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WO2025003105A1 - Modulation de gènes codant pour la glutamate déshydrogénase - Google Patents

Modulation de gènes codant pour la glutamate déshydrogénase Download PDF

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Publication number
WO2025003105A1
WO2025003105A1 PCT/EP2024/067755 EP2024067755W WO2025003105A1 WO 2025003105 A1 WO2025003105 A1 WO 2025003105A1 EP 2024067755 W EP2024067755 W EP 2024067755W WO 2025003105 A1 WO2025003105 A1 WO 2025003105A1
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Prior art keywords
polynucleotide
polypeptide
seq
plant
plant leaf
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English (en)
Inventor
Lucien Bovet
Aurore Ginette Denise HILFIKER
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Philip Morris Products SA
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Philip Morris Products SA
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Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/10—Processes for modifying non-agronomic quality output traits, e.g. for industrial processing; Value added, non-agronomic traits
    • A01H1/101—Processes for modifying non-agronomic quality output traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine or caffeine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01H5/12—Leaves
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/82—Solanaceae, e.g. pepper, tobacco, potato, tomato or eggplant
    • A01H6/823—Nicotiana, e.g. tobacco
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
    • C12N9/0016—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with NAD or NADP as acceptor (1.4.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y104/00—Oxidoreductases acting on the CH-NH2 group of donors (1.4)
    • C12Y104/01—Oxidoreductases acting on the CH-NH2 group of donors (1.4) with NAD+ or NADP+ as acceptor (1.4.1)
    • C12Y104/01002—Glutamate dehydrogenase (1.4.1.2)

Definitions

  • the present invention relates in general to plants having modulated expression or activity of glutamate dehydrogenase.
  • BACKGROUND OF THE INVENTION Heated tobacco products, which heat real tobacco instead of burning it, do not emit side- stream smoke, but smokers exhale aerosols into the atmosphere. The inhalation of ammonia may cause nasopharyngeal and tracheal burns, bronchiolar and alveolar oedema, and airway destruction, resulting in respiratory distress or failure. Yamamoto et al.
  • NtGDH genes from Nicotiana tabacum that are referred to herein as NtGDH are disclosed that are involved in the accumulation of ammonia in leaf during curing. NtGDH genes were investigated to identify those that are particularly expressed during the early curing phase. Unexpectedly, certain NtGDH genes were found not to be expressed during the early curing phase – such as NtGDH4, NtGDH7, NtGDH8 and NtGDH11 – whereas other NtGDH genes were expressed during the early curing phase. In particular, 6 NtGDH genes were expressed during the early curing phase, namely NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10 and NtGDH12.
  • NtGDH6 and NtGDH10 were found to be mainly expressed after 24 hours curing, whereas NtGDH2, NtGDH3, NtGDH9 and NtGDH12 were more induced in the late phase of curing from 48 hours to 192 hours.
  • NtGDH2, NtGDH3, NtGDH9 and NtGDH12 followed similar induction profiles, however, the expression of NtGDH2 and NtGDH3 constantly increased during the curing process and reached the highest expression values (based on Fragments Per Kilobase of transcript per Million mapped reads (FPKM values) after 192 hours, which was not the case for NtGDH9 or NtGDH12.
  • NtGDH genes – such as NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10 and NtGDH12 — can be used change the chemistry of cured leaves not only by limiting the increase of ammonia, but also by changing amino acid content - including proline, aspartate, serine, threonine and arginine content - and sugar content — such as glucose, fructose and lactose.
  • NtGDH genes upregulating (for example, overexpressing) certain NtGDH genes would also change the chemistry of cured leaves by increasing ammonia, increasing total alkaloids, decreasing amino acids – including proline, aspartate, serine, threonine and arginine – and decreasing sugars - such as glucose, fructose and lactose.
  • modulating the expression of more than one NtGDH polynucleotide or modulating the activity of more than one NtGDH polypeptide will result in a stronger modulation of ammonia and thereby a reduced cell toxicity due to ammonium during the senescence process along with additional side effects on the tobacco chemistry including alkaloids and amino acids.
  • the downregulation for example, silencing
  • the downregulation for example, silencing
  • the downregulation does not impact the biomass and height of the modified plants, which suggests that the presence of absence of active NtGDH2 and NtGDH3 proteins does not affect plant growth and development.
  • Cured leaf material of control tobacco and NtGDH-RNAi lines was pooled and subjected to a sensory test. Sensory test results showed that a different and favorable sensory perception was obtained with the NtGDH-RNAi lines. Accordingly, the present invention now provides the possibility to obtain cured tobacco with reduced ammonia which will be less toxic tobacco material for the consumer whilst still being capable of having a favorable sensory perception.
  • the present invention also provides the possibility to allow the blending of ‘high’ ammonia cured tobacco – such as Flavor Burley or some Dark tobaccos – to obtain new tobacco flavors with lower ammonia levels.
  • NtGDH glutamate dehydrogenase
  • the NtGDH comprises, consists, or consists essentially of at least one of a NtGDH2 polynucleotide or a NtGDH3 polynucleotide or a NtGDH6 polynucleotide or a NtGDH9 polynucleotide or a NtGDH10 polynucleotide or a NtGDH12 polynucleotide or a NtGDH2 polypeptide or a NtGDH3 polypeptide or a NtGDH6 polypeptide or a NtGDH9 polypeptid
  • the expression of the NtGDH6 polynucleotide and the NtGDH10 polynucleotide are modulated or the activity of the NtGDH6 polypeptide and the NtGDH10 polypeptide are modulated.
  • the expression of the NtGDH2 polynucleotide and the NtGDH3 polynucleotide are modulated or the activity of the NtGDH2 polypeptide and the NtGDH3 polypeptide are modulated.
  • the expression of the NtGDH9 polynucleotide and the NtGDH12 polynucleotide are modulated or the activity of the NtGDH9 polypeptide and the NtGDH12 polypeptide are modulated.
  • each of the NtGDH2 polynucleotide and the NtGDH3 polynucleotide and the NtGDH9 polynucleotide and the NtGDH12 polynucleotide are modulated or the activity of each of the NtGDH2 polypeptide and the NtGDH3 polypeptide and the NtGDH9 polypeptide and the NtGDH12 polypeptide are modulated.
  • the expression of each of the NtGDH2 polynucleotide and the NtGDH3 polynucleotide and the NtGDH6 polynucleotide and the NtGDH9 polynucleotide and the NtGDH10 polynucleotide and the NtGDH12 polynucleotide or the NtGDH2 polypeptide and the NtGDH3 polypeptide and the NtGDH6 polypeptide and the NtGDH9 polypeptide and the NtGDH10 polypeptide and the NtGDH12 polypeptide are modulated.
  • the expression of one or more of a NtGDH4 polynucleotide or a NtGDH7 polynucleotide or a NtGDH8 polynucleotide or a NtGDH11 polynucleotide or a NtGDH4 polypeptide or a NtGDH7 polypeptide or a NtGDH8 polypeptide or a NtGDH11 polypeptide is not modulated, wherein (i) the NtGDH4 polynucleotide comprises, consists or consists essentially of a sequence having at least 88% sequence identity to SEQ ID NO: 5; or (ii) the NtGDH4 polypeptide is encoded by the polynucleotide set forth in (i); or (iii) the NtGDH4 polypeptide has at least 92% sequence identity to SEQ ID NO:6; or (iv) the NtGDH7 polynucleotide comprises, consists or consists essentially of
  • the plant leaf or part of the plant leaf comprises at least one genetic alteration that modulates the expression or activity of the at least one NtGDH polynucleotide or the at least one NtGDH polypeptide; or wherein the plant leaf or part of the plant leaf comprises one or more of exogenous DNA or exogenous RNA that modulates the expression or activity of the at least one NtGDH polynucleotide or the at least one NtGDH polypeptide; or wherein the plant leaf or part of the plant leaf comprises one or more of a vector or a viral vector or an Agrobacterium vector or a CRISPR vector that modulates the expression or activity of the at least one NtGDH polynucleotide or the at least one NtGDH polypeptide; or wherein the plant leaf or part of the plant leaf comprises at least one modification that is capable of driving one or more of RNA interference or transcriptional gene silencing or virus induced gene silencing that modulates the expression or activity of the at least one NtGDH poly
  • the modulated expression or activity of the at least one NtGDH polynucleotide or NtGDH polypeptide modulates the amount of ammonia and amino acids and sugar and total alkaloids in the plant leaf or part of the plant leaf when cured.
  • the amino acids are proline, aspartate, serine, threonine and arginine.
  • the plant leaf or part thereof is air cured, suitably, wherein the air cured leaf or part thereof is sun cured or fire cured; or wherein the plant leaf or part thereof is air dried, suitably, wherein the air dried leaf or part thereof is sun dried or fire dried.
  • step (b) the expression of the NtGDH6 polynucleotide and the NtGDH10 polynucleotide are modulated or the activity of the NtGDH6 polypeptide and the NtGDH10 polypeptide are modulated.
  • the expression of the NtGDH2 polynucleotide and the NtGDH3 polynucleotide are modulated or the activity of both the NtGDH2 polypeptide and the NtGDH3 polypeptide are modulated.
  • the expression of the NtGDH9 polynucleotide and the NtGDH12 polynucleotide are modulated or the activity of the NtGDH9 polypeptide and the NtGDH12 polypeptide are modulated.
  • the expression of each of the NtGDH2 polynucleotide and the NtGDH3 polynucleotide and the NtGDH9 polynucleotide and the NtGDH12 polynucleotide are modulated or the activity of each of the NtGDH2 polypeptide and the NtGDH3 polypeptide and the NtGDH9 polypeptide and the NtGDH12 polypeptide are modulated.
  • the expression of each of the NtGDH2 polynucleotide and the NtGDH3 polynucleotide and the NtGDH6 polynucleotide and the NtGDH9 polynucleotide and the NtGDH10 polynucleotide and the NtGDH12 polynucleotide or the NtGDH2 polypeptide and the NtGDH3 polypeptide and the NtGDH6 polypeptide and the NtGDH9 polypeptide and the NtGDH10 polypeptide and the NtGDH12 polypeptide are modulated.
  • the expression of one or more of a NtGDH4 polynucleotide or a NtGDH7 polynucleotide or a NtGDH8 polynucleotide or a NtGDH11 polynucleotide or the activity of a NtGDH4 polypeptide or a NtGDH7 polypeptide or a NtGDH8 polypeptide or a NtGDH11 polypeptide is not modulated, wherein (i) the NtGDH4 polynucleotide comprises, consists or consists essentially of a sequence having at least 88% sequence identity to SEQ ID NO: 5; or (ii) the NtGDH4 polypeptide is encoded by the polynucleotide set forth in (i); or (iii) the NtGDH4 polypeptide has at least 92% sequence identity to SEQ ID NO:6; or (iv) the NtGDH7 polynucleotide comprises, consists or consists
  • the Nicotiana plant leaf or part of the plant leaf is a Nicotiana tabacum plant leaf or part of the plant leaf.
  • expression or activity is modulated by genome editing; suitably, wherein the genome editing is selected from CRISPR-mediated genome editing, mutagenesis, zinc finger nuclease-mediated mutagenesis, chemical or radiation mutagenesis, homologous recombination, oligonucleotide-directed mutagenesis and meganuclease-mediated mutagenesis; or wherein in step (b) expression or activity is modulated using an interference polynucleotide.
  • a cured Nicotiana plant leaf or a part of the cured plant leaf wherein: (i) the ammonia content is between about 0.16 ⁇ 0.04 % Dry Weight Basis (DWB) to 0.110.16 ⁇ 0.03 % DWB; and (ii) the glucose, fructose and sucrose content is from 0.51 ⁇ 0.58 % DWB to 1.55 ⁇ 1.10 % DWB; and (iii) the total free amino acid content is from 51.0 ⁇ 6.60 mg/g DWB to 60.1 ⁇ 4.58 mg/g DWB; and (iv) the total alkaloid content is from 2.24 ⁇ 0.8 % DWB to 4.20 ⁇ 0.39 %.
  • DWB Dry Weight Basis
  • a cured tobacco blend comprising at least two different types of cured tobacco, wherein at least one of the cured tobaccos is cured tobacco from the mutant, non-naturally occurring or transgenic Nicotiana plant leaf or part of the plant leaf or the cured mutant, non-naturally occurring or transgenic Nicotiana tabacum plant leaf or a part thereof or the cured Nicotiana plant leaf or a part of the cured plant leaf.
  • at least one other cured tobacco is a Burley tobacco or an Oriental tobacco or a Dark tobacco or a flue cured tobacco or a combination of two or more thereof.
  • a method for producing a tobacco blend having a reduced amount of ammonia comprising: (a) providing a cured Nicotiana plant leaf or a part thereof, wherein the first cured Nicotiana plant leaf or a part thereof is from the mutant, non-naturally occurring or transgenic Nicotiana plant leaf or part of the plant leaf according to any of claims or the cured mutant, non-naturally occurring or transgenic Nicotiana tabacum plant leaf or a part thereof or the cured Nicotiana plant leaf or a part of the cured plant leaf; and (b) blending the first cured Nicotiana plant leaf or a part thereof with at least one second cured Nicotiana plant leaf or a part thereof to produce a tobacco blend in which the sum of ammonia is lower than the sum of ammonia in the at least one second cured Nicotiana plant leaf or a part thereof.
  • a cured tobacco blend obtained or obtainable by the method of claim 26 comprising a cured form of the mutant, non-naturally occurring or transgenic Nicotiana plant leaf or part of the plant leaf or the cured mutant, non-naturally occurring or transgenic Nicotiana tabacum plant leaf or a part thereof or the cured Nicotiana plant leaf or a part of the cured plant leaf according or the cured tobacco blend.
  • SOME ADVANTAGES Modulating the expression and/or activity of certain NtGDHs as described herein can result in cured plant material with lower levels of ammonia resulting in less toxic tobacco material for consumers.
  • Modulating the expression and/or activity of certain NtGDHs as described herein can result in modulated levels of sugars and amino acids in cured plant material. This can result in tobacco with novel aromatic or sensory properties. Modulating the expression and/or activity of certain NtGDHs as described herein can result in modulated levels of total alkaloids in cured plant material. Biomass and plant height are not altered in the modified plants which is valuable for commercial plant production as yields will not be altered.
  • the present invention allows for the blending of ‘high’ ammonia cured tobaccos with lower ammonia cured tobaccos of the present invention to thereby reduce the overall ammonia levels in the blend.
  • non-genetically modified plants can be created which may be more acceptable to consumers.
  • the present disclosure is not restricted to the use of EMS mutant plants.
  • An EMS mutant plant can have less potential to bring improved properties to a crop after breeding.
  • the desirable characteristic(s) of the EMS mutant plant can be lost for different reasons. For example, several mutations may be required, the mutation can be dominant or recessive, and the identification of a point mutation in a gene target can be difficult to reach.
  • the present disclosure exploits the use of NtGDH that can be specifically manipulated to produce plants with a desirable phenotype.
  • FIGURES are a series of graphs showing NtGDH, SAG12 and SGR1 expression during the first 8 curing days (0-192 hours), corresponding to the early curing phase of Stella leaves hung in a barn (also corresponding to the onset of tobacco ammonia accumulation). The data are collected from RNAseq data, expressed as FPKM values. SAG12 and SGR1 are markers of senescence.
  • Figure 2 is a graph showing the expression of NtGDH2 and NtGDH3 in tobacco plant leaf (TN90, Burley background) after 48 hours curing (qPCR) following gene silencing by using a RNAi approach (GATEWAY vector).
  • the lines E459-2, E459-3 and E459-5 were selected (T1 plants) for chemical analysis to be compared with control plants.
  • Figure 3 is a series of box plots to visualise the chemical data presented in Table 1 for ammonia, sum of sugars, aspartic acid and proline.
  • Figure 4 is a series of box plots showing leaf biomass and height in the lines E459-2, E459-3 and E459-5 and control.
  • Leaf biomass was calculated after weighing 4 mature mid-upper leaves at harvest time, no statistical differences being observed between the control and the anti-NtGDH2-3 lines. At harvest time, the height of each plant was also determined, no significant differences being found between the lines.
  • Coding sequence or “polynucleotide encoding” means the nucleotides (RNA or DNA molecule) that comprise a polynucleotide which encodes a polypeptide.
  • the coding sequence can further include initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in the cells of an individual or mammal to which the polynucleotide is administered.
  • the coding sequence may be codon optimized.
  • “Complement” or “complementary” can mean Watson-Crick (for example, A-T/U and C-G) or Hoogsteen base pairing between nucleotides or nucleotide analogues.
  • “Complementarity” refers to a property shared between two polynucleotides, such that when they are aligned antiparallel to each other, the nucleotide bases at each position will be complementary.
  • Construct refers to a double-stranded, recombinant polynucleotide fragment comprising one or more polynucleotides.
  • the construct comprises a "template strand” base-paired with a complementary "sense or coding strand.”
  • a given construct can be inserted into a vector in two possible orientations, either in the same (or sense) orientation or in the reverse (or anti- sense) orientation with respect to the orientation of a promoter positioned within a vector - such as an expression vector.
