Modulating sugar and amino acid content in plant (sultr3)

Abstract

To produce tobacco which provides consumers with new flavors and sensory experiences while still maintaining commercially acceptable yields and traits.SOLUTION: A plant cell comprising (i) a polynucleotide comprising or consisting of a sequence having at least 90% sequence identity to a specified nucleic acid sequence, (ii) a polypeptide encoded by the polynucleotide described in (i), (iii) a polypeptide comprising or consisting of a sequence having at least 90% sequence identity to a specified amino acid sequence, or (iv) a construct, vector, or expression vector comprising the isolated polynucleotide described in (i). Also provided is a tobacco product comprising the plant cell.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority from European Patent Application No. 19200856.3, 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 chloroplast sulfate (SULTR3). [Background technology]

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

[0004] Flavor characteristics are the result of specific flavor compounds or precursors of these compounds present at specific levels in tobacco plants. For example, the altered sugar content in cured tobacco can result in the perception of different flavors and aromas of tobacco. In aerosols and smoke, glucose, and to a lesser extent fructose, can produce Amadori compounds through the Maillard reaction. This can result in flavors like bread, nuts, or popcorn.

[0005] However, because the tobacco varieties available for commercial production are limited, this also means that there are limited opportunities to develop tobacco products with different flavor and aroma profiles, which also applies to the production of reconstituted tobacco materials used in heat-not-burn 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 still maintaining commercially acceptable yields and traits. The present invention seeks to address this and other needs. Summary of the Invention

[0007] The polynucleotide and polypeptide sequences of SULTR3 from Nicotiana tabacum are disclosed herein. Many different genes are considered to be likely sulfate transporters in plants based on structural identity, but the time at which these genes become active in their function as sulfate transporters in plants is typically unknown. In particular, little is known about sulfate transporter gene expression in tobacco, especially during curing. The present inventors have now identified certain NtSULTR3 polynucleotides involved in sulfate transporters in plants that are functionally expressed during curing. Surprisingly, it has been observed that modulating the expression of these certain NtSULTR3 genes or modulating the activity of the proteins encoded thereby can alter the pools of reducing sugars (such as glucose or fructose, or a combination thereof), non-reducing sugars (such as sucrose), free amino acids (such as glutamine, glutamic acid, or aspartic acid, or a combination of two or more thereof), and optionally asparagine produced during leaf curing. It is unexpected that changes to certain sulfate transporter genes can lead to changes in the levels of sugars or free amino acids, and optionally asparagine, 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 different flavors or sensory perceptions of the aerosol or smoke produced when the tobacco blend is heated. Similarly, liquid extracts obtained from tobacco may have different flavors or sensory perceptions. Altering the sugar-amino acid balance may also affect the release of acrylamide in aerosols and smoke.

[0008] Fourteen chloroplast sulfate transporter polynucleotide sequences from Nicotiana tabacum are described, including NtSULTR3;1A-S (SEQ ID NO: 1), NtSULTR3;1A-T (SEQ ID NO: 3), NtSULTR3;1B-S (SEQ ID NO: 5), NtSULTR3;1B-T (SEQ ID NO: 7), NtSULTR3;2-S (SEQ ID NO: 9), NtSULTR3;2-T (SEQ ID NO: 11), NtSULTR3;3 It is disclosed that the sequences include NtSULTR3;-S (SEQ ID NO: 13), NtSULTR3;3-T (SEQ ID NO: 15), NtSULTR3;4A-S (SEQ ID NO: 17), NtSULTR3;4A-T (SEQ ID NO: 19), NtSULTR3;4B-S (SEQ ID NO: 21), NtSULTR3;4B-T (SEQ ID NO: 23), NtSULTR3;5-S (SEQ ID NO: 25) and NtSULTR3;5-T (SEQ ID NO: 27). NtSULTR3;1A-S (SEQ ID NO: 1), NtSULTR3;1A-T (SEQ ID NO: 3), NtSULTR3;1B-S (SEQ ID NO: 5), NtSULTR3;1B-T (SEQ ID NO: 7), NtSULTR3;3-T (SEQ ID NO: 15), NtSULTR3;4A-S (SEQ ID NO: 17), NtSULTR3;4A-T (SEQ ID NO: 19), and NtSULTR3;4B-T (SEQ ID NO: 23) have been shown to be particularly expressed during desiccation. NtSULTR3;1A-S (SEQ ID NO: 1), NtSULTR3;1A-T (SEQ ID NO: 3), and NtSULTR3;3-T (SEQ ID NO: 15) have been shown to play a role in sugar and amino acid metabolism, particularly during desiccation.

[0009] Modifications to the expression or activity of one or more SULTR3s can be combined with modifications to the expression or activity of one or more SUSs to further regulate the levels of sugars and free amino acids in cured leaves. In particular, regulating both SULTR3 and SUSs in plant cells can regulate the level of reducing sugars in cured leaves to a greater extent than regulating either SULTR3 or SUSs in plant cells. NtSUS1-S (SEQ ID NO: 30), NtSUS1-T (SEQ ID NO: 32), NtSUS2-S (SEQ ID NO: 34), NtSUS2-T (SEQ ID NO: 36), NtSUS3-S (SEQ ID NO: 38), NtSUS3-T (SEQ ID NO: 40), NtSUS4-S (SEQ ID NO: 42), NtSUS4-T (SEQ ID NO: 44), NtSUS5-S (SEQ ID NO: 46), NtSUS5-T (SEQ ID NO: 48), NtSUS6-S (SEQ ID NO: 50), and NtSUS6-T (SEQ ID NO: 52) are disclosed. NtSUS2-S (SEQ ID NO: 34), NtSUS2-T (SEQ ID NO: 36), NtSUS3-S (SEQ ID NO: 38), NtSUS3-T (SEQ ID NO: 40), NtSUS4-S (SEQ ID NO: 42), and NtSUS4-T (SEQ ID NO: 44) may play a role in (reducing) sugar metabolism during desiccation. NtSUS2-S (SEQ ID NO: 34), NtSUS3-S (SEQ ID NO: 38), NtSUS3-T (SEQ ID NO: 40), and NtSUS4-S (SEQ ID NO: 42) in particular may play a role in sugar metabolism during desiccation.

[0010] In one aspect, the nucleic acid sequence of the present invention (i) comprises or consists of a sequence having at least 60% sequence identity to SEQ ID NO: 1 (NtSULTR3;1A-S), SEQ ID NO: 3 (NtSULTR3;1A-T), SEQ ID NO: 5 (NtSULTR3;1B-S), SEQ ID NO: 7 (NtSULTR3;1B-T), SEQ ID NO: 15 (NtSULTR3;3-T), SEQ ID NO: 17 (NtSULTR3;4A-S), SEQ ID NO: 19 (NtSULTR3;4A-T) or SEQ ID NO: 23 (NtSULTR3;4B-T); or (ii) a polynucleotide consisting essentially of (i); (ii) a polypeptide encoded by the polynucleotide described in (i); (iii) a polypeptide having at least 87% sequence identity to SEQ ID NO:2 (NtSULTR3;1A-S), or at least 87% sequence identity to SEQ ID NO:4 (NtSULTR3;1A-T), or at least 87% sequence identity to SEQ ID NO:6 (NtSULTR3;1B-S), or at least 87% sequence identity to SEQ ID NO:8 (NtSULTR3;1B-T). or (iv) a polypeptide comprising, consisting of, or consisting essentially of a sequence having 88% sequence identity, or at least 70% sequence identity to SEQ ID NO: 16 (NtSULTR3;3-T), or at least 84% sequence identity to SEQ ID NO: 18 (NtSULTR3;4A-S), or at least 63% or at least 79% sequence identity to SEQ ID NO: 20 (NtSULTR3;4A-T), or at least 87% sequence identity to SEQ ID NO: 24 (NtSULTR3;4B-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 modulates (a) expression or activity of the polynucleotide or (b) expression or activity of the polynucleotide polypeptide, compared to a control plant cell in which expression or activity of the polynucleotide or polypeptide is not modified.

[0011] Preferably, the plant cell comprises a polynucleotide comprising, consisting of, or consisting essentially of a sequence having at least 60% sequence identity to SEQ ID NO:1 (NtSULTR3;1A-S) or SEQ ID NO:3 (NtSULTR3;1A-T); (ii) a polypeptide encoded by the polynucleotide set forth in (i); (iii) a polypeptide comprising, consisting of, or consisting essentially of a sequence having at least 87% sequence identity to SEQ ID NO:2 (NtSULTR3;1A-S) or at least 87% sequence identity to SEQ ID NO:4 (NtSULTR3;1A-T); or (iv) a construct, vector, or expression vector comprising the isolated polynucleotide set forth in (i).

[0012] Preferably, the modulated expression or modulated activity modulates the levels of glucose, fructose and sucrose in dried leaves of a plant comprising the plant cell compared to the levels of glucose, fructose and sucrose in dried leaves of a control.

[0013] Preferably, the glucose level is reduced by at least about 77%, at least about 69%, and at least about 60%, respectively, compared to the control dried leaves. Preferably, the fructose level is reduced by at least about 77%, at least about 69%, and at least about 60%, respectively, compared to the control dried leaves. Preferably, the sucrose level is reduced by at least about 77%, at least about 69%, and at least about 60%, respectively, compared to the control dried leaves. Preferably, the glucose, fructose, and sucrose levels are reduced by at least about 77%, at least about 69%, and at least about 60%, respectively, compared to the control dried leaves. For example, the levels of glucose, fructose, or sucrose, or one or more combinations thereof, are reduced by at least about 80%, at least about 75%, at least about 70%, or at least about 65%.

[0014] Preferably, the modulated expression or modulated activity modulates the levels of the free amino acids glutamine, glutamic acid and aspartic acid in dried leaves of a plant comprising the plant cell compared to the levels of the free amino acids glutamine, glutamic acid and aspartic acid in dried leaves of a control.

[0015] Preferably, the free amino acid levels are increased by at least about 1.5-fold, at least about 2.3-fold, at least about 2.4-fold, and at least about 1.5-fold, respectively, compared to the control dried leaves. Preferably, the glutamine level is increased by at least about 1.5-fold, at least about 2.3-fold, at least about 2.4-fold, and at least about 1.5-fold, respectively, compared to the control dried leaves. Preferably, the glutamic acid level is increased by at least about 1.5-fold, at least about 2.3-fold, at least about 2.4-fold, and at least about 1.5-fold, respectively, compared to the control dried leaves. Preferably, the aspartic acid level is increased by at least about 1.5-fold, at least about 2.3-fold, at least about 2.4-fold, and at least about 1.5-fold, respectively, compared to the control dried leaves. Preferably, the levels of the free amino acids glutamine, glutamic acid and aspartic acid are increased by at least about 1.5-fold, at least about 2.3-fold, at least about 2.4-fold and at least about 1.5-fold, respectively, compared to control dried leaves. Preferably, the dried leaves are from leaves located in the middle of the plant.

[0016] Preferably, there is a minimal effect on the phenotype of the plant comprising the plant cell, for example the phenotype of the plant may be unchanged.

[0017] 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.

[0018] Preferably, at least one modification is a genetic mutation in the polynucleotide.

[0019] Preferably, the plant is Nicotiana tabacum.

[0020] Preferably, the plant cell further comprises (i) at least one modification in an NtSUS polynucleotide or polypeptide encoded thereby, preferably wherein 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 wherein 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, or (ii) further comprises at least one modification in a CLC-Nt2 polynucleotide or polypeptide encoded thereby or an NtCLCe polynucleotide or polypeptide encoded thereby, or a combination thereof, or (iii) a combination of (i) and (ii).

[0021] In a further aspect, there is provided a plant or part thereof comprising a plant cell as described herein.

[0022] In a further aspect there is provided plant material, dried plant material or homogenized plant material derived from or obtained from a plant or part thereof as described herein, 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, suitably the dried plant material is 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.

[0023] In a further aspect, a tobacco product is provided that includes a plant cell described herein, a plant part described herein, or a plant material described herein.

[0024] In a further aspect, there is provided a method for producing 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.

[0025] Preferably, in step (a), the 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, or 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.

[0026] In a further aspect, a method is provided for producing dried plant material having altered levels of glucose, fructose, and sucrose, and altered levels of the free amino acids glutamine, glutamic acid, and aspartic acid, compared to control plant material, comprising the steps of: (a) providing a plant or part thereof or plant material described herein; (b) harvesting the plant material therefrom; and (c) drying the plant material.

[0027] In a further aspect, a method of producing a liquid tobacco extract is provided, the method comprising: (a) preparing a tobacco starting material from a plant or portion thereof comprising plant cells comprising at least one modification that modulates the expression or activity of NtSULTR3 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.

[0028] In a further aspect, a method for producing a liquid tobacco extract is provided, the method comprising the steps of: (a) preparing a first tobacco starting material from a plant or part thereof, the plant cells comprising plant cells comprising at least one modification that modulates expression or activity of NtSULTR3 as described herein; and (b) (i) at least one modification in an NtSUS polynucleotide or polypeptide encoded thereby, wherein 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, and 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. (ii) comprising at least one modification in a CLC-Nt2 polynucleotide or polypeptide encoded thereby or an NtCLCe polynucleotide or polypeptide encoded thereby, or a combination thereof, or (iii) comprising a plant cell that is a combination of (i) and (ii); (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.

[0029] In a further aspect, there is provided a liquid tobacco extract produced, obtained or obtainable by the above method.

[0030] Some advantages Advantageously, altering the sugar-amino acid balance in tobacco may affect the release of acrylamide, a carcinogenic compound that results from the interaction of glucose (fructose) with asparagine, when heated in aerosol and smoke.

[0031] Advantageously, reconstituted tobacco materials for heat-ready stick cigarettes require reducing sugars for proper cast leaf preparation, and the present disclosure may affect the sugar content and balance, thereby affecting cast leaf preparation.

[0032] Advantageously, non-genetically modified plants can be produced that may be more acceptable to consumers.

[0033] Advantageously, the present disclosure is not limited to the use of EMS mutant plants.

[0034] 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 source for sink leaves and seeds. Therefore, sucrose and asparagine must first be transported from the parenchyma (photosynthetic) senescent leaf cells to the phloem and then to the upper sink tissues. Manipulating one or more NtSULTR3 polynucleotides or the polypeptides encoded thereby can affect the levels of reducing sugars, non-reducing sugars, and free amino acids.

[0035] Advantageously, the present disclosure may be combined with modulating the expression of other genes or polypeptides encoded thereby, such as NtSUS, as described herein. [Brief explanation of the drawings]

[0036] [Figure 1]Graph and table showing expression of NtSULTR3 during a Virginia hot air drying time course. Gene expression is analyzed using Tobarray-Affymetrix chips during 2.5 days of drying (A), and using RNAseq at green, mature, and 48 hours after drying (B). [Figure 2] A series of graphs showing the variation of sulfate (A), abscisic acid (ABA) (B), methionine (Met, C), and methionine sulfoxide (Met sulfoxide, D) during the curing time course of dark tobacco. There are no specific concentration values ​​(arbitrary units) within such metabolomic data. [Figure 3] 1 is a graph showing silencing of NtSULTR3;1A-S and NtSULTR3;1A-T using a GATEWAY vector and measurement of expression in Virginia tobacco leaves after 48 hours of curing using qPCR. T1-14, T1-20, and T1-17 are anti-NtSULTR3;1A transgenic lines, while CT1-6, CT1-2, and CT1-10 correspond to unsilenced control lines. [Figure 4] Sugar (glucose, fructose, and sucrose) levels in dried leaves (CT1, n = 8; and T1, n = 6) of the 35S:NtSULTR3;1A-RNAi line (T1-SULTR3) and the control (CT1-SULTR3) are shown. Box plots, as well as T-test statistical analysis, are presented. [Figure 5] Figure 1 shows the free amino acid content in dried leaves (CT1, n = 8; and T1, n = 6) of the 35S:NtSULTR3;1A-RNAi line (T1-SULTR3) and the control (CT1-SULTR3). Box plots, as well as T-test statistical analysis, are presented. [Figure 6] 1 is a bar graph showing the reducing sugar content by variety in Burley, Virginia and Orient tobacco after harvest (maturity), after 2 days of curing (48 hours of curing), and at the end of curing. DETAILED DESCRIPTION OF THE INVENTION

[0037] The section headings used in this disclosure are for organizational purposes and are not intended to be limiting.

[0038] 1.Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. In case of conflict, the present document, including definitions, governs. Preferred methods and materials are described below, but methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. The materials, methods, and examples disclosed herein are merely illustrative and are not intended to be limiting.

[0039] 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.

[0040] The singular forms "a," "and," and "the" include plural references unless the context clearly dictates otherwise.

[0041] The present disclosure contemplates other embodiments "comprising," "consisting of," and "consisting essentially of" the embodiments or elements presented herein, whether explicitly stated or not.

[0042] For purposes of reciting numerical ranges herein, each intermediate number therebetween of the same degree of precision is expressly contemplated. For example, for the range 6 to 9, the numbers 7 and 8 are contemplated in addition to the numbers 6 and 9, and for the range 6.0 to 7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are expressly contemplated.

[0043] As used throughout the specification and claims, the following terms have the following meanings.

[0044] "Coding sequence" or "encoding polynucleotide" refers to a nucleotide (RNA or DNA molecule) comprising a polynucleotide that encodes a polypeptide. The coding sequence may further comprise initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in the cells of an individual or mammal to which the polynucleotide is administered. The coding sequence may be codon-optimized.

[0045] "Complement" or "complementary" can refer to Watson-Crick (e.g., AT / U and CG) or Hoogsteen base pairing between nucleotides or nucleotide analogs. "Complementarity" refers to the property shared between two polynucleotides, e.g., when aligned antiparallel to each other, that the nucleotide bases at each position are complementary.

[0046] A "construct" refers to a double-stranded recombinant polynucleotide fragment comprising one or more polynucleotides. A construct comprises a "template strand" base-paired with a complementary "sense or coding strand." A given construct can be inserted into a vector in either of two possible orientations: the same (or sense) orientation or the opposite (or antisense) orientation relative to the orientation of a promoter placed within the vector (e.g., an expression vector).

[0047] The term "control," in the context of a control plant or control plant cell, refers to a plant or plant cell in which the expression, function, or activity of one or more genes or polypeptides is not altered (e.g., increased or decreased), thereby providing a comparison with a plant in which the expression, function, or activity of one or more genes or polypeptides is altered. A "control plant" is a plant that is substantially equivalent to a test plant or modified plant in all parameters except for the test parameter. For example, when referring to a plant into which a polynucleotide has been introduced, the control plant is an equivalent plant into which such polynucleotide has not been introduced. A control plant can also be an equivalent plant into which a control polynucleotide has been introduced. In such cases, the control polynucleotide is one that is predicted to have little or no phenotypic effect on the plant. A control plant can contain an empty vector. A control plant can correspond to a wild-type plant. A control plant can be a null segregant, in which the T1 segregant no longer carries the transgene.

[0048] The term "reduction" 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 "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 from the same variety of plant processed in the same manner. Thus, in some contexts, wild-type plants of the same variety processed in the same manner are used as a control to determine whether a reduction in amount is obtained.

[0049] "Donor DNA" or "donor template" refers to a double-stranded DNA fragment or molecule that contains at least a portion of a gene of interest. The donor DNA can encode a functional polypeptide.

[0050] An "endogenous gene or polypeptide" refers to a gene or polypeptide that originates in the genome of an organism and has not undergone alteration, such as deletion, gain, or replacement of genetic material. An endogenous gene is subject to normal gene transmission and gene expression. An endogenous polypeptide is subject to normal expression.

[0051] "Enhancer sequences" refer to sequences that can increase gene expression. These sequences can be located upstream, within introns, or downstream of the transcribed region. The transcribed region consists of exons and intervening introns from the promoter to the transcription termination region. Enhancement of gene expression can be through various mechanisms, including increasing transcription efficiency, stabilizing mature mRNA, and enhancing translation.

[0052] "Expression" refers to the production of a functional product. For example, expression of a polynucleotide fragment can refer to transcription of the polynucleotide fragment (e.g., transcription resulting in mRNA or functional RNA), and can include translation of mRNA into a precursor or mature polypeptide. "Overexpression" refers to the production of a gene product in a transgenic organism that exceeds the level of production in a null isolate (or non-transgenic) organism from the same experiment.

[0053] "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.

[0054] A "genetic construct" refers to a DNA or RNA molecule comprising a polynucleotide that encodes a polypeptide. The coding sequence may include initiation and termination signals operably linked to regulatory elements, including a promoter and polyadenylation signal, capable of directing expression.

[0055] " Genome editing " generally refers to the process in which genomic nucleic acid is modified in cells.This can be, for example, by removing, inserting or substituting one or more nucleotides in genomic nucleic acid.Endonucleases can be used to create specific cuts or nicks at defined positions in genome, as further described herein.

[0056] 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 separate polynucleotides being compared is a function of the number of identical or matching nucleotides at comparable positions. Homology or similarity can be determined over the entire length of the subject sequence.

[0057] "Identical" or "identity" in the context of two or more polynucleotides or polypeptides means that the sequences have a specified percentage of residues that are the same across a specific region. The percentage is calculated by optimally aligning the two sequences, comparing the two sequences across a specific region, determining the number of positions where identical residues exist in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the specific region, and multiplying the result by 100 to obtain the percentage of sequence identity. If the two sequences are of different lengths or the alignment results in one or more staggered ends and a specific region of comparison contains only a single sequence, the residues of the single sequence are included in the denominator of the calculation but not in the numerator. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identity can be determined manually or by using a computer sequence algorithm such as ClustalW, ClustalX, BLAST, FASTA, or Smith-Waterman. Suitable parameters for ClustalW can be as follows: For polynucleotide alignments: Gap opening penalty = 15.0, gap extension penalty = 6.66, and matrix = Identity. For polypeptide alignments: Gap opening penalty = 10.0, gap extension penalty = 0.2, and matrix = Gonnet. For DNA and protein alignments: ENDGAP = -1, and GAPDIST = 4.

[0058] The term "increase" or "increased" refers to an increase of about 10% to about 99% in the amount or function or activity, such as, but not limited to, one or more of polypeptide function or activity, transcriptional function or activity, and polypeptide expression, 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 would be found in an unmodified plant or in a product from the same variety of plant 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 determine whether an increase in amount is obtained.

[0059] The term "inhibit" or "inhibited" refers to a reduction of about 98% to about 100%, or at least 98%, at least 99%, but particularly 100%, of the amount or function or activity, such as, but not limited to, one or more of polypeptide function or activity, transcriptional function or activity, and polypeptide expression.

[0060] The term "introduced" means providing a polynucleotide (e.g., a construct) or 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, and includes 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., transfected mRNA).

[0061] 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 a polynucleotide or polypeptide gives rise to essentially one band in an electrophoretic gel. Specifically, this means that the polynucleotide or polypeptide is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure. An isolated polynucleotide can be purified from a naturally occurring host cell. Conventional polynucleotide purification methods known to those skilled in the art can be used to obtain an isolated polynucleotide. The term also encompasses recombinant and chemically synthesized polynucleotides.

[0062] "Liquid tobacco extract" describes the direct product of an extraction process performed on tobacco starting material. The extraction process to produce a liquid tobacco extract can involve heating the tobacco starting material under specific heating conditions and recovering the volatile compounds that are produced. A liquid tobacco extract can contain a mixture of compounds that originate from the tobacco starting material and are removed during the extraction process, typically in combination with a liquid carrier or solvent.

[0063] "Modulate" or "modulation" refers to causing or promoting a qualitative or quantitative change, alteration, or modification in a process, pathway, function, or activity of interest. Without limitation, such a change, alteration, or modification can be an increase or decrease in the relative process, pathway, function, or activity of interest. For example, gene expression or polypeptide expression, or polypeptide function or activity, can be modulated. Typically, the relative change, alteration, or modification will be determined by comparison with a control.

