Regulation of reducing sugar content in plants (INV)

Abstract

[0010] Described herein is a plant cell comprising: (i) a polynucleotide comprising, consisting of, or consisting essentially of a sequence having at least 81% sequence identity to SEQ ID NO:5 (NtINV4-S) or at least 62% sequence identity to SEQ ID NO:7 (NtINV4-T); (ii) a polypeptide encoded by the polynucleotide described in (i); (iii) a polypeptide comprising, consisting of, or consisting essentially of a sequence having at least 85% sequence identity to SEQ ID NO:6 (NtINV4-S) or at least 85% sequence identity to SEQ ID NO:8 (NtINV4-T); or (iv) a construct, vector, or expression vector comprising the isolated polynucleotide described in (i), wherein the plant cell comprises at least one modification that (a) modulates expression or activity of the polynucleotide, or (b) modulates expression or activity of the polynucleotide or polypeptide, compared to a control plant cell in which expression or activity of the polynucleotide or polypeptide is not modified.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority from European Patent Application No. 19200865.4, filed October 1, 2019, the disclosure of which is incorporated herein by reference.

[0002] The present invention relates to plant cells and the like having regulated expression or activity of invertase (INV). [Background technology]

[0003] To produce tobacco products, different types of tobacco are mixed in various ratios to produce blends with specific flavor characteristics. Flue-cured tobacco (e.g., Virginia) is the most widely grown tobacco and is characterized by a high sugar and nitrogen ratio, but has a limited flavor profile. Other varieties of tobacco, such as air-cured (e.g., Burley, Maryland, and Galpao) or flame-cured (e.g., dark) tobacco types, offer alternative flavor profiles. These different flavor profiles are important in the production of blended tobacco products.

[0004] Flavor characteristics are the result of specific flavor compounds present at specific levels in tobacco plants, or the precursors of these compounds. As an example, changes in the sugar content of dried tobacco can lead to different perceptions of tobacco flavor and aroma. In aerosols and smoke, glucose, and to a lesser extent fructose, can produce Amadori compounds via the Maillard reaction. This can result in bready, nutty, or popcorn-like flavors.

[0005] However, because the variety of tobacco available for commercial production is limited, this means that there are also limited opportunities to develop tobacco products with different flavor and aroma profiles. This also applies to the manufacture of reconstituted tobacco materials used in heated tobacco sticks in reduced-risk products.

[0006] There remains a need in the art for improved opportunities to create tobaccos that offer consumers new flavors and sensory experiences while maintaining commercially acceptable yields and traits. The present invention seeks to address this and other needs. Summary of the Invention

[0007] Disclosed herein are polynucleotide and polypeptide sequences of INVs from Nicotiana tabacum. Although many different genes are believed to encode putative INVs in plants based on structural identity, the time at which these genes become functional as INVs in plants is generally unknown. In particular, little is known about INV gene expression in tobacco, particularly during the curing process. The inventors have identified specific NtINV polynucleotides in plants that are functionally expressed during the curing process. Surprisingly, it is observed that by modulating the expression of these specific NtINV genes, or the activity of the proteins encoded thereby, the pool of reducing sugars and sucrose produced during leaf drying can be altered. It is unexpected that alterations in specific INV genes can lead to alterations in the levels of reducing sugars and sucrose produced during leaf drying. Advantageously, this now provides an opportunity to create tobacco blends with new flavor and aroma characteristics. This may also result in a different flavor or sensory perception of the aerosol or smoke produced when heating the tobacco blend. Similarly, liquid extracts obtained from tobacco may have different flavors or sensory perceptions. Altering the balance of reducing sugars and sucrose may also affect the release of acrylamide in aerosols and smoke.

[0008] Several INV genomic polynucleotide sequences from Nicotiana tabacum are described herein, namely NtINV3-S (SEQ ID NO: 1), NtINV3-T (SEQ ID NO: 3), NtINV4-S (SEQ ID NO: 5), and NtINV4-T (SEQ ID NO: 7). Several INV polypeptide sequences from Nicotiana tabacum are also described herein, namely NtINV3-S (SEQ ID NO: 2), NtINV3-T (SEQ ID NO: 4), NtINV4-S (SEQ ID NO: 6), and NtINV4-T (SEQ ID NO: 8). NtINV4-S and NtINV4-T have been shown to play a role in sugar metabolism, particularly during the curing process. During the tobacco curing process, flue-cured tobacco (e.g., Virginia) usually contains at least eight times more reducing sugar than air-cured tobacco (e.g., Burley), which is mainly due to the genetic predisposition to accumulate high levels of starch. After leaf harvest and during the senescence process (yellowing stage), most of the starch is converted first to sucrose and then to reducing sugars that are likely involved in INV.

[0009] NtINV4-S and NtINV4-T polypeptide sequences are very similar, sharing 96% identity. This situation is similar for NtINV3-S and NtINV3-T polypeptide sequences, which are also very similar, sharing 96% identity. Notably, the pair of NtINV4-S and NtINV4-T, and the pair of NtINV3-S and NtINV3-T only share a low identity of about 60%, suggesting different functions or regulation. Surprisingly, only NtINV4-S and NtINV4-T are overexpressed during drying treatment, whereas NtINV3-S and NtINV3-T are not overexpressed during drying. In certain embodiments, the expression or activity of NtINV3-S and NtINV3-T is not regulated. NtINV3-S and NtINV3-T are likely involved in other metabolic pathways, and changes in their expression could result in potentially agronomically deleterious phenotypes (e.g., slow growth). Knowing which INV genes are overexpressed during drought treatments advantageously allows for the selection of plants with changes in only the relevant genes, reducing potential adverse effects on other metabolic processes.

[0010] Modifications to the expression or activity of one or more INVs can be combined with modifications to the expression or activity of one or more sucrose synthases (SUSs) to further regulate the levels of sugars in dried leaves. Thus, combinations of modifications to INVs and SUSs are disclosed. For example, decreasing the expression or activity of one or more INVs described herein can increase or decrease glucose or fructose levels, or a combination thereof, in dried leaves and increase sucrose levels. As a further example, increasing the expression or activity of one or more INVs described herein can increase glucose or fructose levels, or a combination thereof, in dried leaves and decrease sucrose levels. As a further example, decreasing the expression or activity of one or more INVs and one or more SUSs described herein can further decrease glucose or fructose levels, or a combination thereof, in dried leaves compared to decreasing the expression or activity of one or more INVs alone. As a further example, increasing the expression or activity of one or more INVs and one or more SUSs described herein can further increase glucose or fructose levels, or a combination thereof, in dried leaves compared to increasing the expression or activity of one or more INVs alone.

[0011] Disclosed are NtSUS1-S (SEQ ID NO: 10), NtSUS1-T (SEQ ID NO: 12), NtSUS2-S (SEQ ID NO: 14), NtSUS2-T (SEQ ID NO: 16), NtSUS3-S (SEQ ID NO: 18), NtSUS3-T (SEQ ID NO: 20), NtSUS4-S (SEQ ID NO: 22), NtSUS4-T (SEQ ID NO: 24), NtSUS5-S (SEQ ID NO: 26), NtSUS5-T (SEQ ID NO: 28), NtSUS6-S (SEQ ID NO: 30), and NtSUS6-T (SEQ ID NO: 32). The corresponding predicted polypeptide sequences of NtSUS1-S (SEQ ID NO: 11), NtSUS1-T (SEQ ID NO: 13), NtSUS2-S (SEQ ID NO: 15), NtSUS2-T (SEQ ID NO: 17), NtSUS3-S (SEQ ID NO: 19), NtSUS3-T (SEQ ID NO: 21), NtSUS4-S (SEQ ID NO: 23), NtSUS4-T (SEQ ID NO: 25), NtSUS5-S (SEQ ID NO: 27), NtSUS5-T (SEQ ID NO: 29), NtSUS6-S (SEQ ID NO: 31), and NtSUS6-T (SEQ ID NO: 33) are also disclosed. NtSUS2-S, NtSUS2-T, NtSUS3-S, NtSUS3-T, NtSUS4-S, and NtSUS4-T may play a role in sugar metabolism during the desiccation process. NtSUS2-S, NtSUS3-S, NtSUS3-T, and NtSUS4-S in particular may play a role in sugar metabolism during desiccation treatment.

[0012] In one aspect, there is provided a plant cell comprising: (i) a polynucleotide comprising, consisting of, or consisting essentially of a sequence having at least 81% sequence identity to SEQ ID NO:5 (NtINV4-S), or at least 62% sequence identity to SEQ ID NO:7 (NtINV4-T); (ii) a polypeptide encoded by the polynucleotide described in (i); (iii) a polypeptide comprising, consisting of, or consisting essentially of a sequence having at least 84% or at least 85% sequence identity to SEQ ID NO:6 (NtINV4-S), or at least 85% sequence identity to SEQ ID NO:8 (NtINV4-T); or (iv) a construct, vector, or expression vector comprising the isolated polynucleotide described in (i), wherein the plant cell comprises at least one modification that (a) modulates expression or activity of the polynucleotide, or (b) modulates expression or activity of the polynucleotide or polypeptide, compared to a control plant cell in which expression or activity of the polynucleotide or polypeptide has not been modified.

[0013] Preferably, the regulated expression or regulated activity regulates the level of one or more reducing sugars in dried leaves of a plant comprising the plant cells compared to the level of one or more reducing sugars in dried leaves of a control plant containing control plant cells, preferably the reducing sugars are glucose or fructose, or a combination thereof.

[0014] Suitably, the modulated expression or modulated activity also modulates the level of sucrose in dried leaves of a plant comprising the plant cell.

[0015] Preferably, the dried leaves reduce glucose levels by at least about 63% compared to control dried leaves.

[0016] Preferably, the dried leaves have reduced fructose levels by at least about 43% compared to the control dried leaves. Preferably, the dried leaves have reduced glucose and fructose levels by at least about 63% and at least about 43%, respectively, compared to the control dried leaves.

[0017] Preferably the dried leaves are from the middle leaves of the plant.

[0018] Suitably there is a minimal effect on the phenotype of a plant comprising the plant cell, for example the phenotype of the plant may not change.

[0019] Preferably there is no change in total free amino acids compared to a control plant containing control plant cells.

[0020] Suitably, the at least one modification is at least one modification in the genome of the plant cell, or at least one modification in a construct, vector or expression vector, or at least one transgenic modification.

[0021] Preferably, at least one modification is a genetic mutation of the polynucleotide.

[0022] Preferably, the plant is Nicotiana tabacum.

[0023] Preferably, the plant cell further comprises at least one modification in the NtSUS polynucleotide or polypeptide encoded thereby, more preferably, the NtSUS polynucleotide or polypeptide encoded thereby is selected from the group consisting of NtSUS2-T, NtSUS3-S, NtSUS3-T, NtSUS4-S, NtSUS4-T or a combination of two or more thereof, more preferably, the NtSUS polynucleotide or polypeptide encoded thereby is selected from the group consisting of NtSUS2-S, NtSUS3-S, NtSUS3-T and NtSUS4-S or a combination of two or more thereof.

[0024] Suitably, the plant cell comprises at least one mutation in an NtINV4 polynucleotide or an NtINV4 polypeptide, and at least one mutation in an NtSUS polynucleotide or a polypeptide encoded thereby.

[0025] In a further aspect, there is provided a plant or part thereof comprising a plant cell as described herein.

[0026] A plant material, dried plant material or homogenized plant material derived or obtained from a plant or part thereof is disclosed in a further aspect, suitably the plant material is selected from the group consisting of biomass, seeds, stems, flowers or leaves, or a combination of two or more thereof. The dried plant material may be selected from the group consisting of hot air dried plant material, sun dried plant material or air dried plant material, or a combination of two or more thereof.

[0027] In a further aspect, a tobacco product is provided that includes the plant cell, plant part, or plant material.

[0028] In a further aspect, there is provided a method for generating a plant described herein, comprising: (a) providing a plant cell that comprises at least one modification described herein; and (b) propagating the plant cell into a plant.

[0029] Preferably, in step (a), at least one modification is introduced by genome editing, preferably the genome editing is selected from CRISPR-mediated genome editing, zinc finger nuclease-mediated mutagenesis, chemical or radiation mutagenesis, homologous recombination, oligonucleotide-directed mutagenesis, and meganuclease-mediated mutagenesis.

[0030] Preferably, in step (a), at least one modification is introduced using an interfering polynucleotide or by introducing at least one mutation or a combination thereof.

[0031] In a further aspect, a method for producing a dried plant material having an altered amount of reducing sugars compared to a control plant material is disclosed, comprising the steps of: (a) providing a plant or part thereof or plant material as described herein; (b) harvesting the plant material therefrom; and (c) drying the plant material.

[0032] In a further aspect, a method of producing a liquid tobacco extract is provided, the method comprising:

[0033] The method includes the steps of: (a) preparing a tobacco starting material from a plant or part thereof containing plant cells that contain at least one modification that modulates expression or activity of NtINV as described herein; (b) heating the tobacco starting material at a suitable extraction temperature; (c) collecting volatile compounds released from the tobacco starting material during heating; and (d) combining the collected volatile compounds released from the tobacco starting material to form a liquid tobacco extract.

[0034] In a further aspect, a method of producing a liquid tobacco extract is disclosed, the method comprising the steps of: (a) preparing a first tobacco starting material from a plant or part thereof containing plant cells in which expression or activity of NtINV is modified as described herein; (b) preparing a second tobacco starting material from a plant or part thereof containing plant cells in which expression or activity of NtSUS is modified as described herein; (c) heating the first tobacco starting material at a first extraction temperature; (d) heating the second tobacco starting material at a second extraction temperature; (e) collecting volatile compounds released from the first tobacco starting material and the second tobacco starting material during heating; and (f) combining the collected volatile compounds released from the first and second tobacco starting materials and forming a liquid tobacco extract from the combined volatile compounds.

[0035] In a further aspect, disclosed is a liquid tobacco extract produced, obtained, or obtainable by the method of producing a liquid tobacco extract as described herein.

[0036] In further aspects, there are disclosed (i) a polynucleotide comprising, consisting of, or consisting essentially of a sequence having at least 60% sequence identity to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7; a polynucleotide comprising, consisting of, or consisting essentially of a sequence having at least 50% sequence identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8; (ii) a polypeptide encoded by the polynucleotide described in (i), (iii) a polypeptide comprising, consisting of, or consisting essentially of a sequence having at least 80% sequence identity to SEQ ID NO:2 or SEQ ID NO:4, 80% sequence identity to SEQ ID NO:6 or SEQ ID NO:8; (iv) a plant cell comprising a construct, vector, or expression vector comprising the isolated polynucleotide described in (i), wherein the plant cell comprises at least one modification that modulates expression or activity of the polynucleotide or polypeptide compared to a control plant cell in which expression or activity of the polynucleotide or polypeptide is not modified. Effect of the Invention

[0037] Advantageously, altering the sugar-amino acid balance in tobacco can affect the release of flavor compounds and acrylamide, a carcinogenic compound resulting from the interaction of glucose (fructose) with asparagine, when heated in aerosol and smoke.

[0038] Advantageously, reconstituted tobacco materials for heat-not-burn tobacco sticks require reducing sugars for proper cast leaf preparation, and the present disclosure can affect the sugar content and balance, thereby affecting cast leaf preparation.

[0039] Advantageously, non-genetically modified plants can be produced that may be more acceptable to consumers.

[0040] Advantageously, the present disclosure is not limited to the use of EMS mutant plants.

[0041] The present disclosure can be applied to various plant varieties or crops. Normally, senescent leaves (source leaves) produce sucrose as a carbon source and asparagine as an assimilable nitrogen resource for sink leaves and seeds. Therefore, sucrose and asparagine must first be transported from parenchyma (photosynthetic) senescent leaf cells to the phloem and then to the upper sink tissues. Manipulation of NtINV, or the polypeptide encoded by it, can affect the levels of reducing sugars such as glucose and fructose, and less affect free amino acids. This approach may allow the development of novel tobacco varieties with low glucose and fructose content and high sucrose content.

[0042] Advantageously, the present disclosure can be combined with modulating the expression of other genes, such as NtSUS or the polypeptides encoded thereby, as described herein. [Brief description of the drawings]

[0043] [Figure 1] FIG. 1 is a series of graphs showing: (A) the change in reducing sugars (glucose and fructose) during the hot air drying time course of Virginia, (B) the expression of NtINV3-S, NtINV3-T, NtINV4-S, and NtINV4-T (Tobarray-Affymetrix) during the hot air drying time course of Virginia, and (C) the expression of the senescence-related gene SAG12 during the hot air drying time course of Virginia. [Figure 2A] FIG. 2 is a series of graphs showing: (A) glucose change, (B) fructose change, and (C) sucrose change over the air-curing time course of dark tobacco. Data generated from metabolomic analysis by Metabolon (no units available). [Figure 2B] Same as above. [Figure 2C] Same as above. [Diagram 3] Figure 3 shows the results of silencing NtINV4-S and NtINV4-T using the GATEWAY vector and the measured expression (qPCR) of NtINV4-S and NtINV4-T in Virginia tobacco leaves after 48 hours of curing. T0-INV4 is the generated transgenic line, and CT0-INV4 is the corresponding control line. [Figure 4-1] Figure 4 is a series of graphs showing: (A) glucose, (B) fructose, and (C) sucrose content of silenced 35S:INV4-RNAi (INV4-T0) and control (CT0) dried leaves (CT0, n=4, and T0, n=4). Box plots and T-test statistical analysis are presented. [Figure 4-2] Same as above. [Diagram 5] FIG. 5 is a bar graph showing the content of each type of reducing sugar after harvest (maturity), after two days of curing (48 hours of curing), and at the end of curing for Burley, Virginia, and Oriental tobaccos. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0044] The section headings used in this disclosure are for organizational purposes and are not intended to be limiting.

[0045] 1.Definition Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. In case of conflict, the present document, including definitions, governs. Preferred methods and materials are described below, but methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. The materials, methods, and examples disclosed herein are merely illustrative and are not intended to be limiting.

[0046] The terms "comprise," "include," "having," "having," "can," "containing," and variations thereof are intended to be open-ended transitional phrases, terms, or words that do not exclude the possibility of additional acts or configurations.

[0047] The singular forms "a," "and," and "the" include plural references unless the context clearly dictates otherwise.

[0048] The present disclosure also contemplates other embodiments that "comprise," "consist," and "consist essentially of" the embodiments or elements presented herein, whether explicitly stated or not.

[0049] As recitations of numerical ranges herein, each intermediate number therebetween is specifically contemplated with the same degree of precision. For example, in the range 6-9, the numbers 7 and 8 are specifically contemplated in addition to the numbers 6 and 9, and in 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 specifically contemplated.

[0050] As used throughout the specification and claims, the following terms have the following meanings.

[0051] "Coding sequence" or "encoding a polynucleotide" refers to a nucleotide (RNA or DNA molecule) that comprises a polynucleotide that codes for a polypeptide. The coding sequence can further comprise initiation and termination signals operably linked to control elements, including a promoter and polyadenylation signal, capable of directing expression in cells of an individual or mammal to which the polynucleotide is administered. The coding sequence can be codon optimized.

[0052] "Complement" or "complementary" can mean Watson-Crick (e.g., AT / U and CG) or Hoogsteen base pairing between nucleotides or nucleotide analogs. "Complementarity" refers to the property shared between two polynucleotides, e.g., when aligned antiparallel to each other, that the nucleotide bases at every position are complementary.

[0053] "Construct" refers to a double-stranded recombinant polynucleotide fragment that contains one or more polynucleotides. A construct contains a "template strand" bases that are base-paired with a complementary "sense or coding strand." A given construct can be inserted into a vector in either of two possible orientations: the same (or sense) orientation or the opposite (or antisense) orientation relative to the orientation of the promoter placed in the vector (e.g., an expression vector).

[0054] The term "control" in the context of a control plant or control plant cell refers to a plant or plant cell in which the expression, function, or activity of one or more genes or polypeptides is not altered (e.g., increased or decreased), and thus can provide a comparison with a plant in which the expression, function, or activity of one or more genes or polypeptides is altered. A "control plant" is a plant that is substantially equivalent to a test plant or modified plant in all parameters except the test parameter. For example, when referring to a plant into which a polynucleotide has been introduced, the control plant is an equivalent plant in which such a polynucleotide has not been introduced. A control plant can be an equivalent plant into which a control polynucleotide has been introduced. In such cases, the control polynucleotide is one that is expected to have little or no phenotypic effect on the plant. A control plant can include an empty vector. A control plant can correspond to a wild-type plant. A control plant can be a null segregant, in which the T1 segregant no longer carries the transgene.

[0055] The term "reduced" or "reduced" refers to a reduction of about 10% to about 99% in the amount or function, such as polypeptide function, transcription function, or polypeptide expression, or a reduction of at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100%, or at least 150%, or at least 200% or more. The term "reduced" or the phrase "reduced amount" can refer to an amount or function that is less than that which would be found in an unmodified plant or in a product derived from a plant of the same variety processed in the same manner. Thus, in some contexts, a wild-type plant of the same variety processed in the same manner is used as a control to measure whether a reduction in amount is obtained.

[0056] "Donor DNA" or "donor template" refers to a double-stranded DNA fragment or molecule that contains at least a portion of a gene of interest. The donor DNA may encode a functional polypeptide.

[0057] "Endogenous gene or polypeptide" refers to a gene or polypeptide that originates in the genome of an organism and has not been altered, such as by deletion, gain, or replacement of genetic material. An endogenous gene is subject to normal gene transmission and gene expression. An endogenous polypeptide is subject to normal expression.

[0058] "Enhancer sequences" refer to sequences that can increase gene expression. These sequences can be located upstream, within introns, or downstream of the transcribed region. The transcribed region consists of exons and intervening introns from the promoter to the transcription termination region. Increased gene expression can occur through a variety of mechanisms, including increased transcription efficiency, stabilization of mature mRNA, and enhanced translation.

[0059] "Expression" refers to the production of a functional product. For example, expression of a polynucleotide fragment can refer to transcription of the polynucleotide fragment (e.g., transcription resulting in an mRNA or functional RNA), or translation of an mRNA into a precursor or mature polypeptide, or a combination thereof.

[0060] "Overexpression" refers to the production of a gene product in transgenic organisms that exceeds levels of production in null segregating (or non-transgenic) organisms of the same experiment.

[0061] "Functional" describes a polypeptide that has a biological function or activity. A "functional gene" refers to a gene that is transcribed into mRNA that is translated into a functional or active polypeptide.

