions.
  • Suitable promoters include constitutive promoters that can be activated in most cell types without requiring specific inducers.
  • Examples of promoters that can be used to controlpolypeptide expression include the cauliflower mosaic virus 35S (CaMV/35S), SSU, OCS, lib-4, usp, STLS1 , B33, nos or ubiquitin- or phaseolin-promoters. Persons skilled in the art are capable of generating multiple variations of recombinant promoters.
  • Tissue-specific promoters are transcriptional control elements that are only active in particular cells or tissues at specific times during plant development, such as in vegetative tissues or reproductive tissues.
  • tissue-specific promoters under developmental control include promoters that can initiate transcription only (or primarily only) in certain tissues, such as vegetative tissues, for example, roots or leaves, or reproductive tissues, such as fruit,
  • Reproductive tissue-specific promoters may be, for example, anther-specific, ovule-specific, embryo-specific, endospermspecific, integument-specific, seed and seed coat-specific, pollen-specific, petal-specific, sepal-specific, or combinations thereof.
  • Exemplary leaf-specific promoters include pyruvate, orthophosphate dikinase (PPDK) promoter from C4 plant (maize), cab-m1Ca+2 promoter from maize, the Arabidopsis thaliana myb-related gene promoter (AtmybS), the ribulose biphosphate carboxylase (RBCS) promoters (for example, the tomato RBCS 1 , RBCS2 and RBCS3A genes expressed in leaves and light-grown seedlings, RBCS1 and RBCS2 expressed in developing tomato fruits or ribulose bisphosphate carboxylase promoter expressed almost exclusively in mesophyll cells in leaf blades and leaf sheaths at high levels).
  • PPDK orthophosphate dikinase
  • AtmybS Arabidopsis thaliana myb-related gene promoter
  • RBCS ribulose biphosphate carboxylase
  • Exemplary senescence-specific promoters include a tomato promoter active during fruit ripening, senescence and abscission of leaves, a maize promoter of gene encoding a cysteine protease, the promoter of 82E4 and the promoter of SAG genes. Exemplary anther-specific promoters can be used. Exemplary root-preferred promoters known to persons skilled in the art may be selected. Exemplary seed-preferred promoters include both seed-specific promoters (those promoters active during seed development such as promoters of seed storage polypeptides) and seed-germinating promoters (those promoters active during seed germination).
  • inducible promoters include promoters responsive to pathogen attack, anaerobic conditions, elevated temperature, light, drought, cold temperature, or high salt concentration.
  • Pathogen-inducible promoters include those from pathogenesis-related polypeptides (PR polypeptides), which are induced following infection by a pathogen (for example, PR polypeptides, SAR polypeptides, beta-1 , 3-glucanase, chitinase).
  • promoters may be derived from bacterial origin for example, the octopine synthase promoter, the nopaline synthase promoter and other promoters derived from Ti plasmids, or may be derived from viral promoters (for example, 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).
  • CaMV cauliflower mosaic virus
  • CaMV cauliflower mosaic virus
  • CaMV constitutive promoters of tobacco mosaic virus
  • CaMV cauliflower mosaic virus
  • CaMV cauliflower mosaic virus
  • figwort mosaic virus 35S promoter figwort mosaic virus 35S promoter
  • a plant or plant cell comprising at least one mutation in one or more polynucleotides or polypeptides as described herein is disclosed, wherein said mutation results in modulated function or activity of NtSWEET or the polypeptide(s) encoded thereby.
  • a method for identifying a plant with modulated levels of one or more amino acids in the plant or part thereof as compared to the level of the one or more amino acids in the control plant comprising screening a polynucleotide sample from a plant of interest for the presence of one or more mutations in the NtSWEET polynucleotide sequences according to the present disclosure, and optionally correlating the identified mutation(s) with mutation(s) that are known to modulate levels of one or more amino acids.
  • NtSWEET gene There is also disclosed a plant or plant cell that is heterozygous or homozygous for one or more mutations in a NtSWEET gene according to the present disclosure, wherein said mutation results in modulated expression of the NtSWEET gene or function or activity of the NtSWEET polypeptide encoded thereby.
  • a number of approaches can be used to combine mutations in one plant including sexual crossing.
  • a plant having one or more favourable heterozygous or homozygous mutations in a gene according to the present disclosure that modulates expression of the gene or the function or activity of the polypeptide encoded thereby can be crossed with a plant having one or more favourable heterozygous or homozygous mutations in one or more other genes that modulate expression thereof or the function or activity of the polypeptide encoded thereby.
  • crosses are made in order to introduce one or more favourable heterozygous or homozygous mutations within gene according to the present disclosure within the same plant.
  • the function or activity of one or more polypeptides of the present disclosure in a plant is increased or decreased if the function or activity is lower or higher than the function or activity of the same polypeptide(s) in a plant that has not been modified to inhibit the function or activity of that polypeptide and which has been cultured, harvested and cured or dried using the same protocols.
  • the mutation(s) is introduced into a plant or plant cell using a mutagenesis approach, 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 impact gene expression or that interfere with the function of the encoded polypeptide can be determined using methods that are well known in the art. Insertional mutations in gene exons usually result in null-mutants. Mutations in conserved residues can be particularly effective in inhibiting the metabolic function of the encoded polypeptide. It will be appreciated, for example, that a mutation in one or more of the highly conserved regions would likely alter polypeptide function, while a mutation outside of
  • Any plant of interest including a plant cell or plant material can be genetically modified by various methods known to induce mutagenesis, including site-directed mutagenesis, oligonucleotide- directed mutagenesis, chemically-induced mutagenesis, irradiation-induced mutagenesis, mutagenesis utilizing modified bases, mutagenesis utilizing gapped duplex DNA, doublestrand break mutagenesis, mutagenesis utilizing repair-deficient host strains, mutagenesis by total gene synthesis, DNA shuffling and other equivalent methods.
  • Mutations in the polynucleotides and polypeptides described herein can include man-made mutations or synthetic mutations or genetically engineered mutations. Mutations in the polynucleotides and polypeptides described herein can be mutations that are obtained or obtainable via a process which includes an in vitro or an in vivo manipulation step. Mutations in the polynucleotides and polypeptides described herein can be mutations that are obtained or obtainable via a process which includes intervention by man. The function or activity of the mutant polypeptide variant may be higher, lower or about the same as the unmutated polypeptide.
  • Methods that introduce a mutation randomly in a polynucleotide can include chemical mutagenesis and radiation mutagenesis.
  • Chemical mutagenesis involves the use of exogenously added chemicals - such as mutagenic, teratogenic, or carcinogenic organic compounds - to induce mutations.
  • Mutagens that create primarily point mutations and short deletions, insertions, missense mutations, simple sequence repeats, transversions ortransitions, including chemical mutagens or radiation, may be used to create the mutations.
  • Mutagens include ethyl methanesulfonate, methylmethane sulfonate, N-ethyl-N-nitrosurea, triethylmelamine, N-methyl-N-nitrosourea, procarbazine, chlorambucil, cyclophosphamide, diethyl sulfate, acrylamide monomer, melphalan, nitrogen mustard, vincristine, dimethylnitrosamine, N-methyl-N'-nitro-Nitrosoguanidine, nitrosoguanidine, 2-aminopurine, 7,12 dimethyl-benz(a)anthracene, ethylene oxide, hexamethylphosphoramide, bisulfan, diepoxyalkanes (diepoxyoctane, diepoxybutane, and the like), 2-methoxy-6-chloro-9[3-(ethyl- 2-chloro-ethyl)aminopropylamino]acridine di
  • Suitable mutagenic agents can also include, for example, ionising radiation - such as X-rays, gamma rays, fast neutron irradiation and UV radiation.
  • ionising radiation such as X-rays, gamma rays, fast neutron irradiation and UV radiation.
  • the dosage of the mutagenic chemical or radiation is
  • the mutation process may include one or more plant crossing steps.
  • screening can be performed to identify mutations that create premature stop codons or otherwise non-functional genes. After mutation, screening can be performed to identify mutations that create functional genes that are capable of being expressed at increased or decreased levels. Screening of mutants can be carried out by sequencing, or by the use of one or more probes or primers specific to the gene or polypeptide. Specific mutations in polynucleotides can also be created that can result in modulated gene expression, modulated stability of mRNA, or modulated stability of polypeptide. Such plants are referred to herein as "non-naturally occurring" or "mutant" plants.
  • the mutant or non-naturally occurring plants will include at least a portion of foreign or synthetic or manmade nucleotide (for example, DNA or RNA) that was not present in the plant before it was manipulated.
  • the foreign nucleotide may be a single nucleotide, two or more nucleotides, two or more contiguous nucleotides or two or more non-contiguous nucleotides - such as at least 10, 20, 30, 40, 50,100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400 or 1500 or more contiguous or non-contiguous nucleotides.
  • Zinc finger polypeptides can be used to modulate the expression or function or activity of the 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 l-Crel, can be used to modulate the expression or function or activity of one or more of the NtSWEET polynucleotides described herein.
  • the use of meganucleases is described in Curr Gene Then (2011) Feb;11 (1):11-27 and Int J Mol Sci. (2019) 20(16), 4045.
  • Transcription activator-like effector nucleases can be used to modulate the expression or function or activity of one or more of the NtSWEET polynucleotides described herein.
  • TALENs Transcription activator-like effector nucleases
  • the CRISPR system can be used to modulate the expression or function or activity of one or more of the NtSWEET polynucleotides described herein and is a preferred method.
  • This technology is described in, for example, Plant Methods (2016) 12:8; Front Plant Sci. (2016) 7: 506; Biotechnology Advances (2015) 33, 1 , p41-52; Acta Pharmaceutica Sinica B (2017) 7, 3, P292-302; Curr. Op. in Plant Biol. (2017) 36, 1-8 and Int J Mol Sci (2019) 20(16), 4045.
  • the CRISPR editing system generally includes two components: a CRISPR-associated endonuclease (Cas) (for example, Cas9) and a guide RNA (gRNA).
  • Cas forms a double stranded DNA break at a site in the genome that is defined by the sequence of a gRNA molecule bound to Cas. The location at which Cas breaks the DNA is defined by the unique sequence of the gRNA that is bound to it.
  • gRNA is a specifically designed RNA sequence that recognizes the target DNA region of interest and directs the Cas nuclease there for editing.
  • a tracr RNA which serves as a binding scaffold for the Cas nuclease
  • crRNA crispr RNA
  • the exact region of the DNA to be targeted will depend on the specific application.
