Regulation of the gene encoding glutamate dehydrogenase

Abstract

Disclosed are mutant, non-natural, or transgenic tobacco plant leaves or portions of plant leaves in which the expression or activity of glutamate dehydrogenase (NtGDH) is regulated, wherein the NtGDH comprises or essentially consists of at least one of the following: NtGDH2 polynucleotide, or NtGDH3 polynucleotide, or NtGDH6 polynucleotide, or NtGDH9 polynucleotide, or NtGDH10 polynucleotide, or NtGDH12 polynucleotide, or NtGDH2 polypeptide, or NtGDH3 polypeptide, or NtGDH6 polypeptide, or NtGDH9 polypeptide, or NtGDH10 polypeptide, or NtGDH12 polypeptide, and the expression of each of the polynucleotides or polypeptides is regulated compared to a control plant in which the expression of each of the polynucleotides or polypeptides is not regulated.

Description

Technical Field

[0001] The present invention generally relates to plants in which the expression or activity of glutamate dehydrogenase is regulated.

Background Art

[0002] Heated tobacco products heat instead of burning actual tobacco and do not emit sidestream smoke, but smokers exhale the mainstream aerosol into the atmosphere. Inhaling ammonia can cause burns in the nasopharynx and trachea, edema in the bronchioles and alveoli, and airway destruction, leading to dyspnea or respiratory failure. Yamamoto et al. (2022) Toxics Oct 6;10(10):592 showed that the total ammonia emissions increase with the increase in heating temperature regardless of the tobacco heating device used. They concluded that ammonia in the mainstream aerosol was emitted by a general heat process, probably by heat extraction in water vapor from tobacco leaves. The ammonia released into the aerosol correlates with the ammonia (ammonium ions) present in the tobacco material before the heating experience. These data also indicate that the type of drying treatment is a determining factor in the accumulation of ammonia in the tobacco material matrix, and when the tobacco is air-dried, both the ammonia in the tobacco and the ammonia aerosolized into the mainstream are dominant.

[0003] The presence of ammonia in tobacco material is related not only to the type of drying treatment used but also to the type of tobacco. For example, as shown by Lefingwell in Chap. 8, Production, Chemistry, And Technology, D. Layten Davis and Mark T. Nielson, Eds., Blackwell Science (Pub.), 1999, Burley tobacco is known to produce significantly more ammonia than Virginia tobacco. Figure 2 of this specification shows that when fresh Burley leaves are hung in an air-dried barn immediately after harvest, the ammonia content (as a compound toxic to green leaf cells) is very low in the green leaves and only reaches a maximum value after about 15 days (the end of the so-called yellowing process, associated with the induced cellular senescence process of the leaves). Very similar data have already been described by Burton et al. (1983) Rec. Adc. Tob. Sci., 9, 91-153. These data also suggest that ammonia is a byproduct of cellular senescence processes, likely resulting from active proteolysis and subsequent degradation of amine compounds (amino acids) within the leaf matrix (Miyashita & Good (2008) Plant Signal Behav. 3(10), 842-3; Rolny et al. (2016) Acta Physiol Plant 38, 89). However, such catabolic processes leading to ammonia production in dried tobacco have not been identified. The need to reduce ammonia levels in tobacco material remains in the art. This invention attempts to address this and other needs. [Overview of the Initiative]

[0004] This specification discloses that certain GDH genes derived from tobacco (Nicotiana tabacum), referred to as NtGDH, are involved in the accumulation of ammonia in leaves during drying. We investigated NtGDH genes to identify those particularly expressed during the initial drying stage. Unexpectedly, certain NtGDH genes, such as NtGDH4, NtGDH7, NtGDH8, and NtGDH11, were not expressed during the initial drying stage, while other NtGDH genes were. In particular, six NtGDH genes—NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10, and NtGDH12—were expressed during the initial drying stage. Interestingly, NtGDH6 and NtGDH10 were found to be primarily expressed 24 hours after drying, while NtGDH2, NtGDH3, NtGDH9, and NtGDH12 were more heavily induced in the later stages of drying, from 48 to 192 hours. NtGDH2, NtGDH3, NtGDH9, and NtGDH12 followed similar induction profiles, but the expression of NtGDH2 and NtGDH3 steadily increased throughout the drying process, reaching peak expression levels (based on Fragments Per Kilobase of transcript per Million mapped reads (FPKM values)) at 192 hours, whereas this was not the case for NtGDH9 or NtGDH12. The effect of downregulation (e.g., silencing) of specific NtGDH genes on air-dried leaves showed a decrease in ammonia content. Surprisingly, total alkaloid content also decreased, which was negatively correlated with an increase in total amino acids. The amino acids that were primarily increased in the dried leaves were proline, aspartic acid, serine, threonine, and arginine. Sugars were also increased.Therefore, in summary, inactivation or reduction of expression of specific NtGDH genes, e.g., NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10, and NtGDH12, can be used to alter the chemical properties of dried leaves not only by limiting the increase in ammonia, but also by altering the amino acid content—including proline, aspartic acid, serine, threonine, and arginine—and sugar content—glucose, fructose, and lactose, etc. Conversely, upregulation (e.g., overexpression) of specific NtGDH genes is expected to alter the chemical properties of dried leaves by increasing ammonia, increasing total alkaloids, decreasing amino acids—including proline, aspartic acid, serine, threonine, and arginine—and decreasing sugars—glucose, fructose, and lactose, etc. Based on the data presented herein, it is believed that regulating one more NtGDH polynucleotide or regulating the activity of one more NtGDH polypeptide results in a stronger regulation of ammonia, and consequently, a reduction in cytotoxicity due to ammonium during the cellular senescence process, along with additional side effects on the chemistry of tobacco, including alkaloids and amino acids. Advantageously, downregulation (e.g., silencing) of NtGDH2 and NtGDH3 has been shown to have no effect on the biomass and height of modified plants, suggesting that the presence or absence of active NtGDH2 and NtGDH3 proteins does not affect plant growth and development. Dried leaf materials of control tobacco and NtGDH-RNAi lines were pooled and subjected to sensory testing. The results of the sensory testing showed that the NtGDH-RNAi lines yielded different and preferred sensory perceptions. Therefore, the present invention hereby offers the possibility of obtaining ammonia-reduced dried tobacco that is less toxic to consumers while still retaining a preferred sensory perception.The present invention also enables the blending of "high" ammonia-dried tobaccos (such as Flavor Burley or certain dark tobaccos) to obtain new tobacco flavors with lower ammonia levels.

[0005] In one embodiment, a mutant, non-natural, or transgenic tobacco plant leaf or portion of a plant leaf in which the expression or activity of glutamate dehydrogenase (NtGDH) is regulated is disclosed, wherein NtGDH comprises or essentially comprises at least one of the following: NtGDH2 polynucleotide, or NtGDH3 polynucleotide, or NtGDH6 polynucleotide, or NtGDH9 polynucleotide, or NtGDH10 polynucleotide, or NtGDH12 polynucleotide, or NtGDH2 polypeptide, or NtGDH3 polypeptide, or NtGDH6 polypeptide, or NtGDH9 polypeptide, or NtGDH10 polypeptide, or NtGDH12 polypeptide, and (i) the NtGDH2 polynucleotide comprises or essentially comprises a sequence having at least 90% sequence identity to SEQ ID NO: 1, or (ii) the NtGDH2 polypeptide is encoded by the polynucleotide described in (i), or (iii) the NtGDH2 polypeptide has at least 90% sequence identity to SEQ ID NO: 2 (iv) The NtGDH3 polynucleotide consists of or essentially consists of a sequence having at least 90% sequence identity to SEQ ID NO: 3, or (v) The NtGDH3 polypeptide is encoded by the polynucleotide described in (iv), or (vi) The NtGDH3 polypeptide has at least 94% sequence identity to SEQ ID NO: 4, or (vii) The NtGDH6 polynucleotide consists of or essentially consists of a sequence having at least 90% sequence identity to SEQ ID NO: 7, or (viii) The NtGDH6 polypeptide is encoded by the polynucleotide described in (i), or (ix) The NtGDH6 polypeptide has at least 94% sequence identity to SEQ ID NO: 8, or (x) The NtGDH9 polynucleotide consists of or essentially consists of a sequence having at least 85% sequence identity to SEQ ID NO: 13, or (xi) The NtGDH9 polypeptide is encoded by the polynucleotide described in (i),Or (xii)NtGDH9 polypeptide having at least 91% sequence identity to SEQ ID NO: 14, or (xiii)NtGDH10 polynucleotide comprising or essentially comprising a sequence having at least 89% sequence identity to SEQ ID NO: 15, or (xiv)NtGDH10 polypeptide encoded by the polynucleotide described in (i), or (xv)NtGDH10 polypeptide having at least 94% sequence identity to SEQ ID NO: 16, or (xix)NtGDH12 polynucleotide comprising or essentially comprising a sequence having at least 85% sequence identity to SEQ ID NO: 19, or (xx)NtGDH12 polypeptide encoded by the polynucleotide described in (i), or (xxi)NtGDH12 polypeptide having at least 91% sequence identity to SEQ ID NO: 20, provided that NtGDH Compared to control plants where the expression of 2 polynucleotides, NtGDH3 polynucleotides, NtGDH6 polynucleotides, NtGDH9 polynucleotides, NtGDH10 polynucleotides, or NtGDH12 polynucleotides, or the activity of NtGDH2 polypeptides, NtGDH3 polypeptides, NtGDH6 polypeptides, NtGDH9 polypeptides, NtGDH10 polypeptides, or NtGDH12 polypeptides is not regulated, the expression of NtGDH2 polynucleotides, NtGDH3 polynucleotides, NtGDH6 polynucleotides, NtGDH9 polypeptides, NtGDH10 polypeptides, or NtGDH12 polynucleotides, or the activity of NtGDH2 polypeptides, NtGDH3 polypeptides, NtGDH6 polypeptides, NtGDH9 polypeptides, NtGDH10 polypeptides, or NtGDH12 polypeptides is regulated.

[0006] Preferably, the expression of NtGDH6 polynucleotide and NtGDH10 polynucleotide is regulated, or the activity of NtGDH6 polypeptide and NtGDH10 polypeptide is regulated.

[0007] Preferably, the expression of NtGDH2 polynucleotides and NtGDH3 polynucleotides is regulated, or the activity of NtGDH2 polypeptides and NtGDH3 polypeptides is regulated.

[0008] Preferably, the expression of NtGDH9 polynucleotide and NtGDH12 polynucleotide is regulated, or the activity of NtGDH9 polypeptide and NtGDH12 polypeptide is regulated.

[0009] Preferably, the expression of NtGDH2 polynucleotide, NtGDH3 polynucleotide, NtGDH9 polynucleotide, and NtGDH12 polynucleotide is regulated, or the activity of NtGDH2 polypeptide, NtGDH3 polypeptide, NtGDH9 polypeptide, and NtGDH12 polypeptide is regulated.

[0010] Preferably, the expression of NtGDH2 polynucleotide, NtGDH3 polynucleotide, NtGDH6 polynucleotide, NtGDH9 polynucleotide, NtGDH10 polynucleotide, and NtGDH12 polynucleotide, or NtGDH2 polypeptide, NtGDH3 polypeptide, NtGDH6 polypeptide, NtGDH9 polypeptide, NtGDH10 polypeptide, and NtGDH12 polypeptide is regulated.

[0011] Preferably, the expression or activity of one or more of the NtGDH4 polynucleotide, NtGDH7 polynucleotide, NtGDH8 polynucleotide, NtGDH11 polynucleotide, NtGDH4 polypeptide, NtGDH7 polypeptide, NtGDH8 polypeptide, or NtGDH11 polypeptide is not regulated, and (i) the NtGDH4 polynucleotide consists of or essentially consists of a sequence having at least 88% sequence identity to SEQ ID NO: 5, or (ii) the NtGDH4 polypeptide is encoded by the polynucleotide described in (i), or (iii) the NtGDH4 polypeptide has at least 92% sequence identity to SEQ ID NO: 6, or (iv) the NtGDH7 polynucleotide consists of or essentially consists of a sequence having at least 92% sequence identity to SEQ ID NO: 9, or (v) the NtGDH7 polypeptide is (i (v) a polynucleotide encoded by the polynucleotide described in (i), or (vi) an NtGDH7 polypeptide having at least 96% sequence identity to SEQ ID NO: 10, or (vii) an NtGDH8 polynucleotide comprising or essentially comprising a sequence having at least 92% sequence identity to SEQ ID NO: 11, or (viii) an NtGDH8 polypeptide encoded by the polynucleotide described in (i), or (ix) an NtGDH8 polypeptide having at least 95% sequence identity to SEQ ID NO: 12, or (x) an NtGDH11 polynucleotide comprising or essentially comprising a sequence having at least 87% sequence identity to SEQ ID NO: 17, or (xi) an NtGDH11 polypeptide encoded by the polynucleotide described in (i), or (xii) an NtGDH11 polypeptide having at least 92% sequence identity to SEQ ID NO: 18.

[0012] Preferably, the plant leaf or a portion thereof contains at least one genetic modification that modifies the expression or activity of at least one NtGDH polynucleotide or at least one NtGDH polypeptide, or the plant leaf or a portion thereof contains one or more exogenous DNAs or exogenous RNAs that modulate the expression or activity of at least one NtGDH polynucleotide or at least one NtGDH polypeptide, or the plant leaf or a portion thereof contains a vector, viral vector, Agrobacterium vector, or CRISP that modulates the expression or activity of at least one NtGDH polynucleotide or at least one NtGDH polypeptide. A plant leaf or part thereof containing one or more R vectors, or containing at least one modification capable of one or more RNA interference, transcription gene silencing, or virus-induced gene silencing that modulates the expression or activity of at least one NtGDH polynucleotide or at least one NtGDH polypeptide, or a plant leaf or part thereof containing one or more exogenous double-stranded RNA (dsRNA), exogenous hairpin RNA (hpRNA), or exogenous small interfering RNAs, or a combination of two or more thereof, that modulates the expression or activity of at least one NtGDH polynucleotide or at least one NtGDH polypeptide.

[0013] Preferably, the regulated expression or activity of at least one NtGDH polynucleotide or NtGDH polypeptide regulates the amount of ammonia, amino acids, sugars, and total alkaloids in plant leaves or portions of plant leaves after drying.

[0014] Preferably, the amino acids are proline, aspartic acid, serine, threonine, and arginine.

[0015] Preferably, the plant leaves or a part thereof are air-dried; preferably, the air-dried leaves or a part thereof are sun-dried or heat-dried; or the plant leaves or a part thereof are air-dried; preferably, the air-dried leaves or a part thereof are sun-dried or heat-dried.

[0016] Preferably, the tobacco plant leaves or a part of the plant leaves are Nicotiana tabacum plant leaves or a part of the plant leaves.

[0017] In another embodiment, a method is provided for preparing dried tobacco leaves or portions thereof having controlled levels of ammonia and amino acids and sugars and total alkaloids compared to dried tobacco leaves or portions thereof derived from a dried control tobacco plant, the method comprising the steps of: (a) providing a tobacco leaf comprising or essentially comprising NtGDH, which includes at least one of NtGDH2 polynucleotide, or NtGDH3 polynucleotide, or NtGDH6 polynucleotide, or NtGDH9 polynucleotide, or NtGDH10 polynucleotide, or NtGDH12 polynucleotide, or NtGDH2 polypeptide, or NtGDH3 polypeptide, or NtGDH6 polypeptide, or NtGDH9 polypeptide, or NtGDH10 polypeptide, or NtGDH12 polypeptide, wherein (i) the NtGDH2 polynucleotide comprises or essentially comprises a sequence having at least 90% sequence identity with SEQ ID NO: 1, or (ii) NtGDH2 poly The lipeptide consists of, or essentially consists of, a polynucleotide that is encoded by the polynucleotide described in (i), or (iii) the NtGDH2 polypeptide has at least 94% sequence identity to SEQ ID NO: 2, or (iv) the NtGDH3 polynucleotide contains a sequence having at least 90% sequence identity to SEQ ID NO: 3, or (v) the NtGDH3 polypeptide is encoded by the polynucleotide described in (iv), or (vi) the NtGDH3 polypeptide has at least 94% sequence identity to SEQ ID NO: 4, or (vii) the NtGDH6 polynucleotide contains a sequence having at least 90% sequence identity to SEQ ID NO: 7, or (viii) the NtGDH6 polypeptide is encoded by the polynucleotide described in (i), or (ix) the NtGDH6 polypeptide has at least 94% sequence identity to SEQ ID NO: 8, or (x) the NtGDH9 polynucleotide contains a sequence having at least 85% sequence identity to SEQ ID NO: 13,The steps of providing a (x)NtGDH9 polypeptide comprising, or essentially comprising, or (xii)NtGDH9 polypeptide encoded by the polynucleotide described in (i), or (xii)NtGDH9 polypeptide having at least 91% sequence identity to SEQ ID NO: 14, or (xiii)NtGDH10 polynucleotide comprising, or essentially comprising, a sequence containing at least 89% sequence identity to SEQ ID NO: 15, or (xiv)NtGDH10 polypeptide encoded by the polynucleotide described in (i), or (xv)NtGDH10 polypeptide having at least 94% sequence identity to SEQ ID NO: 16, or (xix)NtGDH12 polynucleotide comprising, or essentially comprising, a sequence containing at least 85% sequence identity to SEQ ID NO: 19, or (xx)NtGDH12 polypeptide encoded by the polynucleotide described in (i), or (xxi)NtGDH12 polypeptide having at least 91% sequence identity to SEQ ID NO: 20; and (b) tobacco plant (c) a step of adjusting the expression of at least one NtGDH2 polynucleotide, NtGDH3 polynucleotide, NtGDH6 polynucleotide, NtGDH9 polynucleotide, NtGDH10 polynucleotide, or NtGDH12 polynucleotide, or the activity of NtGDH2 polypeptide, NtGDH3 polypeptide, NtGDH6 polypeptide, NtGDH9 polypeptide, NtGDH10 polypeptide, or NtGDH12 polypeptide in a leaf or a portion of a plant leaf; (d) a step of drying a leaf or a portion of a plant leaf; (e) optionally a step of measuring the level of ammonia and one or more of amino acids, sugars, and total alkaloids in the dried tobacco leaf or a portion of a dried plant leaf; (f) the expression of NtGDH2 polynucleotide, NtGDH3 polynucleotide, NtGDH6 polynucleotide, NtGDH9 polynucleotide, NtGDH10 polynucleotide, or NtGDH12 polynucleotide,Alternatively, a step to obtain dried tobacco plant leaves or a portion of its leaves having regulated levels of ammonia, amino acids, sugars, and total alkaloids compared to a control plant in which the activity of NtGDH2 polypeptide, NtGDH3 polypeptide, NtGDH6 polypeptide, NtGDH9 polypeptide, NtGDH10 polypeptide, or NtGDH12 polypeptide is not regulated.