[0062] "Genetic construct" refers to a DNA or RNA molecule that comprises a polynucleotide that encodes a polypeptide. The coding sequence can include initiation and termination signals that are operably linked to regulatory elements, including a promoter and polyadenylation signal, capable of directing expression.

[0063] "Genome editing" generally refers to a process in which genomic nucleic acid in a cell is modified. This may be, for example, by removing, inserting, or substituting one or more nucleotides in the genomic nucleic acid. Endonucleases can be used to create specific cuts or nicks at predetermined positions in the genome, as further described herein.

[0064] The term "homology" or "similarity" refers to the degree of sequence similarity between two polypeptide molecules or two polynucleotide molecules compared by sequence alignment. The degree of homology between two discontinuous polynucleotides being compared is a function of the number of identical or matching nucleotides at comparable positions. Homology or similarity can be determined over the entire length of the subject sequence.

[0065] "Identical" or "identity" in the context of two or more polynucleotides or polypeptides means that the sequences have a certain ratio of residues that are the same over a particular region. The ratio is calculated by optimally aligning the two sequences, comparing the two sequences over a particular region, determining the number of positions where identical residues exist in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the particular region, and multiplying the result by 100 to obtain the percentage of sequence identity. If the two sequences have different lengths, or if the alignment results in one or more staggered ends and the particular region being compared contains only a single sequence, the residues of the single sequence are included in the denominator of the calculation, but not in the numerator. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identity can be determined manually or by using a computer sequence algorithm such as ClustalW, ClustalX, BLAST, FASTA, or Smith-Waterman. Suitable parameters for ClustalW can be as follows: For polynucleotide alignments: Gap opening penalty = 15.0, Gap extension penalty = 6.66, and matrix = Identity. For polypeptide alignments: Gap opening penalty = 10.0, Gap extension penalty = 0.2, and matrix = Gonnet. For DNA and protein alignments: ENDGAP = -1, and GAPDIST = 4.

[0066] The term "increase" or "increased" refers to an increase of about 10% to about 99% in the amount or function or activity, including, but not limited to, one or more polypeptide functions or activities, transcriptional functions or activities, and polypeptide expression, or an increase of at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, at least 100%, at least 150%, or at least 200% or more. The term "increased" or the phrase "increased amount" can refer to an amount or function or activity in a plant or a product produced from a plant that is greater than that which may be found in an unmodified plant or a product derived from a plant of the same variety processed in the same manner. Thus, in some contexts, a wild-type plant of the same variety processed in the same manner is used as a control to measure whether an increase in amount is obtained.

[0067] The term "inhibit" or "inhibited" refers to a reduction in the amount or function or activity, such as, but not limited to, about 98% to about 100%, or at least 98%, at least 99%, but particularly 100%, of one or more polypeptide functions or activities, transcriptional function or activity, and polypeptide expression.

[0068] The term "introduced" means providing a polynucleotide (e.g., a construct) or a polypeptide to a cell. "Introduced" includes reference to the uptake of a polynucleotide into a eukaryotic cell where the polynucleotide may be integrated into the genome of the cell, including reference to the transient supply of a polynucleotide or polypeptide to a cell. "Introduced" includes reference to stable or transient transformation methods as well as sexual crossing. Thus, "introduced" in the context of inserting a polynucleotide (e.g., a recombinant / expression construct) into a cell means "transfection" or "transformation" or "transduction" and includes reference to the uptake of a polynucleotide into a eukaryotic cell where the polynucleotide may be integrated into the genome of the cell (e.g., a chromosome, a plasmid, a plastid, or a mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., a transfected mRNA).

[0069] The terms "isolated" or "purified" refer to a material that is substantially or essentially free from components that normally accompany it when found in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A polypeptide that is the predominant species present in a preparation is substantially purified. In particular, an isolated polynucleotide is separated from open reading frames that flank the desired gene and encode polypeptides other than the desired polypeptide. The term "purified" means that the polynucleotide or polypeptide gives rise to essentially one band in an electrophoretic gel. Specifically, this means that the polynucleotide or polypeptide is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure. An isolated polynucleotide can be purified from a naturally occurring host cell. Conventional polynucleotide purification methods known to those of skill in the art can be used to obtain an isolated polynucleotide. The term also encompasses recombinant and chemically synthesized polynucleotides.

[0070] "Liquid tobacco extract" describes the direct product of an extraction process performed on a tobacco starting material. The extraction process to produce a liquid tobacco extract can include heating the tobacco starting material under specific heating conditions and collecting the volatile compounds that are produced. A liquid tobacco extract can contain a mixture of compounds that originate from the tobacco starting material and have been removed during the extraction process, typically in combination with a liquid carrier or solvent.

[0071] "Modulate" or "modulation" refers to causing or promoting a qualitative or quantitative change, alteration, or modification in a process, pathway, function, or activity of a subject. Without being limited thereto, such a change, alteration, or modification can be an increase or decrease in the relative process, pathway, function, or activity of a subject. 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 comparing with a control.

[0072] The term "non-natural" describes entities such as polynucleotides, genetic mutations, polypeptides, plants, plant cells, and plant materials that are not naturally formed or do not exist in nature. Such non-natural or artificial entities can be created, synthesized, initiated, modified, intervened, or manipulated by methods described herein or known in the art. Such non-natural or artificial entities can be created, synthesized, initiated, modified, intervened, or manipulated by humans. Thus, by way of example, non-natural plants, non-natural plant cells, or non-natural plant materials can be created using traditional plant breeding techniques (e.g., backcrossing) or by genetic engineering techniques (e.g., antisense RNA, interfering RNA, meganucleases, etc.). As a further example, a non-natural plant, non-natural plant cell, or non-natural plant material may be created by introgressing or transferring one or more genetic mutations (e.g., one or more polymorphisms) from a first plant or plant cell to a second plant or plant cell (which may itself be natural) such that the resulting plant, plant cell, or plant material, or their progeny, contains a genetic configuration (e.g., a genome, chromosome, or a segment thereof) that is not naturally formed or does not exist in nature. The resulting plant, plant cell, or plant material is thus artificial or non-natural. Thus, an artificial or non-natural plant or plant cell may be created by modifying a gene sequence in a first naturally occurring plant or plant cell, even if the resulting gene sequence naturally occurs in a second plant or plant cell that contains a different genetic background from the first plant or plant cell. In certain embodiments, the mutation is not a naturally occurring mutation that occurs in a polynucleotide or polypeptide, such as a gene or polypeptide, in nature. Differences in genetic background can be detected by phenotypic differences or by molecular biology techniques known in the art, such as polynucleotide sequencing, the presence or absence of genetic markers (e.g., microsatellite RNA markers).

[0073] "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. Thus, polynucleotide also encompasses the complementary strand of the depicted single strand. Many variants of a polynucleotide can be used for the same purpose as a given polynucleotide. Thus, polynucleotide also encompasses substantially identical polynucleotides and their complements. A single strand provides a probe that can hybridize to a given sequence under stringent hybridization conditions. Thus, polynucleotide also encompasses probes that hybridize under stringent hybridization conditions. A polynucleotide can be single-stranded or double-stranded, or can contain portions of both double-stranded and single-stranded sequences. Polynucleotides can be DNA, both genomic and cDNA, RNA, or hybrids, and can contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. Polynucleotides can be obtained by chemical synthesis methods or by recombinant methods.

[0074] The specificity of single-stranded DNA for hybridizing complementary fragments is determined by the "stringency" of the reaction conditions (Sambrook et al., Molecular Cloning and Laboratory Manual, Second Ed., Cold Spring Harbor (1989)). Hybridization under "stringent conditions" describes hybridization protocols in which polynucleotide sequences that are at least 60% homologous to each other remain hybridized. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) of the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH, and polynucleotide concentration) at which 50% of the probes complementary to a given sequence hybridize to the given sequence at equilibrium. A given sequence is generally present in excess at the Tm, so that 50% of the probes are occupied at equilibrium.

[0075] Stringent conditions typically include: (1) low ionic strength and high temperature wash, e.g., 15 mM sodium chloride, 1.5 mM sodium citrate, 0.1% sodium dodecyl sulfate at 50°C; (2) denaturing agents during hybridization, e.g., 50% (v / v) formamide, 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer (750 mM sodium chloride, 75 mM sodium citrate, pH 6.5) at 42°C; or (3) 50% formamide. Also, the wash solution typically comprises 5×SSC (0.75 M NaCl, 75 mM sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50 μg / mL), 0.1% SDS, and 10% dextran sulfate at 42° C., with a high stringency wash consisting of 0.2×SSC (sodium chloride / sodium citrate) at 42° C. and 50% formamide at 55° C., followed by a wash in 0.1×SSC with EDTA at 55° C. Suitably, the conditions are such that sequences that are at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous to each other typically remain hybridized to each other.

[0076] "Moderately stringent conditions" use wash solutions and hybridization conditions that are less stringent such that polynucleotides hybridize to the entire polynucleotide, fragments, derivatives, or analogs. One example includes hybridization in 6xSSC, 5xDenhardt's solution, 0.5% SDS, and 100 μg / mL denatured salmon sperm DNA at 55°C, followed by one or more washes in 1xSSC, 0.1% SDS at 37°C. Temperature, ionic strength, etc. can be adjusted to suit experimental factors such as probe length. Other moderate stringency conditions have been described (see Ausubel et al., Current Protocols in Molecular Biology, Volumes 1-3, John Wiley & Sons, Inc., Hoboken, NJ (1993); Kriegler, Gene Transfer and Expression: A Laboratory Manual, Stockton Press, New York, NY (1990); Perbal, A Practical Guide to Molecular Cloning, 2nd edition, John Wiley & Sons, New York, NY (1988)).

[0077] "Low stringency conditions" use wash solutions and hybridization conditions that are less stringent than moderate stringency such that polynucleotides hybridize to the entire polynucleotide, fragments, derivatives, or analogs. Non-limiting examples of low stringency hybridization conditions include hybridization in 35% formamide, 5xSSC, 50mM Tris HCl (pH 7.5), 5mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100μg / mL denatured salmon sperm DNA, 10% (weight / volume) dextran sulfate at 40°C, followed by one or more washes in 2xSSC, 25mM Tris HCl (pH 7.4), 5mM EDTA, and 0.1% SDS at 50°C. Other low stringency conditions, such as cross-species hybridization, have been well described (see Ausubel et al., 1993; Kriegler, 1990).

[0078] "Operably linked" means that the expression of a gene is under the control of a promoter that is spatially linked. The promoter may be located 5' (upstream) or 3' (downstream) of the gene under its control. The distance between the promoter and the gene may be approximately the same as the distance between the promoter and the gene that the promoter controls in the gene from which it is derived. As is known in the art, this distance may be varied without loss of promoter function. "Operably linked" refers to the association of polynucleotide fragments in a single fragment, such that one function is controlled by the other. For example, a promoter is operably linked to a polynucleotide fragment if the promoter is capable of controlling the transcription of the polynucleotide fragment.

[0079] The term "plant" refers to any plant at any stage of its life cycle or development, and its progeny. In one embodiment, the plant is a tobacco plant, 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 their progeny. Plant cells include, but are not limited to, seeds, suspension cultures, embryos, meristematic tissue sites, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and pollen grain cells. Suitable species, cultivars, hybrids, and varieties of tobacco plants are described herein.

[0080] "Plant material" includes leaves, roots, sepals, root tips, petals, flowers, buds, stems, seeds and petioles. Plant material can be viable or non-viable plant material.

[0081] "Polynucleotide," "polynucleotide sequence," or "polynucleotide fragment" are used interchangeably herein and refer to a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural, or altered nucleotide bases. The polynucleotides of this disclosure are set forth in the accompanying sequence listing.

[0082] "Polypeptide" or "polypeptide sequence" refers to a polymer of amino acids in which one or more amino acid residues are artificial chemical analogues of a corresponding naturally occurring amino acid, as well as naturally occurring polymers of amino acids. These terms also include modifications including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation. The polypeptides of the present disclosure are set forth in the accompanying sequence listing.

[0083] "Promoter" refers to a synthetic or naturally occurring molecule capable of conferring, activating, or enhancing expression of a polynucleotide in a cell. The term refers to a polynucleotide element / sequence that is typically located upstream and operably linked to a double-stranded polynucleotide fragment. A promoter may be entirely derived from the region adjacent to a native gene of interest, or may be composed of different elements derived from different native promoters or synthetic polynucleotide segments. A promoter may contain one or more specific transcription control sequences to further enhance expression or to alter spatial or temporal expression. A promoter may also contain distal enhancer or repressor elements, and may be located as many as several thousand base pairs from the start site of transcription. Promoters may be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. A promoter may constitutively or differentially control expression of genetic components with respect to the cell, tissue, or organ in which expression occurs, or with respect to the developmental stage in which expression occurs, or in response to external stimuli such as physiological stress, pathogens, metal ions, or inducers.

[0084] As used interchangeably herein, "tissue-specific promoter" and "tissue-preferential promoter" refer to promoters that are primarily, but not necessarily, expressed in one tissue or organ, but may also be expressed in one particular cell. "Developmentally regulated promoter" refers to a promoter whose function is determined by developmental events. "Constitutive promoter" refers to a promoter that causes a gene to be expressed in most cell types at most times. "Inducible promoter" selectively expresses an operably linked DNA sequence in response to the presence of an endogenous or exogenous stimulus, for example, by a chemical compound (a chemical inducer), or in response to an environmental, hormonal, chemical, or developmental signal, or a combination of two or more thereof. Examples of inducible or regulated promoters include promoters that are regulated by light, heat, pressure, watering or drought, pathogens, plant hormones, wounding, or chemicals such as ethanol, jasmonic acid, salicylic acid, or safeners.

[0085] "Recombinant" refers to the artificial combination of two otherwise separate segments of sequence, for example, by chemical synthesis or by the manipulation of isolated segments of polynucleotides by recombinant genetic 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 natural events (e.g., spontaneous mutation, natural transformation or transduction or transposition), such as those that occur without deliberate human intervention.

[0086] "Recombinant construct" refers to a combination of polynucleotides that are not normally found together in nature. Thus, a recombinant construct may contain control sequences and coding sequences that are derived from different sources, or control sequences and coding sequences that are derived from the same source but arranged in a manner different from that normally found in nature. A recombinant construct may be a recombinant DNA construct.

[0087] As used interchangeably herein, "control sequence" and "control element" refer to polynucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence that affect the transcription, RNA processing or stability, or translation of the associated coding sequence. Control sequences include promoters, translation leader sequences, introns, and polyadenylation recognition sequences. The terms "control sequence" and "control element" are used interchangeably herein.

[0088] The term "tobacco" is used in a collective sense to refer to tobacco crops (e.g., tobacco plants that are grown in a field and are not grown hydroponically), tobacco plants, and parts thereof, including but not limited to roots, stems, leaves, flowers, and seeds, prepared or obtained as described herein. "Tobacco" is understood to include the Nicotiana tabacum plant and its products.

[0089] The term "tobacco products" refers to consumer tobacco products, including, but not limited to, smoking materials (e.g., cigarettes, cigars, and pipe tobacco), snuff, chewing tobacco, gum, and lozenges, as well as components, materials, and ingredients for the manufacture of consumer tobacco products. Preferably, these tobacco products are made from tobacco leaves and stems harvested from tobacco plants and cut, dried, cured, or fermented according to conventional techniques in tobacco preparation.

[0090] "Transcription terminator," "termination sequence," or "terminator" refers to a DNA sequence located downstream of a coding sequence and includes polyadenylation recognition sequences and other sequences that encode regulatory signals that can affect mRNA processing or gene expression. Polyadenylation signals are usually characterized by their effect on the addition of polyadenylic acid sequences to the 3' end of a pre-mRNA.

[0091] "Transgenic" refers to any cell, cell line, callus, tissue, plant part, or plant whose genome has been altered by the presence of a heterologous polynucleotide, such as a recombinant construct, including those original transgenic events, as well as those produced by sexual crossing or asexual propagation from the original transgenic event. The term does not encompass alterations of the genome (chromosomal or extrachromosomal) by conventional plant breeding methods or by natural events (e.g., random cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation).

[0092] "Transgenic plant" refers to a plant that contains one or more heterologous polynucleotides in its genome, i.e., a plant that contains recombinant genetic material not normally found therein and that has been introduced into the plant in question (or an ancestor of that plant) by human manipulation. For example, a heterologous polynucleotide is stably integrated into the genome such that the polynucleotide is transmitted to successive generations. The heterologous polynucleotide can be integrated into the genome alone or as part of a recombinant construct. Commercial development of genetically improved germplasm has also progressed to the stage of introducing multiple traits into crop plants, often referred to as gene stacking techniques. In such techniques, multiple genes that confer different characteristics of interest can be introduced into a plant. Gene stacking can be achieved by many means, including but not limited to co-transformation, re-transformation, and cross breeding with different transgenes. Thus, a plant grown from a plant cell into which recombinant DNA has been introduced by transformation is a transgenic plant, as are all progeny of the plant that contain the introduced transgenes (whether sexually or asexually reproduced). The term transgenic plant is understood to include whole plants or trees as well as parts of plants or trees, such as grains, seeds, flowers, leaves, roots, fruits, pollen, petioles, etc. Each heterologous polynucleotide may confer a different trait to the transgenic plant.

[0093] "Transgene" refers to a gene or genetic material containing a genetic sequence that has been isolated from one organism and introduced into a different organism. This non-native DNA segment may retain the ability to produce RNA or a polypeptide in the transgenic organism, or may alter the normal function of the genetic code of the transgenic organism.

[0094] A "variant" with respect to a polynucleotide means (i) a portion or fragment of the polynucleotide, (ii) the complement of a polynucleotide or a portion thereof, (iii) a polynucleotide that is substantially identical to a reference polynucleotide or its complement, or (iv) a polynucleotide that hybridizes under stringent conditions to a reference polynucleotide, its complement, or a substantially identical polynucleotide.

[0095] "Variant" with respect to a peptide or polypeptide refers to a peptide or polypeptide that differs in sequence by amino acid insertions, deletions, or conservative substitutions, but that retains at least one biological function or activity. Variant can also refer to a polypeptide that retains at least one biological function or activity. Conservative substitutions of amino acids, i.e., replacing one amino acid with a different amino acid that has similar properties (e.g., hydrophilicity, degree, and distribution of charged regions), are recognized in the art as typically involving minor changes.

[0096] The term "variety" refers to a group of plants that share certain characteristics that distinguish them from other plants of the same species. While possessing one or more distinctive traits, varieties are further characterized by very minor overall variations among individuals within the variety. Varieties are often commercially available.

[0097] A "vector" refers to a polynucleotide vehicle that includes a combination of polynucleotide components to allow for the delivery of polynucleotides, polynucleotide constructs, and polynucleotide conjugates, etc. The vector can be a viral vector, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. The vector can be a DNA or RNA vector. Suitable vectors include episomes capable of extrachromosomal replication, such as circular double-stranded nucleotide plasmids, linear double-stranded nucleotide plasmids, and vectors of any other origin. An "expression vector" is a polynucleotide vehicle that includes a combination of polynucleotide components to allow for the expression of polynucleotides, polynucleotide constructs, and polynucleotide conjugates, etc. Suitable expression vectors include episomes capable of extrachromosomal replication, such as circular double-stranded nucleotide plasmids, linear double-stranded nucleotide plasmids, and functionally equivalent expression vectors of any other origin. An expression vector includes at least a promoter, as defined below, located upstream and operably linked to a polynucleotide, polynucleotide construct, or polynucleotide conjugate.

[0098] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those skilled in the art. For example, any nomenclature and techniques used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and polypeptide and polynucleotide chemistry and hybridization described herein are well known and commonly used in the art. The meaning and scope of the terms must be clear, but in the event of any potential ambiguity, the definitions provided herein shall take precedence over any dictionary or external definitions. Furthermore, unless otherwise specified by context, singular terms shall include the plural and plural terms shall include the singular.

[0099] 2. Polynucleotides Disclosed are isolated polynucleotides that comprise, consist of, or consist essentially of a sequence having at least 60% sequence identity to any sequence described herein, including any polynucleotide shown in the sequence listing. Suitably, the isolated polynucleotide comprises, consists of, or consists essentially of a sequence having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, 80%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99% or 100% sequence identity thereto.

[0100] Preferably, the polynucleotides described herein encode active polypeptides having at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 100% or more of the function or activity of a polypeptide shown in the sequence listing.

[0101] In another embodiment, an isolated NtINV polynucleotide is provided that comprises, consists of, or consists essentially of a polynucleotide having at least 60% sequence identity to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9.

[0102] In another embodiment, an isolated NtSUS polynucleotide is provided that comprises, consists of, or consists essentially of a polynucleotide having at least 60% sequence identity to SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, or SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, or SEQ ID NO:32.

[0103] Suitably, the isolated polynucleotide comprises, consists of, or consists essentially of a sequence having at least about 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 to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9.

[0104] Suitably, the isolated polynucleotide comprises, consists of, or consists essentially of a sequence having at least about 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 to SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, or SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, or SEQ ID NO:32.

[0105] Suitably, the isolated polynucleotide comprises, consists of, or consists essentially of a sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9.

[0106] Suitably, the isolated polynucleotide comprises, consists of, or consists essentially of a sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, or SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, or SEQ ID NO:32.

[0107] Suitably, the isolated polynucleotide comprises, consists of, or consists essentially of a sequence having at least about 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:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9.

[0108] Suitably, the isolated polynucleotide comprises, consists of, or consists essentially of a sequence having at least about 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:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, or SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, or SEQ ID NO:32.

[0109] In another embodiment, a polynucleotide is provided that comprises, consists of, or consists essentially of a polynucleotide having substantial homology (i.e., sequence similarity) or substantial identity to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9.

[0110] In another embodiment, a polynucleotide is provided that comprises, consists of, or consists essentially of a polynucleotide having substantial homology (i.e., sequence similarity) or substantial identity to SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, or SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, or SEQ ID NO:32.

[0111] In another embodiment, fragments of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9 are provided, which have substantial homology (i.e., sequence similarity) or substantial identity thereto, having at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, 80%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to the corresponding fragment of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9.

[0112] In another embodiment, fragments of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, or SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, or SEQ ID NO:32 are provided, wherein the fragments are at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, 80%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% identical to the corresponding fragment of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, or SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, or SEQ ID NO:32. having 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, having substantial homology (i.e., sequence similarity) or substantial identity thereto.

[0113] In another embodiment, a polynucleotide comprising a sufficient or substantial degree of identity or similarity to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9 that encodes a polypeptide that functions as an INV is provided.

[0114] In another embodiment, a polynucleotide comprising a sufficient or substantial degree of identity or similarity to SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, or SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, or SEQ ID NO:32 is provided that encodes a polypeptide that functions as a SUS.