  • gRNAs can be targeted to the promoter driving expression of the target polynucleotide.
  • Methods for designing gRNAs are well known in the art, including Chop Chop Harvard. The application of Cas9-based genome editing in Arabidopsis and tobacco is described in, for example, Methods Enzymol. (2014) 546:459-72 and Plant Physiol Biochem.
  • RNA-guided nucleases for use in the CRISPR system have been described, including, Casl, CasIB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, CaslO, Cpfl, Csyl, Csy2, Csy3, Csel, Cse2, Csel, 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.
  • the present disclosure also provides a method of cleaving one or more polynucleotides in a plant cell, comprising introducing a gRNA and an RNA-guided nuclease into the plant cell, wherein the gRNA acts in association with the RNA-guided nuclease to create a strand break in one or more of the polynucleotides described herein.
  • a CRISPR construct comprising: (i) a polynucleotide encoding a CRISPR-associated endonuclease; and (ii) a gRNA including a polynucleotide sequence (typically of about 17-20 nucleotides) complementary to the DNA of the polynucleotide as described herein that is to be targeted.
  • Antisense technology is another well-known method that can be used to modulate the expression or activity of one or more NtSWEET polypeptides described herein. See, for example, Gene (1988) 10;72(1-2):45-50.
  • NtSWEET polynucleotides can be targeted for inactivation by introducing transposons (for example, IS elements or other mobile genetic elements) into the genomes of plants of interest. See, for example, Cytology and Genetics (2006) 40(4):68-81.
  • NtSWEET polynucleotides can be targeted for inactivation by introducing ribozymes derived from a number of small circular RNAs that are capable of self-cleavage and replication in plants. See, for example, FEMS Microbiology Reviews (1999) 23, 3, 257-275.
  • the mutant or non-naturally occurring plants or plant cells can have any combination of one or more modifications (for example, mutations) in one or more of the NtSWEET polynucleotides described herein which result in modulated expression or function or activity of those polynucleotides or their polynucleotide products.
  • the mutant or non- naturally occurring plants or plant cells 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.
  • the mutant or non-naturally occurring plants or plant cells may have one or more modifications in a specific portion of NtSWEET polynucleotide(s) or NtSWEET polypeptide(s) - such as in a region of NtSWEET that encodes an active site of the NtSWEET polypeptide or a portion thereof.
  • the mutant or non-naturally occurring plants or plant cells may have one or more modifications in a region outside of one or more NtSWEET polynucleotide(s) or NtSWEET polypeptide(s) - such as in a region upstream or downstream of the NtSWEET polynucleotide(s) provided that it regulates the function or expression of the NtSWEET.
  • Upstream elements can include promoters,
  • enhancers or transcription factors Some elements - such as enhancers - can be positioned upstream or downstream of the gene it regulates. The element(s) need not be located near to the gene that it regulates since some elements have been found located several hundred thousand base pairs upstream or downstream of the gene that it regulates.
  • the mutant or non- naturally occurring plants or plant cells may have one or more modifications located within the first 100 nucleotides of the gene(s), within the first 200 nucleotides of the gene(s), within the first 300 nucleotides of the gene(s), within the first 400 nucleotides of the gene(s), within the first 500 nucleotides of the gene(s), within the first 600 nucleotides of the gene(s), within the first 700 nucleotides of the gene(s), within the first 800 nucleotides of the gene(s), within the first 900 nucleotides of the gene(s), within the first 1000 nucleotides of the gene(s), within the first 1100 nucleotides of the gene(s), within the first 1200 nucleotides of the gene(s), within the first 1300 nucleotides of the gene(s), within the first 1400 nucleotides of the gene(s) or within the first 1500 nucleotides of the gene(s).
  • the mutant or non-naturally occurring plants or plant cells may have one or more modifications located within the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth or fifteenth set of 100 nucleotides of the gene(s) or combinations thereof.
  • Mutant or non-naturally occurring plants or plant cells comprising the mutant polypeptide variants are disclosed.
  • seeds from plants are mutagenised and then grown into first generation mutant plants.
  • the first-generation plants are then allowed to self-pollinate and seeds from the first generation plant are grown into second generation plants, which are then screened for mutations in their loci.
  • the mutagenized plant material can be screened for mutations, an advantage of screening the second-generation plants is that all somatic mutations correspond to germline mutations.
  • plant materials including but not limited to, seeds, pollen, plant tissue or plant cells, may be mutagenised in order to create the mutant plants.
  • the type of plant material mutagenised may affect when the plant polynucleotide is screened for mutations.
  • the seeds resulting from that pollination are grown into first generation plants. Every cell of the first generation plants will contain mutations created in the pollen; thus these first generation plants may then be screened for mutations instead of waiting until the second generation.
  • NtSWEET polynucleotides from individual plants, plant cells, or plant material can optionally be pooled in order to expedite screening for mutations in the population of plants
  • the mutagenized plant tissue, cells or material originating from the mutagenized plant tissue, cells or material.
  • One or more subsequent generations of plants, plant cells or plant material can be screened.
  • the size of the optionally pooled group is dependent upon the sensitivity of the screening method used.
  • After the samples are optionally pooled they can be subjected to polynucleotide-specific amplification techniques, such as PCR. Any one or more primers or probes specific to the gene or the sequences immediately adjacent to the gene may be utilized to amplify the sequences within the optionally pooled sample.
  • the one or more primers or probes are designed to amplify the regions of the locus where useful mutations are most likely to arise.
  • the primer is designed to detect mutations within regions of the polynucleotide. Additionally, it is preferable for the primer(s) and probe(s) to avoid known polymorphic sites in order to ease screening for point mutations.
  • the one or more primers or probes may be labelled using any conventional labelling method.
  • Primer(s) or probe(s) can be designed based upon the sequences described herein using methods that are well understood in the art.
  • the primer(s) or probe(s) may be labelled using any conventional labelling method. These can be designed based upon the sequences described herein using methods that are well understood in the art.
  • Polymorphisms may be identified by means known in the art and some have been described in the literature.
  • a plant may be regenerated or grown from the plant, plant tissue or plant cell. Any suitable methods for regenerating or growing a plant from a plant cell or plant tissue may be used, such as, without limitation, tissue culture or regeneration from protoplasts.
  • plants may be regenerated by growing transformed plant cells on callus induction media, shoot induction media or root induction media. See, for example, McCormick et al., Plant Cell Reports 5:81-84 (1986). These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting hybrid having expression of the desired phenotypic characteristic identified.
  • transformed seeds refers to seeds that contain the nucleotide construct stably integrated into the plant genome.
  • a method of preparing a mutant plant involves providing at least one cell of a plant comprising one or more NtSWEET genes encoding a functional NtSWEET. Next, the at least one cell of the plant is treated under conditions effective to modulate the function of the NtSWEET polynucleotide(s). The at least
  • the treating step involves subjecting the at least one cell to a chemical mutagenising agent as described above and under conditions effective to yield at least one mutant plant cell.
  • the treating step involves subjecting the at least one cell to a radiation source under conditions effective to yield at least one mutant plant cell.
  • mutant plant includes mutant plants in which the genotype is modified as compared to a control plant, suitably by means other than genetic engineering or genetic modification.
  • the mutant plant, mutant plant cell or mutant plant material may comprise one or more mutations that have occurred naturally in another plant, plant cell or plant material and confer a desired trait.
  • This mutation can be incorporated (for example, introgressed) into another plant, plant cell or plant material (for example, a plant, plant cell or plant material with a different genetic background to the plant from which the mutation was derived) to confer the trait thereto.
  • a mutation that occurred naturally in a first plant may be introduced into a second plant - such as a second plant with a different genetic background to the first plant.
  • the skilled person is therefore able to search for and identify a plant carrying naturally in its genome one or more mutant alleles of the genes described herein which confer a desired trait.
  • the mutant allele(s) that occurs naturally can be transferred to the second plant by various methods including breeding, backcrossing and introgression to produce a lines, varieties or hybrids that have one or more mutations in the genes described herein.
  • the same technique can also be applied to the introgression of one or more non-naturally occurring mutation(s) from a first plant into a second plant.
  • Plants showing a desired trait may be screened out of a pool of mutant plants. Suitably, the selection is carried out utilising the knowledge of the polynucleotide as described herein. Consequently, it is possible to screen for a genetic trait as compared to a control. Such a screening approach may involve the application of conventional amplification or hybridization techniques as discussed herein.
  • a further aspect of the present disclosure relates to a method for identifying a mutant plant comprising: (a) providing a sample comprising one or more NtSWEET polynucleotide(s) from a plant; and (b) determining the sequence of the polynucleotide(s), wherein a difference in the sequence of the polynucleotide(s) as compared to the polynucleotide(s) of a control plant is indicative that said plant is a mutant plant.
  • a method for identifying a mutant plant which accumulates increased or decreased levels of amino acids as compared to a control plant comprising: (a) providing a sample from a plant to be screened; (b) determining if said sample comprises one
  • a method for preparing a mutant plant which has increased or decreased levels of at least one amino acid - as compared to a control plant comprising: (a) providing a sample from a first plant; (b) determining if said sample comprises one or more mutations in one or more NtSWEET polynucleotides described herein that result in modulated levels of the at least one amino acid; and (c) transferring the one or more mutations into a second plant.
  • the level of the at least one amino acid is determined in cured or dried leaves.
  • the mutation(s) can be transferred into the second plant using various methods that are known in the art - such as by genetic engineering, genetic manipulation, introgression, plant breeding, backcrossing and the like.
  • the first plant is a naturally occurring plant.
  • the second plant has a different genetic background to the first plant.
  • a method for preparing a mutant plant which has increased or decreased levels of at least one amino acid as compared to a control plant comprising: (a) providing a sample from a first plant; (b) determining if said sample comprises one or more mutations in one or more of the NtSWEET polynucleotides described herein that results in modulated levels of the at least one amino acid; and (c) introgressing the one or more mutations from the first plant into a second plant.
  • the level of the at least one amino acid is determined in cured or dried leaves.
  • the step of introgressing comprises plant breeding, optionally including backcrossing and the like.
  • the first plant is a naturally occurring plant.
  • the second plant has a different genetic background to the first plant.
  • the first plant is not a cultivar or an elite cultivar.
  • the second plant is a cultivar or an elite cultivar.
  • a further aspect relates to a mutant plant (including a cultivar or elite cultivar mutant plant) obtained or obtainable by the methods described herein.
  • the mutant plant may have one or more mutations localised only to a specific region of the plant - such as within the sequence of the one or more NtSWEET polynucleotide(s) described herein. According to this embodiment, the remaining genomic sequence of the mutant plant will be the same or substantially the same as the plant prior to the mutagenesis.