[0018] Preferably, in step (b), the expression of NtGDH6 polynucleotide and NtGDH10 polynucleotide is regulated, or the activity of NtGDH6 polypeptide and NtGDH10 polypeptide is regulated.

[0019] Preferably, the expression of NtGDH2 polynucleotide and NtGDH3 polynucleotide is regulated, or the activity of both NtGDH2 polypeptide and NtGDH3 polypeptide is regulated.

[0020] Preferably, the expression of NtGDH9 polynucleotide and NtGDH12 polynucleotide is regulated, or the activity of NtGDH9 polypeptide and NtGDH12 polypeptide is regulated.

[0021] Preferably, the expression of NtGDH2 polynucleotide, NtGDH3 polynucleotide, NtGDH9 polynucleotide, and NtGDH12 polynucleotide is regulated, or the activity of NtGDH2 polypeptide, NtGDH3 polypeptide, NtGDH9 polypeptide, and NtGDH12 polypeptide is regulated.

[0022] Preferably, the expression of NtGDH2 polynucleotide, NtGDH3 polynucleotide, NtGDH6 polynucleotide, NtGDH9 polynucleotide, NtGDH10 polynucleotide, and NtGDH12 polynucleotide, or NtGDH2 polypeptide, NtGDH3 polypeptide, NtGDH6 polypeptide, NtGDH9 polypeptide, NtGDH10 polypeptide, and NtGDH12 polypeptide is regulated.

[0023] Preferably, the expression of one or more NtGDH4 polynucleotides, NtGDH7 polynucleotides, NtGDH8 polynucleotides, or NtGDH11 polynucleotides, or the activity of NtGDH4 polypeptides, NtGDH7 polypeptides, NtGDH8 polypeptides, or NtGDH11 polypeptides is not regulated, and (i) the NtGDH4 polynucleotide consists of or essentially consists of a sequence containing at least 88% sequence identity to SEQ ID NO: 5, or (ii) the NtGDH4 polypeptide is encoded by the polynucleotide described in (i), or (iii) the NtGDH4 polypeptide has at least 92% sequence identity to SEQ ID NO: 6, or (iv) the NtGDH7 polynucleotide consists of or essentially consists of a sequence containing at least 92% sequence identity to SEQ ID NO: 9, or (v) the NtGDH7 polypeptide is (iv) a polynucleotide encoded by the polynucleotide described in (iv), or (vi) an NtGDH7 polypeptide having at least 96% sequence identity to SEQ ID NO: 10, or (vii) an NtGDH8 polynucleotide comprising a sequence having at least 92% sequence identity to SEQ ID NO: 11, or (viii) an NtGDH8 polypeptide encoded by the polynucleotide described in (i), or (ix) an NtGDH8 polypeptide having at least 95% sequence identity to SEQ ID NO: 12, or (x) an NtGDH11 polynucleotide comprising a sequence having at least 87% sequence identity to SEQ ID NO: 17, or (xi) an NtGDH11 polypeptide encoded by the polynucleotide described in (i), or (xii) an NtGDH11 polypeptide having at least 92% sequence identity to SEQ ID NO: 18.

[0024] Preferably, the tobacco plant leaves or a part of the plant leaves are Nicotiana tabacum plant leaves or a part of the plant leaves.

[0025] Preferably, in step (b), the expression or activity is regulated by genome editing, and preferably, 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 in step (b), the expression or activity is regulated using an interfering polynucleotide.

[0026] In another aspect, provided are dried processed mutants, non-natural or transgenic Nicotiana tabacum plant leaves or parts thereof, obtainable or available by the methods described herein.

[0027] In another aspect, provided are dried processed tobacco plant leaves or parts of dried processed plant leaves, wherein (i) the ammonia content is between about 0.16 ± 0.04% dry weight basis (DWB) to 0.11 0.16 ± 0.03% DWB, (ii) the glucose, fructose and sucrose content is from 0.51 ± 0.58% DWB to 1.55 ± 1.10% DWB, (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 4.2 ± 0.39%.

[0028] In another aspect, provided is a dried processed tobacco blend comprising at least two different types of dried processed tobacco, wherein at least one of the dried processed tobaccos is a mutant, non-natural or transgenic tobacco plant leaf or a part of such plant leaf, or a dried processed mutant, non-natural or transgenic Nicotiana tabacum plant leaf or a part of such plant leaf, or a dried processed tobacco from a dried processed tobacco plant leaf or a part of a dried processed plant leaf.

[0029] Preferably, at least one other dried tobacco is Burley tobacco, or Oriental tobacco, or Dark tobacco, or Flue-cured tobacco, or a combination of two or more of them.

[0030] In a further aspect, a method for producing a tobacco blend with a reduced amount of ammonia is provided, the method comprising: (a) providing a dried tobacco plant leaf or a part thereof, wherein the first dried tobacco plant leaf or a part thereof is a variant, non-natural or transgenic tobacco plant leaf or a part thereof according to any claim, or a dried variant, non-natural or transgenic Nicotiana tabacum plant leaf or a part thereof, or from a dried tobacco plant leaf or a part of a dried plant leaf; and (b) blending the first dried tobacco plant leaf or a part thereof with at least one second dried tobacco plant leaf or a part thereof to produce a tobacco blend having a total amount of ammonia lower than the total amount of ammonia in the at least one second dried tobacco plant leaf or a part thereof.

[0031] A dried tobacco blend obtainable or available by the method of claim 26.

[0032] In a further aspect, a tobacco product or smoking article is provided, comprising a variant, non-natural or transgenic tobacco plant leaf or a part thereof, or a dried variant, non-natural or transgenic Nicotiana tabacum plant leaf or a part thereof, or a dried tobacco plant leaf or a part of a dried plant leaf, or a dried form of a dried tobacco blend.

[0033] Some advantages Regulating the expression and / or activity of the specific NtGDH described herein results in dried plant material having lower levels of ammonia, and thus a tobacco material that is less toxic to the consumer.

[0034] Modulation of the expression and / or activity of specific NtGDHs described herein may result in regulated levels of sugars and amino acids in dried plant material. This may result in tobacco with novel aromatic or sensory properties.

[0035] Modulation of the expression and / or activity of specific NtGDHs described herein may result in regulated levels of total alkaloids in dried plant material.

[0036] Since modified plants do not change in biomass or plant height, their yields remain unchanged, making them valuable for commercial plant production.

[0037] This invention allows for the blending of "high" ammonia-dried tobacco with the lower ammonia-dried tobacco of the present invention, thereby reducing the overall ammonia level in the blend.

[0038] Advantageously, it becomes possible to create non-genetically modified plants, which may be more acceptable to consumers.

[0039] Advantageously, this disclosure is not limited to the use of EMS mutant plants. EMS mutant plants may have a low probability of resulting in improved traits in the bred crop. Once breeding is initiated, desirable traits of EMS mutant plants may be lost for various reasons. For example, several mutations may be required, and these mutations may be dominant or recessive, and identifying point mutations in gene targets can be difficult. In contrast, this disclosure utilizes the use of NtGDH, which can be specifically manipulated to produce plants with a desired phenotype. [Brief explanation of the drawing]

[0040] [Figure 1]Figure 1 shows a series of graphs illustrating the expression of NtGDH, SAG12, and SGR1 during the first 8 drying days (0–192 hours) of Stella leaves hanging in a barn, corresponding to the initial drying stage (which also marks the onset of tobacco ammonia accumulation). Data were collected from RNA-seq data and are expressed as FPKM values. SAG12 and SGR1 are markers of cellular senescence. [Figure 2] Figure 2 is a graph showing the expression of NtGDH2 and NtGDH3 in tobacco leaves (TN90, Burley background) after 48 hours of drying treatment following gene silencing using an RNAi approach (GATEWAY vector). Lines E459-2, E459-3, and E459-5 (T1 plants) were selected for chemical analysis for comparison with control plants. [Figure 3-1] Figure 3 is a series of box plots to visualize the chemical data presented in Table 1 for ammonia, total sugars, aspartic acid, and proline. [Figure 3-2] Figure 3 is a series of box plots to visualize the chemical data presented in Table 1 for ammonia, total sugars, aspartic acid, and proline. [Figure 4] Figure 4 shows a series of box plots illustrating leaf biomass and height in the E459-2, E459-3, and E459-5 lines and the control. Leaf biomass was calculated after weighing four mature upper-middle leaves at harvest, and no statistically significant differences were observed between the control and the anti-NtGDH2-3 lines. Plant height was also determined at harvest, and no significant differences were found between the lines. [Modes for carrying out the invention]

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art. In case of any conflict, this specification, including its definitions, shall prevail. Preferred methods and materials are described below, but similar or equivalent methods and materials may also be used in carrying out or testing the present invention. The materials, methods and examples disclosed herein are for illustrative purposes only and are not intended to be limiting.

[0042] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and their variations are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional actions or structures.

[0043] Unless the context explicitly indicates otherwise, the singular forms "a," "and," and "the" include multiple references.

[0044] This disclosure also intends other embodiments that "include," "consist of," and "essentially consist of" the embodiments or elements presented herein, whether expressly or not. "Essentially consisting of" is used to mean that further components may exist, but only to the extent that these components do not substantially affect the essential characteristics.

[0045] Where numerical ranges are described herein, individual numbers within that range with the same precision are explicitly intended. For example, for the range 6–9, the digits 7 and 8 are assumed in addition to the digits 6 and 9, and for the range 6.0–7.0, the digits 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly assumed.

[0046] When used throughout the specification and claims, the following terms have the following meanings:

[0047] "Code sequence" or "encoding a polynucleotide" means the nucleotides (RNA or DNA molecules) that make up the polynucleotide encoding the polypeptide. The code sequence may further include start and termination signals functionally bound to a regulatory element containing a promoter and a polyadenylation signal that can direct the expression of the polynucleotide in cells of an individual or mammal to which it is administered. The code sequence may be codon-optimized.

[0048] "Complementary" or "complementary" can refer to Watson-Crick base pairings (e.g., AT / U and CG) or Hoogsteen base pairings between nucleotides or nucleotide analogs. "Complementarity" refers to a property shared between two polynucleotides, where the nucleotide bases at each position are complementary when they are aligned antiparallel to each other.

[0049] A “construct” refers to a double-stranded recombinant polynucleotide fragment containing one or more polynucleotides. A construct includes a “template strand” that is base-paired with a complementary “sense strand or coding strand.” A given construct can be inserted into a vector (such as an expression vector) in one of two possible orientations: the same (or sense) orientation or the opposite (or antisense) orientation with respect to the orientation of the promoter placed within the vector.

[0050] In the context of control plants or control plant cells, the term "control" means a plant or plant cell in which the expression, function, or activity of one or more genes or polypeptides has not been modified (e.g., not increased or decreased), and thus 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 the test plant or modified plant in all parameters except the test parameters. For example, if referring to a plant into which a polynucleotide has been introduced, the control plant is an equivalent plant in which such a polynucleotide has not been introduced. A control plant may be an equivalent plant into which a control polynucleotide has been introduced. In such a case, the control polynucleotide is expected to have little or no phenotypic effect on the plant. A control plant may contain an empty vector. A control plant may correspond to a wild-type plant. A control plant may be a null isolate in which the T1 isolate no longer possesses the introduced gene.

[0051] The term “reduction” or “reduced” refers to a reduction of approximately 10% to approximately 99%, or at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100%, at least 150%, or at least 200% or more in quantity or function (such as polypeptide function, transcriptional function, or polypeptide expression). The term “reduced,” or phrase “reduced amount,” may refer to an amount or function less than what would be found in an unmodified, same-cultivated plant or product of the same variety. Thus, in some contexts, a wild-type plant of the same variety treated in the same manner is used as a control to measure whether a reduction in quantity is obtained.

[0052] "Donor DNA" or "donor template" refers to a double-stranded DNA fragment or molecule containing at least a portion of the target gene. The donor DNA may also encode a functional polypeptide.

[0053] "Endogenous genes or endogenous polypeptides" refer to genes or polypeptides that originate from the genome of an organism and have not undergone changes such as loss, acquisition, or exchange of genetic material. Endogenous genes undergo normal gene transfer and gene expression. Endogenous polypeptides undergo normal expression.

[0054] An "enhancer sequence" refers to a sequence that can increase gene expression. These sequences can be located upstream, within introns, or downstream of the transcription region. The transcription region consists of exons and intervening introns, from the promoter to the transcription termination region. Enhancement of gene expression can be achieved through various mechanisms, including increased transcription efficiency, stabilization of mature mRNA, and improved translation efficiency.

[0055] The term "exogenous" is used interchangeably with "heterogeneous" and refers to polynucleotides artificially supplied to a biological system (such as a plant). Exogenous polynucleotides may be derived from different plants or plant species.

[0056] "Expression" refers to the production of a functional product. For example, the expression of a polynucleotide fragment can refer to the transcription of the polynucleotide fragment (e.g., transcription resulting in mRNA or functional RNA), or the translation of mRNA into a precursor or mature polypeptide, or a combination thereof.

[0057] "Overexpression" refers to the production of a gene product in a transgenic organism that exceeds the production levels in a null isolate (or non-transgenic organism) derived from the same experiment.

[0058] "Functionality" describes polypeptides that possess biological function or activity. "Functional gene" refers to a gene that is transcribed into mRNA and translated into a functional or active polypeptide.

[0059] A "gene construct" refers to a DNA or RNA molecule containing polynucleotides that encode a polypeptide. The coding sequence may include start and termination signals functionally bound to regulatory elements, including a promoter and polyadenylation signal, which can lead to expression.

[0060] "Genome editing" generally refers to the process by which genomic nucleic acids within a cell are modified. This can be done, for example, by removing, inserting, or substituting one or more nucleotides in the genomic nucleic acid. Endonucleases can be used to create specific cuts or nicks at defined locations within the genome, which are further described herein.

[0061] The term "homology" or "similarity" refers to the degree of sequence similarity between two polypeptides or polynucleotide molecules compared by sequence alignment. The degree of homology between two separate polynucleotides being compared is a function of the number of identical or matching nucleotides at comparable positions. Homology or similarity can be determined over the entire length of the sequences in question.

[0062] In the context of two or more polynucleotides or polypeptides, "identical" or "same" means that the sequences have a certain percentage of the same residues across a particular region. The percentage can be calculated by optimally aligning the two sequences, comparing them across a specific region, determining the number of positions where identical residues exist in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the specific region, and multiplying the result by 100 to obtain the percentage of sequence identity. If the two sequences are of different lengths, or if the alignment results in one or more staggered ends and the particular comparison region contains only a single sequence, the residues of the single sequence are included in the denominator of the calculation but not in the numerator. When comparing DNA and RNA, thymine (T) and uracil (U) may be considered equivalent. Identity can be determined manually or using computer sequencing algorithms such as ClustalW, ClustalX, BLAST, FASTA, or Smith-Waterman. The following parameters may be preferred for ClustalW: For polynucleotide alignment, gap open penalty = 15.0, gap elongation penalty = 6.66, and matrix = Identity. For polypeptide alignment, gap open penalty = 10.0, gap elongation penalty = 0.2, and matrix = Gonnet. For DNA and protein alignment, ENDGAP = -1 and GAPDIST = 4.

[0063] The term “increase” or “increased” refers to an increase of approximately 10% to approximately 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 in quantity, function, or activity, and without limitation, an increase in one or more of polypeptide function or activity, transcriptional function or activity, and polypeptide expression. The term “increased” or phrase “increased amount” may refer to an amount, function, or activity in a plant or product derived from a plant that is greater than the amount that would be found in an unmodified, same-cultivated plant or product of the same variety. Thus, in some contexts, a wild-type plant of the same variety treated in the same manner is used as a control to measure whether an increase in quantity is obtained.

[0064] The term “inhibit” or “inhibited” refers to a reduction of approximately 98% to approximately 100%, or at least 98%, at least 99%, and especially 100%, in quantity, function, or activity, including, but not limited to, a reduction of one or more polypeptide functions or activities, transcriptional functions or activities, and polypeptide expression.

[0065] The term “introduced” can mean providing a polynucleotide (e.g., a construct) or polypeptide to a cell. “Introduced” refers to the incorporation of a polynucleotide into a eukaryotic cell, where the polynucleotide may be incorporated into the cell’s genome, and also refers to the transient provision of a polynucleotide or polypeptide to a cell. Introduced includes methods of stable or transient transformation, as well as sexual cross-pollination. Therefore, in the context of the insertion of a polynucleotide (e.g., a recombinant construct / expression construct) into a cell, “introduced” means “transfection,” “transformation,” or “transduction,” referring to the incorporation of a polynucleotide into a eukaryotic cell (e.g., chromosome, plasmid, plastid, or mitochondrial DNA), conversion to an autonomous replicator, or transient expression (e.g., transfected mRNA), where the polynucleotide may be incorporated into the cell’s genome.

[0066] The terms “isolated” or “purified” refer to materials that are substantially or essentially free from the components that would normally accompany them if found in their natural state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high-performance liquid chromatography. The polypeptide that is the dominant species present in the preparation is substantially purified. In particular, isolated polynucleotides are separated from open reading frames that are adjacent to the desired gene and encode polypeptides different from the desired polypeptide. The term “purified” indicates that the polynucleotide or polypeptide produces essentially a single band in an electrophoretic gel. In particular, it means that the polynucleotide or polypeptide is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure. Isolated polynucleotides can be purified from host cells in which they naturally occur. Isolated polynucleotides can be obtained using conventional polynucleotide purification methods known to those skilled in the art. This term also includes recombinant polynucleotides and chemically synthesized polynucleotides.