[0115] In another embodiment, there is provided a polynucleotide polymer comprising, consisting of, or consisting essentially of a polynucleotide designated herein as SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9.

[0116] In another embodiment, there is provided a polynucleotide polymer comprising, consisting of, or consisting essentially of a polynucleotide designated herein as SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, or SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, or SEQ ID NO:32.

[0117] Suitably, the polynucleotides described herein encode members of the INV or SUS family that have INV or SUS activity, respectively.

[0118] A polynucleotide can comprise a polymer of nucleotides that can be unmodified or modified deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Thus, a polynucleotide can be, but is not limited to, genomic DNA, complementary DNA (cDNA), mRNA, or antisense RNA, or fragments thereof. Moreover, a polynucleotide can be single-stranded or double-stranded DNA, a DNA that is a mixture of single-stranded and double-stranded regions, a hybrid molecule that includes DNA and RNA, or a hybrid molecule that includes a mixture of single-stranded and double-stranded regions or fragments thereof. Furthermore, a polynucleotide can be composed of triplex regions that include DNA, RNA, or both, or fragments thereof. A polynucleotide can contain one or more modified bases, such as phosphothioates, and can be a peptide nucleic acid. In general, a polynucleotide can be assembled from isolated or cloned fragments of cDNA, genomic DNA, oligonucleotides, or individual nucleotides, or combinations of the foregoing. Although the polynucleotides described herein are shown as DNA sequences, they include their corresponding RNA sequences, and their complementary (e.g., fully complementary) DNA or RNA sequences, including their reverse complements.

[0119] A fragment of a polynucleotide can range from at least about 25 nucleotides, about 50 nucleotides, about 75 nucleotides, about 100 nucleotides, about 150 nucleotides, about 200 nucleotides, about 250 nucleotides, about 300 nucleotides, about 400 nucleotides, about 500 nucleotides, about 600 nucleotides, about 700 nucleotides, about 800 nucleotides, about 900 nucleotides, about 1000 nucleotides, about 1100 nucleotides, about 1200 nucleotides, about 1300 nucleotides, or about 1400 nucleotides, up to a full-length polynucleotide encoding a polypeptide described herein.

[0120] Polynucleotides generally contain phosphodiester bonds, but in some cases polynucleotide analogs may have alternative backbones, including, for example, phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphosphoramidite linkages, as well as peptide polynucleotide backbones and linkages. Other analog polynucleotides include those with cationic backbones, nonionic backbones, and non-ribose backbones. Modifications of the ribose-phosphate backbone may be made for a variety of reasons, such as to increase the stability and half-life of such molecules in physiological environments or as probes on biochips. Mixtures of natural polynucleotides and analogs, or mixtures of different polynucleotide analogs, as well as mixtures of natural polynucleotides and analogs, may be made.

[0121] A variety of polynucleotide analogs are known, including, for example, phosphoramidate, phosphorothioate, phosphorodithioate, O-methylphosphoramidite linkages, and peptide polynucleotide backbones and linkages. Other analog polynucleotides include those with cationic, nonionic, and non-ribose backbones. Polynucleotides containing one or more carbocyclic sugars are also included.

[0122] Other analogs include peptide polynucleotides, which are peptide polynucleotide analogs.

[0123] Among the uses of the disclosed polynucleotides and fragments thereof are the use of the fragments as probes in hybridization assays or as primers for use in amplification assays.Such fragments generally comprise at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more consecutive nucleotides of DNA sequence.In other embodiments, the DNA fragments comprise at least about 10, 15, 20, 30, 40, 50 or 60 or more consecutive nucleotides of DNA sequence.Therefore, in one aspect, a method for detecting polynucleotides is also provided, comprising the use of probes or primers or both.

[0124] The basic parameters that influence the selection of hybridization conditions, and the guidance for devising suitable conditions, are described by Sambrook, J., EF Fritsch, and T. Maniatis (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). Using knowledge of the genetic code combined with the polypeptide sequences described herein, a set of degenerate oligonucleotides can be prepared. Such oligonucleotides are useful, for example, as primers in polymerase chain reaction (PCR), in which DNA fragments are isolated and amplified.

[0125] At least one modification (eg, mutation) can be included in one or more of NtINV3-S, NtINV3-T, NtINV4-S, and NtINV4-T.

[0126] At least one modification (eg, mutation) can be included in one or more of NtINV4-S and NtINV4-T.

[0127] At least one modification (e.g., mutation) may be included in one or more of NtINV3-S, NtINV3-T, NtINV4-S, and NtINV4-T. Optionally, at least one or more further modifications (e.g., mutations) may be included in one or more of NtSUS1-S, NtSUS1-T, NtSUS2-S, NtSUS2-T, NtSUS3-S, NtSUS3-T, NtSUS4-S, NtSUS4-T, NtSUS5-S, NtSUS5-T, NtSUS6-S, and NtSUS6-T, preferably in one or more of NtSUS2-S, NtSUS2-T, NtSUS3-S, NtSUS3-T, NtSUS4-S, and NtSUS4-T.

[0128] At least one modification (e.g., mutation) can be included in one or more of NtINV3-S, NtINV3-T, NtINV4-S, and NtINV4-T; at least one modification (e.g., mutation) can be included in one or more of NtSUS2-S, NtSUS2-T, NtSUS3-S, NtSUS3-T, NtSUS4-S, and NtSUS4-T, while no modification (e.g., mutation) is included in one or more of NtSUS1-S, NtSUS1-T, NtSUS5-S, NtSUS5-T, NtSUS6-S, and NtSUS6-T.

[0129] At least one modification (e.g., mutation) can be included in one or more of NtINV3-S, NtINV3-T, NtINV4-S, and NtINV4-T, and at least one modification (e.g., mutation) can be included in one or more of NtSUS2-S, NtSUS3-S, NtSUS3-T, and NtSUS4-S, while no modification (e.g., mutation) is included in one or more of NtSUS1-S, NtSUS1-T, NtSUS2-T, NtSUS4-T, NtSUS5-S, NtSUS5-T, NtSUS6-S, and NtSUS6-T.

[0130] At least one modification (e.g., mutation) can be included in one or more of NtINV4-S and NtINV4-T, and at least one modification (e.g., mutation) can be included in one or more of NtSUS2-S, NtSUS2-T, NtSUS3-S, NtSUS3-T, NtSUS4-S and NtSUS4-T, while no modification (e.g., mutation) is included in one or more of NtSUS1-S, NtSUS1-T, NtSUS5-S, NtSUS5-T, NtSUS6-S and NtSUS6-T.

[0131] At least one modification (e.g., mutation) can be included in one or more of NtINV4-S and NtINV4-T, and at least one modification (e.g., mutation) can be included in one or more of NtSUS2-S, NtSUS3-S, NtSUS3-T, and NtSUS4-S, while no modification (e.g., mutation) is included in one or more of NtSUS1-S, NtSUS1-T, NtSUS2-T, NtSUS4-T, NtSUS5-S, NtSUS5-T, NtSUS6-S, and NtSUS6-T.

[0132] 3. Polypeptides Also provided are isolated polypeptides comprising, consisting of, or consisting essentially of a polypeptide having at least 60% sequence identity to any of the polypeptides described herein, including any of the polypeptides shown in the sequence listing. Suitably, the isolated polypeptide comprises, consists of, or consists essentially of a sequence having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, 80%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity thereto.

[0133] Also provided is an NtINV polypeptide comprising, consisting of, or consisting essentially of a sequence having at least 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8.

[0134] Also provided are polypeptides comprising, consisting of, or consisting essentially of a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8.

[0135] Also provided are polypeptides comprising, consisting of, or consisting essentially of a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8.

[0136] Polypeptides encoded by SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8 are also provided.

[0137] The polypeptide can include a sequence that includes a sufficient or substantial degree of identity or similarity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8 to function as an INV.

[0138] at least 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% relative to SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, or SEQ ID NO:31 or SEQ ID NO:33 Also provided are NtSUS polypeptides comprising, consisting of, or consisting essentially of a sequence having 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.

[0139] Also provided is a polypeptide comprising, consisting of, or consisting essentially of a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, or SEQ ID NO:31, or SEQ ID NO:33.

[0140] Also provided is a polypeptide comprising, consisting of, or consisting essentially of a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, or SEQ ID NO:31, or SEQ ID NO:33.

[0141] Also provided are polypeptides encoded by SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, or SEQ ID NO:31, or SEQ ID NO:33.

[0142] The polypeptide can include a sequence that includes a sufficient or substantial degree of identity or similarity to SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, or SEQ ID NO:31, or SEQ ID NO:33, to function as a SUS.

[0143] A fragment of a polypeptide typically retains some or all of the function or activity of the full-length sequence (such as INV or SUS activity). A fragment of a polypeptide can range from at least about 25 amino acids, about 50 amino acids, about 75 amino acids, about 100 amino acids, about 150 amino acids, about 200 amino acids, about 250 amino acids, about 300 amino acids, about 400 amino acids, about 500 amino acids, up to the full-length polypeptides described herein.

[0144] Polypeptides also include mutants produced by the introduction of any type of alteration (e.g., amino acid insertions, deletions, or substitutions; changes in glycosylation status; changes affecting refolding or isomerization, three-dimensional structure, or self-association status), either deliberately engineered or naturally isolated, provided that they still possess some or all of their function or activity. Preferably, this function or activity is modulated.

[0145] Deletion refers to the removal of one or more amino acids from a polypeptide. Insertion refers to the introduction of one or more amino acid residues into a given site within a polypeptide. Insertion may include the insertion within a sequence of single or multiple amino acids. Substitution refers to the replacement of an amino acid of a polypeptide with another amino acid having similar properties (e.g., similar hydrophobicity, hydrophilicity, antigenicity, tendency to form or break alpha-helical or beta-sheet structures). Amino acid substitutions are typically of single residues, but may be clustered depending on the functional constraints imposed on the polypeptide and may range from about 1 to about 10 amino acids. Amino acid substitutions are preferably conservative amino acid substitutions as explained below. Amino acid substitutions, deletions, or insertions can be made using peptide synthesis techniques (e.g., solid phase peptide synthesis) or by recombinant DNA engineering. Methods for manipulating DNA sequences to generate substitution, insertion, or deletion mutants of a polypeptide are well known in the art. Mutants may have modifications that produce silent changes, resulting in functionally equivalent polypeptides. As long as secondary binding of the substance is maintained, intentional amino acid substitutions can be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and amphipathicity of residues.For example, negatively charged amino acids include aspartic acid and glutamic acid, positively charged amino acids include lysine and arginine, and amino acids with uncharged polar head groups with similar hydrophilicity values ​​include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.Conservative substitutions can be made, for example, according to the following table.Amino acids in the same column in the second column and preferably in the same line in the third column can be substituted for each other.

[0146] TIFF0007659547000001.tif66150

[0147] The polypeptide may be a mature or immature polypeptide, or a polypeptide derived from an immature polypeptide. The polypeptide may be linear or may be cyclized using known methods. The polypeptide typically comprises at least 10, at least 20, at least 30, or at least 40 consecutive amino acids.

[0148] At least one modification (eg, mutation) can be included in one or more of NtINV3-S, NtINV3-T, NtINV4-S, and NtINV4-T.

[0149] At least one modification (eg, mutation) can be included in one or more of NtINV4-S and NtINV4-T.

[0150] At least one modification (e.g., mutation) may be included in one or more of NtINV3-S, NtINV3-T, NtINV4-S, and NtINV4-T. Optionally, at least one or more further modifications (e.g., mutations) may be included in one or more of NtSUS1-S, NtSUS1-T, NtSUS2-S, NtSUS2-T, NtSUS3-S, NtSUS3-T, NtSUS4-S, NtSUS4-T, NtSUS5-S, NtSUS5-T, NtSUS6-S, and NtSUS6-T, preferably in one or more of NtSUS2-S, NtSUS2-T, NtSUS3-S, NtSUS3-T, NtSUS4-S, and NtSUS4-T.

[0151] At least one modification (e.g., mutation) can be included in one or more of NtINV3-S, NtINV3-T, NtINV4-S, and NtINV4-T; at least one modification (e.g., mutation) can be included in one or more of NtSUS2-S, NtSUS2-T, NtSUS3-S, NtSUS3-T, NtSUS4-S, and NtSUS4-T, while no modification (e.g., mutation) is included in one or more of NtSUS1-S, NtSUS1-T, NtSUS5-S, NtSUS5-T, NtSUS6-S, and NtSUS6-T.

[0152] At least one modification (e.g., mutation) can be included in one or more of NtINV3-S, NtINV3-T, NtINV4-S, and NtINV4-T, and at least one modification (e.g., mutation) can be included in one or more of NtSUS2-S, NtSUS3-S, NtSUS3-T, and NtSUS4-S, while no modification (e.g., mutation) is included in one or more of NtSUS1-S, NtSUS1-T, NtSUS2-T, NtSUS4-T, NtSUS5-S, NtSUS5-T, NtSUS6-S, and NtSUS6-T.

[0153] At least one modification (e.g., mutation) can be included in one or more of NtINV4-S and NtINV4-T, and at least one modification (e.g., mutation) can be included in one or more of NtSUS2-S, NtSUS2-T, NtSUS3-S, NtSUS3-T, NtSUS4-S and NtSUS4-T, while no modification (e.g., mutation) is included in one or more of NtSUS1-S, NtSUS1-T, NtSUS5-S, NtSUS5-T, NtSUS6-S and NtSUS6-T.

[0154] At least one modification (e.g., mutation) can be included in one or more of NtINV4-S and NtINV4-T, and at least one modification (e.g., mutation) can be included in one or more of NtSUS2-S, NtSUS3-S, NtSUS3-T, and NtSUS4-S, while no modification (e.g., mutation) is included in one or more of NtSUS1-S, NtSUS1-T, NtSUS2-T, NtSUS4-T, NtSUS5-S, NtSUS5-T, NtSUS6-S, and NtSUS6-T.

[0155] 4. Plant Modification A. Transformation Recombinant constructs can be used to transform plants or plant cells to modulate the expression, function, or activity of a polypeptide. A recombinant polynucleotide construct can include a polynucleotide encoding one or more of the polynucleotides described herein, operably linked to a control region suitable for expressing the polypeptide. Thus, the polynucleotide can include a coding sequence encoding a polypeptide described herein. A plant or plant cell in which the expression, function, or activity of a polypeptide is modulated can include mutant, non-natural, transgenic, man-made, or genetically modified plants or plant cells. Suitably, a transgenic plant or plant cell includes a genome that has been altered by stable integration of recombinant DNA. Recombinant DNA includes DNA that has been genetically modified and constructed outside the cell, and includes DNA that contains natural DNA or cDNA, or synthetic DNA. A transgenic plant can include a plant regenerated from an initially transformed plant cell, or a progeny transgenic plant from a later generation or cross of a transformed plant. Suitably, the transgenic modification alters the expression or function or activity of a polynucleotide or a polypeptide described herein compared to a control plant.

[0156] The polypeptide encoded by the recombinant polynucleotide may be a native polypeptide or may be heterologous to the cell. In some cases, the recombinant construct contains a polynucleotide that regulates expression and is operably linked to a control region. Examples of suitable control regions are described herein.

[0157] Also provided is a vector that contains recombinant polynucleotide constructs, such as those described herein.Suitable vector backbones include those that are routinely used in the art, such as plasmid, virus, artificial chromosome, bacterial artificial chromosome, yeast artificial chromosome, or bacteriophage artificial chromosome.Suitable expression vectors include, but are not limited to, plasmid and viral vectors derived from bacteriophage, baculovirus, and retrovirus.Many vectors and expression systems are commercially available.

[0158] A vector can include, for example, an origin of replication, a scaffold attachment region, or a marker. A marker gene can confer a selectable phenotype to a plant cell. For example, a marker can confer biocide resistance, such as resistance to an antibiotic (e.g., kanamycin, G418, bleomycin, or hygromycin) or a herbicide (e.g., glyphosate, chlorsulfuron, or phosphinothricin). In addition, an expression vector can include a tag sequence designed to facilitate manipulation or detection (e.g., purification or localization) of the expressed polypeptide. Tag sequences, such as luciferase, beta-glucuronidase, green fluorescent polypeptide, glutathione S-transferase, polyhistidine, c-myc, or hemagglutinin sequences, are typically expressed as a fusion with the encoded polypeptide. Such tags can be inserted anywhere within the polypeptide, including either the carboxyl or amino terminus.

[0159] A plant or plant cell can be transformed by having a recombinant polynucleotide integrated into its genome so that it is stably transformed. The plant or plant cell described herein can be stably transformed. A stably transformed cell typically retains the introduced polynucleotide at every cell division. A plant or plant cell can be transiently transformed so that the recombinant polynucleotide is not integrated into its genome. A transiently transformed cell typically loses all or part of the introduced recombinant polynucleotide at every cell division so that the introduced recombinant polynucleotide is not detected in daughter cells after a sufficient number of cell divisions.

[0160] Many methods for transforming plant cells are available in the art, including biolistec, gene gun techniques, Agrobacterium-mediated transformation, viral vector-mediated transformation, freeze-thaw methods, particle bombardment, direct DNA uptake, sonication, microinjection, plant virus-mediated transduction, and electroporation.

[0161] If cells or tissue cultures are used as recipient tissues for transformation, plants can be regenerated from the transformed cultures, if desired, by techniques known to those skilled in the art.

[0162] The selection of the control region to be included in the recombinant construct depends on several factors, including but not limited to efficiency, selectability, inducibility, desired expression level, and cell- or tissue-preferential expression. It is a routine matter for those skilled in the art to regulate the expression of a coding sequence by appropriately selecting and positioning a control region relative to the coding sequence. The transcription of a polynucleotide can be regulated in a similar manner. Some suitable control regions initiate transcription only in a specific cell type, or mainly in a specific cell type. Methods for identifying and characterizing control regions in plant genomic DNA are known in the art.

[0163] Exemplary promoters include tissue-specific promoters that are recognized by tissue-specific factors that are present in different tissues or cell types (e.g., root-specific promoters, shoot-specific promoters, xylem-specific promoters), or that are present during different developmental stages, or that are present in response to different environmental conditions. Suitable promoters include constitutive promoters that can be activated in most cell types without the need for specific inducers. Examples of promoters that can be used to control the expression of a polypeptide include the cauliflower mosaic virus 35S (CaMV / 35S), SSU, OCS, lib4, usp, STLS1, B33, nos, or ubiquitin or phaseolin promoters. Those skilled in the art can generate multiple variations of recombinant promoters.

[0164] Tissue-specific promoters are transcriptional control elements that are only active in certain cells or tissues, such as plant tissues or reproductive tissues, at certain times during plant development. Examples of tissue-specific promoters under developmental control include promoters that can initiate transcription only (or mainly) in certain tissues, such as plant tissues (e.g., roots or leaves) or reproductive tissues (e.g., fruit, ovule, seed, pollen, pistil, flower, or any embryonic tissue). Reproductive tissue-specific promoters can be, for example, anther-specific, ovule-specific, embryo-specific, endosperm-specific, integument-specific, seed and seed coat-specific, pollen-specific, petal-specific, sepal-specific, or combinations thereof.

[0165] Exemplary leaf-specific promoters include pyruvate, orthophosphate dikinase (PPDK) promoter from C4 plants (maize), cab-m1Ca+2 promoter from maize, Arabidopsis myb-related gene promoter (Atmyb5), ribulose-pyrrolidone carboxylase (RBCS) promoter (e.g., tomato RBCS1, RBCS2, and RBCS3A genes expressed in leaves and seedlings grown under artificial light, RBCS1 and RBCS2 expressed in developing tomato fruit, or the ribulose-pyrrolidone carboxylase promoter expressed at high levels almost exclusively in mesophyll cells of leaf blades and leaf sheaths).

[0166] Exemplary senescence-specific promoters include the tomato promoter active during fruit maturation, senescence, and leaf abscission, the corn promoter of the gene encoding cysteine ​​protease, the promoter of 82E4, and the promoter of the SAG gene.Exemplary anther-specific promoters can be used.Exemplary root-preferential promoters known to those skilled in the art can be selected.Exemplary seed-preferential promoters include both seed-specific promoters (promoters active during seed development, such as promoters of seed storage polypeptides) and seed germination promoters (promoters active during seed germination).

[0167] Examples of inducible promoters include promoters that respond to pathogen attack, anaerobic conditions, high temperature, light, drought, low temperature, or high salinity. Pathogen-inducible promoters include those derived from pathogenesis-related polypeptides (PR polypeptides), which are induced following infection by a pathogen (e.g., PR polypeptides, SAR polypeptides, beta-1,3-glucanases, chitinases).

[0168] In addition to plant promoters, other suitable promoters may be derived from bacterial origins, such as the octopine synthase promoter, the nopaline synthase promoter, and other promoters from the Ti plasmid, or from viral promoters, such as the 35S and 19S RNA promoters of the cauliflower mosaic virus (CaMV), the constitutive promoters of the tobacco mosaic virus, the cauliflower mosaic virus (CaMV) 19S and 35S promoters, or the Figwort mosaic virus 35S promoter.

[0169] B mutation Disclosed herein are plants or plant cells comprising at least one mutation in one or more of the polynucleotides or polypeptides described herein, which mutation results in modulated function or activity of NtINV or a polypeptide encoded thereby, or modulated function or activity of NtINV and NtSUS or a polypeptide encoded thereby. Combinations of such mutations are discussed herein.

[0170] A method is provided for regulating the level of an NtSUS polypeptide in a (cured) plant or in a (cured) tobacco plant material, the method comprising introducing into the genome of the plant one or more mutations that regulate expression of at least one NtINV gene, or at least one NtINV gene and at least one NtSUS gene, wherein the at least one gene is selected from any sequence according to the present disclosure.

[0171] Also provided is a method for identifying plants having regulated levels of reducing sugars, comprising screening a polynucleotide sample from a plant of interest for the presence of one or more mutations in a sequence according to the present disclosure (such as NtINV, or NtINV and NtSUS, or a combination thereof) and, optionally, correlating the identified mutations with mutations known to regulate the levels of reducing sugars.

[0172] Also disclosed herein are plants or plant cells that are heterozygous or homozygous for one or more mutations in the NtINV gene, or the NtINV gene and the NtSUS gene, in accordance with the present disclosure, which mutations result in modulation of expression of the gene or the function or activity of the NtINV polypeptide, or the NtINV and NtSUS polypeptides encoded thereby.