  • the mutant plants may have one or more mutations localised in more than one genomic region of the plant - such as within the sequence of one or more of the NtSWEET polynucleotides described herein and in one or more further regions of the genome. According to this embodiment, the remaining genomic sequence of the mutant plant will not be the same or will not be substantially the same as the plant prior to the mutagenesis.
  • the mutant plants may not have one or more mutations in one or more, two or more, three or more, four or more or five or more exons of the NtSWEET polynucleotide(s) described herein; or may not have one or more mutations in one or more, two or more, three or more, four or more or five or more introns of the NtSWEET polynucleotide(s) described herein; or may not have one or more mutations in a promoter of the NtSWEET polynucleotide(s) described herein; or may not have one or more mutations in the 3’ untranslated region of the NtSWEET polynucleotide(s) described herein; or may not have one or more mutations in the 5’ untranslated region of the NtSWEET polynucleotide(s) described herein; or may not have one or more mutations in the coding region of the NtSWEET polynucleotide(s) described herein; or may be
  • a method of identifying a plant, a plant cell or plant material comprising a mutation in a gene encoding a NtSWEET polynucleotide described herein comprising: (a) subjecting a plant, a plant cell or plant material to mutagenesis; (b) obtaining a sample from said plant, plant cell or plant material or descendants thereof; and (c) determining the polynucleotide sequence of the NtSWEET gene(s) or a variant or a fragment thereof, wherein a difference in said sequence is indicative of one or more mutations therein.
  • This method also allows the selection of plants having mutation(s) that occur(s) in genomic regions that affect the expression of the NtSWEET gene in a plant cell, such as a transcription initiation site, a start codon, a region of an intron, a boundary of an exon-intron, a terminator, Plants suitable for use in the present disclosure include monocotyledonous and dicotyledonous plants and plant cell systems and include members of the genera Nicotiana.
  • Various embodiments are directed to mutant tobacco, non-naturally occurring tobacco or transgenic tobacco plants or tobacco plant cells and can be applied to any species of the genus Nicotiana, including N. rustica and N. tabacum (for example, LA B21 , LN KY171 , Tl 1406, Basma, Galpao, Perique, Beinhart 1000-1 , and Petico).
  • Other species include N. acaulis, N. acuminata, N. africana, N. alata, N. ameghinoi, N. amplexicaulis, N. arentsii, N. attenuate, N. azambujae, N. benavidesii, N. benthamiana, N. bigelovii, N.
  • the plant is N. tabacum.
  • the transgenic, non-naturally occurring or mutant plant may therefore be a tobacco variety or elite tobacco cultivar that comprises one or more transgenes, or one or more genetic mutations or a combination thereof.
  • the genetic mutation(s) (for example, one or more polymorphisms) can be mutations that do not exist naturally in the individual tobacco variety or tobacco cultivar (for example, elite tobacco cultivar) or can be genetic mutation(s) that do occur naturally provided that the mutation does not occur naturally in the individual tobacco variety or tobacco cultivar (for example, elite tobacco cultivar).
  • Nicotiana tabacum varieties include Burley type, dark type, flue-cured type, and Oriental type tobaccos.
  • varieties or cultivars are: BD 64, CC 101 , CC 200, CC 27, CC 301 , CC 400, CC 500, CC 600, CC 700, CC 800, CC 900, Coker 176, Coker 319, Coker 371 Gold, Coker 48, CD 263, DF911 , DT 538 LC Galpao tobacco, GL 26H, GL 350, GL 600, GL 737, GL 939, GL 973, HB 04P, HB 04P LC, HB3307PLC, Hybrid 403LC, Hybrid 404LC, Hybrid 501 LC, K 149, K 326, K 346, K 358, K394, K 399, K 730, KDH 959, KT 200, KT204LC, KY10, KY14, KY 160, KY 17, KY 171
  • Embodiments are also directed to compositions and methods for producing mutant plants, non-naturally occurring plants, hybrid plants, or transgenic plants that have been modified to modulate the expression or function of one or more NtSWEET polynucleotide(s) described herein (or any combination thereof as described herein).
  • the mutant plants, non-naturally occurring plants, hybrid plants, or transgenic plants that are obtained may be similar or substantially the same in overall appearance to control plants.
  • Various phenotypic characteristics such as degree of maturity, number of leaves per plant, stalk height, leaf insertion angle, leaf size (width and length), internode distance, and lamina-midrib ratio can be assessed by field observations.
  • One aspect relates to a seed of a mutant plant, a non-naturally occurring plant, a hybrid plant or a transgenic plant described herein.
  • the seed is a tobacco seed.
  • a further aspect relates to pollen or an ovule of a mutant plant, a non-naturally occurring plant, a hybrid plant or a transgenic plant that is described herein.
  • a mutant plant, a non-naturally occurring plant, a hybrid plant or a transgenic plant as described herein which further comprises a polynucleotide conferring male sterility.
  • the regenerable cells include cells from leaves, pollen, embryos, cotyledons, hypocotyls, roots, root tips, anthers, flowers and a part thereof, ovules, shoots, stems, stalks, pith and capsules or callus or protoplasts derived therefrom.
  • the plant material that is described herein can be cured or dried tobacco material.
  • the CORESTA recommendation for tobacco curing is described in: CORESTA Guide N°17, April 2016, Sustainability in Leaf Tobacco Production.
  • the expression of one or more of SWEET12-S or SWEET12-T or SWEET15-S or SWEET15-T or the activity of one or more of SWEET12-S or SWEET12-T or SWEET15-S or SWEET15-T is modulated as compared to a control plant.
  • the expression of SWEET12-S or the activity of SWEET12-S is modulated as compared to a control plant.
  • the expression of SWEET12-T or the activity of SWEET12-T is modulated as compared to a control plant.
  • the expression of SWEET12-S and SWEET12-T or the activity of SWEET12-S and SWEET12- T is modulated as compared to a control plant.
  • the expression of SWEET15-S or the activity of SWEET15-S is modulated as compared to a control plant.
  • the expression of SWEET15-T or the activity of SWEET15-T is modulated as compared to a control plant.
  • the expression of SWEET15-S and SWEET15-T or the activity of SWEET15-S and SWEET15-T is modulated as compared to a control plant.
  • the expression of SWEET12-S and SWEET15-S or the activity of SWEET12-S and SWEET15-S is modulated as compared to a control plant.
  • SWEET12-S and SWEET15-T are modulated as compared to a control plant.
  • SWEET12-T and SWEET15-S are modulated as compared to a control plant.
  • SWEET12-T and SWEET15-T are modulated as compared to a control plant.
  • 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 a control plant.
  • 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 a control plant.
  • SWEET12-S and SWEET15-S and SWEET15- T or the activity of SWEET12-S and SWEET15-S and SWEET15-T is modulated as compared to a control plant.
  • 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 a control plant.
  • 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 a control plant.
  • 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 development during the vegetative phase of the plant as compared to the control plant and/or it can modulate the flowering time of the plant as compared to the control plant.
  • modulating (suitably, decreasing) the expression of SWEET12-S and/or SWEET12-T or the activity of SWEET12-S and/or SWEET12-T can modulate (suitably, increase) plant height during the vegetative phase of
  • modulating (suitably, decreasing) the expression of SWEET15-S and/or SWEET15-T or the activity of SWEET15-S and/or SWEET15-T can modulate (suitably, increase) plant height during the vegetative phase of growth and can lead to accelerated flowering time.
  • modulating (suitably, increasing) the expression of SWEET15-T and/or SWEET15- S or the activity of SWEET15-T and/or SWEET15-S can modulate (for example, 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 profile of cured or dried leaf derived from the plant.
  • the chemical profile can include the sugar profile and/or the amino acid profile.
  • modulating (suitably, increasing) the expression of SWEET12-S and/or SWEET12-T or the activity of SWEET12-S and/or SWEET12-T can modulate (suitably, decrease) the fructose, glucose and sucrose content of cured or dried leaf.
  • modulating (suitably, increasing) the expression of SWEET 15-T or the activity of SWEET15-T can modulate (suitably, decrease) the fructose, glucose and sucrose content of cured or dried leaf.
  • modulating (suitably, increasing) the expression of SWEET15-S and SWEET15-T or the activity of SWEET15-S and SWEET 15-T can modulate (suitably, decrease) the fructose, glucose and sucrose content of cured or dried leaf.
  • 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 amino acid profile of cured or dried leaf derived from the plant.
  • the amino acid profile can include asparagine and tryptophan and proline or asparagine, tryptophan, proline, phenylalanine, glycine, methionine and glutamic acid.
  • the amino acid profile is modulated, the levels of one or more amino acids can be increased or decreased.
  • the levels of asparagine and tryptophan are increased and the level of proline is 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.
  • modulating can modulate the levels of asparagine and tryptophan and proline and phenylalanine and glycine and methionine and glutamic acid, suitably, increase the levels of asparagine, tryptophan, phenylalanine, glycine and methionine and decrease the levels of glutamic acid and proline.
  • modulating can modulate the levels of asparagine, tryptophan, phenylalanine, glycine and methionine and decrease the levels of glutamic acid and proline.
  • SWEET15-T and/or SWEET15-S or the activity of SWEET15-T and/or SWEET15-S can modulate the levels of asparagine and tryptophan and proline, suitably, increase the levels of asparagine and tryptophan and decrease the level of proline.
  • SWEET 12-S and/or SWEET 12-T or the activity of SWEET 12- S and/or can SWEET12-T is decreased, no significant changes in the levels of sugars or amino acids are observed, however the sensory profile of the cured or dried tobacco is 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 a control plant can modulate the amount of leaf biomass.
  • modulating (suitably, increasing) the expression of SWEET12-S and/or SWEET12-T or the activity of SWEET12-S and/or SWEET12-T can modulate (suitably, decrease) the amount of leaf biomass.
  • modulating (suitably, increasing) the expression of SWEET15-S and/or SWEET15-T or the activity of SWEET15-S and/or SWEET15-T can modulate (suitably, decrease) the amount of leaf biomass.
  • there is no significant change in the amount of leaf biomass - such as when the expression of SWEET12-S and/or SWEET12-T or the activity of SWEET12-S and/or SWEET12-T is decreased or when the expression of SWEET15-S and/or SWEET15-T or the activity of SWEET15-S and/or SWEET15-T is decreased.
  • it is preferred that the amount of leaf biomass is not decreased as this will maintain the yields that can be obtained during the commercial production of tobacco.