[0067] "Liquid tobacco extract" describes the direct product of an extraction process performed on tobacco starting material. An extraction process for producing a liquid tobacco extract may include heating the tobacco starting material under specific heating conditions and collecting the resulting volatile compounds. A liquid tobacco extract may typically contain a mixture of compounds derived from the tobacco starting material and removed during the extraction process, often in combination with a liquid carrier or solvent.

[0068] "To regulate" or "to modulate" means to cause or facilitate a qualitative or quantitative change, alteration, or modification in the process, pathway, function, or activity of interest. Without limitation, such change, alteration, or modification may be an increase (e.g., upregulation) or decrease (e.g., downregulation) in the relative process, pathway, function, or activity of interest of interest. For example, gene expression or polypeptide expression or polypeptide function or activity may be regulated. Typically, relative change, alteration, or modification is determined by comparison with a control.

[0069] The term “non-natural” refers to entities (such as polynucleotides, genetic mutations, polypeptides, plants, plant cells, and plant materials) that are not formed naturally or do not exist in nature. Such non-natural or artificial entities may be produced, synthesized, initiated, modified, intervened in, or manipulated by methods described herein or by methods known in the art. Such non-natural or artificial entities may be produced, synthesized, initiated, modified, intervened in, or manipulated by humans. Thus, non-natural plants may not be produced using biological processes in themselves. For example, non-natural plants, non-natural plant cells, or non-natural plant materials may be produced using conventional plant breeding techniques (such as backcrossing) or genetic engineering techniques (such as antisense RNA, interfering RNA, and meganucleases). As a further example, non-natural plants, non-natural plant cells, or non-natural plant materials may be produced by the introduction or transfer of one or more genetic variations (e.g., one or more polymorphisms) from a first plant or plant cell to a second plant or plant cell (which may itself be naturally occurring), and the resulting plant, plant cell, plant material, or offspring shall contain a genetic configuration (e.g., genome, chromosome, or part thereof) that is not formed naturally or does not exist in nature. The resulting plant, plant cell, or plant material is therefore artificial or non-natural. Thus, artificial or non-natural plants or plant cells may be produced by altering the gene sequence in a first natural plant or plant cell, even if the resulting gene sequence is naturally occurring in a second plant or plant cell having a different genetic background than the first plant or plant cell. In certain embodiments, the variations are not naturally occurring variations that exist naturally in polynucleotides or polypeptides (such as genes or polypeptides). Differences in genetic background can be detected by differences in phenotype or by molecular biological techniques known in the art (such as polynucleotide sequencing, the presence or absence of genetic markers (e.g., microsatellite RNA markers)).

[0070] An "oligonucleotide" or "polynucleotide" means that at least two nucleotides are linked by a covalent bond. A single-stranded description also defines the sequence of the complementary strand. Therefore, a polynucleotide also includes the complementary strand of the described single-stranded sequence. Many variants of a polynucleotide can be used for the same purposes as a given polynucleotide. Therefore, a polynucleotide also includes substantially identical polynucleotides and their complements. A single strand provides a probe that can hybridize to a given sequence under stringent hybridization conditions. Therefore, a polynucleotide also includes probes that hybridize under stringent hybridization conditions. A polynucleotide may be single-stranded or double-stranded, or may contain portions of both double-stranded and single-stranded sequences. Polynucleotides may be both genomic DNA and cDNA, RNA, or hybrids, and may include combinations of deoxyribonucleotides and ribonucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. Polynucleotides can be obtained by chemical synthesis or recombinant methods.

[0071] The specificity of single-strand DNA to hybridize to 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)). Hybridizing under "stringent conditions" describes a hybridization protocol in which polynucleotides that are at least 60% homologous to each other remain hybridized. Generally, stringent conditions are selected to be about 5°C lower than the thermal fusion temperature (Tm) of a particular sequence at a defined ionic strength and pH. Tm is the temperature at which 50% of the probes complementary to a given sequence (at a defined ionic strength, pH, and polynucleotide concentration) hybridize to the given sequence at equilibrium. Since a given sequence is generally in excess, at Tm, 50% of the probes are occupied at equilibrium. Stringent conditions typically include (1) low ionic strength and high-temperature washing, e.g., 15 mM sodium chloride, 1.5 mM sodium citrate, and 0.1% sodium dodecyl sulfate at 50°C; (2) denaturing agents during hybridization at 42°C, e.g., 50% (v / v) formamide, 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, and 50 mM sodium phosphate buffer (750 mM sodium chloride, 75 mM sodium citrate; pH 6.5); or (3) 50% formamide. Washing also typically includes washing in 0.2×SSC (sodium chloride / sodium citrate) at 42°C, washing in 50% formamide at 55°C, and subsequent high-stringency washing in EDTA-containing 0.1×SSC at 55°C, comprising 5×SSC (0.75M NaCl, 75mM sodium citrate), 50mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt solution, sonicated salmon sperm DNA (50 μg / mL), 0.1% SDS, and 10% dextran sulfate, at 42°C. Preferably, the conditions are such that sequences that are at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous to each other typically remain hybridized.

[0072] "Moderately stringent conditions" refer to conditions where the washing solution and hybridization conditions are low-stringency, and where polynucleotides hybridize to whole polynucleotides, fragments, derivatives, or analogues. One example includes hybridization in 6×SSC, 5×Denhardt solution, 0.5% SDS, and 100 μg / mL denatured salmon sperm DNA at 55°C, followed by one or more washes in 1×SSC, 0.1% SDS at 37°C. Temperature, ionic strength, etc., can be adjusted to accommodate experimental factors such as probe length. Other moderate stringency conditions are described (see Ausubel et al., Current Protocols in Molecular Biology, Volumes 1-3, John Wiley & Sons, Inc., Hoboken, NJ (1993); Kriegler, Gene Transfer and Expression: A Laboratory Manual, Stockton Press, New York, NY (1990); Perbal, A Practical Guide to Molecular Cloning, 2nd edition, John Wiley & Sons, New York, NY (1988)).

[0073] "Low-stringent conditions" refer to conditions in which the washing solution and hybridization conditions are lower than moderate stringency, and in which polynucleotides hybridize into whole polynucleotides, fragments, derivatives, or analogues. Non-limiting examples of low-stringency hybridization conditions include hybridization in 35% formamide, 5×SSC, 50 mM Tris HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg / mL denatured salmon sperm DNA, and 10% (g / vol) dextran sulfate at 40°C, followed by one or more washes in 2×SSC, 25 mM Tris HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS at 50°C. Other low-stringency conditions, such as interspecies hybridization, have been well documented (see Ausubel et al., 1993; Kriegler, 1990).

[0074] "Functionally coupled" means that the expression of a gene is under the control of a promoter that is spatially coupled to it. The promoter may be located 5' (upstream) or 3' (downstream) of the gene it controls. The distance between the promoter and the gene may be approximately the same as the distance between the promoter and the gene it controls in the gene from which the promoter originates. As is known in the art, variations in this distance can be accommodated without loss of promoter function. "Functionally coupled" refers to the association of polynucleotide fragments in a single fragment, resulting in the regulation of one function by the other. For example, if a promoter can regulate the transcription of a polynucleotide fragment, then the promoter is functionally coupled to the polynucleotide fragment.

[0075] The term “plant” refers to any plant and its offspring at any stage of its life cycle or development. In one embodiment, plant is tobacco, which refers to plants belonging to the genus Nicotiana, preferably Nicotiana tabacum. The term refers to the whole plant, plant organs, plant tissues (such as leaves or parts thereof), plant reproductive bodies, plant seeds, plant cells and their offspring. Plant cells include, but are not limited to, cells from seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, stems, gametophytes, sporophytes, pollen, and microspores. Preferred species, cultivars, hybrids, and varieties of tobacco plants are described herein.

[0076] "Plant material" includes leaves, roots, sepals, root tips, petals, flowers, shoots, stems, seeds, and stalks. Plant material may be viable or non-viable plant material.

[0077] "Polynucleotide," "polynucleotide sequence," or "polynucleotide fragment" are used interchangeably herein and refer to polymers of single-stranded or double-stranded RNA or DNA, optionally comprising synthetic, unnatural, or modified nucleotide bases. The polynucleotides of this disclosure are listed in the accompanying sequence list.

[0078] A "polypeptide" or "polypeptide sequence" refers to a polymer of amino acids in which one or more amino acid residues are artificial chemical analogs of corresponding naturally occurring amino acids, as well as polymers of naturally occurring amino acids. The term also includes, without limitation, modifications including glycosylation, lipid addition, sulfated, gamma-carboxylated, hydroxylated, and ADP-ribosylated glutamate residues. The polypeptides of this disclosure are listed in the attached sequence list.

[0079] A “promoter” refers to a synthetic or naturally occurring molecule that can confer, activate, or enhance the expression of a polynucleotide in a cell. The term typically refers to a functionally linked polynucleotide element / sequence located upstream of a double-stranded polynucleotide fragment. A promoter may be entirely derived from a region adjacent to the native gene of interest, or it may consist of different elements derived from different native promoters or synthetic polynucleotide segments. A promoter may include one or more specific transcriptional regulatory sequences to further enhance expression, alter spatial expression, or alter temporal expression. A promoter may also include distal enhancer or repressor elements that can be located thousands of base pairs away from the transcription start site. Promoters may be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. A promoter may constitutively or differentially regulate the expression of a gene component with respect to the cell, tissue, or organ in which expression occurs, or with respect to the developmental stage in which expression occurs, or it may be regulated in response to external stimuli such as physiological stress, pathogens, metal ions, or inducers.

[0080] As used interchangeably herein, “tissue-specific promoter” and “tissue-preferential promoter” refer to promoters that are primarily expressed in a single tissue or organ, but not necessarily exclusively, and may also be expressed in a single specific cell. “Developmentally regulated promoter” refers to a promoter whose function is determined by developmental events. “Constitutive promoter” refers to a promoter that expresses a gene in most cases in most cell types. “Inducible promoter” selectively expresses a functionally bound DNA sequence in response to the presence of endogenous or exogenous stimuli (e.g., by chemical compounds (chemical inducers), or in response to the environment, hormones, chemical or developmental signals, or a combination of these). Examples of inducible or regulated promoters include promoters regulated by light, heat, stress, flooding or drought, pathogens, phytohormones, injury, or chemicals such as ethanol, jasmonic acid, salicylic acid, or safeners.

[0081] "Recombination" refers to the artificial combination of two separately separated sequence segments, such as by chemical synthesis or by manipulating isolated polynucleotide segments using genetic engineering techniques. This term also includes cells or vectors modified by the introduction of polynucleotides of a different species, or cells derived from such modified cells, but does not include modifications of cells or vectors by naturally occurring events that occur without intentional human intervention (e.g., spontaneous mutation, spontaneous transformation, or transduction or translocation).

[0082] A "recombinant construct" refers to a combination of polynucleotides that are not typically found together in nature. Therefore, a recombinant construct may include regulatory and coding sequences from different sources, or regulatory and coding sequences from the same source but arranged in a manner different from that typically found in nature. A recombinant construct may also be a recombinant DNA construct.

[0083] As used interchangeably herein, “regulatory sequence” and “regulatory element” refer to polynucleotide sequences located upstream (5' non-coding sequence), within, or downstream (3' non-coding sequence) of a coding sequence that affect the transcription, RNA processing, stability, or translation of the associated coding sequence. Regulatory sequences include promoters, translational leader sequences, introns, and polyadenylation recognition sequences. The terms “regulatory sequence” and “regulatory element” are used interchangeably herein.

[0084] The term “tobacco” is used in a collective sense to refer to tobacco crops (e.g., multiple tobacco plants grown in fields, not hydroponically), tobacco plants and parts thereof, including but not limited to the preparations or obtained herein, including roots, stems, leaves, flowers, and seeds. “Tobacco” is understood to refer to plants belonging to the genus Nicotiana and their products, including the Nicotiana tabacum plant and its products.

[0085] The term “tobacco products” refers to consumer tobacco products, including but not limited to smoking materials (e.g., cigarettes, cigars, and pipe tobacco), snuff, chewing tobacco, gum, and lozenges, as well as components, materials, and ingredients for the manufacture of consumer tobacco products. Preferably, these tobacco products are manufactured from tobacco leaves and stems harvested from the tobacco plant and cut, dried, desiccated, or fermented according to conventional techniques in tobacco preparation.

[0086] A "transcription terminator," "termination sequence," or "terminator" refers to a DNA sequence located downstream of a coding sequence, including a polyadenylation recognition sequence and other sequences that encode regulatory signals that can affect mRNA processing or gene expression. Polyadenylation signals are typically characterized by their influence on the addition of polyadenylate to the 3' end of mRNA precursors.

[0087] "Transgenic" refers to any cell, cell line, callus, tissue, plant part, or plant whose genome has been modified by the presence of heterologous polynucleotides, such as recombinant constructs, and includes those produced by the initial transgenic event and those produced by sexual hybridization or asexual reproduction from the initial transgenic event. This term does not include modifications of the genome (chromosome or extrachromosome) by conventional plant breeding methods or naturally occurring events (such as random cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation). Therefore, in embodiments, the transgenic plant or part thereof is not produced using essentially biological processes.

[0088] A "transgenic plant" is a plant whose genome contains one or more heterologous polynucleotides, i.e., a plant containing recombinant genetic material that would not normally be found, introduced into the plant (or its ancestors) through human (artificial) manipulation. For example, heterologous polynucleotides can be stably integrated into the genome so that the polynucleotides are passed on to subsequent generations. Heterologous polynucleotides can be integrated into the genome alone or as part of a recombinant construct. The commercial development of genetically improved germplasm has also progressed to the stage of introducing multiple traits into crop plants, often called the gene stacking approach. In this approach, multiple genes conferring different characteristics of interest can be introduced into a plant. Gene stacking can be achieved by many means, including but not limited to cotransformation, retransformation, and crossbreeding of lines with different transgenes. Therefore, a plant grown from plant cells into which recombinant DNA has been introduced by transformation is a transgenic plant, and all of its offspring (whether produced sexually or asexually) containing the introduced transgenes are also transgenic plants. The term "transgenic plant" is understood to include the entire plant or tree, as well as parts of the plant or tree, such as grains, seeds, flowers, leaves, roots, fruits, pollen, and stems. Each heterologous polynucleotide can confer different traits to the transgenic plant.

[0089] A "transgene" refers to a gene or genetic material containing a gene sequence that has been isolated from one organism and introduced into another organism. This foreign DNA segment may retain the ability to produce RNA or polypeptides in the transgenic organism, or it may alter the normal function of the transgenic organism's genetic code.

[0090] With respect to polynucleotides, “variant” means (i) a part or fragment of a polynucleotide, (ii) a complement of a polynucleotide or a part thereof, (iii) a polynucleotide substantially identical to the polynucleotide in question or its complement, or (iv) a polynucleotide that hybridizes to the polynucleotide in question, its complement, or a polynucleotide substantially identical thereto under stringent conditions.

[0091] With respect to peptides or polypeptides, a “variant” means a peptide or polypeptide whose sequence differs due to an insertion, deletion, or conservative substitution of amino acids, but which retains at least one biological function or activity. A variant may also mean a polypeptide that retains at least one biological function or activity. Conservative substitutions of amino acids, i.e., substitutions with different amino acids that have similar properties (e.g., hydrophilicity, degree and distribution of charged regions), are typically recognized in the art as resulting in minor changes.

[0092] The term "cultivar" refers to a group of plants that share certain characteristics that distinguish them from other plants of the same species. While possessing one or more distinctive traits, cultivars are further characterized by very little overall variation among individuals within that cultivar. Cultivars are often sold commercially.

[0093] "Vector" refers to a polynucleotide medium containing a combination of polynucleotide components to enable the transport of polynucleotides, polynucleotide constructs, and polynucleotide complexes, etc. A vector may be a viral vector, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. A vector may be a DNA or RNA vector. Suitable vectors include episomes that enable extrachromosomal replication, such as circular double-stranded nucleotide plasmids, linear double-stranded nucleotide plasmids, and other vectors of any origin. "Expression vector" is a polynucleotide medium containing a combination of polynucleotide components to enable the expression of polynucleotides, polynucleotide constructs, and polynucleotide complexes, etc. Suitable expression vectors include episomes that enable extrachromosomal replication, such as circular double-stranded nucleotide plasmids, linear double-stranded nucleotide plasmids, and other functionally equivalent expression vectors of any origin. An expression vector includes at least a promoter located upstream of the polynucleotide, polynucleotide construct, or polynucleotide complex and functionally coupled, as defined below.

[0094] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have meanings generally understood by those skilled in the art. For example, any nomenclature and techniques used in connection with cell and tissue culture, molecular biology, plant biology, microbiology, genetics, and polypeptide and polynucleotide chemistry, as well as hybridization, as described herein, are well known and commonly used in the art. The meaning and scope of terms should be clear, but in the event of any potential ambiguity, the definitions provided herein shall prevail over any dictionary or external definitions. Furthermore, unless otherwise required by context, singular terms shall include plural terms and plural terms shall include singular terms.

[0095] Disclosed are isolated polynucleotides comprising, or essentially comprising, a sequence having at least 60% sequence identity to any of the sequences described herein, including any polynucleotides shown in the sequence list. Preferably, an isolated polynucleotide comprises, or essentially comprises, 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 to it. Preferably, the isolated polynucleotide consists of, or essentially consists 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. Preferably, the isolated polynucleotide consists of, or essentially consists of, a sequence having at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto.

[0096] Preferably, the polynucleotides described herein encode active polypeptides having at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or more of the NtGDH function or activity of the polypeptide shown in the sequence list.