  • control in the context of a control plant or control plant cells means a plant or plant cells in which the expression, function or activity of one or more genes or polypeptides has not been modified (for example, increased or decreased) and so it can provide a comparison with a plant in which the expression, function or activity of the same one or more genes or polypeptides has been modified.
  • a “control plant” is a plant that is substantially equivalent to a test plant or modified plant in all parameters with the exception of the test parameters. For example, when referring to a plant into which a polynucleotide has been introduced, a control plant is an equivalent plant into which no such polynucleotide has been introduced.
  • a control plant can be an equivalent plant into which a control polynucleotide has been introduced.
  • the control polynucleotide is one that is expected to result in little or no phenotypic effect on the plant.
  • the control plant may comprise an empty vector.
  • the control plant may correspond to a wild-type plant.
  • the control plant may be a null segregant wherein the T1 segregant no longer possesses a transgene.
  • decrease refers to a decrease of from about 10% to about 99%, or a decrease of at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% or, or at least 150%, or at least 200% more of a quantity or a function - such as polypeptide function, transcriptional function, or polypeptide expression.
  • a decreased amount can refer to a quantity or a function that is less than what would be found in a plant or a product from the same variety of plant processed in the same manner, which has not been modified. Thus, in some contexts, a wild-type plant of the same variety that has been processed in the same manner is used as a control by which to measure whether a decrease in quantity is obtained.
  • Donor DNA or “donor template” refers to a double-stranded DNA fragment or molecule that includes at least a portion of the gene of interest. The donor DNA may encode a functional polypeptide.
  • Endogenous gene or polypeptide refers to a gene or polypeptide that originates from the genome of an organism and has not undergone a change, such as a loss, gain, or exchange of genetic material. An endogenous gene undergoes normal gene transmission and gene expression. An endogenous polypeptide undergoes normal expression.
  • Endogenous sequences refer to the sequences that can increase gene expression. These sequences can be located upstream, within introns or downstream of the transcribed region. The transcribed region is comprised of the exons and the intervening introns, from the promoter to the transcription termination region. The enhancement of gene expression can be through various mechanisms including increasing transcriptional efficiency, stabilization of mature mRNA and translational enhancement.
  • Exogenous is used interchangeably with the term “heterologous” and refers to a polynucleotide that has been artificially supplied to a biological system – such as a plant.
  • An exogenous polynucleotide may be a polynucleotide that comes from a different plant or species of plant.
  • “Expression” refers to the production of a functional product.
  • expression of a polynucleotide fragment may refer to transcription of the polynucleotide fragment (for example, transcription resulting in mRNA or functional RNA) or translation of mRNA into a precursor or mature polypeptide, or a combination thereof.
  • “Overexpression” refers to the production of a gene product in transgenic organisms that exceeds levels of production in a null segregating (or non-transgenic) organism from the same experiment. “Functional” describes a polypeptide that has biological function or activity.
  • a “functional gene” refers to a gene transcribed to mRNA, which is translated to a functional or active polypeptide.
  • “Genetic construct” refers to DNA or RNA molecules that comprise a polynucleotide that encodes a polypeptide. The coding sequence can include initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression. “Genome editing” generally refers to the process by which genomic nucleic acid in a cell is altered.
  • the terms "homology” or “similarity” refer to the degree of sequence similarity between two polypeptides or between two polynucleotide molecules compared by sequence alignment. The degree of homology between two discrete polynucleotides being compared is a function of the number of identical, or matching, nucleotides at comparable positions. Homology or similarity can be determined across the full length of a subject sequence.
  • Identity in the context of two or more polynucleotides or polypeptides means that the sequences have a specified percentage of residues that are the same over a specified region. The percentage may be calculated by optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which the identical residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to yield the percentage of sequence identity.
  • the term "increase” or “increased” refers to an increase of from about 10% to about 99%, or an increase of at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, at least 100%, at least 150%, or at least 200% or more or more of a quantity or a function or an activity, such as but not limited to one or more of polypeptide function or activity, transcriptional function or activity and polypeptide expression.
  • the term “increased,” or the phrase “an increased amount” can refer to a quantity or a function or an activity in a plant or a product generated from the plant that is more than what would be found in a plant or a product from the same variety of plant processed in the same manner, which has not been modified.
  • a wild-type plant of the same variety that has been processed in the same manner is used as a control by which to measure whether an increase in quantity is obtained.
  • inhibitor refers to a decrease of from about 98% to about 100%, or a decrease of at least 98%, at least 99%, but particularly of 100%, of a quantity or a function or an activity, such as but not limited to one or more of polypeptide function or activity, transcriptional function or activity and polypeptide expression.
  • introduction can mean providing a polynucleotide (for example, a construct) or polypeptide into a cell.
  • Introduced includes reference to the incorporation of a polynucleotide into a eukaryotic cell where the polynucleotide may be incorporated into the genome of the cell, and includes reference to the transient provision of a polynucleotide or polypeptide to the cell. Introduced includes reference to stable or transient transformation methods, as well as sexually crossing.
  • intraduced in the context of inserting a polynucleotide (for example, a recombinant construct/expression construct) into a cell, means “transfection” or “transformation” or “transduction” and includes reference to the incorporation of a polynucleotide into a eukaryotic cell where the polynucleotide may be incorporated into the genome of the cell (for example, chromosome, plasmid, plastid or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (for example, transfected mRNA).
  • 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.
  • 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” denotes that a polynucleotide or polypeptide gives rise to essentially one band in an electrophoretic gel. Particularly, it means that the polynucleotide or polypeptide is at least 85% pure, more suitably at least 95% pure, and most suitably at least 99% pure.
  • Isolated polynucleotides may be purified from a host cell in which they naturally occur. Conventional polynucleotide purification methods known to skilled artisans may be used to obtain isolated polynucleotides. The term also embraces recombinant polynucleotides and chemically synthesized polynucleotides.
  • “Liquid tobacco extract” describes the direct product of an extraction process carried out on a tobacco starting material. The extraction process for producing the liquid tobacco extract can comprise heating the tobacco starting material under specific heating conditions and collecting the volatile compounds generated. The liquid tobacco extract can contain a mixture of compounds that have derived from the tobacco starting material and have been removed during the extraction process, typically in combination with a liquid carrier or solvent.
  • Modulate refers to causing or facilitating a qualitative or quantitative change, alteration, or modification in a process, pathway, function or activity of interest.
  • a change, alteration, or modification may be an increase (for example, upregulation) or decrease (for example, downregulation) in the relative process, pathway, function or activity of interest.
  • gene expression or polypeptide expression or polypeptide function or activity can be modulated.
  • the relative change, alteration, or modification will be determined by comparison to a control.
  • non-naturally occurring describes an entity – such as a polynucleotide, a genetic mutation, a polypeptide, a plant, a plant cell and plant material - that is not formed by nature or that does not exist in nature.
  • Such non-naturally occurring entities or artificial entities may be made, synthesized, initiated, modified, intervened, or manipulated by methods described herein or that are known in the art.
  • Such non-naturally occurring entities or artificial entities may be made, synthesized, initiated, modified, intervened, or manipulated by man.
  • a non-naturally occurring plant may not be produced using an essentially biological process.
  • a non-naturally occurring plant a non-naturally occurring plant cell or non-naturally occurring plant material may be made using traditional plant breeding techniques - such as backcrossing - or by genetic manipulation technologies - such as antisense RNA, interfering RNA, meganuclease and the like.
  • a non-naturally occurring plant, a non-naturally occurring plant cell or non-naturally occurring plant material may be made by introgression of or by transferring one or more genetic mutations (for example one or more polymorphisms) from a first plant or plant cell into a second plant or plant cell (which may itself be naturally occurring), such that the resulting plant, plant cell or plant material or the progeny thereof comprises a genetic constitution (for example, a genome, a chromosome or a segment thereof) that is not formed by nature or that does not exist in nature.
  • the resulting plant, plant cell or plant material is thus artificial or non- naturally occurring.
  • an artificial or non-naturally occurring plant or plant cell may be made by modifying a genetic sequence in a first naturally occurring plant or plant cell, even if the resulting genetic sequence occurs naturally in a second plant or plant cell that comprises a different genetic background from the first plant or plant cell.
  • a mutation is not a naturally occurring mutation that exists naturally in a polynucleotide or a polypeptide – such as a gene or a polypeptide.
  • Differences in genetic background can be detected by phenotypic differences or by molecular biology techniques known in the art - such as polynucleotide sequencing, presence or absence of genetic markers (for example, microsatellite RNA markers).
  • “Oligonucleotide” or “polynucleotide” means at least two nucleotides covalently linked together.
  • the depiction of a single strand also defines the sequence of the complementary strand.
  • a polynucleotide also encompasses the complementary strand of a depicted single strand.
  • Many variants of a polynucleotide may be used for the same purpose as a given polynucleotide.
  • a polynucleotide also encompasses substantially identical polynucleotides and complements thereof.
  • a single strand provides a probe that may hybridize to a given sequence under stringent hybridization conditions.
  • a polynucleotide also encompasses a probe that hybridizes under stringent hybridization conditions.
  • Polynucleotides may be single stranded or double stranded, or may contain portions of both double stranded and single stranded sequence.
  • the polynucleotide may be DNA, both genomic and cDNA, RNA, or a hybrid, where the polynucleotide may contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine.
  • Polynucleotides may be obtained by chemical synthesis methods or by recombinant methods.
  • the specificity of single-stranded DNA to hybridize complementary fragments is determined by the "stringency" of the reaction conditions (Sambrook et al., Molecular Cloning and Laboratory Manual, Second Ed., Cold Spring Harbor (1989)).
  • stringent conditions describes hybridization protocols in which polynucleotides at least 60% homologous to each other remain hybridized.
  • stringent conditions are selected to be about 5oC lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.
  • the Tm is the temperature (under defined ionic strength, pH, and polynucleotide concentration) at which 50% of the probes complementary to the given sequence hybridize to the given sequence at equilibrium. Since the given sequences are generally present at excess, at Tm, 50% of the probes are occupied at equilibrium.
  • Stringent conditions typically comprise: (1) low ionic strength and high temperature washes, for example 15 mM sodium chloride, 1.5 mM sodium citrate, 0.1% sodium dodecyl sulfate, at 50oC; (2) a denaturing agent during hybridization, for example, 50% (v/v) formamide, 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer (750 mM sodium chloride, 75 mM sodium citrate; pH 6.5), at 42oC; or (3) 50% formamide.
  • low ionic strength and high temperature washes for example 15 mM sodium chloride, 1.5 mM sodium citrate, 0.1% sodium dodecyl sulfate, at 50oC
  • a denaturing agent during hybridization for example, 50% (v/v) formamide, 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer (
  • Washes typically also comprise 5xSSC (0.75 M NaCl, 75 mM sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5xDenhardt's solution, sonicated salmon sperm DNA (50 ⁇ g/mL), 0.1% SDS, and 10% dextran sulfate at 42oC, with a wash at 42oC in 0.2xSSC (sodium chloride/sodium citrate) and 50% formamide at 55oC, followed by a high- stringency wash consisting of 0.1xSSC containing EDTA at 55oC.
  • the conditions are such that sequences at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous to each other typically remain hybridized to each other.
  • “Moderately stringent conditions” use washing solutions and hybridization conditions that are less stringent, such that a polynucleotide will hybridize to the entire, fragments, derivatives, or analogs of the polynucleotide.
  • One example comprises hybridization in 6xSSC, 5xDenhardt's solution, 0.5% SDS and 100 ⁇ g/mL denatured salmon sperm DNA at 55oC, followed by one or more washes in 1xSSC, 0.1% SDS at 37oC.
  • the 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, N.J. (1993); Kriegler, Gene Transfer and Expression: A Laboratory Manual, Stockton Press, New York, N.Y. (1990); Perbal, A Practical Guide to Molecular Cloning, 2nd edition, John Wiley & Sons, New York, N.Y. (1988)).
  • Low stringent conditions use washing solutions and hybridization conditions that are less stringent than those for moderate stringency, such that a polynucleotide will hybridize to the entire, fragments, derivatives, or analogs of the polynucleotide.
  • a non-limiting example of low stringency hybridization conditions includes hybridization in 35% formamide, 5xSSC, 50 mM Tris HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 ⁇ g/mL denatured salmon sperm DNA, 10% (wt/vol) dextran sulfate at 40oC, followed by one or more washes in 2xSSC, 25 mM Tris HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS at 50oC.
  • “Operably linked” means that expression of a gene is under the control of a promoter with which it is spatially connected.
  • a promoter may be positioned 5' (upstream) or 3' (downstream) of a gene under its control.
  • the distance between the promoter and a gene may be approximately the same as the distance between that promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, variation in this distance may be accommodated without loss of promoter function.
  • Promoter means a synthetic or naturally-derived molecule which is capable of conferring, activating or enhancing expression of a polynucleotide in a cell.
  • the term refers to a polynucleotide element/sequence, typically positioned upstream and operably-linked to a double-stranded polynucleotide fragment.
  • a promoter may regulate the expression of a gene component constitutively or differentially with respect to cell, the tissue or organ in which expression occurs or, with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, pathogens, metal ions, or inducing agents.
  • tissue-specific promoter and tissue-preferred promoter as used interchangeably herein refer to a promoter that is expressed predominantly but not necessarily exclusively in one tissue or organ, but that may also be expressed in one specific cell.
  • a “developmentally regulated promoter” refers to a promoter whose function is determined by developmental events.
  • a “constitutive promoter” refers to a promoter that causes a gene to be expressed in most cell types at most times.
  • an “inducible promoter” selectively express an operably linked DNA sequence in response to the presence of an endogenous or exogenous stimulus, for example by chemical compounds (chemical inducers) or in response to environmental, hormonal, chemical or developmental signals or a combination of two or more thereof.
  • inducible or regulated promoters include promoters regulated by light, heat, stress, flooding or drought, pathogens, phytohormones, wounding, or chemicals such as ethanol, jasmonate, salicylic acid, or safeners.
  • “Recombinant” refers to an artificial combination of two otherwise separated segments of sequence – such as by chemical synthesis or by the manipulation of isolated segments of polynucleotides by genetic engineering techniques.
  • the term also includes reference to a cell or vector, that has been modified by the introduction of a heterologous polynucleotide or a cell derived from a cell so modified, but does not encompass the alteration of the cell or vector by naturally occurring events (for example, spontaneous mutation, natural transformation or transduction or transposition) such as those occurring without deliberate human intervention.
  • "Recombinant construct” refers to a combination of polynucleotides that are not normally found together in nature. Accordingly, a recombinant construct may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that normally found in nature.
  • the recombinant construct can be a recombinant DNA construct.
  • regulatory sequences and “regulatory elements” as used interchangeably herein refer to polynucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences include promoters, translation leader sequences, introns, and polyadenylation recognition sequences. The terms “regulatory sequence” and “regulatory element” are used interchangeably herein.
  • tobacco is used in a collective sense to refer to tobacco crops (for example, a plurality of tobacco plants grown in the field and not hydroponically grown tobacco), tobacco plants and parts thereof, including but not limited to, roots, stems, leaves, flowers, and seeds prepared or obtained, as described herein, and parts thereof. It is understood that “tobacco” refers to plants that belong to the Nicotiana genus and products thereof and includes Nicotiana tabacum plants and products thereof.
  • tobacco products refers to consumer tobacco products, including but not limited to, smoking materials (for example, cigarettes, cigars, and pipe tobacco), snuff, chewing tobacco, gum, and lozenges, as well as components, materials and ingredients for manufacture of consumer tobacco products.
  • these tobacco products are manufactured from tobacco leaves and stems harvested from tobacco and cut, dried, cured, or fermented according to conventional techniques in tobacco preparation.
  • Transcription terminator refers to DNA sequences located downstream of a coding sequence, including polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression.
  • the polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor.
  • Transgenic refers to any cell, cell line, callus, tissue, plant part or plant, the genome of which has been altered by the presence of a heterologous polynucleotide, such as a recombinant construct, including those initial transgenic events as well as those created by sexual crosses or asexual propagation from the initial transgenic event.
  • the term does not encompass the alteration of the genome (chromosomal or extra-chromosomal) by conventional plant breeding methods or by naturally occurring events - such as random cross-fertilization, non- recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation.
  • the transgenic plant or part thereof is not produced using an essentially biological process.
  • Transgenic plant refers to a plant which comprises within its genome one or more heterologous polynucleotides, that is, a plant that contains recombinant genetic material not normally found therein and which has been introduced into the plant in question (or into progenitors of the plant) by human (artificial) manipulation.
  • the heterologous polynucleotide can be stably integrated within the genome such that the polynucleotide is passed on to successive generations.
  • the heterologous polynucleotide can be integrated into the genome alone or as part of a recombinant construct.
  • the commercial development of genetically improved germplasm has also advanced to the stage of introducing multiple traits into crop plants, often referred to as a gene stacking approach.
  • transgenic plant encompasses the entire plant or tree and parts of the plant or tree, for instance grains, seeds, flowers, leaves, roots, fruit, pollen, stems and the like. Each heterologous polynucleotide may confer a different trait to the transgenic plant.
  • Transgene refers to a gene or genetic material containing a gene sequence that has been isolated from one organism and is introduced into a different organism. This non-native segment of DNA may retain the ability to produce RNA or polypeptide in the transgenic organism, or it may alter the normal function of the transgenic organism's genetic code.