[0173] Many techniques can be used to combine mutations in one plant, including sexual crossing. A plant having one or more favorable heterozygous or homozygous mutations in a gene according to the present disclosure that regulates the expression of the gene or the function or activity of the polypeptide encoded thereby can be crossed with a plant having one or more favorable heterozygous or homozygous mutations in one or more other genes that regulate the expression or the function or activity of the polypeptide encoded thereby. In one embodiment, crossing is performed to introduce one or more favorable heterozygous or homozygous mutations in a gene according to the present disclosure in the same plant.

[0174] The function or activity of one or more of the polypeptides of the present disclosure in a plant is increased or decreased when the function or activity is lower or higher than the function or activity of the same polypeptide in a plant that has not been modified to inhibit the function or activity of the polypeptide and that has been grown, harvested, and dried using the same protocols.

[0175] In some embodiments, mutations are introduced into plants or plant cells using mutagenesis techniques, and the introduced mutations are identified or selected using methods known to those skilled in the art, such as Southern blot analysis, DNA sequencing, PCR analysis, or phenotypic analysis. Mutations that affect gene expression or disrupt the function of the encoded polypeptide can be determined using methods well known in the art. Insertional mutations in gene exons usually result in null mutants. Mutations in conserved residues can be particularly effective in suppressing the metabolic function of the encoded polypeptide. For example, it will be understood that mutations in one or more highly conserved regions are likely to alter polypeptide function, while mutations outside of those highly conserved regions are likely to have little or no effect on polypeptide function. Furthermore, mutations in single nucleotides can create stop codons, which can result in truncated polypeptides and loss of function depending on the extent of truncation.

[0176] Also disclosed is the method for obtaining mutant polynucleotides and polypeptides.Any plant of interest, including 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, radiation-induced mutagenesis, mutagenesis using modified bases, mutagenesis using gapped duplex DNA, double-strand break mutagenesis, mutagenesis using repair-deficient host lineage, mutagenesis by total gene synthesis, DNA shuffling and other equivalent methods.

[0177] 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 can be obtained by processes that include in vitro or in vivo engineering steps. Mutations in the polynucleotides and polypeptides described herein can be mutations that are obtained or can be obtained by processes that include human intervention. The function or activity of the mutant polypeptide variants can be higher, lower, or nearly the same as that of the unmutated polypeptide.

[0178] Methods for randomly introducing mutations into polynucleotides can include chemical mutagenesis and radiation mutagenesis. Chemical mutagenesis involves the use of exogenously added chemicals, such as mutagenic, teratogenic, or carcinogenic organic compounds, to induce mutations. Mutagens that primarily cause point mutations, as well as short deletions, insertions, missense mutations, simple sequence repeats, transversions, or transitions, can be used to induce mutations, including chemical mutagens or radiation. Mutagens include ethyl methanesulfonate, methyl methanesulfonate, N-ethyl-N-nitrosourea, triethylmelamine, N-methyl-N-nitrosourea, procarbazine, chlorambucil, cyclophosphamide, diethyl sulfate, acrylamide monomer, melphalan, nitrogen mustard, vincristine, dimethylnitrosamine, N-methyl-N'-nitro-nitrosoguanidine, nitrosoguanidine, 2-aminopurine, 7,12 dimethyl-benz(a)anthracene, ethylene oxide, hexamethylphosphoramide, bisulfane, diepoxyalkanes (diepoxyoctane, diepoxybutane, etc.), 2-methoxy-6-chloro-9[3-(ethyl-2-chloro-ethyl)aminopropylamino]acridine dihydrochloride, and formaldehyde.

[0179] Accidental mutations at loci that may not have been directly caused by mutagens are also contemplated if they result in the desired phenotype. Suitable mutagenic agents can also include, for example, ionizing radiation (e.g., X-rays, gamma rays, fast neutron irradiation, and UV radiation). The dosage of mutagenic chemicals or radiation is experimentally determined for each type of plant tissue to obtain a mutation frequency that is below the threshold level characterized by lethality or sterility. Any method of preparing plant polynucleotides known to those skilled in the art can be used to prepare plant polynucleotides for mutation screening.

[0180] The mutation process may involve one or more plant cross-breeding steps.

[0181] After mutation, screening can be performed to identify mutations that create premature stop codons or non-functional genes. After mutation, screening can be performed to identify mutations that create functional genes that can be expressed at elevated or decreased levels. Screening for mutants can be performed by sequencing or by using one or more probes or primers specific for the gene or polypeptide. Specific mutations in polynucleotides can also be made that can result in modulation of gene expression, modulation of mRNA stability, or modulation of polypeptide stability. Such plants are referred to herein as "non-natural" or "mutant" plants. Typically, mutant or non-natural plants will contain at least a portion of foreign or synthetic or man-made nucleotides (e.g., DNA or RNA) that were not present in the plant before engineering. The exogenous nucleotide can be a single nucleotide, two or more nucleotides, two or more contiguous nucleotides, or two or more non-contiguous nucleotides, for example, at least 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or 1500 or more contiguous or non-contiguous nucleotides.

[0182] C. Transgenics and genome editing Sequence-specific polynucleotides capable of disrupting the transcription of one or more endogenous genes, sequence-specific polynucleotides capable of disrupting the translation of RNA transcripts (e.g., double-stranded RNA, siRNA, ribozymes), sequence-specific polypeptides capable of disrupting the stability of one or more polypeptides, sequence-specific polynucleotides capable of disrupting the enzymatic function of one or more polypeptides or the binding function of one or more polypeptides with respect to a substrate or regulatory polypeptide, antibodies that exhibit specificity for one or more polypeptides, small molecule compounds capable of disrupting the stability of one or more polypeptides or the enzymatic function of one or more polypeptides or the binding function of one or more polypeptides, zinc finger polypeptides that bind one or more polynucleotides, and meganucleases that have a function for one or more polynucleotides can be used to regulate the expression or function or activity of one or more of the polynucleotides or polypeptides described herein. Genome editing techniques are well known in the art and are discussed further below.

[0183] D zinc finger nuclease Zinc finger polypeptides can be used to regulate the expression or function or activity of one or more of the NtINV or NtINV and NtSUS polynucleotides described herein. The use of zinc finger nucleases is described in Nature Rev. Genet. (2010) 11(9):636-646).

[0184] e. Meganuclease Meganucleases, such as I-CreI, can be used to regulate the expression or function or activity of one or more of the NtINV, or NtINV and NtSUS polynucleotides described herein. Use of meganucleases is described in Curr Gene Ther. (2011) Feb; 11(1): 11-27 and Int J Mol Sci. (2019) 20(16), 4045.

[0185] f.TALEN Transcription activator-like effector nucleases (TALENs) can be used to regulate the expression or function or activity of one or more of the NtINV or NtINV and NtSUS polynucleotides described herein. The use of TALENs is described in Nature Rev. Mol. Cell Biol. (2013) 14: 49-55 and Int J Mol Sci. (2019) 20 (16), 4045.

[0186] g.CRISPR CRISPR system can be used to regulate the expression or function or activity of one or more of NtINV or NtINV and NtSUS polynucleotides described herein, and is a preferred method.This technology is described, for example, in Plant Methods (2016) 12:8; Front Plant Sci.(2016)7:506, Biotechnology Advances(2015)33,1,p41-52, Acta Pharmaceutica Sinica B(2017)7,3,p292-302, Curr.Op.in Plant Biol.(2017)36,1-8 and Int J Mol Sci(2019)20(16),4045.As is well known in the art, CRISPR editing system generally includes two components: CRISPR-associated endonuclease (Cas) (e.g., Cas9) and guide RNA (gRNA). Cas creates double-stranded DNA breaks at sites in the genome defined by the sequence of the gRNA molecule bound to Cas. The location at which Cas cuts the DNA is defined by the unique sequence of the gRNA bound to it. The gRNA is an RNA sequence specifically designed to recognize a target DNA region of interest and direct Cas nuclease there to edit it. It has two sections: (i) a tracer RNA that serves as a binding scaffold for the Cas nuclease, and (ii) a crisper RNA (crRNA) of 17-20 nucleotide sequence that is complementary to the target DNA. The exact region of DNA that is targeted depends on the particular application. For example, gRNA can be targeted to a promoter that drives expression of a target polynucleotide to activate or repress the target polynucleotide. Methods for designing gRNAs are well known in the art, including Chop Chop Harvard.

[0187] The application of Cas9-based genome editing in Arabidopsis and tobacco is described, for example, in Methods Enzymol. (2014) 546: 459-72 and Plant Physiol Biochem. (2018) 131: 37-46. CRISPR technology has been widely introduced into plants (see, for example, WO2015 / 189693).

[0188] In addition to Cas9, other RNA-guided nucleases have been described for use in CRISPR systems, including Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, CaslO, Cpfl, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3 and Csf4. In some embodiments, the use of Cas9 is preferred.

[0189] The present disclosure further provides a CRISPR-based genome editing system comprising an RNA-guided nuclease and a gRNA, the CRISPR-based genome editing system modulating the activity of one or more of the polynucleotides described herein. The present disclosure also provides a method of cleaving one or more polynucleotides in a plant cell, the method comprising introducing a gRNA and an RNA-guided nuclease into the plant cell, the gRNA acting in conjunction with the RNA-guided nuclease to generate strand breaks in one or more of the polynucleotides described herein. Also disclosed is a CRISPR construct comprising (i) a polynucleotide encoding a CRISPR-associated endonuclease, and (ii) a gRNA comprising a polynucleotide sequence (typically about 17-20 nucleotides) complementary to the DNA of the polynucleotide described herein to be targeted.

[0190] h. Antisense modification Antisense technology is another well-known method that can be used to regulate the expression or activity of one or more of the NtINV polypeptides, or one or more of the NtINV and NtSUS polypeptides.See, e.g., Gene (1988) 10;72(1-2):45-50.

[0191] i. Mobile genetic elements Alternatively, genes can be targeted for inactivation by introducing transposons (e.g., IS elements) into the genome of a plant of interest. See, e.g., Cytology and Genetics (2006) 40(4):68-81.

[0192] j Ribozyme Alternatively, NtINV, or NtINV and NtSUS polynucleotides, can be targeted for inactivation by introducing into the plant ribozymes derived from a number of small circular RNAs capable of self-cleavage and replication. See, e.g., FEMS Microbiology Reviews (1999) 23, 3, 257-275.

[0193] 5.Plants A mutant or non-naturally occurring plant or plant cell can have any combination of one or more modifications (e.g., mutations) in NtINV or NtINV and one or more of NtSUS, or NtINV or NtINV and one or more of NtSUS, that result in regulating the expression or function or activity of the polynucleotide or its polynucleotide product. For example, a mutant or non-naturally occurring plant or plant cell can have a single modification in a single NtINV or a single NtINV and a single NtSUS polynucleotide or polypeptide; multiple modifications in a single NtINV or a single NtINV and a single NtSUS polynucleotide or polypeptide; a single modification in two or more, three or more, or four or more NtINV or NtINV and NtSUS polynucleotide or polypeptide; or multiple modifications in two or more, three or more, or four or more NtINV or NtINV and NtSUS polynucleotide or polypeptide. As a further example, a mutant or non-natural plant or plant cell may have one or more modifications in a particular portion of an NtINV or NtINV and NtSUS polynucleotide or polypeptide, such as in a region of NtINV or NtINV and NtSUS that encodes an active site of an NtINV or NtSUS polypeptide or a portion thereof. As a further example, a mutant or non-natural plant or plant cell may have one or more modifications in a region outside of one or more NtINV or NtINV and NtSUS polynucleotides or polypeptides, for example, in a region upstream or downstream of the NtINV or NtINV and NtSUS polynucleotide that it regulates, provided that they regulate the function or expression of NtINV or NtINV and NtSUS p. Upstream elements may include promoters, enhancers, or transcription factors. Some elements (e.g., enhancers) may be located upstream or downstream of the gene they regulate.An element need not be located near the gene it regulates, as some elements have been found to be located hundreds of thousands of base pairs upstream or downstream of the gene it regulates. A mutant or non-naturally occurring plant or plant cell may have one or more modifications located within the first 100 nucleotides of the gene, the first 200 nucleotides of the gene, the first 300 nucleotides of the gene, the first 400 nucleotides of the gene, the first 500 nucleotides of the gene, the first 600 nucleotides of the gene, the first 700 nucleotides of the gene, the first 800 nucleotides of the gene, the first 900 nucleotides of the gene, the first 1000 nucleotides of the gene, the first 1100 nucleotides of the gene, the first 1200 nucleotides of the gene, the first 1300 nucleotides of the gene, the first 1400 nucleotides of the gene, or the first 1500 nucleotides of the gene. A mutant or non-naturally occurring plant or plant cell can have one or more modifications located within the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, or fifteenth set of 100 nucleotides of a gene, or a combination thereof. Disclosed are mutant or non-naturally occurring plants or plant cells (such as mutant, non-naturally occurring, or transgenic plants or plant cells as described herein) that comprise mutant polypeptide variants.

[0194] In one embodiment, seeds from the plant are mutagenized and then grown into first generation mutant plants. The first generation plants are then self-pollinated, and seeds from the first generation plants are grown into second generation plants, which are then screened for mutations at the locus. Mutagenized plant material can be screened for mutations, but the advantage of screening second generation plants is that all somatic mutations correspond to germline mutations. Those skilled in the art will understand that a variety of plant materials, including but not limited to seeds, pollen, plant tissues, or plant cells, can be mutagenized to create mutant plants. However, the type of mutagenized plant material may affect when the plant's polynucleotides are screened for mutations. For example, if pollen is subject to mutagenesis before pollination of a non-mutagenized plant, the seeds resulting from that pollination will grow into first generation plants. All cells of the first generation plants will contain the mutations created in the pollen, and therefore these first generation plants can then be screened for mutations without waiting until the second generation.

[0195] 6. Preparation, Screening, and Hybridization of Modified Plants The NtINV or NtINV and NtSUS polynucleotides prepared from individual plants, plant cells, or plant materials can be optionally pooled to facilitate screening for mutations within a population of plants originating from mutagenic plant tissues, cells, or materials. One or more subsequent generations of plants, plant cells, or plant materials can be screened. The size of the group that is optionally pooled depends on the sensitivity of the screening method used.

[0196] 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 genes or sequences immediately adjacent to the gene can be utilized to amplify sequences in the optionally pooled samples. Preferably, one or more primers or probes are designed to amplify the region of the locus where useful mutations are most likely to occur. Most preferably, primers are designed to detect mutations within the region of the polynucleotide. Furthermore, primers and probes preferably avoid known polymorphic sites to facilitate screening for point mutations. To facilitate detection of the amplification product, one or more primers or probes can be labeled using any conventional labeling method. Primers or probes can be designed based on the sequences described herein using methods well understood in the art.

[0197] To facilitate detection of the amplification products, primers or probes may be labeled using any conventional labeling method, which can be designed based on the sequences described herein using methods well understood in the art.

[0198] Polymorphisms can be identified by means known in the art, several of which have been described in the literature.

[0199] In some embodiments, plants can be regenerated or cultivated from plants, plant tissues, or plant cells. Any suitable method for regenerating or cultivating plants from plant cells or plant tissues can be used, including but not limited to tissue culture or regeneration from protoplasts. Preferably, plants can be regenerated by cultivating transformed plant cells on callus induction medium, shoot induction medium, or root induction medium. See, for example, McCormick et al., Plant Cell Reports 5:81-84 (1986). These plants are then cultivated and pollinated with either the same transformed line or a different line, and the resulting hybrids with expression of the desired phenotypic traits are identified. Two or more generations are cultivated to ensure that expression of the desired phenotypic traits is stably maintained and inherited, and then seeds are harvested to ensure that expression of the desired phenotypic traits is achieved. Thus, a "transformed seed" refers to a seed that contains a nucleotide construct that is stably integrated into the plant genome.

[0200] Thus, in a further aspect, a method of preparing a mutant plant is provided. The method involves providing at least one cell of a plant comprising an NtINV or NtINV and NtSUS gene encoding a functional polynucleotide as described herein (or any combination thereof as described herein). The at least one cell of the plant is then treated under conditions effective to modulate the function of the polynucleotide. The at least one mutant plant cell is then grown into a mutant plant, the mutant plant having a modulated level of the NtINV or NtINV and NtSUS polypeptide as described herein (or any combination thereof as described herein) compared to that of a control plant. In one embodiment of the method of making a mutant plant, the treating step involves subjecting at least one cell to a chemical mutagen as described above and under conditions effective to obtain at least one mutant plant cell. In another embodiment of the method, the treating step involves subjecting at least one cell to a radiation source under conditions effective to obtain at least one mutant plant cell. The term "mutated plant" includes mutant plants whose genotype has been altered compared to a control plant, preferably which has been altered by means other than genetic engineering or genetic modification.

[0201] In certain embodiments, the mutant plant, mutant plant cell, or mutant plant material may contain one or more mutations that naturally occur in another plant, plant cell, or plant material and that confer a desired characteristic. The mutation can be incorporated (e.g., introgressed) into another plant, plant cell, or plant material (e.g., a plant, plant cell, or plant material having a different genetic background than the plant in which the mutation was induced) to confer the characteristic thereto. Thus, as an example, a naturally occurring mutation in a first plant can be introduced into a second plant (e.g., a second plant having a different genetic background than the first plant). Thus, one skilled in the art can search for and identify plants that naturally carry in their genome one or more mutant alleles of the genes described herein that confer the desired characteristic. The naturally occurring mutant alleles can be transferred to a second plant by various methods, including breeding, backcrossing, and introgression, to generate a line, variety, or hybrid having one or more mutations in the genes described herein. The same technique can be applied to the introgression of one or more non-natural mutations from a first plant to a second plant. Plants exhibiting desired characteristics can be screened out from a pool of mutant plants. Advantageously, the selection is performed using knowledge of the polynucleotide sequence as described herein. As a result, screening for genetic characteristics compared to a control is possible. Such screening techniques may involve the application of traditional amplification or hybridization techniques as discussed herein. Thus, a further aspect of the present disclosure relates to a method for identifying a mutant plant, comprising the steps of (a) providing a sample comprising one or more of NtINV or NtINV and NtSUS polynucleotides from a plant, and (b) determining the polynucleotide sequence, wherein a difference in the sequence of the polynucleotide compared to the polynucleotide of a control plant indicates that said plant is a mutant plant.In another aspect, a method is provided for identifying mutant plants that accumulate increased or decreased levels of reducing sugars compared to a control plant, comprising the steps of: (a) providing a sample from a plant to be screened; (b) determining whether said sample contains one or more mutations in one or more of the NtINV or NtINV and NtSUS polynucleotides described herein; and (c) determining the level of at least one reducing sugar, preferably glucose or fructose or a combination thereof, of said plant. Preferably, the level of at least one reducing sugar is determined in dried leaves. In another aspect, a method is provided for preparing mutant plants that have increased or decreased levels of at least one reducing sugar, preferably glucose or fructose or a combination thereof, compared to a control plant, comprising the steps of: (a) providing a sample from a first plant; (b) determining whether said sample contains one or more mutations in one or more of the NtINV or NtINV and NtSUS polynucleotides described herein that result in a regulated level of at least one reducing sugar; and (c) introducing one or more mutations into a second plant. Preferably, the level of at least one reducing sugar is determined in dried leaves. The mutation can be transferred to the second plant using various methods known in the art, such as genetic recombination, genetic engineering, introgression, plant breeding, backcrossing, etc. In one embodiment, the first plant is a wild-type plant. In one embodiment, the second plant has a different genetic background than the first plant.In another aspect, a method is provided for preparing a mutant plant having an increased or decreased level of at least one reducing sugar, preferably glucose or fructose or a combination thereof, compared to a control plant, comprising the steps of: (a) providing a sample from a first plant; (b) determining whether said sample contains one or more mutations in one or more of the NtINV or NtINV and NtSUS polynucleotides described herein that result in an adjusted level of at least one reducing sugar; and (c) introgressing the one or more mutations from the first plant to a second plant. Preferably, the level of the at least one reducing sugar is determined in dried leaves. In one embodiment, the introgression step comprises plant breeding, optionally including backcrossing, etc. In one embodiment, the first plant is a wild type plant. In one embodiment, the second plant has a different genetic background than the first plant. In one embodiment, the first plant is not a cultivar or elite cultivar. In one embodiment, the second plant is a cultivar or elite cultivar. Further aspects relate to mutant plants (including mutant plants of cultivars or elite cultivars) obtained or obtainable by the methods described herein. In certain embodiments, mutant plants may have one or more mutations localized only to a specific region of the plant, such as within the sequence of one or more NtINV or NtINV and NtSUS polynucleotides described herein. According to this embodiment, the remaining genomic sequence of the mutant plant will be identical or substantially identical to the plant prior to mutagenesis.

[0202] In certain embodiments, the mutant plant may have one or more mutations localized within multiple genomic regions of the plant, for example, within the sequence of one or more of the NtINV or NtINV and NtSUS polynucleotides described herein and within one or more additional regions of the genome. According to this embodiment, the remaining genomic sequence of the mutant plant will not be identical or substantially identical to the plant before mutagenesis. In certain embodiments, the mutant plant may not have one or more mutations in one or more, two or more, three or more, four or more, or five or more exons of the NtINV or NtINV and NtSUS polynucleotides described herein, or may not have one or more mutations in one or more, two or more, three or more, four or more, or five or more introns of the NtINV or NtINV and NtSUS polynucleotides described herein, or may not have one or more mutations in the promoter of the NtINV or NtINV and NtSUS polynucleotides described herein, or may not have one or more mutations in the promoter of the NtINV or NtINV and NtSUS polynucleotides described herein. The 3' untranslated region of the NtINV and NtSUS polynucleotides may not have one or more mutations, or the 5' untranslated region of the NtINV or NtINV and NtSUS polynucleotides described herein may not have one or more mutations, or the coding region of the NtINV or NtINV and NtSUS polynucleotides described herein may not have one or more mutations, or the non-coding region of the NtINV or NtINV and NtSUS polynucleotides described herein may not have one or more mutations, or any combination of two or more, three or more, four or more, five or more, or six or more of these portions.

[0203] In a further aspect, a method is provided for identifying a plant, plant cell, or plant material comprising a mutation in a gene encoding an NtINV or NtINV and NtSUS polynucleotide as described herein, the method comprising: (a) subjecting a plant, plant cell, or plant material to mutagenesis; (b) obtaining a sample from the plant, plant cell, or plant material, or a progeny thereof; and (c) determining the polynucleotide sequence of the NtINV or NtINV and NtSUS gene or a variant or fragment thereof, wherein the sequence difference indicates one or more mutations therein. The method also allows for the selection of plants having a mutation occurring in a genomic region that affects the expression of the NtINV or NtINV and NtSUS gene in plant cells, such as a transcription start site, a start codon, a region of an intron, an exon-intron boundary, a terminator, or a stop codon.