[0097] In another embodiment, sequence number 1 (NtGHD2) has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or sequence number 3 (NtGDH3) has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or sequence number 5 (NtGDH4) has at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or sequence number 7 (NtGDH6) has at least 90%, 91%, 92%, 93%, 94%, 95% Sequence identity of 96%, 97%, 98%, 99%, or 100%, or at least 92%, 93%, 94%, or 95% in sequence number 9 (NtGDH7) Sequence identity of 96%, 97%, 98%, 99%, or 100%, or at least 92%, 93%, 94%, or 95% in sequence number 11 (NtGDH8) Sequence identity of 96%, 97%, 98%, 99%, or 100%, or at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95% in sequence number 13 (NtGDH9) Sequence identity of 96%, 97%, 98%, 99%, or 100%, or at least 89%, 90%, 91%, 92%, 93%, 94%, or 95% in sequence number 15 (NtGDH10) Sequence identity of 96%, 97%, 98%, 99%, or 100%, or at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95% in sequence number 17 (NtGDH11) Sequence identity of 96%, 97%, 98%, 99%, or 100%, or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95% in sequence number 19 (NtGDH12) Isolated NtGDH polynucleotides from Nicotiana tabacum, comprising, or essentially comprising, polynucleotides having 96%, 97%, 98%, 99%, or 100% sequence identity, are provided.

[0098] In another embodiment, a polynucleotide is provided comprising, or essentially comprising, a polynucleotide having substantial homology (i.e., sequence similarity) or substantial identity with SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 19.

[0099] In another embodiment, the corresponding fragment 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 and at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% A fragment of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 19 is provided, having substantial homology (i.e., sequence similarity) or substantial identity with respect to SEQ ID NO: 1, SEQ ID NO: 19, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 19, having substantial homology (i.e., sequence similarity) or substantial identity with respect to SEQ ID NO: 1, SEQ ID NO: 199, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 19, SEQ ID NO: 19, SEQ ID NO: 19, SEQ ID NO: 19, SEQ ID NO: 19, SEQ ID NO: 19, SEQ ID NO: 19, SEQ ID NO: 19, SEQ ID NO: 19, SEQ ID NO: 19, SEQ ID NO: 11, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 19, SEQ ID NO: 19

[0100] In another embodiment, a fragment of sequence number 1 is provided that has substantial homology (i.e., sequence similarity) or substantial identity with respect to the fragment corresponding to sequence number 1, having 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 with respect to the fragment of sequence number 1.

[0101] In another embodiment, a fragment of sequence number 3 is provided that has substantial homology (i.e., sequence similarity) or substantial identity with respect to the corresponding fragment of sequence number 3, having 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 with respect to the corresponding fragment of sequence number 3.

[0102] In another embodiment, a fragment of sequence number 5 is provided that has substantial homology (i.e., sequence similarity) or substantial identity with respect to the corresponding fragment of sequence number 5, having 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 with respect to the corresponding fragment of sequence number 5.

[0103] In another embodiment, a fragment of sequence number 7 is provided that has substantial homology (i.e., sequence similarity) or substantial identity with respect to the corresponding fragment of sequence number 7, having 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 with respect to the corresponding fragment of sequence number 7.

[0104] In another embodiment, a fragment of sequence number 9 is provided that has substantial homology (i.e., sequence similarity) or substantial identity with respect to the corresponding fragment of sequence number 9, having 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 with respect to the corresponding fragment of sequence number 9.

[0105] In another embodiment, a fragment of sequence number 11 is provided that has substantial homology (i.e., sequence similarity) or substantial identity with respect to the corresponding fragment of sequence number 11, having 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 with respect to the corresponding fragment of sequence number 11.

[0106] In another embodiment, a fragment of sequence number 13 is provided that has substantial homology (i.e., sequence similarity) or substantial identity with respect to the corresponding fragment of sequence number 13, having 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 with respect to the corresponding fragment of sequence number 13.

[0107] In another embodiment, a fragment of sequence number 15 is provided that has substantial homology (i.e., sequence similarity) or substantial identity with respect to the corresponding fragment of sequence number 15, having 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 with respect to the corresponding fragment of sequence number 15.

[0108] In another embodiment, a fragment of sequence number 17 is provided that has substantial homology (i.e., sequence similarity) or substantial identity with respect to the corresponding fragment of sequence number 17, having 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 with respect to the corresponding fragment of sequence number 17.

[0109] In another embodiment, a fragment of sequence number 19 is provided that has substantial homology (i.e., sequence similarity) or substantial identity with respect to the corresponding fragment of sequence number 19, having 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 with respect to the corresponding fragment of sequence number 19.

[0110] In another embodiment, a polynucleotide is provided that has a sufficient or substantial degree of identity or similarity to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 19, which encodes a polypeptide that functions as NtGDH.

[0111] In another embodiment, a polymer of polynucleotides comprising, or essentially comprising, a polynucleotide designated herein as SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 19 is provided.

[0112] In accordance with the present invention, the expression of at least one NtGDH2 polynucleotide comprising, or essentially comprising, a sequence having at least 90% sequence identity with SEQ ID NO: 1, or at least 90% sequence identity with SEQ ID NO: 3, or at least 90% sequence identity with SEQ ID NO: 7, or at least 85% sequence identity with SEQ ID NO: 13, or at least 89% sequence identity with SEQ ID NO: 15, or at least 85% sequence identity with SEQ ID NO: 19, or a combination of two or more of these, is regulated.

[0113] In one embodiment, the expression of NtGDH6 polynucleotide and NtGDH10 polynucleotide is regulated.

[0114] In one embodiment, the expression of NtGDH2 polynucleotide and NtGDH3 polynucleotide is regulated.

[0115] In one embodiment, the expression of NtGDH9 polynucleotide and NtGDH12 polynucleotide is regulated.

[0116] In one embodiment, the expression of NtGDH2 polynucleotide, NtGDH3 polynucleotide, NtGDH9 polynucleotide, and NtGDH12 polynucleotide is regulated.

[0117] In one embodiment, the expression of NtGDH2 polynucleotide, NtGDH3 polynucleotide, NtGDH6 polynucleotide, NtGDH9 polynucleotide, NtGDH10 polynucleotide, and NtGDH12 polynucleotide is regulated.

[0118] In one embodiment, the expression of one or more NtGDH4 polynucleotides, NtGDH7 polynucleotides, NtGDH8 polynucleotides, or NtGDH11 polynucleotides is not regulated, and the NtGDH4 polynucleotide consists of or essentially consists of a sequence having at least 88% sequence identity with SEQ ID NO: 5, the NtGDH7 polynucleotide consists of or essentially consists of a sequence having at least 92% sequence identity with SEQ ID NO: 9, the NtGDH8 polynucleotide consists of or essentially consists of a sequence having at least 92% sequence identity with SEQ ID NO: 11, and the NtGDH11 polynucleotide consists of or essentially consists of a sequence having at least 87% sequence identity with SEQ ID NO: 17.

[0119] Preferably, the polynucleotides described herein encode NtGDH polypeptides having NtGDH activity.

[0120] Polynucleotides may include polymers of unmodified or modified deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Therefore, polynucleotides may, without limitation, be genomic DNA, complementary DNA (cDNA), mRNA, or antisense RNA, or fragments thereof. Furthermore, polynucleotides may be single-stranded or double-stranded DNA, DNA which is a mixture of single-stranded and double-stranded regions, hybrid molecules containing DNA and RNA, or hybrid molecules having a mixture of single-stranded and double-stranded regions, or fragments thereof. Furthermore, polynucleotides may consist of triple-stranded regions containing DNA, RNA, or both, or fragments thereof. Polynucleotides may contain one or more modified bases, such as phosphothioates, and may be peptide nucleic acids. Generally, polynucleotides may be constructed from isolated or cloned cDNA, genomic DNA, oligonucleotides, or fragments of individual nucleotides, or combinations thereof. The polynucleotides described herein are shown as DNA sequences, but include their corresponding RNA sequences and their complementary (e.g., fully complementary) DNA or RNA sequences, including their reverse complements.

[0121] The polynucleotide fragments can range from at least about 25 nucleotides, about 50 nucleotides, about 75 nucleotides, about 100 nucleotides, about 150 nucleotides, about 200 nucleotides, about 250 nucleotides, about 300 nucleotides, about 400 nucleotides, about 500 nucleotides, about 600 nucleotides, about 700 nucleotides, about 800 nucleotides, about 900 nucleotides, about 1000 nucleotides, about 1100 nucleotides, about 1200 nucleotides, about 1300 nucleotides, or about 1400 nucleotides to full-length polynucleotides encoding the polypeptides described herein.

[0122] Polynucleotides generally contain phosphodiester bonds, but in some cases, polynucleotide analogs may have alternative skeletons, such as phosphoramide, phosphorothioate, phosphorodithioate, or O-methylphosphoramidite bonds. Other analog polynucleotides include those with positively charged skeletons, nonionic skeletons, and non-ribose skeletons. Modification of the ribose-phosphate skeleton may be done for various reasons, such as to increase the stability and half-life of such molecules in a physiological environment, or as probes on biochips. Mixtures of naturally occurring polynucleotides and analogs can be prepared, or mixtures of different polynucleotide analogs, and mixtures of naturally occurring polynucleotides and analogs can be prepared.

[0123] Various polynucleotide analogs are known, including, for example, those linked to phosphoramidates, phosphorothioates, phosphorodithioates, O-methylphosphoamidite bonds, and peptide polynucleotide backbones. Other analog polynucleotides include those having cationic, nonionic, and non-ribose backbones. Polynucleotides containing one or more carbocyclic sugars are also mentioned.

[0124] Other analogs include peptide polynucleotide analogs, which are peptide polynucleotides.

[0125] Among the uses of the disclosed polynucleotides and their fragments is the use of fragments as probes or primers in hybridization assays for use in amplification assays. Such fragments generally contain at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more consecutive nucleotides of a DNA sequence. In other embodiments, the DNA fragment contains at least about 10, 15, 20, 30, 40, 50, or 60 or more consecutive nucleotides of a DNA sequence. Thus, in one embodiment, a method for detecting polynucleotides is also provided, which includes the use of probes or primers or both.

[0126] Basic parameters influencing the selection of hybridization conditions and guidelines for devising preferred conditions are described by Sambrook, J., E.Fritsch, and T. Maniatis (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). Using the knowledge of the genetic code combined with the polypeptide sequences described herein, a set of degenerate oligonucleotides can be prepared. Such oligonucleotides are useful, for example, as primers in polymerase chain reaction (PCR), thereby isolating and amplifying DNA fragments.

[0127] At least one modification (e.g., a mutation) may be included in one or more of SEQ ID NOs. 1, 3, 5, 7, 9, 11, 13, 15, 17, or 19.

[0128] Isolated NtGDH polypeptides encoded by the polynucleotides described herein are provided.

[0129] An isolated NtGDH polypeptide is provided, comprising or essentially comprising a polypeptide having at least 60% sequence identity to any polypeptide described herein, including any polypeptide shown in the sequence list. Preferably, the isolated polypeptide comprises or essentially comprises 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 to it. Preferably, the isolated NtGDH polypeptide consists of, or essentially consists 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. Preferably, the isolated NtGDH polypeptide consists of, or essentially consists 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.

[0130] Also provided are NtGDH polypeptides comprising, or essentially comprising, a sequence having at least approximately 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 in Sequence ID No. 2.

[0131] Also provided are NtGDH polypeptides comprising, or essentially comprising, a sequence having at least approximately 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 in sequence number 4.

[0132] Also provided are NtGDH polypeptides comprising, or essentially comprising, 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 in sequence number 6.

[0133] Also provided are NtGDH polypeptides comprising, or essentially comprising, a sequence having at least approximately 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 in sequence number 8.

[0134] Also provided are NtGDH polypeptides comprising, or essentially comprising, 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 in sequence number 10.

[0135] Also provided are NtGDH polypeptides comprising, or essentially comprising, 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 in sequence number 12.

[0136] Also provided are NtGDH polypeptides comprising, or essentially comprising, 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 in sequence number 14.

[0137] Also provided are NtGDH polypeptides comprising, or essentially comprising, a sequence having at least approximately 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 sequence number 16.

[0138] Also provided are NtGDH polypeptides comprising, or essentially comprising, 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 in sequence number 18.

[0139] Also provided are NtGDH polypeptides comprising, or essentially comprising, a sequence having at least approximately 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 in sequence number 20.

[0140] Also provided are NtGDH polypeptides comprising, or essentially comprising, 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 in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 19.

[0141] Polypeptides encoded by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 19 are also provided.

[0142] In order to function as NtGDH, the polypeptide may contain sequences that have a sufficient or substantial degree of identity or similarity to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 19.

[0143] In accordance with the present invention, the activity of at least one NtGDH polypeptide is regulated. The at least one NtGDH polypeptide may be an NtGDH2 polypeptide that is composed of or essentially composed of an NtGDH2 polynucleotide containing a sequence having at least 90% sequence identity in SEQ ID NO: 1, or an NtGDH2 polypeptide having at least 94% sequence identity in SEQ ID NO: 2; or an NtGDH3 polypeptide that is composed of or essentially composed of an NtGDH3 polynucleotide that is composed of a sequence having at least 90% sequence identity in SEQ ID NO: 3, or an NtGDH3 polypeptide having at least 94% sequence identity in SEQ ID NO: 4; or an NtGDH6 polypeptide that is composed of or essentially composed of an NtGDH6 polynucleotide that is composed of a sequence having at least 90% sequence identity in SEQ ID NO: 7, or an NtGDH6 polypeptide having at least 94% sequence identity in SEQ ID NO: 8. It may also be an NtGDH9 polypeptide comprising or essentially comprising an NtGDH9 polynucleotide containing a sequence having at least 85% sequence identity in SEQ ID NO: 13, or having at least 91% sequence identity in SEQ ID NO: 14; or an NtGDH10 polypeptide comprising or essentially comprising an NtGDH10 polynucleotide containing a sequence having at least 89% sequence identity in SEQ ID NO: 15, or having at least 94% sequence identity in SEQ ID NO: 16; or an NtGDH12 polypeptide comprising or essentially comprising an NtGDH12 polynucleotide containing a sequence having at least 85% sequence identity in SEQ ID NO: 19, or having at least 91% sequence identity in SEQ ID NO: 20.

[0144] In one embodiment, the activity of the NtGDH6 polypeptide and the NtGDH10 polypeptide is regulated.

[0145] In one embodiment, the activity of the NtGDH2 polypeptide and the NtGDH3 polypeptide is regulated.

[0146] In one embodiment, the activity of the NtGDH9 polypeptide and the NtGDH12 polypeptide is regulated.

[0147] In one embodiment, the activity of the NtGDH2 polypeptide, NtGDH3 polypeptide, NtGDH9 polypeptide, and NtGDH12 polypeptide is regulated.

[0148] In one embodiment, the activity of the NtGDH2 polypeptide, NtGDH3 polypeptide, NtGDH6 polypeptide, NtGDH9 polypeptide, NtGDH10 polypeptide, and NtGDH12 polypeptide is regulated.

[0149] In one embodiment, the activity of the NtGDH4 polypeptide, NtGDH7 polypeptide, NtGDH8 polypeptide, or NtGDH11 polypeptide is not regulated, or a combination of two or more thereof is not regulated, and the NtGDH4 polypeptide is encoded by a polynucleotide consisting of, or essentially consisting of, a sequence having at least 88% sequence identity to SEQ ID NO: 5, or the NtGDH4 polypeptide has at least 92% sequence identity to SEQ ID NO: 6, or the NtGDH7 polypeptide consists of, or essentially consists of, a sequence having at least 92% sequence identity to SEQ ID NO: 9. The NtGDH8 polypeptide is encoded by an NtGDH7 polynucleotide, or has at least 96% sequence identity to SEQ ID NO: 10, or the NtGDH8 polypeptide is encoded by an NtGDH8 polynucleotide consisting of or essentially consisting of an NtGDH8 polynucleotide, which contains a sequence having at least 92% sequence identity to SEQ ID NO: 11, or has at least 95% sequence identity to SEQ ID NO: 12, or the NtGDH11 polypeptide is encoded by an NtGDH11 polynucleotide consisting of or essentially consisting of an NtGDH8 polynucleotide, which contains a sequence having at least 87% sequence identity to SEQ ID NO: 17, or has at least 92% sequence identity to SEQ ID NO: 18.

[0150] Polypeptide fragments described herein are also intended. Polypeptide fragments typically retain some or all of the function or activity of the full-length sequence (such as NtGDH activity). Polypeptide fragments can range from at least about 25 amino acids, about 50 amino acids, about 75 amino acids, about 100 amino acids, about 150 amino acids, about 200 amino acids, about 250 amino acids, about 300 amino acids, about 400 amino acids, about 500 amino acids, to the full-length polypeptides described herein.

[0151] Polypeptides include mutants produced by introducing any type of modification (e.g., amino acid insertion, deletion, or substitution, changes in glycosylation state, refolding or isomerization, changes affecting three-dimensional structure, or changes affecting self-assembly state), but both intentionally engineered mutants and naturally isolated mutants, provided that they still possess some or all of their function or activity. Preferably, this function or activity is regulated.

[0152] Deletion refers to the removal of one or more amino acids from a polypeptide. Insertion refers to the introduction of one or more amino acid residues into a predetermined site in a polypeptide. Insertions can include intrasequence insertions of one or more amino acids. Substitution refers to the replacement of an amino acid in a polypeptide with another amino acid having similar properties (such as similar hydrophobicity, hydrophilicity, antigenicity, or a tendency to form or disrupt α-helix or β-sheet structures). Amino acid substitutions are typically single residues, but may be clustered depending on the functional constraints imposed on the polypeptide, and may range from about 1 to about 10 amino acids. Amino acid substitutions are preferably conserved amino acid substitutions, such as those described below. Amino acid substitutions, deletions, or insertions can be performed by peptide synthesis techniques (such as solid-phase peptide synthesis) or recombinant DNA manipulation. Methods for manipulating DNA sequences to produce polypeptide substitution, insertion, or deletion variants are well known in the art. Variants may have modifications that result in silent changes and yield functionally equivalent polypeptides. As long as the secondary bonds of the substances are preserved, intentional amino acid substitutions can be made based on the similarity of the polarity, charge, solubility, hydrophobicity, hydrophilicity, and amphiphilicity of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid, positively charged amino acids include lysine and arginine, and amino acids with uncharged polar head groups having similar hydrophilic values ​​include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine. Conservative substitutions can be made, for example, according to the table below. Amino acids in the same block in the second column, and preferably amino acids in the same family in the third column, can be substituted for each other. JPEG2026522599000002.jpg39127

[0153] Polypeptides may be mature polypeptides or immature polypeptides or polypeptides derived from immature polypeptides. Polypeptides can be converted to linear or cyclic forms using known methods. Polypeptides typically contain at least 10, at least 20, at least 30, or at least 40 consecutive amino acids.