  • “Variant” with respect to a polynucleotide means: (i) a portion or fragment of a polynucleotide; (ii) the complement of a polynucleotide or portion thereof; (iii) a polynucleotide that is substantially identical to a polynucleotide of interest or the complement thereof; or (iv) a polynucleotide that hybridizes under stringent conditions to the polynucleotide of interest, complement thereof, or a polynucleotide substantially identical thereto.
  • “Variant” with respect to a peptide or polypeptide means a peptide or polypeptide that differs in sequence by the insertion, deletion, or conservative substitution of amino acids, but retain at least one biological function or activity. Variant may also mean a polypeptide that retains at least one biological function or activity.
  • a conservative substitution of an amino acid that is, replacing an amino acid with a different amino acid of similar properties (for example, hydrophilicity, degree and distribution of charged regions) is recognized in the art as typically involving a minor change.
  • the term "variety” refers to a population of plants that share constant characteristics which separate them from other plants of the same species. While possessing one or more distinctive traits, a variety is further characterized by a very small overall variation between individuals within that variety.
  • Vector refers to a polynucleotide vehicle that comprises a combination of polynucleotide components for enabling the transport of polynucleotides, polynucleotide constructs and polynucleotide conjugates and the like.
  • a vector may be a viral vector, bacteriophage, bacterial artificial chromosome or yeast artificial chromosome.
  • a vector may be a DNA or RNA vector. Suitable vectors include episomes capable of extra-chromosomal replication such as circular, double-stranded nucleotide plasmids; linearized double-stranded nucleotide plasmids; and other vectors of any origin.
  • An "expression vector” is a polynucleotide vehicle that comprises a combination of polynucleotide components for enabling the expression of polynucleotide(s), polynucleotide constructs and polynucleotide conjugates and the like.
  • Suitable expression vectors include episomes capable of extra-chromosomal replication such as circular, double-stranded nucleotide plasmids; linearized double-stranded nucleotide plasmids; and other functionally equivalent expression vectors of any origin.
  • An expression vector comprises at least a promoter positioned upstream and operably-linked to a polynucleotide, polynucleotide constructs or polynucleotide conjugate, as defined below.
  • an isolated polynucleotide comprising, consisting or consisting essentially of a sequence having at least 60% sequence identity to any of the sequences described herein, including any of polynucleotides shown in the sequence listing.
  • the isolated polynucleotide comprises, consists or consists essentially of a sequence having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto.
  • the isolated polynucleotide comprises, consists or consists essentially of a sequence having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto.
  • the isolated polynucleotide comprises, consists or consists essentially of a sequence having at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto.
  • the polynucleotide(s) described herein encode an active polypeptide that has at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 100% or more of the NtGDH function or activity of the polypeptide(s) shown in the sequence listing.
  • an isolated NtGDH polynucleotide from Nicotiana tabacum comprising, consisting or consisting essentially of a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 (NtGHD2), or at least 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 3 (NtGDH3), or at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 5 (NtGDH4), or at least 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 7 (NtGDH6), or at least 92%, 93%,
  • a polynucleotide comprising, consisting or consisting essentially of a polynucleotide with substantial homology (that is, sequence similarity) or substantial identity to SEQ ID NO: 1 or SEQ ID NO: 3 or SEQ ID NO: 5 or SEQ ID NO: 7 or SEQ ID NO: 9 or SEQ ID NO: 11 or SEQ ID NO: 13 or SEQ ID NO: 15 or SEQ ID NO: 17 or SEQ ID NO: 19.
  • fragments of SEQ ID NO: 1 with substantial homology (that is, sequence similarity) or substantial identity thereto that have at least about 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 fragments of SEQ ID NO: 1.
  • fragments of SEQ ID NO: 3 with substantial homology (that is, sequence similarity) or substantial identity thereto that have at least about 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 fragments of SEQ ID NO: 3.
  • fragments of SEQ ID NO: 5 with substantial homology (that is, sequence similarity) or substantial identity thereto that have at least about 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 fragments of SEQ ID NO: 5.
  • fragments of SEQ ID NO: 7 with substantial homology (that is, sequence similarity) or substantial identity thereto that have at least about 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 fragments of SEQ ID NO: 7.
  • fragments of SEQ ID NO: 9 with substantial homology (that is, sequence similarity) or substantial identity thereto that have at least about 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 fragments of SEQ ID NO: 9.
  • fragments of SEQ ID NO: 11 with substantial homology (that is, sequence similarity) or substantial identity thereto that have at least about 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 fragments of SEQ ID NO: 11.
  • fragments of SEQ ID NO: 13 with substantial homology (that is, sequence similarity) or substantial identity thereto that have at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to the corresponding fragments of SEQ ID NO: 13.
  • fragments of SEQ ID NO: 15 with substantial homology (that is, sequence similarity) or substantial identity thereto that have at least about 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 fragments of SEQ ID NO: 15.
  • fragments of SEQ ID NO: 17 with substantial homology (that is, sequence similarity) or substantial identity thereto that have at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to the corresponding fragments of SEQ ID NO: 17.
  • fragments of SEQ ID NO: 19 with substantial homology (that is, sequence similarity) or substantial identity thereto that have at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to the corresponding fragments of SEQ ID NO: 19.
  • polynucleotides comprising a sufficient or substantial degree of identity or similarity to SEQ ID NO: 1 or SEQ ID NO: 3 or SEQ ID NO: 5 or SEQ ID NO: 7 or SEQ ID NO: 9 or SEQ ID NO: 11 or SEQ ID NO: 13 or SEQ ID NO: 15 or SEQ ID NO: 17 or SEQ ID NO: 19 that encode a polypeptide that functions as a NtGDH.
  • a polymer of polynucleotides which comprises, consists or consists essentially of a polynucleotide designated herein as SEQ ID NO: 1 or SEQ ID NO: 3 or SEQ ID NO: 5 or SEQ ID NO: 7 or SEQ ID NO: 9 or SEQ ID NO: 11 or SEQ ID NO: 13 or SEQ ID NO: 15 or SEQ ID NO: 17 or SEQ ID NO: 19.
  • the expression of at least one of the NtGDH2 polynucleotide comprising, consisting or consisting essentially of a sequence having at least 90% sequence identity to SEQ ID NO: 1; or at least 90% sequence identity to SEQ ID NO: 3; or at least 90% sequence identity to SEQ ID NO: 7; or at least 85% sequence identity to SEQ ID NO: 13; or at least 89% sequence identity to SEQ ID NO: 15; or at least 85% sequence identity to SEQ ID NO: 19 or a combination of two ore or thereof is modulated.
  • the expression of the NtGDH6 polynucleotide and the NtGDH10 polynucleotide are modulated.
  • the expression of the NtGDH2 polynucleotide and the NtGDH3 polynucleotide are modulated.
  • the expression of the NtGDH9 polynucleotide and the NtGDH12 polynucleotide are modulated.
  • the expression of each of the NtGDH2 polynucleotide and the NtGDH3 polynucleotide and the NtGDH9 polynucleotide and the NtGDH12 polynucleotide are modulated.
  • the expression of each of the NtGDH2 polynucleotide and the NtGDH3 polynucleotide and the NtGDH6 polynucleotide and the NtGDH9 polynucleotide and the NtGDH10 polynucleotide and the NtGDH12 polynucleotide are modulated.
  • the expression of one or more of a NtGDH4 polynucleotide or a NtGDH7 polynucleotide or a NtGDH8 polynucleotide or a NtGDH11 polynucleotide are not modulated, wherein the NtGDH4 polynucleotide comprises, consists or consists essentially of a sequence having at least 88% sequence identity to SEQ ID NO: 5; and the NtGDH7 polynucleotide comprises, consists or consists essentially of a sequence having at least 92% sequence identity to SEQ ID NO: 9; and the NtGDH8 polynucleotide comprises, consists or consists essentially of a sequence having at least 92% sequence identity to SEQ ID NO: 11; and the NtGDH11 polynucleotide comprises, consists or consists essentially of a sequence having at least 87% sequence identity to SEQ ID NO: 17.
  • polynucleotides described herein encode NtGDH polypeptides that have NtGDH activity.
  • a polynucleotide can include a polymer of nucleotides, which may be unmodified or modified deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Accordingly, a polynucleotide can be, without limitation, a genomic DNA, complementary DNA (cDNA), mRNA, or antisense RNA or a fragment(s) thereof.
  • a polynucleotide can be single-stranded or double-stranded DNA, DNA that is a mixture of single-stranded and double-stranded regions, a hybrid molecule comprising DNA and RNA, or a hybrid molecule with a mixture of single-stranded and double- stranded regions or a fragment(s) thereof.
  • the polynucleotide can be composed of triple-stranded regions comprising DNA, RNA, or both or a fragment(s) thereof.
  • a polynucleotide can contain one or more modified bases, such as phosphothioates, and can be a peptide nucleic acid.
  • polynucleotides can be assembled from isolated or cloned fragments of cDNA, genomic DNA, oligonucleotides, or individual nucleotides, or a combination of the foregoing.
  • polynucleotides described herein are shown as DNA sequences, they include their corresponding RNA sequences, and their complementary (for example, completely complementary) DNA or RNA sequences, including the reverse complements thereof.
  • Fragments of a polynucleotide may range from at least about 25 nucleotides, about 50 nucleotides, about 75 nucleotides, about 100 nucleotides about 150 nucleotides, about 200 nucleotides, about 250 nucleotides, about 300 nucleotides, about 400 nucleotides, about 500 nucleotides, about 600 nucleotides, about 700 nucleotides, about 800 nucleotides, about 900 nucleotides, about 1000 nucleotides, about 1100 nucleotides, about 1200 nucleotides, about 1300 nucleotides or about 1400 nucleotides and up to the full-length polynucleotide encoding the polypeptides described herein.
  • a polynucleotide will generally contain phosphodiester bonds, although in some cases, polynucleotide analogues are included that may have alternate backbones, comprising, for example, phosphoramidate, phosphorothioate, phosphorodithioate, or O- methylphophoroamidite linkages; and peptide polynucleotide backbones and linkages.
  • Other analogue polynucleotides include those with positive backbones; non-ionic backbones, and non-ribose backbones. Modifications of the ribose-phosphate backbone may be done for a variety of reasons, for example, to increase the stability and half-life of such molecules in physiological environments or as probes on a biochip.
  • Mixtures of naturally occurring polynucleotides and analogues can be made; alternatively, mixtures of different polynucleotide analogues, and mixtures of naturally occurring polynucleotides and analogues may be made.
  • a variety of polynucleotide analogues are known, including, for example, phosphoramidate, phosphorothioate, phosphorodithioate, O-methylphophoroamidite linkages and peptide polynucleotide backbones and linkages.
  • Other analogue polynucleotides include those with positive backbones, non-ionic backbones and non-ribose backbones.
  • Polynucleotides containing one or more carbocyclic sugars are also included.
  • Other analogues include peptide polynucleotides which are peptide polynucleotide analogues.
  • fragments generally comprise at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more contiguous nucleotides of a DNA sequence.
  • a DNA fragment comprises at least about 10, 15, 20, 30, 40, 50 or 60 or more contiguous nucleotides of a DNA sequence.
  • a method for detecting a polynucleotide comprising the use of the probes or primers or both.
  • the basic parameters affecting the choice 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, N.Y.).
  • sets of degenerate oligonucleotides can be prepared.
  • Such oligonucleotides are useful as primers, for example, in polymerase chain reactions (PCR), whereby DNA fragments are isolated and amplified.
  • At least one modification can be included in one or more of SEQ ID NO: 1 or SEQ ID NO: 3 or SEQ ID NO: 5 or SEQ ID NO: 7 or SEQ ID NO: 9 or SEQ ID NO: 11 or SEQ ID NO: 13 or SEQ ID NO: 15 or SEQ ID NO: 17 or SEQ ID NO: 19.
  • NtGDH polypeptide encoded by the polynucleotide(s) described herein.
  • NtGDH polypeptide comprising, consisting or consisting essentially of a polypeptide having at least 60% sequence identity to any of the polypeptides described herein, including any of the polypeptides shown in the sequence listing.
  • the isolated polypeptide comprises, consists or consists essentially of a sequence having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, 80%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity thereto.
  • the isolated NtGDH polypeptide comprises, consists or consists essentially of a sequence having at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity thereto.
  • the isolated NtGDH polypeptide comprises, consists or consists essentially of a sequence having at least 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.
  • NtGDH polypeptide comprising, consisting or consisting essentially of a sequence having at least about 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.
  • NtGDH polypeptide comprising, consisting or consisting essentially of a sequence having at least about 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: 4.
  • NtGDH polypeptide comprising, consisting or consisting essentially of a sequence having at least 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: 6.
  • NtGDH polypeptide comprising, consisting or consisting essentially of a sequence having at least about 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: 8.
  • NtGDH polypeptide comprising, consisting or consisting essentially of a sequence having at least 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to SEQ ID NO: 10.
  • NtGDH polypeptide comprising, consisting 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: 12.
  • NtGDH polypeptide comprising, consisting or consisting essentially of a sequence having at least 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: 14.
  • NtGDH polypeptide comprising, consisting or consisting essentially of a sequence having at least about 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: 16.
  • NtGDH polypeptide comprising, consisting or consisting essentially of a sequence having at least 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: 18.
  • NtGDH polypeptide comprising, consisting or consisting essentially of a sequence having at least about 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: 20.
  • the polypeptide can include sequences comprising a sufficient or substantial degree of identity or similarity to SEQ ID NO: 1 or SEQ ID NO: 3 or SEQ ID NO: 5 or SEQ ID NO: 7 or SEQ ID NO: 9 or SEQ ID NO: 11 or SEQ ID NO: 13 or SEQ ID NO: 15 or SEQ ID NO: 17 or SEQ ID NO: 19 to function as a NtGDH.
  • the activity of at least one NtGDH polypeptide is modulated.
  • the at least one NtGDH polypeptide can be a NtGDH2 polypeptide encoded by a NtGDH2 polynucleotide comprising, consisting or consisting essentially of a sequence having at least 90% sequence identity to SEQ ID NO: 1 or has at least 94% sequence identity to SEQ ID NO:2; or can be a NtGDH3 polypeptide encoded by a NtGDH3 polynucleotide comprising, consisting or consisting essentially of a sequence having at least 90% sequence identity to SEQ ID NO: 3 or has at least 94% sequence identity to SEQ ID NO: 4; or can be a NtGDH6 polypeptide encoded by a NtGDH6 polynucleotide comprising, consisting or consisting essentially of a sequence having at least 90% sequence identity to SEQ ID NO: 7 or has at least 94% sequence identity to SEQ ID NO:8; or
  • the activity of each of the NtGDH2 polypeptide and the NtGDH3 polypeptide and the NtGDH6 polypeptide and the NtGDH9 polypeptide and the NtGDH10 polypeptide and the NtGDH12 polypeptide are modulated.
  • the activity of the NtGDH4 polypeptide or the NtGDH7 polypeptide or the NtGDH8 polypeptide or the NtGDH11 polypeptide is not modulated or a combination of two or more thereof are not modulated, wherein the NtGDH4 polypeptide is encoded by a polynucleotide comprising, consisting or consisting essentially of a sequence having at least 88% sequence identity to SEQ ID NO: 5; or a NtGDH4 polypeptide having at least 92% sequence identity to SEQ ID NO:6; or wherein the NtGDH7 polypeptide is encoded by a NtGDH7 polynucleotide comprising, consisting or consisting essentially of a sequence having at least 92% sequence identity to SEQ ID NO: 9 or has at least 96% sequence identity to SEQ ID NO: 10; or the NtGDH8 polypeptide is encoded by a NtGDH8 polynucleotide comprising, consisting or consisting essentially
  • Fragment of the polypeptides described herein are also contemplated.
  • the fragments of the polypeptide(s) typically retain some or all of the function or activity of the full length sequence - such as NtGDH activity.
  • Fragments of a polypeptide may range from at least about 25 amino acids, about 50 amino acids, about 75 amino acids, about 100 amino acids about 150 amino acids, about 200 amino acids, about 250 amino acids, about 300 amino acids, about 400 amino acids, about 500 amino acids, and up to the full-length polypeptide described herein.
  • the polypeptides also include mutants produced by introducing any type of alterations (for example, insertions, deletions, or substitutions of amino acids; changes in glycosylation states; changes that affect refolding or isomerization, three-dimensional structures, or self- association states), which can be deliberately engineered or isolated naturally provided that they still have some or all of their function or activity. Suitably, this function or activity is modulated.
  • a deletion refers to removal of one or more amino acids from a polypeptide.
  • An insertion refers to one or more amino acid residues being introduced into a predetermined site in a polypeptide. Insertions may comprise intra-sequence insertions of single or multiple amino acids.
  • a substitution refers to the replacement of amino acids of the polypeptide with other amino acids having similar properties (such as similar hydrophobicity, hydrophilicity, antigenicity, propensity to form or break a-helical structures or ⁇ -sheet structures).
  • Amino acid substitutions are typically of single residues, but may be clustered depending upon functional constraints placed upon the polypeptide and may range from about 1 to about 10 amino acids. The amino acid substitutions are suitably conservative amino acid substitutions as described below. Amino acid substitutions, deletions or insertions can be made using peptide synthetic techniques - such as solid phase peptide synthesis or by recombinant DNA manipulation.