[0204] 7. Plant families, species, varieties, seeds, and tissue cultures Suitable plants for use in the present disclosure include monocotyledonous and dicotyledonous plants and plant cell lines, and may include members of the genus Camellia, Cannabis or Nicotiana.Suitable species of Camellia and Cannabis include Camellia sinensis (tea), Cannabis sativa, Cannabis indica and Cannabis ruderalis.

[0205] Various embodiments are directed to mutant, non-natural or transgenic tobacco plants or tobacco plant cells and may be applied to any species of the genus Nicotiana, including N. rustica and N. tabacum (e.g., LA B21, LN KY171, TI 1406, Basma, Galpao, Perique, Beinhart 1000-1, and Petico).The species include N. acaulis, N. acuminata, N. africana, N. alata, N. ameghinoi, N. amplexicaulis, N. arentsii, N. attenuata, N. azambujae, and N. benavidesii N. benthamiana, N. bigelovii, N. bonariensis, N. cavicola, N. clevelandii, N. cordifolia, N. corymbosa, N. debneyi, N. excelsior, N. forgetia na、N.fragrans、N.glauca、N.glutinosa、N.goodspeedii、N.gossei、N.hybrid、N.ingulba、N.kawakamii、N.knightiana、N.langsdorffii、N.line aris、N.longiflora、N.maritima、N.megalosiphon、N.miersii、N.noctiflora、N.nudicaulis、N.obtusifolia、N.occidentalis、N.occidentalis subsp.hesperis、N.otophora、N.paniculata、N.pauciflora、N.petunioides、N.plumbaginifolia、N.quadrivalvis、N.raimondii、N.repanda、N.rosulata subsp.ingulba、N.rotundifolia、N.setchellii、N.simulans、N.solanifolia、N.spegazzinii、N.stocktonii、N.suaveolens、N.sylvestr is, N. thyrsiflora, N. tomentosa, N. tomentosiformis, N. trigonophylla, N. umbratica, N. undulata, N. velutina, N. wigandioides, Nx sanderae has a slightly different variety than N.tabacum.

[0206] The use of tobacco cultivars and elite tobacco cultivars is also contemplated herein. Thus, a transgenic, non-natural, or mutant plant can be a tobacco cultivar or elite tobacco cultivar that contains one or more transgenes, or one or more genetic mutations, or a combination thereof. The genetic mutation (e.g., one or more polymorphisms) can be a mutation that does not naturally occur in a particular tobacco cultivar or tobacco cultivar (e.g., elite tobacco cultivar), or can be a naturally occurring genetic mutation if the mutation does not naturally occur in a particular tobacco cultivar or tobacco cultivar (e.g., elite tobacco cultivar).

[0207] Particularly useful Nicotiana tabacum varieties include Burley, Dark, Flue-cured, and Orient tobacco. Non-limiting examples of varieties or cultivars include BD64, CC101, CC200, CC27, CC301, CC400, CC500, CC600, CC700, CC800, CC900, Coker 176, Coker 319, Coker 371 Gold, Coker 48, CD263, DF911, 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, K149, K326, K346, K 358, K394, K399, K730, KDH 959, KT200, KT204LC, KY10, KY14, KY 160, KY 17, KY 171, KY 907, KY907LC, KY14xL8 LC, Little Crittenden, McNair 373, McNair 944, msKY 14xL8, Narrow Leaf Madole, Narrow Leaf Madole LC, NBH 98, N-126, N-777LC, N-7371LC, NC 100, NC 102, NC 2000, NC 291, NC 297, NC 299, NC 3, NC 4, NC 5, NC 6, NC7, NC 606, NC 71, NC 72, NC 810, NC BH 129, NC 2002, Neal Smith Madole, Oxford 207, PD 7302 LC, PD 7309 LC, PD 7312 LC, "Perique" tobacco, PVH03, PVH09, PVH19, PVH50, PVH51, R 610, R 630, R 7-11, R 7-12, RG 17, RG 81, RG H51, RGH 4, RGH 51, RS1410, Speight 168, Speight 172, Speight 179, Speight 210, Speight 220, Speight 225, Speight 227, Speight 234, Speight G-28, Speight G-70, Speight H-6, Speight H20, Speight NF3, TI 1406, TI 1269, TN 86, TN86LC, TN 90, TN 97, TN97LC, TN D94, TN D950, TR (Tom Rosson), Madore, VA 309, VA359, AA 37-1, B13P, Xanthi (Mitchell-Mor), Bel-W3, 79-615, Samsung Homes Holmes NN, KTRDC No. 2 Hybrid 49, Burley 21, KY8959, KY9, MD609, PG01, PG04, PO1, PO2, PO3, RG11, RG 8, VA509, AS44, Banket A1, Basma Drama B84 / 31, Basma I Zichna ZP4 / B, Basma Xanthi BX 2A, Batek, Besuki Jember C104, Coker 347, Criollo Misionero, Delcrest, Djebel 81, DVH 405, Galpao Comum, HB04P, Hicks Broadleaf Broadleaf, Kabakulak Elassona, Kutsage E1, LA BU 21, NC 2326, NC 297, PVH 2110, Red Russian, Samsun, Saplak, Simmaba, Talgar 28, Wislica, Yayaldag, Prilep HC-72, Prilep P23, Prilep PB 156 / 1, Prilep P12-2 / 1, Yaka JK-48, Yaka JB 125 / 3, TI-1068, KDH-960, TI-1070, TW136, Basma, TKF 4028, L8, TKF2002, GR141, Bass Maxanti, GR149, GR153, Petit Havana. Low converter variants of the above are also contemplated, even if not specifically identified herein.

[0208] The embodiments are also directed to compositions and methods for generating mutant, non-natural, hybrid, or transgenic plants modified to regulate the expression or function of one or more NtINV or NtINV and NtSUS polynucleotides described herein (or any combination thereof as described herein). Advantageously, the resulting mutant, non-natural, hybrid, or transgenic plants may be similar or substantially identical in overall appearance to the control plant. Various phenotypic characteristics can be assessed by field observation, such as maturity, number of leaves per plant, stem height, leaf insertion angle, leaf size (width and length), internode distance, and leaf blade to midvein ratio.

[0209] One aspect relates to the seed of the mutant plant, non-natural plant, hybrid plant, or transgenic plant described herein.Preferably, the seed is a tobacco seed.A further aspect relates to the pollen or ovule of the mutant plant, non-natural plant, hybrid plant, or transgenic plant described herein.Furthermore, there is provided a mutant plant, non-natural plant, hybrid plant, or transgenic plant described herein, further comprising a polynucleotide that confers male sterility.

[0210] Also provided are tissue cultures of regenerable cells of the mutant, non-naturally occurring, hybrid, or transgenic plants described herein, or parts thereof, which upon culture regenerate plants capable of expressing all of the morphological and physiological characteristics of the parent. Regenerable cells include cells derived from leaves, pollen, embryos, cotyledons, hypocotyls, roots, root tips, anthers, flowers and parts thereof, ovules, shoots, stems, stalks, pith and capsules, or callus or protoplasts derived therefrom.

[0211] The plant material described herein may be a cured tobacco material (e.g., a Virginia-type or Oriental-type cured tobacco material). The cured tobacco material may be an air-cured or sun-cured or air-dried tobacco material.

[0212] CORESTA recommendations regarding tobacco curing processes are contained in CORESTA Guide N°17, April 2016, Sustainability in Leaf Tobacco Production.

[0213] 8. Regulation of sugar content The mutant, transgenic, or non-naturally occurring plants or parts thereof of the present disclosure exhibit regulated levels of at least one reducing sugar, such as glucose or fructose or a combination thereof, within the plant material (e.g., in dried leaves). In certain embodiments, reduced levels of glucose or fructose or a combination thereof may result in increased levels of sucrose.

[0214] Preferably, the regulated level of at least one reducing sugar is observed in at least dried leaves, preferably in fully dried leaves. Preferably, the dried leaves are taken from leaves at the middle position of the plant. Preferably, there is no or negligible phenotypic variation such as visual plant fitness compared to the control plant. Preferably, there is no or negligible variation in total free amino acids compared to the control plant. In certain embodiments, the acrylamide level in smoke obtained by heating dried leaves or products derived therefrom can be regulated.

[0215] A further aspect relates to a mutant, non-naturally occurring, or transgenic plant or cell described herein, in which the level of at least one reducing sugar is reduced by at least 5% compared to a control plant in which expression or function of the NtINV or NtINV and NtSUS polypeptides is not regulated.

[0216] In certain embodiments, glucose or fructose levels, or a combination thereof, are reduced by about 30% or more, e.g., about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or more, compared to a control plant.

[0217] In certain embodiments, glucose or fructose levels, or a combination thereof, are reduced by about 40% or more, e.g., about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or more, compared to a control plant.

[0218] In certain embodiments, glucose levels are reduced by at least 60% or at least 63% and fructose levels are reduced by at least about 40% or at least 63% compared to a control plant.

[0219] In certain embodiments, glucose levels are reduced by at least 60% or at least 63% compared to a control plant, fructose levels are reduced by at least about 40% or at least 63% and sucrose levels are increased by at least 2-fold, at least 3-fold, or at least 4-fold.

[0220] In certain embodiments, glucose and fructose levels are increased as compared to a control plant.

[0221] In certain embodiments, the level of glucose is increased, the level of fructose is increased, and the level of sucrose is decreased as compared to a control plant.

[0222] The increase can be about a 25%, 50%, 100%, 250%, or 500% or greater increase compared to a control plant. The decrease can be about a 25%, 50%, or 75% or greater decrease compared to a control plant.

[0223] Still further embodiments relate to cured plant material (such as cured leaves or cured tobacco) derived or derivable from a mutant, non-naturally occurring, or transgenic plant or cell, wherein expression of one or more of the NtINV or NtINV and NtSUS polynucleotides described herein or the function of the NtINV or NtINV and NtSUS polypeptides encoded thereby is modulated and glucose, fructose and optionally sucrose levels are modulated as described above compared to a control plant.

[0224] Embodiments are also directed to compositions and methods for generating mutant, non-naturally occurring, or transgenic plants or plant cells that have been modified to modulate expression or function of one or more NtINV or NtINV and NtSUS polynucleotides or NtINV or NtINV and NtSUS polypeptides described herein, which can result in plants or plant components (e.g., leaves, such as dried leaves) or plant cells having modulated glucose, fructose and optionally sucrose content.

[0225] In one embodiment, the phenotype of the mutant, non-native, or transgenic plant is substantially the same as that of the control plant. In one embodiment, the leaf weight of the mutant, non-native, or transgenic plant is substantially the same as that of the control plant. In one embodiment, the leaf number of the mutant, non-native, or transgenic plant is substantially the same as that of the control plant. In one embodiment, the leaf weight and leaf number of the mutant, non-native, or transgenic plant are substantially the same as that of the control plant. In one embodiment, the stem height of the mutant, non-native, or transgenic plant is substantially the same as that of the control plant, for example, at 1 month, 2 months, or 3 months or more after field transplantation, or at 10 days, 20 days, 30 days, or 36 days or more after reaching maximum height. For example, the stem height of the mutant, non-native, or transgenic plant is not less than that of the control plant. In another embodiment, the chlorophyll content of the mutant, non-native, or transgenic plant is substantially the same as that of the control plant. In another embodiment, the stem height of the mutant, non-naturally occurring, or transgenic plant is substantially the same as that of the control plant, and the chlorophyll content of the mutant, non-naturally occurring, or transgenic plant is substantially the same as that of the control plant, hi other embodiments, the leaf size or shape or number or color of the mutant, non-naturally occurring, or transgenic plant is substantially the same as that of the control plant.

[0226] In another aspect, a method for modulating the amount of at least one reducing sugar in at least a part of a plant (e.g., a leaf (such as dried leaf) or tobacco) is provided, comprising the steps of: (i) modulating the expression or function of one or more of the NtINV or NtINV and NtSUS polypeptides described herein (or any combination thereof as described herein), preferably wherein the NtINV or NtINV and NtSUS polypeptides are encoded by corresponding NtINV or NtINV and NtSUS polynucleotides described herein; (ii) measuring the level of at least one reducing sugar (e.g., glucose or fructose) and optionally at least one non-reducing sugar such as sucrose in at least a part of the mutant, non-native, or transgenic plant obtained in step (i) (e.g., a leaf (such as dried leaf) or tobacco or smoke); and (iii) identifying mutant, non-native, or transgenic plants in which the level of the at least one reducing sugar and optionally at least one non-reducing sugar is modulated compared to a control plant.

[0227] In another aspect, there is provided a method for modulating the amount of at least one reducing sugar in at least a portion of a dried material (such as dried leaves), comprising the steps of: (i) modulating expression or function of one or more of NtINV or NtINV and NtSUS polypeptides (or any combination thereof as described herein), suitably wherein the NtINV or NtINV and NtSUS polypeptides are encoded by corresponding NtINV or NtINV and NtSUS polynucleotides described herein; (ii) harvesting and drying the plant material (such as one or more of the leaves) for a period of time; (iii) measuring the level of at least one reducing sugar (e.g. glucose and fructose) and optionally at least one non-reducing sugar such as sucrose in at least a portion of the dried plant material obtained in or during step (ii); and (iv) identifying a dried plant material in which the level of the at least one reducing sugar and optionally at least one non-reducing sugar is modulated compared to a control plant.

[0228] The increase in expression compared to a control can be from about 5% to about 100%, or at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, or an increase of 100% or more (such as 200%, 300%, 500%, 1000% or more), including transcription function, or NtINV or NtINV and NtSUS polynucleotide expression, or NtINV or NtINV and NtSUS polypeptide expression, or a combination thereof.

[0229] The increase in function or activity compared to a control can be from about 5% to about 100%, or at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, or 100% or more (such as 200%, 300%, 500%, 1000% or more) increase, including transcription function, or NtINV or NtINV and NtSUS polynucleotide expression or NtINV or NtINV and NtSUS polypeptide expression, or a combination thereof.

[0230] The reduction in expression compared to a control can be from about 5% to about 100%, or at least a 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, or 100% reduction, including transcription function, or NtINV or NtINV and NtSUS polynucleotide expression or NtINV or NtINV and NtSUS polypeptide expression, or a combination thereof.

[0231] The decrease in function or activity compared to a control can be from about 5% to about 100%, or at least a 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, or 100% decrease, including transcription function, or NtINV or NtINV and NtSUS polynucleotide expression or NtINV or NtINV and NtSUS polypeptide expression, or a combination thereof.

[0232] The polynucleotides and recombinant constructs described herein can be used to modulate the expression or function or activity of NtINV or NtINV and NtSUS polynucleotides or NtINV or NtINV and NtSUS polypeptides described herein in a plant species of interest, preferably tobacco.

[0233] Numerous polynucleotide-based methods can be used to increase gene expression in plants and plant cells. As an example, a construct, vector, or expression vector compatible with the plant to be transformed can be prepared, which contains the gene of interest together with an upstream promoter capable of overexpressing the gene in the plant or plant cells. Exemplary promoters are described herein. After transformation and when grown under suitable conditions, the promoter can drive expression to regulate the levels of NtINV or NtINV and NtSUS in the plant, or in specific tissues thereof. In one exemplary embodiment, a vector carrying one or more of the NtINV or NtINV and NtSUS polynucleotides described herein (or any combination thereof as described herein) is generated to overexpress the gene in the plant or plant cells. The vector carries a suitable promoter, such as the cauliflower mosaic virus CaMV 35S promoter, upstream of the transgene that drives its constitutive expression in all tissues of the plant. The vector also carries an antibiotic resistance gene to provide for selection of transformed callus and cell lines.

[0234] Expression of the sequence from the promoter can be enhanced by including expression control sequences that are well known in the art.Signals associated with aging and signals that are active during the desiccation process are specifically shown.

[0235] Thus, various embodiments are directed to a method for modulating the expression level of one or more of the NtINV or NtINV and NtSUS polynucleotides described herein (or any combination thereof as described herein) by incorporating multiple copies of the NtINV or NtINV and NtSUS polynucleotide into a plant genome, comprising transforming a plant cell host with an expression vector comprising a promoter operably linked to one or more of the NtINV or NtINV and NtSUS polynucleotides described herein. The polypeptide encoded by the recombinant polynucleotide may be a native polypeptide or may be heterologous to the cell.

[0236] In one embodiment, the plant for use in the present disclosure is a hot air drying treated plant, such that the plant has a high reducing sugar content (when grown in a field, the dry weight is greater than about 14% at the end of the drying treatment). A mutant, transgenic, or non-native plant or part thereof that has been hot air drying treated can have a reducing sugar content of less than about 14% dry weight at the end of the drying treatment when grown in a field, for example, less than about 10% dry weight at the end of the drying treatment when grown in a field, or less than about 5% dry weight at the end of the drying treatment when grown in a field, or less than about 1% dry weight at the end of the drying treatment when grown in a field.

[0237] In one embodiment, the plant used in the present disclosure is a sun-dried plant such that such plant has a reducing sugar content (greater than about 6.8% dry weight at the end of the drying process when field-grown). A sun-dried mutant, transgenic, or non-native plant or part thereof can have a reducing sugar content of less than about 5% dry weight at the end of the drying process when field-grown, for example, less than about 2.5% dry weight at the end of the drying process when field-grown, or less than about 1% dry weight at the end of the drying process when field-grown.

[0238] In one embodiment, the plant used in the present disclosure is an air-dried plant.Such a plant has a reducing sugar content of more than about 1.7% dry mass at the end of drying treatment when grown in field.Sun-dried mutant, transgenic or non-natural plant or its part can have a reducing sugar content of less than about 1.5% dry mass at the end of drying treatment when grown in field, for example, less than about 1% dry mass at the end of drying treatment when grown in field, or less than about 0.5% dry mass at the end of drying treatment when grown in field.

[0239] In certain embodiments, the use of hot air or sun dried plants is preferred.

[0240] 9. Breeding Plants carrying mutant alleles of one or more of the NtINV or NtINV and NtSUS polynucleotides described herein (or any combination thereof as described herein) can be used in plant breeding programs to generate useful lines, varieties, and hybrids. In particular, mutant alleles can be introgressed into commercially important varieties as described above. Thus, a method of plant breeding is provided that includes crossing a mutant, non-native, or transgenic plant described herein with a plant containing a different genetic identity. The method may further include crossing the progeny plant with another plant, and optionally repeating the crossing until progeny with the genetic trait or genetic background are obtained. One purpose served by such breeding methods is to introduce desirable genetic characteristics into other varieties, breeding lines, hybrids, or cultivars, especially those of commercial interest. Another purpose is to facilitate stacking of genetic modifications of different genes in a single plant variety, line, hybrid, or cultivar. Intra-species as well as inter-species crossings are contemplated. The progeny plants resulting from such crosses, also referred to as breeding lines, are examples of non-naturally occurring plants of the present disclosure.

[0241] In one embodiment, a method for producing a non-natural plant is provided, comprising: (a) crossing a mutant or transgenic plant with a second plant to obtain progeny tobacco seeds; (b) cultivating the progeny tobacco seeds under plant cultivation conditions to obtain a non-natural plant. The method may further comprise: (c) crossing the non-natural plant of the previous generation with itself or another plant to obtain progeny tobacco seeds; (d) cultivating the progeny tobacco seeds of step (c) under plant cultivation conditions to obtain additional non-natural plants; and (e) repeating the crossing and cultivating steps of (c) and (d) multiple times to generate additional generations of the non-natural plant. The method may optionally comprise, prior to step (a), providing a parent line that includes a characterized genetic identity and is not identical to the mutant or transgenic plant. In some embodiments, depending on the breeding program, the crossing and cultivating steps are repeated 0-2 times, 0-3 times, 0-4 times, 0-5 times, 0-6 times, 0-7 times, 0-8 times, 0-9 times, or 0-10 times to generate a generation of non-naturally occurring plants. Backcrossing is an example of such a method in which the progeny is crossed with one of its parents or another plant genetically similar to the parent to obtain a next generation progeny plant with a genetic identity closer to that of one of the parents. Plant breeding, particularly plant breeding techniques, are well known and can be used in the methods of the present disclosure. The present disclosure further provides non-naturally occurring plants generated by these methods. In certain embodiments, the step of selecting the plant is excluded.

[0242] In some embodiments of the methods described herein, the lines resulting from breeding and screening of mutant genes are evaluated in the field using standard field procedures. A control genotype is included, including the original non-mutagenized parent, and entries are arranged in the field in a randomized complete block design or other suitable field design. For tobacco, standard agronomic methods are used, e.g., tobacco is harvested, weighed, and sampled for chemical and other general testing before and during curing. Statistical analysis of the data is performed to confirm the similarity of the selected lines to the parent lines. Cytogenetic analysis of the selected plants is optionally performed to confirm chromosome complement and chromosome pairing relationships.

[0243] DNA fingerprinting, single nucleotide polymorphism, microsatellite markers, or similar techniques can be used in marker-assisted selection (MAS) breeding programs to transfer or breed mutant alleles of genes into other tobacco plants as described herein. For example, breeders can create segregating populations from hybridizing genotypes containing mutant alleles with agronomically desirable genotypes. F2 or backcross generation plants can be screened using markers developed from genomic sequences or fragments thereof using one of the techniques listed herein. Plants identified as carrying mutant alleles can be backcrossed or self-pollinated to create a second population to be screened. Depending on the expected inheritance pattern or MAS technique used, it may be necessary to self-pollinated selected plants before each cycle of backcrossing to help identify the desired individual plants. Backcrossing or other breeding procedures can be repeated until the desired phenotype of the recurrent parent is restored.

[0244] According to the present disclosure, in a breeding program, successful crosses produce F1 plants that are fertile. The selected F1 plants can be crossed with one of the parents, and the first generation plants of the backcross are self-pollinated to generate a population that is screened again for mutant gene expression (e.g., a null version of the gene). The backcross, self-pollinated, and screening process is repeated, for example, at least four times until a final screen produces a plant that is fertile and reasonably similar to the recurrent parent. If desired, the plant is self-pollinated, and then the progeny is screened again to confirm that the plant shows mutant gene expression. In some embodiments, the F2 generation plant population is screened for mutant gene expression, for example, by using PCR methods with primers based on the polynucleotide sequence information of the polynucleotides described herein (or any combination thereof described herein) according to standard methods to identify plants that do not express the polypeptide due to a defect in the gene.