[0154] At least one modification (e.g., a mutation) may be included in one or more of SEQ ID NOs. 1, 3, 5, 7, 9, 11, 13, 15, 17, or 19.

[0155] Recombinant constructs may be used to transform plants or plant cells to modulate polypeptide expression, function, or activity. Recombinant polynucleotide constructs may contain polynucleotides encoding one or more polynucleotides described herein, functionally bound to a regulatory region suitable for polypeptide expression. Thus, polynucleotides may contain coding sequences encoding polypeptides described herein. Plants or plant cells with modulated polypeptide expression, function, or activity include mutants, non-natural types, transgenic, artificially produced, or genetically modified plants or plant cells. Preferably, transgenic plants or plant cells contain a genome modified by stable integration of recombinant DNA. Recombinant DNA may include genetically modified, extracellularly constructed DNA, and DNA including naturally occurring DNA or cDNA or synthetic DNA. Transgenic plants may include plants regenerated from original transformed plant cells and offspring transgenic plants or hybrids of transformed plants from subsequent generations. Preferably, the transgenic modification modifies the expression, function, or activity of polynucleotides or polypeptides described herein compared to a control plant.

[0156] The polypeptide encoded by the recombinant polynucleotide may be a native polypeptide or of heterologous origin to the cell. In some cases, the recombinant construct includes an expression-regulating polynucleotide functionally bound to a regulatory region. Examples of preferred regulatory regions are described herein.

[0157] Vectors containing recombinant polynucleotide constructs as described herein are also provided. Suitable vector backbones include, for example, plasmids, viruses, artificial chromosomes, bacterial artificial chromosomes, yeast artificial chromosomes, or bacteriophage artificial chromosomes, which are commonly used in the art. Suitable expression vectors include, but are not limited to, plasmids and viral vectors derived from, for example, bacteriophages, baculoviruses, and retroviruses. Numerous vectors and expression systems are commercially available.

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

[0159] Plants or plant cells can be transformed by being stably transformed by the integration of recombinant polynucleotides into their genome. The plants or plant cells described herein can be stably transformed. Stably transformed cells typically retain the polynucleotides introduced at each cell division. Plants or plant cells can be transiently transformed so that recombinant polynucleotides are not integrated into their genome. Transiently transformed cells typically lose all or part of the recombinant polynucleotides introduced at each cell division, and as a result, the recombinant polynucleotides introduced in daughter cells cannot be detected after a sufficient number of cell divisions.

[0160] In this field, many methods are available for transforming plant cells, including bioristics, gene gun technology, Agrobacterium-mediated transformation, viral vector-mediated transformation, freeze-thaw methods, microparticle bombardment, direct DNA incorporation, sonication, microinjection, plant virus-mediated transfer, and electroporation.

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

[0162] The selection of regulatory regions to be included in recombinant constructs depends on several factors, including but not limited to efficiency, selectivity, inducibility, desired expression levels, and cell or tissue preferential expression. Regulating the expression of coding sequences by appropriately selecting and positioning regulatory regions relative to the coding sequence is a routine matter for those skilled in the art. Transcription of polynucleotides can be regulated in a similar manner. Some suitable regulatory regions initiate transcription only in specific cell types, or primarily in specific cell types. Methods for identifying and characterizing regulatory regions in plant genomic DNA are known in the art.

[0163] Exemplary promoters include tissue-specific promoters recognized by tissue-specific factors present in different tissues or cell types (e.g., root-specific promoters, stem-specific promoters, xylem-specific promoters), or promoters present at different developmental stages, or promoters present in response to different environmental conditions. Preferred 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 cauliflower mosaic virus 35S (CaMV / 35S), SSU, OCS, lib4, usp, STLS1, B33, nos, or ubiquitin or phaseolin promoters. Those skilled in the art can generate multiple variants of recombinant promoters.

[0164] Tissue-specific promoters are transcriptional regulatory elements that are active only in specific cells or tissues at specific times during plant development, for example, in vegetative or reproductive tissues. Examples of tissue-specific promoters under developmental control include promoters that can initiate transcription only in (or primarily in) specific tissues, such as vegetative tissues (e.g., roots or leaves) or reproductive tissues (e.g., fruits, ovules, seeds, pollen, pistils, 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 a combination thereof.

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

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

[0167] Examples of inducible promoters include promoters that respond to pathogen attack, anaerobic conditions, high temperatures, light, drought, low temperatures, or high salt concentrations. Pathogen-inducible promoters include those derived from pathogen-associated polypeptides (PR polypeptides) induced after pathogen infection (e.g., PR polypeptides, SAR polypeptides, β-1,3-glucanase, chitinase).

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

[0169] Disclosed herein are plants or plant cells comprising at least one genetic modification (e.g., mutation) in one or more polynucleotides or polypeptides described herein, wherein the genetic modification results in a regulated function or activity of NtGDH or the polypeptide encoded thereby.

[0170] Further methods are provided for regulating levels of NtGDH polypeptide in dried plants or dried plant materials, the methods comprising introducing one or more genetic modifications (e.g., mutations) into the plant genome that regulate the expression of at least one NtGDH gene selected from one or more sequences in accordance with this disclosure.

[0171] A method is also provided for identifying dried tobacco leaves or portions of those leaves that have regulated levels of ammonia compared to ammonia levels in a control plant, the method comprising screening polynucleotide samples from dried tobacco leaves or portions of those leaves for the presence of one or more genetic modifications (e.g., mutations) in the NtGDH polynucleotide sequence in accordance with this disclosure, and optionally correlating them with identified genetic modifications known to regulate ammonia levels in dried tobacco leaves or portions of those leaves. Preferably, the amounts of amino acids, sugars and total alkaloids in dried tobacco leaves or portions of those leaves are also regulated.

[0172] Also disclosed are plants or plant cells that are heterozygous or homozygous to one or more genetic modifications (e.g., mutations) in the NtGDH gene in accordance with this disclosure, the genetic modifications resulting in regulated expression of the NtGDH gene or the function or activity of the NtGDH polypeptide encoded thereby.

[0173] Many approaches can be used to combine genetic modifications (e.g., mutations) in a single plant, including sexual hybridization. A plant having one or more favorable heterozygous or homozygous genetic modifications in genes that regulate gene expression or the function or activity of the polypeptides they encode in accordance with this disclosure can be hybridized with a plant having one or more favorable heterozygous or homozygous genetic modifications in one or more other genes that regulate the expression or the function or activity of the polypeptides they encode. In one embodiment, hybridization is performed to introduce one or more favorable heterozygous or homozygous genetic modifications in genes in accordance with this disclosure into the same plant.

[0174] The function or activity of one or more polypeptides in a plant according to this disclosure is increased or decreased if it is lower or higher than the function or activity of the same polypeptide in a plant that has not been modified to inhibit the function or activity of that polypeptide, and has been cultured, harvested, and dried using the same protocol.

[0175] In some embodiments, genetic modifications are introduced into plants or plant cells using mutagenic approaches, and the introduced mutations are identified or selected using methods known to those skilled in the art, such as Southern blotting, DNA sequencing, PCR analysis, or phenotypic analysis. Mutations that affect gene expression or interfere with the function of the encoded polypeptide can be determined using methods well known in the art. Insertion mutations in gene exons typically result in null mutants. Mutations in conserved residues may be particularly effective in inhibiting the metabolic function of the encoded polypeptide. For example, it will be understood that mutations in one or more highly conserved regions are likely to alter polypeptide function, while mutations outside these highly conserved regions are likely to have little to no effect on polypeptide function. Furthermore, mutations in a single nucleotide may produce a stop codon, resulting in a cleaved polypeptide with loss of function depending on the degree of cleavage.

[0176] Methods for obtaining modified polynucleotides and polypeptides are also disclosed. Any target plant, including plant cells or plant material, can be genetically modified by a variety of methods known to induce mutagenesis, including site-directed mutagenesis, oligonucleotide-directed mutagenesis, chemically induced mutagenesis, radiation-induced mutagenesis, mutagenesis utilizing modified bases, mutagenesis utilizing gap double-strand DNA, mutagenesis utilizing double-strand breaks, mutagenesis utilizing repair-deficient host strains, mutagenesis by whole gene synthesis, DNA shuffling, and other equivalent methods.

[0177] Modifications in polynucleotides and polypeptides described herein may include artificially produced, synthetic, or genetically modified modifications. Modifications in polynucleotides and polypeptides described herein may be obtained or available through processes involving in vitro or in vivo manipulation steps. Modifications in polynucleotides and polypeptides described herein may be obtained or available through processes involving human intervention. The function or activity of the polypeptide variant may be higher, lower, or about the same as that of the unmodified polypeptide.

[0178] Methods for introducing random modifications to polynucleotides include chemical mutagenesis and radiomutagenesis. Chemical mutagenesis involves the use of exogenously added chemicals, such as mutagenic, teratogenic, or carcinogenic organic compounds, to induce mutations. Mutagens, including chemical mutagens or radiation, that primarily produce point mutations and short deletions, insertions, missense mutations, simple repeat sequences, transversions, or transitions may be used to create mutations. Mutagens include ethylmethanesulfonate, methylmethanesulfonate, N-ethyl-N-nitrosourea, triethylmelamine, N-methyl-N-nitrosourea, procarbazine, chlorambucyl, cyclophosphamide, diethyl sulfate, acrylamide monomer, melphalan, nitrogen mustard, vincristine, dimethylnitrosamine, N-methyl-N'-nitro-nitrosoguanidine, nitrosoguanidine, 2-aminopurine, 7,12-dimethylbenz(a)anthracene, ethylene oxide, hexamethylphosphoramide, busulfan, diepoxyalkanes (such as diepoxyoctane and diepoxybutane), 2-methoxy-6-chloro-9[3-(ethyl-2-chloro-ethyl)aminopropylamino]acrylidine dihydrochloride, and formaldehyde.

[0179] Spontaneous mutations at loci that may not be directly caused by the mutagens are also intended, provided they result in the desired phenotype. Suitable mutagenic substances may include, for example, ionizing radiation such as X-rays, gamma rays, fast neutron irradiation, and UV radiation. The dose of the mutagenic chemical or radiation is determined experimentally for each type of plant tissue so that a mutation frequency below a threshold level characterized by lethality or reproductive sterility is obtained. Plant polynucleotides can be prepared for mutation screening using any plant polynucleotide preparation method known to those skilled in the art.

[0180] The mutation process may include one or more plant hybridization steps.

[0181] After mutation, screening can be performed to identify mutations that create premature stop codons or other non-functional genes. After mutation, screening can be performed to identify mutations that create functional genes that have the ability to be expressed at increased or decreased levels. Screening of mutants can be performed by sequencing or by using one or more probes or primers specific to the gene or polypeptide. Certain mutations in polynucleotides that can result in regulated gene expression, regulated mRNA stability, or regulated polypeptide stability can also be created. Such plants are referred to herein as “non-natural” or “mutant” plants. Typically, mutant or non-natural plants will contain at least some foreign, synthetic, or artificially produced nucleotides (e.g., DNA or RNA) that were not present in the plant before it was manipulated. The foreign nucleotide may be a single nucleotide, two or more nucleotides, two or more consecutive nucleotides, or two or more discontinuous 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 consecutive or discontinuous nucleotides).

[0182] The expression, function, or activity of one or more NtGDH polynucleotides or NtGDH polypeptides described herein can be regulated using sequence-specific polynucleotides that can interfere with the transcription of one or more endogenous NtGDH genes; sequence-specific polynucleotides (e.g., double-stranded RNA, siRNA, ribozymes) that can interfere with the translation of NtGDH RNA transcripts; sequence-specific polypeptides that can interfere with the stability of one or more NtGDH polypeptides; sequence-specific polynucleotides that can interfere with the enzymatic function of one or more NtGDH polypeptides or the binding function of one or more NtGDH polypeptides to substrate or regulatory polypeptides; antibodies that exhibit specificity to one or more NtGDH polypeptides; small molecule compounds that can interfere with the stability of one or more NtGDH polypeptides or the enzymatic function of one or more NtGDH polypeptides or the binding function of one or more NtGDH polypeptides; zinc finger polypeptides that bind to one or more NtGDH polynucleotides; and meganucleases that function to one or more polynucleotides. Genome editing technologies are well known in the industry and are discussed further below.

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

[0184] Meganucleases such as I-CreI can be used to modulate the expression, function, or activity of one or more 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.

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

[0186] The CRISPR system can be used and is a preferred method to modulate the expression, function, or activity of one or more NtGDH polynucleotides described herein. This technique is described, for example, in Plant Methods (2016) 12:8; Front Plant Sci. (2016) 7:506; Biotechnology Advances (2015) 33,1,p41-52; Acta Pharmaceutica Sinica B (2017) 7,3,p292-302; Curr. Op. in Plant Biol. (2017) 36,1-8 and Int J Mol Sci (2019) 20(16),4045. As is well known in the art, CRISPR editing systems generally consist of two components: a CRISPR-associated endonuclease (Cas) (e.g., Cas9) and a guide RNA (gRNA). Cas forms a double-strand DNA break at a site in the genome defined by the sequence of the gRNA molecule bound to Cas. The site where Cas cleaves DNA is defined by the specific sequence of the gRNA bound to it. A gRNA is an RNA sequence specifically designed to recognize a target DNA region and direct the Cas nuclease there for editing. It consists of two sections: (i) a tracr RNA that acts as a binding scaffold for the Cas nuclease, and (ii) a crispr RNA (crRNA) of 17-20 nucleotides complementary to the target DNA. The exact region of DNA to be targeted depends on the specific application. For example, to activate or repress a target polynucleotide, the gRNA may target a promoter that drives the expression of the target polynucleotide. Methods for designing gRNAs are well-known in the industry, including those by Chop Chop Harvard. Applications of Cas9-based genome editing in Arabidopsis thaliana and tobacco are described, for example, in Methods Enzymol. (2014) 546:459-72 and Plant Physiol Biochem. (2018) 131:37-46. CRISPR technology is widely used in plants (see, for example, WO2015 / 189693).In addition to Cas9, other RNA-inducible nucleases for use in CRISPR systems are described, including Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas10, Cpf1, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, and Csf4. In certain embodiments, the use of Cas9 is preferred. This disclosure further provides a CRISPR-based genome editing system comprising an RNA-induced nuclease and a gRNA, wherein the CRISPR-based genome editing system modulates the activity of one or more polynucleotides described herein. This disclosure also provides a method for cleaving one or more polynucleotides in plant cells, comprising introducing a gRNA and an RNA-induced nuclease into plant cells, wherein the gRNA acts in conjunction with the RNA-induced nuclease to create a strand break in one or more polynucleotides described herein. A CRISPR construct is also disclosed comprising: (i) a polynucleotide encoding a CRISPR-associated endonuclease; and (ii) a gRNA containing a polynucleotide sequence (typically about 17-20 nucleotides) complementary to the DNA of the polynucleotide to be targeted, as described herein.

[0187] Antisense techniques are other well-known methods 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.

[0188] NtGDH polynucleotides can be targeted for inactivation by introducing transposons (e.g., IS elements or other mobile genetic elements) into the genome of the target plant. See, for example, Cytology and Genetics (2006) 40(4):68-81.

[0189] NtGDH polynucleotides can be targeted for inactivation by introducing ribozymes derived from many small circular RNAs that can self-cleave and replicate in plants. See, for example, FEMS Microbiology Reviews (1999) 23, 3, 257-275.

[0190] Mutant or non-natural plants or plant cells may have any combination of one or more modifications (e.g., mutations) in one or more NtGDH polynucleotides described herein, resulting in regulated expression or function or activity of those polynucleotides or their polynucleotide products. For example, mutant or non-natural 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, single modifications in two or more NtGDH polynucleotides or NtGDH polypeptides, or multiple modifications in two or more NtGDH polynucleotides or NtGDH polypeptides. As a further example, mutant or non-natural plants or plant cells may have one or more modifications in specific parts of an NtGDH polynucleotide or NtGDH polypeptide, such as a region of NtGDH encoding the active site or a part of the NtGDH polypeptide. As a further example, mutant or non-natural plant or plant cell may have one or more modifications in the outer regions of one or more NtGDH polynucleotides or NtGDH polypeptides, such as upstream or downstream regions of NtGDH polynucleotides, provided that they regulate the function or expression of NtGDH. Upstream elements include promoters, enhancers, or transcription factors. Some elements, such as enhancers, may be located upstream or downstream of the gene they regulate. Since some elements have been found to be located hundreds of thousands of base pairs upstream or downstream of the gene they regulate, elements do not need to be located close to the gene they regulate.A mutant or non-natural plant or plant cell may have one or more modifications located within the first 100 nucleotides, first 200 nucleotides, first 300 nucleotides, first 400 nucleotides, first 500 nucleotides, first 600 nucleotides, first 700 nucleotides, first 800 nucleotides, first 900 nucleotides, first 1000 nucleotides, first 1100 nucleotides, first 1200 nucleotides, first 1300 nucleotides, first 1400 nucleotides, or first 1500 nucleotides of a gene. A mutant or non-natural plant or plant cell may have one or more modifications or combinations thereof located within the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, or fifteenth set of 100 nucleotides of a gene. A variant or non-natural plant or plant cell containing a variant of the mutant polypeptide (e.g., the variant, non-natural or transgenic plant or plant cell described herein) is disclosed.