  • Modulating the expression of SWEET12-S and/or SWEET12-T or the activity of SWEET12-S or SWEET12-T as compared to a control plant can modulate the levels of ammonia.
  • modulating (suitably, increasing) the expression of SWEET12-S and/or SWEET12-T or the activity of SWEET12-S and/or SWEET12-T can modulate (suitably, increase) the levels of ammonia.
  • Ammonium compounds are known to react with sugars during tobacco processing and smoking to form flavour components that alter the taste of tobacco smoke. Accordingly, modulating the level of ammonia is expected to alter the flavour of tobacco.
  • the modulated levels are observed in at least cured or dried leaves, suitably fully cured or dried leaves.
  • Tobacco is considered to be fully cured when the leaf's central rib is free of moisture, resulting in leaves that are light tan to reddish-brown to deep brown in colour.
  • the cured leaves are taken from mid-position leaves of a plant.
  • a further aspect relates to a mutant, non-naturally occurring or transgenic plant or cell in which the expression of one or more NtSWEET polynucleotides or the activity of one or more
  • NtSWEET polypeptide(s) has been modulated as compared to a control plant or part thereof in which the expression of NtSWEET or the activity of NtSWEET has not been modulated.
  • a still further aspect relates to cured or dried plant material - such as cured or dried leaf or cured or dried tobacco - derived or derivable from the mutant, non-naturally occurring or transgenic plant or cell, wherein expression of one or more of the one or more NtSWEET polynucleotides described herein or the function of the NtSWEET polypeptide(s) encoded thereby is modulated as compared to a control plant or part thereof.
  • Embodiments are also directed to compositions and methods for producing mutant, non- naturally occurring or transgenic plants or plant cells that have been modified to modulate the expression or activity of one or more of the NtSWEET polynucleotides or NtSWEET polypeptides described herein.
  • the phenotype of the mutant, non-naturally occurring or transgenic plant is substantially the same as the control plant or part thereof.
  • the leaf weight of the mutant, non-naturally occurring or transgenic plant is substantially the same as the control plant or part thereof.
  • the leaf number of the mutant, non- naturally occurring or transgenic plant is substantially the same as the control plant or part thereof.
  • the leaf weight and the leaf number of the mutant, non-naturally occurring or transgenic plant is substantially the same as the control plant.
  • the stalk height of the mutant, non-naturally occurring or transgenic plants is substantially the same as the control plants or parts thereof at, for example, one, two or three or more months after field transplant or 10, 20, 30 or 36 or more days after topping.
  • the stalk height of the mutant, non-naturally occurring or transgenic plants is not less than the stalk height of the control plants or parts thereof.
  • a method for modulating the amount of at least one amino acid and/or at least one sugar in at least a part of a plant comprising: (i) modulating the expression or function of an one or more of the NtSWEET polypeptides described herein, suitably, wherein the NtSWEET polypeptide(s) is encoded by the corresponding NtSWEET polynucleotides described herein; (ii) measuring the level of the at least one amino acid and/or at least one sugar in at least a part (for example, the leaves - such as cured or dried leaves - or tobacco or in smoke) of the mutant, non-naturally occurring or transgenic plant obtained in step (i); and (iii) identifying a mutant, non-naturally occurring or transgenic plant or part thereof in which the level of the at least one amino acid and/or sugar has been modulated in comparison to a control plant or part thereof.
  • a method for modulating the amount of at least one amino acid and/or at least one sugar in cured or dried plant material - such as cured or dried leaf - comprising: (i) modulating the expression or function of an one or more of the NtSWEET polypeptides (or any combination thereof as described herein), suitably, wherein the NtSWEET polypeptide(s) is encoded by the corresponding NtSWEET polynucleotides described herein; (ii) harvesting plant material - such as one or more of the leaves - and curing for a period of time; (iii) measuring the level of the at least one amino acid and/or at least one sugar in cured or dried plant material obtained in step (ii) or during step (ii); and (iv) identifying cured or dried plant material in which the level of the at least one amino acid and/or at least one sugar has been modulated in comparison to a control plant or part thereof.
  • An increase in expression as compared to the control 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, which includes an increase in transcriptional function or NtSWEET polynucleotide expression or NtSWEET polypeptide expression.
  • An increase in function or activity as compared to a control 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, which includes an increase in transcriptional function or NtSWEET polynucleotide expression or NtSWEET polypeptide expression or a combination thereof.
  • a decrease in expression as compared to a control may be from about 5 % to about 100 %, or a decrease 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 %, which includes a decrease in transcriptional function or NtSWEET polynucleotide expression or NtSWEET polypeptide expression or a combination thereof.
  • a decrease in function or activity as compared to a control may be from about 5 % to about 100 %, or a decrease 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 %, which includes a decrease in transcriptional function or NtSWEET polynucleotide expression or NtSWEET polypeptide expression or a combination thereof.
  • Polynucleotides and recombinant constructs described herein can be used to modulate the expression or function or activity of the NtSWEET polynucleotides or NtSWEET polypeptides described herein in a plant species of interest, suitably tobacco.
  • a number of polynucleotide based methods can be used to increase gene expression in plants and plant cells.
  • a construct, vector or expression vector that is compatible with the plant to be transformed can be prepared which comprises the gene of interest together with an upstream promoter that is capable of overexpressing the gene in the plant or plant cell.
  • Exemplary promoters are described herein. Following transformation and when grown under suitable conditions, the promoter can drive expression in order to modulate the levels of NtSWEET in the plant, or in a specific tissue thereof.
  • 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 its constitutive expression in all tissues of the plant.
  • the vector also carries an antibiotic resistance gene in order to confer selection of the transformed calli and cell lines.
  • sequences from promoters can be enhanced by including expression control sequences, which are well known in the art. Signals associated with senescence and signals which are active during the curing procedure are specifically indicated.
  • Various embodiments are therefore directed to methods for modulating the expression level of one or more NtSWEET polynucleotides described herein (or any combination thereof as described herein) by integrating multiple copies of the NtSWEET polynucleotide(s) into a plant genome, comprising: transforming a plant cell host with an expression vector that comprises a promoter operably-linked to one or more NtSWEET polynucleotides described herein.
  • the polypeptide encoded by a recombinant polynucleotide can be a native polypeptide, or can be heterologous to the cell.
  • the plant for use in the present disclosure is a mutant, non-naturally occurring or transgenic plant that is flue-cured.
  • the plant for use in the present disclosure is a mutant, non-naturally occurring or transgenic plant that is sun-cured.
  • the plant for use in the present disclosure is a mutant, non-naturally occurring or transgenic plant that is air-cured.
  • the plant for use in the present disclosure is a mutant, non-naturally occurring or transgenic Virginia tobacco plant that is cured, for example, flue-cured.
  • the plant for use in the present disclosure is a mutant, non-naturally occurring or transgenic Burley tobacco plant that is cured, for example, air-cured.
  • the plant for use in the present disclosure is a mutant, non-naturally occurring or transgenic Dark tobacco plant that is cured, for example, fire-cured.
  • the sensory profile of tobacco can be advantageously modified as can be appreciated from Table 6.
  • a plant carrying a mutant allele of one or more NtSWEET polynucleotides described herein can be used in a plant breeding program to create useful lines, varieties and hybrids.
  • a mutant allele can be introgressed into commercially important varieties described herein.
  • methods for breeding plants that comprise crossing a mutant plant, a non-naturally occurring plant or a transgenic plant as described herein with a plant comprising a different genetic identity.
  • the method may further comprise crossing the progeny plant with another plant, and optionally repeating the crossing until a progeny with the desirable genetic traits or genetic background is obtained.
  • One purpose served by such breeding methods is to introduce a desirable genetic trait into other varieties, breeding lines, hybrids or cultivars, particularly those that are of commercial interest.
  • Another purpose is to facilitate stacking of genetic modifications of different genes in a single plant variety, lines, hybrids or cultivars. Intraspecific as well as interspecific matings are contemplated. The progeny plants that arise from such crosses, also referred to as breeding lines, are examples of non-naturally occurring plants of the disclosure.
  • a method for producing a non-naturally occurring plant comprising: (a) crossing a mutant or transgenic plant with a second plant to yield progeny tobacco seed; (b) growing the progeny tobacco seed, under plant growth conditions, to yield the non-naturally occurring plant.
  • the method may further comprises: (c) crossing the previous generation of non-naturally occurring plant with itself or another plant to yield progeny tobacco seed; (d) growing the progeny tobacco seed of step (c) under plant growth conditions, to yield additional non-naturally occurring plants; and (e) repeating the crossing and growing steps of (c) and (d) multiple times to generate further generations of non-naturally occurring plants.
  • the method may optionally comprises prior to step (a), a step of providing a parent plant which comprises a genetic identity that is characterized and that is not identical to the mutant or transgenic plant.
  • the crossing and growing steps are repeated from 0 to 2 times, from 0 to 3 times, from 0 to 4 times, 0 to 5 times, from 0 to 6 times, from 0 to 7 times, from 0 to 8 times, from 0 to 9 times or from 0 to 10 times, in order to generate generations of non-naturally occurring plants.
  • Backcrossing is an example of such a method wherein a progeny is crossed with one of its parents or another
  • lines resulting from breeding and screening for variant genes are evaluated in the field using standard field procedures.
  • Control genotypes including the original unmutagenized parent are included and entries are arranged in the field in a randomized complete block design or other appropriate field design.
  • standard agronomic practices are used, for example, the tobacco is harvested, weighed, and sampled for chemical and other common testing before and during curing.
  • Statistical analyses of the data are performed to confirm the similarity of the selected lines to the parental line. Cytogenetic analyses of the selected plants are optionally performed to confirm the chromosome complement and chromosome pairing relationships.
  • DNA fingerprinting, single nucleotide polymorphism, microsatellite markers, or similar technologies may be used in a marker-assisted selection (MAS) breeding program to transfer or breed mutant alleles of a gene into other tobaccos, as described herein.
  • MAS marker-assisted selection
  • a breeder can create segregating populations from hybridizations of a genotype containing a mutant allele with an agronomically desirable genotype. Plants in the F2 or backcross generations can be screened using a marker developed from a genomic sequence or a fragment thereof, using one of the techniques listed herein. Plants identified as possessing the mutant allele can be backcrossed or self-pollinated to create a second population to be screened.
  • successful crosses yield F1 plants that are fertile.
  • Selected F1 plants can be crossed with one of the parents, and the first backcross generation plants are self-pollinated to produce a population that is again screened for variant gene expression (for example, the null version of the gene).