[0245] Hybrid tobacco varieties can be produced by preventing self-pollination of the female parent (i.e., seed parent) of a first variety and allowing pollen from the male parent of a second variety to fertilize the female parent, resulting in F1 hybrid seeds being formed in the female plant. Self-pollination of the female plant can be prevented by removing the stamens of the flower at an early stage of flower development. Alternatively, pollen formation can be prevented in the female parent using the formation of male sterility. For example, male sterility can be produced by cytoplasmic male sterility (CMS) or transgenic male sterility, where the transgene inhibits microspore formation or pollen formation, or self-incompatibility. Female parent plants containing CMS are particularly useful. In embodiments where the female parent is CMS, pollen is harvested from the male fertile plant and manually applied to the stigma of the CMS female parent, and the resulting F1 seeds are harvested.

[0246] The varieties and lines described herein can be used to form single-cross tobacco F1 hybrids. In such an embodiment, the plants of the parent varieties can be grown as substantially homogenous adjacent populations to promote natural cross-pollination from the male parent to the female parent. The F1 seeds formed in the female parent are selectively harvested by conventional means. The two parent varieties can also be grown in large quantities to harvest a mixture of F1 hybrid seeds formed in the female parent and seeds formed in the male parent as a result of self-pollination. Alternatively, a three-way cross can be performed, in which a single-cross F1 hybrid is used as the female parent and crossed with a different male parent. As another alternative, a double-cross hybrid can be made, in which the F1 progeny of two different single crosses are crossed between themselves.

[0247] A population of mutant, non-natural, or transgenic plants can be screened or selected for members of the population that have a desired characteristic or phenotype. For example, a population of progeny of a single transformation event can be screened for plants that have a desired level of expression or function of the polypeptide encoded thereby. Physical and biochemical methods can be used to identify expression or activity levels. These include Southern analysis or PCR amplification for detection of polynucleotides; Northern blots, S1 RNase protection, primer extension, or RT-PCR amplification for detection of RNA transcripts; enzymatic assays for detection of enzyme or ribozyme activity of polypeptides and polynucleotides; and polypeptide gel electrophoresis, Western blots, immunoprecipitation, and enzyme immunoassays for detection of polypeptides. Other techniques, such as in situ hybridization, enzyme staining, and immunostaining and enzyme assays, can also be used to detect the presence or expression, function, or activity of NtINV or NtINV and NtSUS polypeptides or polynucleotides.

[0248] Described herein are mutant, non-native, or transgenic plant cells and plants that comprise one or more recombinant polynucleotides, one or more polynucleotide constructs, one or more double-stranded RNAs, one or more conjugates, or one or more vectors / expression vectors.

[0249] 10. Modification of other genes Without being limited thereto, the plants and parts thereof described herein may be modified either before or after the expression, function or activity of one or more NtINV or NtINV and NtSUS polynucleotides or NtINV or NtINV and NtSUS polypeptides according to the present disclosure is modulated.

[0250] One or more of the following genetic modifications may be present in a mutant, non-native, or transgenic plant:

[0251] One or more genes involved in the conversion of nitrogenous metabolic intermediates can be modified to result in lower levels of at least one tobacco specific nitrosamine (TSNA). Non-limiting examples of such genes include those encoding nicotine demethylases, such as CYP82E4, CYP82E5, and CYP82E10, as described in WO2006 / 091194, WO2008 / 070274, WO2009 / 064771, and WO2011 / 088180, and nitrate reductases, as described in WO2016 / 046288.

[0252] One or more genes involved in heavy metal uptake or heavy metal transport can be modified to produce lower heavy metal content.Non-limiting examples include genes belonging to the multidrug resistance-related polypeptide family, the cation diffusion facilitator (CDF) family, the Zrt-Irt-like polypeptide (ZIP) family, the cation exchanger (CAX) family, the copper transporter (COPT) family, the heavy metal ATPases family (e.g., HMA as described in WO2009 / 074325 and WO2017 / 129739), the macrophage polypeptide related to natural resistance (e.g., NRAMP) homolog family, and other members of the ATP-binding cassette (ABC) transporter family (e.g., MRP) as described in WO2012 / 028309, which are involved in the transport of heavy metals such as cadmium.

[0253] Other exemplary modifications can result in plants with regulated expression or function of isopropylmalate synthase, resulting in changes in sucrose ester composition that can be used to alter the beneficial profile (see WO2013 / 029799).

[0254] Other exemplary modifications can result in plants with modulated expression or function of threonine synthase, in which methionine levels can be regulated (see WO2013 / 029800).

[0255] 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 and alter flavor profile (see WO2013 / 064499).

[0256] Other exemplary modifications can result in plants in which expression or function of members of the CLC family of chloride channels is modulated to regulate nitrate levels therein (see WO2014 / 096283 and WO2015 / 197727).

[0257] Other exemplary modifications can result in plants in which the expression or function of one or more asparagine synthetases are modulated to regulate the levels of asparagine in the leaves and the levels of acrylamide in the aerosol produced upon heating or burning of the leaves (see WO2017 / 129739).

[0258] Other exemplary modifications may result in plants having modulated protease activity during desiccation treatment (see WO2016 / 009006).

[0259] Another exemplary modification may result in plants with reduced nitrate levels by altering the gene expression of nitrate reductase (e.g., Nia2), or the activity of the protein encoded thereby (see WO2016 / 046288).

[0260] Other exemplary modifications may result in plants with altered alkaloid levels by altering the gene expression of, or the activity of the proteins encoded thereby, the putative ABC-2 transporters NtABCGl-T and NtABCGl-S (see WO2019 / 086609).

[0261] Other exemplary modifications may result in plants that regulate the time to flowering by altering the genetic expression of the gene encoding terminal flowering 1 (TFL1), or the activity of the protein encoded thereby (see WO2018 / 114641). Other exemplary modifications may result in plants that have regulated expression or function of one or more asparagine synthases to regulate the levels of asparagine in leaves, and plants that regulate the levels of acrylamide in aerosols produced upon heating or burning leaves (see WO2017 / 042162).

[0262] Other examples of modifications include herbicide resistance, for example glyphosate is the active ingredient of many broad spectrum herbicides. Glyphosate-tolerant transgenic plants have been developed by introduction of the aroA gene (glyphosate EPSP synthase from Salmonella typhimurium and E. coli). Sulfonylurea-tolerant plants have been generated by transformation of mutant ALS (acetolactate synthase) genes from Arabidopsis thaliana. Atrazine-tolerant transgenic plants have been generated by introducing mutant Amaranthus hybridus photosystem II OB polypeptides into plants, and bromoxynil-tolerant transgenic plants have been generated by incorporating the bxn gene from Klebsiella pneumoniae bacteria.

[0263] Another exemplary modification results in plants that are resistant to insects. Bacillus thuringiensis (Bt) toxins can provide an effective way to delay the emergence of Bt-resistant pests, as recently demonstrated in broccoli, where the cry1Ac and cry1CBt genes in the cone eradicated diamondback moths that were resistant to either single polypeptide and significantly delayed the evolution of resistant insects.

[0264] Another exemplary modification results in plants that are resistant to diseases caused by pathogens (e.g., viruses, bacteria, fungi). Plants expressing the Xa21 gene (resistant to bacterial spot) have been engineered along with plants expressing both a Bt fusion gene and a chitinase gene (resistant to the wheat grain moth and resistant to coleoptera).

[0265] Another exemplary modification results in altered fertility, such as male sterility.

[0266] Another exemplary modification results in plants that are tolerant to abiotic stresses (e.g., drought, temperature, salinity); tolerant transgenic plants have been created by introducing the acylglycerol phosphate enzymes from Arabidopsis, genes encoding mannitol dehydrogenase and sorbitol dehydrogenase, involved in the synthesis of mannitol and sorbitol, improving tolerance to drought.

[0267] Another exemplary modification results in a plant in which the activity of one or more nicotine N-demethylases is modulated such that the levels of nornicotine and nornicotine metabolites formed during the drying process can be regulated (see WO2015169927).

[0268] Other exemplary modifications can result in plants with improved polypeptide and oil storage capacity, increased photosynthetic efficiency, extended shelf life, increased carbohydrate content, and fungal resistance. Transgenic plants with regulated expression of S-adenosyl-L-methionine (SAM) or cystathionine gamma-synthase (CGS), or a combination thereof, are also contemplated.

[0269] One or more genes involved in the nicotine synthesis pathway can be modified to result in a plant or part of a plant that has a regulated level of nicotine when subjected to a dry treatment. The nicotine synthesis gene can be selected from the group consisting of A622, BBLa, BBLb, JRE5L1, JRE5L2, MATE1, MATE2, MPO1, MPO2, MYC2a, MYC2b, NBB1, nic1, nic2, NUP1, NUP2, PMT1, PMT2, PMT3, PMT4, and QPT, or one or more combinations thereof.

[0270] One or more genes involved in controlling the amount of one or more alkaloids can be modified to result in a plant or part of a plant producing regulated levels of alkaloids. The alkaloid level control genes can be selected from the group consisting of BBLa, BBLb, JRE5L1, JRE5L2, MATE1, MATE2, MYC2a, MYC2b, nic1, nic2, NUP1, and NUP2, or a combination of two or more thereof.

[0271] One or more such traits can be introgressed from another cultivar into, or directly transformed into, a mutant, non-native, or transgenic plant.

[0272] Various embodiments provide mutant, non-naturally occurring, or transgenic plants, as well as biomass, that modulate the expression level of one or more polynucleotides and thereby the levels of polypeptides encoded thereby in accordance with the present disclosure.

[0273] 11.Consumable products The plant parts described herein, particularly the blades and midribs of such plants, can be incorporated into or used to manufacture various consumable products, including, but not limited to, aerosol-forming materials, aerosol-forming devices, smoking articles, smokable articles, smokeless products, medical or cosmetic products, intravenous preparations, tablets, powders, and tobacco products. Examples of aerosol-forming materials include tobacco compositions, tobacco, tobacco extracts, cut tobacco, cut fillers, flue-cured tobacco, expanded tobacco, homogenized tobacco, reconstituted tobacco, and pipe tobacco. Smoking articles and smokable articles are types of aerosol-forming devices. Examples of smoking articles or smokable articles include cigarettes, cigarillos, and cigars. Examples of smokeless products include chewing tobacco and snuff. In certain aerosol-forming devices, rather than combustion, the tobacco composition or another aerosol-forming material is heated by one or more electric heating elements to generate an aerosol. In another type of heated aerosol-forming device, the aerosol is generated by transferring heat from a combustible fuel element or heat source to a physically separated aerosol-forming material that may be located within, around, or downstream of the heat source. Smokeless tobacco products and various tobacco-containing aerosol-forming materials may contain tobacco in any form, including dry particles, pieces, granules, powders, or slurries, as well as deposited on, mixed with, surrounded by, or combined with other components in any form, such as flakes, films, tabs, foams, or beads. The term "smoke" is used to describe the type of aerosol generated by smoking articles such as cigarettes or by burning aerosol-forming materials.

[0274] In one embodiment, dried plant material derived from the mutant, transgenic, and non-natural plants described herein is also provided. Processes for drying fresh tobacco leaves are known to those skilled in the art and include, but are not limited to, air drying, flame drying, hot air tube drying, and sun drying, as described herein.

[0275] In another embodiment, described herein is a tobacco product that includes a tobacco-containing aerosol-forming material, including plant material (e.g., leaves, preferably dried leaves) derived from a mutant tobacco plant, a transgenic tobacco plant, or a non-natural tobacco plant described herein. The tobacco product described herein can be a blended tobacco product, which can further include unmodified tobacco.

[0276] 12. Products and methods for crop management and agriculture Mutant, non-native, or transgenic plants may have other uses, for example, in agriculture.

[0277] The present disclosure also provides a method for producing seeds, including cultivating mutant, non-natural, or transgenic plants as described herein, and collecting seeds from cultivated plants.Seeds from plants as described herein can be prepared by means known in the art and packaged in packaging material to form an article of manufacture.Packaging materials such as paper and cloth are well known in the art.The seed package can have a label, such as a tag or label fixed to the packaging material, a label printed on the package that describes the nature of the seeds contained therein, and the like.

[0278] Compositions, methods, and kits for genotyping plants for identification, selection, or breeding can include means for detecting the presence of NtINV or NtINV and NtSUS polynucleotides in a sample of polynucleotides. Thus, compositions are described as comprising one or more primers for specifically amplifying at least a portion of one or more NtINV or NtINV and NtSUS polynucleotides, and optionally one or more probes, and optionally one or more reagents for amplification or detection.

[0279] Thus, gene-specific oligonucleotide primers or probes are disclosed that comprise about 10 or more contiguous polynucleotides corresponding to the NtINV or NtINV and NtSUS polynucleotides described herein. Such primers or probes may comprise or consist of more than about 15, 20, 25, 30, 40, 45, or 50 contiguous polynucleotides that hybridize (e.g., specifically hybridize) to the NtINV or NtINV and NtSUS polynucleotides described herein. In some embodiments, the primers or probes may comprise or consist of about 10-50 contiguous nucleotides, about 10-40 contiguous nucleotides, about 10-30 contiguous nucleotides, or about 15-30 contiguous nucleotides that may be used in sequence-dependent methods of gene identification (e.g., Southern hybridization) or isolation (e.g., in situ hybridization of bacterial colonies or bacteriophage plaques), or gene detection (e.g., as one or more amplification primers in amplification or detection). One or more specific primers or probes can be designed and used to amplify or detect some or all of the polynucleotides. As a specific example, two primers can be used in a PCR protocol to amplify a polynucleotide fragment. PCR can also be performed using one primer derived from the polynucleotide sequence and a second primer that hybridizes to a sequence upstream or downstream of the polynucleotide sequence (e.g., a promoter sequence, the 3' end of a pre-mRNA, or a sequence derived from a vector). Examples of temperature and isothermal techniques useful for in vitro amplification of polynucleotides are well known in the art. The sample can be or can be derived from a plant, plant cell, or plant material, or a tobacco product produced or derived from a plant, plant cell, or plant material as described herein.

[0280] In a further aspect, a method for detecting NtINV or NtINV and NtSUS polynucleotides (or any combination thereof as described herein) in a sample is also provided, the method comprising the steps of: (a) providing a sample containing or suspected to contain a polynucleotide; (b) contacting said sample with one or more primers or one or more probes to specifically detect at least a portion of the NtINV or NtINV and NtSUS polynucleotide; and (c) detecting the presence of an amplification product, the presence of which indicates the presence of the NtINV or NtINV and NtSUS polynucleotide in the sample. In a further aspect, the use of one or more primers or probes to specifically detect at least a portion of the NtINV or NtINV and NtSUS polynucleotide is also provided. A kit for detecting at least a portion of the NtINV or NtINV and NtSUS polynucleotide is also provided, which comprises one or more primers or probes to specifically detect at least a portion of the NtINV or NtINV and NtSUS polynucleotide. The kits may include reagents for polynucleotide amplification (e.g., PCR) or probe hybridization detection techniques (e.g., Southern blot, Northern blot, in situ hybridization, or microarray). The kits may include reagents for antibody binding detection techniques such as Western blot, ELISA, SELDI mass spectrometry, or test strips. The kits may include reagents for DNA sequencing. The kits may include the reagents and instructions for using the kit.

[0281] In some embodiments, the kits may include instructions for one or more of the described methods. The described kits may be useful for determining genetic identity, phylogenetic studies, genotyping, haplotypes, pedigree analysis, or plant breeding, particularly in co-dominance assessments.

[0282] The present disclosure also provides a method for genotyping plants, plant cells, or plant materials containing NtINV or NtINV and NtSUS polynucleotides described herein. Genotyping provides a means of distinguishing between chromosomal pair homologs and can be used to identify segregants in a population of plants. Molecular marker methods can be used for phylogenetic studies, characterizing genetic relationships between crop varieties, identifying hybrids or somatic hybrids, localizing segments of chromosomes that affect single genetic traits, map-based cloning, and quantitative inheritance studies. Specific methods of genotyping may employ any number of molecular marker analysis techniques, including amplified fragment length polymorphism (AFLP). AFLP is the product of allelic differences between amplified fragments due to polynucleotide variability. Thus, the present disclosure further provides a means for tracking the segregation of one or more genes or polynucleotides, as well as chromosomal sequences genetically related to these genes or polynucleotides, using techniques such as AFLP analysis.

[0283] 13. Tobacco extract Also disclosed herein are methods of producing a liquid tobacco extract, and the liquid tobacco extract produced by the methods.

[0284] A particular extraction temperature is preferably selected for a tobacco starting material based on at least the reducing sugar content and optionally the nicotine content of the tobacco starting material. The extraction temperature is typically selected 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 some control over the composition of the extract derived from the tobacco starting material. Preferably, each tobacco starting material is heated at the extraction temperature for at least about 90 minutes, more preferably at least about 120 minutes. The heating step is typically carried out in an inert atmosphere. Preferably, a flow of inert gas, such as nitrogen, is passed through the tobacco starting material during the heating step. Volatile tobacco compounds are released into the inert gas flow during the heating step such that the inert gas acts as a carrier for the volatile components. The inert gas flow may have a flow rate of at least about 25 liters / minute, more preferably at least about 30 liters / minute. A relatively high flow rate of the inert gas may advantageously improve the efficiency of the extraction from the tobacco starting material. Optionally, the heating step may be carried out under vacuum. Suitable heating methods for carrying out the heating of the tobacco starting material are known to those skilled in the art and include carbonization, steam distillation, vacuum distillation, flash distillation, and thin film steam distillation.

[0285] If the volatile compounds are collected by absorption in a liquid solvent, the step of forming a liquid tobacco extract can include drying the solution of the volatile compounds in the liquid solvent to concentrate the solution. Drying can be carried out using any suitable means, including, but not limited to, desiccation, molecular sieving, freeze-drying, phase separation, distillation, membrane permeation, controlled crystallization and filtration of water, reverse hygroscopicity, ultracentrifugation, liquid chromatography, reverse osmosis, or chemical drying.

[0286] 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 is disclosed that includes the composition or formulation or gel composition. In such an aerosol generating system, the composition or formulation or gel is typically heated in an aerosol generating device, such as a device that includes a heater element that interacts with the composition or formulation or gel incorporating the liquid tobacco extract to generate an aerosol. During use, volatile compounds are released by heat transfer and are entrained in the air drawn through the aerosol generating device. As the released compounds cool, they condense to form an aerosol, which is inhaled by the consumer.

[0287] The present invention is further described in the following examples, which are provided to further illustrate the invention. These examples describe preferred modes currently contemplated for carrying out the invention and are intended to illustrate, but not limit, the invention. EXAMPLES

[0288] Example 1 - Materials and Methods DNA extracts and plant genotypes Leaf samples are extracted using BioSprint 96 (Qiagen, Hilden, Germany) in conjunction with the BioSprint 96 DNA Plant Kit (Qiagen, Hilden, Germany). To determine the plant genotype, DNA samples are used in TaqMan reactions. TaqMan is performed using an ABI PRISM 7900HT Sequence Detection System (Applied Biosystems, Life Technologies, Foster City, CA, USA) and TaqMan Fast Advanced Master Mix (Applied Biosystems, Life Technologies, Foster City, CA, USA).

[0289] Determination of free amino acid content Amino acid content can be measured using various methods known in the art. One such method is Method MP 1471 rev 5 2011, Resana, Italy: Chelab Silliker Srl, Merieux NutriSciences Company. For amino acid determination in dried plant leaves, the dried leaf pieces are optionally dried at 40°C for 2-3 days after removing the mid-vein. The tobacco material is then ground to a fine powder (about 100uM) before analysis of amino acid content. Another method for measuring amino acid content in plant material is described in UNI EN ISO 13903:2005. Measurement of free amino acid content can be performed according to UNI EN ISO 13903:2005.

[0290] Determination of reducing sugar content Reducing sugar content can be measured using a segmented flow colorimetric method developed for the analysis of tobacco samples as adapted by Skalar Instrument Co (West Chester, PA) and described in Tobacco Science 20:139-144 (1976). Measurement of reducing sugar content is also described in Coresta Recommended Method 38, CRM38, CRM and ISO 15154:2003. For reducing sugar determination in dried leaves, the dried leaf blades are dried at 40°C for 2-3 days as required after removal of the midvein. The tobacco material is then ground to a fine powder (approximately 100uM) before analysis of reducing sugars. Measurement of reducing sugar content is performed according to ISO 15154:2003.

[0291] Example 2 - Analysis of expression of NtINV4-S and NtINV4-T in plant tissues Table 1 shows that NtINV4-S and NtINV4-T are expressed in tissues throughout the plant, especially in the petals, and to a lesser extent in immature flowers, sepals, lower leaves, middle leaves, and upper leaves in field-grown Virginia tobacco plants. In contrast, NtINV3-S and NtINV3-T have very low expression in the tissues examined in field-grown Virginia tobacco plants, with very low but only significant expression in immature flowers.

[0292] Example 3 - Release of reducing sugars (glucose and fructose) during Virginia hot air drying time During the curing process of Virginia (flue-cured) tobacco, reducing sugars, mainly glucose and fructose, are strongly increased in yellowing leaves, reaching maximum levels after 1-2 days (see Figure 1A). Interestingly, expression of NtINV4-S and NtINV4-T increases approximately two-fold (log2) and reaches a plateau after 1 or 2 days (see Figure 1A). NtINV4-T is more extensively expressed during curing than NtINV4-S (see Figure 1B and Table 2). Both copies of NtINV3 are not significantly expressed in cured leaves (see Figure 2B) or green leaves (see Table 1). As a control, SAG12, a general marker of senescence in plants, is fully expressed after 1 day of curing (see Figure 1C).

[0293] Example 4 - Release of reducing sugars (glucose and fructose) during air-curing time in dark tobacco To determine whether the induction of NtINV4-S and NtINV4-T is not specific to Virginia tobacco, we study the expression data in dark tobacco. In this case, leaves are air-dried. During the first 120 h, samples are collected after 24 h, 48 h, 96 h, and 120 h, frozen, lyophilized, and submitted for metabolic analysis. As seen in Virginia tobacco leaves, glucose and fructose (reducing sugars) increase 4-6 fold from 0 h to 120 h of drying (end of the yellowing stage) (see Figure 2). In fully dried leaves (about 10 days after the start of drying), glucose and fructose levels decrease in the leaf matrix as in flue-cured tobacco (compare Figures 1 and 2), but to a greater extent. Sucrose remains almost stable during the first 120 h of drying, thereby suggesting that the supply of sucrose molecules during leaf yellowing is maintained in dark tobacco. At the end of drying, sucrose is fully hydrolyzed and therefore likely fully metabolized (see Figure 2C).