[0191] In one embodiment, seeds from a plant are mutagenerated and then grown into first-generation mutant plants. The first-generation plants then self-pollinate, and seeds from the first-generation plants are grown into second-generation plants, which are then screened for mutations at their gene loci. While mutagenerated plant material can be screened for mutations, the advantage of screening second-generation plants is that all somatic mutations correspond to germline mutations. Those skilled in the art will understand that various plant materials, including but not limited to seeds, pollen, plant tissue, or plant cells, can be mutagenerated to produce mutant plants. However, the type of mutagenerated plant material may affect when plant polynucleotides are screened for mutations. For example, if pollen is mutagenerated before pollination of a non-mutagenetic plant, the resulting seeds are grown into first-generation plants. All cells of the first-generation plants contain the mutations produced in the pollen, and therefore these first-generation plants can be screened for mutations without waiting until the second generation.

[0192] NtGDH polynucleotides prepared from individual plants, plant cells, or plant materials may be optionally pooled to screen for mutations in plant populations derived from mutagenic plant tissues, cells, or materials. One or more subsequent generations of plants, plant cells, or plant materials may be screened. The size of the optionally pooled group depends on the sensitivity of the screening method used. After the samples have been optionally pooled, they may be subjected to polynucleotide-specific amplification techniques such as PCR. Sequences in the optionally pooled samples can be amplified using any one or more primers or probes specific to a gene or a sequence directly adjacent to a gene. Preferably, one or more primers or probes are designed to amplify the region of the locus where useful mutations are most likely to occur. Most preferably, the primers are designed to detect mutations within a region of polynucleotide. Furthermore, to facilitate the screening of point mutations, it is preferable that the primers and probes avoid known polymorphic sites. To facilitate the detection of the amplification product, one or more primers or probes may be labeled using any conventional labeling method. Primers or probes may be designed based on the sequences described herein using methods well understood in the art. To facilitate the detection of amplification products, primers or probes may be labeled using any conventional labeling method. These can be designed based on the sequences described herein using methods well understood in the art.

[0193] Polymorphisms can be identified by means known in the art, some of which are described in the literature.

[0194] In some embodiments, plants can be regenerated or grown from plants, plant tissues, or plant cells. Any suitable method for regenerating or growing plants from plant cells or plant tissues can be used, and may include, without limitation, methods such as regeneration from tissue culture or protoplasts. Preferably, plants can be regenerated by growing transformed plant cells on callus-inducing medium, shoot-inducing medium, or root-inducing medium. See, for example, McCormick et al., Plant Cell Reports (1986) 5:81-84. These plants are then grown and pollinated with the same or different transformed lines to identify the resulting hybrids having the expression of the desired phenotypic features. Two or more generations are grown to ensure that the expression of the desired phenotypic features is stably maintained and inherited, and then seeds are harvested to ensure that the expression of the desired phenotypic features has been achieved. Thus, “transformed seeds” refer to seeds containing nucleotide constructs stably integrated into the plant genome.

[0195] Accordingly, in a further embodiment, a method for preparing mutant plants is provided. The method comprises providing at least one cell of a plant containing one or more NtGDH genes encoding functional NtGDH. The at least one cell of the plant is then treated under conditions effective for modulating the function of the NtGDH polynucleotide. The at least one mutant plant cell is then propagated into a mutant plant, where the mutant plant has a regulated level of the NtGDH polypeptide described herein compared to a control plant. In one embodiment of the method for producing this mutant plant, the treatment step comprises providing at least one cell to the chemical mutagen described above under conditions effective for producing at least one mutant plant cell. In another embodiment of the method, the treatment step comprises providing at least one cell to a radiation source under conditions effective for producing at least one mutant plant cell. The term “mutant plant” preferably includes mutant plants whose genotype has been modified compared to a control plant by means other than genetic engineering or genetic modification.

[0196] In certain embodiments, a mutant plant, mutant plant cell, or mutant plant material may contain one or more naturally occurring mutations in another plant, plant cell, or plant material that confer a desired trait. This mutation can be incorporated (e.g., genetically modified) into another plant, plant cell, or plant material (e.g., a plant, plant cell, or plant material having a different genetic background than the plant from which the mutation originated) and confer the trait to it. Therefore, for example, a naturally occurring mutation in a first plant can be introduced into a second plant (such as a second plant having a different genetic background than the first plant). Thus, those skilled in the art can search for and identify plants that naturally possess one or more mutant alleles of the genes described herein in their genome and confer a desired trait. The naturally occurring mutant alleles can be transferred to the second plant by various methods, including breeding, backcrossing, and genetic modification, to produce a lineage, variety, or hybrid having one or more mutations in the genes described herein. The same technique can also be applied to the genetic modification of one or more non-natural mutations from a first plant to a second plant. Plants exhibiting the desired trait can be selected from a pool of mutant plants. Preferably, selection is carried out using the polynucleotide knowledge described herein. As a result, it is possible to screen for genetic traits compared to controls. Such a screening approach may include the application of conventional amplification or hybridization techniques, as discussed herein.

[0197] Accordingly, further aspects of the present disclosure relate to a method for identifying mutant plants, the method comprising (a) providing a sample containing one or more NtGDH polynucleotides from a plant, and (b) determining the sequence of the polynucleotides, indicating that the plant is a mutant plant if there is a difference in the sequence of the polynucleotides compared to a control plant.

[0198] In another embodiment, a method is provided for identifying mutant plants that accumulate increased or decreased levels of ammonia compared to a control plant, the method comprising (a) providing a sample from a plant to be screened, (b) determining whether the sample contains one or more mutations in one or more NtGDH polynucleotides described herein, and (c) determining the level of ammonia in the plant. Preferably, the level of ammonia is determined in a dried leaf. Preferably, the levels of amino acids, sugars and total alkaloids in the dried leaf are also determined.

[0199] In another embodiment, a method is provided for preparing mutant plants having increased or decreased levels of ammonia compared to a control plant, the method comprising: (a) providing a sample from a first plant; (b) determining whether the sample contains one or more mutations in one or more NtGDH polynucleotides described herein that result in a controlled level of ammonia; and (c) transferring one or more mutations into a second plant. Preferably, the ammonia level is determined in a dried leaf. Preferably, the levels of amino acids, sugars and total alkaloids in the dried leaf are also determined.

[0200] Mutations can be introduced into a second plant using various methods known in the art, such as genetic engineering, genetic manipulation, gene transfer, plant breeding, and backcrossing. In one embodiment, the first plant is a naturally occurring plant. In one embodiment, the second plant has a different genetic background from the first plant. In another embodiment, a method is provided for preparing mutant plants having increased or decreased levels of ammonia compared to a control plant, the method comprising (a) providing a sample from the first plant, (b) determining whether the sample contains one or more mutations in one or more NtGDH polynucleotides described herein that result in regulated levels of ammonia, and (c) gene-transferring one or more mutations from the first plant into the second plant. Preferably, the ammonia level is determined in dried leaves. Preferably, the levels of amino acids, sugars, and total alkaloids in dried leaves are also determined.

[0201] In one embodiment, the gene transfer process optionally includes plant breeding, such as backcrossing. In one embodiment, the first plant is a naturally occurring plant. In one embodiment, the second plant has a different genetic background from the first plant. In one embodiment, the first plant is not a cultivar or superior cultivar. In one embodiment, the second plant is a cultivar or superior cultivar.

[0202] Further embodiments relate to mutant plants (including cultivar or superior cultivar mutant plants) obtained or available by the methods described herein. In certain embodiments, the mutant plant may have one or more mutations localized only to specific regions within the sequences of one or more NtGDH polynucleotides described herein. According to this embodiment, the rest of the genome sequence of the mutant plant will be the same as or substantially the same as that of the plant before mutagenesis.

[0203] In certain embodiments, the mutant plant may have one or more mutations localized within the sequence of one or more NtGDH polynucleotides described herein, and in more than one genomic region of the plant, such as one or more further regions of the genome. According to this embodiment, the rest of the genome sequence of the mutant plant will not be the same as, or substantially the same as, the plant before mutagenesis. In certain embodiments, the mutant plant lacks one or more mutations in one, two or more, three or more, four or more, or five or more exons of the NtGDH polynucleotide described herein, or lacks one or more mutations in one, two or more, three or more, four or more, or five or more introns of the NtGDH polynucleotide described herein, or lacks one or more mutations in the promoter of the NtGDH polynucleotide described herein, or lacks one or more mutations in the 3' untranslated region of the NtGDH polynucleotide described herein, or lacks one or more mutations in the 5' untranslated region of the NtGDH polynucleotide described herein, or lacks one or more mutations in the coding region of the NtGDH polynucleotide described herein, or lacks one or more mutations in the non-coding region of the NtGDH polynucleotide described herein.

[0204] In a further embodiment, a method is provided for identifying plants, plant cells, or plant material containing mutations in the gene encoding the NtGDH polynucleotide described herein, the method comprising (a) subjecting a plant, plant cell, or plant material to mutagenesis; (b) obtaining a sample from the plant, plant cell, or plant material or its offspring; and (c) determining the polynucleotide sequence of the NtGDH gene or its variant or fragment, such that a difference in the sequence indicates one or more mutations therein. The method also enables the selection of plants having mutations occurring in genomic regions affecting the expression of the NtGDH gene in plant cells, such as transcription start sites, start codons, intron regions, exon-intron boundaries, and terminators.

[0205] Suitable plants for use in this disclosure include monocots and dicots, and plant cell lines, including members of the genus Nicotiana.

[0206] Various embodiments are directed to mutant tobacco, non-natural tobacco, or transgenic tobacco plants or tobacco plant cells and can be applied to any species of the genus Nicotiana, including N. rustica and N. tabacum (e.g., LA B21, LN KY171, TI 1406, Basma, Galpao, Perique, Beinhart 1000-1, and Petico).The species include N. acaulis, N. acuminata, N. africana, N. alata, N. ameghinoi, N. amplexicaulis, N. arentsii, N. attenuata, N. azambujae, and N. benavidesii N. benthamiana, N. bigelovii, N. bonariensis, N. cavicola, N. clevelandii, N. cordifolia, N. corymbosa, N. debneyi, N. excelsior, N. forgetia na、N.fragrans、N.glauca、N.glutinosa、N.goodspeedii、N.gossei、N.hybrid、N.ingulba、N.kawakamii、N.knightiana、N.langsdorffii、N.line aris、N.longiflora、N.maritima、N.megalosiphon、N.miersii、N.noctiflora、N.nudicaulis、N.obtusifolia、N.occidentalis、N.occidentalis subsp.hesperis、N.otophora、N.paniculata、N.pauciflora、N.petunioides、N.plumbaginifolia、N.quadrivalvis、N.raimondii、N.repanda、N.rosulata subsp.ingulba、N.rotundifolia、N.setchellii、N.simulans、N.solanifolia、N.spegazzinii、N.stocktonii、N.suaveolens、N.sylvestr is, N. thyrsiflora, N. tomentosa, N. tomentosiformis, N. trigonophylla, N. umbratica, N. undulata, N. velutina, N. wigandioides, Nx sanderae and a slightly different variety than N.tabacum.

[0207] The use of tobacco cultivars and superior tobacco cultivars is also intended herein. Therefore, transgenic, non-natural, or mutant plants may be tobacco cultivars or superior tobacco cultivars containing one or more transgenes, or one or more genetic mutations, or combinations thereof. The genetic mutation (e.g., one or more polymorphisms) may be a mutation that does not naturally occur in individual tobacco cultivars or tobacco cultivars (e.g., superior tobacco cultivars), or a genetic mutation that occurs naturally provided that the mutation does not naturally occur in individual tobacco cultivars or tobacco cultivars (e.g., superior tobacco cultivars).

[0208] Particularly useful Nicotiana tabacum varieties include Burley, Dark, Flu-Cured, and Oriental tobaccos. Non-exclusive examples of varieties or cultivars include BD 64, CC 101, CC 200, CC 27, CC 301, CC 400, CC 500, CC 600, CC 700, CC 800, CC 900, Coker 176, Coker 319, Coker 371 Gold, Coker 48, CD 263, DF911, DT 538 LC Galpao tobacco, GL 26H, GL 350, GL 600, GL 737, GL 939, GL 973, HB 04P, HB 04P LC, HB3307PLC, Hybrid 403LC, Hybrid 404LC, Hybrid 501 LC, K 149, K 326, K 346, K 358, K394, K 399, K 730, KDH 959, KT 200, KT204LC, KY10, KY14, KY 160, KY 17, KY 171, KY 907, KY907LC, KY14xL8 LC, Little Crittenden, McNair 373, McNair 944, msKY 14xL8, Narrow Leaf Madole, Narrow Leaf Madole LC, NBH 98, N-126, N-777LC, N-7371LC, NC 100, NC 102, NC 2000, NC 291, NC 297, NC 299, NC 3, NC 4, NC 5, NC 6, NC7, NC 606, NC 71, NC 72, NC 810, NC BH 129, N.C. 2002, Neal Smith Madole, OXFORD 207, PD 7302 LC, PD 7309 LC, PD 7312 LC, 'Perique' Tobacco, PVH03, PVH09, PVH19, PVH50, PVH51, R 610, R 630, R 7-11, R 7-12, RG 17, RG 81, RG H51, RGH 4, RGH 51, RS 1410, Speight 168, Speight 172, Speight 179, Speight 210, Speight 220, Speight 225, Speight 227, Speight 234, Speight G-28, Speight G-70, SpeightH-6, Speight H20, Speight NF3, TI 1406, TI 1269, TN 86, TN86LC, TN 90, TN 97, TN97LC, TN D94, TN D950, TR(Tom Rosson)Madole, VA 309, VA359, AA 37-1, B13P, Xanthi(Mitchell-Mor), Bel-W3, 79-615, Samsun Holmes NN, KTRDC number 2 Hybrid 49, Burley 21, KY8959, KY9, MD 609, PG01, PG04, PO1, PO2, PO3, RG11, RG 8, VA509, AS44, Bank A1, Basma Drama B84 / 31, Basma I Lesson ZP4 / B, Basma Xanthi BX 2A, Batek, Besuki Jember, C104, Coker 347, Creole Missionary, Delcrest, Djebel 81, DVH 405, Galpao Comum, HB04P, Hicks Broadleaf, Kabakulak Elassona, Kutsage E1, LA BU 21, NC 2326, NC 297, PVH 2110, Red Russian, Samsun, Saplak, Simmaba, Talgar 28, Wislica, Yayaldag, Prilep HC-72, Prilep P23, Prilep PB 156 / 1, Prilep P12-2 / 1、Yaka JK-48、Yaka JB 125 / 3、TI-1068、KDH-960、TI-1070、TW136、Basma、TKF 4028、L8、TKF 2002、GR141、Basma xanthine GR149 GR153 Petit Let's go to Havana in the early days Thanks for watching the snowflakes.

[0209] Embodiments also relate to compositions and methods for producing mutant plants, non-natural plants, hybrid plants, or transgenic plants modified to modulate the expression or function of one or more NtGDH polynucleotides described herein (or any combination thereof described herein). Advantageously, the resulting mutant plants, non-natural plants, hybrid plants, or transgenic plants may be similar to, or substantially identical to, the control plants in overall appearance. Various phenotypic characteristics, such as maturity, number of leaves per plant, stem height, leaf insertion angle, leaf size (width and length), internode distance, and leaf blade-to-midrib ratio, can be evaluated by field observation.

[0210] One embodiment relates to the seeds of a mutant plant, non-natural plant, hybrid plant, or transgenic plant described herein. Preferably, the seeds are tobacco seeds. Further embodiments relate to the pollen or ovules of a mutant plant, non-natural plant, hybrid plant, or transgenic plant described herein. Further, a mutant plant, non-natural plant, hybrid plant, or transgenic plant described herein is provided, further comprising a polynucleotide that confers male sterility.

[0211] Tissue cultures of regenerative cells of mutant plants, non-natural plants, hybrid plants, or transgenic plants or parts thereof, as described herein, are also provided, which regenerate plants capable of expressing all the morphological and physiological characteristics of the parent. Regenerative cells include cells from leaves, pollen, embryos, cotyledons, hypocotyls, roots, root tips, anthers, flowers and parts thereof, ovules, buds, stems, petioles, pith, and capsules or callus or protoplasts derived therefrom.

[0212] The plant materials described herein may also be dried tobacco materials. CORESTA recommendations regarding tobacco drying are described in CORESTA Guide No. 17, April 2016, Sustainability in Leaf Tobacco Production. Dried tobacco materials may be fully dried tobacco materials, or tobacco materials that have been dried for a period of time such as at least 24 hours, at least 48 hours, at least 96 hours, or at least 192 hours.