  • the process of backcrossing, self-pollination, and screening is repeated, for example, at least 4 times until the final screening produces a plant that is fertile and reasonably similar to the recurrent parent.
  • This plant if desired, is self-pollinated and the progeny are subsequently screened again to confirm that the plant exhibits variant gene expression.
  • a plant population in the F2 generation is screened for variant gene expression, for example, a plant is identified
  • Hybrid tobacco varieties can be produced by preventing self-pollination of female parent plants (that is, seed parents) of a first variety, permitting pollen from male parent plants of a second variety to fertilize the female parent plants, and allowing F1 hybrid seeds to form on the female plants.
  • Self-pollination of female plants can be prevented by emasculating the flowers at an early stage of flower development.
  • pollen formation can be prevented on the female parent plants using a form of male sterility.
  • male sterility can be produced by cytoplasmic male sterility (CMS), or transgenic male sterility wherein a transgene inhibits microsporogenesis or pollen formation, or self-incompatibility.
  • CMS cytoplasmic male sterility
  • transgenic male sterility wherein a transgene inhibits microsporogenesis or pollen formation, or self-incompatibility.
  • Female parent plants containing CMS are particularly useful. In embodiments in which the female parent plants are CMS, pollen is harvested from male fertile plants
  • Varieties and lines described herein can be used to form single-cross tobacco F1 hybrids.
  • the plants of the parent varieties can be grown as substantially homogeneous adjoining populations to facilitate natural cross-pollination from the male parent plants to the female parent plants.
  • the F1 seed formed on the female parent plants is selectively harvested by conventional means.
  • One also can grow the two parent plant varieties in bulk and harvest a blend of F1 hybrid seed formed on the female parent and seed formed upon the male parent as the result of self-pollination.
  • three-way crosses can be carried out wherein a single-cross F1 hybrid is used as a female parent and is crossed with a different male parent.
  • double-cross hybrids can be created wherein the F1 progeny of two different single-crosses are themselves crossed.
  • a population of mutant, non-naturally occurring or transgenic plants can be screened or selected for those members of the population that have a desired trait or phenotype. For example, a population of progeny of a single transformation event can be screened for those plants having a desired level of expression or function of the polypeptide(s) encoded thereby. Physical and biochemical methods can be used to identify expression or activity levels.
  • Mutant, non-naturally occurring or transgenic plant cells and plants are described herein comprising one or more recombinant polynucleotides, one or more polynucleotide constructs, one or more double-stranded RNAs, one or more conjugates or one or more vectors/expression vectors.
  • plants and parts thereof described herein can be modified either before or after the expression, function or activity of the one or more NtSWEET polynucleotides or NtSWEET polypeptides according to the present disclosure have been modulated.
  • One or more of the following further genetic modifications can be present in the mutant, non- naturally occurring or transgenic plants and parts thereof.
  • One or more genes that are involved in the conversion of nitrogenous metabolic intermediates can be modified resulting in lower levels of at least one tobacco-specific nitrosamine (TSNA).
  • TSNA tobacco-specific nitrosamine
  • Non-limiting examples of such genes include those encoding nicotine demethylase - such as CYP82E4, CYP82E5 and CYP82E10 as described in W02006/091194, W02008/070274, W02009/064771 and WO2011/088180 - and nitrate reductase, as described in WO2016/046288.
  • One or more genes that are involved in heavy metal uptake or heavy metal transport can be modified resulting in lower heavy metal content.
  • Non-limiting examples include genes in the family of multidrug resistance associated polypeptides, the family of cation diffusion facilitators (CDF), the family of Zrt- 1 rt-like polypeptides (ZIP), the family of cation exchangers (CAX), the family of copper transporters (COPT), the family of heavy-metal ATPases (for example, HMAs, as described in W02009/074325 and WO2017/129739), the family of homologs of natural resistance-associated macrophage polypeptides (NRAMP), and other members of the family of ATP-binding cassette (ABC) transporters (for example, MRPs), as described in WO20 12/028309, which participate in transport of heavy metals - such as cadmium.
  • CDF family of cation diffusion facilitators
  • ZIP Zrt- 1 rt-like polypeptides
  • CAX family of cation exchangers
  • COX copper transporters
  • COPD copper transporters
  • HMAs family
  • exemplary modifications can result in plants with modulated expression or function of isopropylmalate synthase which results in a change in sucrose ester composition which can be used to alter favour profile (see WO2013/029799).
  • Other exemplary modifications can result in plants with modulated expression or function of threonine synthase in which levels of methional can be modulated (see WO2013/029800).
  • Other exemplary modifications can result in plants with modulated expression or function of one or more of neoxanthin synthase, lycopene beta cyclase and 9-cis-epoxycarotenoid dioxygenase to modulate beta- damascenone content to alter flavour profile (see WO2013/064499).
  • Other exemplary modifications can result in plants with modulated expression or function of members of the
  • exemplary modifications can result in plants having modified alkaloid levels by altering the gene expression of putative ABC-2 transporters NtABCGI-T and NtABCGI-S or the activity of the protein encoded thereby (see WO20 19/086609).
  • Other exemplary modifications can result in plants having modulated time to flowering by altering the gene expression of genes encoding Terminal Flower 1 (TFL1) or the activity of the protein encoded thereby (see WO2018/114641).
  • Other exemplary modifications can result in plants with modulated expression or function of one or more asparagine synthetases to modulate levels of asparagine in leaf and modulated levels of acrylamide in aerosol produced upon heating or combusting the leaf (see WO2017/042162).
  • Glyphosate resistant transgenic plants have been developed by transferring the aroA gene (a glyphosate EPSP synthetase from Salmonella typhimurium and E.coli). Sulphonylurea resistant plants have been produced by transforming the mutant ALS (acetolactate synthetase) gene from Arabidopsis. OB polypeptide of photosystem II from mutant Amaranthus hybridus has been transferred in to plants to produce atrazine resistant transgenic plants; and bromoxynil resistant transgenic plants have been produced by incorporating the bxn gene from the bacterium Klebsiella pneumoniae.
  • aroA gene a glyphosate EPSP synthetase from Salmonella typhimurium and E.coli
  • Sulphonylurea resistant plants have been produced by transforming the mutant ALS (acetolactate synthetase) gene from Arabidopsis.
  • OB polypeptide of photosystem II from mutant Amaranthus hybridus has been transferred in to plants
  • Bacillus thuringiensis (Bt) toxins can provide an effective way of delaying the emergence of Bt-resistant pests, as recently illustrated in broccoli where pyramided crylAc and cry1C Bt genes controlled diamondback moths resistant to either single polypeptide and significantly delayed the evolution of resistant insects.
  • Another exemplary modification results in plants that are resistant to diseases caused by pathogens (for example, viruses, bacteria, fungi). Plants expressing the Xa21 gene (resistance to bacterial blight) with plants expressing both a Bt fusion gene and a chitinase gene (resistance to yellow stem borer and tolerance to sheath) have been engineered.
  • Another exemplary modification results in altered reproductive capability, such as male sterility.
  • Another exemplary modification results in plants that are resistant to insects.
  • Bacillus thuringiensis (Bt) toxins can provide an effective way of delaying the emergence of Bt-resistant pests, as recently illustrated in broccoli where pyramided crylA
  • abiotic stress for example, drought, temperature, salinity
  • tolerant transgenic plants have been produced by transferring acyl glycerol phosphate enzyme from Arabidopsis; genes coding mannitol dehydrogenase and sorbitol dehydrogenase which are involved in synthesis of mannitol and sorbitol 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 levels of nornicotine and metabolites of nornicotine - that are formed during curing can be modulated (see WO2015169927).
  • T ransgenic 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.
  • SAM S-adenosyl- L-methionine
  • CGS cystathionine gamma-synthase
  • One or more genes that are involved in the nicotine synthesis pathway can be modified resulting in plants or parts of plants that when cured or dried, produce modulated levels of nicotine.
  • the nicotine synthesis genes can be selected from the group consisting of: A622, BBLa, BBLb, JRE5L1, JRE5L2, MATE1, MATE 2, MP01, MP02, MYC2a, MYC2b, NBB1, nic1, nic2, NUP1, NUP2, PMT1, PMT2, PMT3, PMT4 and QPT or a combination of one or more thereof.
  • One or more genes that are involved in controlling the amount of one or more alkaloids can be modified resulting in plants or parts of plants that produce modulated levels of alkaloid.
  • Alkaloid level controlling genes can be selected from the group consisting of; BBLa, BBLb, JRE5L1, JRE5L2, MATE1, MATE 2, MYC2a, MYC2b, nic1, nic2, NLIP1 and NUP2 or a combination of two or more thereof.
  • the further genetic modification concerns asparagine synthetase (ASN) genes as described in WO2017042162.
  • ASN asparagine synthetase
  • Modulating the expression of ASN genes for example, one or more of NtASAN1-S, NIASN1-T, NIASN5-S and MASN5-T as described in WO2017042162
  • the activity of ASN for example, NtASN1-S, NtASN1-T, NtASN5-S and NtASNS-T as described in WO2017042162
  • modulating the expression and/or activity of the combination of ASN and NtSWEET may have the potential to rearrange the chemistry of cured or dried tobacco leaf (particularly the amino acid chemistry of Burley or Dark tobacco) and thereby alter the sensory properties.
  • DAPAT diaminopimelate aminotransferase
  • AAT aspartate amino transferases
  • SULTR3 - such as NtSULTR3;1A-S, NtSULTR3;1A-T and NtSULTR3;3-T - play a role in sugar and amino acid metabolism during curing (see WO2021/063863).
  • the further genetic modification can concern DAPAT and/or AAT (for example, one or more of NtAATI-S, NtAAT1-T, NtAA T2-S, NtAAT2-T, NtAA T3-S, NtAAT3-T, NtAA T4-S or NtAAT4-T as described in WO2017042162) and/or one or more of NtSULTR3;1A-S, NtSULTR3;1A-T and NtSULTR3;3-T as described in see WO2021/063863.
  • AAT for example, one or more of NtAATI-S, NtAAT1-T, NtAA T2-S, NtAAT2-T, NtAA T3-S, NtAAT3-T, NtAA T4-S or NtAAT4-T as described in WO2017042162
  • Modulating the expression and/or activity of the combination of DAPAT and/or AAT and/or SULTR3 and NtSWEET may have the potential to rearrange the chemistry of cured or dried tobacco leaf and thereby alter the sensory properties.