[0064] The term "non-natural" describes entities such as polynucleotides, genetic mutations, polypeptides, plants, plant cells, and plant materials that are not naturally occurring or do not exist in nature. Such non-natural or artificial entities can be created, synthesized, initiated, modified, intervened, or manipulated by methods described herein or known in the art. Such non-natural or artificial entities can be created, synthesized, initiated, modified, intervened, or manipulated by humans. Thus, for example, non-natural plants, non-natural plant cells, or non-natural plant materials can be created using traditional plant breeding techniques such as backcrossing, or by genetic engineering techniques such as antisense RNA, interfering RNA, and meganucleases. As a further example, a non-natural plant, non-natural plant cell, or non-natural plant material can be created by introgressing or transferring one or more genetic mutations (e.g., one or more polymorphisms) from a first plant or plant cell to a second plant or plant cell (which itself may be natural) so that the resulting plant, plant cell, or plant material, or its progeny, contains a genetic configuration (e.g., a genome, chromosome, or segment thereof) that is not naturally formed or does not exist in nature. The resulting plant, plant cell, or plant material is therefore artificial or non-natural. Thus, an artificial or non-natural plant or plant cell can be created by modifying a genetic sequence in a first natural plant or plant cell, even if the resulting genetic sequence naturally exists in a second plant or plant cell that contains a genetic background different from that of the first plant or plant cell. In certain embodiments, the mutation is not a naturally occurring mutation that occurs in a polynucleotide or polypeptide, such as a gene or polypeptide. Differences in genetic background can be detected by phenotypic differences or by molecular biology techniques known in the art, such as polynucleotide sequencing, the presence or absence of genetic markers (e.g., microsatellite RNA markers).

[0065] "Oligonucleotide" or "polynucleotide" refers to at least two nucleotides covalently linked together. A depiction of a single strand also defines the sequence of the complementary strand. Thus, a polynucleotide also encompasses the complementary strand of a depicted single strand. Many variants of polynucleotides can be used for the same purpose as a given polynucleotide. Thus, a polynucleotide also encompasses substantially identical polynucleotides and their complements. A single strand provides a probe that can hybridize to a given sequence under stringent hybridization conditions. Thus, a polynucleotide also encompasses probes that hybridize under stringent hybridization conditions. A polynucleotide can be single-stranded or double-stranded, or can contain portions of both double-stranded and single-stranded sequence. A polynucleotide can be DNA, both genomic and cDNA, RNA, or a hybrid, and can contain combinations of deoxyribonucleotides and ribonucleotides, as well as combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. Polynucleotides can be obtained by chemical synthesis or by recombinant methods.

[0066] The specificity of single-stranded DNA for hybridizing complementary fragments is determined by the "stringency" of the reaction conditions (Sambrook et al., Molecular Cloning and Laboratory Manual, Second Ed., Cold Spring Harbor (1989)). Hybridization under "stringent conditions" describes a hybridization protocol in which polynucleotides at least 60% homologous to each other remain hybridized. Generally, stringent conditions are selected to be approximately 5°C lower than the thermal melting point (Tm) of a specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH, and polynucleotide concentration) at which 50% of the probes complementary to a given sequence hybridize to the given sequence at equilibrium. Since a given sequence is generally present in excess at the Tm, 50% of the probes are occupied at equilibrium.

[0067] Stringent conditions typically include: (1) a low ionic strength and high temperature wash, e.g., 15 mM sodium chloride, 1.5 mM sodium citrate, 0.1% sodium dodecyl sulfate at 50°C; (2) a denaturing agent during hybridization, e.g., 50% (v / v) formamide, 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer (750 mM sodium chloride, 75 mM sodium citrate; pH 6.5) at 42°C; or (3) 50% formamide. Washes also typically involve a wash in 0.2×SSC (sodium chloride / sodium citrate) at 42° C. containing 5×SSC (0.75 M NaCl, 75 mM sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50 μg / mL), 0.1% SDS, and 10% dextran sulfate, and a high stringency wash consisting of 50% formamide at 55° C., followed by 0.1×SSC containing EDTA at 55° C. Suitably, conditions are such that sequences at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous to each other typically remain hybridized to each other.

[0068] "Moderately stringent conditions" use wash solutions and hybridization conditions that are less stringent, such that polynucleotides hybridize to the entire polynucleotide, fragments, derivatives, or analogs. One example includes hybridization in 6xSSC, 5xDenhardt's solution, 0.5% SDS, and 100 μg / mL denatured salmon sperm DNA at 55°C, followed by one or more washes in 1xSSC, 0.1% SDS at 37°C. Temperature, ionic strength, etc., can be adjusted to accommodate experimental factors such as probe length. Other moderate stringency conditions have been described (see Ausubel et al., Current Protocols in Molecular Biology, Volumes 1-3, John Wiley & Sons, Inc., Hoboken, NJ (1993); Kriegler, Gene Transfer and Expression: A Laboratory Manual, Stockton Press, New York, NY (1990); Perbal, A Practical Guide to Molecular Cloning, 2nd edition, John Wiley & Sons, New York, NY (1988)).

[0069] "Low stringency conditions" use wash solutions and hybridization conditions that are less stringent than those for moderate stringency, such that polynucleotides hybridize to the entire polynucleotide, fragments, derivatives, or analogs. Non-limiting examples of low stringency hybridization conditions include hybridization in 35% formamide, 5xSSC, 50 mM Tris HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg / mL denatured salmon sperm DNA, 10% (wt / vol) dextran sulfate at 40°C, followed by one or more washes in 2xSSC, 25 mM Tris HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS at 50°C. Other conditions of low stringency, such as those for cross-species hybridization, have been well described (see Ausubel et al., 1993; Kriegler, 1990).

[0070] "Operably linked" means that the expression of a gene is under the control of a spatially linked promoter. The promoter can be located 5' (upstream) or 3' (downstream) of the gene under its control. The distance between the promoter and the gene can be approximately the same as the distance between the promoter and the gene it controls in the gene from which it is derived. As is known in the art, variations in this distance can be adjusted without loss of promoter function. "Operably linked" refers to the association of polynucleotide fragments in a single fragment, such that one function is controlled by the other. For example, a promoter is operably linked to a polynucleotide fragment when it is capable of controlling the transcription of the polynucleotide fragment.

[0071] The term "plant" refers to any plant at any stage of its life cycle or development, and its progeny. In one embodiment, the plant is a tobacco plant and refers to a plant belonging to the genus Nicotiana. The term includes reference to whole plants, plant organs, plant tissues, plant propagules, plant seeds, plant cells, and their progeny. Plant cells include, but are not limited to, cells from seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and pollen grains. Suitable species, cultivars, hybrids, and varieties of tobacco plants are described herein.

[0072] "Plant material" includes leaves, roots, sepals, root tips, petals, flowers, shoots, stems, seeds and stalks. Plant material can be viable or non-viable plant material.

[0073] "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.

[0074] "Polypeptide" or "polypeptide sequence" refers to a polymer of amino acids in which one or more amino acid residues are artificial chemical analogues of a corresponding naturally occurring amino acid, as well as to naturally occurring polymers of amino acids. These terms also include modifications, including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation. The polypeptides of the present disclosure are set forth in the accompanying sequence listing.

[0075] "Promoter" refers to a synthetic or naturally occurring molecule capable of conferring, activating, or enhancing expression of a polynucleotide in a cell. The term typically refers to a polynucleotide element / sequence located upstream and operably linked to a double-stranded polynucleotide fragment. A promoter can be derived entirely from the region adjacent to a native gene of interest or can be composed of different elements derived from different native promoters or synthetic polynucleotide segments. A promoter can contain one or more specific transcription control sequences to further enhance expression, alter spatial expression, or alter temporal expression. A promoter can also contain distal enhancer or repressor elements and can be located as many as several thousand base pairs from the start site of transcription. Promoters can be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. A promoter can constitutively or differentially control the expression of gene components depending on the cell, tissue, or organ in which expression occurs, the developmental stage in which expression occurs, or in response to external stimuli such as physiological stress, pathogens, metal ions, or inducers.

[0076] As used interchangeably herein, "tissue-specific promoter" and "tissue-preferred promoter" refer to a promoter that is primarily, but not necessarily exclusively, expressed in one tissue or organ, but can also be expressed in one particular cell. A "developmentally regulated promoter" refers to a promoter whose function is determined by developmental events. A "constitutive promoter" refers to a promoter that causes a gene to be expressed in most cell types at most times. An "inducible promoter" selectively expresses an operably linked DNA sequence in response to the presence of endogenous or exogenous stimuli, for example, chemical compounds (chemical inducers), or in response to environmental, hormonal, chemical, and / or developmental signals. Examples of inducible or regulated promoters include promoters regulated by light, heat, pressure, waterlogging or drought, pathogens, plant hormones, wounding, or chemicals such as ethanol, jasmonic acid, salicylic acid, or safeners.

[0077] "Recombinant" refers to the artificial combination of two otherwise separate segments of sequence, either by chemical synthesis or by the manipulation of isolated segments of polynucleotides by genetic engineering techniques. The term also includes reference to a cell or vector that has been modified by the introduction of a heterologous polynucleotide, or a cell derived from a cell so modified, but does not encompass the alteration of a cell or vector by naturally occurring events (e.g., spontaneous mutation, natural transformation or transduction or transposition), such as those that occur without deliberate human intervention.

[0078] "Recombinant construct" refers to a combination of polynucleotides that are not normally found together in nature. Thus, a recombinant construct may contain control and coding sequences from different sources, or control and coding sequences 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.

[0079] As used interchangeably herein, "control sequence" and "control element" refer to polynucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence that influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Control sequences include promoters, translation leader sequences, introns, and polyadenylation recognition sequences. The terms "control sequence" and "control element" are used interchangeably herein.

[0080] The term "tobacco" is used collectively to refer to tobacco crops (e.g., tobacco plants that are field-grown and not hydroponically grown), tobacco plants, and parts thereof, including, but not limited to, roots, stems, leaves, flowers, and seeds, prepared or obtained as described herein. "Tobacco" is understood to include the Nicotiana tabacum plant and its products.

[0081] 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 for tobacco preparation.

[0082] "Transcription terminator," "termination sequence," or "terminator" refers to a DNA sequence located downstream of a coding sequence and includes polyadenylation recognition sequences and other sequences encoding regulatory signals that can affect mRNA processing or gene expression. Polyadenylation signals are usually characterized by affecting the addition of polyadenylic acid moieties to the 3' end of a pre-mRNA.

[0083] "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 the original transgenic event, as well as those produced from the original transgenic event by sexual crossing or asexual propagation. The term does not encompass alterations of the genome (chromosomal or extrachromosomal) by conventional plant breeding methods or by natural events (e.g., random cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation).

[0084] A "transgenic plant" refers to a plant that contains one or more heterologous polynucleotides in its genome, i.e., a plant containing recombinant genetic material not normally found therein and that has been introduced into the plant (or an ancestor of the plant) through human manipulation. For example, a heterologous polynucleotide can be stably integrated into the genome so that the polynucleotide is transmitted to successive generations. A 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 introduction of multiple traits into crop plants, often referred to as gene stacking. In this approach, 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, retransformation, and cross-breeding lines carrying different transgenes. Thus, a plant grown from a plant cell into which recombinant DNA has been introduced by transformation is a transgenic plant, as are all progeny of the plant that contain the introduced transgenes (whether produced sexually or asexually). The term transgenic plant is understood to encompass whole plants or trees as well as parts of the plants or trees, such as grains, seeds, flowers, leaves, roots, fruit, pollen, stems, etc. Each heterologous polynucleotide may confer a different trait to the transgenic plant.

[0085] "Transgene" refers to a gene or genetic material containing a genetic sequence that has been isolated from one organism and introduced into a different organism. This non-native segment of DNA may retain the ability to produce RNA or polypeptides in the transgenic organism, or may alter the normal function of the transgenic organism's genetic code.

[0086] "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.

[0087] A "variant" with respect to a peptide or polypeptide refers to a peptide or polypeptide that differs in sequence by amino acid insertion, deletion, or conservative substitution, but retains at least one biological function or activity. A variant can also refer to a polypeptide that retains at least one biological function or activity. Conservative amino acid substitutions, i.e., replacing one amino acid with a different amino acid with similar properties (e.g., hydrophilicity, degree and distribution of charged regions), are recognized in the art as typically resulting in minor changes.

[0088] The term "variety" refers to a group of plants that share certain characteristics that distinguish them from other plants of the same species. While possessing one or more distinctive traits, varieties are further characterized by very little overall variation among individuals within the variety. Varieties are often sold commercially.

[0089] A "vector" refers to a polynucleotide vehicle containing a combination of polynucleotide components to enable the delivery of polynucleotides, polynucleotide constructs, polynucleotide conjugates, and the like. A vector can be a viral vector, bacteriophage, bacterial artificial chromosome, or yeast artificial chromosome. A vector can be a DNA or RNA vector. Suitable vectors include episomes capable of extrachromosomal replication, such as circular double-stranded nucleotide plasmids, linear double-stranded nucleotide plasmids, and other vectors of any origin. An "expression vector" is a polynucleotide vehicle containing a combination of polynucleotide components to enable the expression of polynucleotides, polynucleotide constructs, polynucleotide conjugates, and the like. Suitable expression vectors include episomes capable of extrachromosomal replication, such as circular double-stranded nucleotide plasmids, linear double-stranded nucleotide plasmids, and other functionally equivalent expression vectors of any origin. An expression vector contains at least a promoter, as defined below, located upstream and operably linked to a polynucleotide, polynucleotide construct, or polynucleotide conjugate.

[0090] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those skilled in the art. For example, any nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and polypeptide and polynucleotide chemistry and hybridization described herein, as well as the techniques thereof, are well known and commonly used in the art. The meaning and scope of terms should be clear, but in the event of any potential ambiguity, the definitions provided herein shall take precedence over any dictionary or external definitions. Furthermore, unless otherwise required by context, singular terms shall include the plural, and plural terms shall include the singular.

[0091] 2. Polynucleotides Disclosed are isolated polynucleotides comprising, consisting of, or consisting essentially of a sequence having at least 60% sequence identity to any of the sequences described herein, including any polynucleotide set forth in the Sequence Listing. Preferably, the isolated polynucleotide comprises, consists of, or consists essentially of a sequence having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, 80%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto.

[0092] 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 the polypeptides set forth in the sequence listing.

[0093] In one embodiment, an isolated NtSULTR3 polynucleotide is provided, comprising, consisting of, or consisting 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, SEQ ID NO:9, 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, or SEQ ID NO:27, preferably SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, or SEQ ID NO:23, more preferably SEQ ID NO:1 or SEQ ID NO:3.

[0094] In another embodiment, there is provided an isolated NtSUS polynucleotide comprising, consisting of, or consisting essentially of a polynucleotide having at least 60% sequence identity to SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42 or SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50 or SEQ ID NO:52, preferably SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42 or SEQ ID NO:44, more preferably SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:40 or SEQ ID NO:42.

[0095] Preferably, the isolated NtSULTR3 polynucleotide has a sequence identity similar to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, 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, or SEQ ID NO:27, preferably SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, or SEQ ID NO:23, more preferably SEQ ID NO:1 or SEQ ID NO:3. 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.

[0096] Preferably, the isolated NtSUS polynucleotide has a sequence identity similar to SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42 or SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50 or SEQ ID NO:52, preferably SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42 or SEQ ID NO:44, more preferably SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:40 or SEQ ID NO:42. 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.

[0097] Preferably, the isolated NtSULTR3 polynucleotide has a sequence similar to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, 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, or SEQ ID NO:27, preferably SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, or SEQ ID NO:23, more preferably SEQ ID NO:1 or SEQ ID NO:3. 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.

[0098] Preferably, the isolated NtSUS polynucleotide has an identity at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% identity to SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42 or SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50 or SEQ ID NO:52, preferably SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42 or SEQ ID NO:44, more preferably SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:40 or SEQ ID NO:42. 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.

[0099] Preferably, the isolated NtSULTR3 polypeptide 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, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19 or SEQ ID NO:23, more preferably SEQ ID NO:1 or SEQ ID NO:3.

[0100] Preferably, the isolated NtSUS polypeptide 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:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42 or SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50 or SEQ ID NO:52, preferably SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42 or SEQ ID NO:44, more preferably SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:40 or SEQ ID NO:42.

[0101] In another embodiment, there is provided an NtSULTR3 polynucleotide comprising, consisting of, or consisting 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, SEQ ID NO:9, 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 or SEQ ID NO:27, preferably SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19 or SEQ ID NO:23, more preferably SEQ ID NO:1 or SEQ ID NO:3.

[0102] In another embodiment, there is provided an NtSUS polynucleotide comprising, consisting of, or consisting essentially of a polynucleotide having substantial homology (i.e., sequence similarity) or substantial identity to SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42 or SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50 or SEQ ID NO:52, preferably SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42 or SEQ ID NO:44, more preferably SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:40 or SEQ ID NO:42.

[0103] In another embodiment, fragments of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, 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 or SEQ ID NO:27 are provided, wherein the fragments have a sequence similar to that of the corresponding fragment of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, 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 or SEQ ID NO:27, and ... having substantial homology (i.e., sequence similarity) or substantial identity thereto, having 96%, 97%, 98%, 99%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity.

[0104] In another embodiment, fragments of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, or SEQ ID NO:23 are provided, wherein the fragments have a sequence similar to that of the corresponding fragment of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, or SEQ ID NO:23, and 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% or more of the sequence of the corresponding fragment of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, or SEQ ID NO:23. having substantial homology (i.e., sequence similarity) or substantial identity thereto, having 96%, 97%, 98%, 99%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity.

[0105] In another embodiment, fragments of SEQ ID NO:1 or SEQ ID NO:3 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 or SEQ ID NO:3.

[0106] In another embodiment, fragments of SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42 or SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50 or SEQ ID NO:52 are provided, wherein the fragments have a sequence similar to that of the corresponding fragment of SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42 or SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50 or SEQ ID NO:52, and 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% or more of the sequence of the corresponding fragment of SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42 or SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50 or SEQ ID NO:52. having substantial homology (i.e., sequence similarity) or substantial identity thereto, having 96%, 97%, 98%, 99%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity.

[0107] In another embodiment, fragments of SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42 or SEQ ID NO:44 are provided, wherein the fragments have a sequence similar to that of the corresponding fragment of SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42 or SEQ ID NO:44, and 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% or more of the sequence similar to that of the corresponding fragment of SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42 or SEQ ID NO:44. having substantial homology (i.e., sequence similarity) or substantial identity thereto, having 96%, 97%, 98%, 99%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity.

[0108] In another embodiment, fragments of SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:40 or SEQ ID NO:42 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:34, SEQ ID NO:38, SEQ ID NO:40 or SEQ ID NO:42.

[0109] In another embodiment, there is provided an NtSULTR3 polynucleotide that comprises 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, SEQ ID NO:9, 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 or SEQ ID NO:27 that encodes a polynucleotide that functions as a chloroplast sulfate transporter, preferably 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, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19 or SEQ ID NO:23 that encodes a polynucleotide that functions as a chloroplast sulfate transporter, more preferably a sufficient or substantial degree of identity or similarity to SEQ ID NO:1 or SEQ ID NO:3 that encodes a polynucleotide that functions as a chloroplast sulfate transporter.

[0110] In another embodiment, there is provided an NtSUS polynucleotide that encodes a polypeptide that functions as a SUS, comprising a sufficient or substantial degree of identity or similarity to SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42 or SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50 or SEQ ID NO:52, preferably a polynucleotide that functions as a SUS, comprising a sufficient or substantial degree of identity or similarity to SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42 or SEQ ID NO:44, more preferably a polynucleotide that functions as a SUS, comprising a sufficient or substantial degree of identity or similarity to SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:40 or SEQ ID NO:42.

[0111] In another embodiment, there is provided a polymer of NtSULTR3 polynucleotides 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, SEQ ID NO:9, 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, or SEQ ID NO:27, preferably SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, or SEQ ID NO:23, more preferably SEQ ID NO:1 or SEQ ID NO:3.

[0112] In another embodiment there is provided a polymer of NtSUS polynucleotides comprising, consisting of or consisting essentially of a polynucleotide designated herein as SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42 or SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50 or SEQ ID NO:52, preferably SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42 or SEQ ID NO:44, more preferably SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:40 or SEQ ID NO:42.

[0113] Suitably, the polynucleotides described herein encode a member of the SULTR3 family having chloroplast sulfate transporter activity or a member of the SUS family having SUS activity.

[0114] A polynucleotide can comprise a polymer of nucleotides, which can be unmodified or modified deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Thus, a polynucleotide can be, but is not limited to, genomic DNA, complementary DNA (cDNA), mRNA, or antisense RNA, or fragments thereof. Furthermore, a polynucleotide can be single-stranded or double-stranded DNA, a DNA that is a mixture of single-stranded and double-stranded regions, a hybrid molecule containing DNA and RNA, or a hybrid molecule containing a mixture of single-stranded and double-stranded regions or fragments thereof. Additionally, a polynucleotide can be composed of triple-stranded regions containing DNA, RNA, or both, or fragments thereof. A polynucleotide can contain one or more modified bases, such as phosphothioates, and can be a peptide nucleic acid. Generally, a polynucleotide can be assembled from isolated or cloned fragments of cDNA, genomic DNA, oligonucleotides, or individual nucleotides, or combinations of the foregoing. Although the polynucleotides described herein are presented as DNA sequences, they also include their corresponding RNA sequences and their complementary (e.g., perfectly complementary) DNA or RNA sequences, including their reverse complements.

[0115] Fragments of polynucleotides can range from at least about 25 nucleotides, about 50 nucleotides, about 75 nucleotides, about 100 nucleotides, about 150 nucleotides, about 200 nucleotides, about 250 nucleotides, about 300 nucleotides, about 400 nucleotides, about 500 nucleotides, about 600 nucleotides, about 700 nucleotides, about 800 nucleotides, about 900 nucleotides, about 1000 nucleotides, about 1100 nucleotides, about 1200 nucleotides, about 1300 nucleotides, or about 1400 nucleotides, up to a full-length polynucleotide encoding a polypeptide described herein.

[0116] Polynucleotides generally contain phosphodiester bonds, but in some cases, polynucleotide analogs may have alternative backbones, including, for example, phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphosphoramidite linkages, as well as peptide polynucleotide backbones and linkages. Other analog polynucleotides include those with cationic, non-ionic, and non-ribose backbones. Modifications of the ribose-phosphate backbone may be made for a variety of reasons, such as to increase the stability and half-life of such molecules in physiological environments or as probes on biochips. Mixtures of natural polynucleotides and analogs may be made; alternatively, mixtures of different polynucleotide analogs and mixtures of natural polynucleotides and analogs may be made.

[0117] 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.

[0118] Other analogs include peptide polynucleotides, which are peptide polynucleotide analogs.

[0119] Among the uses of the disclosed polynucleotides and their fragments are the use of the fragments as probes in hybridization assays or primers for use in amplification assays. Such fragments generally contain at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more consecutive nucleotides of a DNA sequence. In other embodiments, the DNA fragments contain at least about 10, 15, 20, 30, 40, 50, or 60 or more consecutive nucleotides of a DNA sequence. Thus, in one aspect, methods for detecting polynucleotides are also provided, including the use of probes or primers, or both.

[0120] The basic parameters that influence the selection of hybridization conditions, and guidance for devising suitable conditions, are described by Sambrook, J., E.F. Fritsch, and T. Maniatis (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). Using knowledge of the genetic code combined with the polypeptide sequences described herein, a set of degenerate oligonucleotides can be prepared. Such oligonucleotides are useful, for example, as primers in polymerase chain reactions (PCR), in which DNA fragments are isolated and amplified.

[0121] A method for achieving moderate and high stringency conditions is described herein.At least one modification (for example, mutation) can be included in one or more of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, 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 or SEQ ID NO:27, preferably at least one modification (for example, mutation) can be included in one or more of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19 or SEQ ID NO:23, more preferably at least one modification (for example, mutation) can be included in one or more of SEQ ID NO:1 or SEQ ID NO:3.

[0122] At least one modification (e.g., mutation) may be included in one or more of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19 or SEQ ID NO:23, and at least one or more further modifications (e.g., mutations) may be included in one or more of SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42 or SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50 or SEQ ID NO:52.

[0123] At least one modification (e.g., mutation) may be included in one or more of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19 or SEQ ID NO:23, and at least one modification (e.g., mutation) may be included in one or more of SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42 and SEQ ID NO:44, more preferably in one or more of SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:40 and SEQ ID NO:42.

[0124] At least one modification (e.g., mutation) may be included in one or more of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, or SEQ ID NO:23; at least one modification (e.g., mutation) may be included in one or more of SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, and SEQ ID NO:44, but no modification (e.g., mutation) is included in one or more of SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52; more preferably, at least one modification (e.g., mutation) may be included in one or more of SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:40, and SEQ ID NO:42, but no modification (e.g., mutation) is included in one or more of SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:36, and one or more of SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52.

[0125] At least one modification (e.g., mutation) may be included in one or more of SEQ ID NO:1 or SEQ ID NO:3, and at least one or more additional modifications (e.g., mutations) may be included in one or more of SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42 or SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50 or SEQ ID NO:52.