[0294] In the same samples, frozen leaf material is used to isolate RNA and analyze the expression of NtINV3-S, NtINV3-T, NtINV4-S, and NtINV4-T. As observed in flue-cured tobacco, the NtINV3 gene is not expressed in harvested leaves, nor is it upregulated during air-curing of dark tobacco. On the other hand, NtINV4-S and NtINV4-T are upregulated by a factor of more than 10-fold, reaching a maximum after 5 days of curing (120 h, see Table 2). As observed in flue-cured tobacco, SAG12 is more rapidly upregulated and then decreases after 5 days of curing (120 h).

[0295] Example 5 - Silencing of NtINV4 in flue-cured tobacco Silencing of NtINV4 in flue-cured tobacco is investigated to determine whether these genes contribute to the reduction of reducing sugar levels in cured tobacco leaves. Specific DNA fragments (SEQ ID NO: 9) within the coding sequences of both NtINV4-S and NtINV4-T are cloned with a strong constitutive Mirabilis mosaic virus (MMV) promoter in a Gateway vector. The NtINV4 gene fragment is flanked between the MMV and the 3'nos terminal sequence of the nopaline synthase gene of Agrobacterium tumefaciens. Tobacco line K326 is transformed using a standard Agrobacterium-mediated transformation protocol. Results regarding INV4 expression levels of four control and four transgenic 35S:INV4-RNAi lines are shown in Figure 3. To allow the selection of plants with low reducing sugar content, after 48 hours of curing treatment, independent T0 plant leaves and the respective control lines are analyzed to determine the effect on the reduction of reducing sugar content. The best T0 line showing the lowest level of NtINV4 expression compared to the control line is selected by qPCR. Seeds are harvested from these best TO lines. Manipulation of the NtINV4 gene (e.g., using either a constitutive promoter or a specific senescence promoter such as SAG12 or E4) can alter the chemistry of tobacco cured leaves. Similarly, knocking out NtINV4 genes using genome editing strategies (e.g., CRISPR or mutant selection) can alter the amino acid leaf chemistry of major commercial tobacco varieties.

[0296] Example 6 - Analysis of glucose, fructose, and sucrose in drought leaves of four control and four transgenic 35S:INV4-RNAi lines Five mid-position leaves of four control and four transgenic 35S:INV4-RNAi lines illustrated in Figure 3 are collected at maturity and subjected to hot air drying. Sugars (glucose, fructose, and sucrose) are analyzed in complete dried leaves. Data presented in Figure 4 show a strong and significant reduction of glucose and fructose in anti-INV4 plants. Glucose and fructose levels are reduced by about 63% and about 43%, respectively. Interestingly, sucrose is significantly 4.1-fold higher in the 35S:INV4-RNAi lines, thereby suggesting that a portion of the sucrose pool that accumulates during leaf drying is not hydrolyzed by the INV4 gene in the silenced lines. No impact on visual plant fitness and variation in total free amino acids is observed between the control and 35S:INV4-RNAi lines.

[0297] Knocking out or downregulating the expression of NtINV4-S and NtINV4-T may contribute to the reduction of reducing sugar content in dried leaves. To further reduce the amount of reducing sugars, a combination of knocking out or downregulating NtSUS and NtINV may be considered. To increase the pool of reducing sugars in dried leaves, overexpression of NtINV4-S, or NtINV4-T, or a combination thereof, using senescence-inducible promoters such as SAG12 or E4 may be considered (the use of constitutive promoters may change the plant metabolism at the vegetative stage).

[0298] Example 7 - Identification of SUS genes in Burley, Virginia, and Orient tobacco leaves after curing treatment To identify key functions contributing to sucrose metabolism during early curing processing times in Burley, Virginia, and Orient tobacco leaves, an overrepresentation analysis (log2 fold change >2, adjusted p-value <0.05) of the functions of upregulated genes in cured leaves after 48 hours of curing processing compared to mature leaves at harvest is performed in Burley, Virginia, and Orient tobacco. Genes involved in the production of reducing sugars that are active after 48 hours of curing processing are identified, independent of the type of curing processing and tobacco variety. Tobacco genes involved in the production of reducing sugars are identified.

[0299] The major genes directly involved in the production of reducing sugars during early desiccation in leaves belong to the SUS gene family, which is likely to be the key enzyme for promoting the accumulation of reducing sugars in desiccation-treated detached leaves.

[0300] The tobacco genome has been found to have 12 NtSUS gene products distributed into six families with one S and one T copy from each ancestor: NtSUS1-S (SEQ ID NO:10), NtSUS1-T (SEQ ID NO:12), NtSUS2-S (SEQ ID NO:14), NtSUS2-T (SEQ ID NO:16), NtSUS3-S (SEQ ID NO:18), NtSUS3-T (SEQ ID NO:20), NtSUS4-S (SEQ ID NO:22), NtSUS4-T (SEQ ID NO:24), NtSUS5-S (SEQ ID NO:26), NtSUS5-T (SEQ ID NO:28), NtSUS6-S (SEQ ID NO:30), and NtSUS6-T (SEQ ID NO:32).

[0301] The SUS transcripts are derived from the genomic sequences NtSUS2-S (SEQ ID NO: 14), NtSUS3-S (SEQ ID NO: 18), NtSUS3-T (SEQ ID NO: 20), and NtSUS4-S (SEQ ID NO: 22). These genes are upregulated during leaf desiccation treatment (senescence) as shown in Table 3. This confirms that the S copies are specifically involved in the chemical modification of early desiccation leaves, in this particular case, the increase in glucose and fructose.

[0302] Although lower reducing sugar levels were found in burley dried leaves compared to Virginia and Oriental, the NtSUS gene was nevertheless activated in burley (see Table 3), likely as a structural response, to ensure an available carbon source for amino acid synthesis during the early drought treatment stages.

[0303] In both burley (BU) and Virginia (FC), NtSUS1-S and NtSUS1-T, which are not expressed during early drought treatment (see Table 3), are specifically expressed in roots and stems, indicating possible specific functions in these tissues to deliver carbohydrates for cell wall synthesis or to supply carbon sources under anoxia (see Table 4). On the other hand, NtSUS3-S, NtSUS3-T, and NtSUS4-S, which are induced during early leaf drought treatment, are also expressed in all organs, whereas NtSUS2-S and NtSUS2-T are mainly expressed in immature flowers and petals. NtSUS5-S, NtSUS5-T, NtSUS6-S, and NtSUS6-T are expressed at low levels in all analyzed plant tissues (see Table 4).

[0304] To increase the pool of reducing sugars in dried leaves, overexpression of NtSUS2-S, NtSUS3-S, NtSUS3-T, or NtSUS4-S, or combinations thereof, using senescence-inducible promoters such as SAG12 or E4 could be considered (the use of constitutive promoters may strongly alter plant metabolism). On the other hand, knocking out NtSUS2-S, NtSUS3-S, NtSUS3-T, and / or NtSUS4-S could contribute to a decrease in the content of reducing sugars in dried leaves.

[0305] Example 8 - Silencing of NtSUS expression in Virginia tobacco leaves Silencing of NtSUS in burley tobacco is investigated to determine whether these genes contribute to the reduction of reducing sugar content in cured Virginia tobacco leaves. Specific DNA fragments within the coding sequences of both NtSUS are cloned under the strong constitutive Mirabilis mosaic virus (MMV) promoter in the Gateway vector. The NtSUS gene fragments are flanked between the MMV and the 3'nos terminal sequence of the Agrobacterium tumefaciens nopaline synthase gene.

[0306] To allow the selection of plants with low reducing sugar content, after 60 hours of drought treatment, independent T0 plant leaves and each control line are analyzed to determine the effect on reducing sugar content. The best T0 lines that show the lowest levels of reducing sugars are selected. Seeds are harvested from these best T0 lines. T1 progeny are analyzed by qPCR to determine the efficiency of the NtSUS silencing event in relation to the reduction of reducing sugar content.

[0307] Manipulation of the NtSUS gene (e.g., using either a constitutive promoter or a specific senescence promoter such as SAG12 or E4) can alter the chemistry of dried tobacco leaves. Similarly, knocking out the NtSUS gene using genome editing strategies (e.g., CRISPR or mutant selection) can alter the amino acid leaf chemistry of major commercial tobacco varieties.

[0308] Example 9 - Producing liquid tobacco extracts from NtINV4 and NtSUS modified tobacco plants, each with regulated reducing sugar content The tobacco starting material is prepared from dried leaves of an NtINV4 modified tobacco plant or an NtSUS modified tobacco plant according to the present disclosure. The tobacco material is cut to form tobacco pieces having dimensions of about 2.5 millimeters by about 2.5 millimeters, and the tobacco pieces are loaded into an extraction chamber without compression. The tobacco starting material is heated in the extraction chamber. During heating, a flow of nitrogen passes through the extraction chamber at a flow rate of about 40 liters per minute. For each tobacco starting material, the volatile compounds released during the heating process are collected by absorbing them into a liquid solvent formed from propylene glycol at minus 10 degrees Celsius and agitation of 750 rpm. The solution of propylene glycol with the collected volatile compounds is dried in a drying process to reduce the moisture level of the solution to about 15 percent. A concentrated solution of volatiles recovered from the tobacco starting material is collected.

[0309] A combined liquid tobacco extract can be prepared. For each of the tobacco starting materials processed as described above, the first tobacco starting material is heated at a different temperature and duration than the second tobacco starting material. For each tobacco starting material, the volatile compounds released during the heating process are collected and dried. The resulting concentrated solutions of volatiles collected from the first and second tobacco starting materials can be combined in a defined ratio to produce a liquid tobacco extract.

[0310] Any publications cited or described herein provide relevant information disclosed prior to the filing date of this application. Nothing herein should be construed as an admission that the inventors are not entitled to prior to such disclosure. All publications mentioned in the above specification are incorporated herein by reference. Various modifications and variations of the present invention will become apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with certain preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described methods for carrying out the invention that are apparent to those skilled in the art of cell biology, molecular biology, and plant biology, or related fields, are intended to be within the scope of the following claims.

[0311] [Table 1]

[0312] [Table 2]

[0313] [Table 3]

[0314] [Table 4]