  • Modifications to combinations of NtSWEET and one or more, or two or more, or three of more or four or more of ASN and DAPAT and AAT and SULTR3 are disclosed, including NtSWEET and ASN; NtSWEET and DAPAT; NtSWEET and AAT; NtSWEET and ASN and DAPAT; NtSWEET and ASN and AAT; NtSWEET and ASN and DAPAT and AAT; NtSWEET and SULTR3; NtSWEET and ASN and SULTR3; NtSWEET and DAPAT and SULTR3; NtSWEET and AAT and SULTR3; NtSWEET and ASN and DAPAT and SULTR3; NtSWEET and AAT and SULTR3; NtSWEET
  • One or more traits may be introgressed into the mutant, non-naturally occurring or transgenic plants from another cultivar or may be directly transformed into it.
  • Various embodiments provide mutant plants, non-naturally occurring plants or transgenic plants, as well as biomass in which the expression level of one or more polynucleotides according to the present disclosure are modulated to thereby modulate the level of polypeptide(s) encoded thereby.
  • parts of the plants described herein, particularly the leaf lamina and/or stalks and/or midrib of such plants, can be incorporated into or used in making various consumable products including but not limited to aerosol forming materials, aerosol forming devices, smoking articles, smokable articles, smokeless products, medicinal or cosmetic products, intravenous preparations, tablets, powders, and tobacco products.
  • aerosol forming materials include tobacco compositions, tobaccos, tobacco extract, cut tobacco, cut filler, cured or dried tobacco, expanded tobacco, homogenized tobacco, reconstituted tobacco, and pipe tobaccos.
  • Smoking articles and smokable articles are types of aerosol forming devices. Examples of
  • smoking articles or smokable articles include cigarettes, cigarillos, and cigars.
  • smokeless products comprise chewing tobaccos, and snuffs.
  • a tobacco composition or another aerosol forming material is heated by one or more electrical heating elements to produce an aerosol.
  • an aerosol is produced by the transfer of heat from a combustible fuel element or heat source to a physically separate aerosol forming material, which may be located within, around or downstream of the heat source.
  • Smokeless tobacco products and various tobacco-containing aerosol forming materials may contain tobacco in any form, including as dried particles, shreds, granules, powders, or a slurry, deposited on, mixed in, surrounded by, or otherwise combined with other ingredients in any format, such as flakes, films, tabs, foams, or beads.
  • the term ‘smoke’ is used to describe a type of aerosol that is produced by smoking articles, such as cigarettes, or by combusting an aerosol forming material.
  • cured or dried plant material from the mutant, transgenic and non-naturally occurring plants described herein.
  • Processes of curing green tobacco leaves are known by those having skills in the art and include without limitation aircuring, fire-curing, flue-curing and sun-curing as described herein.
  • tobacco products including tobacco-containing aerosol forming materials comprising plant material - such as leaves, suitably cured or dried leaves - from the mutant tobacco plants, transgenic tobacco plants or non-naturally occurring tobacco plants described herein.
  • plant material - such as leaves, suitably cured or dried leaves - from the mutant tobacco plants, transgenic tobacco plants or non-naturally occurring tobacco plants described herein.
  • the tobacco products described herein can be a blended tobacco product which may further comprise unmodified tobacco.
  • the mutant, non-naturally occurring or transgenic plants may have other uses in, for example, agriculture.
  • the disclosure also provides methods for producing seeds comprising cultivating the mutant plant, non-naturally occurring plant, or transgenic plant described herein, and collecting seeds from the cultivated plants.
  • Seeds from plants described herein can be conditioned and bagged in packaging material by means known in the art to form an article of manufacture.
  • Packaging material such as paper and cloth are well known in the art.
  • a package of seed can have a label, for example, a tag or label secured to the packaging material, a label printed on the package that describes the nature of the seeds therein.
  • compositions, methods and kits for genotyping plants for identification, selection, or breeding can comprise a means of detecting the presence of a NtSWEET polynucleotide(s) in a sample of polynucleotide. Accordingly, a composition is described comprising one or more primers for specifically amplifying at least a portion of one or more of the NtSWEET polynucleotides and
  • probe 54 optionally one or more probes and optionally one or more reagents for conducting the amplification or detection.
  • gene specific oligonucleotide primers or probes comprising about 10 or more contiguous polynucleotides corresponding to the NtSWEET polynucleotide(s) described herein are disclosed.
  • Said primers or probes may comprise or consist of about 15, 20, 25, 30, 40, 45 or 50 more contiguous polynucleotides that hybridise (for example, specifically hybridise) to the NtSWEET polynucleotide(s) described herein.
  • the primers or probes may comprise or consist of about 10 to 50 contiguous nucleotides, about 10 to 40 contiguous nucleotides, about 10 to 30 contiguous nucleotides or about 15 to 30 contiguous nucleotides that may be used in sequence-dependent methods of gene identification (for example, Southern hybridization) or isolation (for example, in situ hybridization of bacterial colonies or bacteriophage plaques) or gene detection (for example, as one or more amplification primers in amplification or detection).
  • the one or more specific primers or probes can be designed and used to amplify or detect a part or all of the polynucleotide(s).
  • two primers may be used in a PCR protocol to amplify a polynucleotide fragment.
  • the PCR may also be performed using one primer that is derived from a polynucleotide sequence and a second primer that hybridises to the sequence upstream or downstream of the polynucleotide sequence - such as a promoter sequence, the 3' end of the mRNA precursor or a sequence derived from a vector.
  • Examples of thermal and isothermal techniques useful for in vitro amplification of polynucleotides are well known in the art.
  • the sample may be or may be derived from a plant, a plant cell or plant material or a tobacco product made or derived from the plant, the plant cell or the plant material as described herein.
  • a method of detecting a NtSWEET polynucleotide(s) described herein (or any combination thereof as described herein) in a sample comprising the step of: (a) providing a sample comprising, or suspected of comprising, a polynucleotide; (b) contacting said sample with one or more primers or one or more probes for specifically detecting at least a portion of the NtSWEET polynucleotide(s); and (c) detecting the presence of an amplification product, wherein the presence of an amplification product is indicative of the presence of the NtSWEET polynucleotide(s) in the sample.
  • kits for detecting at least a portion of the NtSWEET polynucleotide(s) are also provided which comprise one or more primers or probes for specifically detecting at least a portion of the NtSWEET polynucleotide(s).
  • the kit may comprise reagents for polynucleotide amplification - such as PCR - or reagents for probe
  • the kit may comprise reagents for antibody binding-detection technology such as Western Blots, ELISAs, SELDI mass spectrometry or test strips.
  • the kit may comprise reagents for DNA sequencing.
  • the kit may comprise reagents and instructions for using the kit.
  • kits may comprise instructions for one or more of the methods described.
  • the kits described may be useful for genetic identity determination, phylogenetic studies, genotyping, haplotyping, pedigree analysis or plant breeding particularly with codominant scoring.
  • the present disclosure also provides a method of genotyping a plant, a plant cell or plant material comprising a NtSWEET polynucleotide as described herein.
  • Genotyping provides a means of distinguishing homologs of a chromosome pair and can be used to differentiate segregants in a plant population.
  • Molecular marker methods can be used for phylogenetic studies, characterizing genetic relationships among crop varieties, identifying crosses or somatic hybrids, localizing chromosomal segments affecting monogenic traits, map based cloning, and the study of quantitative inheritance.
  • the specific method of genotyping may employ any number of molecular marker analytic techniques including amplification fragment length polymorphisms (AFLPs).
  • AFLPs amplification fragment length polymorphisms
  • AFLPs are the product of allelic differences between amplification fragments caused by polynucleotide variability.
  • the present disclosure further provides a means to follow segregation of one or more genes or polynucleotides as well as chromosomal sequences genetically linked to these genes or polynucleotides using such techniques as AFLP analysis.
  • a specific extraction temperature is selected for the tobacco starting material.
  • the extraction temperature(s) is typically selected from within 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(s).
  • the tobacco starting material(s) is heated at the extraction temperature for at least about 90 minutes, more suitably at least about 120 minutes.
  • the heating step is typically carried out in an inert atmosphere.
  • a flow of an inert gas - such as nitrogen - is passed through the starting tobacco material during the heating step.
  • the volatile tobacco compounds are released into the flow of inert gas during the heating step such that the inert gas acts as a carrier for the volatile components.
  • the flow of inert gas can be at a flow rate of at least about 25 litres per minute, more suitably at least about 30 litres per
  • the heating step may be carried out under vacuum. Suitable heating methods for carrying out the heating of the tobacco starting material are known to the skilled person and include: dry distillation, hydrodistillation, vacuum distillation, flash distillation and thin film hydrodistillation.
  • the step of forming the liquid tobacco extract can comprise drying the solution of the volatile compounds in the liquid solvent in order to concentrate the solution. Drying may be carried out using any suitable means, including but not limited to desiccation, molecular sieves, freeze drying, phase separation, distillation, membrane permeation, controlled crystallisation of water and filtering, reverse hygroscopicity, ultracentrifugation, liquid chromatography, reverse osmosis or chemical drying.
  • the liquid tobacco extract is particularly suitable for producing a composition or formulation or gel composition, for use in an aerosol-generating system.
  • An aerosol-generating system comprising the composition or formulation or gel composition is disclosed.
  • the composition or formulation or gel is typically heated within an aerosolgenerating device - such as a device comprising a heater element that interacts with the composition or formulation or gel incorporating the liquid tobacco extract to produce an aerosol.
  • 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.
  • seeds Prior to germination, seeds are sterilized with vapor chlorine gas. A 5% final chlorine solution is placed together with glass tubes containing seeds in a bell jar. Hydrochloric acid (37%) is then added to the solution, and the seeds are incubated for 2 h. Then, the seeds are placed on Murashige & Skoog (Murashige and Skoog, 1962) growth medium and transferred to a plant growth room (24°C, 16 h light / 20°C, 8 h dark) for 4 weeks. Well-developed plantlets are transferred to a greenhouse and cultivated in 10 L pots until fully grown. Artificial light is applied
  • Flowering time is evaluated in 15-weeks-old plants grown in standard flue-cured fertilization conditions. Flowering time is quantified as the percentage of plant presenting flower primordia or developed flowers, or no inflorescence at all.
  • Amino acid content is measured using Method MP 1471 rev 5 2011 , Resana, Italy: Chelab Silliker S.r.l, Merieux NutriSciences Company. For amino acid determination in cured plant leaves, after mid-rib removal, cured lamina is dried at 40 °C for 2-3 days, if required. Tobacco material is then ground in fine powder (-100 uM) before the analysis of amino acid content. Alternatively, amino acid content is measured in plant material as described in UNI EN ISO 13903:2005.