  • variants may have alterations which produce a silent change and result in a functionally equivalent polypeptide.
  • Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and the amphipathic nature of the residues as long as the secondary binding of the substance is retained.
  • negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine. Conservative substitutions may be made, for example according to the Table below.
  • polypeptide may be a mature polypeptide or an immature polypeptide or a polypeptide derived from an immature polypeptide.
  • Polypeptides may be in linear form or cyclized using known methods. Polypeptides typically comprise at least 10, at least 20, at least 30, or at least 40 contiguous amino acids.
  • At least one modification can be included in one or more of SEQ ID NO: 1 or SEQ ID NO: 3 or SEQ ID NO: 5 or SEQ ID NO: 7 or SEQ ID NO: 9 or SEQ ID NO: 11 or SEQ ID NO: 13 or SEQ ID NO: 15 or SEQ ID NO: 17 or SEQ ID NO: 19.
  • Recombinant constructs can be used to transform plants or plant cells in order to modulate polypeptide expression, function or activity.
  • a recombinant polynucleotide construct can comprise a polynucleotide encoding one or more polynucleotides as described herein, operably linked to a regulatory region suitable for expressing the polypeptide.
  • a polynucleotide can comprise a coding sequence that encodes the polypeptide as described herein.
  • Plants or plant cells in which polypeptide expression, function or activity are modulated can include mutant, non-naturally occurring, transgenic, man-made or genetically engineered plants or plant cells.
  • the transgenic plant or plant cell comprises a genome that has been altered by the stable integration of recombinant DNA.
  • Recombinant DNA includes DNA which has been genetically engineered and constructed outside of a cell and includes DNA containing naturally occurring DNA or cDNA or synthetic DNA.
  • a transgenic plant can include a plant regenerated from an originally-transformed plant cell and progeny transgenic plants from later generations or crosses of a transformed plant.
  • the transgenic modification alters the expression or function or activity of the polynucleotide or the polypeptide described herein as compared to a control plant.
  • the polypeptide encoded by a recombinant polynucleotide can be a native polypeptide, or can be heterologous to the cell.
  • the recombinant construct contains a polynucleotide that modulates expression, operably linked to a regulatory region. Examples of suitable regulatory regions are described herein. Vectors containing recombinant polynucleotide constructs such as those described herein are also provided.
  • Suitable vector backbones include, for example, those routinely used in the art such as plasmids, viruses, artificial chromosomes, bacterial artificial chromosomes, yeast artificial chromosomes, or bacteriophage artificial chromosomes.
  • Suitable expression vectors include, without limitation, plasmids and viral vectors derived from, for example, bacteriophage, baculoviruses, and retroviruses. Numerous vectors and expression systems are commercially available.
  • the vectors can include, for example, origins of replication, scaffold attachment regions or markers.
  • a marker gene can confer a selectable phenotype on a plant cell.
  • a marker can confer biocide resistance, such as resistance to an antibiotic (for example, kanamycin, G418, bleomycin, or hygromycin), or an herbicide (for example, glyphosate, chlorsulfuron or phosphinothricin).
  • an expression vector can include a tag sequence designed to facilitate manipulation or detection (for example, purification or localization) of the expressed polypeptide.
  • Tag sequences such as luciferase, beta- glucuronidase, green fluorescent polypeptide, glutathione S-transferase, polyhistidine, c-myc or hemagglutinin sequences typically are expressed as a fusion with the encoded polypeptide.
  • a plant or plant cell can be transformed by having the recombinant polynucleotide integrated into its genome to become stably transformed.
  • the plant or plant cell described herein can be stably transformed.
  • Stably transformed cells typically retain the introduced polynucleotide with each cell division.
  • a plant or plant cell can be transiently transformed such that the recombinant polynucleotide is not integrated into its genome. Transiently transformed cells typically lose all or some portion of the introduced recombinant polynucleotide with each cell division such that the introduced recombinant polynucleotide cannot be detected in daughter cells after a sufficient number of cell divisions.
  • a number of methods are available in the art for transforming a plant cell including biolistics, gene gun techniques, Agrobacterium-mediated transformation, viral vector-mediated transformation, freeze-thaw method, microparticle bombardment, direct DNA uptake, sonication, microinjection, plant virus-mediated transfer, and electroporation. If a cell or cultured tissue is used as the recipient tissue for transformation, plants can be regenerated from transformed cultures if desired, by techniques known to those skilled in the art.
  • the choice of regulatory regions to be included in a recombinant construct depends upon several factors, including, but not limited to, efficiency, selectability, inducibility, desired expression level, and cell- or tissue-preferential expression.
  • telomeres e.g., telomeres
  • exemplary promoters include tissue-specific promoters recognized by tissue-specific factors present in different tissues or cell types (for example, root-specific promoters, shoot-specific promoters, xylem-specific promoters), or present during different developmental stages, or present in response to different environmental conditions.
  • Suitable promoters include constitutive promoters that can be activated in most cell types without requiring specific inducers.
  • Examples of promoters that can be used to control polypeptide expression include the cauliflower mosaic virus 35S (CaMV/35S), SSU, OCS, lib4, usp, STLS1, B33, nos or ubiquitin- or phaseolin-promoters.
  • CaMV/35S cauliflower mosaic virus 35S
  • SSU cauliflower mosaic virus 35S
  • OCS OCS
  • lib4 usp
  • STLS1, B33 nos or ubiquitin- or phaseolin-promoters.
  • Persons skilled in the art are capable of generating multiple variations of recombinant promoters.
  • Tissue-specific promoters are transcriptional control elements that are only active in particular cells or tissues at specific times during plant development, such as in vegetative tissues or reproductive tissues.
  • tissue-specific promoters under developmental control include promoters that can initiate transcription only (or primarily only) in certain tissues, such as vegetative tissues, for example, roots or leaves, or reproductive tissues, such as fruit, ovules, seeds, pollen, pistols, flowers, or any embryonic tissue.
  • Reproductive tissue-specific promoters may be, for example, anther-specific, ovule-specific, embryo-specific, endosperm- specific, integument-specific, seed and seed coat-specific, pollen-specific, petal-specific, sepal-specific, or combinations thereof.
  • Exemplary leaf-specific promoters include pyruvate, orthophosphate dikinase (PPDK) promoter from C4 plant (maize), cab-m1Ca+2 promoter from maize, the Arabidopsis thaliana myb-related gene promoter (Atmyb5), the ribulose biphosphate carboxylase (RBCS) promoters (for example, the tomato RBCS 1, RBCS2 and RBCS3A genes expressed in leaves and light-grown seedlings, RBCS1 and RBCS2 expressed in developing tomato fruits or ribulose bisphosphate carboxylase promoter expressed almost exclusively in mesophyll cells in leaf blades and leaf sheaths at high levels).
  • PPDK orthophosphate dikinase
  • Atmyb5 the Arabidopsis thaliana myb-related gene promoter
  • RBCS ribulose biphosphate carboxylase
  • Exemplary senescence-specific promoters include a tomato promoter active during fruit ripening, senescence and abscission of leaves, a maize promoter of gene encoding a cysteine protease, the promoter of 82E4 and the promoter of SAG genes. Exemplary anther-specific promoters can be used. Exemplary root-preferred promoters known to persons skilled in the art may be selected. Exemplary seed-preferred promoters include both seed-specific promoters (those promoters active during seed development such as promoters of seed storage polypeptides) and seed-germinating promoters (those promoters active during seed germination).
  • inducible promoters include promoters responsive to pathogen attack, anaerobic conditions, elevated temperature, light, drought, cold temperature, or high salt concentration.
  • Pathogen-inducible promoters include those from pathogenesis-related polypeptides (PR polypeptides), which are induced following infection by a pathogen (for example, PR polypeptides, SAR polypeptides, beta-1,3-glucanase, chitinase).
  • promoters may be derived from bacterial origin for example, the octopine synthase promoter, the nopaline synthase promoter and other promoters derived from Ti plasmids, or may be derived from viral promoters (for example, 35S and 19S RNA promoters of cauliflower mosaic virus (CaMV), constitutive promoters of tobacco mosaic virus, cauliflower mosaic virus (CaMV) 19S and 35S promoters, or figwort mosaic virus 35S promoter).
  • CaMV cauliflower mosaic virus
  • CaMV cauliflower mosaic virus
  • CaMV constitutive promoters of tobacco mosaic virus
  • CaMV cauliflower mosaic virus
  • CaMV cauliflower mosaic virus
  • figwort mosaic virus 35S promoter figwort mosaic virus 35S promoter
  • a plant or plant cell comprising at least one genetic modification (for example, mutation) in one or more polynucleotides or polypeptides as described herein is disclosed, wherein said genetic modification results in modulated function or activity of NtGDH or the polypeptide(s) encoded thereby.
  • a method for modulating the level of a NtGDH polypeptide in a (cured) plant or in (cured) plant material comprising introducing into the genome of said plant one or more genetic modifications (for example, mutations) that modulate expression of at least one NtGDH, wherein said at least one NtGDH gene is selected from one or more of the sequences according to the present disclosure.
  • a method for identifying a cured Nicotiana plant leaf or part of the plant leaf with modulated levels of ammonia therein as compared to the level of ammonia in the control plant comprising screening a polynucleotide sample from a cured Nicotiana plant leaf or part of the plant leaf for the presence of one or more genetic modifications (for example, mutations) in the NtGDH polynucleotide sequences according to the present disclosure, and optionally correlating the identified genetic modification(s) that are known to modulate levels of ammonia in a cured Nicotiana plant leaf or part of the plant leaf.
  • the amount of amino acids and sugar and total alkaloids in the cured Nicotiana plant leaf or part of the plant leaf will also be modulated.
  • a plant or plant cell that is heterozygous or homozygous for one or more genetic modifications (for example, mutations) in a NtGDH gene according to the present disclosure, wherein said genetic modification(s) results in modulated expression of the NtGDH gene or function or activity of the NtGDH polypeptide encoded thereby.
  • a number of approaches can be used to combine genetic modification(s) (for example, mutations) in one plant including sexual crossing.
  • a plant having one or more favourable heterozygous or homozygous genetic modifications in a gene according to the present disclosure that modulates expression of the gene or the function or activity of the polypeptide encoded thereby can be crossed with a plant having one or more favourable heterozygous or homozygous genetic modifications in one or more other genes that modulate expression thereof or the function or activity of the polypeptide encoded thereby.
  • crosses are made in order to introduce one or more favourable heterozygous or homozygous genetic modifications within the gene according to the present disclosure within the same plant.
  • the function or activity of one or more polypeptides of the present disclosure in a plant is increased or decreased if the function or activity is lower or higher than the function or activity of the same polypeptide(s) in a plant that has not been modified to inhibit the function or activity of that polypeptide and which has been cultured, harvested and cured using the same protocols.
  • the genetic modification(s) is introduced into a plant or plant cell using a mutagenesis approach, and the introduced mutation is identified or selected using methods known to those of skill in the art - such as Southern blot analysis, DNA sequencing, PCR analysis, or phenotypic analysis. Mutations that impact gene expression or that interfere with the function of the encoded polypeptide can be determined using methods that are well known in the art.
  • Mutations in conserved residues can be particularly effective in inhibiting the metabolic function of the encoded polypeptide. It will be appreciated, for example, that a mutation in one or more of the highly conserved regions would likely alter polypeptide function, while a mutation outside of those highly conserved regions would likely have little to no effect on polypeptide function.
  • a mutation in a single nucleotide can create a stop codon, which would result in a truncated polypeptide and, depending on the extent of truncation, loss of function.
  • Any plant of interest including a plant cell or plant material can be genetically modified by various methods known to induce mutagenesis, including site-directed mutagenesis, oligonucleotide- directed mutagenesis, chemically-induced mutagenesis, irradiation-induced mutagenesis, mutagenesis utilizing modified bases, mutagenesis utilizing gapped duplex DNA, double- strand break mutagenesis, mutagenesis utilizing repair-deficient host strains, mutagenesis by total gene synthesis, DNA shuffling and other equivalent methods.
  • Modifications in the polynucleotides and polypeptides described herein can include man-made modifications or synthetic modifications or genetically engineered modifications.
  • Modifications in the polynucleotides and polypeptides described herein can be modifications that are obtained or obtainable via a process which includes an in vitro or an in vivo manipulation step. Modifications in the polynucleotides and polypeptides described herein can be modifications that are obtained or obtainable via a process which includes intervention by man. The function or activity of the polypeptide variant may be higher, lower or about the same as the unmodified polypeptide.
  • Methods that introduce a modification randomly in a polynucleotide can include chemical mutagenesis and radiation mutagenesis. Chemical mutagenesis involves the use of exogenously added chemicals – such as mutagenic, teratogenic, or carcinogenic organic compounds – to induce mutations.
  • Mutagens that create primarily point mutations and short deletions, insertions, missense mutations, simple sequence repeats, transversions ortransitions, including chemical mutagens or radiation, may be used to create the mutations.
  • Mutagens include ethyl methanesulfonate, methylmethane sulfonate, N-ethyl-N-nitrosurea, triethylmelamine, N-methyl-N-nitrosourea, procarbazine, chlorambucil, cyclophosphamide, diethyl sulfate, acrylamide monomer, melphalan, nitrogen mustard, vincristine, dimethylnitrosamine, N-methyl-N'-nitro-Nitrosoguanidine, nitrosoguanidine, 2-aminopurine, 7,12 dimethyl-benz(a)anthracene, ethylene oxide, hexamethylphosphoramide, bisulfan, diepoxyalkanes (diepoxyoctan
  • Suitable mutagenic agents can also include, for example, ionising radiation – such as X-rays, gamma rays, fast neutron irradiation and UV radiation.
  • the dosage of the mutagenic chemical or radiation is determined experimentally for each type of plant tissue such that a mutation frequency is obtained that is below a threshold level characterized by lethality or reproductive sterility. Any method of plant polynucleotide preparation known to those of skill in the art may be used to prepare the plant polynucleotide for mutation screening.
  • the mutation process may include one or more plant crossing steps.
  • screening can be performed to identify mutations that create premature stop codons or otherwise non-functional genes. After mutation, screening can be performed to identify mutations that create functional genes that are capable of being expressed at increased or decreased levels. Screening of mutants can be carried out by sequencing, or by the use of one or more probes or primers specific to the gene or polypeptide. Specific mutations in polynucleotides can also be created that can result in modulated gene expression, modulated stability of mRNA, or modulated stability of polypeptide. Such plants are referred to herein as "non-naturally occurring" or "mutant" plants.
  • the mutant or non-naturally occurring plants will include at least a portion of foreign or synthetic or man- made nucleotide (for example, DNA or RNA) that was not present in the plant before it was manipulated.
  • the foreign nucleotide may be a single nucleotide, two or more nucleotides, two or more contiguous nucleotides or two or more non-contiguous nucleotides – such as at least 10, 20, 30, 40, 50,100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400 or 1500 or more contiguous or non-contiguous nucleotides.
  • Zinc finger polypeptides can be used to modulate the expression or function or activity of the one or more NtGDH polynucleotides described herein.
  • the use of zinc finger nucleases is described in Nature Rev. Genet. (2010) 11 (9): 636-646).
  • Meganucleases such as I-CreI, can be used to modulate the expression or function or activity of one or more of the NtGDH 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.
  • TALENs Transcription activator-like effector nucleases
  • the use of TALENs is described in Nature Rev. Mol. Cell Biol. (2013) 14: 49-55 and Int J Mol Sci. (2019) 20(16), 4045.
  • the CRISPR system can be used to modulate the expression or function or activity of one or more of the NtGDH polynucleotides described herein and is a preferred method. This technology is described in, for example, Plant Methods (2016) 12:8; Front Plant Sci.
  • the CRISPR editing system generally includes two components: a CRISPR-associated endonuclease (Cas) (for example, Cas9) and a guide RNA (gRNA).
  • Cas forms a double stranded DNA break at a site in the genome that is defined by the sequence of a gRNA molecule bound to Cas. The location at which Cas breaks the DNA is defined by the unique sequence of the gRNA that is bound to it.
  • gRNA is a specifically designed RNA sequence that recognizes the target DNA region of interest and directs the Cas nuclease there for editing. It has two sections: (i) a tracr RNA, which serves as a binding scaffold for the Cas nuclease; and (ii) crispr RNA (crRNA), a 17-20 nucleotide sequence complementary to the target DNA.
  • crRNA crispr RNA
  • the exact region of the DNA to be targeted will depend on the specific application. For example, to activate or repress a target polynucleotide, gRNAs can be targeted to the promoter driving expression of the target polynucleotide. Methods for designing gRNAs are well known in the art, including Chop Chop Harvard.
  • RNA-guided nucleases for use in the CRISPR system have been described, including, Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, CaslO, Cpfl, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3 and Csf4.
  • the present disclosure further provides a CRISPR based genome editing system comprising an RNA-guided nuclease and a gRNA, where the CRISPR based genome editing system modulates the activity of one or more of the polynucleotides described herein.
  • the present disclosure also provides a method of cleaving one or more polynucleotides in a plant cell, comprising introducing a gRNA and an RNA-guided nuclease into the plant cell, wherein the gRNA acts in association with the RNA-guided nuclease to create a strand break in one or more of the polynucleotides described herein.