[0126] At least one modification (e.g., mutation) may be included in one or more of SEQ ID NO:1 or SEQ ID NO:3, and at least one modification (e.g., mutation) may be included in one or more of SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42 and SEQ ID NO:44, more preferably in one or more of SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:40 and SEQ ID NO:42.

[0127] At least one modification (e.g., mutation) may be included in one or more of SEQ ID NO:1 or SEQ ID NO:3, at least one modification (e.g., mutation) may be included in one or more of SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42 and SEQ ID NO:44, but no modification (e.g., mutation) is included in one or more of SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50 and SEQ ID NO:52, and more preferably at least one modification (e.g., mutation) may be included in one or more of SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:40 and SEQ ID NO:42, but no modification (e.g., mutation) is included in one or more of SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:36 and one or more of SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50 and SEQ ID NO:52.

[0128] 3. Polypeptides Also provided is an isolated polypeptide comprising, consisting of, or consisting essentially of a polypeptide having at least 60% sequence identity to any of the polypeptides described herein, including any of the polypeptides set forth in the Sequence Listing. Preferably, the isolated polypeptide comprises, consists of, or consists essentially of a sequence having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, 80%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity thereto. 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% of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, 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, SEQ ID NO:24, SEQ ID NO:26 and SEQ ID NO:28 Also provided are NtSULTR3 polypeptides comprising, consisting of, or consisting essentially of a sequence with 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.

[0129] Also provided are NtSULTR3 polypeptides comprising, consisting of, or consisting essentially of a sequence having at least 80%, 81%, 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, SEQ ID NO:8, 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, SEQ ID NO:24, SEQ ID NO:26 and SEQ ID NO:28.

[0130] Also provided are NtSULTR3 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, SEQ ID NO:8, 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, SEQ ID NO:24, SEQ ID NO:26 and SEQ ID NO:28.

[0131] Also provided are NtSULTR3 polypeptides encoded by SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, 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, SEQ ID NO:24, SEQ ID NO:26 and SEQ ID NO:28.

[0132] The NtSULTR3 polypeptide can comprise a sequence that comprises a sufficient or substantial degree of identity or similarity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, 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, SEQ ID NO:24, SEQ ID NO:26, and SEQ ID NO:28 to function as a chloroplast sulfate transporter.

[0133] 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% of SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51 or SEQ ID NO:53 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.

[0134] at least 80%, 81%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% to SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51 or SEQ ID NO:53, or more preferably to SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43 or SEQ ID NO:45, or more preferably to SEQ ID NO:35, SEQ ID NO:39, SEQ ID NO:41 or SEQ ID NO:43 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.

[0135] Also provided is an NtSUS 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:31, SEQ ID NO:33, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, or SEQ ID NO:53, preferably SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43 or SEQ ID NO:45, more preferably SEQ ID NO:35, SEQ ID NO:39, SEQ ID NO:41 or SEQ ID NO:43.

[0136] Also disclosed are polypeptides comprising, consisting of, or consisting essentially of a sequence having at least 88% sequence identity to SEQ ID NO:2 or SEQ ID NO:4, 81% sequence identity to SEQ ID NO:2 or SEQ ID NO:4 and SEQ ID NO:6 or SEQ ID NO:8, or 69% sequence identity to SEQ ID NO:2 or SEQ ID NO:4 and SEQ ID NO:6 or SEQ ID NO:8 and SEQ ID NO:10 or SEQ ID NO:12.

[0137] Also provided is an NtSUS polypeptide encoded by SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51 or SEQ ID NO:53, preferably SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43 or SEQ ID NO:45, more preferably SEQ ID NO:35, SEQ ID NO:39, SEQ ID NO:41 or SEQ ID NO:43.

[0138] The NtSUS polypeptide may comprise a sequence comprising a sufficient or substantial degree of identity or similarity to SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51 or SEQ ID NO:53 to function as a SUS, preferably SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43 or SEQ ID NO:45 to function as a SUS, more preferably SEQ ID NO:35, SEQ ID NO:39, SEQ ID NO:41 or SEQ ID NO:43 to function as a SUS.

[0139] Fragments of polypeptides typically retain some or all of the function or activity of the full-length sequence (such as chloroplast sulfate transporter or SUS activity). Fragments of polypeptides can range from at least about 25 amino acids, about 50 amino acids, about 75 amino acids, about 100 amino acids, about 150 amino acids, about 200 amino acids, about 250 amino acids, about 300 amino acids, about 400 amino acids, about 500 amino acids, up to the full-length polypeptides described herein.

[0140] Polypeptides also include variants produced by introducing 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), whether intentionally engineered or naturally isolated, provided that they still possess some or all of their function or activity. Preferably, this function or activity is modulated.

[0141] Deletion refers to the removal of one or more amino acids from a polypeptide. Insertion refers to the introduction of one or more amino acid residues into a predetermined site within a polypeptide. Insertion can include the intrasequence insertion of single or multiple amino acids. Substitution refers to the replacement of an amino acid in a polypeptide with another amino acid having similar properties (e.g., similar hydrophobicity, hydrophilicity, antigenicity, tendency to form or disrupt an α-helical or β-sheet structure). Amino acid substitutions are typically of single residues but can be clustered depending on the functional constraints imposed on the polypeptide and can range from about 1 to about 10 amino acids. Amino acid substitutions are preferably conservative amino acid substitutions, as described below. Amino acid substitutions, deletions, or insertions can be made using peptide synthesis techniques (e.g., solid-phase peptide synthesis) or by recombinant DNA engineering. Methods for manipulating DNA sequences to produce substitution, insertion, or deletion variants of polypeptides are well known in the art. Variants can have alterations that produce silent changes, resulting in functionally equivalent polypeptides. As long as the secondary binding of the substance is maintained, intentional amino acid substitutions can be made based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and amphipathic properties of the residues.For example, negatively charged amino acids include aspartic acid and glutamic acid, positively charged amino acids include lysine and arginine, and amino acids with uncharged polar head groups with similar hydrophilicity values ​​include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.Conservative substitutions can be made, for example, according to the following table.Amino acids in the same column in the second column and preferably in the same line in the third column can be substituted for each other.

[0142] JPEG2025186246000001.jpg62150

[0143] The polypeptide may be a mature or immature polypeptide, or a polypeptide derived from an immature polypeptide. The polypeptide may be linear or cyclized using known methods. The polypeptide typically contains at least 10, at least 20, at least 30, or at least 40 consecutive amino acids.

[0144] At least one modification (e.g., mutation) may be included in one or more of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, 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, SEQ ID NO:24, SEQ ID NO:26 and SEQ ID NO:28.

[0145] At least one modification (eg, mutation) may be included in one or more of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, and SEQ ID NO:24.

[0146] At least one modification (eg, mutation) may be included in one or more of SEQ ID NO:2 and SEQ ID NO:4.

[0147] At least one modification (e.g., mutation) may be included in one or more of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, 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, SEQ ID NO:24, SEQ ID NO:26 and SEQ ID NO:28, and optionally at least one further modification (e.g., mutation) may be included in one or more of SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51 or SEQ ID NO:53, preferably in one or more of SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43 and SEQ ID NO:45, or more preferably in one or more of SEQ ID NO:35, SEQ ID NO:39, SEQ ID NO:41 or SEQ ID NO:43.

[0148] At least one modification (e.g., mutation) may be included in one or more of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:16, SEQ ID NO:18, and SEQ ID NO:20 and SEQ ID NO:24, and optionally at least one further modification (e.g., mutation) may be included in one or more of SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51 or SEQ ID NO:53, preferably in one or more of SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43 and SEQ ID NO:45, or more preferably in one or more of SEQ ID NO:35, SEQ ID NO:39, SEQ ID NO:41 or SEQ ID NO:43.

[0149] At least one modification (e.g., mutation) may be included in one or more of SEQ ID NO:2 and SEQ ID NO:4, and optionally at least one or more further modifications (e.g., mutations) may be included in one or more of SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51 or SEQ ID NO:53, preferably in one or more of SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43 and SEQ ID NO:45, or more preferably in one or more of SEQ ID NO:35, SEQ ID NO:39, SEQ ID NO:41 or SEQ ID NO:43.

[0150] At least one modification (e.g., mutation) may be included in one or more of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, 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, SEQ ID NO:24, SEQ ID NO:26, and SEQ ID NO:28; at least one modification (e.g., mutation) may be included in one or more of SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, and SEQ ID NO:45, but no modification (e.g., mutation) is included in one or more of SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, and SEQ ID NO:53; more preferably, at least one modification (e.g., mutation) may be included in one or more of SEQ ID NO:35, SEQ ID NO:39, SEQ ID NO:41, and SEQ ID NO:43, but no modification (e.g., mutation) is included in one or more of SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:37, and SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, and SEQ ID NO:53.

[0151] At least one modification (e.g., mutation) may be included in one or more of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, and SEQ ID NO:24; at least one modification (e.g., mutation) may be included in one or more of SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, and SEQ ID NO:45, but no modification (e.g., mutation) is included in one or more of SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, and SEQ ID NO:53; more preferably, at least one modification (e.g., mutation) may be included in one or more of SEQ ID NO:35, SEQ ID NO:39, SEQ ID NO:41, and SEQ ID NO:43, but no modification (e.g., mutation) is included in one or more of SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:37, and SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, and SEQ ID NO:53.

[0152] At least one modification (e.g., mutation) may be included in one or more of SEQ ID NO:2 and SEQ ID NO:4, at least one modification (e.g., mutation) may be included in one or more of SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43 and SEQ ID NO:45, but no modification (e.g., mutation) is included in one or more of SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51 and SEQ ID NO:53, and more preferably, at least one modification (e.g., mutation) may be included in one or more of SEQ ID NO:35, SEQ ID NO:39, SEQ ID NO:41 and SEQ ID NO:43, but no modification (e.g., mutation) is included in one or more of SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:37, and SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51 and SEQ ID NO:53.

[0153] 4. Plant Modification a. Transformation Recombinant constructs can be used to transform plants or plant cells to modulate polypeptide expression, function, or activity. Recombinant polynucleotide constructs can include polynucleotides encoding one or more of the polynucleotides described herein, operably linked to regulatory regions suitable for polypeptide expression. Thus, polynucleotides can include coding sequences encoding the polypeptides described herein. Plants or plant cells in which polypeptide expression, function, or activity is modulated can include mutant, non-natural, transgenic, artificial, or genetically modified plants or plant cells. Preferably, transgenic plants or plant cells contain genomes that have been altered by stable integration of recombinant DNA. Recombinant DNA includes DNA that has been genetically engineered and constructed outside the cell, including DNA containing natural DNA, cDNA, or synthetic DNA. Transgenic plants can include plants regenerated from the originally transformed plant cell, as well as progeny transgenic plants from subsequent generations or crosses of the transformed plant. Preferably, the transgenic modification alters the expression, function, or activity of the polynucleotides or polypeptides described herein compared to a control plant.

[0154] 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.

[0155] Also provided is a vector containing a recombinant polynucleotide construct such as those described herein.Suitable vector backbones include those commonly used in the art, such as plasmids, viruses, artificial chromosomes, bacterial artificial chromosomes, yeast artificial chromosomes, or bacteriophage artificial chromosomes.Suitable expression vectors include, but are not limited to, plasmids and viral vectors derived from, for example, bacteriophages, baculoviruses, and retroviruses.Many vectors and expression systems are commercially available.

[0156] A vector may contain, for example, an origin of replication, a scaffold attachment region, or a marker. Marker genes can confer a selectable phenotype to plant cells. For example, markers can confer biocide resistance, such as resistance to antibiotics (e.g., kanamycin, G418, bleomycin, or hygromycin) or herbicides (e.g., glyphosate, chlorsulfuron, or phosphinothricin). In addition, expression vectors can contain tag sequences designed to facilitate manipulation or detection (e.g., purification or localization) of the expressed polypeptide. Tag sequences, such as luciferase, beta-glucuronidase, green fluorescent polypeptide, glutathione S-transferase, polyhistidine, c-myc, or hemagglutinin sequences, are typically expressed as a fusion with the encoded polypeptide. Such tags can be inserted anywhere within the polypeptide, including at either the carboxyl or amino terminus.

[0157] A plant or plant cell can be transformed by having a recombinant polynucleotide integrated into its genome so that it is stably transformed. The plants or plant cells described herein can be stably transformed. Stably transformed cells typically retain the introduced polynucleotide with each cell division. A plant or plant cell can be transiently transformed so that the recombinant polynucleotide is not integrated into its genome. Transiently transformed cells typically lose all or part of the introduced recombinant polynucleotide with each cell division, such that the introduced recombinant polynucleotide cannot be detected in daughter cells after a sufficient number of cell divisions. Numerous methods for transforming plant cells are available in the art, including biolistic, gene gun techniques, Agrobacterium-mediated transformation, viral vector-mediated transformation, freeze-thawing, microparticle bombardment, direct DNA uptake, sonication, microinjection, plant virus-mediated transduction, and electroporation.

[0158] If cells or cultured tissues are used as recipient tissues for transformation, plants can be regenerated from the transformed cultures, if desired, by techniques known to those skilled in the art.

[0159] The selection of a regulatory region to be included in a recombinant construct depends on several factors, including, but not limited to, efficiency, selectability, inducibility, desired expression level, and cell- or tissue-preferential expression. It is a routine matter for those skilled in the art to regulate the expression of a coding sequence by appropriately selecting and positioning a regulatory region relative to the coding sequence. Transcription of polynucleotides can be regulated in a similar manner. Some suitable regulatory regions initiate transcription only in certain cell types, or primarily in certain cell types. Methods for identifying and characterizing regulatory regions in plant genomic DNA are known in the art.

[0160] Exemplary promoters include tissue-specific promoters recognized by tissue-specific factors present in different tissues or cell types (e.g., root-specific promoters, shoot-specific promoters, xylem-specific promoters), or present during different developmental stages, or present in response to different environmental conditions. Suitable promoters include constitutive promoters that can be activated in most cell types without the need for specific inducers. Examples of promoters that can be used to control polypeptide expression include the cauliflower mosaic virus 35S (CaMV / 35S), SSU, OCS, lib4, usp, STLS1, B33, nos, or ubiquitin or phaseolin promoters. Those skilled in the art can generate multiple variants of recombinant promoters. Tissue-specific promoters are transcriptional control elements that are active only in specific cells or tissues at specific times during plant development, such as in vegetative or reproductive tissues. Examples of tissue-specific promoters under developmental control include promoters that can initiate transcription only (or primarily only) in certain tissues, such as vegetative tissues, e.g., roots or leaves, or reproductive tissues, such as fruit, ovules, seeds, pollen, pistils, flowers, or any embryonic tissue. Reproductive tissue-specific promoters can be, for example, anther-specific, ovule-specific, embryo-specific, endosperm-specific, integument-specific, seed and testa-specific, pollen-specific, petal-specific, sepal-specific, or combinations thereof.

[0161] Exemplary leaf-specific promoters include the bilbert orthophosphate dikinase (PPDK) promoter from C4 plants (maize), the cab-m1Ca+2 promoter from maize, the Arabidopsis thaliana myb-related gene promoter (Atmyb5), the ribulose biphosphate carboxylase (RBCS) promoter (e.g., the tomato RBCS1, RBCS2, and RBCS3A genes expressed in leaves and light-grown seedlings, RBCS1 and RBCS2 expressed in developing tomato fruit, or the ribulose biphosphate carboxylase promoter expressed at high levels almost exclusively in mesophyll cells in leaf blades and sheaths).

[0162] Suitable senescence-specific promoters include tomato promoters active during fruit ripening, senescence and leaf abscission, maize promoters of genes encoding cysteine ​​proteases, 82E4 promoters and SAG gene promoters.Exemplary anther-specific promoters can be used.Exemplary root-preferential promoters known to those skilled in the art can be selected.Exemplary seed-preferential promoters include both seed-specific promoters (those promoters active during seed development, such as promoters of seed storage polypeptides) and seed germination promoters (promoters active during seed germination).

[0163] Examples of inducible promoters include promoters that respond to pathogen attack, anaerobic conditions, high temperature, light, drought, low temperature, or high salinity. Pathogen-inducible promoters include those from pathogenesis-related polypeptides (PR polypeptides) and are induced following infection by a pathogen (e.g., PR polypeptides, SAR polypeptides, beta-1,3-glucanases, chitinases).

[0164] In addition to plant promoters, other suitable promoters may be derived from bacterial origin, such as the octopine synthase promoter, the nopaline synthase promoter, and other promoters derived from Ti plasmids, or may be derived from viral promoters (e.g., the 35S and 19S RNA promoters of the cauliflower mosaic virus (CaMV), the constitutive promoters of the tobacco mosaic virus, the cauliflower mosaic virus (CaMV) 19S and 35S promoters, or the figwort mosaic virus 35S promoter).

[0165] 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, wherein the mutation results in the regulated function or activity of NtSULTR3 or the polypeptide encoded thereby, or the regulated function or activity of NtSULTR3 and NtSUS or the polypeptide encoded thereby. Combinations of such mutations are contemplated herein.

[0166] A method is provided for regulating the level of NtSULTR3 polypeptide or NtSULTR3 polypeptide and NtSUS polypeptide in a (dried) plant or (dried) plant material, the method comprising introducing into the genome of the plant one or more mutations that regulate the expression of at least one NtSULTR3 gene or at least one NtSULTR3 gene and at least one NtSUS gene, wherein the at least one gene is selected from any of the sequences according to the present disclosure.

[0167] Also provided is a method for identifying plants with regulated levels of reducing sugars, the Gao method 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 (e.g., NtSULTR3 or NtSULTR3 and NtSUS, or a combination thereof), and optionally correlating the identified mutations with mutations known to regulate the level of reducing sugars.

[0168] Also disclosed are plants or plant cells that are heterozygous or homozygous for one or more mutations in the NtSULTR3 gene or the NtSULTR3 gene and the NtSUS gene according to the present disclosure, which mutations result in modulation of the expression of the gene or the function or activity of the NtSULTR3 polypeptide or the NtSULTR3 and NtSUS polypeptides encoded thereby.

[0169] Numerous methods, including sexual crossing, can be used to combine mutations in one plant.A plant having one or more favorable heterozygous or homozygous mutations in a gene according to the present disclosure, which 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, which regulate the expression of the gene or the function or activity of the polypeptide encoded thereby.In one embodiment, crossing is carried out to introduce one or more favorable heterozygous or homozygous mutations in a gene according to the present disclosure in the same plant.

[0170] The function or activity of one or more polypeptides of the present disclosure in a plant is increased or decreased if the function or activity is lower or higher than the function or activity of the same polypeptide in a plant that has not been modified to inhibit the function or activity of the polypeptide and that has been grown, harvested, and dried using the same protocols.

[0171] In some embodiments, mutations are introduced into plants or plant cells using mutagenesis techniques, and the introduced mutations are identified or selected using methods known to those skilled in the art (e.g., Southern blot analysis, DNA sequencing, PCR analysis, or phenotypic analysis). Mutations that affect gene expression or disrupt the function of the encoded polypeptide can be determined using methods well known in the art. Insertional mutations in gene exons usually result in null mutants. Mutations in conserved residues can be particularly effective in disrupting the metabolic function of the encoded polypeptide. For example, it will be understood that mutations in one or more highly conserved regions are likely to alter polypeptide function, while mutations outside those highly conserved regions are likely to have little or no effect on polypeptide function. In addition, mutations in a single nucleotide can create a stop codon, resulting in a truncated polypeptide and, depending on the extent of truncation, loss of function.

[0172] Also disclosed are methods for obtaining mutant polynucleotides and polypeptides.Any plant of interest, including plant cells or plant materials, can be genetically modified by various known methods for inducing mutagenesis, including site-directed mutagenesis, oligonucleotide-directed mutagenesis, chemically induced mutagenesis, radiation-induced mutagenesis, mutagenesis using modified bases, mutagenesis using gapped duplex DNA, double-strand break mutagenesis, mutagenesis using repair-deficient host strains, mutagenesis by total gene synthesis, DNA shuffling and other equivalent methods.

[0173] The mutations in the polynucleotides and polypeptides described herein may include man-made mutations, synthetic mutations, or genetically engineered mutations. The mutations in the polynucleotides and polypeptides described herein may be mutations that have been obtained or can be obtained through a process involving in vitro or in vivo manipulation steps. The mutations in the polynucleotides and polypeptides described herein may be mutations that have been obtained or can be obtained through a process involving human intervention. The function or activity of the mutant polypeptide variant may be higher, lower, or approximately the same as that of the unmutated polypeptide.

[0174] 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 produce point mutations, as well as short deletions, insertions, missense mutations, simple sequence repeats, transversions, or transitions, including chemical mutagens or radiation, can be used to create mutations. Mutagens include ethyl methanesulfonate, methyl methanesulfonate, N-ethyl-N-nitrosourea, triethylmelamine, N-methyl-N-nitrosourea, procarbazine, chlorambucil, cyclophosphamide, diethyl sulfate, acrylamide monomer, melphalan, nitrogen mustard, vincristine, dimethylnitrosamine, N-methyl-N'-nitro-nitrosoguanidine, nitrosoguanidine, 2-aminopurine, 7,12 dimethyl-benz(a)anthracene, ethylene oxide, hexamethylphosphoramide, bisulfane, diepoxyalkanes (diepoxyoctane, diepoxybutane, etc.), 2-methoxy-6-chloro-9[3-(ethyl-2-chloro-ethyl)aminopropylamino]acridine dihydrochloride, and formaldehyde.

[0175] Spontaneous mutations at loci that may not be directly caused by mutagens are also contemplated, provided that they result in the desired phenotype. Suitable mutagens may also include, for example, ionizing radiation (e.g., X-rays, gamma rays, fast neutron irradiation, and UV radiation). The dosage of mutagenic chemicals or radiation is experimentally determined for each type of plant tissue to obtain a mutation frequency that is below the threshold level characterized by lethality or reproductive sterility. Any method of preparing plant polynucleotides known to those skilled in the art can be used to prepare plant polynucleotides for mutation screening.

[0176] The mutation process may involve one or more plant cross-breeding steps.

[0177] After mutation, screening can be performed to identify mutations that create premature stop codons or otherwise non-functional genes. After mutation, screening can be performed to identify mutations that create functional genes that can be expressed at increased or decreased levels. Screening for mutants can be performed by sequencing or by using one or more probes or primers specific to the gene or polypeptide. Specific mutations in polynucleotides can also be created that can result in regulated gene expression, regulated mRNA stability, or regulated polypeptide stability. Such plants are referred to herein as "non-natural" or "mutant" plants. Typically, mutant or non-natural plants will contain at least a portion of exogenous, synthetic, or man-made nucleotides (e.g., DNA or RNA) that were not present in the plant before manipulation. The exogenous nucleotide can be a single nucleotide, two or more nucleotides, two or more consecutive nucleotides, or two or more non-contiguous nucleotides (e.g., at least 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or 1500 or more consecutive or non-contiguous nucleotides).

[0178] c. Transgenics and genome editing Sequence-specific polynucleotides that can interfere with the transcription of one or more endogenous genes, sequence-specific polynucleotides that can interfere with the translation of RNA transcripts (for example, double-stranded RNA, siRNA, ribozymes), sequence-specific polypeptides that can interfere with the stability of one or more polypeptides, sequence-specific polynucleotides that can interfere with the enzymatic function of one or more polypeptides or the binding function of one or more polypeptides with respect to substrates or regulatory polypeptides, antibodies that show specificity for one or more polypeptides, small molecule compounds that can interfere with the stability of one or more polypeptides or the enzymatic function of one or more polypeptides or the binding function of one or more polypeptides, zinc finger polypeptides that bind to one or more polynucleotides, and meganucleases that have 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 will be further discussed below.

[0179] D. zinc finger nuclease Zinc finger polypeptides can be used to regulate the expression, function, or activity of one or more of the NtSULTR3 or NtSULTR3 and NtSUS polynucleotides described herein. The use of zinc finger nucleases is described in Nature Rev. Genet. (2010) 11(9):636-646).

[0180] e. meganuclease Meganucleases such as I-CreI can be used to regulate the expression, function, or activity of one or more of the NtSULTR3 or NtSULTR3 and NtSUS polynucleotides described herein. The use of meganucleases is described in Curr Gene Ther. (2011) Feb; 11 (1): 11-27 and Int J Mol Sci. (2019) 20 (16), 4045.

[0181] f.TALEN Transcription activator-like effector nuclease (TALEN) can be used to regulate the expression or function or activity of one or more of the NtSULTR3 or NtSULTR3 and NtSUS polynucleotides described herein.The use of TALEN is described in Nature Rev.Mol.Cell Biol.(2013)14:49-55 and Int J Mol Sci.(2019)20(16),4045.