[0213] In a further embodiment, a method is provided for preparing dried tobacco leaves or a portion of tobacco leaves having controlled levels of ammonia, amino acids, sugars, and total alkaloids compared to tobacco leaves or a portion of tobacco leaves derived from a control dried tobacco plant, the method comprising the steps of: (a) providing a tobacco plant comprising or essentially comprising NtGDH, wherein the NtGDH2 polynucleotide, or NtGDH3 polynucleotide, or NtGDH6 polynucleotide, or NtGDH9 polynucleotide, or NtGDH10 polynucleotide, or NtGDH12 polynucleotide, or NtGDH2 polypeptide, or NtGDH3 polypeptide, or NtGDH6 polypeptide, or NtGDH9 polypeptide, or NtGDH10 polypeptide, or NtGDH12 polypeptide, wherein (i) the NtGDH2 polynucleotide comprises or essentially comprises a sequence having at least 90% sequence identity with SEQ ID NO: 1, or (ii) the NtGDH2 polypeptide is as described in (i) (iii) an NtGDH2 polypeptide having at least 94% sequence identity to SEQ ID NO: 2, or (iv) an NtGDH3 polynucleotide comprising or essentially comprising a sequence having at least 90% sequence identity to SEQ ID NO: 3, or (v) an NtGDH3 polypeptide being encoded by the polynucleotide described in (iv), or (vi) an NtGDH3 polypeptide having at least 94% sequence identity to SEQ ID NO: 4, or (vii) an NtGDH6 polynucleotide A nucleotide comprising or essentially comprising a sequence having at least 90% sequence identity in SEQ ID NO: 7, or (viii) an NtGDH6 polypeptide encoded by the polynucleotide described in (i), or (ix) an NtGDH6 polypeptide having at least 94% sequence identity in SEQ ID NO: 8, or (x) an NtGDH9 polynucleotide comprising or essentially comprising a sequence having at least 85% sequence identity in SEQ ID NO: 13, or (xi) an NtGDH9 polypeptide comprising,(i) A providing step comprising: (xii)NtGDH9 polypeptide having at least 91% sequence identity to SEQ ID NO: 14, or (xiii)NtGDH10 polynucleotide comprising a sequence having at least 89% sequence identity to SEQ ID NO: 15, or (xiv)NtGDH10 polypeptide having at least 94% sequence identity to SEQ ID NO: 16, or (xix)NtGDH12 polynucleotide comprising a sequence having at least 85% sequence identity to SEQ ID NO: 19, or (xx)NtGDH12 polypeptide having at least 91% sequence identity to SEQ ID NO: 20; and (b) At least one NtGDH2 in a tobacco plant leaf or a portion of a tobacco plant leaf. (c) a step of adjusting the expression of polynucleotides or NtGDH3 polynucleotides or NtGDH6 polynucleotides or NtGDH9 polynucleotides or NtGDH10 polynucleotides or NtGDH12 polynucleotides, or the activity of NtGDH2 polypeptides or NtGDH3 polypeptides or NtGDH6 polypeptides or NtGDH9 polypeptides or NtGDH10 polypeptides or NtGDH12 polypeptides; (c) a step of harvesting plant leaves or a portion of plant leaves from a tobacco plant; (d) a step of drying the plant leaves or a portion of plant leaves; (e) optionally a step of measuring the levels of ammonia and one or more of amino acids, sugars and total alkaloids in the dried tobacco plant leaves or a portion of the dried plant leaves; (f) an expression of NtGDH2 polynucleotides or NtGDH3 polynucleotides or NtGDH6 polynucleotides or NtGDH9 polynucleotides or NtGDH10 polynucleotides or NtGDH12 polynucleotides;Alternatively, a process to obtain dried tobacco plant leaves or a portion of its leaves having controlled levels of ammonia, amino acids, sugars, and total alkaloids compared to a control plant in which the activity of NtGDH2 polypeptide, NtGDH3 polypeptide, NtGDH6 polypeptide, NtGDH9 polypeptide, NtGDH10 polypeptide, or NtGDH12 polypeptide is not regulated.

[0214] Preferably, in step (b), the expression of NtGDH6 polynucleotide and NtGDH10 polynucleotide is regulated, or the activity of NtGDH6 polypeptide and NtGDH10 polypeptide is regulated.

[0215] Preferably, the expression of NtGDH2 polynucleotide and NtGDH3 polynucleotide is regulated, or the activity of both NtGDH2 polypeptide and NtGDH3 polypeptide is regulated.

[0216] Preferably, the expression of NtGDH9 polynucleotide and NtGDH12 polynucleotide is regulated, or the activity of NtGDH9 polypeptide and NtGDH12 polypeptide is regulated.

[0217] Preferably, the expression of NtGDH2 polynucleotide, NtGDH3 polynucleotide, NtGDH9 polynucleotide, and NtGDH12 polynucleotide is regulated, or the activity of NtGDH2 polypeptide, NtGDH3 polypeptide, NtGDH9 polypeptide, and NtGDH12 polypeptide is regulated.

[0218] Preferably, the expression of NtGDH2 polynucleotide, NtGDH3 polynucleotide, NtGDH6 polynucleotide, NtGDH9 polynucleotide, NtGDH10 polynucleotide, and NtGDH12 polynucleotide, or NtGDH2 polypeptide, NtGDH3 polypeptide, NtGDH6 polypeptide, NtGDH9 polypeptide, NtGDH10 polypeptide, and NtGDH12 polypeptide is regulated.

[0219] Preferably, the expression of one or more NtGDH4 polynucleotides, NtGDH7 polynucleotides, NtGDH8 polynucleotides, or NtGDH11 polynucleotides, or the activity of NtGDH4 polypeptides, NtGDH7 polypeptides, NtGDH8 polypeptides, or NtGDH11 polypeptides is not regulated, and (i) the NtGDH4 polynucleotide consists of or essentially consists of a sequence containing at least 88% sequence identity to SEQ ID NO: 5, or (ii) the NtGDH4 polypeptide is encoded by the polynucleotide described in (i), or (iii) the NtGDH4 polypeptide has at least 92% sequence identity to SEQ ID NO: 6, or (iv) the NtGDH7 polynucleotide consists of or essentially consists of a sequence containing at least 92% sequence identity to SEQ ID NO: 9, or (v) the NtGDH7 polypeptide is (iv) a polynucleotide encoded by the polynucleotide described in (iv), or (vi) an NtGDH7 polypeptide having at least 96% sequence identity to SEQ ID NO: 10, or (vii) an NtGDH8 polynucleotide comprising a sequence having at least 92% sequence identity to SEQ ID NO: 11, or (viii) an NtGDH8 polypeptide encoded by the polynucleotide described in (i), or (ix) an NtGDH8 polypeptide having at least 95% sequence identity to SEQ ID NO: 12, or (x) an NtGDH11 polynucleotide comprising a sequence having at least 87% sequence identity to SEQ ID NO: 17, or (xi) an NtGDH11 polypeptide encoded by the polynucleotide described in (i), or (xii) an NtGDH11 polypeptide having at least 92% sequence identity to SEQ ID NO: 18.

[0220] Preferably, in step (d), the plant leaves or a portion of the plant leaves are dried until they are completely dried, or for a period of time such as at least 24 hours, at least 48 hours, at least 96 hours, or at least 192 hours.

[0221] The mutants, transgenic or non-natural plants or parts thereof of the disclosed plant material, e.g., dried leaves, exhibit controlled levels of ammonia. Preferably, the plant material, e.g., dried leaves, also exhibit controlled levels of amino acids, sugars, and total alkaloids.

[0222] Preferably, controlled levels of ammonia, and optionally, controlled levels of amino acids, sugars, and total alkaloids, are observed in at least dried leaves, preferably fully dried leaves. Dried tobacco is considered fully dried if the central veins of the leaf contain no moisture, resulting in leaves ranging in color from light yellowish-brown to reddish-brown to deep brown.

[0223] Preferably, the dried leaves are taken from leaves located in the middle of the plant. Preferably, there is no effect on the leaf phenotype compared to leaves from a control plant.

[0224] In one embodiment, glucose levels are also adjusted compared to a control plant or a portion thereof. Preferably, glucose levels are increased when the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10, and NtGDH12 is decreased. Preferably, glucose levels are decreased when the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10, and NtGDH12 is increased.

[0225] In one embodiment, the fructose level is also adjusted compared to a control plant or a portion thereof. Preferably, the fructose level is increased when the expression or activity of one or more NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10, and NtGDH12 is decreased. Preferably, the fructose level is decreased when the expression or activity of one or more NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10, and NtGDH12 is increased.

[0226] In one embodiment, the sucrose level is also adjusted compared to a control plant or a portion thereof. Preferably, the sucrose level is increased when the expression or activity of one or more NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10, and NtGDH12 is decreased. Preferably, the sucrose level is decreased when the expression or activity of one or more NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10, and NtGDH12 is increased.

[0227] In one embodiment, the total amount of sugar is also adjusted compared to a control plant or a portion thereof. Preferably, the level of total sugar is increased when the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10, and NtGDH12 is decreased. Preferably, the level of total sugar is decreased when the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10, and NtGDH12 is increased.

[0228] In one embodiment, the nitrate level is also adjusted compared to a control plant or a portion thereof. Preferably, the nitrate level is reduced when the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10, and NtGDH12 is reduced. Preferably, the nitrate level is increased when the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10, and NtGDH12 is increased.

[0229] In one embodiment, the total alkaloid level is also adjusted compared to a control plant or a portion thereof. Preferably, the total alkaloid level is increased when the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10, and NtGDH12 is decreased. Preferably, the total alkaloid level is decreased when the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10, and NtGDH12 is increased.

[0230] In one embodiment, the level of total free amino acids is also adjusted compared to a control plant or a portion thereof. Preferably, the level of total free amino acids is increased when the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10, and NtGDH12 is decreased. Preferably, the level of total free amino acids is decreased when the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10, and NtGDH12 is increased.

[0231] In one embodiment, the level of aspartic acid is also adjusted compared to a control plant or a portion thereof. Preferably, the level of aspartic acid is increased when the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10, and NtGDH12 is decreased. Preferably, the level of aspartic acid is decreased when the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10, and NtGDH12 is increased.

[0232] In one embodiment, the level of asparagine is also adjusted compared to a control plant or a portion thereof. Preferably, the level of asparagine is increased when the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10, and NtGDH12 is decreased. Preferably, the level of asparagine is decreased when the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10, and NtGDH12 is increased.

[0233] In one embodiment, the level of glutamic acid is also adjusted compared to a control plant or a portion thereof.

[0234] In one embodiment, the level of glutamine is also adjusted compared to a control plant or a portion thereof. Preferably, the level of glutamine is increased when the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10, and NtGDH12 is reduced. Preferably, the level of glutamine is decreased when the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10, and NtGDH12 is reduced.

[0235] In one embodiment, the proline level is also adjusted compared to a control plant or a portion thereof. Preferably, the proline level is increased when the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10, and NtGDH12 is reduced. Preferably, the glutamine level is decreased when the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10, and NtGDH12 is reduced.

[0236] In one embodiment, serine levels are also adjusted compared to a control plant or a portion thereof. Preferably, serine levels are increased when the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10, and NtGDH12 is reduced. Preferably, glutamine levels are decreased when the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10, and NtGDH12 is reduced.

[0237] In one embodiment, the threonine level is also adjusted compared to a control plant or a portion thereof. Preferably, the threonine level is increased when the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10, and NtGDH12 is reduced. Preferably, the threonine level is decreased when the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10, and NtGDH12 is reduced.

[0238] In one embodiment, lysine levels are also adjusted compared to a control plant or a portion thereof. Preferably, lysine levels are increased when the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10, and NtGDH12 is reduced. Preferably, lysine levels are decreased when the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10, and NtGDH12 is reduced.

[0239] In one embodiment, the arginine level is also adjusted compared to a control plant or a portion thereof. Preferably, the arginine level is increased when the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10, and NtGDH12 is decreased. Preferably, the arginine level is decreased when the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10, and NtGDH12 is increased.

[0240] In one embodiment, the isoleucine level is also adjusted compared to a control plant or a portion thereof. Preferably, the isoleucine level is reduced when the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10, and NtGDH12 is reduced. Preferably, the arginine level is increased when the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10, and NtGDH12 is increased.

[0241] In one embodiment, the histidine level is also adjusted compared to a control plant or a portion thereof. Preferably, the histidine level is reduced when the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10, and NtGDH12 is reduced. Preferably, the histidine level is increased when the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10, and NtGDH12 is increased.

[0242] In one embodiment, the level of methionine is also adjusted compared to a control plant or a portion thereof. Preferably, the level of methionine is reduced when the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10, and NtGDH12 is reduced. Preferably, the level of methionine is increased when the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10, and NtGDH12 is increased.

[0243] In one embodiment, the level of citrulline is also adjusted compared to a control plant or a portion thereof. Preferably, the level of citrulline is reduced when the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10, and NtGDH12 is reduced. Preferably, the level of citrulline is increased when the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10, and NtGDH12 is increased.

[0244] In one embodiment, the leucine level is not adjusted compared to a control plant or a portion thereof.

[0245] In one embodiment, tyrosine levels are also adjusted compared to a control plant or a portion thereof. Preferably, tyrosine levels are increased when the expression or activity of one or more NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10, and NtGDH12 is decreased. Preferably, tyrosine levels are decreased when the expression or activity of one or more NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10, and NtGDH12 is increased.

[0246] In one embodiment, the level of valine is not adjusted compared to a control plant or a part thereof.

[0247] In one embodiment, the level of tryptophan is also adjusted compared to a control plant or a portion thereof. Preferably, the level of tryptophan is reduced when the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10, and NtGDH12 is reduced. Preferably, the level of tryptophan is increased when the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10, and NtGDH12 is increased.

[0248] In one embodiment, the level of alanine is also adjusted compared to a control plant or a portion thereof.

[0249] In one embodiment, the level of GABA is also adjusted compared to a control plant or a portion thereof. Preferably, the level of GABA is reduced when the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10, and NtGDH12 is reduced. Preferably, the level of GABA is increased when the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10, and NtGDH12 is increased.

[0250] In one embodiment, the level of phenylalanine is also adjusted compared to a control plant or a portion thereof. Preferably, the level of phenylalanine is reduced when the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10, and NtGDH12 is reduced. Preferably, the level of phenylalanine is increased when the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10, and NtGDH12 is increased.

[0251] In one embodiment, the glycine level is also adjusted compared to a control plant or a portion thereof. Preferably, the glycine level is reduced when the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10, and NtGDH12 is reduced. Preferably, the glycine level is increased when the expression or activity of one or more of NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10, and NtGDH12 is increased.

[0252] In one embodiment, when the expression or activity of one or more NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10, and NtGDH12 is reduced, the levels of glucose, fructose, sucrose, total sugars, total free amino acids, aspartic acid, asparagine, glutamine, proline, serine, threonine, lysine, arginine, and tyrosine are increased. In another embodiment, when the expression or activity of one or more NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10, and NtGDH12 is increased, the levels of glucose, fructose, sucrose, total sugars, total free amino acids, aspartic acid, asparagine, glutamine, proline, serine, threonine, lysine, arginine, and tyrosine are reduced.

[0253] In one embodiment, when the expression or activity of one or more NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10, and NtGDH12 is decreased, the levels of ammonia, nitrates, total alkaloids, isoleucine, histidine, methionine, citrulline, tryptophan, GABA, phenylalanine, and glycine are decreased. In another embodiment, when the expression or activity of one or more NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10, and NtGDH12 is increased, the levels of ammonia, nitrates, total alkaloids, isoleucine, histidine, methionine, citrulline, tryptophan, GABA, phenylalanine, and glycine are increased.

[0254] Further aspects of this disclosure relate to dried tobacco plant leaves or portions of dried plant leaves having an ammonia content between approximately 0.16 ± 0.04% DWB and 0.11 ± 0.16 ± 0.03% DWB. The dry weight basis can be calculated after drying at 105°C until a certain mass is reached, which is essentially 100 percent solids.

[0255] Further aspects of this disclosure relate to dried tobacco leaves or portions of dried plant leaves having (i) an ammonia content between approximately 0.16±0.04% DWB and 0.11±0.16±0.03% DWB, and (ii) a glucose, fructose, and sucrose content between 0.51±0.58% DWB and 1.55±1.10% DWB, or (iii) a total free amino acid content between 51.0±6.60 mg / g DWB and 60.1±4.58 mg / g DWB, or (iv) a total alkaloid content between 2.24±0.8% DWB and 4.20±0.39%.

[0256] Further aspects of this disclosure relate to dried tobacco leaves or portions of dried plant leaves having (i) an ammonia content between approximately 0.16±0.04%DWB and 0.11±0.16±0.03%DWB, (ii) a glucose, fructose, and sucrose content between 0.51±0.58%DWB and 1.55±1.10%DWB, (iii) a total free amino acid content between 51.0±6.60 mg / g DWB and 60.1±4.58 mg / g DWB, and (iv) a total alkaloid content between 2.24±0.8%DWB and 4.20±0.39%.

[0257] Further embodiments relate to mutant, non-natural, or transgenic plants or cells in which the expression of one or more NtGDH polynucleotides or the activity of one or more NtGDH polypeptides is reduced, and ammonia levels are reduced compared to a control plant or a portion thereof in which the expression of NtGDH or the activity of NtGDH is not reduced.

[0258] Further embodiments relate to desiccated plant materials, such as desiccated leaves or desiccated tobacco, obtained from or available from mutant, non-natural, or transgenic plants or cells, wherein the expression of one or more NtGDH polynucleotides described herein or the function of the NtGDH polypeptide encoded thereby is reduced, wherein the level of ammonia is reduced compared to a control plant or a portion thereof.

[0259] The embodiments also relate to compositions and methods for producing mutant, non-natural, or transgenic plants or plant cells modified to regulate, preferably reduce, the expression or activity of one or more NtGDH polynucleotides or NtGDH polypeptides described herein, thereby resulting in plants or plant parts (leaves, e.g., dried leaves) or plant cells having a regulated, preferably reduced ammonia content.

[0260] In one embodiment, the phenotype of the mutant, non-natural, or transgenic plant is substantially the same as that of the control plant or a part thereof. In one embodiment, the leaf weight of the mutant, non-natural, or transgenic plant is substantially the same as that of the control plant or a part thereof. In one embodiment, the number of leaves of the mutant, non-natural, or transgenic plant is substantially the same as that of the control plant or a part thereof. In one embodiment, the leaf weight and number of leaves of the mutant, non-natural, or transgenic plant are substantially the same as those of the control plant. In one embodiment, the stem height of the mutant, non-natural, or transgenic plant is substantially the same as that of the control plant or a part thereof, for example, one month, two months, or three months or more after transplanting to the field, or 10, 20, 30, or 36 days or more after pinching. For example, the stem height of the mutant, non-natural, or transgenic plant is not lower than the stem height of the control plant or a part thereof.

[0261] In another embodiment, a method is provided for regulating the amount of ammonia in at least a portion of a plant (such as leaves, e.g., dried leaves), comprising: (i) regulating the expression or function of one or more NtGDH polypeptides described herein, preferably the NtGDH polypeptide being encoded by a corresponding NtGDH polynucleotide described herein; (ii) measuring the level of ammonia in at least a portion of the mutant, non-natural, or transgenic plant obtained in step (i) (such as leaves, e.g., dried leaves); and (iii) identifying the mutant, non-natural, or transgenic plant or a portion thereof in which the level of ammonia is regulated compared to a control plant or a portion thereof. Preferably, the levels of one or more amino acids, sugars, and total alkaloids are also regulated.