  • a CRISPR construct comprising: (i) a polynucleotide encoding a CRISPR-associated endonuclease; and (ii) a gRNA including a polynucleotide sequence (typically of about 17-20 nucleotides) complementary to the DNA of the polynucleotide as described herein that is to be targeted.
  • Antisense technology is another well-known method that can be used to modulate the expression or activity of one or more NtGDH polypeptides described herein. See, for example, Gene (1988) 10;72(1-2):45-50.
  • NtGDH polynucleotides can be targeted for inactivation by introducing transposons (for example, IS elements or other mobile genetic elements) into the genomes of plants of interest. See, for example, Cytology and Genetics (2006) 40(4):68-81. NtGDH polynucleotides can be targeted for inactivation by introducing ribozymes derived from a number of small circular RNAs that are capable of self-cleavage and replication in plants. See, for example, FEMS Microbiology Reviews (1999) 23, 3, 257–275.
  • the mutant or non-naturally occurring plants or plant cells can have any combination of one or more modifications (for example, mutations) in one or more of the NtGDH polynucleotides described herein which result in modulated expression or function or activity of those polynucleotides or their polynucleotide products.
  • the mutant or non-naturally occurring plants or plant cells may have a single modification in a single NtGDH polynucleotide or NtGDH polypeptide; multiple modifications in a single NtGDH polynucleotide or NtGDH polypeptide; a single modification in two or more NtGDH polynucleotides or NtGDH polypeptides; or multiple modifications in two or more NtGDH polynucleotides or NtGDH polypeptides.
  • the mutant or non-naturally occurring plants or plant cells may have one or more modifications in a specific portion of NtGDH polynucleotide(s) or NtGDH polypeptide(s) – such as in a region of NtGDH that encodes an active site of the NtGDH polypeptide or a portion thereof.
  • the mutant or non-naturally occurring plants or plant cells may have one or more modifications in a region outside of one or more NtGDH polynucleotide(s) or NtGDH polypeptide(s) – such as in a region upstream or downstream of the NtGDH polynucleotide(s) provided that it regulates the function or expression of the NtGDH.
  • Upstream elements can include promoters, enhancers or transcription factors. Some elements – such as enhancers – can be positioned upstream or downstream of the gene it regulates. The element(s) need not be located near to the gene that it regulates since some elements have been found located several hundred thousand base pairs upstream or downstream of the gene that it regulates.
  • the mutant or non-naturally occurring plants or plant cells may have one or more modifications located within the first 100 nucleotides of the gene(s), within the first 200 nucleotides of the gene(s), within the first 300 nucleotides of the gene(s), within the first 400 nucleotides of the gene(s), within the first 500 nucleotides of the gene(s), within the first 600 nucleotides of the gene(s), within the first 700 nucleotides of the gene(s), within the first 800 nucleotides of the gene(s), within the first 900 nucleotides of the gene(s), within the first 1000 nucleotides of the gene(s), within the first 1100 nucleotides of the gene(s), within the first 1200 nucleotides of the gene(s), within the first 1300 nucleotides of the gene(s), within the first 1400 nucleotides of the gene(s) or within the first 1500 nucleotides of the gene(s).
  • the mutant or non-naturally occurring plants or plant cells may have one or more modifications located within the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth or fifteenth set of 100 nucleotides of the gene(s) or combinations thereof.
  • Mutant or non-naturally occurring plants or plant cells comprising the mutant polypeptide variants are disclosed.
  • seeds from plants are mutagenised and then grown into first generation mutant plants.
  • the first generation plants are then allowed to self-pollinate and seeds from the first generation plant are grown into second generation plants, which are then screened for mutations in their loci.
  • the mutagenized plant material can be screened for mutations, an advantage of screening the second generation plants is that all somatic mutations correspond to germline mutations.
  • plant materials including but not limited to, seeds, pollen, plant tissue or plant cells, may be mutagenised in order to create the mutant plants.
  • the type of plant material mutagenised may affect when the plant polynucleotide is screened for mutations. For example, when pollen is subjected to mutagenesis prior to pollination of a non-mutagenized plant the seeds resulting from that pollination are grown into first generation plants.
  • Every cell of the first generation plants will contain mutations created in the pollen; thus these first generation plants may then be screened for mutations instead of waiting until the second generation.
  • Prepared NtGDH polynucleotides from individual plants, plant cells, or plant material can optionally be pooled in order to expedite screening for mutations in the population of plants originating from the mutagenized plant tissue, cells or material.
  • One or more subsequent generations of plants, plant cells or plant material can be screened.
  • the size of the optionally pooled group is dependent upon the sensitivity of the screening method used. After the samples are optionally pooled, they can be subjected to polynucleotide-specific amplification techniques, such as PCR.
  • any one or more primers or probes specific to the gene or the sequences immediately adjacent to the gene may be utilized to amplify the sequences within the optionally pooled sample.
  • the one or more primers or probes are designed to amplify the regions of the locus where useful mutations are most likely to arise.
  • the primer is designed to detect mutations within regions of the polynucleotide.
  • the one or more primers or probes may be labelled using any conventional labelling method.
  • Primer(s) or probe(s) can be designed based upon the sequences described herein using methods that are well understood in the art.
  • the primer(s) or probe(s) may be labelled using any conventional labelling method. These can be designed based upon the sequences described herein using methods that are well understood in the art. Polymorphisms may be identified by means known in the art and some have been described in the literature.
  • a plant may be regenerated or grown from the plant, plant tissue or plant cell. Any suitable methods for regenerating or growing a plant from a plant cell or plant tissue may be used, such as, without limitation, tissue culture or regeneration from protoplasts.
  • plants may be regenerated by growing transformed plant cells on callus induction media, shoot induction media or root induction media. See, for example, McCormick et al., Plant Cell Reports (1986) 5:81-84. These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting hybrid having expression of the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure expression of the desired phenotypic characteristic has been achieved.
  • "transformed seeds” refers to seeds that contain the nucleotide construct stably integrated into the plant genome.
  • a method of preparing a mutant plant involves providing at least one cell of a plant comprising one or more NtGDH genes encoding a functional NtGDH. Next, the at least one cell of the plant is treated under conditions effective to modulate the function of the NtGDH polynucleotide(s). The at least one mutant plant cell is then propagated into a mutant plant, where the mutant plant has modulated levels of NtGDH polypeptide(s) described herein as compared to that of a control plant.
  • the treating step involves subjecting at least one cell to a chemical mutagenising agent as described above and under conditions effective to yield at least one mutant plant cell.
  • the treating step involves subjecting the at least one cell to a radiation source under conditions effective to yield at least one mutant plant cell.
  • mutant plant includes mutant plants in which the genotype is modified as compared to a control plant, suitably by means other than genetic engineering or genetic modification.
  • the mutant plant, mutant plant cell or mutant plant material may comprise one or more mutations that have occurred naturally in another plant, plant cell or plant material and confer a desired trait. This mutation can be incorporated (for example, introgressed) into another plant, plant cell or plant material (for example, a plant, plant cell or plant material with a different genetic background to the plant from which the mutation was derived) to confer the trait thereto.
  • a mutation that occurred naturally in a first plant may be introduced into a second plant – such as a second plant with a different genetic background to the first plant.
  • the skilled person is therefore able to search for and identify a plant carrying naturally in its genome one or more mutant alleles of the genes described herein which confer a desired trait.
  • the mutant allele(s) that occurs naturally can be transferred to the second plant by various methods including breeding, backcrossing and introgression to produce a lines, varieties or hybrids that have one or more mutations in the genes described herein.
  • the same technique can also be applied to the introgression of one or more non-naturally occurring mutation(s) from a first plant into a second plant.
  • Plants showing a desired trait may be screened out of a pool of mutant plants.
  • the selection is carried out utilising the knowledge of the polynucleotide as described herein. Consequently, it is possible to screen for a genetic trait as compared to a control.
  • Such a screening approach may involve the application of conventional amplification or hybridization techniques as discussed herein.
  • a further aspect of the present disclosure relates to a method for identifying a mutant plant comprising: (a) providing a sample comprising one or more NtGDH polynucleotide(s) from a plant; and (b) determining the sequence of the polynucleotide(s), wherein a difference in the sequence of the polynucleotide(s) as compared to the polynucleotide(s) of a control plant is indicative that said plant is a mutant plant.
  • a method for identifying a mutant plant which accumulates increased or decreased levels of ammonia as compared to a control plant comprising: (a) providing a sample from a plant to be screened; (b) determining if said sample comprises one or more mutations in one or more NtGDH polynucleotides described herein; and (c) determining the level of ammonia in said plant.
  • the level of ammonia is determined in cured leaves.
  • the level of amino acids and sugar and total alkaloids is also determined in cured leaves.
  • a method for preparing a mutant plant which has increased or decreased levels of ammonia - as compared to a control plant comprising: (a) providing a sample from a first plant; (b) determining if said sample comprises one or more mutations in one or more NtGDH polynucleotides described herein that result in modulated levels of ammonia; and (c) transferring the one or more mutations into a second plant.
  • the level of ammonia is determined in cured leaves.
  • the level of amino acids and sugar and total alkaloids is also determined in cured leaves.
  • the mutation(s) can be transferred into the second plant using various methods that are known in the art – such as by genetic engineering, genetic manipulation, introgression, plant breeding, backcrossing and the like.
  • the first plant is a naturally occurring plant.
  • the second plant has a different genetic background to the first plant.
  • a method for preparing a mutant plant which has increased or decreased levels of ammonia as compared to a control plant comprising: (a) providing a sample from a first plant; (b) determining if said sample comprises one or more mutations in one or more of the NtGDH polynucleotides described herein that results in modulated levels of ammonia; and (c) introgressing the one or more mutations from the first plant into a second plant.
  • the level of ammonia is determined in cured leaves.
  • the level of amino acids and sugar and total alkaloids is also determined in cured leaves.
  • the step of introgressing comprises plant breeding, optionally including backcrossing and the like.
  • the first plant is a naturally occurring plant.
  • the second plant has a different genetic background to the first plant.
  • the first plant is not a cultivar or an elite cultivar.
  • the second plant is a cultivar or an elite cultivar.
  • a further aspect relates to a mutant plant (including a cultivar or elite cultivar mutant plant) obtained or obtainable by the methods described herein.
  • the mutant plant may have one or more mutations localised only to a specific region of the plant – such as within the sequence of the one or more NtGDH polynucleotide(s) described herein. According to this embodiment, the remaining genomic sequence of the mutant plant will be the same or substantially the same as the plant prior to the mutagenesis.
  • the mutant plants may have one or more mutations localised in more than one genomic region of the plant – such as within the sequence of one or more of the NtGDH polynucleotides described herein and in one or more further regions of the genome. According to this embodiment, the remaining genomic sequence of the mutant plant will not be the same or will not be substantially the same as the plant prior to the mutagenesis.
  • the mutant plants may not have one or more mutations in one or more, two or more, three or more, four or more or five or more exons of the NtGDH polynucleotide(s) described herein; or may not have one or more mutations in one or more, two or more, three or more, four or more or five or more introns of the NtGDH polynucleotide(s) described herein; or may not have one or more mutations in a promoter of the NtGDH polynucleotide(s) described herein; or may not have one or more mutations in the 3’ untranslated region of the NtGDH polynucleotide(s) described herein; or may not have one or more mutations in the 5’ untranslated region of the NtGDH polynucleotide(s) described herein; or may not have one or more mutations in the coding region of the NtGDH polynucleotide(s) described herein; or may be
  • a method of identifying a plant, a plant cell or plant material comprising a mutation in a gene encoding a NtGDH polynucleotide described herein comprising: (a) subjecting a plant, a plant cell or plant material to mutagenesis; (b) obtaining a sample from said plant, plant cell or plant material or descendants thereof; and (c) determining the polynucleotide sequence of the NtGDH gene(s) or a variant or a fragment thereof, wherein a difference in said sequence is indicative of one or more mutations therein.
  • This method also allows the selection of plants having mutation(s) that occur(s) in genomic regions that affect the expression of the NtGDH gene in a plant cell, such as a transcription initiation site, a start codon, a region of an intron, a boundary of an exon-intron, a terminator, Plants suitable for use in the present disclosure include monocotyledonous and dicotyledonous plants and plant cell systems and include members of the genera Nicotiana. Various embodiments are directed to mutant tobacco, non-naturally occurring tobacco or transgenic tobacco plants or tobacco plant cells and can be applied to any species of the genus Nicotiana, including N. rustica and N.
  • N. acaulis for example, 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, N. benavidesii, N. benthamiana, N. bigelovii, N. bonariensis, N. cavicola, N. clevelandii, N. cordifolia, N. corymbosa, N. debneyi, N. excelsior, N.
  • the plant is N. tabacum.
  • the use of tobacco cultivars and elite tobacco cultivars is also contemplated herein.
  • the transgenic, non-naturally occurring or mutant plant may therefore be a tobacco variety or elite tobacco cultivar that comprises one or more transgenes, or one or more genetic mutations or a combination thereof.
  • the genetic mutation(s) (for example, one or more polymorphisms) can be mutations that do not exist naturally in the individual tobacco variety or tobacco cultivar (for example, elite tobacco cultivar) or can be genetic mutation(s) that do occur naturally provided that the mutation does not occur naturally in the individual tobacco variety or tobacco cultivar (for example, elite tobacco cultivar).
  • Nicotiana tabacum varieties include Burley type, dark type, flue-cured type, and Oriental type tobaccos.
  • varieties or cultivars are: BD 64, CC 101, CC 200, CC 27, CC 301, CC 400, CC 500, CC 600, CC 700, CC 800, CC 900, Coker 176, Coker 319, Coker 371 Gold, Coker 48, CD 263, DF911, DT 538 LC Galpao tobacco, GL 26H, GL 350, GL 600, GL 737, GL 939, GL 973, HB 04P, HB 04P LC, HB3307PLC, Hybrid 403LC, Hybrid 404LC, Hybrid 501 LC, K 149, K 326, K 346, K 358, K394, K 399, K 730, KDH 959, KT 200, KT204LC, KY10, KY14, KY 160, KY 17, KY 171, KY 907, KY
  • Embodiments are also directed to compositions and methods for producing mutant plants, non-naturally occurring plants, hybrid plants, or transgenic plants that have been modified to modulate the expression or function of one or more NtGDH polynucleotide(s) described herein (or any combination thereof as described herein).
  • the mutant plants, non- naturally occurring plants, hybrid plants, or transgenic plants that are obtained may be similar or substantially the same in overall appearance to control plants.
  • Various phenotypic characteristics such as degree of maturity, number of leaves per plant, stalk height, leaf insertion angle, leaf size (width and length), internode distance, and lamina-midrib ratio can be assessed by field observations.
  • One aspect relates to a seed of a mutant plant, a non-naturally occurring plant, a hybrid plant or a transgenic plant described herein.
  • the seed is a tobacco seed.
  • a further aspect relates to pollen or an ovule of a mutant plant, a non-naturally occurring plant, a hybrid plant or a transgenic plant that is described herein.
  • a mutant plant, a non-naturally occurring plant, a hybrid plant or a transgenic plant as described herein which further comprises a polynucleotide conferring male sterility.
  • the regenerable cells include cells from leaves, pollen, embryos, cotyledons, hypocotyls, roots, root tips, anthers, flowers and a part thereof, ovules, shoots, stems, stalks, pith and capsules or callus or protoplasts derived therefrom.
  • the plant material that is described herein can be cured tobacco material.
  • the CORESTA recommendation for tobacco curing is described in: CORESTA Guide No. 17, April 2016, Sustainability in Leaf Tobacco Production.
  • the cured tobacco material can be fully cured tobacco material or it can be tobacco material that has been cured for a period time – such as for at least 24 hours, for at least 48 hours, for at least 96 hours or for at least 192 hours.
  • a method of preparing a cured Nicotiana plant leaf or a part of the plant leaf with modulated levels of ammonia and amino acids and sugar and total alkaloids as compared to a Nicotiana plant leaf or a part of the plant leaf from a control cured Nicotiana plant comprising the steps of: (a) providing a Nicotiana plant comprising a NtGDH that comprises, consists, or consists essentially of at least one of a NtGDH2 polynucleotide or a NtGDH3 polynucleotide or a NtGDH6 polynucleotide or a NtGDH9 polynucleotide or a NtGDH10 polynucleotide or
  • step (b) the expression of the NtGDH6 polynucleotide and the NtGDH10 polynucleotide are modulated or the activity of the NtGDH6 polypeptide and the NtGDH10 polypeptide are modulated.
  • the expression of the NtGDH2 polynucleotide and the NtGDH3 polynucleotide are modulated or the activity of both the NtGDH2 polypeptide and the NtGDH3 polypeptide are modulated.
  • the expression of the NtGDH9 polynucleotide and the NtGDH12 polynucleotide are modulated or the activity of the NtGDH9 polypeptide and the NtGDH12 polypeptide are modulated.
  • the expression of each of the NtGDH2 polynucleotide and the NtGDH3 polynucleotide and the NtGDH9 polynucleotide and the NtGDH12 polynucleotide are modulated or the activity of each of the NtGDH2 polypeptide and the NtGDH3 polypeptide and the NtGDH9 polypeptide and the NtGDH12 polypeptide are modulated.