[0182] g.CRISPR CRISPR system can be used to regulate the expression or function or activity of one or more of NtSULTR3 or NtSULTR3 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) (for example, Cas9) and guide RNA (gRNA). Cas creates double-stranded DNA breaks at sites in the genome defined by the sequence of the gRNA molecule bound to it. The location at which Cas cleaves DNA is defined by the unique sequence of the gRNA bound to it. The gRNA is a specifically designed RNA sequence that recognizes a target DNA region and directs Cas nuclease there for editing. It has two sections: (i) a tracer RNA that serves as a binding scaffold for the Cas nuclease, and (ii) a 17-20 nucleotide sequence of Crisp RNA (crRNA) that is complementary to the target DNA. The exact region of DNA targeted will depend on the specific application. For example, to activate or repress a target polynucleotide, the gRNA can be targeted to a promoter that drives expression of the target polynucleotide. Methods for designing gRNAs are well known in the art and include Chop Chop Harvard.

[0183] 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).

[0184] In addition to Cas9, other RNA-guided nucleases have been described for use in CRISPR systems, including Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, CaslO, Cpfl, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3 and Csf4. In certain embodiments, the use of Cas9 is preferred.

[0185] The present disclosure provides a CRISPR-based genome editing system comprising an RNA-guided nuclease and a gRNA, which regulates the activity of one or more of the polynucleotides described herein. The present disclosure also provides a method for cleaving one or more polynucleotides in a plant cell, comprising introducing a gRNA and an RNA-guided nuclease into the plant cell, wherein the gRNA acts in conjunction with the RNA-guided nuclease to create a strand break in one or more of the polynucleotides described herein. A CRISPR construct is also disclosed, which comprises (i) a polynucleotide encoding a CRISPR-associated endonuclease, and (ii) a gRNA comprising a polynucleotide sequence (typically about 17-20 nucleotides) complementary to the DNA of the targeted polynucleotide described herein.

[0186] h. Antisense modification Antisense technology is another well-known method that can be used to regulate the expression or activity of one or more NtSULTR3 polypeptides or one or more of the NtSULTR3 and NtSUS polypeptides. See, e.g., Gene (1988) 10;72(1-2):45-50.

[0187] i. Mobile genetic elements Alternatively, genes can be targeted for inactivation by introducing transposons (e.g., IS elements) into the genome of the plant of interest. See, e.g., Cytology and Genetics (2006) 40(4):68-81.

[0188] j. ribozyme Alternatively, NtSULTR3 or NtSULTR3 and NtSUS polynucleotides can be targeted for inactivation by introducing into plants ribozymes derived from a number of small circular RNAs capable of self-cleavage and replication. See, e.g., FEMS Microbiology Reviews (1999) 23, 3, 257-275.

[0189] 5.Plants A mutant or non-naturally occurring plant or plant cell may have one or more modifications (e.g., mutations) in one or more of NtSULTR3 or NtSULTR3 and NtSUS, or any combination of the polypeptides encoded thereby that result in regulated expression, function, or activity of the polynucleotides or their polynucleotide products. For example, a mutant or non-naturally occurring plant or plant cell may have a single modification in a single NtSULTR3 polynucleotide or polypeptide, or a single NtSULTR3 polynucleotide or polypeptide and a single NtSUS polynucleotide or polypeptide; multiple modifications in a single NtSULTR3 polynucleotide or polypeptide, or a single NtSULTR3 and a single NtSUS polynucleotide or polypeptide; two or more, three or more, or four or more NtSULTR3 polynucleotides or polypeptides, or a single modification in NtSULTR3 and NtSUS polynucleotides or polypeptides; or multiple modifications in two or more, three or more, or four or more NtSULTR3 or NtSULTR3 and NtSUS polynucleotides or polypeptides. As a further example, a mutant or non-naturally occurring plant or plant cell can have one or more modifications in a particular portion of an NtSULTR3 or NtSULTR3 and NtSUS polynucleotide or polypeptide (e.g., in a region of an NtSULTR3 or NtSULTR3 and NtSUS that encodes the active site of an NtSULTR3 or NtSUS polypeptide or portion thereof). As a further example, a mutant or non-naturally occurring plant or plant cell can have one or more modifications in a region outside of one or more NtSULTR3 or NtSULTR3 and NtSUS polynucleotides or polypeptides (e.g., in a region upstream or downstream of an NtSULTR3 or NtSULTR3 and NtSUS polynucleotide that it regulates, provided that it regulates the function or expression of the NtSULTR3 or NtSULTR3 and NtSUS p). Upstream elements can include promoters, enhancers, or transcription factors.Some elements, such as enhancers, can be located upstream or downstream of the gene they regulate.Some elements have been found to be located hundreds of thousands of base pairs upstream or downstream of the gene they regulate, so elements do not need to be located near the gene they regulate.Mutant or non-natural plant or plant cell can have one or more modifications located within the first 100 nucleotides of a gene, the first 200 nucleotides of a gene, the first 300 nucleotides of a gene, the first 400 nucleotides of a gene, the first 500 nucleotides of a gene, the first 600 nucleotides of a gene, the first 700 nucleotides of a gene, the first 800 nucleotides of a gene, the first 900 nucleotides of a gene, the first 1000 nucleotides of a gene, the first 1100 nucleotides of a gene, the first 1200 nucleotides of a gene, the first 1300 nucleotides of a gene, the first 1400 nucleotides of a gene, or the first 1500 nucleotides of a gene. The mutant or non-naturally occurring plant or plant cell can have one or more modifications located within the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, or fifteenth set of 100 nucleotides of the gene, or a combination thereof. Disclosed are mutant or non-naturally occurring plants or plant cells (such as the mutant, non-naturally occurring, or transgenic plants or plant cells described herein) that comprise mutant polypeptide variants.

[0190] In one embodiment, seeds from the plant are mutagenized and then grown into first-generation mutant plants. The first-generation plants are then self-pollinated, and seeds from the first-generation plants are grown into second-generation plants and then screened for mutations at those loci. While mutagenized plant material can be screened for mutations, the advantage of screening second-generation plants is that all somatic mutations correspond to germline mutations. Those skilled in the art will understand that a variety of plant materials, including but not limited to seeds, pollen, plant tissues, or plant cells, can be mutagenized to create mutant plants. However, the type of mutagenized plant material can affect the screening of plant polynucleotides for mutations. For example, if pollen is subjected to mutagenesis before pollination of a non-mutagenized plant, the seeds resulting from that pollination will grow into first-generation plants. All cells of the first-generation plants will contain the mutations created in the pollen, and therefore these first-generation plants can then be screened for mutations without waiting until the second generation.

[0191] 6. Preparation, Screening, and Hybridization of Modified Plants The NtSULTR3 or NtSULTR3 and NtSUS polynucleotides prepared from individual plants, plant cells, or plant materials can optionally be pooled to facilitate screening for mutations in a population of plants derived from the mutagenized plant tissues, cells, or materials. One or more subsequent generations of plants, plant cells, or plant materials can be screened. The size of the optionally pooled group depends on the sensitivity of the screening method used.

[0192] After the samples are optionally pooled, they can be subjected to polynucleotide-specific amplification techniques such as PCR. Any one or more primers or probes specific to the gene or sequence immediately adjacent to the gene can be used to amplify the sequence in the optionally pooled sample. 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. Additionally, primers and probes preferably avoid known polymorphic sites to facilitate screening for point mutations. To facilitate detection of amplification products, 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.

[0193] To facilitate detection of the amplification products, primers or probes may be labeled using any conventional labeling method, which may be designed based on the sequences described herein using methods well understood in the art.

[0194] Polymorphisms can be identified by means known in the art, several of which are described in the literature.

[0195] In some embodiments, plants may be regenerated or grown from plants, plant tissues, or plant cells. Any suitable method for regenerating or growing plants from plant cells or plant tissues may be used, including, but not limited to, tissue culture or regeneration from protoplasts. Preferably, plants may be regenerated by growing transformed plant cells on callus, shoot, or root induction media. See, for example, McCormick et al., Plant Cell Reports 5:81-84 (1986). These plants are then grown and pollinated with either the same transformed line or a different line, and the resulting hybrids with the desired phenotypic characteristics are identified. Two or more generations may be grown to ensure that the expression of the desired phenotypic characteristics is stably maintained and inherited, and the seeds may then be harvested to ensure that the desired phenotypic characteristics have been achieved. Thus, "transformed seeds" refer to seeds containing a nucleotide construct stably integrated into the plant genome.

[0196] Thus, in a further aspect, a method for preparing a mutant plant is provided. The method involves providing at least one cell of a plant containing an NtSULTR3 or NtSULTR3 and NtSUS gene (or any combination thereof described herein) encoding a functional polynucleotide 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, and the mutant plant has a modulated level of the NtSULTR3 or NtSULTR3 and NtSUS polypeptide (or any combination thereof described herein) described herein compared to that of a control plant. In one embodiment of this method for producing a mutant plant, the treating step involves subjecting at least one cell to a chemical mutagen, as described above, and under conditions effective to obtain at least one mutant plant cell. In another embodiment of this method, the treating step involves subjecting at least one cell to a radiation source under conditions effective to obtain at least one mutant plant cell. The term "mutant plant" includes mutant plants whose genotype is altered compared to a control plant, preferably by means other than genetic engineering or genetic modification.

[0197] In certain embodiments, a mutant plant, mutant plant cell, or mutant plant material may contain one or more mutations that naturally occur in another plant, plant cell, or plant material and confer a desired trait. This mutation may be incorporated (e.g., introgressed) into another plant, plant cell, or plant material (e.g., a plant, plant cell, or plant material having a different genetic background than the plant from which the mutation originated) to confer the trait thereto. Thus, by way of example, a naturally occurring mutation in a first plant may be introduced into a second plant (e.g., a second plant having a different genetic background than the first plant). Thus, one skilled in the art can search for and identify plants that naturally harbor one or more mutant alleles of the genes described herein in their genome that confer a desired trait. Naturally occurring mutant alleles may be transferred to a second plant by various methods, including breeding, backcrossing, and introgression, to produce a line, variety, or hybrid having one or more mutations in the genes described herein. The same 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 traits can be screened out from a pool of mutant plants. Preferably, selection is performed using knowledge of the polynucleotides described herein. Consequently, it is possible to screen for genetic traits compared to controls. Such screening procedures can involve the application of traditional amplification or hybridization techniques as discussed herein. Accordingly, a further aspect of the present disclosure relates to a method for identifying mutant plants, comprising: (a) providing a sample containing one or more NtSULTR3 or NtSULTR3 and NtSUS polynucleotides from a plant; and (b) determining the sequence of the polynucleotides, wherein a difference in the sequence of the polynucleotide compared to the polynucleotide of a control plant indicates that the plant is a mutant plant.In another aspect, a method is provided for identifying mutant plants that accumulate increased or decreased levels of reducing sugars, non-reducing sugars, and free amino acids compared to a control plant, comprising the steps of: (a) providing a sample from a plant to be screened; (b) determining whether the sample contains one or more mutations in one or more NtSULTR3 or NtSULTR3 and NtSUS polynucleotides described herein; and (c) determining the levels of at least one reducing sugar, at least one non-reducing sugar, and at least one free amino acid. Preferably, the levels of at least one reducing sugar, non-reducing sugar, and free amino acid are determined in dried leaves. In another aspect, a method is provided for preparing a mutant plant having increased or decreased levels of at least one reducing sugar, at least one non-reducing sugar, and at least one free amino acid compared to a control plant, the method comprising: (a) providing a sample from a first plant; (b) determining whether the sample contains one or more mutations in one or more NtSULTR3 or NtSULTR3 and NtSUS polynucleotides described herein, which result in adjusted levels of at least one reducing sugar, at least one non-reducing sugar, and at least one free amino acid; and (c) transferring the 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 (e.g., by genetic engineering, genetic manipulation, introgression, plant breeding, backcrossing, etc.). In one embodiment, the first plant is a wild-type plant. In one embodiment, the second plant has a different genetic background from the first plant.In another aspect, a method is provided for preparing a mutant plant having increased or decreased levels of at least one reducing sugar, at least one non-reducing sugar, and at least one free amino acid compared to a control plant, comprising: (a) providing a sample from a first plant; (b) determining whether the sample contains one or more mutations in one or more of the NtSULTR3 or NtSULTR3 and NtSUS polynucleotides described herein that result in adjusted levels of at least one reducing sugar, at least one non-reducing sugar, and at least one free amino acid; and (c) introgressing the one or more mutations from the first plant into a second plant. Preferably, the levels of at least one reducing sugar, at least one non-reducing sugar, and at least one free amino acid are determined in dried leaves. In one embodiment, the introgressing step involves 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 from the first plant. In one embodiment, the first plant is not a cultivar or elite cultivar. In one embodiment, the second plant is a cultivar or elite cultivar. A further aspect relates to mutant plants (including mutant plants of cultivars or elite cultivars) obtained or obtainable by the methods described herein. In certain embodiments, the mutant plant may have one or more mutations localized only to a specific region of the plant, such as within the sequences of one or more NtSULTR3 or NtSULTR3 and NtSUS polynucleotides described herein. According to this embodiment, the remaining genomic sequence of the mutant plant is the same or substantially the same as that of the plant before mutagenesis.

[0198] In certain embodiments, the mutant plant may have one or more mutations localized in multiple genomic regions of the plant (e.g., within the sequence of one or more of the NtSULTR3 or NtSULTR3 and NtSUS polynucleotides described herein and in one or more additional regions of the genome). In accordance with this embodiment, the remaining genomic sequence of the mutant plant will be identical or substantially different from that of the plant prior to mutagenesis. In certain embodiments, the mutant plant may not have one or more mutations in one or more, two or more, three or more, four or more, or five or more exons of the NtSULTR3 or NtSULTR3 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 NtSULTR3 or NtSULTR3 and NtSUS polynucleotides described herein, or may not have one or more mutations in the promoter of the NtSULTR3 or NtSULTR3 and NtSUS polynucleotides described herein, or may not have one or more mutations in the promoter of the NtSULTR3 or NtSULTR3 and NtSUS polynucleotides described herein. or may not have one or more mutations in the 3' untranslated region of the NtSULTR3 or NtSULTR3 and NtSUS polynucleotides described herein, or may not have one or more mutations in the 5' untranslated region of the NtSULTR3 or NtSULTR3 and NtSUS polynucleotides described herein, or may not have one or more mutations in the coding region of the NtSULTR3 or NtSULTR3 and NtSUS polynucleotides described herein, or may not have one or more mutations in the non-coding region of the NtSULTR3 or NtSULTR3 and NtSUS polynucleotides described herein, or any combination of two or more, three or more, four or more, five or more, or six or more thereof.

[0199] In a further aspect, there is provided a method for identifying a plant, plant cell, or plant material containing a mutation in a gene encoding the NtSULTR3 or NtSULTR3 and NtSUS polynucleotide described herein, comprising: (a) subjecting the plant, plant cell, or plant material to mutagenesis; (b) obtaining a sample from the plant, plant cell, or plant material, or a progeny thereof; and (c) determining the polynucleotide sequence of the NtSULTR3 or NtSULTR3 and NtSUS gene, or a variant or fragment thereof, wherein differences in the sequence indicate one or more mutations therein. This method also allows for the selection of plants with mutations occurring in genomic regions that affect the expression of the NtSULTR3 or NtSULTR3 and NtSUS gene in plant cells, such as transcription start sites, start codons, intronic regions, exon-intron boundaries, terminators, or stop codons.

[0200] 7. Plant families, species, cultivars, seeds, and tissue cultures Suitable plants for use in the present disclosure include monocotyledonous and dicotyledonous plants and plant cell lines.Suitable plants for use in the present disclosure include members of the genus Camellia, Cannabis, and Nicotiana.Suitable species of Camellia and Cannabis can include Camellia sinensis (tea), Cannabis sativa, Cannabis indica, and Cannabis ruderalis.

[0201] Various embodiments relate to mutant, non-native, or transgenic tobacco plants or plant cells. The disclosed compositions and methods can be applied to any species of the genus Nicotiana, including N. rustica and N. tabacum (e.g., LA B21, LN KY171, TI 1406, Basma, Galpao, Perique, Beinhart 1000-1, and Petico).Other species include N. acaulis, N. acuminata, N. africana, N. alata, N. ameghinoi, N. amplexicaulis, N. arentsii, N. attenuata, N. azambujae, and N. 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 is a common species of N.tabacum.

[0202] The use of tobacco cultivars and elite tobacco cultivars is also contemplated herein.Therefore, transgenic, non-natural type or mutant plants can be tobacco cultivars or elite tobacco cultivars that contain one or more introduced genes, or one or more genetic mutations, or a combination thereof.Genetic mutations (for example, one or more polymorphisms) can be mutations that do not naturally occur in individual tobacco cultivars or tobacco cultivars (for example, elite tobacco cultivars), or can be genetic mutations that occur naturally, provided that the mutations do not naturally occur in individual tobacco cultivars or tobacco cultivars (for example, elite tobacco cultivars).

[0203] Particularly useful Nicotiana tabacum varieties include burley-type, dark-type, flue-cured-type, and orient-type tobaccos. Non-limiting examples of varieties or cultivars include BD 64, CC 101, CC 200, CC 27, CC 301, CC 400, CC 500, CC 600, CC 700, CC 800, CC 900, Coker 176, Coker 319, Coker 371 Gold, Coker 48, CD 263, DF911, DT 538 LC Galpao tobacco, GL 26H, GL 350, GL 600, GL 737, GL 939, GL 973, HB 04P, HB 04P LC, HB3307PLC, Hybrid 403LC, Hybrid 404LC, Hybrid 501 LC, K 149, K 326, K 346, K 358, K394, K 399, K 730, KDH 959, KT 200, KT204LC, KY10, KY14, KY 160, KY 17, KY 171, 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" cigarettes, 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, RS 1410, Speight 168, Speight 172, Speight 179, Speight 210, Speight 220, Speight 225, Speight 227, Speight 234, Speight G-28, Speight G-70, SpeightH-6, Speight H20, Speight NF3, TI 1406, TI 1269, TN 86, TN86LC, TN 90, TN 97, TN97LC, TN D94, TN D950, TR(Tom Rosson)Madole, VA 309, VA359, AA 37-1, B13P, Xanthi(Mitchell-Mor), Bel-W3, 79-615, Samsun Holmes NN, KTRDC2, KTRDC2, PO3, RG11, KY8959, KY9, MD 609, PG01, PG04, PO1, PO2, PO3, RG11, RG 8. VA509 81, DVH 405, Galpao Comum, HB04P, Hicks Broadleaf, Kabakulak Elassona, Kutsage E1, LA BU 21, NC 2326, NC 297, PVH 2110, Red Russian, Samsun, Saplak, Simmaba, Talgar 28, Wislica, Yayaldag, Prilep HC-72, Prilep P23, Prilep PB 156 / 1, Prilep P12-2 / 1, Yaka JK-48, Yaka JB 125 / 3、TI-1068、KDH-960、TI-1070、TW136、Basma、TKF 4028、L8、TKF 2002、GR141、Basma xanthi、GR149、GR153、Petit Havana is a nice place to stay with a lot of snow (low converter) is a selection.

[0204] Embodiments also relate to compositions and methods for producing mutant plants, non-naturally occurring plants, hybrid plants, or transgenic plants that have been modified to regulate the expression or function of one or more NtSULTR3 or NtSULTR3 and NtSUS polynucleotides described herein (or any combination thereof described herein). Advantageously, the resulting mutant plants, non-naturally occurring plants, hybrid plants, or transgenic plants can be similar or substantially identical to control plants in overall appearance. Various phenotypic characteristics, such as the degree of maturity, number of leaves per plant, stalk height, leaf insertion angle, leaf size (width and length), internode distance, and leaf blade-midrib ratio, can be evaluated by field observation.

[0205] One aspect relates to the seeds of the mutant plants, non-natural plants, hybrid plants, or transgenic plants described herein. Preferably, the seeds are tobacco seeds. A further aspect relates to the pollen or ovules of the mutant plants, non-natural plants, hybrid plants, or transgenic plants described herein. In addition, the mutant plants, non-natural plants, hybrid plants, or transgenic plants described herein are provided, which further comprise a polynucleotide that confers male sterility.

[0206] Also provided are tissue cultures of regenerable cells of the mutant, non-naturally occurring, hybrid, or transgenic plants described herein, or parts thereof, which cultures regenerate plants capable of expressing all the morphological and physiological characteristics of the parent. Regenerable cells include cells from leaves, pollen, embryos, cotyledons, hypocotyls, roots, root tips, anthers, flowers and their parts, ovules, shoots, stems, stalks, pith, and capsules, or callus or protoplasts derived therefrom.

[0207] The plant material described herein can be cured tobacco material (e.g., cured tobacco material from a Virginia or Orient type). The cured tobacco material can be flue-cured, sun-cured, or air-cured tobacco material.

[0208] CORESTA recommendations for tobacco curing are set out in CORESTA Guide No. 17, April 2016, Sustainability in Leaf Tobacco Production.

[0209] 8. Regulation of sugar and amino acid content The mutant, transgenic, or non-naturally occurring plants or parts thereof of the present disclosure exhibit regulated sugar and amino acid content in dried leaves, preferably fully dried leaves. Preferably, the dried leaves are taken from leaves at the middle position of the plant. Preferably, the mutant, transgenic, or non-naturally occurring plants or parts thereof have substantially the same visual appearance as a control plant.

[0210] A plant part (e.g., dried leaves) of the present disclosure may have a reduced level of at least one reducing sugar by at least 50%, a reduced level of at least one non-reducing sugar by at least 50%, and an increased level of at least one free amino acid by at least 1.5-fold compared to a control plant in which the expression or function of the NtSULTR3 or NtSULTR3 and NtSUS polypeptides is not regulated. For example, a plant part (e.g., dried leaves) may have a reduced level of at least one reducing sugar by at least 60%, a reduced level of at least one non-reducing sugar by at least 60%, and an increased level of at least one free amino acid by at least 1.5-fold. As a further example, a plant part (e.g., dried leaves) may have a reduced level of at least one reducing sugar by at least 69%, a reduced level of at least one non-reducing sugar by at least 60%, and an increased level of at least one free amino acid by at least 1.5-fold.

[0211] In certain embodiments, glucose and fructose levels are reduced by at least about 55% or more (e.g., at least about 60% or more, or at least about 65% or more), and sucrose levels are reduced by at least about 55% or more (e.g., at least about 60% or more) compared to control plants.

[0212] In certain embodiments, glucose levels are reduced by at least 55% or more, or at least 65% or more, or at least 70% or more, or at least 75% or more, or at least 77% or more, fructose levels are reduced by at least 55% or more, or at least 60% or more, or at least 65% or more, or at least 69% or more, and sucrose levels are reduced by at least 55% or at least 60% or more, compared to the control plant.

[0213] In certain embodiments, glucose levels are reduced by at least 75% or more, fructose levels are reduced by at least 65% or more, and sucrose levels are reduced by at least about 55% or more compared to a control plant.

[0214] In certain embodiments, glucose levels are reduced by at least 77% or more, fructose levels are reduced by at least 69% or more, and sucrose levels are reduced by at least about 60% or more compared to a control plant.

[0215] In certain embodiments, the level of at least one free amino acid is increased by at least 1.5 times compared to a control plant. In certain embodiments, the levels of glutamine, glutamic acid, and aspartic acid are increased by at least 2 times compared to a control plant. In certain embodiments, the levels of glutamine, glutamic acid, and aspartic acid are increased by at least 2.3 times, at least 2.4 times, and at least 2 times, respectively, compared to a control plant.

[0216] In certain embodiments, compared to a control plant, glucose levels are reduced by at least 75% or more, fructose levels are reduced by at least 65% or more, sucrose levels are reduced by at least about 55% or more, and glutamine, glutamic acid, and aspartic acid levels are increased by at least 2-fold.

[0217] In certain embodiments, compared to a control plant, glucose levels are reduced by at least 77% or more, fructose levels are reduced by at least 69%, sucrose levels are reduced by at least about 60%, and glutamine, glutamic acid, and aspartic acid levels are increased by at least 2.3-fold, at least 2.4-fold, and at least 2-fold, respectively.

[0218] The amount of asparagine can be increased by at least about 1.5-fold compared to the control plant.