[0315] Sequence Listing SEQ ID NO:1: Polynucleotide sequence of NtINV3-S SEQ ID NO:2: Polypeptide sequence of NtINV3-S MAETNNSVPYTQLPAEDNNTSVNSPAGCRLRPKRVSFIVLTGLVAALLLFVAVKYGKNEAEDVNPGPVPPQETVCNMLGSNLMPLTSMKTVARGVAEGVSAKSRGRFLGLRPFPWTKQMLAWQRTSFHFQPKKNWMNDPNGPLFYKGWYHLFYQYNPEAAVW GNIVWGHAVSRDLIHWQHLPVAMVADQWYDINGVWTGSATILPDGKLVMLYTGSTNESVQVQNLAYPADPSDPLLIKWVKYEGNPVLVPPPGIAAKDFRDPTTAWTTPQGKWRITIGSKVNKTGISLVYDTIDFKNFELLDGVLHGVSGTGMWECVDFYPVS KVVENGLDTSDNGPAVKHVLKSSLDDDRNDYYALGTYDAVAGKWVPDNPTIDVGIGLRYDYGNFYASKTFYDQEKKRRVLWAWITESDSEAADICKGWASLQPIPRTIKYDKKTGSNIITWPVAEVENLRFNSKEFDKVEVKPGNVVPLEVGTATQLDIMAE FEVDPKVLEKLEGSNATYECRSSGGSAERGALGPFGLLVLTDKGLSEQTPIYFYIAKDAAGNFTTFFCNDLTRSSEATDVRKLIYGSTVPVLQGEKLSLRTLVDHSIVESFAQNGRTAITSRLYPTKAIYEDAKLYLFNNATDVTITASVKIWQIHSANIQSS SEQ ID NO: 4: Polypeptide sequence of NtINV3-T MAETNNSVPYTQLPAEDNNTSSNSPAKCRRRPKRVSFIVLTGLVAALLLFVAVKYGNNEAEDVNPGPVPPQETVCNMLGSNLMPLTTMRTVARGVAEGVSAKSRGRFLGLRPFPWTKQMLAWQRTSFHFQPKKNWMNGPLFYKGWYHLFYQYNPEAAVWGN IVWGHAVSRDLIHWQHLPVAMVADQWYDINGVWTGSATILPDGKLVMLYTGSTNESVQVQNLAYPADPSDPLLRKWVKYEGNPVLVPPPGIATKDFRDPTTAWTTPQGKWRITIGSKVNKTGISLVYDTIDFKKFELLDGVLHGVPGTGMWECVDFYPVSK VVENGLDTSDNGPAVKHVLKSSLDDDRNDYYALGTYDAVAGKWIPDNPTIDVGIGLRYDYGNFYASKTFYDQEKKRRVLWAWITEGDSEAADICKGWASLQPIPRTIKYDKKTGSNIITWPVAEVENLRLNSKEFDKVEVKPGSVFPLEVGTATQLDIMAE FEIDPKVLERLEGNNATYECRSSGGSAERGALGPFGLLVLTDKGLSEQTPIYFYIAKDAAGNFTTFFCNDLTRSSEATDVRKLIYGSTVPVLQGEKLSLRTLVDHSIVESFAQSGRTAITSRVYPTKAIYEDAKLYLFNNATDVSTASLKIWQMNSANIQSS SEQ ID NO: 5: Polynucleotide sequence of NtINV4-S SEQ ID NO: 6: Polypeptide sequence of NtINV4-S MATHHSHYDPENSTTHYTVLPDQPESAGAGRRKSLKVVSGILLSSFFLLSLVFVILNQSSDLSQENSRSSETLTPALSRGVSQGVSEKTFKDVSGRSLSYYPWTNAMLTWQRTAYHFQPQKNWMNDPNGPLYHKGWYHLFYQYNPDSAVWGNITWGHAIS TDLIHWLYLPFAMVPDQWYDINGVWTGSATILPDGQIMMLYTGDTNDYVQVQNLAYPANLSDPLLIDWVKYQDNPVMVPPPGIGVKDFRDPTTAWTGPQNGQWLLTIGSKIGKTGIALVYDTSNFTNFKLLDGVLHAVPGTGMWECVDFYPVSTVEANGLD TSYNGPGIKHVLKASLDDDKHDYYAIGTYDPVKNKWTPDNPELDVGIGLRLDYGKYYASKTFYDPKEQRRILWGWIGETDSEAADLLKGWASVQSIPRTLLYDKETRTHVLQWPVKEIESLRIGDPLVKQVNLQPGSIELVHVDSAAQLDVEASFEVDKA ALAGTIEADVGFNCSTSGGAAKRGILGPFGVVVIADQTLSELTPVYFYIAKGTGGRAETYFCADETRSSEAPGVAKQVYGSSVPVLDGEQHSMRLLVDHSIVESFAQGGRTVITSRIYPTKAINGAARFVFNNATGASVTASLKIWSLKSADIRSFPLDQL SEQ ID NO: 7: Polynucleotide sequence of NtINV4-T SEQ ID NO: 8: Polypeptide sequence of NtINV4-T MATHHSHYDPENSTTHYTVLPDQPESAGSGHRKSLKVVSGILLSSFFLLSLVFVIVNQSSDLSQKNSHSSETLTPALSRGVSQGVSEKTFRDVSGGSLSYYPWTNAMLTWQRTAYHFQPQKNWMNGPLYHKGWYHLFYQYNPDSAIWGNITWGHAISTD LIHWLYLPFALVPDQWYDINGVWTGSATFLPDGQIMMLYTGDTNDYVQVQNLAYPANLSDPLLIDWVKYRGNPVMVPPPGIGVKDFRDPTTAWTGPQNGQWLLTIGSKIGKTGIAIVYGTSNFTNFKLLDGVLHAVPGTGMWECVDFYPVSTDEANGLDT SYNGPGIKHVLKASLDDDKHDYYAIGTYDPVKNKWTPDNPQLDVGIGLRLDYGKYYASKTFYDPKEQRRILWGWIGETDSEAADLLKGWASVQSIPRTVLYDKETRTHVLQWPVKEIESLRIGDPLVKRVNLQPGSIELVHVDSAAQLDVEASFEVDKA ALEGTIEADVGFNCSTSGGAAKRGILGPFGVVVIADQTLSELTPVYFYIAKGPDGRAETYFCADETRSSEAPGVAKQVYGSSVPVLDDEQHSMRLLVDHSIVESFAQGGRTVITSRIYPTKAINGAARLFVFNNATRSVTASLKIWSLESADIRSFPLDQ SEQ ID NO: 9: Nucleotide sequence used for silencing NtINV4-T and NtINV4-S ggtttcaactgcagtactagtggaggtgctgctaaaagaggcattttgggaccatttggtgtcgttgtaattgctgatcaaacgctttctgagctaac SEQ ID NO: 10: Polynucleotide sequence of NtSUS1-S SEQ ID NO: 11: Polypeptide sequence of NtSUS1-S MAASGLSIKKSLEESILAHPDEILALKSRIETEGKGVMKPLDLLNHLVSVTSKTNGVNIVPSALVEVLSCSQEAVIVPPKLALAVRPRPGVWEYLSLNLKTKKVAELSIPEYLQLKENTVDESGNILELDFEPFTTVTPPKTLSDSIGNGLEFLNRHIASKMFHDKEISRCLLDFLRNHNYKGKSLMVKESIQSLESFQL VLKKAEEHLCTLNPETPYSNFESKFEEIGLERGWGNTAERVQDTISHLLHLLEAPNASSLENFLGRIPLVFNVVILTPHGYFAQDNVLGYPDTGGQVVYILDQVPAMEREMLHRMKLQGLDDIIPRILVVTRLLPDAVGTTCGERMEKVYGAEHSHIIRVPFRTEKGMLRKWISRFEVWPYMETFTEDVAEELVKELQAKP DLIIGNYSEGNLAASLLAKKFGATQCTIAHALEKTKYPNSDLNWKKFDDKYHFSSQFTADLFAMNHTDFIITSTFQEIAGSKNTVGQYESHTAFTMPGLYRVVHGIDSFDPKFNIVSPGADMSIYFPYTEKEKRLTNFHPEIEELLYSPVENKDHLCVLKDRNKPILFTMARLDRVKNLTGLVEWYAKNARLRELVNLVVV GGDRRKESKDLEEQAEMKKMYDLIETYNLNGQFRWISSQMNRVRNGELYRYIADTRGAFVQPAFYEAFGLTVVESMTCGLPTFATCNGGPFEIIVNGKSGFHIDPNQGDKAADMLVNFFEKSKEDPSYWDAISKGGLQRILEKYTWQIYSQKVITLSGIYGFWKYATKNDKVASAKKRYLEMFYELGFKKSAEKVPLAIDE SEQ ID NO: 12: Polynucleotide sequence of NtSUS1-T SEQ ID NO: 13: Polypeptide sequence of NtSUS1-T MAGSGLSIKESLEESILAHPDEILALKSRIETEGKGVMKPVDLLNHLVSVTSKTNGVNVVPSALVEVLSCSQEAVIVPPKLALAVRPRPGVWEYLSLNLKTKKVAELSIPEYLQLKENTVDESGNILELDFEPFTTVTTPKTLSDSIGNGLEFLNRHIASKMFLDKEIAKCLLDFLRNHNYKGKSLMVKESIQSLESFQL VLKKAEEYLHTLNPETPYSKFESKFEEIGLERGWGNTAERVQDTISHLLHLLEAPNASSLENFLGRIPLVFNVVILTPHGYFAQDNVLGYPDTGGQVVYILDQVPAMEREMLHRMKLQGLDDIIPRILVVTRLLPDAVGTTCGEWMEKVYGAEHSHIIRVPFRTEKGMLRKWISRFEVWPYMETFTEDVAEELVKELQAKP DLIIGNYSEGNLAASLLAKKFGATQCTIAHALEKTKYPNSDLNWKKFDDKYHFSSQFTADLFAMNHTDFIITSTFQEIAGSKNTVGQYESHTAFTMPGLYRVVHGINSFDPKFNIVSPGADMSIYFPYTEKEKRLTNFHPEIEELLYSPVENKDHLCVLKDQNKPILFTMARLDRVKNLTGLVEWYAKNARLRELVNLVVV GGDRRKESKDLEEQAEMKKMYDLIETYNLNGQFRWISSQMNRVRNGELYRYIADTRGAFVQPAFYEAFGLTVVESMTCGLPTFATCNGGPFEIIVNGKSGFHIDPNQGDKAADMLVNFFEKSKEDPSYWDTISKGGLQRILEKYTWQIYSQKVITLSGIYGFWKYATKNDKVASAKKRYLEMFYEFGFKKSAEKVPLAIDE SEQ ID NO: 14: Polynucleotide sequence of NtSUS2-S SEQ ID NO: 15: Polypeptide sequence of NtSUS2-S MAERALTRVHSLRERLDATLAAHRNEILLFLSRIESHGKGILKPHQLLAEFDAIRQDDKKKLNDHAFEELLKSTQEAIVLPPWVALAIRLRPGVWEYVRVNVNALVVEELTVPEYLHFKEELVDGTSNGNFVLELDFEPFTASFPKPTLTKSIGNGVEFLNRHLSAKMFHDKESMTPLLEFLRVHNYKGKTMMLNDRIQNL TTLQNVLRKAEEYLIMLPPETPFSEFEHKFQEIGLEKGWGDTAERVLEMICMLLDLLEAPDSCTLEKFLGRIPMVFNVVILSPHGYFAQENVLGYPDTGGQVVYILDQVPALEREMLKRLKEQGLDITPRILIVTRLLPDAVGTTCGQRLEKVYGAEHSHILRVPFRTEKGIVRKWISRFEVWPYMETFTEDVAKELAAEL QAKPDLIIGNYGNLVASLLAHKLGVTQCTIAHALEKTKYPDSDIYWKKFDEKYHFSSQFTADLIAMNHTDFIITSTFQEIAGSKDTVGQYESHQAFTMPGLYRVVHGIDVFDPKFNIVSPGADINLYFPYSEKEKRLTALHPEIEELLYSDVENEEHLCVLKDRNKPILFTMARLDRVKNLTGLVEWYAKNARLRELVN LVVVGGDRRKESKDLEEQAEMKKMYELIKTHNLNGQFRWISSQMNRVRNGELYRYIADTRGAFVQPAFYEAFGLTVVEAMTCGLPTFATNHGGPAEIIVNGKSGFHIDPYHGEQAADLLADFFEKCKTEPSHWETISTGGLKRIQEKYTWQIYSERLLTLAAVYGFWKHVSKLDRLEIRRYLEMFYALKYRKMAEAVPLAAE SEQ ID NO: 16: Polynucleotide sequence of NtSUS2-T SEQ ID NO: 17: Polypeptide sequence of NtSUS2-T MLFMGLKQLSEDFSPAESTAMAERVLTRVHSLRERLDATLAAHRNEILLFLSRIESHGKGILKPHQLLAEFDAIRQDDKKKLNDHAFEELLKSTQEAIVLPPWVALAIRLRPGVWEYVRVNVNALVVEELTVPEYLHFKEELVDGTSNGNFVLELDFEPFTASFPKPTLTKSIGNGVEFLNRHLSAKMFHDKESMTPLLEFLRVHN YKGKTMMLNDRIQNLTTLQNVLRKAEEYLIMLPPETPFSEFEHKFQEIGLEKGWGDTAERVLEMICMLLDLLEAPDSCTLEKFLGRIPMVFNVVILSPHGYFA QENVLGYPDTGGQVVYILDQVPALEREMLKRLKEQGLDITPRILIVTRLLPDAVGTTCGQRLEKVYGAEHSHILRVPFRTEKGIVRKWISRFEVWPYMETFTE DVAKELAAELQAKPDLIIGNYSEGNLVASLLAHKLGVTQCTIAHALEKTKYPDSDIYWKKFDEKYHFSSQFTADLIAMNHTDFIITSTFQEIAGSKDTVGQYESHQAFTMPGLYRVVHGIDVFDPKFNIVSPGADINLYFPYSEKEKRLTALHPEIEELLYSDIENEEHLCVLKDRNKPILFTMARLDRVKNLTGLVEWYAKNARL RELVNLVVVGGDRRKESKDLEEQTEMKKMYELIKTHNLNGQFRWISSQMNRVRNGELYRYIADTRGAFVQPAFYEAFGLTVVEAMTCGLPTFATNHGGPAEII VNGKSGFHIDPYHGEQAADLLADFFEKCKTEPSHWETISTGGLKRIQEKYTWQIYSERLLTLAAVYGFWKHVSKLDRLEIRRYLEMFYALKYRKMAEAVPLAAE SEQ ID NO: 18: Polynucleotide sequence of NtSUS3-S SEQ ID NO: 19: Polypeptide sequence of NtSUS3-S MANPKFTRVPSMRERVEDTLSAHRNQLVALLSRYVAQGKGILQPHHLIDEFNNAVCDDTACEKLKDGPFSEVLKATQEAIVLPPFVAIAVRPRPGVWEYVRVNVYDLSVEQLTVPEYLHFKEELVDGEGNNHFVLELDFEPFNASVPRPSRSSSIGNGVQFLNRHLSSIMFRSKDSLDPLLDFLRGHCHKGNVLMLNDRIQR ISRLESALSKAEDYLSKLSPDTSYNEFEYALQEMGFERGWGDTARRVLETMHLLSDILQAPDPSTLETFLGRLPMVFNVVILSPHGYFGQANVLGLPDTGGQVVYILDQVRALEAEMLLRIKQQGLNFKPRILVVTRLIPDAKGTMCNQRLERISGTEYSHILRVPFRTEKGILHKWISRFDVWPYLEKFTEDVASEMTAELQ GKPDLIIGNYSDGNLVASLLAYKMGVTQCTIAHALEKTKYPDSDIYWKKFEEKYHFSCQFTADLAMNSDFIITSTYQEIAGTKNTVGQYESHTAFTLPGLYRVVHGIDVFDPKFNIVSPGADMTIYFPYSDKEKRLTSLHGSIEKLLFDPAQNEEHIGNLNDKSKPIIFSMARLDHVKNITGLVECYAKNATLRELANLV VVAGYNDVKKSSDREEITEIEKMHALIKEHKLDGQFRWVSAQTNRARNGELYRYIADQRGIFVQPAFYEAFGLTVVEAMTCGLPTFATCHGGPNEIIEPGVSGFHIDPYHPDKAAELMSEFFQRCKQDPTHWEKISASGLRRILERYTWKIYSERLMTLSGVYGFWKLVSKLERRETRRYLEMFYILKFRELAKSVPLAIDDK SEQ ID NO: 20: Polynucleotide sequence of NtSUS3-T SEQ ID NO: 21: Polypeptide sequence of NtSUS3-T MFTWLKLNIKNKGRKNTVGQYESHTAFTLPGLYRVVHGIDVFDPKFNIVSPGADMTIYFPYSDKEKRLTSLHGSIEKLLFDPAQNEEHIGNLNDKSKPIIFSMARLDHVKNITGLVECYAKNATLRELANLVVVAGYNDVKKSSDREEIAEIEKMHALIKEHKLDGQ FRWIAAQTNRARNGELYRYIADKRGIFVQPAFYEAFGLTVVEAMTCGLPTFATCHGGPNEIIEHGVSGFHIDPYHPDKAAELMAEFFQRCKQDPTHWEKISASGLRRILERYTWKIYSERLMTLSGVYGFWKLVSKLERRETRRYLEMFYILKFRELAKSVPLAIDDK SEQ ID NO: 22: Polynucleotide sequence of NtSUS4-S SEQ ID NO: 23: Polypeptide sequence of NtSUS4-S MAERVLTRVHSLRERLDATLAAHRNEILLFLSRIESHGKGILKPHQLLAEFDSIHKEDKNKLNDHAFEEVLKSTQEAIVLSPWVALAIRLRPGVWEYVRVNVNALVVEELTVPEYLQFKEELVNGTSHDNFVLELDFEPFTASFPKPTLTKSIGNGVEFLNRHLSAKMFHDKESMTPLLEFLRVHHYKGKTMMLNDRIQDLN TLQNVLRKAEEYLTTLSPETSYSAFEHKFQEIGLERGWGDTAERVLEMICMLLDLLEAPDSCTLEKFLGRIPMVFNVVILSPHGYFAQENVLGYPDTGGQVVYILDQVPALEREMLKRIKEQGLDIKPRILIVTRLLPDAVGTTCGQRLEKVFGTEHSHILRVPFRTEKGIVRKWISRFEVWPYMETFTEDVAKEIAAELQA KPDLIIGNYSEGNLAASLLAHKLGVTQCTIAHALEKTKYPDSDIYLKKFDEKYHFSAQFTADLIAMNHTDFIITSTFQEIAGSKDTVGQYESHMAFTMPGLYRVVHGIDVFDPKFNIVSPGADMNLYFPYYEKEKRLTAYHPEIEELLFSDVENDEHMCVLKNRNKPIIFTMARLDRVKNLTGLVELYAKNPRLRELVNLVV VGGDRRKESKDLEEQAEMKKMYELIKTHNLNGQFRWISSQMNRVRNGELYRYIADTRGAFVQPAFYEAFGLTVVEAMTCGLPTFATNHGGPAEIIVHGKSGFHIDPYHGDQAAELLADFFEKCKKEPSHWEAISEGGLKRIQEKYTWQIYSDRLLTLAAVYGFWKHVSKLDRLEIRRYLEMFYALKFRKLVSFIAFCTPAIV SEQ ID NO: 24: Polynucleotide sequence of NtSUS4-T SEQ ID NO: 25: Polypeptide sequence of NtSUS4-T MAERVLTRVHSLRERLDATLAAHRNEILLFLSRIESHGKGILKPHQLLAEFESIHKEDKNKLNDHAFEEVLKSTQEAIVLSPWVALAIRLRPGVWEYVRVNVNALIVEELTVPEYLQFKEELVNGTSNDNFVLELDFEPFTASFPKPTLTKSIGNGVEFLNRHLSAKMFHDKESMTPLLEFLRVHHYKGKTMMLNDRVQDL NTLQNVLRKAEEYLTTLSPETSYSVFEHKFQEIGLERGWGDNAERVLEMICMLLDLLEAPDSCTLEKFLGRIPMVFNVVILSPHGYFAQENVLGYPDTGGQVVYILDQVPALEREMLKRIKEQGLDIKPRILIVTRLLPDAVGTTCGQRLEKVFGTEHSHILRVPFRTEKGIVRKWISRFEVWPYMETFTEDVAKEIAAEL QAKPDLIIGNYSEGNLAASLLAHKLGVTQCTIAHALEKTKYPDSDIYLKKFDEKYHFSAQFTADLIAMNHTDFIITSTFQEIAGSKDTVGQYESHMAFTMPGLYRVVHGIDVFDPKFNIVSPGADMNLYFPYFEKEKRLTAYHPEIEELLFSDVENDEHMCVLKDRNKPIIFTMARLDRVKNLTGLVELYAKNPRLRELVN LVVVGGDRRKESKDLEEQAEMKKMYELIKTHNLNGQFRWISSQMNRVRNGELYRYIADTRGAFVQPAFYEAFGLTVVEAMTCGLPTFATNHGGPAEIIVHGKSGFHIDPYHGEQAAELLADFFERCKKEPSHWEAISEGGLKRIQEKYTWQIYSDRLLTLAAVYGFWKHVSKLDRLEIRRYLEMFYALKFRKLAEAVPLAVE SEQ ID NO: 26: Polynucleotide sequence of NtSUS5-S SEQ ID NO: 27: Polypeptide sequence of NtSUS5-S MASTVADSMPDALKQSRYHMKRCFARFIAMGRRLMKLKHLTEEIEETIEDKAERTRILEGSLGKIMSSTQEAAVVPPYVAFAVRHNPGFWDYVKVNAETLSVEAI SAREYLKFKEMIFDEDWAKDDNALEVDFGAFDYSNPRLALSSSVGNGLNFISKVLSSKFGGKPEDAQPLLDYLLALNHQGENLMINENLNGVAKLQAALIVAEVF VSSFPKDTPYKDFEHKLKEWGFDKGWGHNAGRVRETMRLLSEIIQAPDPINMESFFSKLPTTFNIVIFSIHGYFGQADVLGLPDTGGQVVYILDQVRALEEEMLQ RIKQQGLNVKPKILVVSRLIPDARGTTCNQEMEPILNSSHSHILRIPFRTEKGVLRQWDASAKILELMEGKPDLIIGNYTDGNLVASLLANKLGVTQGTIAHALE KTKYEDSDVKWKQFDPKYHFSCQFTADLLAMNAADFIITSTYQEIAGSETRPGQYESHTAFTMPGLYRAVSGINVFDPKFNIAAPGAEQSTYFPFTEKQKRFSTFRPAINELLYSNEENNEHIGFLADRKKPIIFSMARFDTVKNLSGLTEWYGKNKKLRNLVNLVIVGGFFDPSKSKDREEAAEIKKMHELIEKYQLKGQMRWIAAQTD KYRNSELYRTIADTKGAFVQPALYEAFGLTVIEAMDCGLPTFATNQGGPAEIIVDGVSGFHIDPYNGDESSKKIADFFEKCKVDSKYWNRISEGGLKRIEECYTW KIYANKVLNMGSIYGFWRQFNVGQKQAKQRYFEMFYNPLFRKLAKSVPIPHEEPLPLATSDSTQSQELKLPLPVPAAVAKVLPLTRHAFNLITSLPRVTGKVDVK SEQ ID NO: 28: Polynucleotide sequence of NtSUS5-T SEQ ID NO: 29: Polypeptide sequence of NtSUS5-T MASTVAGSMPDALKQSRYHMKRCFARFIAMGRRLMKLKHLTEEIEKTIEDKAERTKILEGSLGKIMSSTQEAAVVPPYVAFAVRHNPGFWDYVKVDAETLSVEAISARDYLKFKEMIFDEDWAKDENALEVDFGAFDYSNHRLALSSSVGNGLNFISKVLSSKFGGKAEDAQPLLDYLLALNH QGENLMINENLNGVSKLQAALIVAEVFVSSFPKDTPYKDFEHKLKEWGFEKGWGHNAGRVRETMRLLSEIIQAPDPINMESFFSRLPTTFNIVIFSIHGYFGQADVLGLPDTGGQVVYILDQVRALEEEMLQRIKQQGLNVKPKILVVTRLIPDARGTTCNQEMEPILNSSHSHILRIPFRTE KGVLRQWDASAKILELMEGKPDLIIGNYTDGNLVASLLANKLGVTQGTIAHALEKTKYEDSDVKWKQFDSKYHFSCQFTADLLAMNAADFIITSTYQEIAGSETRPGQYESHTAFTMPGLYRAVSGINVFDPKFNIAAPGAEQSAYFPFTEKQKRFSAFRPAIEELLYSNEQNNEHIGFLADR KKPIIFSMARFDTVKNLSGLTEWYGKNKKLRNLVNLVIVGGFFDPSKSKDREEAAEIKKMHELIEKYKLKGQMRWIAAQTDKYQNSELYRTIADTKGAFVQPALYEAFGLTVIEAMNCGLPTFATNQGGPAEIIVDGVSGFHIDPYNGDESSKKIADFFEKCKVDSKYWNKICGGGLKRIEEW SEQ ID NO: 30: Polynucleotide sequence of NtSUS6-S SEQ ID NO: 31: Polypeptide sequence of NtSUS6-S MATAPALNRSESIADSMPEALRQSRYHMKKCFAKYIEQGKRMMKLHNLMDELEKVIDDPAERNHVLEGLLGYILCTTMEAAVVPPYIAFATRQNPGFWEYVKVNAND LSVEGITATEYLKFKEMIVDECWAKDEYALEIDFGAVDFSTPRLTLSSSIGNGLSYVSKFLTSKLNATSASAQCLVDYLLTLNHQGDKLMINETLSTVSKLQAALVVA EASISSLPTDTPYESFELRFKQWGFEKGWGDTAERVSDTMRTLSEVLQAPDPLNIQKFFGRVPTVFNIVLFSVHGYFGQADVLGLPDTGGQVVYVLDQVVAFEEEML QRIKQQGLNIKPQILVLTRLIPDAKGTKCNQELEPIKNTKHSHILRVPFRTEKGVLNQWVSRFDIYPYLERYTQDAADKIVELMEGKPDLIIGNYTDGNLVASLMARK LGITLGTIAHALEKTKYEDSDIKLKELDPKYHFSCQFTADLIAMNSADFIITSTYQEIAGSKDKPGQYESHSAFTLPGLYRVASGINVFDPKFNIAAPGADQSVYFP YTEKQKRLTAFRPAIEELLFSKVDNDEHVGYLEDRKKPILFTMARLDTVKNTSGLTEWYGKNKRLRSLVNLVVVGGSFDPTKSKDREEAAEIKKMHMLIEKYQLKGQI RWIAAQTDRYRNSELYRTIADSKGAFVQPALYEAFGLTVIEAMNCGLPTFATNQGGPAEIIVDGVSGFHIDPNNGDESSNKIANFFQKCREDPEYWNRISVQGLNRI YECYTWKIYANKVLNMGSIYTFWRTLYRDQKQAKQRYIETFYNLEFRNLVKNVPIRKDETPQGPKEREKVKPQISQRHALKLLPTVFQETLALTIKVAEVRLDKIATA SEQ ID NO: 32: Polynucleotide sequence of NtSUS6-T SEQ ID NO: 33: Polypeptide sequence of NtSUS6-T MATAPALKRSESIADSMPEALRQSRYHMKKCFAKYIEQGKRMMKLHNLLMDELEKVIDDPAERNHVLEGLLGYILCTTMEAAVVPPYIAFATRQNPGFWEYVKVNANDLSVEGIT ATDYLKFKEMIVDESWAKDEYALEIDFGAVDFSTPRLTLSSSIGNGLSYVSKFLTSKLNATSASAQCLVDYLLTLNHQGDKLMINETLGTVSKLQAALVVAEASISSLPTDTPY QSFELRFKQWGFEKGWGDTAERVRDTMRTLSEVLQAPDPLNIEKFFGRVPTVFNIVLFSVHGYFGQANVLGLPDTGGQVVYVLDQVVAFEEEMLQRIKQQGLNIKPQILVLTRL IPDAKGTKCNQELEPIKNTKHSHILRVPFRTEKGVLNQWVSRFDIYPYLERYTQDAADKIIELMEGKPDLIIGNYTDGNLVASLMARKLGITLGTIAHALEKTKYEDSDIKLKE LDPKYHFSCQFTADLIAMNSADFIITSTYQEIAGSKDRPGQYESHSAFTLPGLYRVASGINVFDPKFNIAAPGADQSVYFPYTEKQTRLTAFRPAIEELLFSKVDNDEHIGYLE DRKKPILFTMARLDTVKNTSGLTEWYGKNKRLRSLVNLVVVGGSFDPTKSKDREEAAEIKKMHMLIEKYQLKGQIRWIAAQTDRYRNSELYRTIADSKGAFVQPALYEAFGLTV IEAMNCGLPTFATNQGGPAEIIVDGVSGFHIDPNNGDESSNKVANFFQKCREDPEYWNRISVQGLNRIYECYTWKIYANKVLNMGSIYTFWRTLYRDQKQAKQRYIETFYNLEF RNLVKNVPIRQDETPQGPKERREKVKPQISQRHALKLLPIVFQETLVYSSTKLELYSMQLASAVHLSKSPVMSIKLTKSINSAFPFPMLDCYYSYFVYGSLPSLFSPVLLLLQLY

Claims

1. A plant cell comprising: (i) a polynucleotide comprising a sequence having at least 90% sequence identity to SEQ ID NO:5 (NtINV4-S), or at least 90% sequence identity to SEQ ID NO:7 (NtINV4-T); (ii) a polypeptide encoded by the polynucleotide described in (i); (iii) a polypeptide comprising a sequence having at least 90% sequence identity to SEQ ID NO:6 (NtINV4-S), or at least 90% sequence identity to SEQ ID NO:8 (NtINV4-T); or (iv) a construct, vector, or expression vector comprising the polynucleotide described in (i), A plant cell, wherein the plant cell contains at least one modification that modulates (a) the expression or activity of the polynucleotide or (b) the expression or activity of the polypeptide, compared to a control plant cell in which the expression or activity of the polynucleotide or polypeptide is not modified.

2. The plant cell of claim 1, wherein the regulated expression or regulated activity regulates the level of one or more reducing sugars in dried leaves of a plant comprising the plant cell compared to the level of one or more reducing sugars in dried leaves of a control plant containing the control plant cell.

3. The plant cell described in claim 2, wherein the reducing sugar is glucose or fructose, or a combination thereof.

4. the intermediate dried leaves reduced glucose levels by at least 63% compared to the intermediate dried control leaves; or the intermediate dried leaves reduced fructose levels by at least 43% compared to the intermediate dried control leaves; or 4. The plant cell of claim 2 or 3, wherein the intermediate dried leaves have at least 63% reduced glucose levels and at least 43% reduced fructose levels, respectively, compared to the control intermediate dried leaves.

5. The plant cell according to any one of claims 1 to 4, wherein said at least one modification is a genetic mutation in said polynucleotide and said plant is Nicotiana tabacum.

6. The plant cell of any one of claims 1 to 5, further comprising at least one modification in the NtSUS polynucleotide or the polypeptide encoded thereby.

7. The plant cell described in claim 6, wherein the NtSUS polynucleotide or the polypeptide encoded thereby is selected from the group consisting of NtSUS2-T, NtSUS3-S, NtSUS3-T, NtSUS4-S, NtSUS4-T and combinations of two or more thereof.

8. The plant cell described in claim 6, wherein the NtSUS polynucleotide or the polypeptide encoded thereby is selected from the group consisting of NtSUS2-S, NtSUS3-S, NtSUS3-T, NtSUS4-S and combinations of two or more thereof.

9. A plant cell described in claim 6, 7 or 8, comprising at least one mutation in an NtINV4 polynucleotide described in (i) of claim 1, or an NtINV4 polypeptide described in (ii) of claim 1 or (iii) of claim 1, and at least one mutation in the NtSUS polynucleotide or polypeptide encoded thereby.

10. A plant or a part thereof comprising the plant cell according to any one of claims 1 to 9.

11. 11. A plant material, dried plant material or homogenized plant material derived or obtained from the plant or part thereof according to claim 10.

12. The plant material, dried plant material or homogenized plant material described in claim 11, wherein the plant material is selected from the group consisting of biomass, seeds, stems, flowers, leaves, and combinations of two or more thereof.

13. The plant material, dried plant material or homogenized plant material of claim 11, wherein the dried plant material is selected from the group consisting of flue-cured plant material, sun-dried plant material, air-dried plant material, and combinations of two or more thereof.

14. A tobacco product comprising a plant cell according to any one of claims 1 to 9, a part of a plant according to claim 10, or a plant material according to claim 12 or 13.

15. 11. A method for producing the plant of claim 10, comprising: (a) providing a plant cell comprising at least one modification according to any one of claims 1 to 9; (b) propagating the plant cell into a plant.

16. The method of claim 15, wherein in step (a), the at least one modification is introduced by genome editing.

17. The method described in claim 16, wherein the genome editing is selected from CRISPR-mediated genome editing, zinc finger nuclease-mediated mutagenesis, chemical or radiation mutagenesis, homologous recombination, oligonucleotide-directed mutagenesis and meganuclease-mediated mutagenesis.

18. 18. The method of claim 15, 16 or 17, wherein in step (a), the at least one modification is introduced using an interfering polynucleotide or by introducing at least one mutation, or a combination thereof.

19. 1. A method for producing a dried plant material having an altered amount of reducing sugars compared to a control plant material, comprising: (a) providing a plant or part thereof according to claim 10, or a plant material according to claim 11, 12 or 13; (b) harvesting the plant material therefrom; (c) drying the plant material.

20. 1. A method for producing a liquid tobacco extract, comprising: (a) preparing a first tobacco starting material from a plant or part thereof containing a plant cell according to any one of claims 1 to 5; (b) preparing a second tobacco starting material from a plant or part thereof containing plant cells that include at least one modification in an NtSUS polynucleotide or polypeptide encoded thereby; (c) heating the first tobacco starting material at a first extraction temperature; (d) heating the second tobacco starting material at a second extraction temperature; and (e) recovering volatile compounds released from the first and second tobacco starting materials during heating; and (f) combining the recovered volatile compounds released from the first and second tobacco starting materials and forming a liquid tobacco extract from the combined volatile compounds.

21. A method for producing a liquid tobacco extract as described in claim 20, wherein in step (b), the NtSUS polynucleotide or the polypeptide encoded thereby is selected from the group consisting of NtSUS2-T, NtSUS3-S, NtSUS3-T, NtSUS4-S, NtSUS4-T and combinations of two or more thereof.

22. A method for producing a liquid tobacco extract as described in claim 20, wherein in step (b), the NtSUS polynucleotide or the polypeptide encoded thereby is selected from the group consisting of NtSUS2-S, NtSUS3-S, NtSUS3-T, NtSUS4-S and combinations of two or more thereof.

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