  • the expression of each of the NtGDH2 polynucleotide and the NtGDH3 polynucleotide and the NtGDH6 polynucleotide and the NtGDH9 polynucleotide and the NtGDH10 polynucleotide and the NtGDH12 polynucleotide or the NtGDH2 polypeptide and the NtGDH3 polypeptide and the NtGDH6 polypeptide and the NtGDH9 polypeptide and the NtGDH10 polypeptide and the NtGDH12 polypeptide are modulated.
  • the expression of one or more of a NtGDH4 polynucleotide or a NtGDH7 polynucleotide or a NtGDH8 polynucleotide or a NtGDH11 polynucleotide or the activity of a NtGDH4 polypeptide or a NtGDH7 polypeptide or a NtGDH8 polypeptide or a NtGDH11 polypeptide is not modulated, wherein (i) the NtGDH4 polynucleotide comprises, consists or consists essentially of a sequence having at least 88% sequence identity to SEQ ID NO: 5; or (ii) the NtGDH4 polypeptide is encoded by the polynucleotide set forth in (i); or (iii) the NtGDH4 polypeptide has at least 92% sequence identity to SEQ ID NO:6; or (iv) the NtGDH7 polynucleotide comprises, consists or consists
  • the plant leaf or part of the plant leaf is cured until it is fully cured or is cured for a period time – such as for at least 24 hours, for at least 48 hours, for at least 96 hours or for at least 192 hours.
  • the mutant, transgenic or non-naturally occurring plants or parts thereof of the present disclosure exhibit modulated levels of ammonia in the plant material, for example, in cured leaves.
  • modulated level of amino acids and sugar and total alkaloids are also exhibited in the plant material, for example, in cured leaves.
  • the modulated levels ammonia, and optionally, amino acids and sugar and total alkaloids are observed in at least cured leaves, suitably fully cured leaves.
  • Tobacco is considered to be fully cured when the leaf's central rib is free of moisture, resulting in leaves that are light tan to reddish-brown to deep brown in colour.
  • the cured leaves are taken from mid-position leaves of a plant.
  • the level of glucose is also modulated as compared to a control plant or part thereof.
  • the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10 and NtGDH12 is decreased then the level of glucose is increased.
  • the level of glucose is decreased.
  • the level of fructose is also modulated as compared to a control plant or part thereof.
  • the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10 and NtGDH12 is decreased then the level of fructose is increased.
  • the level of fructose is decreased.
  • the level of sucrose is also modulated as compared to a control plant or part thereof.
  • the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10 and NtGDH12 is decreased then the level of sucrose is increased.
  • the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10 and NtGDH12 is increased then the level of sucrose is decreased.
  • the sum of sugars is also modulated as compared to a control plant or part thereof.
  • the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10 and NtGDH12 is decreased then the level of the sums of sugars is increased.
  • the level of nitrate is also modulated as compared to a control plant or part thereof.
  • the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10 and NtGDH12 is decreased then the level of nitrate is decreased.
  • the level of total alkaloids is also modulated as compared to a control plant or part thereof.
  • the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10 and NtGDH12 is decreased then the level of total alkaloids is increased.
  • the level of total alkaloids is decreased.
  • the level of total free amino acids is also modulated as compared to a control plant or part thereof.
  • the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10 and NtGDH12 is decreased then the level of total free amino acids is increased.
  • the level of aspartic acid is also modulated as compared to a control plant or part thereof.
  • the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10 and NtGDH12 is decreased then the level of aspartic acid is increased.
  • the level of aspartic acid is decreased.
  • the level of asparagine is also modulated as compared to a control plant or part thereof.
  • the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10 and NtGDH12 is decreased then the level of asparagine is increased.
  • the level of asparagine is decreased.
  • the level of glutamic acid is also modulated as compared to a control plant or part thereof.
  • the level of glutamine is also modulated as compared to a control plant or part thereof.
  • the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10 and NtGDH12 is decreased then the level of glutamine is increased.
  • the level of proline is also modulated as compared to a control plant or part thereof.
  • the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10 and NtGDH12 is decreased then the level of proline is increased.
  • the level of glutamine is decreased.
  • the level of serine is also modulated as compared to a control plant or part thereof.
  • the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10 and NtGDH12 is decreased then the level of serine is increased.
  • the level of glutamine is decreased.
  • the level of threonine is also modulated as compared to a control plant or part thereof.
  • the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10 and NtGDH12 is decreased then the level of threonine is increased.
  • the level of lysine is also modulated as compared to a control plant or part thereof.
  • the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10 and NtGDH12 is decreased then the level of lysine is increased.
  • the level of arginine is also modulated as compared to a control plant or part thereof.
  • the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10 and NtGDH12 is decreased then the level of arginine is increased.
  • the level of isoleucine is also modulated as compared to a control plant or part thereof.
  • the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10 and NtGDH12 is decreased then the level of isoleucine is decreased.
  • the level of histidine is also modulated as compared to a control plant or part thereof.
  • the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10 and NtGDH12 is decreased then the level of histidine is decreased.
  • the level of histidine is increased.
  • the level of methionine is also modulated as compared to a control plant or part thereof.
  • the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10 and NtGDH12 is decreased then the level of methionine is decreased.
  • the level of citrulline is also modulated as compared to a control plant or part thereof.
  • the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10 and NtGDH12 is decreased then the level of citrulline is decreased.
  • the level of tyrosine is decreased.
  • the level of valine is not modulated as compared to a control plant or part thereof.
  • the level of tryptophan is also modulated as compared to a control plant or part thereof.
  • the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10 and NtGDH12 is decreased then the level of tryptophan is decreased.
  • the level of tryptophan is increased.
  • the level of alanine is also modulated as compared to a control plant or part thereof.
  • the level of GABA is also modulated as compared to a control plant or part thereof.
  • the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10 and NtGDH12 is decreased then the level of GABA is decreased.
  • the level of GABA is increased.
  • the level of phenylalanine is also modulated as compared to a control plant or part thereof.
  • the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10 and NtGDH12 is decreased then the level of phenylalanine is decreased.
  • the level of glycine is also modulated as compared to a control plant or part thereof.
  • the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10 and NtGDH12 is decreased then the level of glycine is decreased.
  • the level of glycine is increased.
  • the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10 and NtGDH12 is decreased then the level of glucose, fructose, sucrose, sum of sugars, total free amino acids, aspartic acid, asparagine, glutamine, proline, serine, threonine, lysine, arginine and tyrosine is increased.
  • NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10 and NtGDH12 is increased then the level of glucose, fructose, sucrose, sum of sugars, total free amino acids, aspartic acid, asparagine, glutamine, proline, serine, threonine, lysine, arginine and tyrosine is decreased.
  • NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10 and NtGDH12 is decreased then the level of ammonia, nitrate, total alkaloids, isoleucine, histidine, methionine, citrulline, tryptophan, GABA, phenylalanineand glycine is decreased.
  • NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10 and NtGDH12 is increased then the level of ammonia, nitrate, total alkaloids, isoleucine, histidine, methionine, citrulline, tryptophan, GABA, phenylalanine and glycine is increased.
  • a further aspect of the present disclosure relates to cured Nicotiana plant leaf or a part of the cured plant leaf in which the ammonia content is between about 0.16 ⁇ 0.04 % DWB to 0.11 0.16 ⁇ 0.03 % DWB.
  • a further aspect of the present disclosure relates to cured Nicotiana plant leaf or a part of the cured plant leaf in which the ammonia content is between about 0.16 ⁇ 0.04 % DWB to 0.11 0.16 ⁇ 0.03 % DWB; and (ii) the glucose, fructose and sucrose content is from 0.51 ⁇ 0.58 % DWB to 1.55 ⁇ 1.10 % DWB; and (iii) the total free amino acid content is from 51.0 ⁇ 6.60 mg/g DWB to 60.1 ⁇ 4.58 mg/g DWB; and (iv) the total alkaloid content is from 2.24 ⁇ 0.8 % DWB to 4.20 ⁇ 0.39 %.
  • a further aspect relates to a mutant, non-naturally occurring or transgenic plant or cell in which the expression of one or more NtGDH polynucleotides or the activity of one or more NtGDH polypeptide(s) has been decreased, that has decreased levels of ammonia as compared to a control plant or part thereof in which the expression of NtGDH or the activity of NtGDH has not been decreased.
  • a still further aspect relates to cured plant material – such as cured leaf or cured tobacco - derived or derivable from the mutant, non-naturally occurring or transgenic plant or cell, wherein expression of one or more of the one or more NtGDH polynucleotides described herein or the function of the NtGDH polypeptide(s) encoded thereby is decreased, and wherein the level of ammonia decreased, as compared to a control plant or part thereof.
  • Embodiments are also directed to compositions and methods for producing mutant, non- naturally occurring or transgenic plants or plant cells that have been modified to modulate, suitably decrease, the expression or activity of one or more of the NtGDH polynucleotides or NtGDH polypeptides described herein which can result in plants or plant parts (for example, leaves – such as cured leaves) or plant cells with modulated, suitably decreased ammonia content.
  • the phenotype of the mutant, non-naturally occurring or transgenic plant is substantially the same as the control plant or part thereof.
  • the leaf weight of the mutant, non-naturally occurring or transgenic plant is substantially the same as the control plant or part thereof.
  • the leaf number of the mutant, non- naturally occurring or transgenic plant is substantially the same as the control plant or part thereof.
  • the leaf weight and the leaf number of the mutant, non-naturally occurring or transgenic plant is substantially the same as the control plant.
  • the stalk height of the mutant, non-naturally occurring or transgenic plants is substantially the same as the control plants or parts thereof at, for example, one, two or three or more months after field transplant or 10, 20, 30 or 36 or more days after topping.
  • the stalk height of the mutant, non-naturally occurring or transgenic plants is not less than the stalk height of the control plants or parts thereof.
  • a method for modulating the amount of ammonia in at least a part of a plant comprising: (i) modulating the expression or function of an one or more of the NtGDH polypeptides described herein, suitably, wherein the NtGDH polypeptide(s) is encoded by the corresponding NtGDH polynucleotides described herein; (ii) measuring the level of ammonia in at least a part (for example, the leaves – such as cured leaves) of the mutant, non-naturally occurring or transgenic plant obtained in step (i); and (iii) identifying a mutant, non-naturally occurring or transgenic plant or part thereof in which the level of ammonia has been modulated in comparison to a control plant or part thereof.
  • the levels of one or more of amino acids and sugars and total alkaloids is also modulated.
  • a method for modulating the amount of at least one amino acid in cured plant material – such as cured leaf - comprising: (i) modulating the expression or function of an one or more of the NtGDH polypeptides (or any combination thereof as described herein), suitably, wherein the NtGDH polypeptide(s) is encoded by the corresponding NtGDH polynucleotides described herein; (ii) harvesting plant material – such as one or more of the leaves - and curing for a period of time; (iii) measuring the level of the ammonia in cured plant material obtained in step (ii) or during step (ii); and (iv) identifying cured plant material in which the level of ammonia has been modulated in comparison to a control plant or part thereof.
  • the levels of one or more of amino acids and sugars and total alkaloids is also modulated.
  • An increase in expression as compared to the control may be from about 5 % to about 100 %, or an increase of at least 10 %, at least 20 %, at least 25 %, at least 30 %, at least 40 %, at least 50 %, at least 60 %, at least 70 %, at least 75 %, at least 80 %, at least 90 %, at least 95 %, at least 98 %, or 100 % or more – such as 200%, 300%, 500%, 1000% or more, which includes an increase in transcriptional function or NtGDH polynucleotide expression or NtGDH polypeptide expression.
  • An increase in function or activity as compared to a control may be from about 5 % to about 100 %, or an increase of at least 10 %, at least 20 %, at least 25 %, at least 30 %, at least 40 %, at least 50 %, at least 60 %, at least 70 %, at least 75 %, at least 80 %, at least 90 %, at least 95 %, at least 98 %, or 100 % or more - such as 200%, 300%, 500%, 1000% or more, which includes an increase in transcriptional function or NtGDH polynucleotide expression or NtGDH polypeptide expression or a combination thereof.
  • a decrease in expression as compared to a control may be from about 5 % to about 100 %, or a decrease of at least 10 %, at least 20 %, at least 25 %, at least 30 %, at least 40 %, at least 50 %, at least 60 %, at least 70 %, at least 75 %, at least 80 %, at least 90 %, at least 95 %, at least 98 %, or 100 %, which includes a decrease in transcriptional function or NtGDH polynucleotide expression or NtGDH polypeptide expression or a combination thereof.
  • a decrease in function or activity as compared to a control may be from about 5 % to about 100 %, or a decrease of at least 10 %, at least 20 %, at least 25 %, at least 30 %, at least 40 %, at least 50 %, at least 60 %, at least 70 %, at least 75 %, at least 80 %, at least 90 %, at least 95 %, at least 98 %, or 100 %, which includes a decrease in transcriptional function or NtGDH polynucleotide expression or NtGDH polypeptide expression or a combination thereof.
  • Polynucleotides and recombinant constructs described herein can be used to modulate the expression or function or activity of the NtGDH polynucleotides or NtGDH polypeptides described herein in a plant species of interest, suitably tobacco.
  • a number of polynucleotide based methods can be used to increase gene expression in plants and plant cells.
  • a construct, vector or expression vector that is compatible with the plant to be transformed can be prepared which comprises the gene of interest together with an upstream promoter that is capable of overexpressing the gene in the plant or plant cell. Exemplary promoters are described herein.
  • the promoter can drive expression in order to modulate the levels of NtGDH in the plant, or in a specific tissue thereof.
  • a vector carrying one or more NtGDH polynucleotides described herein (or any combination thereof as described herein) is generated to overexpress the gene in a plant or plant cell.
  • the vector carries a suitable promoter – such as the cauliflower mosaic virus CaMV 35S promoter - upstream of the transgene driving its constitutive expression in all tissues of the plant.
  • the vector also carries an antibiotic resistance gene in order to confer selection of the transformed calli and cell lines.
  • the expression of sequences from promoters can be enhanced by including expression control sequences, which are well known in the art.
  • Various embodiments are therefore directed to methods for modulating the expression level of one or more NtGDH polynucleotides described herein (or any combination thereof as described herein) by integrating multiple copies of the NtGDH polynucleotide(s) into a plant genome, comprising: transforming a plant cell host with an expression vector that comprises a promoter operably-linked to one or more NtGDH polynucleotides described herein.
  • the polypeptide encoded by a recombinant polynucleotide can be a native polypeptide, or can be heterologous to the cell.
  • the plant for use in the present disclosure is a mutant, non-naturally occurring or transgenic plant that is flue-cured. In one embodiment, the plant for use in the present disclosure is a mutant, non-naturally occurring or transgenic plant that is sun-cured. In one embodiment, the plant for use in the present disclosure is a mutant, non-naturally occurring or transgenic plant that is air-cured. In one embodiment, the plant for use in the present disclosure is a mutant, non-naturally occurring or transgenic Virginia tobacco plant that is cured, for example, flue-cured. In one embodiment, the plant for use in the present disclosure is a mutant, non-naturally occurring or transgenic Burley tobacco plant that is cured, for example, air-cured.
  • the plant for use in the present disclosure is a mutant, non-naturally occurring or transgenic Dark tobacco plant that is cured, for example, fire-cured.
  • the sensory profile of tobacco can be modified.
  • modified Burely tobacco has a decreased harshness, makes the aerosol more round and smooth with less typical dark notes, but with more animalic and nutty notes (less trigeminals impact).
  • a plant carrying a mutant allele of one or more NtGDH polynucleotides described herein can be used in a plant breeding program to create useful lines, varieties and hybrids. For example, a mutant allele can be introgressed into commercially important varieties described herein.
  • methods for breeding plants comprise crossing a mutant plant, a non- naturally occurring plant or a transgenic plant as described herein with a plant comprising a different genetic identity.
  • the method may further comprise crossing the progeny plant with another plant, and optionally repeating the crossing until a progeny with the desirable genetic traits or genetic background is obtained.
  • One purpose served by such breeding methods is to introduce a desirable genetic trait into other varieties, breeding lines, hybrids or cultivars, particularly those that are of commercial interest.
  • Another purpose is to facilitate stacking of genetic modifications of different genes in a single plant variety, lines, hybrids or cultivars. Intraspecific as well as interspecific matings are contemplated.
  • a method for producing a non-naturally occurring plant comprising: (a) crossing a mutant or transgenic plant with a second plant to yield progeny tobacco seed; (b) growing the progeny tobacco seed, under plant growth conditions, to yield the non-naturally occurring plant.
  • the method may further comprises: (c) crossing the previous generation of non-naturally occurring plant with itself or another plant to yield progeny tobacco seed; (d) growing the progeny tobacco seed of step (c) under plant growth conditions, to yield additional non-naturally occurring plants; and (e) repeating the crossing and growing steps of (c) and (d) multiple times to generate further generations of non-naturally occurring plants.
  • the method may optionally comprises prior to step (a), a step of providing a parent plant which comprises a genetic identity that is characterized and that is not identical to the mutant or transgenic plant.