[0219] Plant parts (e.g., dried leaves) of the present disclosure may have an increased level of at least one reducing sugar, an increased level of at least one non-reducing sugar, and a decreased level of at least one free amino acid compared to a control plant in which the expression or function of the NtSULTR3 or NtSULTR3 and NtSUS polynucleotide or polypeptide encoded thereby is not modulated.

[0220] A further embodiment relates to dried plant material (e.g., dried leaves or dried tobacco) derived or capable of being derived from a mutant, non-naturally occurring, or transgenic plant or cell described herein, wherein the levels of at least one reducing sugar and at least one non-reducing sugar and free amino acids are adjusted as discussed above compared to a control. The amount of asparagine may be adjusted compared to a control plant.

[0221] A further aspect relates to dried plant material (e.g., dried leaves or dried tobacco) that is derived from or can be derived from a mutant, non-naturally occurring, or transgenic plant or cell described herein, and the levels of glucose, fructose, and sucrose, as well as the levels of glutamine, glutamic acid, and aspartic acid, are adjusted as discussed above compared to a control, as compared to a control. The amount of asparagine can be adjusted compared to a control plant.

[0222] Embodiments also relate to compositions and methods for producing the mutant, non-native, or transgenic plants or plant cells described herein having modulated levels of at least one reducing sugar and at least one non-reducing sugar and free amino acid, as discussed above. The amount of asparagine may be modulated compared to a control plant.

[0223] In one embodiment, the phenotype of the mutant, non-native or transgenic plant is substantially the same as that of a control plant. In one embodiment, the leaf weight of the mutant, non-native or transgenic plant is substantially the same as that of a control plant. In one embodiment, the leaf number of the mutant, non-native or transgenic plant is substantially the same as that of a 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 a control plant. In one embodiment, the stalk height of the mutant, non-native or transgenic plant is substantially the same as that of a control plant, for example, 1 month, 2 months, or 3 months or more after field transplantation, or 10 days, 20 days, 30 days, or 36 days or more after topping. For example, the stalk 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 a 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.

[0224] In another aspect, a method for producing a mutant, non-native or transgenic plant comprising the steps of: (i) modulating the expression or function of one or more of the NtSULTR3 or NtSULTR3 and NtSUS polypeptides described herein (or any combination thereof described herein), suitably wherein the NtSULTR3 or NtSULTR3 and NtSUS polypeptides are encoded by corresponding NtSULTR3 or NtSULTR3 and NtSUS polynucleotides described herein; and (ii) detecting at least one mutation in at least a portion of the mutant, non-native or transgenic plant obtained in step (i) (e.g., in leaves such as dried leaves or in tobacco or smoke). and (iii) identifying a mutant, non-native, or transgenic plant in which the levels of at least one reducing sugar, at least one non-reducing sugar, and at least one free amino acid are modulated compared to a control plant.

[0225] In another aspect, a method for producing a plant comprising the steps of: (i) modulating the expression or function of one or more NtSULTR3 or NtSULTR3 and NtSUS polypeptides (or any combination thereof described herein), suitably wherein the NtSULTR3 or NtSULTR3 and NtSUS polypeptides are encoded by corresponding NtSULTR3 or NtSULTR3 and NtSUS polynucleotides described herein; (ii) harvesting and drying plant material, such as one or more of the leaves, for a period of time; and (iii) harvesting at least one of the dried plant material obtained in or during step (ii). Provided is a method for modulating the amount of at least one reducing sugar and at least one non-reducing sugar and at least one free amino acid in at least a portion of dried plant material, such as dried leaves, comprising: (i) measuring, at least in part, the levels of at least one reducing sugar (e.g., glucose and fructose), at least one non-reducing sugar (e.g., sucrose), and at least one free amino acid (e.g., glutamine, glutamic acid, and aspartic acid); and (ii) identifying dried plant material in which the levels of at least one reducing sugar and at least one non-reducing sugar and at least one free amino acid are modulated compared to a control plant.

[0226] 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 100% or more (e.g., 200%, 300%, 500%, 1000% or more) increase in transcriptional function, or NtSULTR3 or NtSULTR3 and NtSUS polynucleotide expression or NtSULTR3 or NtSULTR3 and NtSUS polypeptide expression, or a combination thereof.

[0227] 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 (e.g., 200%, 300%, 500%, 1000% or more) increase in transcriptional function, or NtSULTR3 or NtSULTR3 and NtSUS polynucleotide expression or NtSULTR3 or NtSULTR3 and NtSUS polypeptide expression, or a combination thereof.

[0228] The reduction 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 100% reduction, and includes a reduction in transcriptional function, or NtSULTR3 or NtSULTR3 and NtSUS polynucleotide expression or NtSULTR3 or NtSULTR3 and NtSUS polypeptide expression, or a combination thereof.

[0229] The decrease in function or activity compared to a control can be from about 5% to about 100%, or at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, or 100% reduction, and includes a decrease in transcriptional function, or NtSULTR3 or NtSULTR3 and NtSUS polynucleotide expression or NtSULTR3 or NtSULTR3 and NtSUS polypeptide expression, or a combination thereof.

[0230] The polynucleotides and recombinant constructs described herein can be used to modulate the expression or function or activity of NtSULTR3 or NtSULTR3 and NtSUS polynucleotides or NtSULTR3 or NtSULTR3 and NtSUS polypeptides described herein in a plant species of interest, preferably tobacco.

[0231] Numerous polynucleotide-based methods can be used to increase gene expression in plants and plant cells. As an example, a construct, vector, or expression vector compatible with the plant to be transformed can be prepared, containing a gene of interest along with an upstream promoter capable of overexpressing the gene in the plant or plant cell. Exemplary promoters are described herein. After transformation and when grown under suitable conditions, the promoter can drive expression to regulate the levels of NtSULTR3 or NtSULTR3 and NtSUS in the plant or specific tissues thereof. In one exemplary embodiment, a vector carrying one or more NtSULTR3 or NtSULTR3 and NtSUS polynucleotides described herein (or any combination thereof described herein) is generated to overexpress genes in plants or plant cells. The vector carries a suitable promoter, such as the cauliflower mosaic virus (CaMV) 35S promoter, upstream of the transgene, driving its constitutive expression in all tissues of the plant. The vector also carries an antibiotic resistance gene to confer selection for transformed callus and cell lines.

[0232] 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 drying procedure are specifically indicated.

[0233] Thus, various embodiments relate to methods for modulating the expression levels of one or more NtSULTR3 or NtSULTR3 and NtSUS polynucleotides described herein (or any combination thereof described herein), by integrating multiple copies of the NtSULTR3 or NtSULTR3 and NtSUS polynucleotides into the plant genome, comprising transforming a plant cell host with an expression vector comprising a promoter operably linked to one or more NtSULTR3 or NtSULTR3 and NtSUS polynucleotides described herein. The polypeptide encoded by the recombinant polynucleotide may be a native polypeptide or may be heterologous to the cell.

[0234] In one embodiment, plants for use in the present disclosure are plants that are hot-air dried so that such plants have a high reducing sugar content (greater than about 14% dry weight at the end of drying when field-grown). Mutant, transgenic, or non-native plants or portions thereof that are hot-air dried may have a reducing sugar content that is less than about 14% dry weight at the end of drying when field-grown (e.g., less than about 10% dry weight at the end of drying when field-grown, or less than about 5% dry weight at the end of drying when field-grown, or less than about 1% dry weight at the end of drying when field-grown).

[0235] In one embodiment, the plants used in the present disclosure are sun-dried plants such that the plants have a reducing sugar content (greater than about 6.8% dry weight at the end of drying when field-grown). A sun-dried mutant, transgenic, or non-native plant or portion thereof may have a reducing sugar content, when field-grown, that is less than about 5% dry weight at the end of drying (e.g., less than about 2.5% dry weight at the end of drying when field-grown, or less than about 1% dry weight at the end of drying when field-grown).

[0236] 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 weight when grown in the field at the end of drying.Sun-dried mutant, transgenic or non-natural plant or part thereof can have a reducing sugar content of less than about 1.5% dry weight when grown in the field at the end of drying (for example, less than about 1% dry weight when grown in the field at the end of drying, or less than about 0.5% dry weight when grown in the field at the end of drying).

[0237] In certain embodiments, the use of plants that are hot air dried or sun dried is preferred.

[0238] 9. Breeding Plants carrying mutant alleles of one or more NtSULTR3 or NtSULTR3 and NtSUS polynucleotides described herein (or any combination thereof described herein) can be used in plant breeding programs to create useful lines, varieties, and hybrids. In particular, mutant alleles can be introgressed into commercially important varieties described above. Accordingly, provided are methods of plant breeding that include crossing a mutant, non-native, or transgenic plant described herein with a plant containing a different genetic identity. The method can further include crossing the progeny plant with another plant, and optionally repeating the crossing until progeny possessing desirable genetic traits or genetic backgrounds are obtained. One goal served by such breeding methods is to introduce desirable genetic traits into other varieties, breeding lines, hybrids, or cultivars, particularly those of commercial interest. Another goal is to facilitate stacking of genetic modifications of different genes in a single plant variety, line, hybrid, or cultivar. Both interspecific and intraspecific crosses are contemplated. The progeny plants resulting from such crosses are also referred to as breeding lines and are examples of the disclosed non-naturally occurring plants.

[0239] In one embodiment, a method for producing a non-naturally occurring plant is provided, comprising: (a) crossing a mutant or transgenic plant with a second plant to obtain progeny tobacco seeds; (b) growing the progeny tobacco seeds under plant growth conditions to obtain a non-naturally occurring plant. The method may further comprise: (c) crossing the previous generation non-naturally occurring plant with itself or another plant to obtain progeny tobacco seeds; (d) growing the progeny tobacco seeds of step (c) under plant growth conditions to obtain additional non-naturally occurring plants; and (e) repeating the crossing and growing steps of (c) and (d) multiple times to generate additional generations of non-naturally occurring plants. The method may optionally comprise, prior to step (a), providing a parent plant that comprises a characterized genetic identity and is not identical to the mutant or transgenic plant. In some embodiments, depending on the breeding program, the crossing and growing steps are repeated 0-2, 0-3, 0-4, 0-5, 0-6, 0-7, 0-8, 0-9, or 0-10 times to generate generations of non-naturally occurring plants. Backcrossing is an example of such a method in which progeny is crossed with one of its parents or another plant genetically similar to that parent to obtain a next-generation progeny plant with a genetic identity closer to that of one of the parents. Plant breeding, particularly plant breeding techniques, are well known and can be used in the disclosed methods. The present disclosure further provides non-naturally occurring plants produced by these methods. Certain embodiments exclude the step of selecting plants.

[0240] In some embodiments of the methods described herein, lines resulting from breeding and screening for variant genes are evaluated in the field using standard field procedures. Control genotypes, including the original, unmutagenized parents, are included, and entries are arranged in the field in a randomized complete block design or other suitable field design. For tobacco, standard agronomic methods are used, e.g., the tobacco is harvested, weighed, and sampled for chemical and other common tests before and during curing. Statistical analysis of the data is performed to confirm the similarity of the selected lines to the parent lines. Cytogenetic analysis of the selected plants is optionally performed to confirm chromosome complement and chromosome pairing relationships.

[0241] DNA fingerprinting, single nucleotide polymorphism, microsatellite markers, or similar techniques can be used in marker-assisted selection (MAS) breeding programs to introgress or breed mutant alleles of genes into other tobacco plants, as described herein. For example, breeders can create segregating populations from hybridization of genotypes containing mutant alleles with agronomically desirable genotypes. F2 or backcross generation plants can be screened using markers developed from the genomic sequence or a fragment thereof using one of the techniques listed herein. Plants identified as carrying the mutant allele can be backcrossed or self-pollinated to create a second population to be screened. Depending on the expected inheritance pattern or MAS technique used, it may be necessary to self-pollinated selected plants before each 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.

[0242] According to the present disclosure, in a breeding program, successful crosses produce fertile F1 plants. The selected F1 plants can be crossed with one of the parents, and the first backcross generation plants are self-pollinated to produce a population that is again screened for variant gene expression (e.g., a null version of the gene). The backcross, self-pollination, and screening process is repeated, for example, at least four times until the final screening produces a plant that is fertile and reasonably similar to the recurrent parent. If desired, the plant is self-pollinated, and then the progeny is again screened to confirm that the plant exhibits variant gene expression. In some embodiments, the F2 generation plant population is screened for variant gene expression, for example, by using PCR methods with primers based on polynucleotide sequence information for the polynucleotides described herein (or any combination thereof described herein) according to standard methods, to identify plants that do not express the polypeptide due to a gene defect.

[0243] Hybrid tobacco varieties can be produced by preventing self-pollination of a female parent plant (i.e., seed parent) of a first variety, allowing pollen from a male parent plant of a second variety to pollinate the female parent plant, resulting in the formation of F1 hybrid seeds in the female plant. Self-pollination of female plants can be prevented by removing the stamens of flowers at an early stage of flower development. Alternatively, pollen formation can be prevented in female parent plants using the formation of male sterility. For example, male sterility can be produced by cytoplasmic male sterility (CMS) or transgenic male sterility, where the introduced gene inhibits microspore production and / or pollen formation, or self-incompatibility. Female parent plants containing CMS are particularly useful. In embodiments where the female parent plant is CMS, pollen is harvested from a male fertile plant and manually applied to the stigma of a CMS female parent plant, and the resulting F1 seed is harvested.

[0244] The varieties and lines described herein can be used to form single-cross tobacco F1 hybrids. In such embodiments, the parental varieties' plants can be grown as a substantially homogeneous, adjacent population to promote natural cross-pollination from the male parent plants to the female parent plants. The F1 seeds formed in the female parent plants can be selectively harvested by conventional means. Two parent plant varieties can also be grown in large quantities, and a blend of F1 hybrid seeds formed in the female parent and seeds formed in the male parent as a result of self-pollination can be harvested. Alternatively, a three-way cross can be performed, in which a single-cross F1 hybrid is used as the female parent and crossed with a different male parent. As another alternative, a double-cross hybrid can be created, in which the F1 progeny of two different single crosses are crossed with themselves.

[0245] A population of mutant, non-native, or transgenic plants can be screened or selected for those members of the population that have the desired trait or phenotype. For example, a population of progeny from a single transformation event can be screened for those plants that have the desired level of expression or function of the polypeptide encoded thereby. Physical and biochemical methods can be used to identify expression or activity levels. These include Southern analysis or PCR amplification for detecting polynucleotides; Northern blots, S1 RNase protection, primer extension, or RT-PCR amplification for detecting RNA transcripts; enzymatic assays for detecting enzyme or ribozyme activity of polypeptides and polynucleotides; and polypeptide gel electrophoresis, Western blots, immunoprecipitation, and enzyme immunoassays for detecting polypeptides. Other techniques, such as in situ hybridization, enzyme staining, and immunostaining and enzyme assays, can also be used to detect the presence or expression, function, or activity of NtSULTR3 or NtSULTR3 and NtSUS polypeptides or polynucleotides.

[0246] 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.

[0247] 10. Modification of other genes Without limitation, the plants and parts thereof described herein can be modified either before or after the expression, function or activity of one or more NtSULTR3 or NtSULTR3 and NtSUS polynucleotides and / or NtSULTR3 or NtSULTR3 and NtSUS polypeptides in accordance with the present disclosure are modulated.

[0248] One or more of the following additional genetic modifications may be present in mutant, non-native or transgenic plants and parts thereof:

[0249] 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.

[0250] One or more genes involved in heavy metal uptake or heavy metal transport can be modified to result in lower heavy metal content. Non-limiting examples include genes in the family of polypeptides associated with multidrug resistance, the family of cation diffusion facilitators (CDFs), the family of Zrt-Irt-like polypeptides (ZIPs), the family of cation exchangers (CAXs), the family of copper transporters (COPTs), the family of heavy metal ATPases (e.g., HMAs described in WO2009 / 074325 and WO2017 / 129739), the family of macrophage polypeptides related to natural resistance (NRAMPs), and other members of the ATP-binding cassette (ABC) transporter family (e.g., MRPs) described in WO2012 / 028309, which are involved in the transport of heavy metals such as cadmium.

[0251] Another exemplary modification 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).

[0252] Another exemplary modification can result in a plant with regulated expression or function of threonine synthase, such that levels of methionine can be regulated (see WO2013 / 029800).

[0253] Other exemplary modifications may result in plants with modulated expression or function of one or more of neoxanthin synthase, lycopene beta cyclase, and 9-cis-epoxycarotenoid dioxygenase, which modulate beta-damascenone content and alter flavor profiles (see WO2013 / 064499).

[0254] Another exemplary modification may result in plants having regulated expression or function of members of the CLC family of chloride channels that regulate nitrate levels therein (see WO2014 / 096283 and WO2015 / 197727).

[0255] Other exemplary modifications may result in plants having regulated expression or function of one or more asparagine synthetases, which regulate the levels of asparagine in leaves, and regulated levels of acrylamide in the aerosol produced upon heating or burning of leaves (see WO2017 / 129739).

[0256] Another exemplary modification may result in plants with modulated protease activity during desiccation (see WO2016 / 009006).

[0257] Another exemplary modification can result in plants with reduced nitrate levels by altering the gene expression of nitrate reductase (e.g., Nia2) or the activity of the protein encoded thereby (see WO2016 / 046288).

[0258] Another exemplary modification may result in plants with altered alkaloid levels by altering the gene expression of the putative ABC-2 transporters NtABCGl-T and NtABCGl-S, or the activity of the proteins encoded thereby (see WO2019 / 086609). Another exemplary modification may result in plants with a regulated time to flowering by altering the gene expression of the gene encoding Terminal Flowering 1 (TFL1), or the activity of the protein encoded thereby (see WO2018 / 114641).

[0259] Other exemplary modifications may result in plants having regulated expression or function of one or more asparagine synthetases, which regulate the levels of asparagine in leaves, and regulated levels of acrylamide in the aerosol produced upon heating or burning of leaves (see WO2017 / 042162).

[0260] Other examples of modifications include modulating herbicide tolerance; for example, glyphosate is the active ingredient in numerous broad-spectrum herbicides. Glyphosate-tolerant transgenic plants have been developed by transferring the aroA gene (glyphosate EPSP synthase from Salmonella typhimurium and E. coli). Sulfonylurea-tolerant plants have been produced by transforming mutant ALS (acetolactate synthase) genes from Arabidopsis. Mutant photosystem II OB polypeptides from Amaranthus hybridus have been transferred to plants to produce atrazine-tolerant transgenic plants, and bromoxynil-tolerant transgenic plants have been produced by incorporating the bxn gene from Klebsiella pneumoniae bacteria.

[0261] Another exemplary modification results in plants that are resistant to insects. Bacillus thuringiensis (Bt) toxins can provide an effective method for delaying the emergence of Bt-resistant pests, as recently demonstrated in broccoli, where the cry1Ac and cry1C Bt genes in cones controlled diamondback moths resistant to either single polypeptide, significantly slowing the evolution of resistant insects.

[0262] Another exemplary modification results in plants that are resistant to diseases caused by pathogens (e.g., viruses, bacteria, fungi). Plants expressing the Xa21 gene (resistance to bacterial leaf spot) have been engineered along with plants expressing both a Bt fusion gene and a chitinase gene (resistance to yellow stem borer and pod disease).

[0263] Another exemplary modification results in altered fertility, such as male sterility.

[0264] Another exemplary modification results in plants that are tolerant to abiotic stresses (e.g., drought, temperature, salinity); tolerant transgenic plants are produced by transferring acylglycerol phosphate enzymes from Arabidopsis, and genes encoding mannitol dehydrogenase and sorbitol dehydrogenase, involved in the synthesis of mannitol and sorbitol, improve drought tolerance.

[0265] Another exemplary modification results in a plant in which the activity of one or more nicotine N-demethylases is modulated so that the levels of nornicotine and nornicotine metabolites formed during drying can be controlled (see WO2015 / 169927).

[0266] Other exemplary modifications may result in plants with improved polypeptide and oil storage, plants with enhanced photosynthetic efficiency, plants with extended shelf life, plants with increased carbohydrate content, and plants that are resistant to fungi. Transgenic plants in which the expression of S-adenosyl-L-methionine (SAM) and / or cystathionine gamma-synthase (CGS) is regulated are also contemplated.

[0267] One or more genes involved in the nicotine synthesis pathway can be modified to result in a plant or plant part that produces controlled levels of nicotine when dried. The nicotine synthesis genes can be selected from the group consisting of A622, BBLa, BBLb, JRE5L1, JRE5L2, MATE1, MATE2, MPO1, MPO2, MYC2a, MYC2b, NBBl, nic1, nic2, NUP1, NUP2, PMT1, PMT2, PMT3, PMT4, and QPT, or one or more combinations thereof.

[0268] One or more genes involved in controlling the amount of one or more alkaloids can be modified to result in a plant or plant part that produces regulated levels of alkaloids. The alkaloid level control 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.

[0269] One or more such traits can be introgressed from another cultivar into, or directly transformed into, a mutant, non-native or transgenic plant.

[0270] Various embodiments provide mutant plants, non-naturally occurring plants, or transgenic plants, as well as biomass in which the expression level of one or more polynucleotides in accordance with the present disclosure is modulated, thereby modulating the level of the polypeptide encoded thereby.

[0271] 11.Consumable products The plant parts described herein, particularly the leaf blades and midribs of such plants, can be incorporated into or used in the manufacture of 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 produce an aerosol. In another type of heated aerosol-forming device, the aerosol is produced by the transfer of heat from a combustible fuel element or heat source to a physically separated aerosol-forming material that may be located within, around, or downstream of the heat source. Smokeless tobacco products and various tobacco-containing aerosol-forming materials may contain tobacco in any form, including as dry particles, pieces, granules, powders, or slurries, or deposited on, mixed with, surrounded by, or combined with any form of other component, such as flakes, films, tabs, foams, or beads. The term "smoke" is used to describe the type of aerosol produced by a smoking article, such as a cigarette, or by burning an aerosol-forming material.

[0272] In one embodiment, dried plant material from the mutant, transgenic, and non-naturally occurring plants described herein is also provided. Processes for curing green tobacco leaves are known to those skilled in the art and include, but are not limited to, air-curing, flame-curing, hot-air curing, and sun-curing, as described herein.

[0273] In another embodiment, a tobacco product is described that includes a tobacco-containing aerosol-forming material, including plant material (e.g., leaves, preferably cured leaves) 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.

[0274] 12. Crop Management and Agricultural Products and Methods Mutant, non-native or transgenic plants may have other uses, for example in agriculture.

[0275] The present disclosure also provides a method for producing seeds, including cultivating a mutant plant, a non-natural plant, or a transgenic plant described herein and collecting seeds from the cultivated plant.Seeds from the plants described herein can be prepared by means known in the art and packaged in packaging material to form an article of manufacture.Packaging materials such as paper and cloth are well known in the art.The seed package has a label, for example, a tag or label fixed to the packaging material, and a label describing the nature of the seeds therein is printed on the package.

[0276] Compositions, methods, and kits for genotyping plants for identification, selection, or breeding can include means for detecting the presence of NtSULTR3 or NtSULTR3 and NtSUS polynucleotides in a polynucleotide sample. Thus, compositions are described that include one or more primers for specifically amplifying at least a portion of one or more of the NtSULTR3 or NtSULTR3 and NtSUS polynucleotides, and optionally one or more probes, and optionally one or more reagents for amplification or detection.

[0277] Thus, gene-specific oligonucleotide primers or probes are disclosed that comprise about 10 or more contiguous polynucleotides corresponding to the NtSULTR3 or NtSULTR3 and NtSUS polynucleotides described herein. The 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 NtSULTR3 or NtSULTR3 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, which 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 part or all of a polynucleotide.As a specific example, two primers can be used in a PCR protocol to amplify a polynucleotide fragment.PCR can also be performed using one primer derived from a polynucleotide sequence and a second primer that hybridizes to the upstream or downstream sequence of the polynucleotide sequence (for example, a promoter sequence, the 3' end of a mRNA precursor, 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 be derived from a plant, a plant cell or plant material produced from a plant or derived from a plant, or a tobacco product, the plant cell or plant material described herein.

[0278] In a further aspect, there is also provided a method of detecting an NtSULTR3 or NtSULTR3 and NtSUS polynucleotide described herein (or any combination thereof described herein) in a sample, comprising the steps of: (a) providing a sample containing or suspected of containing a polynucleotide; (b) contacting the sample with one or more primers or one or more probes to specifically detect at least a portion of the NtSULTR3 or NtSULTR3 and NtSUS polynucleotide; and (c) detecting the presence of an amplification product, wherein the presence of the amplification product indicates the presence of the NtSULTR3 or NtSULTR3 and NtSUS polynucleotide in the sample. In a further aspect, there is also provided the use of one or more primers or probes to specifically detect at least a portion of the NtSULTR3 or NtSULTR3 and NtSUS polynucleotide. Kits for detecting NtSULTR3 or at least a portion of NtSULTR3 and NtSUS polynucleotides are also provided, and include one or more primers or probes for specifically detecting NtSULTR3 or at least a portion of NtSULTR3 and NtSUS polynucleotides. 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.