  • Reducing sugar content is measured using a segmented-flow colorimetric method developed for analysis of tobacco samples as adapted by Skalar Instrument Co (West Chester, PA) and described in Tobacco Science 20: 139-144 (1976). The measurement of reducing sugar content is also described in Coresta Recommended Method 38, CRM38, CRM and ISO 15154: 2003.
  • cured lamina is dried at 40°C for 2-3 days, if required. Tobacco material is then ground in fine powder (-100 uM) before the analyses of reducing sugars.
  • reducing sugar content is measured according to ISO 15154: 2003.
  • Nitrate content is measured using a Lachet QuikChem 8500 instrument in accordance with the manufacturer’s protocol (Lachat QuikChem method 12-107-04-1 -J, Lachet Instruments, Loveland, CO, USA). Alternatively, nitrate content is measured according to ISO 15517:2003. Ammonia content is measured using ion chromatography according to ISO 21045:2018. Gene expression analysis
  • Sequencing data generated is demultiplexed using Illumina BaseSpace® Clarity LIMS ( ⁇ Illumina, Inc.) and subsequently imported to Qiagen CLC Genomics Workbench version 12.0.1 (CLC bio, a QIAGEN Company).
  • the DNA fragment SEQ ID NO: 9 is selected for suppressing the expression of both copies of SWEET12 (SWEET12-S and SWEET12-T) and is cloned between the strong constitutive MMV promoter and the 3' nos terminator sequence of the nopaline synthase gene of Agrobacterium tumefaciens (Cheng et al. (1997) Plant Physiol. 115(3): 971-980).
  • Flue-cured Virginia tobacco (Nicotiana tabacum L.) variety K326 is transformed using standard Agrobacterium-mediated transformation protocols (Horsch et al. (1985) Science, 227, 1229- 1232). Seeds are harvested from independent TO lines exhibiting the strongest SWEET12 silencing.
  • T1 plants from those TO lines are grown in the greenhouse under standard agricultural practices and selected by RT-qPCR experiments to assess SWEET12 gene expression levels using the following primers (5 1 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).
  • SWEET15-T-F1 SEQ ID NO: 15
  • SWEET15-T- R1 SEQ ID NO: 16
  • SWEET15-S-F1 SEQ ID NO: 17
  • SWEET15-S-R1 SEQ ID NO: 18
  • SWEET12-S SEQ ID NO: 1
  • SWEET15-T SEQ ID NO: 7
  • the polynucleotide sequences for SWEET12-S (SEQ ID NO: 1) and SWEET15-T (SEQ ID NO: 7) is cloned between the strong constitutive 35S promoter and the 3' nos terminator sequence of the nopaline synthase gene of Agrobacterium tumefaciens (Cheng et al., 1997 Plant Physiol. 115(3) :971 -980).
  • Virginia tobacco (Nicotiana tabacum L.) variety K326 is transformed using standard Agrobacterium-mediated transformation protocols (Horsch et al. (1985) Science, 227, 1229-1232). Seeds are harvested from independent TO lines exhibiting the strongest SWEET12-S and SWEET15-T.
  • T 1 plants from those TO lines are grown in the greenhouse under standard agricultural practices and selected by RT-qPCR experiments to assess SWEET12 gene expression levels using the primers described in the previous section.
  • Example 2 Gene expression analysis of SWEET12-S (SEQ ID NO: 1) and SWEET12-T (SEQ ID NO: 3)
  • SWEET s could contribute to the sugar accumulation changes that occur when curing tobacco leaves.
  • 25 genes and related gene products are identified in Nicotiana tabacum L. that are associated with putative SWEET function based on their identity with corresponding Arabidopsis and Tomato orthologs.
  • transcriptom ic analyses shows that out of these 25 genes and related gene products, SWEET12-S (SEQ ID NO: 1)
  • SWEET12-T (SEQ ID NO: 3) gene expressions are strongly induced in detached leaves of Virginia tobacco plants that are cured for 72 hours in a flue-curing barn.
  • the expression of those genes rises rapidly after 24h post-curing and keep increasing at 48h and 72h post-curing in leaves detached from lower stalk positions on the plants (X and C) while decreasing slightly in leaves from higher stalk positions (B and T), transcript levels being still very high compared to non-cured tissues (Oh time-point).
  • SWEET12-S and -T have a dedicated function in sugar transport at the early stages of curing, the so-called yellowing phase during which 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. (eds) The Tobacco Plant Genome. Compendium of Plant Genomes. Springer). Since many Senescence-Associated Genes (SAGs) are induced during the yellowing phase of curing (Bovet et al. (2019) Plants (Basel) 11 ;8(11 ):492), the expression of the putative senescence-associated target genes, SWEET15-S (SEQ ID NO: 5) and -T (SEQ ID NO: 7), is investigated. The transcript levels of those genes are not significant during the time-course of flue-curing with transcript levels being lower than 5 FPKM.
  • SWEET12-S and -T are highly expressed in both lamina and midrib tissues of plant leaves, with a higher expression in lamina than in the midrib tissues, as shown in Figure 2. Similar trends are observed for SWEET15-S and -T gene expression, however, the transcript expression levels can be considered below significant levels. Some SWEETs are not only transcriptionally regulated but can also be regulated by phosphorylation, and this post- transcriptional regulation is critical to their biological function (Anjali et al. (2020) Plant Physiol Biochem.156-.1-6). Therefore, a minor change in the low expression level of SWEET15-S and -T does not preclude the possibility for them to actively function during curing.
  • SWEET12-S and -T the expression of SWEET12-S and -T, as well as SWEET15-S and -T in the plant organs are shown. These data show that the four transcripts are mainly present in immature flowers and petals, attesting to active sugar requirements in such tissues for energy supply and seed storage. SWEET15-S exhibits very low expression in all organs, and SWEET12-S is also expressed in stem, possibly indicating sugar transport in phloem.
  • RNAi plants are generated using the insert SEQ ID NO: 9, as a RNAi-construct for silencing of SWEET 12 -S and -T.
  • flue-cured Virginia tobacco (Nicotiana tabacum L.) var. K326 plants is transformed with a construct that places the coding region of SWEET 15-T cDNA under the control of p35s, and RNAi plants are generated using the insert SEQ ID NO: 10 for down-regulation of SWEET 15-S and -T.
  • TO putative transformants is done by RT-qPCR screening on mature leaves. Seeds from each independent p35s: SWEET15-T and p35: SWEET12-S TO progeny presenting an up-regulation of their expression and conversely, seeds from RNAi-SWEET12 and RNAi-SWEET15 TO progeny presenting a silencing of their expression are collected to produce the T1 plant lines. T1 independent lines are selected again by RT-qPCR screening on leaves that are detached from the plants and flue-cured for 48 hrs.
  • T1 lines are compared to their corresponding wild-type (WT) control lines.
  • WT wild-type
  • the seed germination and the emergence of the first true leaves of seedlings are similar between transformed lines and controls suggesting that SWEET12 and SWEET 15 do not play a major role in germination, growth and development during the early juvenile phase.
  • Figure 4 shows a significant delay and reduction in growth of p35s; SWEET12-S lines compared to the control lines.
  • RNAi-SWEET12 lines are taller than control lines.
  • SWEET12-S contributes to the growth and development during the vegetative phase. Although no differences are observed in the growth and development of p35s; SWEET15-T lines when compared to control plants, RNAi-SWEET15 lines are taller than their respective controls, suggesting that SWEET15 may also contribute to vegetative growth and development.
  • Nicotiana tabacum produces its leaves from a single erect stem with a terminal inflorescence
  • SWEET15 lines 75% of p35s; SWEET15 15-week-old plants showed flowers primordia or developed flowers while only 20% of RNAi-SWEET15 lines are flowering. This suggests that, unlike SWEET12, SWEET15 acts as a positive regulator of flowering time.
  • SWEETs are known to fine-tune the sugar balance between cells and plant tissues (see above), we hypothesized that SWEETs could alter the chemistry of tobacco leaf during curing.
  • SWEET15 in the accumulation of sugars during curing.
  • the discordance between the absence of SWEET15 -S and -T gene expressions and the phenotypical changes in sugar accumulation in SWEET15 transformants during the early phase of curing could be either caused by (i) a late induction of SWEET15 genes after 72 hours that is not evaluated in our experimental design (ii) a regulation of sugar accumulation by SWEET15 prior to curing, with changes that persist during curing independently of its expression, (iii) the phenotypical changes observed do not depend on transcriptional regulation and are triggered by post-transcriptional regulation of SWEET transporters.
  • SWEET15 genes are lately (after 4 curing days) induced during air-curing Burley tobacco (low sugar tobacco) particularly support the points (i, ii).
  • SWEET12-S compared to WT controls (Student t-test, ns: non-significant, ** P ⁇ 0.05, ** P ⁇ 0.01 , *** P ⁇ 0.001).
  • the data in Table 2 shows that SWEET12-S lines display a significantly higher asparagine, tryptophan, phenylalanine, glycine and methionine content and a reduced glutamic acid and proline content compared to control lines.
  • the sugar reduction in P35S-SWEET is accompanied by the accumulation of more amino acids, attesting of a more active consumption of carbon resources for amino acid synthesis.
  • the asparagine and tryptophan contents are higher in SWEET15-T lines while the proline content is lower when compared to WT controls as shown in Table 3.
  • the free amino acid content is measured in 18-week-old plants grown in standard flue-cured fertilization conditions and flue-cured under standard agronomic conditions. The results represent the content in a pool of T1 leaves detached from the stalk (C position) and flue cured. Data are summarized in mg/kg. No strong differences are observed in RNAi lines (see Tables 2 and 3).
  • proline is known as an osmoprotectant, it suggests that water stress during curing (senescence) is different in the transgenic lines compared to CT1.
  • proline is known to form the amadori compounds, prolinofructose generating “popcorn” when heated by producing acetyl-pyrroline (Wei et al. (2017) Food Chem. 232:531-544).
  • the results represent the average content — expressed in mg/kg for ammonia, mg/kg as NOS for nitrate and g/100g for alkaloids — in leaves detached from the stalk (C position) and flue- cured. Data are collected from at least three biological replicates. Statistics indicate significant differences in the accumulation of ammonia in p35s: SWEET12-S compared to WT controls (Student t-test, ns: non-significant, * P ⁇ 0.05). In Table 5, The free amino acid content is measured in 18-week-old plants grown in standard flue-cured fertilization conditions and flue- cured under standard agronomic conditions. The results represent the content in a pool of T 1 leaves detached from the stalk (C position) and flue cured.