  • the crossing and growing steps are repeated from 0 to 2 times, from 0 to 3 times, from 0 to 4 times, 0 to 5 times, from 0 to 6 times, from 0 to 7 times, from 0 to 8 times, from 0 to 9 times or from 0 to 10 times, in order to generate generations of non-naturally occurring plants.
  • Backcrossing is an example of such a method wherein a progeny is crossed with one of its parents or another plant genetically similar to its parent, in order to obtain a progeny plant in the next generation that has a genetic identity which is closer to that of one of the parents.
  • Techniques for plant breeding, particularly plant breeding are well known and can be used in the methods of the disclosure.
  • the disclosure further provides non-naturally occurring plants produced by these methods. Certain embodiments exclude the step of selecting a plant.
  • lines resulting from breeding and screening for variant genes are evaluated in the field using standard field procedures. Control genotypes including the original unmutagenized parent are included and entries are arranged in the field in a randomized complete block design or other appropriate field design.
  • standard agronomic practices are used, for example, the tobacco is harvested, weighed, and sampled for chemical and other common testing before and during curing.
  • Statistical analyses of the data are performed to confirm the similarity of the selected lines to the parental line. Cytogenetic analyses of the selected plants are optionally performed to confirm the chromosome complement and chromosome pairing relationships.
  • DNA fingerprinting, single nucleotide polymorphism, microsatellite markers, or similar technologies may be used in a marker-assisted selection (MAS) breeding program to transfer or breed mutant alleles of a gene into other tobaccos, as described herein.
  • MAS marker-assisted selection
  • a breeder can create segregating populations from hybridizations of a genotype containing a mutant allele with an agronomically desirable genotype. Plants in the F2 or backcross generations can be screened using a marker developed from a genomic sequence or a fragment thereof, using one of the techniques listed herein. Plants identified as possessing the mutant allele can be backcrossed or self-pollinated to create a second population to be screened.
  • a plant population in the F2 generation is screened for variant gene expression, for example, a plant is identified that fails to express a polypeptide due to the absence of the gene according to standard methods, for example, by using a PCR method with primers based upon the polynucleotide sequence information for the polynucleotide(s) described herein (or any combination thereof as described herein).
  • Hybrid tobacco varieties can be produced by preventing self-pollination of female parent plants (that is, seed parents) of a first variety, permitting pollen from male parent plants of a second variety to fertilize the female parent plants, and allowing F1 hybrid seeds to form on the female plants.
  • Self-pollination of female plants can be prevented by emasculating the flowers at an early stage of flower development.
  • pollen formation can be prevented on the female parent plants using a form of male sterility.
  • male sterility can be produced by cytoplasmic male sterility (CMS), or transgenic male sterility wherein a transgene inhibits microsporogenesis or pollen formation, or self-incompatibility.
  • CMS cytoplasmic male sterility
  • transgenic male sterility wherein a transgene inhibits microsporogenesis or pollen formation, or self-incompatibility.
  • Female parent plants containing CMS are particularly useful.
  • the female parent plants are CMS
  • pollen is harvested from male fertile plants and applied manually to the stigmas of CMS female parent plants, and the resulting F1 seed is harvested.
  • Varieties and lines described herein can be used to form single-cross tobacco F1 hybrids.
  • the plants of the parent varieties can be grown as substantially homogeneous adjoining populations to facilitate natural cross-pollination from the male parent plants to the female parent plants.
  • the F1 seed formed on the female parent plants is selectively harvested by conventional means.
  • three-way crosses can be carried out wherein a single-cross F1 hybrid is used as a female parent and is crossed with a different male parent.
  • double-cross hybrids can be created wherein the F1 progeny of two different single-crosses are themselves crossed.
  • a population of mutant, non-naturally occurring or transgenic plants can be screened or selected for those members of the population that have a desired trait or phenotype.
  • a population of progeny of a single transformation event can be screened for those plants having a desired level of expression or function of the polypeptide(s) encoded thereby.
  • Physical and biochemical methods can be used to identify expression or activity levels.
  • RNA transcripts include Southern analysis or PCR amplification for detection of a polynucleotide; Northern blots, S1 RNase protection, primer-extension, or RT-PCR amplification for detecting RNA transcripts; enzymatic assays for detecting enzyme or ribozyme function of polypeptides and polynucleotides; and polypeptide gel electrophoresis, Western blots, immunoprecipitation, and enzyme-linked immunoassays to detect polypeptides.
  • Other techniques such as in situ hybridization, enzyme staining, and immunostaining and enzyme assays also can be used to detect the presence or expression, function or activity of NtGDH polypeptides or polynucleotides.
  • Mutant, non-naturally occurring or transgenic plant cells and plants are described herein comprising one or more recombinant polynucleotides, one or more polynucleotide constructs, one or more double-stranded RNAs, one or more conjugates or one or more vectors/expression vectors.
  • the plants and parts thereof described herein can be modified either before or after the expression, function or activity of the one or more NtGDH polynucleotides or NtGDH polypeptides according to the present disclosure have been modulated.
  • One or more of the following further genetic modifications can be present in the mutant, non- naturally occurring or transgenic plants and parts thereof.
  • One or more genes that are involved in the conversion of nitrogenous metabolic intermediates can be modified resulting in lower levels of at least one tobacco-specific nitrosamine (TSNA).
  • TSNA tobacco-specific nitrosamine
  • Non-limiting examples of such genes include those encoding nicotine demethylase - such as CYP82E4, CYP82E5 and CYP82E10 as described in WO2006/091194, WO2008/070274, WO2009/064771 and WO2011/088180 – and nitrate reductase, as described in WO2016/046288.
  • One or more genes that are involved in heavy metal uptake or heavy metal transport can be modified resulting in lower heavy metal content.
  • Non-limiting examples include genes in the family of multidrug resistance associated polypeptides, the family of cation diffusion facilitators (CDF), the family of Zrt- Irt-like polypeptides (ZIP), the family of cation exchangers (CAX), the family of copper transporters (COPT), the family of heavy-metal ATPases (for example, HMAs, as described in WO2009/074325 and WO2017/129739), the family of homologs of natural resistance-associated macrophage polypeptides (NRAMP), and other members of the family of ATP-binding cassette (ABC) transporters (for example, MRPs), as described in WO2012/028309, which participate in transport of heavy metals - such as cadmium.
  • CDF family of cation diffusion facilitators
  • ZIP Zrt- Irt-like polypeptides
  • CAX family of cation exchangers
  • COX copper transporters
  • COPD copper transporters
  • HMAs high-metal
  • exemplary modifications can result in plants with modulated expression or function of isopropylmalate synthase which results in a change in sucrose ester composition which can be used to alter favour profile (see WO2013/029799).
  • Other exemplary modifications can result in plants with modulated expression or function of threonine synthase in which levels of methional can be modulated (see WO2013/029800).
  • Other exemplary modifications can result in plants with modulated expression or function of one or more of neoxanthin synthase, lycopene beta cyclase and 9-cis-epoxycarotenoid dioxygenase to modulate beta- damascenone content to alter flavour profile (see WO2013/064499).
  • exemplary modifications can result in plants with modulated expression or function of members of the CLC family of chloride channels to modulate nitrate levels therein (see WO2014/096283 and WO2015/197727).
  • Other exemplary modifications can result in plants with modulated expression or function of one or more asparagine synthetases to modulate levels of asparagine in leaf and modulated levels of acrylamide in aerosol produced upon heating or combusting the leaf (see WO2017/129739).
  • Other exemplary modifications can result in plants with modulated protease activity during curing (see WO2016/009006).
  • exemplary modifications can result in plants having decreased nitrate levels by altering the gene expression of nitrate reductase (for example, Nia2) or the activity of the protein encoded thereby (see WO2016/046288).
  • exemplary modifications can result in plants having modified alkaloid levels by altering the gene expression of putative ABC-2 transporters NtABCGl-T and NtABCGl-S or the activity of the protein encoded thereby (see WO2019/086609).
  • exemplary modifications can result in plants having modulated time to flowering by altering the gene expression of genes encoding Terminal Flower 1 (TFL1) or the activity of the protein encoded thereby (see WO2018/114641).
  • exemplary modifications can result in plants with modulated expression or function of one or more asparagine synthetases to modulate levels of asparagine in leaf and modulated levels of acrylamide in aerosol produced upon heating or combusting the leaf (see WO2017/042162).
  • examples of other modifications include modulating herbicide tolerance, for example, glyphosate is an active ingredient of many broad spectrum herbicides.
  • Glyphosate resistant transgenic plants have been developed by transferring the aroA gene (a glyphosate EPSP synthetase from Salmonella typhimurium and E.coli). Sulphonylurea resistant plants have been produced by transforming the mutant ALS (acetolactate synthetase) gene from Arabidopsis. OB polypeptide of photosystem II from mutant Amaranthus hybridus has been transferred in to plants to produce atrazine resistant transgenic plants; and bromoxynil resistant transgenic plants have been produced by incorporating the bxn gene from the bacterium Klebsiella pneumoniae. Another exemplary modification results in plants that are resistant to insects.
  • Bacillus thuringiensis (Bt) toxins can provide an effective way of delaying the emergence of Bt-resistant pests, as recently illustrated in broccoli where pyramided cry1Ac and cry1C Bt genes controlled diamondback moths resistant to either single polypeptide and significantly delayed the evolution of resistant insects.
  • Another exemplary modification results in plants that are resistant to diseases caused by pathogens (for example, viruses, bacteria, fungi). Plants expressing the Xa21 gene (resistance to bacterial blight) with plants expressing both a Bt fusion gene and a chitinase gene (resistance to yellow stem borer and tolerance to sheath) have been engineered.
  • Another exemplary modification results in altered reproductive capability, such as male sterility.
  • Another exemplary modification results in plants that are tolerant to abiotic stress (for example, drought, temperature, salinity), and tolerant transgenic plants have been produced by transferring acyl glycerol phosphate enzyme from Arabidopsis; genes coding mannitol dehydrogenase and sorbitol dehydrogenase which are involved in synthesis of mannitol and sorbitol improve drought resistance.
  • abiotic stress for example, drought, temperature, salinity
  • tolerant transgenic plants have been produced by transferring acyl glycerol phosphate enzyme from Arabidopsis; genes coding mannitol dehydrogenase and sorbitol dehydrogenase which are involved in synthesis of mannitol and sorbitol improve drought resistance.
  • Another exemplary modification results in plants in which the activity of one or more nicotine N-demethylases is modulated such that the levels of nornicotine and metabolites of nornicotine - that are formed during
  • SAM S-adenosyl- L-methionine
  • CGS cystathionine gamma-synthase
  • One or more genes that are involved in the nicotine synthesis pathway can be modified resulting in plants or parts of plants that when cured, produce modulated levels of nicotine.
  • the nicotine synthesis genes can be selected from the group consisting of: A622, BBLa, BBLb, JRE5L1, JRE5L2, MATE1, MATE 2, MPO1, MPO2, MYC2a, MYC2b, NBB1, nic1, nic2, NUP1, NUP2, PMT1, PMT2, PMT3, PMT4 and QPT or a combination of one or more thereof.
  • One or more genes that are involved in controlling the amount of one or more alkaloids can be modified resulting in plants or parts of plants that produce modulated levels of alkaloid.
  • Alkaloid level controlling genes can be selected from the group consisting of; BBLa, BBLb, JRE5L1, JRE5L2, MATE1, MATE 2, MYC2a, MYC2b, nic1, nic2, NUP1 and NUP2 or a combination of two or more thereof.
  • Other exemplary modifications can result in plants with modulated amino acid content (see WO2019/185703 and WO2021/063863) or with modulated sugar content (see WO2019/185699 and WO2021/063860 and WO2021/063863) or with modulated nitrate levels (see WO2020/141062) or with modulated sugar and amino acid content (see WO2021/063863).
  • the further genetic modification concerns asparagine synthetase (ASN) genes as described in WO2017042162.
  • ASN asparagine synthetase
  • Modulating the expression of ASN genes for example, one or more of NtASN1-S, NtASN1-T, NtASN5-S and NtASN5-T as described in WO2017042162
  • the activity of ASN for example, NtASN1-S, NtASN1-T, NtASN5-S and NtASN5-T as described in WO2017042162
  • modulating the expression and/or activity of the combination of ASN and NtGDH may have the potential to rearrange the chemistry of cured tobacco leaf (particularly the amino acid chemistry of Burley or Dark tobacco) and thereby alter the sensory properties.
  • other genes and enzymes play a role in the reorganization of amino acids and/or sugars during leaf yellowing - such as diaminopimelate aminotransferase (DAPAT), which is involved in both catabolism and anabolism of lysine, and aspartate amino transferases (AAT), and which is expressed during senescence and has the potential to change leaf chemistry after curing (WO2019/185703).
  • DAPAT diaminopimelate aminotransferase
  • AAT aspartate amino transferases
  • the chloroplast sulphate transporter SULTR3 – such as NtSULTR3;1A-S, NtSULTR3;1A-T and NtSULTR3;3-T - play a role in sugar and amino acid metabolism during curing (see WO2021/063863).
  • the further genetic modification can concern DAPAT and/or AAT (for example, one or more of NtAATI-S, NtAAT1-T, NtAAT2-S, NtAAT2-T, NtAAT3-S, NtAAT3-T, NtAAT4-S or NtAAT4-T as described in WO2017042162) and/or one or more of NtSULTR3;1A-S, NtSULTR3;1A-T and NtSULTR3;3-T as described in see WO2021/063863.
  • AAT for example, one or more of NtAATI-S, NtAAT1-T, NtAAT2-S, NtAAT2-T, NtAAT3-S, NtAAT3-T, NtAAT4-S or NtAAT4-T as described in WO2017042162
  • Modulating the expression and/or activity of the combination of DAPAT and/or AAT and/or SULTR3 and NtGDH may have the potential to rearrange the chemistry of cured tobacco leaf and thereby alter the sensory properties.
  • Modifications to combinations of NtGDH and one or more, or two or more, or three of more or four or more of ASN and DAPAT and AAT and SULTR3 are disclosed, including NtGDH and ASN; NtGDH and DAPAT; NtGDH and AAT; NtGDH and ASN and DAPAT; NtGDH and ASN and AAT; NtGDH and ASN and DAPAT and AAT; NtGDH and SULTR3; NtGDH and ASN and SULTR3; NtGDH and DAPAT and SULTR3; NtGDH and AAT and SULTR3; NtGDH and ASN and DAPAT and SULTR3; NtGDH and AAT and SULTR3; NtGDH and A
  • One or more traits may be introgressed into the mutant, non-naturally occurring or transgenic plants from another cultivar or may be directly transformed into it.
  • Various embodiments provide mutant plants, non-naturally occurring plants or transgenic plants, as well as biomass in which the expression level of one or more polynucleotides according to the present disclosure are modulated to thereby modulate the level of polypeptide(s) encoded thereby.
  • the present invention also provides a cured tobacco blend in which the level of ammonia is lowered.
  • the tobacco blend can contain at least a first cured Nicotiana plant leaf or part thereof according to the present invention and a second cured Nicotiana plant leaf or part thereof in which the sum of ammonia is lower than the sum of ammonia in the at least second cured Nicotiana plant leaf or a part thereof.
  • Blended cigarettes typically use two or three main tobacco types selected from Virginia, Burley, and Oriental.
  • the second cured Nicotiana plant leaf or part thereof is a Burley tobacco or an Oriental tobacco or a Dark tobacco or a flue cured tobacco or a combination of two or more thereof.
  • a method for producing a tobacco blend having a reduced amount of ammonia comprising: providing a cured Nicotiana plant leaf or a part thereof, wherein the first cured Nicotiana plant leaf or a part thereof is from the mutant, non-naturally occurring or transgenic Nicotiana plant leaf or part of the plant leaf; and (b) blending the first cured Nicotiana plant leaf or a part thereof with at least one second cured Nicotiana plant leaf or a part thereof to produce a tobacco blend in which the sum of ammonia is lower than the sum of ammonia in the at least one second cured Nicotiana plant leaf or a part thereof.
  • the tobacco blend can be finely cut before being dried to reduce moisture.
  • the blend is ready for manufacturing.
  • Parts of the plants described herein, particularly the leaf lamina and/or stalks and/or midrib of such plants, can be incorporated into or used in making various consumable products including but not limited to aerosol forming materials, aerosol forming devices, smoking articles, smokable articles, smokeless products, medicinal or cosmetic products, intravenous preparations, tablets, powders, and tobacco products.
  • aerosol forming materials include tobacco compositions, tobaccos, tobacco extract, cut tobacco, cut filler, cured tobacco, expanded tobacco, homogenized tobacco, reconstituted tobacco, and pipe tobaccos.
  • Smoking articles and smokable articles are types of aerosol forming devices.
  • smoking articles or smokable articles include cigarettes, cigarillos, and cigars.
  • smokeless products comprise chewing tobaccos, and snuffs.
  • a tobacco composition or another aerosol forming material is heated by one or more electrical heating elements to produce an aerosol.
  • an aerosol is produced by the transfer of heat from a combustible fuel element or heat source to a physically separate aerosol forming material, which may be located within, around or downstream of the heat source.
  • Smokeless tobacco products and various tobacco-containing aerosol forming materials may contain tobacco in any form, including as dried particles, shreds, granules, powders, or a slurry, deposited on, mixed in, surrounded by, or otherwise combined with other ingredients in any format, such as flakes, films, tabs, foams, or beads.