[0279] In some embodiments, the kits may include instructions for one or more of the described methods. The described kits may be useful for genetic identity determination, phylogenetic studies, genotyping, haplotyping, genealogy analysis, or plant breeding, particularly using co-dominant scoring.

[0280] The present disclosure also provides methods for genotyping plants, plant cells, or plant material containing the NtSULTR3 or NtSULTR3 and NtSUS polynucleotides described herein. Genotyping provides a means of distinguishing between chromosome pair homologs and can be used to identify segregants in plant populations. Molecular marker methods can be used for phylogenetic studies, characterizing genetic relationships between crop varieties, identifying crosses or somatic hybrids, localizing chromosomal segments affecting single genetic traits, map-based cloning, and quantitative inheritance studies. Specific methods of genotyping can employ any number of molecular marker analysis techniques, including amplified fragment length polymorphism (AFLP). AFLP is the product of allelic differences between amplified fragments caused by polynucleotide variability. Thus, the present disclosure further provides a means for tracking the segregation of one or more genes or polynucleotides, as well as chromosomal sequences genetically related to these genes or polynucleotides, using techniques such as AFLP analysis.

[0281] 13. Tobacco extract Also disclosed herein are methods for producing liquid tobacco extracts, and the liquid tobacco extracts produced by the methods.

[0282] A specific 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 a range of about 100°C to about 160°C. The duration of the heating step can optionally be controlled to provide some control over the composition of the extract derived from the tobacco starting material. Preferably, the tobacco starting material is heated at the extraction temperature for at least about 90 minutes, more preferably at least about 120 minutes. The heating step is typically carried out in an inert atmosphere. Preferably, a stream of inert gas, such as nitrogen, is passed through the tobacco starting material during the heating step. Volatile tobacco compounds are released into the inert gas stream during the heating step, so that the inert gas acts as a carrier for the volatile components. The inert gas flow can be at 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 can advantageously improve the efficiency of extraction from the tobacco starting material. Optionally, the heating step can be carried out under vacuum. Suitable heating methods for carrying out the heating of the tobacco starting material are known to those skilled in the art and include dry distillation, steam distillation, vacuum distillation, flash distillation, and thin film steam distillation.

[0283] If the volatile compounds are recovered by absorption in a liquid solvent, the step of forming a liquid tobacco extract may include drying the solution of the volatile compounds in the liquid solvent to concentrate the solution. Drying may be carried out using any suitable means, including, but not limited to, desiccation, molecular sieving, freeze-drying, phase separation, distillation, membrane permeation, controlled water crystallization and filtration, reverse hygroscopicity, ultracentrifugation, liquid chromatography, reverse osmosis, or chemical drying.

[0284] Liquid tobacco extracts are particularly suitable for producing compositions, formulations, or gel compositions for use in aerosol-generating systems. Aerosol-generating systems comprising the compositions, formulations, or gel compositions are disclosed. In such aerosol-generating systems, the compositions, formulations, or gels are typically heated in an aerosol-generating device (e.g., a device including a heater element that interacts with the composition, formulation, or gel incorporating the liquid tobacco extract to produce an aerosol). During use, volatile compounds are released by heat transfer and are entrained in air drawn through the aerosol-generating device. As the released compounds cool, they condense to form an aerosol, which is inhaled by the consumer.

[0285] The present invention is further described in the following examples, which are provided to describe the invention in more detail. These examples describe preferred modes presently contemplated for carrying out the invention and are intended to illustrate, but not limit, the invention. [Example]

[0286] Example 1 - Materials and Methods DNA extraction and plant genotyping Leaf samples are extracted using BioSprint 96 (Qiagen, Hilden, Germany) with the BioSprint 96 DNA Plant Kit (Qiagen, Hilden, Germany). To determine plant genotypes, 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, Foster City, CA, USA).

[0287] Measurement 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, after midrib removal, dried leaf pieces are optionally dried at 40°C for 2-3 days. The tobacco material is then ground into a fine powder (approximately 100 μM) 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.

[0288] 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, after midrib removal, dried leaf pieces are dried at 40°C for 2-3 days, if necessary. The tobacco material is then ground to a fine powder (approximately 100 μM) before analysis of reducing sugars. Measurement of reducing sugar content is performed according to ISO 15154:2003.

[0289] Example 2 - Analysis of NtSULTR gene expression Table 1 shows that NtSULTR3;3-T is expressed in all plant tissues, especially in petals. Interestingly, the copy NtSULTR3;3-S is not expressed in Virginia tobacco, but is expressed in some other tobaccos, such as TN90. Apparently, the NtSULTR3;3-S genomic sequence is either not identified in the Virginia genome or is altered in Virginia and dark tobaccos (see Sierro et al. (2014) Nat Commun. May 8;5:3833, Tables 3 and 4). The NtSULTR3;3-S genomic and polypeptide sequences were inferred from the TN90 sequencing library. Other SULTR3 genes expressed in petals are NtSULTR3;1A-S, NtSULTR3;1A-T, and NtSULTR3;1B-S. NtSULTR3;4A-T is apparently more specific to stems. NtSULTR3;2-S is expressed in sepals and roots. Interestingly, some SULTR3 genes are not expressed or are poorly expressed in green leaves, namely, NtSULTR3;1A-S, NtSULTR3;1A-T, NtSULTR3;3-S, NtSULTR3;4A-S, NtSULTR3;4B-S, NtSULTR3;4B-T, NtSULTR3;5-S, and NtSULTR3;5-T.

[0290] Example 3 - Expression of the NtSULTR gene during desiccation During Virginia (flue-cured) tobacco curing, reducing sugars, glucose, and fructose increase approximately threefold in etiolated leaves, reaching maximum levels after one or two days of drying after leaf harvest. Free amino acids also increase approximately fourfold after one or two days of drying, indicating that leaf etiolation activity affects the production of sugars, reducing sugars, and free amino acids. Among all NtSULTR3 transcripts, NtSULTR3;1A-S and NtSULTR3;3-T expression increased nearly threefold (log2) after two days of drying (transcriptome data from Affymetric Tobarray chips, see Figure 1A). This suggests that expression of these two genes may activate chloroplast transport of sulfate during the early stages of drying. RNA-seq data (see Figure 1B) confirm the data presented in Figure 1A. Other SULTR3 genes (log2>3) may also play a role in sulfate transport into chloroplasts during desiccation, particularly NtSULTR3;1A-T, NtSULTR3;1B-S, NtSULTR3;1B-T, NtSULTR3;4A-S, and NtSULTR3;4A-T.

[0291] Example 4 - Sulfate Levels During Drying Metabolomic data collected from a drying time course of dark tobacco (freeze-dried leaf material) showed that the major pool of sulfate was unaffected during the etiolation phase, suggesting that a small portion of the total sulfate pool was reduced during drying (see Figure 2A). Meanwhile, ABA, a known marker of leaf senescence, increased during the drying time course (see Figure 2B). After 48 h, 30% more methionine and three times more methionine sulfoxide were detected in leaf blade tissue (see Figures 2C and 2D). Methionine sulfoxide is a breakdown product of methionine resulting from ROS activity. ROS activity is known to increase during leaf senescence (Jajic et al. (2015) Plants 4:393-411. doi:10.3390 / plants4030393).

[0292] Example 5 - Expression of SULTR3 and SAG12 during desiccation From the same sample (dark, air-cured tobacco), frozen leaf material was also used to isolate RNA and analyze the expression of the SULTR3 gene and SAG12. SAG12 is a transcriptional marker of leaf senescence and is expressed 60-fold more after 96 hours of leaf etiolation. Concurrently, NtSULTR3;1A-S is the major SULTR3 gene expressed during leaf desiccation (see Figure 1) and is expressed approximately 20-fold more after 96 hours of desiccation (see Table 2). As previously observed in Figure 1, other members of the SULTR3 family are also expressed during leaf desiccation, including NtSULTR3;1A-T, NtSULTR3;1B-S, NtSULTR3;1B-T, NtSULTR3;3-T, NtSULTR3;4A-S, NtSULTR3;4A-T, and NtSULTR3;4B-T.

[0293] Example 6 - Silencing of NtSULTR3;1A-S and NtSULTR3;1A-T As the major SULTR3-induced gene during leaf curing, silencing of NtSULTR3;1A (both S and T copies, SEQ ID NOs: 1 and 3) was investigated in flue-cured tobacco to determine whether the NtSULTR3;1A gene contributes to altering reducing sugar and free amino acid levels in cured tobacco leaves. Specific DNA fragments within the coding sequences of both NtSULTR3;1A-S and NtSULTR3;1A-T were cloned under the strong constitutive Mirabilis mosaic virus (MMV) promoter in a Gateway vector. The NtSULTR3;1A gene fragment was flanked between the MMV and the 3' end sequence of the Agrobacterium tumefaciens nopaline synthase gene. Tobacco line K326 was transformed using a standard Agrobacterium-mediated transformation protocol. Leaves of independent T1 plants and their respective control lines are analyzed by qPCR after 48 hours of desiccation to confirm silencing of NtSULTR3;1A (see Figure 3).

[0294] Example 7 - Analysis of glucose, fructose and sucrose levels in NtSULTR3;1A-S and NtSULTR3;1A-T silenced plants Mid-leaves of control and transgenic 35S:NtSULTR3;1A-RNAi lines were harvested at maturity and subjected to hot air drying. Sugars (glucose, fructose, and sucrose) were analyzed in completely dried leaves (see Figure 4). The data presented in Figure 4 show a strong and significant reduction in glucose, fructose, and sucrose in anti-NtSULTR3;1A plants. Glucose, fructose, and sucrose levels were reduced by 77%, 69%, and 60%, respectively. No effects on visual plant fitness or chlorophyll degradation were observed in anti-NtSULTR3;1A plants grown under greenhouse conditions.

[0295] Example 8 - Analysis of free amino acid levels in NtSULTR3;1A-S and NtSULTR3;1A-T silenced plants Total free amino acids (left panel) were analyzed in completely dried leaves. The data presented in Figure 5 show a strong and significant increase in free amino acids in dried anti-NtSULTR3;1A plants compared to control plants, which was accompanied by a decrease in sugars (Figure 4). The major amino acids that increased during drying of anti-NtSULTR3;1A plants were glutamine, glutamic acid, and aspartic acid. The free amino acids glutamine, glutamic acid, and aspartic acid were approximately 1.5-fold, 2.3-fold, 2.4-fold, and 2-fold higher in the 35S:NtSULTR3;1A-RNAi lines, respectively. Asparagine also increased significantly (1.5-fold) compared to control plants, albeit at a limited significance level (P<0.05, n=6).

[0296] Example 9 - Identification of SUS genes in Burley, Virginia and Orient tobacco leaves after curing To identify key functions contributing to sucrose metabolism during the early drying time of Burley, Virginia, and Orient tobacco leaves, an overrepresentation analysis (log2 fold change >2, adjusted p-value <0.05) of the functions of up-regulated genes in cured leaves after 48 hours of curing 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 are identified, regardless of curing type and tobacco variety. Tobacco genes involved in the production of reducing sugars are identified.

[0297] 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 main enzyme for driving the accumulation of reducing sugars in dry detached leaves.

[0298] The tobacco genome is found to have 12 NtSUS gene products distributed into six families with one S and one T copy from each ancestor: 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.

[0299] The SUS transcripts are from the genome sequences NtSUS2-S, NtSUS3-S, NtSUS3-T, and NtSUS4-S. These genes are upregulated during leaf desiccation (senescence), as shown in Table 3. This confirms that the S transcripts are specifically involved in chemical modifications of early-drying leaves, in this particular case, the increase in glucose and fructose.

[0300] Although lower levels of reducing sugars are found in the drying leaves of burley compared to Virginia and Orient, the NtSUS gene is nevertheless activated in burley (see Table 3), likely also as a structural response, to ensure an available carbon source for amino acid synthesis during the early drying stage.

[0301] In both burley (BU) and Virginia (FC), NtSUS1-S and NtSUS1-T, which are not expressed during early desiccation (see Table 3), are specifically expressed in roots and stems, suggesting their possible specific functions in these tissues to deliver carbohydrates for cell wall synthesis or to provide carbon resources under anoxia (see Table 4). On the other hand, NtSUS3-S, NtSUS3-T, and NtSUS4-S, which are induced during early leaf desiccation, are also expressed in all organs, whereas NtSUS2-S and NtSUS2-T are primarily 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).

[0302] To increase the pool of reducing sugars in dried leaves, overexpression of NtSUS2-S, NtSUS3-S, NtSUS3-T, or NtSUS4-S, or a combination of one or more of them, using a senescence-inducible promoter such as SAG12 or E4, may be considered (the use of a constitutive promoter may strongly alter plant metabolism). On the other hand, knockout of NtSUS2-S, NtSUS3-S, NtSUS3-T, or NtSUS4-S, or a combination of one or more of them, may contribute to reducing the content of reducing sugars in dried leaves.

[0303] Example 10 - Silencing of NtSUS expression in Virginia tobacco leaves Silencing of NtSUS in burley tobacco was 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 genes were cloned under the strong constitutive Mirabilis mosaic virus (MMV) promoter in a Gateway vector. The NtSUS gene fragments were flanked by the MMV promoter and the 3' end sequence of the Agrobacterium tumefaciens nopaline synthase gene.

[0304] To allow for the selection of plants with low reducing sugar content, independent TO plant leaves and each control line are analyzed after 60 hours of desiccation to determine the effect on reducing sugar content. The best TO lines showing the lowest levels of reducing sugars are selected. Seeds are harvested from these best TO lines. T1 progeny are assayed by qPCR to determine the efficiency of the NtSUS silencing event in reducing reducing sugar content.

[0305] Manipulation of the NtSUS gene (e.g., with either a constitutive promoter or a specific senescence promoter (e.g., SAG12 or E4)) can alter the chemistry of tobacco cured leaves. Similarly, knocking out the NtSUS gene using gene editing strategies such as CRISPR or mutant selection can alter the amino acid leaf chemistry of major commercial tobacco varieties.

[0306] Example 11 - Producing liquid tobacco extracts from NtSULTR3 and NtSUS modified tobacco plants, each with modulated reducing sugar content. A first tobacco starting material is prepared from dried leaves of an NtSULTR3 modified tobacco plant, and a second tobacco starting material is prepared from dried leaves of an NtSUS modified tobacco plant according to the present disclosure. The tobacco material is cut to form tobacco pieces having dimensions of 2.5 millimeters x 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 nitrogen flow passes through the extraction chamber at a flow rate of approximately 40 liters per minute. For each tobacco starting material, the volatile compounds released during the heating step are recovered by absorption into a liquid solvent formed from propylene glycol at minus 10 degrees Celsius and with agitation of 750 rpm. The propylene glycol solution containing the recovered volatile compounds is dried in a drying process to reduce the moisture level of the solution to approximately 15 percent. A concentrated solution of the recovered volatiles from the tobacco starting material is recovered.

[0307] 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 for a different period of time than the second tobacco starting material. For each tobacco starting material, the volatile compounds released during the heating step are collected and dried. The resulting concentrated solutions of the collected volatiles from the first and second tobacco starting materials can be combined in a defined ratio to produce a liquid tobacco extract.

[0308] Any publications cited or described herein provide relevant information disclosed prior to the filing date of the present application. Nothing herein should be construed as an admission that the inventors are not entitled to antedate such disclosure. All publications mentioned in the above specification are incorporated herein by reference. Various modifications and variations of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the present invention has been described in connection with specific 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 obvious to those skilled in the art of cell biology, molecular biology, plant biology, or related fields are intended to be within the scope of the following claims.

[0309] Table 1 Expression of the NtSULTR3 gene in roots, stems, central leaves, immature flowers, sepals, and petals of field-grown Virginia plants (RNAseq, FPKM). [Table 1]

[0310] [Table 2]