  • SWEETs are described as key targets which could be used for improving yield, or in response to environmental stresses. This example demonstrates the importance of SWEET genes, modifying the chemistry of tobacco leaves during curing and hence, possibly the aromatic profile of tobacco. By also impacting leaf biomass and flowering time (growth and development), SWEETs are key targets that can be investigated to enhance the leaf yield or modulate plant maturity.
  • SWEET12-S or SWEET 12-T or SWEET 15-S or SWEET 15-T comprising or consisting or consisting essentially of: (i) a SWEET12-S polynucleotide sequence comprising, consisting 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 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 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 or consisting essentially of a sequence having at least 70 % sequence identity to SEQ ID NO: 5; or (iv) a SWEET15-T polynucleotide sequence
  • the plant comprises at least one genetic alteration in a regulatory region or in the coding sequence of one or more of SWEET12-S or SWEET12-T or SWEET15-S or SWEET15-T; and/or wherein the at least one genetic alteration comprises one or more of exogenous DNA or exogenous RNA; and/or wherein the at least one genetic alteration comprises one or more of a vector or a viral vector or an Agrobacterium vector or a CRISPR vector; and/or wherein the at least one genetic alteration is capable of driving one or more of RNA interference or transcriptional gene silencing or virus induced gene silencing; and/or wherein the at least one genetic alteration is capable of expressing one or more of double stranded RNA (dsRNA) or hairpin RNA (hpRNA) or small interfering RNA; and/or wherein the at least one genetic alteration is capable of constitutively expressing one or more of
  • 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 decreased as compared to cured or dried leaf derived from the control plant; or wherein the expression and/or activity of SWEET15-S and SWEET15-T is decreased and wherein at least the fructose, glucose and sucrose content is decreased as compared to cured or dried leaf derived from the control plant; or wherein the expression and/or activity of SWEET12-S is increased and wherein asparagine, tryptophan, phenylalanine, glycine and methionine content is increased and wherein glutamic acid and proline content is decreased as compared to cured or dried leaf derived from the control plant; suitably, wherein ammonia content is increased as compared to cured or dried leaf derived from the control plant; and/or wherein the expression and/or activity of SWEET15-T is increased and where wherein
  • a method of preparing a plant with modulated flowering time and/or modulated amino acid levels and/or modulated sugar levels comprising: (a)providing a plant having modulated expression or activity of one or more of SWEET12-S or SWEET12-T or SWEET15- S or SWEET15-T comprising or consisting or consisting essentially of: (i) a SWEET12-S polynucleotide sequence comprising, consisting 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 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 or consisting essentially of a sequence having at least 70 % sequence identity to SEQ ID NO: 5; or (iv) a SWEET15-T polyn
  • SWEET15-S polypeptide having at least 70 % sequence identity to SEQ ID NO: 4; or (vi) a SWEET15-S polypeptide having at least 70 % sequence identity to SEQ ID NO: 4; or (vi) a SWEET15-S polypeptide having at least 70 % sequence identity to SEQ ID NO: 4; or (vi) a SWEET15-S polypeptide having at least 70 % sequence identity to SEQ ID NO: 4; or (vi) a SWEET15-S polypeptide having at least 70 % sequence identity to SEQ ID
  • SWEET15-T polypeptide having at least 70 % sequence identity to SEQ ID NO: 6; or (vii) a SWEET15-T polypeptide having at least 70 % sequence identity to SEQ ID NO: 6; or (vii) a SWEET15-T polypeptide having at least 70 % sequence identity to SEQ ID NO: 6; or (vii) a SWEET15-T polypeptide having at least 70 % sequence identity to SEQ ID NO: 6; or (vii) a SWEET15-T polypeptide having at least 70 % sequence identity to SEQ ID
  • step (b) the at least one modification is introduced by genome editing; suitably, wherein the genome editing is selected from CRISPR- mediated genome editing, mutagenesis, zinc finger nuclease-mediated mutagenesis, chemical or radiation mutagenesis, homologous recombination, oligonucleotide-directed mutagenesis and meganuclease-mediated mutagenesis; or wherein in step (b) the at least one modification is introduced using an interference polynucleotide; or wherein in step (b) the at least one modification is a promoter located 5’ to the polynucleotide.
  • a method of producing cured plant material having modulated amino acid levels and/or modulated sugar levels comprising: (a) preparing a plant according to paragraph 10 or paragraph 11 or providing the plant of paragraph 12; (b) harvesting plant material (for example, leaf) from the plant; and (c) curing the plant material.
  • a plant product comprising the plant material, the cured plant material, or the homogenised plant material according to paragraph 9 or comprising the cured plant material according to paragraph 14; suitably, wherein the plant product is a tobacco product from Nicotiana tabacum 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.
  • results of sensory analysis for tobacco in which SWEET-12 expression or SWEET-12 activity has been decreased (‘T1 SWEET-RNAi’) or increased (‘T1 SWEET 35S (OE)’) as compared to a control in SWEET-12 expression or SWEET-12 activity has not been modulated (‘Control CT).
  • ‘55% SWEET(K326)/45% FC BR’ refers to 55% Sweet modified tobacco from the greenhouse blended with 45% flue cured tobacco (FC) from Brazil (BR), which is a base tobacco added to cast leaf.
  • SEQ ID NO: 1 polynucleotide coding sequence of SWEET12-S from Nicotiana tabacum atggccatatttgatctccaccatccatggctatttgtgtttggagccttaggaaacattatttccatattcgtc ttcttagctccagtgccaacatttcgccgaatctacaaagaaaaatcaaccatgggctttcaatcagtcccttac gtggtagcactgttttcatctatgctctggatgtattatgcatttatcaagaaaaatgctattctctcatctcc atcaactccttcggttgcattgtcgagacaatttacatctccattttccttctacgcatccaaggaggctagg aggcagacggtgaacttgtttg
  • SEQ ID NO: 2 polypeptide sequence related to SEQ ID NO: 1
  • SEQ ID NO: 3 polynucleotide coding sequence of SWEET12-T from Nicotiana tabacum atggccatatttgacctccaccatccatggctatttgtgttcggagtcttaggaaacattatttccatattcgtc ttcttagctccagtgccaacctttcgccgaatctacaaagaaaaatcaaccatgggttttcaatcagtcccctac gtggtagcactgttttcatccatgctggatgtattatgcatttatcaagaaaaatgccactctctcatctct atcaactccttcggttgcattgtcgagaccatttacatctccattttccttctacgcatccaaggaggctagg aggcagacggtgaggctagg aggcagacgg
  • SEQ ID NO: 4 polypeptide sequence related to SEQ ID NO: 3
  • SEQ ID NO: 5 polynucleotide coding sequence of SWEET15-S from Nicotiana tabacum
  • SEQ ID NO: 6 polypeptide sequence related to SEQ ID NO: 5
  • SEQ ID NO: 7 polynucleotide coding sequence of SWEET15-T from Nicotiana tabacum atggctatcttcactgcttctcatttggctttttgtttttggcgttcttggaaatggggtgtcgttcttggtgtac ttgtctccaataccgactttctataggatatataagagaaaatcaacggaaggattccagtctataccctattcg gttgcactattcagtgccatgctctacttgtactatgcttatctcaaggagaagaatgggattttgct cattact attaacagcttcggaactgccatcgaattcatatatctcacaatcttcttgatgtatgctacccgagaggccaag atttacactacgaagctggt
  • SEQ ID NO: 8 polypeptide sequence related to SEQ ID NO: 7
  • SEQ ID NO: 9 polynucleotide sequence selected to silence (RNAi) of both SWEET 12-S and
  • SEQ ID NO: 10 polynucleotide sequence selected to silence (RNAi) both SWEET 15-S and -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

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  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Abstract

Il est divulgué une plante mutante, non naturelle ou transgénique Nicotiana tabacum comprenant au moins une modification pouvant moduler l'expression ou l'activité d'un ou de plusieurs SWEET12-S ou SWEET12-T ou SWEET15-S ou SWEET15-T comprenant ou consistant ou consistant essentiellement en : (i) une séquence polynucléotidique SWEET12-S comprenant, consistant ou consistant essentiellement en une séquence présentant une identité de séquence d'au moins 70 % avec SEQ ID NO : 1; ou (ii) une séquence polynucléotidique SWEET12-T comprenant, consistant ou consistant essentiellement en une séquence présentant au moins 70% d'identité de séquence avec SEQ ID NO : 3; ou (iii) une séquence polynucléotidique SWEET 15-S comprenant, consistant ou consistant essentiellement en une séquence présentant au moins 70% d'identité de séquence avec SEQ ID NO : 5 ; ou (iv) une séquence polynucléotidique SWEET15-T comprenant, consistant ou consistant essentiellement en une séquence présentant une identité de séquence d'au moins 70 % avec SEQ ID NO : 7 ; ou (v) un polypeptide codé par le polynucléotide énoncé en (i) ou (ii) ou (iii) ou (iv) ; ou (vi) un polypeptide SWEET12-S présentant une identité de séquence d'au moins 97 % avec SEQ ID NO : 2 ; ou (vii) un polypeptide SWEET12-T présentant une identité de séquence d'au moins 93 % avec SEQ ID NO : 4 ; ou (viii) un polypeptide SWEET15-S présentant une identité de séquence d'au moins 97 % avec SEQ ID NO : 6 ; ou (ix) un polypeptide SWEET15-T présentant une identité de séquence d'au moins 97 % avec SEQ ID NO : 8 ; ladite plante ou partie de plante comprenant au moins une modification susceptible de moduler : (a) l'expression du ou des polynucléotides dans la plante ou une partie de celle-ci; ou (b) l'activité du ou des polypeptides dans la plante ou une partie de celle-ci, par comparaison avec une plante témoin Nicotiana tabacum ou une partie de celle-ci dans laquelle l'expression du ou des polynucléotides ou l'activité du ou des polypeptides n'a pas été modifiée, et l'expression ou l'activité d'un ou plusieurs des SWEET12-s ou SWEET12-T ou SWEET15-s ou SWEET15-T étant modulée par rapport à une plante témoin Nicotiana tabacum dans laquelle l'expression ou l'activité d'un ou plusieurs des SWEET12-s ou SWEET12-T ou SWEET15-s ou SWEET15-T n'est pas modulée.
PCT/EP2024/052307 2023-02-02 2024-01-31 Modulation de transporteurs de sucre WO2024160864A1 (fr)

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