  • the term ‘smoke’ is used to describe a type of aerosol that is produced by smoking articles, such as cigarettes, or by combusting an aerosol forming material.
  • tobacco products including tobacco-containing aerosol forming materials comprising plant material – such as leaves, suitably cured leaves - from the mutant tobacco plants, transgenic tobacco plants or non-naturally occurring tobacco plants described herein.
  • the tobacco products described herein can be a blended tobacco product which may further comprise unmodified tobacco.
  • the mutant, non-naturally occurring or transgenic plants may have other uses in, for example, agriculture.
  • the disclosure also provides methods for producing seeds comprising cultivating the mutant plant, non-naturally occurring plant, or transgenic plant described herein, and collecting seeds from the cultivated plants.
  • Seeds from plants described herein can be conditioned and bagged in packaging material by means known in the art to form an article of manufacture.
  • Packaging material such as paper and cloth are well known in the art.
  • a package of seed can have a label, for example, a tag or label secured to the packaging material, a label printed on the package that describes the nature of the seeds therein.
  • Compositions, methods and kits for genotyping plants for identification, selection, or breeding can comprise a means of detecting the presence of a NtGDH polynucleotide(s) in a sample of polynucleotide.
  • composition comprising one or more primers for specifically amplifying at least a portion of one or more of the NtGDH polynucleotides and optionally one or more probes and optionally one or more reagents for conducting the amplification or detection.
  • primers or probes comprising about 10 or more contiguous polynucleotides corresponding to the NtGDH polynucleotide(s) described herein are disclosed.
  • Said primers or probes may comprise or consist of about 15, 20, 25, 30, 40, 45 or 50 more contiguous polynucleotides that hybridise (for example, specifically hybridise) to the NtGDH polynucleotide(s) described herein.
  • the primers or probes may comprise or consist of about 10 to 50 contiguous nucleotides, about 10 to 40 contiguous nucleotides, about 10 to 30 contiguous nucleotides or about 15 to 30 contiguous nucleotides that may be used in sequence-dependent methods of gene identification (for example, Southern hybridization) or isolation (for example, in situ hybridization of bacterial colonies or bacteriophage plaques) or gene detection (for example, as one or more amplification primers in amplification or detection).
  • the one or more specific primers or probes can be designed and used to amplify or detect a part or all of the polynucleotide(s).
  • two primers may be used in a PCR protocol to amplify a polynucleotide fragment.
  • the PCR may also be performed using one primer that is derived from a polynucleotide sequence and a second primer that hybridises to the sequence upstream or downstream of the polynucleotide sequence – such as a promoter sequence, the 3' end of the mRNA precursor or a sequence derived from a vector.
  • a promoter sequence such as a promoter sequence, the 3' end of the mRNA precursor or a sequence derived from a vector.
  • the sample may be or may be derived from a plant, a plant cell or plant material or a tobacco product made or derived from the plant, the plant cell or the plant material as described herein.
  • a method of detecting a NtGDH polynucleotide(s) described herein (or any combination thereof as described herein) in a sample comprising the step of: (a) providing a sample comprising, or suspected of comprising, a polynucleotide; (b) contacting said sample with one or more primers or one or more probes for specifically detecting at least a portion of the NtGDH polynucleotide(s); and (c) detecting the presence of an amplification product, wherein the presence of an amplification product is indicative of the presence of the NtGDH polynucleotide(s) in the sample.
  • kits for detecting at least a portion of the NtGDH polynucleotide(s) are also provided which comprise one or more primers or probes for specifically detecting at least a portion of the NtGDH polynucleotide(s).
  • the kit may comprise reagents for polynucleotide amplification - such as PCR - or reagents for probe hybridization- detection technology - such as Southern Blots, Northern Blots, in-situ hybridization, or microarray.
  • the kit may comprise reagents for antibody binding-detection technology such as Western Blots, ELISAs, SELDI mass spectrometry or test strips.
  • the kit may comprise reagents for DNA sequencing.
  • the kit may comprise reagents and instructions for using the kit.
  • a kit may comprise instructions for one or more of the methods described.
  • the kits described may be useful for genetic identity determination, phylogenetic studies, genotyping, haplotyping, pedigree analysis or plant breeding particularly with co- dominant scoring.
  • the present disclosure also provides a method of genotyping a plant, a plant cell or plant material comprising a NtGDH polynucleotide as described herein.
  • Genotyping provides a means of distinguishing homologs of a chromosome pair and can be used to differentiate segregants in a plant population.
  • Molecular marker methods can be used for phylogenetic studies, characterizing genetic relationships among crop varieties, identifying crosses or somatic hybrids, localizing chromosomal segments affecting monogenic traits, map based cloning, and the study of quantitative inheritance.
  • the specific method of genotyping may employ any number of molecular marker analytic techniques including amplification fragment length polymorphisms (AFLPs).
  • AFLPs are the product of allelic differences between amplification fragments caused by polynucleotide variability.
  • the present disclosure further provides a means to follow segregation of one or more genes or polynucleotides as well as chromosomal sequences genetically linked to these genes or polynucleotides using such techniques as AFLP analysis.
  • a specific extraction temperature is selected for the tobacco starting material.
  • the extraction temperature(s) is typically selected from within the range of about 100 degrees Celsius to about 160 degrees Celsius.
  • the duration of the heating step may optionally be controlled to provide a degree of control over the composition of the extract derived from the tobacco starting material(s).
  • the tobacco starting material(s) is heated at the extraction temperature for at least about 90 minutes, more suitably at least about 120 minutes.
  • the heating step is typically carried out in an inert atmosphere.
  • a flow of an inert gas - such as nitrogen - is passed through the starting tobacco material during the heating step.
  • the volatile tobacco compounds are released into the flow of inert gas during the heating step such that the inert gas acts as a carrier for the volatile components.
  • the flow of inert gas can be at a flow rate of at least about 25 litres per minute, more suitably at least about 30 litres per minute.
  • a relatively high flow rate of inert gas may advantageously improve the efficiency of extraction from the tobacco starting material.
  • the heating step may be carried out under vacuum.
  • Suitable heating methods for carrying out the heating of the tobacco starting material are known to the skilled person and include: dry distillation, hydrodistillation, vacuum distillation, flash distillation and thin film hydrodistillation.
  • the step of forming the liquid tobacco extract can comprise drying the solution of the volatile compounds in the liquid solvent in order to concentrate the solution. Drying may be carried out using any suitable means, including but not limited to desiccation, molecular sieves, freeze drying, phase separation, distillation, membrane permeation, controlled crystallisation of water and filtering, reverse hygroscopicity, ultracentrifugation, liquid chromatography, reverse osmosis or chemical drying.
  • the liquid tobacco extract is particularly suitable for producing a composition or formulation or gel composition, for use in an aerosol-generating system.
  • An aerosol-generating system comprising the composition or formulation or gel composition is disclosed.
  • the composition or formulation or gel is typically heated within an aerosol- generating device - such as a device comprising a heater element that interacts with the composition or formulation or gel incorporating the liquid tobacco extract to produce an aerosol.
  • volatile compounds are released by heat transfer and entrained in air drawn through the aerosol generating device. As the released compounds cool they condense to form an aerosol that is inhaled by the consumer.
  • cured lamina is dried at 40 °C for 2-3 days, if required. Tobacco material is then ground in fine powder (-100 uM) before the analysis of amino acid content.
  • amino acid content is measured in plant material as described in UNI EN ISO 13903:2005 Determination of amino acids content. The determination of free (synthetic and natural) and total (peptide-bound and free) amino acids is achieved using an amino acid analyser or HPLC equipment. Methods for determining sugar content Sugar content is measured using a segmented-flow colorimetric method developed for analysis of tobacco samples as adapted by Skalar Instrument Co (West Chester, PA) and described in Tobacco Science 20: 139-144 (1976).
  • sugar content is also described in Coresta Recommended Method 38, CRM38, CRM and ISO 15154: 2003.
  • cured lamina is dried at 40°C for 2-3 days, if required.
  • Tobacco material is then ground in fine powder (-100 uM) before the analyses of sugars.
  • sugar content is measured according to ISO 15154: 2003, which specifies a method for the determination of the content of reducing carbohydrates in tobacco by continuous-flow analysis.
  • Method for determining ammonia content Ammonia is measured using Coresta Recommended Method No.79 (2016) Determination of ammonia in tobacco and tobacco products by ion chromatographic analysis. Briefly, the ammonia content is determined by extraction of the tobacco sample into a sulfuric acid solution.
  • Ion chromatographic analysis is used to separate ammonium ion from other cationic species.
  • the response of ammonium ion is measured using a conductivity detector and is quantified against an external standard calibration.
  • Method for determining alkaloid content Alkaloids are measured using Coresta Recommended Method No.35 (2010) Determination of total alkaloids (as nicotine) in tobacco by continuous flow analysis. Briefly, an aqueous extract of tobacco is prepared and the total alkaloids (as nicotine) content of the extract is determined by reaction with sulphanilic acid and cyanogen chloride. Cyanogen chloride is generated in situ by the reaction of potassium cyanide and chloramine T. The developed colour is measured at 460 nm.
  • Nitrate is measured using Coresta Recommended Method No. 36 (2015) Determination of nitrate in tobacco and smokeless tobacco products by reduction to nitrite and continuous flow analysis. Briefly, an aqueous extract of tobacco or smokeless tobacco product is prepared and the nitrate content of the extract is determined by reduction of the nitrate to nitrite with hydrazinium sulphate in the presence of a copper catalyst, followed by reaction with sulphanilamide to form the diazo compound. This is coupled with N-1- naphthylethylenediamine dihydrochloride to form a coloured complex, for which the absorbance is measured at 520 nm. Dry weight basis Data can be reported in % DWB ⁇ SD.
  • Dry weight basis is calculated after drying at 105 °C until reaching a constant mass, which is essentially 100 percent solids content.
  • Gene expression analysis Sequencing data generated is demultiplexed using Illumina BaseSpace® Clarity LIMS ( ⁇ Illumina, Inc.) and subsequently imported to Qiagen CLC Genomics Workbench version 12.0.1 (CLC bio, a QIAGEN Company).
  • RNAi procedure The DNA fragment SEQ ID NO: 21 is selected for suppressing the expression of NtGDH and is cloned between the strong constitutive MMV promoter and the 3′ nos terminator sequence of the nopaline synthase gene of Agrobacterium tumefaciens (Cheng et al. (1997) Plant Physiol.115(3):971-980). Nicotiana tabacum is transformed using standard Agrobacterium- mediated transformation protocols (Horsch et al.
  • transcript data Frragments Per Kilobase of transcript per Million mapped reads
  • RNAseq data see Figure 1
  • the typical markers of senescence SAG12 and SGR1 are strongly expressed during this phase, exhibiting a peak of expression at about 96h curing.
  • Some NtGDH genes are not expressed during the early curing phase like NtGDH7, NtGDH8, NtGDH4 and NtGDH11.
  • the other 6 NtGDH genes are expressed during the early curing phase, namely the pairs NtGDH6-10, NtGDH2-3 and NtGDH9-12.
  • NtGDH6 and NtGDH10 are mainly expressed after 24h curing, whereas the couple NtGDH2-3 and NtGDH9-12 are more induced in the late phase of curing from 48h to 192h. Both NtGDH2-3 and NtGDH9-12 follow similar induction profiles, however, the expression of NtGDH2 and NtGDH3 constantly increases during the curing process and reaches the highest FPKM values after 192h, which is not the case for NtGDH9-12. Based on the expression data presented in Figure 1, NtGDH2 and NtGDH3 are silenced using a RNAi approach to determine the impact on the ammonia accumulation within the cured leaf.
  • Example 3 RNAi silencing of NtGDH2-3
  • SEQ ID NO 21 The inserted sequence in GATEWAY vector to specifically generate anti-GDH2-3 plants is presented below as SEQ ID NO 21.
  • T0 After plant transformation (T0), three plants E459-2, E459-3 and E459-5 exhibiting a positive reduction of NtGDH2-3 expression are selected.
  • the strongest silencing impact is found in plants E459-3 and E459-5 after 48 h leaf curing when compared to control plant leaf (C1-3), the line E459-2 being only poorly silenced (see Figure 2).
  • Table 1 the chemical impact of NtGDH2-3 silencing on air-cured leaves is shown.
  • Ammonia content is reduced by around 50% (when considering the two lines E459-3 and E459-5 efficiently silenced for NtGDH2-3, see Figure 2).
  • the ammonia content in line E459-2 is also lower than the controls, but to a lesser extent compared to E459-3 and E459-5. Nevertheless, altogether in the three lines E459-2, E459-3 and E459- 5, the ammonia reduction is still statistically relevant.
  • content of total alkaloids is also significantly reduced by about 30% and anti-correlated with a 30% increase in total amino acids (when the two lines E459-3 and E459-5 efficiently silenced for NtGDH2-3 are considered).
  • the amino acids mainly increasing statistically in the cured leaves of the NtGDH2-3 silenced lines compared to the WT are proline (4.6x more when considering the two lines E459-3 and E459-5 efficiently silencing NtGDH2-3), aspartate (1.8x more when considering the two lines E459-3 and E459-5 efficiently silencing NtGDH2-3), as well as serine, threonine and arginine (respectively, 1.4x, 1.8x and 2.4x more when considering the two lines E459-3 and E459-5 efficiently silencing NtGDH2-3). It is noteworthy that the amino acids aspartate and arginine increasing in anti-NtGDH lines are also reported to be the substrate for nicotine synthesis in the roots.
  • Figure 3 shows a box plot representation of the key metabolic changes on ammonia, sugars, aspartic acid and proline induced by the silencing of NtGDH2-3. All these modifications are significant compared to the control (WT). The most striking impact being on ammonia, reaching 50% reduction in the cured tobacco. Inactivating NtGDH2-3 changes the chemistry of cured leaves not only by limiting the increase of ammonia, but also by changing the amino acids and sugar contents. It is credible that knocking-out additional NtGDH genes like NtGDH9-12 results in a stronger reduction of ammonia and thereby a reduced cell toxicity due to ammonium during the senescence process.
  • Example 4 Impact of silencing on biomass and plant height Silencing of NtGDH2 and NtGDH3 does not impact the biomass and the height of the plants (see Figure 4). This suggests that the presence of absence of active NtGDH2 and NtGDH3 proteins does not affect plant growth and development. This indirectly confirms a specific function in leaves to recycle N from amine groups. This may have a key role under stress or senescence conditions to trigger the synthesis of novel amino acids, like for instance proline under drought stress.
  • Example 5 Sensory analysis Leaf material of the control tobacco (TN90) and the NtGDH -RNAi lines described above (mid- upper leaves) are pooled and subjected to a RRP sensory test in IQOS. Sticks are prepared in a cast-leaf mode corresponding to 60% test tobacco supplemented with 40% Flue cured background tobacco. Four trained panelists tested the samples and the results are depicted in Table 2.
  • TN90 is developing dark, leathery and animalic fresh notes with some harshness.
  • the lack of NtGDH2-3 is drastically changing the sensory perception, providing some clear differentiation. Less dark notes are perceived in the aerosol with additional complex aromatic notes providing thus a good balance with some warm notes and less harshness.

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Abstract

L'invention concerne une feuille de plante Nicotiana mutante, non naturelle ou transgénique ou une partie de la feuille de plante ayant une expression ou une activité modulée de la glutamate déshydrogénase (NtGDH), la NtGDH comprenant, consistant en, ou consistant essentiellement en au moins un des éléments suivants : un polynucléotide NtGDH2 ou un polynucléotide NtGDH3 ou un polynucléotide NtGDH6 ou un polynucléotide NtGDH9 ou un polynucléotide NtGDH10 ou un polynucléotide NtGDH12 ou un polypeptide NtGDH2 ou un polypeptide NtGDH3 ou un polypeptide NtGDH6 ou un polypeptide NtGDH9 ou un polypeptide NtGDH10 ou un polypeptide NtGDH12, l'expression du polynucléotide NtGDH2 ou du polynucléotide NtGDH3 ou du polynucléotide NtGDH6 ou du polynucléotide NtGDH9 ou du polynucléotide NtGDH10 ou du polynucléotide NtGDH12 polynucléotide ou du polypeptide NtGDH2 ou du polypeptide NtGDH3 ou du polypeptide NtGDH6 ou du polypeptide NtGDH9 ou du polypeptide NtGDH10 ou du polypeptide NtGDH12 étant modulée par rapport à une plante témoin dans laquelle l'expression du polynucléotide NtGDH2 ou du polynucléotide NtGDH3 ou du polynucléotide NtGDH6 ou du polynucléotide NtGDH9 ou du polynucléotide NtGDH10 ou du polynucléotide NtGDH12 polynucléotide ou du polypeptide NtGDH2 ou du polypeptide NtGDH3 ou du polypeptide NtGDH6 ou du polypeptide NtGDH9 ou du polypeptide NtGDH10 ou du polypeptide NtGDH12 n'est pas modulée.
PCT/EP2024/067755 2023-06-29 2024-06-25 Modulation de gènes codant pour la glutamate déshydrogénase Pending WO2025003105A1 (fr)

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