[0311] Sequence Listing SEQ ID NO: 1 Polynucleotide sequence of NtSULTR3;1A-S SEQ ID NO: 2 Polypeptide sequence of NtSULTR3;1A-S MGNKDYEYPASMNGESRKTQPVEIPPPQPFFKSLKNTVKETLFPDDPLRQFKNQPPRKKFILGLQYLFPIFEWGPRYTLDFFKSDLIAGITIASLAIPQGISYAKLANLPPILGLYSSFVPPLVYAVMGSSRDLAVGTVAVASLLISSMLGDEVNPIENPTLYLHL AFTATFFSGMFEAALGIFRLGFIVDFLSHATIVGFMGGAATVVILQQLKGILGLDHFTQSTDVISVLRSVFTQTHEWRWESAVLGFCFLFYLLGSRFLSQKRPKLFWISAMAPLMSVILGTIFVYFTHAEKHGVQVIGKLKKGLNPVSIMDLSFGAPYVSTSIKTG IITGVVSLAEGIAVGRSFAMFKNYHIDGNKEMIAFGMMNIVGSCTSCYLTTGANQKSESAGPFSRSAVNFNAGCKTAVSNIVMALAVMVTLLVLTPLFHYTPLVVLSSIIISAMLGLIDYNAAIHLWHVDKFDFLVCISAYLGVVFASVEIGLVIAVGLSLLRVLL FVARPRTLVLGNIPDSKIYRNVEQYTNTDTVPGVLILDLGAPIYFANASYLRERISRWIDDEEDKLNSSGETLQYVILDMGAVGNIDTSGISMLEEVKKNLDRRDLKLVLANPGAEVMKKLNKSKFIETIGQEWIFLTVGEAVESCNYMLHSCKPKSAIDGSFSNNV SEQ ID NO: 3 Polynucleotide sequence of NtSULTR3;1A-T SEQ ID NO: 4 Polypeptide sequence of NtSULTR3;1A-T MGNKDYEYPSSMNGESRKTHAVEIPPPQPFFKSLKNTVKETLFPDDPLRQFKNQPPRKKFILGLQYIFPIFEWGPRYTLDFFKSDLIAGITIASLAIPQGISYAKLANLPPILGLYSSFVPPLVYAVMGSSRDLAVGTVAVASLLISSMLGDEVNPTDN PTLYLHLAFTATFFSGIFEAALGIFRLGFIVDFLSHATIVGFMGGAATVVILQQLKGILGLHHFTQSTDVISVLRSVFTQTHESQKRPKLFWISAMAPLMSVILGTIFVYFTHAEKHGVQVIGELKKGLNPVSIMDLSFGAPYLSTAIKTGIVTGVVSLA EGIAVGRSFAMFKNYHIDGNKEMIAFGMMNIVGSCTSCYLTTEDMILDRQVCGLGIRIEGCKTAVSNIVMALAVMVTLLVLTPLFHYTPLVVLSSIIISAMFGLIDYNAAIHLWHVDKFDFLVCISAYFGVVFASVEIGLVIAVALSLLRVLLFVARPRM LVLGNIPDSKIYRNVEQYTNTDTVPGVLILDLGAPIYFANASYLRERISRWIDDEEDKLNSSGETLQYVILDMGAVGNIDTSGISMLEEVKKNLDRRDLKLVLANPGAEVMKKLNKSNFIETIGQEWIFLTVGEAVESCNYMLHSCKPKSSTDGSFSNNV SEQ ID NO: 5 Polynucleotide sequence of NtSULTR3;1B-S SEQ ID NO: 6 Polypeptide sequence of NtSULTR3;1B-S MGNADYEYPSIMNGESTGIGIHRVEIPPPQPFFKSLKNTVKETLFPDDPLRQFKNQTPLRKFILGVQYFFPIFEWGSRYNFGFFKSDLIAGITIASLAIPQGISYAKLANLPPILGLYSSFVPPLVYAIMGSSRDLAVGTVAVGSLLMASMIGNEVNATENPALY LHLAFTATFFAGLFELALGFFRLGFIVDFLSHATIVGFMGGAATVVILQQLKGILGLEHFTHATDVVSVLRSVFTQIHQWRWESAVLGFCFLFYLMMAKFFSQKRPKLFWISAMAPLTSVILGTILVYLTHAEKHGVAVIGELKKGLNPPSIMDLSFGSAYMTTAI KTGIVTGVISLAEGIAVGRSFAMFKNYHIDGNKEMIAFGMMNIVGSCTSCYLTTGPFSRSAVNFNAGCKTAVSNIVMALAVMVTLLLLTPLFHFTPLVVLSSIIISAMLGLIDYNAAIHLWHVDKFDFLVCISAYIGVVFANIEIGLVLAVGLSLLLRVLLFIARP RTLVLGNIPDSMIYRNVEHYPNTNNVPGVLILDIGAPIYFANSSYLRERISRWIDEEEDKLKSSGETTLQYVILDMGAVGNIDTSGISMLEEVKKNLDRRDYKLVLANPGAEVMKKLNKSKFIETLGQEWIFLTVGEAVGACNFMLHSCKPKSTTDEASQKWSNNV SEQ ID NO: 7 Polynucleotide sequence of NtSULTR3;1B-T SEQ ID NO: 8 Polypeptide sequence of NtSULTR3;1B-T MGNADYEYPSIMNGESAGTGIHRVEIPPPQPFFKSLKNTVKETLFPDDPLRQFKNQTPLRKFILGLQYFFPIFEWGSRYNFGFFKSDLIAGITIASLAIPQGISYAKLANLPPILGLYSSFVPPLVYAIMGSSRDLAVGTVAVGSLLMASMIGNEVNATE NPALYLHLAFTATFFAGLFELALGFFRLGFIVDFLSHATIVGFMGGAATVVILQQLKGILGLEHFTHATDVVSVLRSVFTQTQQSQKRPKLFWISAMAPLTSVILGTILVYVTHAEKHGVAVIGELKKGLNPPSIMDLSFGSAYMTTAIKTGIVTGVISL AEGIAVGRSFAMFKNYHIDGNKEMIAFGMMNIVGSCTSCYLTTGPFSRSAVNFNAGCKTAVSNIVMALAVMVTLLLLTPLFHFTPLVVLSSIIISAMLGLIDYNAAIHLWHVDKFDFLVCISAYIGVVFANIEIGLVLAVGLSLLLRVLLFIARPRTLVLG NIPDSMIYRNVEHYPNTNNVPGVLILDIGAPIYFANSSYLRERISRWIDEEEDKLKFSGETTLQYVILDMGAVGNIDTSGISMLEEVKKNLDRRDYKLVLANPGAEVMKKLNKSKFIETLGQEWIFLTVGEAVGACNFMLHSCKPKSTTDDASQKWSNNV SEQ ID NO: 9 Polynucleotide sequence of NtSULTR3;2-S SEQ ID NO: 10 Polypeptide sequence of NtSULTR3;2-S MGNAHFDDQYSHQKVEIPAPKPFLKTLKSCVKETLFPDDPFRKFKNQSLTKKLALGLQYFVPILDWAPRYTFQLFKADFIAGITIASLAVPQGISYAGLANLPPVIGLYSSFVPPMVYAMLGSSKHLAIGNVAVPSLLISAMLGRVVNPHDNPKLYLQLVFTA TFFAGVFQASLGLLLRLGFIVDFLSHATILGFMGGAATVVCLQQLKGILGLVHFTHETDIVSVMRSIFSQLHQWRWESGVLGCCFLFFLLLTRYFSKKKPAFFWISCMAPLTSVILGSVLVYFTHAEKNGVQVIGHLKKGINPPSYSELAFSSQYLTTAIKTGI VTGVIAMAEGIAVGRSFAIVENYHIDGNKEMIAFGMMNIAGSCTSCYLTTGPFSRTAVNFNAGCKTAVSNIVMATAVMITLLLLTPLFHYTPLVVLSSIIISAMLGIIDYNAAIHLWKVDKYDFLVCISSFIGVVFGSVEVGLIVAVAMSLLRILLFVARPKT FVLGKIPNSMTYRNTEQYSAASSVPGILIIHIDAPIYFANASYLRERISRWIDEEEEKQRTLSEIELQYVILDMSAVGNIDTSGISMLEEVKRNADRRCLKLLLANPGGEVMKKLDKSNFIDTIGKEWIYLTVGEAVNACNYILHTCKFQSKRIESSTIPDDNV SEQ ID NO: 11 Polynucleotide sequence of NtSULTR3;2-T tataaggaggctatgagtgttcctggttttctcattttaagtattgaagctccaatcaactttgccaatgcaacttatcttaaagaaaggttagtattagttgaactgctgcattgaccattctatctttcatttttcttctttttttcttctttccatatttatttaggtctttttatttgccccgaaaaaaaaggatttcaagatggatagaagactacgatgcagagggagaaaaaaacaagaaagagtcggggcttagatttgtggtccttgatttgtctggtaagttcatagagacgttctcaatattgtcatttattcccaatttggcataactggcaaagttgttgtcatgtgaccaggaggtcacaggttcgagccgtgaaaataatatcttgcagaaatgcaggataagattgcgtacaataaatcattgtggtccggctcttctccgggtcccgcgcatagtggaagtttagtgcaccgggctgcccttacccctacttttagataataccaagaaacagtcaggacatcaagaaattcccataaataaaacaaattaatttcaacttgaaaagtgattgtggcttgttttttattcttcagctgtgactgccattgatacaagtggagtctcattgttcaaggatttgagtatggcaatggaaaagaaaggccttgaggcaagtgtactttagcttttagaagaccattttgttttctatttattctgatattatgtgagtatttatttccttaatgattttggcattgcagtttgtattggtgaatccaataggagaagtactggaaaaattacagagggctgatgaaactaaagatatgatgagaccagattgcctttttttaacagtcgaagaagcagtagcttcactttcctcaacaataaaatatcaaataccagacaatgtttga Polypeptide sequence of SEQ ID NO: 12, NtSULTR3;2-T MGNADFDDQYSHQKVEIPPPKPFLKTLKSCVKETLFPDDPFRKFKKQPLTKKLTLGLQYFVPILDWAPRYTFQLFKADFIAGITIASLAVPQGISYAGLANLPPVIGLYSSFVPPMVYAMLGSSKHLAIGNVAVPSLLISAMLGRVVNPHDNPKLYLQLVFTAT FFAGVFQASLGLLRLGFIVDFLSHATILGFMGGAATVVCLQQLKGILGLVHFTHETDIVSVMRSIFSQLHQWRWESGVLGCCFLFFLLLTRYFSKKKPAFFWISCMAPLTSVILGSVLVYFTHAEKNGVQVIGHLKKGINPPSYSELAFSSQYLTTAIKTGIVTG VIAMAEGIAVGRSFAIVENYHIDGNKEMIAFGMMNIAGSCTSCYLTTGPFSRTAVNFNAGCKTAASNIVMATAVMITLLLLTPLFHYTPLVVLSSIIISAMLGIIDYNAAIHLWKVDKYDFLVCICSFIGVVFSSVEVGLIVAVAMSLLRILLFVARPKTFVLG KIPNSMTYRNTEQYSAASRVPGVLIIHIDAPIYFANASYLRERISRWIEEEEEEEEEEKQRTSTEIELQYVILDMSAVGNIDTSGISMLEEVKRNADRRCLKLVLANPGGEVMKKLDKSNFIDKIGKEWIYLTVGEAVNACNYILHTCKFQSERIESSTIPDDNV SEQ ID NO: 13 Polynucleotide sequence of NtSULTR3;3-S aacctctgtggtccggctcttccctggaccttgcgcatagcgggagcttagtgcaccgggctgcccttcccccctacttttggataataccaagaaacagtcaggacatcaagaaattcccacaaataaaacaaattaatttaccaagaaacagtcaggacatcaagaaattcccacaaataaaacaaactaatttcaacttgacaagtaattgtggattgtttttttaatcttcagctgtgactgccattgatacaagtggagtctcattgttcaaggatttgagtatggcaatggaaaagaaaggctttgaggtaagtgtactttagcttttagagtcactatttctttccaacaacaacaacaacaacaacccagtataatcccacttagtggggtctggggagcgtagtgtgtacgcagaccttacccctaccctagggtagagagactgtttccaaatagacccccggcatccttccctccaagaacttcccaccttgctcttggagagactcgaactcacagcctttccttccctccaacaatccactatttctttccaaatgaagtcaaaatcctcaagaccattttgttttctatttattctgatattatgtgagtatttatttccttaatgattttggcattgcagtttgtattggtgaatccaataggagaagtactggaaaaattacagagggctgatgaaactaaagatatgatgagaccagattgcctctttttaacagtcgaagaagcagtagcttcactttcctcaacaataaaataccaaataccagacaatgtttga Polypeptide sequence of SEQ ID NO: 14, NtSULTR3;3-S MEPNNENRVIDITAMEVHKVVSPPHRSTFQKLKNRLKETFFPDDPLRQFKGQPLKQKLVLGAQYVFPILEWGPNYSFKLFKSDIVSGLTIASLAIPQGISYAKLANLPPIVGLYSSFVPPLVYAVLGSSRDLAVGPVSIASLVLGSMLREVVSPTKDPILFL QLAFSSTFFAGLFQASLGFLRLGFIIDFLSKATLIGFMAGAAVIVSLQQLKSLLGITNFTKQMAIVPVLSSVFHRTNEWSWQTILMAFCFLGFLLLTRHISMRKPKLFWISAGAPLLSVIISTLLVFAMKGQKHGISIIGKLQEGLNPPSWNMLHFSGSYLG LVIKTGIITGILSLTEGIAVGRTFAALKNYQVDGNKEMIAIGVMNIVGSSTSCYVTTGAFSRSAVNHNAGSKTAVSNIVMAVTVMVTLLFLMPLFQYTPNVVLGAIIVTAVVGLIDVPAAYQIWKIDKFDFLVLLCAFFGVIFISVQNGLAIAIGISILKVL LQITRPKTVMLGNIPGTGIYRNLDHYKEAMSVPGFLILSIEAPINFANATYLKERISRWIEDYDAEGGKNKKQSGLRFVVLDLSAVTAIDTSGVSLFKDLSMAMEKKGFEFVLVNPIGEVLEKLQRADETKDMMRPDCLFLTVEEAVASLSSTIKYQIPDNV SEQ ID NO: 15 Polynucleotide sequence of NtSULTR3;3-T attcttcagctgtgactgccattgatacaagtggagtctcattgttcaaggatttgagtatggcaatggaaaagaaaggccttgaggcaagtgtactttagcttttagaagaccattttgtttctatttattctgatattatgtgagtatttatttccttaatgattt tggcattgcagtttgtattggtgaatccaataggagaagtactggaaaaattacagaggggctgatgaaactaaagatatgatgagaccagattgcctttttttaacagtcgaagaagcagtagcttcactttcctcaacaataaaatatcaaataccagacaatgtttga SEQ ID NO: 16: Polypeptide sequence of NtSULTR3;3-T MEPNNENRVIDITAMEVHKVVSPPHRSTFQKLKNRLKETFFPDDPLRQFKGQPLKQKLILGAQYVFPILEWGPNYSFKLFKSDIISGLTIASLAIPQGISYAKLANLPPIVGLYSSFVPPLVYAVLGSSRDLAVGPVSIASLVLGSMLREVVSPTKDPILFL QLAFSSTFFAGLFQASLGFLRLGFIIDFLSKATLIGFMAGAAVIVSLQQLKSLLGITNFTKQMAIVPVLSSVFHRTNEWSWQTILMAFCFLVFLLLTRHISMRKPKLFWVSAGAPLLSVIISTLLVFAMKGQKHGISIIGKLQEGLNPPSWNMLHFSGSYLG LVIKTGIVTGILSLTEGIAVGRTFAALKNYQVDGNKEMIAIGVMNIVGSSTSCYVTTGAFSRSAVNHNAGSKTAVSNIVMAVTVMVTLLFLMPLFQYTPNVVLGAIIVTAVVGLIDIPAAYQIWKIDKFDFLVLLCAFFGVIFISVQNGLAIAIGISILKVL LQITRPKTVMLGNIPGTGIYRNLDHYKEAMSVPGFLILSIEAPINFANATYLKERISRWIEDYDAEGEKNKKESGLRFVVLDLSAVTAIDTSGVSLFKDLSMAMEKKGLEFVLVNPIGEVLEKLQRADETKDMMRPDCLFLTVEEAVASLSSTIKYQIPDNV SEQ ID NO: 17 Polynucleotide sequence of NtSULTR3;4A-S SEQ ID NO: 18: Polypeptide sequence of NtSULTR3;4A-S MGLSSNRVEDLSGHACNETIVTISTTTTTELQISSNPPFEVHRVCLPPHKTTLQKLRQRLLEVFFPDDPLHKFKNQTWLMKLVLGLQFFFFPVFEWGPQYNLKLLRADIISGLTIASLAIPQGISYAKLANLPPIVGLYSSFVPPL IYSVLGSSKHLAVGPVSIASLVMGTMLSEAVSYTEEPVLYLQLAFTATLFAGLFQASLGFFRLGFIIDFLSKATLVGFMAGAAVIVSLQQLKGLLGIVHFTSQMQIIPVLSSVFQHKDEWSWQTIVMGVCFLAFLLTTRQISTRNPK LFWLSAASPLASVILSTLVVALLKSNAHGIQTIGHLQKGLNPPSLNMLYLSGPYLPLAIKTGIVSGILALTEGIAVGRTFAALRNYQIDGNKEMMAIGLMNMAGSCSSCYVTTGSFSRSAVNYNAGAQTVFSNIIMATAVLITLLF LMPLFYYTPIVILAAIIITAVIGLIDYQAAFRLWKVDKLDFLACLCSFFGVLFISVPLGLAIAVGVSVFKILLHVTRPNTSVLGNIPGTQVYQNLSRYRTAVRIPSFLILAVEAPIYFANSTYLKERLVQTSNTECRFSILLFANFN SEQ ID NO: 19: Polynucleotide sequence of NtSULTR3;4A-T SEQ ID NO: 20: Polypeptide sequence of NtSULTR3;4A-T MGLSSNRVEDLSGHACNETIITISTTSTELHISNNQPFEVHRVCLPPHKTTLQKLRQRLLEIFFPDDPLHKFKNQTWLMKLVLGLQFFFPVFEWGPQYNLKLLRADVISGLTIASLAIPQGISYAKLANLPPIVGLYSSFVPPLIYSVLGSSKHLAVGPVSIASLVM GTMLSEAVSYTEEPVLYLQLAFTATLFAGLFQSSLGFFRLGFIIDFLSKATLVGFMAGAAVIVSLQQLKGLLGIVHFTSQMQIVPVLSSVFQHKNEWSWQTIVMGVCFLAFLLTTRQISTRNPKLFWLSAASPLASVILSTLVVTLLKSKAHGIQTIGHLQKGLNPP SLNMLYLSGPYLPLAIKTGIVSGILALTEGIAVGRTFAALKNYQVDGNKEMMAIGLMNMAGSCSSCYVTTGSFSRSAVNYNAGAQTVVSNIIMATAVLITLLFLMPLFYYTPIVILAAIIITAVIGLIDYQAAFRLWKVDKLDFLACLCSFFGVLFISVPLGLAIAV GVSVFKILLHVTRPNTSVLGNIPGTQVYQNLSRYRTAVRIPSFLILAVEAPIYFANSTYLKERILRWIREEEEWIVANKETAIKCVIIDMTAVSSIDSSGIDTICELRKTLDKRSLKLVMANPGGNVMEKLHQSNTLDAFGLNGIYLTVSEAVADISSLWKSEPESSI SEQ ID NO: 21: Polynucleotide sequence of NtSULTR3;4B-S SEQ ID NO: 22: Polypeptide sequence of NtSULTR3;4B-S MTLNSIKVEDSSCNATEGESATSSSMQSSGVHKVCLPPYRTTFQKLRQRLSEIFFPDDPLHKFKKQTGLRKFVLGLQFFFPVFEWGPLYSFKLLRSDIISGLTIASLAIPQGISYAKLANLPPIIGLYSSFVPPLIYSILGSSRHLAVGPVSIASLVMGTMLS QAVSYSQEPILYLQLAFTATLFAGLLQASLGFFRLGFIIDFLSKATLLGFMAGAAVIVSLQQLKGLLGISHFTNQMQIVPVLSSVFTHKDEWSWQTIVMGVSFLIFLLATRQISTRKPKLFWISAAAPLVSVILSTIIVFLLKSKTIQTIGHLPKGINPPSLNM LHFSGPHIALAIKIGIITGVLSLTEGIAVGRTFAAMQNYQVDGNKEMIAIGLMMNMAGSCASCFVTTGSFSRSAVNYNAGAKTVVSNIIMAATVLITLLFLMPLFHYTPNLILAAIIITAVIGLIDYQAAFRLWKVDKLDFVACLSSFFGVLFISVPLGLAIAV GVSVFKILLHVTRPNTNVLGYIPGTQSFQSLSRYSTAVRVPSFLIIAVEAPFYFANSTYLQERTLRWIREEEERIEVKRETAIKCVILDMTAVTAIDTSGIDTICELRRILEKRSLKLVLANPVGNVMEKLHNSHALEAFGLDGLYLTVSEAVADISSSWKPEA SEQ ID NO: 23 Polynucleotide sequence of NtSULTR3;4B-T SEQ ID NO: 24: Polypeptide sequence of NtSULTR3;4B-T MTLNSIKVEDSSCNATETEAVTSSSMQSSGVHKVCLPPYRTTFQKLRQRLSEIFFPDDPLHKFKNQTGLRKFVLGLQFFFPVFEWGPLYSFKLVRSDIISGL TIASLAIPQGISYAKLANLPPIIGLYSSFVPPLIYSILGSSRHLAVGPVSIASLVMGTMLSQAVSYSQEPILYLQLAFTATLFAGLLQASLGFFRLGFIIDF LSKATLLGFMAGAAVIVSLQQLKGLLGISHFTNQMQIVPVLSSVFTHKDEWSWQTIVMGVSFLIFLLATRQISTRKPKLFWISAAAPLVSVILSTIIVFLLKSKTIQTIGHLPKGINPPSLNMLHFSGPHLALAIKTGIVTGVLSLTEGIAVGRTFAAMQNYQVDGNKEMIAIGLMNMAGSCASCFVTTGSFSRSAVNYNAGAKT SEQ ID NO: 25: Polynucleotide sequence of NtSULTR3;5-S SEQ ID NO: 26: Polypeptide sequence of NtSULTR3;5-S MTSSPQSLHRVNYAAPRSFGTLLKANLKETLFPDDPFHEIKNEPISRRFLKGAQYFVPIFEWLPKYNFKLFKYDLLAGITIASLAIPQGISYAKLANIPPIIGLYSSFVPPLIYAVFGSSKHLAVGTVAACSLLIAAIIEGKVNASDNMPLYLSLVFTAT LFSGLVQTALGLLRLGILVDFLSHSTITGFMGGTAIIICLQQLKGMLGLKHFTTHTDVVSVLRAIFHNRKEWKWESAVVGIIFLTFLQFTRFVKNKKPKLFWVSAIAPMVTVIVGCLFAYFAHAEKHGIQIVGHLSKGINPSSIHLLNFDPKYISAPIKAG VIAAMISLAEGIAIGRSFAIIRNEQIDGNKEMIAIGLMNIFGSFTSCYLTTGPFSKTAVNFNAGCKTAMSNVVMSICMMLTLLFLAPLFSYTPLVSLSAIIMSAMLGLIDYDKAYHLFKTDKFDFCICMAAFFGVSFISMDIGLMLSVGLALIRALLYIA RPATCKLGLISETGLYRDVEQYPDANGIAGFLILKLGSPIYFANCNYVREILRWIRDERSHTISKGNEIEFLLLELGGITSIDITGVETLLEIRRCVQAKGIKMILVNPRLGVLEKLMVTESIDTITKESVFLTIEDAIDACRFSLKCSDHIKTENLAIV SEQ ID NO: 27: Polynucleotide sequence of NtSULTR3;5-T ttgaagacgcaattgatgcttgcagattttcactcaaatgttcagatcaaatgaaaagagaaaaccttgcaatagtttag SEQ ID NO: 28: Polypeptide sequence of NtSULTR3;5-T MTSSPQSLHRVNYAAPRSFGTLLKANLKETLFPDDPFHEIKNEPISRRFLKGAQYFVPIFEWLPKYSFKLFKYDLLAGITIASLAIPQGISYAKLANIPPIIGLYSSFVPPLIYAVFGSSKHLAVGTVAACSLLIAAIIEGKVNANDNMPLYLSLVFTAT LFSGLVQTALGLLRLGILVDFLSHSTITGFMGGTAIIICLQQLKGMLGLKHFTTHTDVASVLRAIFHNRKEWKWESAVVGIIFLTFLQFTRFVKNKKPKLFWVSAIAPMVTVIVGCLFAYFAHADKHGIQIVGHLSKGINPSSIHLLNFDPKYISAPIKAG VIAAMISLAEGIAIGRSFAIIRNEQIDGNKEMIAIGLMNIFGSFASCYLTTGPFSKTAVNFNAGCKTAMSNVVMSICMMLTLLFLAPLFSYTPLVSLSAIIMSAMLGLIDYDKAYHLFKTDKFDFCICMAAFFGVSFISMDIGLMLSVGLALIRALLYIA RPATCKLGLISETGLYRDVEQYPDANGIAGILILKLGSPIYFANCNYIRERILRWIRDERSLTISEGNEIEFLLLELGGITSIDITGVETLLEIRRCVEAKGIKMILVNPRLGVLEKLMVTESIDTVTKESVFLTIEDAIDACRFSLKCSDQMKRENLAIV SEQ ID NO: 29: Nucleotide sequence used to silence NtSULTR3:1A-S and NtSULTR3:1A-T gtaggcaacattgatactagcggaattagcatgctagaagaggtcaagaagaatcttgatagaagagatctcaagcttgtgctggcaaatccaggggcagaggtaatgaagaagctgaacaagtccaa SEQ ID NO: 30: Polynucleotide sequence of NtSUS1-S SEQ ID NO: 31: Polypeptide sequence of NtSUS1-S MAASGLSIKKSLEESILAHPDEILALKSRIETEGKGVMKPLDLLNHLVSVTSKTNGVNIVPSALVEVLSCSQEAVIVPPKLALAVRPRPGVWEYLSLNLKTKKVAELSIPEYLQLKENTVDESGNILELDFEPFTTVTPPKTLSDSIGNGLEFLNRHIASKMFHDKEISRCLLDFLRNHNYKGKSLMVKESIQSLESFQL VLKKAEEHLCTLNPETPYSNFESKFEEIGLERGWGNTAERVQDTISHLLHLLEAPNASSLENFLGRIPLVFNVVILTPHGYFAQDNVLGYPDTGGQVVYILDQVPAMEREMLHRMKLQGLDDIIPRILVVTRLLPDAVGTTCGERMEKVYGAEHSHIIRVPFRTEKGMLRKWISRFEVWPYMETFTEDVAEELVKELQAKP DLIIGNYSEGNLAASLLAKKFGATQCTIAHALEKTKYPNSDLNWKKFDDKYHFSSQFTADLFAMNHTDFIITSTFQEIAGSKNTVGQYESHTAFTMPGLYRVVHGIDSFDPKFNIVSPGADMSIYFPYTEKEKRLTNFHPEIEELLYSPVENKDHLCVLKDRNKPILFTMARLDRVKNLTGLVEWYAKNARLRELVNLVVV GGDRRKESKDLEEQAEMKKMYDLIETYNLNGQFRWISSQMNRVRNGELYRYIADTRGAFVQPAFYEAFGLTVVESMTCGLPTFATCNGGPFEIIVNGKSGFHIDPNQGDKAADMLVNFFEKSKEDPSYWDAISKGGLQRILEKYTWQIYSQKVITLSGIYGFWKYATKNDKVASAKKRYLEMFYELGFKKSAEKVPLAIDE SEQ ID NO: 32: Polynucleotide sequence of NtSUS1-T SEQ ID NO: 33: Polypeptide sequence of NtSUS1-T MAGSGLSIKESLEESILAHPDEILALKSRIETEGKGVMKPVDLLNHLVSVTSKTNGVNVVPSALVEVLSCSQEAVIVPPKLALAVRPRPGVWEYLSLNLKTKKVAELSIPEYLQLKENTVDESGNILELDFEPFTTVTTPKTLSDSIGNGLEFLNRHIASKMFLDKEIAKCLLDFLRNHNYKGKSLMVKESIQSLESFQL VLKKAEEYLHTLNPETPYSKFESKFEEIGLERGWGNTAERVQDTISHLLHLLEAPNASSLENFLGRIPLVFNVVILTPHGYFAQDNVLGYPDTGGQVVYILDQVPAMEREMLHRMKLQGLDDIIPRILVVTRLLPDAVGTTCGEWMEKVYGAEHSHIIRVPFRTEKGMLRKWISRFEVWPYMETFTEDVAEELVKELQAKP DLIIGNYSEGNLAASLLAKKFGATQCTIAHALEKTKYPNSDLNWKKFDDKYHFSSQFTADLFAMNHTDFIITSTFQEIAGSKNTVGQYESHTAFTMPGLYRVVHGINSFDPKFNIVSPGADMSIYFPYTEKEKRLTNFHPEIEELLYSPVENKDHLCVLKDQNKPILFTMARLDRVKNLTGLVEWYAKNARLRELVNLVVV GGDRRKESKDLEEQAEMKKMYDLIETYNLNGQFRWISSQMNRVRNGELYRYIADTRGAFVQPAFYEAFGLTVVESMTCGLPTFATCNGGPFEIIVNGKSGFHIDPNQGDKAADMLVNFFEKSKEDPSYWDTISKGGLQRILEKYTWQIYSQKVITLSGIYGFWKYATKNDKVASAKKRYLEMFYEFGFKKSAEKVPLAIDE SEQ ID NO: 34: Polynucleotide sequence of NtSUS2-S SEQ ID NO: 35: Polypeptide sequence of NtSUS2-S MAERALTRVHSLRERLDATLAAHRNEILLFLSRIESHGKGILKPHQLLAEFDAIRQDDKKKLNDHAFEELLKSTQEAIVLPPWVALAIRLRPGVWEYVRVNVNALVVEELTVPEYLHFKEELVDGTSNGNFVLELDFEPFTASFPKPTLTKSIGNGVEFLNRHLSAKMFHDKESMTPLLEFLRVHNYKGKTMMLNDRIQNL TTLQNVLRKAEEYLIMLPPETPFSEFEHKFQEIGLEKGWGDTAERVLEMICMLLDLLEAPDSCTLEKFLGRIPMVFNVVILSPHGYFAQENVLGYPDTGGQVVYILDQVPALEREMLKRLKEQGLDITPRILIVTRLLPDAVGTTCGQRLEKVYGAEHSHILRVPFRTEKGIVRKWISRFEVWPYMETFTEDVAKELAAEL QAKPDLIIGNYGNLVASLLAHKLGVTQCTIAHALEKTKYPDSDIYWKKFDEKYHFSSQFTADLIAMNHTDFIITSTFQEIAGSKDTVGQYESHQAFTMPGLYRVVHGIDVFDPKFNIVSPGADINLYFPYSEKEKRLTALHPEIEELLYSDVENEEHLCVLKDRNKPILFTMARLDRVKNLTGLVEWYAKNARLRELVN LVVVGGDRRKESKDLEEQAEMKKMYELIKTHNLNGQFRWISSQMNRVRNGELYRYIADTRGAFVQPAFYEAFGLTVVEAMTCGLPTFATNHGGPAEIIVNGKSGFHIDPYHGEQAADLLADFFEKCKTEPSHWETISTGGLKRIQEKYTWQIYSERLLTLAAVYGFWKHVSKLDRLEIRRYLEMFYALKYRKMAEAVPLAAE SEQ ID NO: 36: Polynucleotide sequence of NtSUS2-T taaatggccaattcagatggatttcttcacagatgaaccgagtgaggaacggtgaactctaccgatacattgctgacactagaggagctttcgtgcagcctgcattctacgaggctttcggtttgactgttgttgaggccatgacctgtggtttgcctacatttgcaactaatcatggcggtccagctgagatcatcgttaacggaaaatctggcttccacatcgatccatatcacggtgagcaagctgctgatctgctagctgatttctttgagaaatgtaagacagaaccttctcattgggaaaccatttcaacgggtggcctgaagcgcatccaagagaagtaagcaactctttcttgactctagtcattgaaattaactttcttgactctagtcattgaaattaactcgggatttgaggcgtagttgattgatattttatcgcgtctctactactgatatatacaggtacacgtggcaaatctactcggagaggctattgacattggctgctgtttacgggttctggaaacatgtttctaagcttgatcgtctagaaatccgtcgatatcttgaaatgttttatgctctcaaataccgcaagatggtgagttcctcttcttccttgcccttctcctagtgtttaagatacaatataattgattgcattatcttagagaatcattaatgttaaattttcttaattcttgaatctgttaatgaagtttttctcttggtttttgtttaggctgaagctgttccattggctgctgagtga SEQ ID NO: 37: Amino acid sequence of NtSUS2-T MLFMGLKQLSEDFSPAESTAMAERVLTRVHSLRERLDATLAAHRNEILLFLSRIESHGKGILKPHQLLAEFDAIRQDDKKKLNDHAFEELLKSTQEAIVLPPWVALAIRLRPGVWEYVRVNVNALVVEELTVPEYLHFKEELVDGTSNGNFVLELDFEPFTASFPKPTLTKSIGNGVEFLNRHLSAKMFHDKESMTPLLEFLRVHN YKGKTMMLNDRIQNLTTLQNVLRKAEEYLIMLPPETPFSEFEHKFQEIGLEKGWGDTAERVLEMICMLLDLLEAPDSCTLEKFLGRIPMVFNVVILSPHGYFA QENVLGYPDTGGQVVYILDQVPALEREMLKRLKEQGLDITPRILIVTRLLPDAVGTTCGQRLEKVYGAEHSHILRVPFRTEKGIVRKWISRFEVWPYMETFTE DVAKELAAELQAKPDLIIGNYSEGNLVASLLAHKLGVTQCTIAHALEKTKYPDSDIYWKKFDEKYHFSSQFTADLIAMNHTDFIITSTFQEIAGSKDTVGQYESHQAFTMPGLYRVVHGIDVFDPKFNIVSPGADINLYFPYSEKEKRLTALHPEIEELLYSDIENEEHLCVLKDRNKPILFTMARLDRVKNLTGLVEWYAKNARL RELVNLVVVGGDRRKESKDLEEQTEMKKMYELIKTHNLNGQFRWISSQMNRVRNGELYRYIADTRGAFVQPAFYEAFGLTVVEAMTCGLPTFATNHGGPAEII VNGKSGFHIDPYHGEQAADLLADFFEKCKTEPSHWETISTGGLKRIQEKYTWQIYSERLLTLAAVYGFWKHVSKLDRLEIRRYLEMFYALKYRKMAEAVPLAAE SEQ ID NO: 38: Polynucleotide sequence of NtSUS3-S SEQ ID NO: 39: Polypeptide sequence of NtSUS3-S MANPKFTRVPSMRERVEDTLSAHRNQLVALLSRYVAQGKGILQPHHLIDEFNNAVCDDTACEKLKDGPFSEVLKATQEAIVLPPFVAIAVRPRPGVWEYVRVNVYDLSVEQLTVPEYLHFKEELVDGEGNNHFVLELDFEPFNASVPRPSRSSSIGNGVQFLNRHLSSIMFRSKDSLDPLLDFLRGHCHKGNVLMLNDRIQR ISRLESALSKAEDYLSKLSPDTSYNEFEYALQEMGFERGWGDTARRVLETMHLLSDILQAPDPSTLETFLGRLPMVFNVVILSPHGYFGQANVLGLPDTGGQVVYILDQVRALEAEMLLRIKQQGLNFKPRILVVTRLIPDAKGTMCNQRLERISGTEYSHILRVPFRTEKGILHKWISRFDVWPYLEKFTEDVASEMTAELQ GKPDLIIGNYSDGNLVASLLAYKMGVTQCTIAHALEKTKYPDSDIYWKKFEEKYHFSCQFTADLAMNNSDFIITSTYQEIAGTKNTVGQYESHTAFTLPGLYRVVHGIDVFDPKFNIVSPGADMTIYFPYSDKEKRLTSLHGSIEKLLFDPAQNEEHIGNLNDKSKPIIFSMARLDHVKNITGLVECYAKNATLRELANLV VVAGYNDVKKSSDREEITEIEKMHALIKEHKLDGQFRWVSAQTNRARNGELYRYIADQRGIFVQPAFYEAFGLTVVEAMTCGLPTFATCHGGPNEIIEPGVSGFHIDPYHPDKAAELMSEFFQRCKQDPTHWEKISASGLRRILERYTWKIYSERLMTLSGVYGFWKLVSKLERRETRRYLEMFYILKFRELAKSVPLAIDDK SEQ ID NO: 40: Polynucleotide sequence of NtSUS3-T SEQ ID NO: 41: Polypeptide sequence of NtSUS3-T MFTWLKLNIKNKGRKNTVGQYESHTAFTLPGLYRVVHGIDVFDPKFNIVSPGADMTIYFPYSDKEKRLTSLHGSIEKLLFDPAQNEEHIGNLNDKSKPIIFSMARLDHVKNITGLVECYAKNATLRELANLVVVAGYNDVKKSSDREEIAEIEKMHALIKEHKLDGQ FRWIAAQTNRARNGELYRYIADKRGIFVQPAFYEAFGLTVVEAMTCGLPTFATCHGGPNEIIEHGVSGFHIDPYHPDKAAELMAEFFQRCKQDPTHWEKISASGLRRILERYTWKIYSERLMTLSGVYGFWKLVSKLERRETRRYLEMFYILKFRELAKSVPLAIDDK SEQ ID NO: 42: Polynucleotide sequence of NtSUS4-S SEQ ID NO: 43: Polypeptide sequence of NtSUS4-S MAERVLTRVHSLRERLDATLAAHRNEILLFLSRIESHGKGILKPHQLLAEFDSIHKEDKNKLNDHAFEEVLKSTQEAIVLSPWVALAIRLRPGVWEYVRVNVNALVVEELTVPEYLQFKEELVNGTSHDNFVLELDFEPFTASFPKPTLTKSIGNGVEFLNRHLSAKMFHDKESMTPLLEFLRVHHYKGKTMMLNDRIQDLN TLQNVLRKAEEYLTTLSPETSYSAFEHKFQEIGLERGWGDTAERVLEMICMLLDLLEAPDSCTLEKFLGRIPMVFNVVILSPHGYFAQENVLGYPDTGGQVVYILDQVPALEREMLKRIKEQGLDIKPRILIVTRLLPDAVGTTCGQRLEKVFGTEHSHILRVPFRTEKGIVRKWISRFEVWPYMETFTEDVAKEIAAELQA KPDLIIGNYSEGNLAASLLAHKLGVTQCTIAHALEKTKYPDSDIYLKKFDEKYHFSAQFTADLIAMNHTDFIITSTFQEIAGSKDTVGQYESHMAFTMPGLYRVVHGIDVFDPKFNIVSPGADMNLYFPYYEKEKRLTAYHPEIEELLFSDVENDEHMCVLKNRNKPIIFTMARLDRVKNLTGLVELYAKNPRLRELVNLVV VGGDRRKESKDLEEQAEMKKMYELIKTHNLNGQFRWISSQMNRVRNGELYRYIADTRGAFVQPAFYEAFGLTVVEAMTCGLPTFATNHGGPAEIIVHGKSGFHIDPYHGDQAAELLADFFEKCKKEPSHWEAISEGGLKRIQEKYTWQIYSDRLLTLAAVYGFWKHVSKLDRLEIRRYLEMFYALKFRKLVSFIAFCTPAIV SEQ ID NO: 44: Polynucleotide sequence of NtSUS4-T SEQ ID NO: 45: Polypeptide sequence of NtSUS4-T MAERVLTRVHSLRERLDATLAAHRNEILLFLSRIESHGKGILKPHQLLAEFESIHKEDKNKLNDHAFEEVLKSTQEAIVLSPWVALAIRLRPGVWEYVRVNVNALIVEELTVPEYLQFKEELVNGTSNDNFVLELDFEPFTASFPKPTLTKSIGNGVEFLNRHLSAKMFHDKESMTPLLEFLRVHHYKGKTMMLNDRVQDL NTLQNVLRKAEEYLTTLSPETSYSVFEHKFQEIGLERGWGDNAERVLEMICMLLDLLEAPDSCTLEKFLGRIPMVFNVVILSPHGYFAQENVLGYPDTGGQVVYILDQVPALEREMLKRIKEQGLDIKPRILIVTRLLPDAVGTTCGQRLEKVFGTEHSHILRVPFRTEKGIVRKWISRFEVWPYMETFTEDVAKEIAAEL QAKPDLIIGNYSEGNLAASLLAHKLGVTQCTIAHALEKTKYPDSDIYLKKFDEKYHFSAQFTADLIAMNHTDFIITSTFQEIAGSKDTVGQYESHMAFTMPGLYRVVHGIDVFDPKFNIVSPGADMNLYFPYFEKEKRLTAYHPEIEELLFSDVENDEHMCVLKDRNKPIIFTMARLDRVKNLTGLVELYAKNPRLRELVN LVVVGGDRRKESKDLEEQAEMKKMYELIKTHNLNGQFRWISSQMNRVRNGELYRYIADTRGAFVQPAFYEAFGLTVVEAMTCGLPTFATNHGGPAEIIVHGKSGFHIDPYHGEQAAELLADFFERCKKEPSHWEAISEGGLKRIQEKYTWQIYSDRLLTLAAVYGFWKHVSKLDRLEIRRYLEMFYALKFRKLAEAVPLAVE SEQ ID NO: 46: Polynucleotide sequence of NtSUS5-S SEQ ID NO: 47: Polypeptide sequence of NtSUS5-S MASTVADSMPDALKQSRYHMKRCFARFIAMGRRLMKLKHLTEEIEETIEDKAERTRILEGSLGKIMSSTQEAAVVPPYVAFAVRHNPGFWDYVKVNAETLSVEAI SAREYLKFKEMIFDEDWAKDDNALEVDFGAFDYSNPRLALSSSVGNGLNFISKVLSSKFGGKPEDAQPLLDYLLALNHQGENLMINENLNGVAKLQAALIVAEVF VSSFPKDTPYKDFEHKLKEWGFDKGWGHNAGRVRETMRLLSEIIQAPDPINMESFFSKLPTTFNIVIFSIHGYFGQADVLGLPDTGGQVVYILDQVRALEEEMLQ RIKQQGLNVKPKILVVSRLIPDARGTTCNQEMEPILNSSHSHILRIPFRTEKGVLRQWDASAKILELMEGKPDLIIGNYTDGNLVASLLANKLGVTQGTIAHALE KTKYEDSDVKWKQFDPKYHFSCQFTADLLAMNAADFIITSTYQEIAGSETRPGQYESHTAFTMPGLYRAVSGINVFDPKFNIAAPGAEQSTYFPFTEKQKRFSTFRPAINELLYSNEENNEHIGFLADRKKPIIFSMARFDTVKNLSGLTEWYGKNKKLRNLVNLVIVGGFFDPSKSKDREEAAEIKKMHELIEKYQLKGQMRWIAAQTD KYRNSELYRTIADTKGAFVQPALYEAFGLTVIEAMDCGLPTFATNQGGPAEIIVDGVSGFHIDPYNGDESSKKIADFFEKCKVDSKYWNRISEGGLKRIEECYTW KIYANKVLNMGSIYGFWRQFNVGQKQAKQRYFEMFYNPLFRKLAKSVPIPHEEPLPLATSDSTQSQELKLPLPVPAAVAKVLPLTRHAFNLITSLPRVTGKVDVK SEQ ID NO: 48: Polynucleotide sequence of NtSUS5-T SEQ ID NO: 49: Polypeptide sequence of NtSUS5-T MASTVAGSMPDALKQSRYHMKRCFARFIAMGRRLMKLKHLTEEIEKTIEDKAERTKILEGSLGKIMSSTQEAAVVPPYVAFAVRHNPGFWDYVKVDAETLSVEAISARDYLKFKEMIFDEDWAKDENALEVDFGAFDYSNHRLALSSSVGNGLNFISKVLSSKFGGKAEDAQPLLDYLLALNH QGENLMINENLNGVSKLQAALIVAEVFVSSFPKDTPYKDFEHKLKEWGFEKGWGHNAGRVRETMRLLSEIIQAPDPINMESFFSRLPTTFNIVIFSIHGYFGQADVLGLPDTGGQVVYILDQVRALEEEMLQRIKQQGLNVKPKILVVTRLIPDARGTTCNQEMEPILNSSHSHILRIPFRTE KGVLRQWDASAKILELMEGKPDLIIGNYTDGNLVASLLANKLGVTQGTIAHALEKTKYEDSDVKWKQFDSKYHFSCQFTADLLAMNAADFIITSTYQEIAGSETRPGQYESHTAFTMPGLYRAVSGINVFDPKFNIAAPGAEQSAYFPFTEKQKRFSAFRPAIEELLYSNEQNNEHIGFLADR KKPIIFSMARFDTVKNLSGLTEWYGKNKKLRNLVNLVIVGGFFDPSKSKDREEAAEIKKMHELIEKYKLKGQMRWIAAQTDKYQNSELYRTIADTKGAFVQPALYEAFGLTVIEAMNCGLPTFATNQGGPAEIIVDGVSGFHIDPYNGDESSKKIADFFEKCKVDSKYWNKICGGGLKRIEEW SEQ ID NO: 50: Polynucleotide sequence of NtSUS6-S SEQ ID NO: 51: Polypeptide sequence of NtSUS6-S MATAPALNRSESIADSMPEALRQSRYHMKKCFAKYIEQGKRMMKLHNLMDELEKVIDDPAERNHVLEGLLGYILCTTMEAAVVPPYIAFATRQNPGFWEYVKVNAND LSVEGITATEYLKFKEMIVDECWAKDEYALEIDFGAVDFSTPRLTLSSSIGNGLSYVSKFLTSKLNATSASAQCLVDYLLTLNHQGDKLMINETLSTVSKLQAALVVA EASISSLPTDTPYESFELRFKQWGFEKGWGDTAERVSDTMRTLSEVLQAPDPLNIQKFFGRVPTVFNIVLFSVHGYFGQADVLGLPDTGGQVVYVLDQVVAFEEEML QRIKQQGLNIKPQILVLTRLIPDAKGTKCNQELEPIKNTKHSHILRVPFRTEKGVLNQWVSRFDIYPYLERYTQDAADKIVELMEGKPDLIIGNYTDGNLVASLMARK LGITLGTIAHALEKTKYEDSDIKLKELDPKYHFSCQFTADLIAMNSADFIITSTYQEIAGSKDKPGQYESHSAFTLPGLYRVASGINVFDPKFNIAAPGADQSVYFP YTEKQKRLTAFRPAIEELLFSKVDNDEHVGYLEDRKKPILFTMARLDTVKNTSGLTEWYGKNKRLRSLVNLVVVGGSFDPTKSKDREEAAEIKKMHMLIEKYQLKGQI RWIAAQTDRYRNSELYRTIADSKGAFVQPALYEAFGLTVIEAMNCGLPTFATNQGGPAEIIVDGVSGFHIDPNNGDESSNKIANFFQKCREDPEYWNRISVQGLNRI YECYTWKIYANKVLNMGSIYTFWRTLYRDQKQAKQRYIETFYNLEFRNLVKNVPIRKDETPQGPKEREKVKPQISQRHALKLLPTVFQETLALTIKVAEVRLDKIATA SEQ ID NO: 52: Polynucleotide sequence of NtSUS6-T SEQ ID NO: 53: Polypeptide sequence of NtSUS6-T MATAPALKRSESIADSMPEALRQSRYHMKKCFAKYIEQGKRMMKLHNLLMDELEKVIDDPAERNHVLEGLLGYILCTTMEAAVVPPYIAFATRQNPGFWEYVKVNANDLSVEGIT ATDYLKFKEMIVDESWAKDEYALEIDFGAVDFSTPRLTLSSSIGNGLSYVSKFLTSKLNATSASAQCLVDYLLTLNHQGDKLMINETLGTVSKLQAALVVAEASISSLPTDTPY QSFELRFKQWGFEKGWGDTAERVRDTMRTLSEVLQAPDPLNIEKFFGRVPTVFNIVLFSVHGYFGQANVLGLPDTGGQVVYVLDQVVAFEEEMLQRIKQQGLNIKPQILVLTRL IPDAKGTKCNQELEPIKNTKHSHILRVPFRTEKGVLNQWVSRFDIYPYLERYTQDAADKIIELMEGKPDLIIGNYTDGNLVASLMARKLGITLGTIAHALEKTKYEDSDIKLKE LDPKYHFSCQFTADLIAMNSADFIITSTYQEIAGSKDRPGQYESHSAFTLPGLYRVASGINVFDPKFNIAAPGADQSVYFPYTEKQTRLTAFRPAIEELLFSKVDNDEHIGYLE DRKKPILFTMARLDTVKNTSGLTEWYGKNKRLRSLVNLVVVGGSFDPTKSKDREEAAEIKKMHMLIEKYQLKGQIRWIAAQTDRYRNSELYRTIADSKGAFVQPALYEAFGLTV IEAMNCGLPTFATNQGGPAEIIVDGVSGFHIDPNNGDESSNKVANFFQKCREDPEYWNRISVQGLNRIYECYTWKIYANKVLNMGSIYTFWRTLYRDQKQAKQRYIETFYNLEF RNLVKNVPIRQDETPQGPKERREKVKPQISQRHALKLLPIVFQETLVYSSTKLELYSMQLASAVHLSKSPVMSIKLTKSINSAFPFPMLDCYYSYFVYGSLPSLFSPVLLLLQLY

Claims

1. A plant cell, (i) a polynucleotide comprising, consisting of, or consisting essentially of a sequence having at least 60% sequence identity to SEQ ID NO:1 (NtSULTR3;1A-S), SEQ ID NO:3 (NtSULTR3;1A-T), SEQ ID NO:5 (NtSULTR3;1B-S), SEQ ID NO:7 (NtSULTR3;1B-T), SEQ ID NO:15 (NtSULTR3;3-T), SEQ ID NO:17 (NtSULTR3;4A-S), SEQ ID NO:19 (NtSULTR3;4A-T), or SEQ ID NO:23 (NtSULTR3;4B-T); (ii) a polypeptide encoded by the polynucleotide described in (i); (iii) at least 87% sequence identity to SEQ ID NO:2 (NtSULTR3; 1A-S), or at least 87% sequence identity to SEQ ID NO:4 (NtSULTR3; 1A-T), or at least 87% sequence identity to SEQ ID NO:6 (NtSULTR3; 1B-S), or at least 88% sequence identity to SEQ ID NO:8 (NtSULTR3; 1B-T), or at least 88% sequence identity to SEQ ID NO:16 (NtSULTR3; 3- a polypeptide comprising, consisting of, or consisting essentially of a sequence having at least 70% sequence identity to SEQ ID NO: 18 (NtSULTR3; 4A-S), or at least 84% sequence identity to SEQ ID NO: 18 (NtSULTR3; 4A-S), or at least 79% sequence identity to SEQ ID NO: 20 (NtSULTR3; 4A-T), or at least 87% sequence identity to SEQ ID NO: 24 (NtSULTR3; 4B-T); (iv) a construct, vector, or expression vector comprising the isolated 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 polynucleotide or polypeptide, compared to a control plant cell in which the expression or activity of the polynucleotide or polypeptide is not modified.

2. (i) a polynucleotide comprising, consisting of, or consisting essentially of a sequence having at least 60% sequence identity to SEQ ID NO: 1 (NtSULTR3; 1A-S) or SEQ ID NO: 3 (NtSULTR3; 1A-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 87% sequence identity to SEQ ID NO:2 (NtSULTR3; 1A-S), or at least 87% sequence identity to SEQ ID NO:4 (NtSULTR3; 1A-T); or (iv) The plant cell of claim 1, comprising a construct, vector, or expression vector comprising the isolated polynucleotide described in (i).

3. 3. The plant cell of claim 1, wherein the regulated expression or regulated activity regulates the levels of glucose, fructose and sucrose in cured leaves of a plant comprising the plant cell compared to the levels of glucose, fructose and sucrose in cured leaves of a control.

4. the level of glucose is reduced by at least about 77%, at least about 69%, and at least about 60%, respectively, compared to control dried leaves; or the level of fructose is reduced by at least about 77%, at least about 69%, and at least about 60%, respectively, compared to control dried leaves; or the level of sucrose is reduced by at least about 77%, at least about 69%, and at least about 60%, respectively, compared to control dried leaves; or 4. The plant cell of claim 3, wherein the levels of glucose, fructose, and sucrose are reduced by at least about 77%, at least about 69%, and at least about 60%, respectively, compared to control dried leaves.

5. 10. A plant cell according to any one of the preceding claims, wherein the regulated expression or regulated activity regulates the levels of free amino acids glutamine, glutamic acid and aspartic acid in dried leaves of a plant comprising said plant cell compared to the levels of free amino acids glutamine, glutamic acid and aspartic acid in dried leaves of a control.

6. the levels of free amino acids are increased by at least about 1.5-fold, at least about 2.3-fold, at least about 2.4-fold and at least about 1.5-fold, respectively, compared to control dried leaves; or the level of glutamine is increased by at least about 1.5-fold, at least about 2.3-fold, at least about 2.4-fold, and at least about 1.5-fold, respectively, compared to control dried leaves; or the level of glutamic acid is increased by at least about 1.5-fold, at least about 2.3-fold, at least about 2.4-fold, and at least about 1.5-fold, respectively, compared to control dried leaves; or the level of aspartic acid is increased by at least about 1.5-fold, at least about 2.3-fold, at least about 2.4-fold, and at least about 1.5-fold, respectively, compared to control dried leaves; or 6. The plant cell of claim 5, wherein the levels of the free amino acids glutamine, glutamic acid and aspartic acid are increased by at least about 1.5-fold, at least about 2.3-fold, at least about 2.4-fold and at least about 1.5-fold, respectively, compared to control dried leaves.

7. 10. The plant cell of claim 1, wherein the at least one modification is a genetic mutation in the polynucleotide and the plant is Nicotiana tabacum.

8. (i) at least one modification in an NtSUS polynucleotide or polypeptide encoded thereby, Preferably, 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, or a combination of two or more thereof; More preferably, the NtSUS polynucleotide or the polypeptide encoded thereby further comprises at least one modification selected from the group consisting of NtSUS2-S, NtSUS3-S, NtSUS3-T and NtSUS4-S, or a combination of two or more thereof; or (ii) further comprising at least one modification in a CLC-Nt2 polynucleotide or polypeptide encoded thereby, or an NtCLCe polynucleotide or polypeptide encoded thereby, or a combination thereof; (iii) A plant cell according to any one of the preceding claims, which is a combination of (i) and (ii).

9. A plant or part thereof comprising a plant cell according to any one of claims 1 to 8.

10. 10. Plant material, dried plant material or homogenized plant material derived from or obtained from a plant or part thereof according to claim 9, preferably the plant material is selected from the group consisting of biomass, seeds, stems, flowers, or leaves, or a combination of two or more thereof; and optionally 1. The plant material, dried plant material, or homogenized plant material, wherein the dried plant material is selected from the group consisting of flue-cured plant material, sun-dried plant material, or air-dried plant material, or a combination of two or more thereof.

11. A tobacco product comprising a plant cell according to any one of claims 1 to 8, a plant part according to claim 9, or a plant material according to claim 10.

12. 10. A method for producing the plant of claim 9, comprising: (a) providing a plant cell comprising at least one modification according to any one of claims 1 to 8; (b) propagating the plant cell into a plant.

13. In step (a), the 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; or 13. The method of claim 12, 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.

14. 1. A method for producing dried plant material having altered levels of glucose, fructose and sucrose and altered levels of the free amino acids glutamine, glutamic acid and aspartic acid compared to a control plant material, comprising: (a) providing a plant or part thereof according to claim 9 or a plant material according to claim 10; (b) harvesting said plant material therefrom; (c) drying the plant material.

15. 1. A method for producing a liquid tobacco extract, comprising: (a) preparing a first tobacco starting material from a plant or part thereof comprising the plant cells of any one of claims 1 to 8; (b) preparing a second tobacco starting material from a plant or part thereof comprising plant cells, (i) at least one modification in an NtSUS polynucleotide or a polypeptide encoded thereby, wherein preferably the NtSUS polynucleotide or the polypeptide encoded thereby comprises at least one modification 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 the polypeptide encoded thereby comprises at least one modification selected from the group consisting of NtSUS2-S, NtSUS3-S, NtSUS3-T, and NtSUS4-S, or a combination of two or more thereof; or (ii) further comprising at least one modification in a CLC-Nt2 polynucleotide or polypeptide encoded thereby, or an NtCLCe polynucleotide or polypeptide encoded thereby, or a combination thereof; (iii) preparing a combination of (i) and (ii); (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 and second tobacco starting materials during heating; (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.

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