[0262] In another embodiment, a method is provided for regulating the amount of at least one amino acid in a dried plant material (such as a dried leaf), comprising: (i) regulating the expression or function of one or more NtGDH polypeptides (or any combination thereof as described herein), preferably the NtGDH polypeptide being encoded by a corresponding NtGDH polynucleotide as described herein; (ii) harvesting a plant material, such as one or more leaves, and drying it for a certain period of time; (iii) measuring the level of ammonia in the dried plant material obtained in or during step (ii); and (iv) identifying the dried plant material in which the level of ammonia has been regulated compared to a control plant or a portion thereof. Preferably, the levels of one or more amino acids, sugars, and total alkaloids are also regulated.

[0263] The increase in expression compared to the control may be approximately 5% to approximately 100%, or at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, or an increase of 100% or more, such as an increase of 200%, 300%, 500%, 1000% or more, and this includes an increase in transcriptional function or NtGDH polynucleotide expression or NtGDH polypeptide expression.

[0264] The increase in function or activity compared to the control may be approximately 5% to approximately 100%, or at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, or an increase of 100% or more, such as an increase of 200%, 300%, 500%, 1000% or more, and this includes an increase in transcriptional function or NtGDH polynucleotide expression or NtGDH polypeptide expression or a combination thereof.

[0265] The reduction in expression compared to the control may be approximately 5% to 100%, or at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, or 100%, and this includes a reduction in transcriptional function or NtGDH polynucleotide expression or NtGDH polypeptide expression or a combination thereof.

[0266] The reduction in function or activity compared to the control may be approximately 5% to approximately 100%, or at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, or 100%, and this includes a reduction in transcriptional function or NtGDH polynucleotide expression or NtGDH polypeptide expression or a combination thereof.

[0267] The polynucleotides and recombinant constructs described herein can be used to modulate the expression, function, or activity of the NtGDH polynucleotides or NtGDH polypeptides described herein in target plant species, preferably tobacco.

[0268] Many polynucleotide-based methods can be used to increase gene expression in plants and plant cells. For example, constructs, vectors, or expression vectors compatible with the plant to be transformed can be prepared, which include the gene of interest along with an upstream promoter that can overexpress the gene in the plant or plant cells. Exemplary promoters are described herein. After transformation, when grown under favorable conditions, the promoter can drive expression to regulate NtGDH levels in the plant or its particular tissues. In one exemplary embodiment, a vector carrying one or more NtGDH polynucleotides described herein (or any combination thereof) is produced to overexpress the gene in a plant or plant cell. The vector has a suitable promoter, such as the cauliflower mosaic virus CaMV 35S promoter, upstream of the transgene that drives its constitutive expression in all tissues of the plant. The vector also has an antibiotic resistance gene to give selection of transformed callus and cell lines.

[0269] The expression of sequences from promoters can be enhanced by including expression regulatory sequences well known in the art. Activated signals are specifically shown during cellular senescence-related signals and drying processes.

[0270] Accordingly, various embodiments are directed toward methods for modulating the expression levels of one or more NtGDH polynucleotides (or any combination thereof) described herein by integrating multiple copies of one or more NtGDH polynucleotides into a plant genome, the methods comprising transforming a plant cell host with an expression vector comprising a promoter functionally conjugated to one or more NtGDH polynucleotides described herein. The polypeptide encoded by the recombinant polynucleotide may be a native polypeptide or may be heterologous to the cell.

[0271] In one embodiment, the plants used in this disclosure are flucurated mutants, non-natural types, or transgenic plants.

[0272] In one embodiment, the plants used in the present disclosure are sun-dried mutants, non-natural types, or transgenic plants.

[0273] In one embodiment, the plants used in this disclosure are air-dried mutants, non-natural types, or transgenic plants.

[0274] In one embodiment, the plants used in the present disclosure are dried, for example, fl-cured mutants, non-natural types, or transgenic Virginia tobacco plants.

[0275] In one embodiment, the plants used in the present disclosure are desiccated, for example, air-dried mutant, non-natural, or transgenic Burley tobacco plants.

[0276] In one embodiment, the plants used in the present disclosure are desiccated, for example, heat-dried mutant, non-natural, or transgenic dark tobacco plants.

[0277] Advantageously, the sensory profile of tobacco can be modified by regulating NtGDH expression and / or activity. For example, as can be seen from Table 2, modified Burley tobacco has reduced irritation, a rounder and smoother mainstream aerosol, less of the typical dark aroma, and more animalistic and nutty aromas (less impact on the trigeminal nerve).

[0278] Plants having one or more NtGDH polynucleotide mutant alleles described herein can be used in plant breeding programs to produce useful lines, varieties, and hybrids. For example, mutant alleles can be introduced into commercially important varieties described herein. Thus, methods for breeding plants are provided, including crossing mutant plants, non-natural type plants, or transgenic plants described herein with plants having different genetic identities. The methods may further include crossing the offspring plants with another plant and, optionally, repeating the cross until offspring having the desired genetic trait or genetic background are obtained. One objective achieved by such breeding methods is to introduce desired genetic traits into other varieties, breeding lines, hybrids, or cultivars, particularly those of commercial interest. Another objective is to facilitate the stacking of genetic modifications of different genes in a single plant variety, line, hybrid, or cultivar. Intraspecific and interspecific crosses are intended. Offspring plants resulting from such crosses are also called breeding lines and are examples of non-natural type plants in this disclosure.

[0279] In one embodiment, a method is provided for producing non-natural plants, comprising (a) crossing a mutant or transgenic plant with a second plant to produce offspring tobacco seeds, and (b) growing the offspring tobacco seeds under plant growth conditions to produce non-natural plants. The method may further include (c) crossing the previous generation of non-natural plants with itself or another plant to produce offspring tobacco seeds, (d) growing the offspring tobacco seeds from step (c) under plant growth conditions to produce additional non-natural plants, and (e) repeating the crossing and growing steps (c) and (d) multiple times to produce further generations of non-natural plants. The method optionally includes, prior to step (a), a step of providing a parent plant that is characterized and has genetic identity that is not identical to the mutant or transgenic plant. In some embodiments, depending on the breeding program, the crossing and growth steps are repeated 0-2, 0-3, 0-4, 0-5, 0-6, 0-7, 0-8, 0-9, or 0-10 times to produce a generation of non-natural type plants. Backcrossing is an example of such a method, in which a descendant is crossed with one of its parents or another plant genetically similar to a parent in order to obtain a descendant plant having the same genetic identity as one of the parents in the next generation. Plant breeding, and in particular plant breeding techniques, are well known and can be used in the methods of this disclosure. This disclosure further provides non-natural type plants produced by these methods. Certain embodiments exclude the step of selecting plants.

[0280] In some embodiments of the methods described herein, lines resulting from breeding and variant gene screening are evaluated in the field using standard field procedures. A control genotype, including the original, unmutated parent, is included, and entries are placed in the field in a randomized full block design or other appropriate field design. For tobacco, standard agricultural practices are used; for example, tobacco is harvested, weighed, and sampled for chemical and other general tests before and during drying. Statistical analysis of the data is performed to confirm the similarity of the selected lines to the parent lines. Cytogenetic analysis of the selected plants is optionally performed to confirm chromosomal complementarity and chromosomal pairing relationships.

[0281] DNA fingerprinting, single nucleotide polymorphisms, microsatellite markers, or similar techniques can be used in marker-assisted selection (MAS) breeding programs to transfer or breed mutant alleles of genes to other tobacco plants, as described herein. For example, a breeder can create a population that is isolated from a cross between a genotype containing a mutant allele and an agriculturally desirable genotype. F2 or backcross generation plants can be screened using one of the techniques described herein, with markers developed from the genome sequence or fragments thereof. Plants identified as carrying a mutant allele can be backcrossed or self-pollinated to create a second population to be screened. Depending on the expected mode of inheritance or the MAS technique used, it may be necessary to self-pollinate the selected plants before each cycle of backcrossing to facilitate the identification of the desired individual plants. Backcrossing or other breeding procedures can be repeated until the desired phenotype of the repeating parent is restored.

[0282] In accordance with this disclosure, in a breeding program, a successful cross produces fertile F1 plants. The selected F1 plants are crossed with one of the parents, and the first backcross generation plants are self-pollinated to produce a population that is screened again for variant gene expression (e.g., a null version of the gene). The process of backcrossing, self-pollination, and screening is repeated at least four times, for example, until the final screening produces plants that are fertile and reasonably similar to the repeating parent. These plants are, if desired, self-pollinated, and the offspring are then screened again to confirm that the plants exhibit variant gene expression. In some embodiments, the F2 generation plant population is screened for variant gene expression, and plants that do not express the polypeptide due to the absence of the gene are identified by standard methods, such as a PCR method using primers based on polynucleotide sequence information as described herein (or any combination thereof as described herein).

[0283] Hybrid tobacco varieties can be produced by preventing self-pollination of the female parent plant (i.e., seed parent) of the first variety, and fertilizing the female parent plant with pollen from the male parent plant of the second variety, thereby forming F1 hybrid seeds on the female plant. Self-pollination of the female plant can be prevented by emasculating the flowers at an early stage of flower development. Alternatively, pollen formation on the female parent plant can be prevented using a form of male sterility. For example, male sterility can be produced by cytoplasmic male sterility (CMS), or transgenic male sterility in which a transgene inhibits microspore formation or pollen formation, or by self-incompatibility. Female parent plants containing CMS are particularly useful. In embodiments where the female parent plant is CMS, pollen is harvested from a male-fertile plant and manually applied to the stigma of the CMS female parent plant, and the resulting F1 seeds are harvested.

[0284] The varieties and lines described herein can be used to form monohybrid tobacco F1 hybrids. In such embodiments, the parent varieties may be grown as substantially homogeneous adjacent populations to facilitate natural cross-pollination from male to female parent plants. The F1 seeds formed on the female parent plants are selectively harvested by conventional means. Alternatively, two parent varieties may be grown together, and a blend of F1 hybrid seeds formed on the female parent and seeds formed on the male parent as a result of self-pollination may be harvested. Or, a triway cross can be performed in which a monohybrid F1 hybrid is used as the female parent and crossed with a different male parent. As another alternative, a multihybrid can be created in which the F1 offspring of two different monohybrids cross with each other.

[0285] Populations of mutants, non-natural types, or transgenic plants can be screened or selected for members of the population possessing a desired trait or phenotype. For example, a population of offspring of a single transformation event can be screened for plants possessing a desired level of expression or function of the polypeptide it encodes. Physical and biochemical methods can be used to determine the level of expression or activity. These include Southern spectroscopy or PCR amplification for the detection of polynucleotides; Northern blotting, S1 RNase protection, primer extension, or RT-PCR amplification for the detection of RNA transcripts; enzyme assays for detecting the enzymatic or ribozyme function of polypeptides and polynucleotides; and polypeptide gel electrophoresis, Western blotting, immunoprecipitation, and enzyme-linked immunosorbent assays for detecting polypeptides. Other techniques such as in situ hybridization, enzyme staining, and immunostaining, as well as enzyme assays, can also be used to detect the presence or expression, function, or activity of NtGDH polypeptides or polynucleotides.

[0286] The following are described herein: mutants comprising one or more recombinant polynucleotides, one or more polynucleotide constructs, one or more double-stranded RNAs, one or more complexes, or one or more vectors / expression vectors, non-natural or transgenic plant cells and plants.

[0287] Without limitation, plants and parts thereof described herein may be modified before or after the expression, function, or activity of one or more NtGDH polynucleotides or NtGDH polypeptides is regulated in accordance with this disclosure.

[0288] One or more of the following further genetic modifications may be present in mutants, non-natural or transgenic plants and parts thereof: One or more genes involved in the conversion of nitrogen metabolic intermediates may be modified to result in lower levels of at least one tobacco-specific nitrosamine (TSNA). Non-limiting examples of such genes include genes encoding nicotine demethylases such as CYP82E4, CYP82E5, and CYP82E10, as described in WO2006 / 091194, WO2008 / 070274, WO2009 / 064771, and WO2011 / 088180, and nitrate reductases as described in WO2016 / 046288. One or more genes involved in heavy metal uptake or heavy metal transport may be modified to result in lower heavy metal content. Non-limiting examples include the multidrug resistance-related polypeptide family, the cation diffusion promoter family (CDF), the Zrt-Irt-like polypeptide family (ZIP), the cation exchanger family (CAX), the copper transporter family (COPT), the heavy metal ATPase family (e.g., HMAs described in WO2009 / 074325 and WO2017 / 129739), homologs of the native resistance-related macrophage polypeptide family (NRAMP), and other members of the ATP-binding cassette transporter family (ABC transporters) (e.g., MRPs described in WO2012 / 028309) involved in the transport of heavy metals (such as cadmium).

[0289] Other exemplary modifications may result in plants having regulated expression or function of isopropylmalate synthase, resulting in alterations to the sucrose ester composition that can be used to modify the flavor profile (see WO2013 / 029799). Other exemplary modifications may result in plants having regulated expression or function of threonine synthase, in which the level of methional can be regulated (see WO2013 / 029800). Other exemplary modifications may result in plants having regulated expression or function of one or more neoxanthine synthase, lycopene betacyclase, and 9-cis-epoxycarotenoid dioxygenase, thereby regulating the beta-damascenone content to alter the flavor profile (see WO2013 / 064499). Other exemplary modifications may result in plants having regulated expression or function of a member of the CLC family of chloride channels to regulate the level of nitrates (see WO2014 / 096283 and WO2015 / 197727). Other exemplary modifications can result in plants having regulated expression or function of one or more asparagine synthetases that regulate the level of asparagine in the leaves, and regulated levels of acrylamide in aerosols produced when the leaves are heated or burned (see WO2017 / 129739). Other exemplary modifications can result in plants having regulated protease activity during drying treatment (see WO2016 / 009006). Other exemplary modifications can result in plants with reduced nitrate levels by altering the gene expression of a nitrate reductase (e.g., Nia2) or the activity of the protein encoded by it (see WO2016 / 046288). Other 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 proteins encoded by them (see WO2019 / 086609).Other exemplary modifications can result in plants with regulated time to flowering by altering the gene expression of the gene encoding Terminal Flower 1 (TFL1) or the activity of the protein it encodes (see WO2018 / 114641). Other exemplary modifications can result in plants with regulated expression or function of one or more asparagine synthetases to regulate asparagine levels in leaves and result in plants with regulated levels of acrylamide in the aerosol produced when the leaves are heated or burned (see WO2017 / 042162). Examples of other modifications include the regulation of herbicide resistance, for example, glyphosate is the active ingredient in many broad-spectrum herbicides. Glyphosate-resistant transgenic plants have been developed by introducing the aroA gene (glyphosate EPSP synthase from Salmonella typhimurium and E. coli). Sulfonylurea-resistant plants have been produced by transforming with the mutant ALS (acetolactic acid synthetase) gene from Arabidopsis thaliana. The OB polypeptide of photosystem II from the mutant Amaranthus hybridus has been introduced into 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. Other exemplary modifications result in plants resistant to insects. The Bacillus thuringiensis (Bt) toxin may provide an effective method for delaying the emergence of Bt-resistant pests, as recently demonstrated in broccoli where pyramidal cry1Ac and cry1C Bt genes control diamondback moths resistant to a single polypeptide, significantly slowing the evolution of resistant insects. Other exemplary modifications result in plants resistant to diseases caused by pathogens (e.g., viruses, bacteria, fungi). Plants expressing the Xa21 gene (resistance to bacterial canker) and plants expressing both the Bt fusion gene and the chitinase gene (resistance to yellow stem worm and rice sheath blight) have been engineered.Other exemplary modifications result in altered reproductive capacity, such as male sterility. Other exemplary modifications result in plants tolerant to abiotic stress (e.g., drought, temperature, salinity), and tolerant transgenic plants have been produced by transferring acylglycerol phosphate enzymes from Arabidopsis thaliana, with genes encoding mannitol dehydrogenase and sorbitol dehydrogenase, which are involved in the synthesis of mannitol and sorbitol, improving drought tolerance. Other exemplary modifications result in plants in which the activity of one or more nicotine N-demethylases is regulated, thereby enabling the regulation of levels of nornicotine and nornicotine metabolites formed during drought treatment (see WO2015169927). Other exemplary modifications can result in plants with improved storage polypeptides and oils, plants with enhanced photosynthetic efficiency, plants with extended shelf life, plants with enhanced carbohydrate content, and plants resistant to fungi. Transgenic plants in which the expression of S-adenosyl-L-methionine (SAM) or cystathionine gamma-synthase (CGS) or a combination thereof is regulated are also intended. One or more genes involved in the nicotine synthesis pathway can be modified to result in a plant or part of a plant that produces regulated levels of nicotine upon drying. Nicotine synthesis genes can be selected from the group consisting of A622, BBLa, BBLb, JRE5L1, JRE5L2, MATE1, MATE2, MPO1, MPO2, MYC2a, MYC2b, NBB1, nic1, nic2, NUP1, NUP2, PMT1, PMT2, PMT3, PMT4, and QPT or a combination of one or more thereof. One or more genes involved in controlling the amount of one or more alkaloids can be modified to result in a plant or part of a plant that produces regulated levels of alkaloids. Alkaloid level regulatory genes can be selected from the group consisting of BBLa, BBLb, JRE5L1, JRE5L2, MATE1, MATE2, MYC2a, MYC2b, nic1, nic2, NUP1, and NUP2, or two or more combinations thereof.

[0290] Other exemplary modifications may result in plants having a controlled amino acid content (see WO2019 / 185703 and WO2021 / 063863), a controlled sugar content (see WO2019 / 185699 and WO2021 / 063860 and WO2021 / 063863), a controlled nitrate level (see WO2020 / 141062), or a controlled sugar and amino acid content (see WO2021 / 063863).

[0291] In preferred embodiments, further genetic modifications relate to the asparagine synthetase (ASN) gene described in WO2017042162. Modification of ASN gene expression (e.g., one or more of NtASN1-S, NtASN1-T, NtASN5-S, and NtASN5-T described in WO2017042162) or ASN activity (e.g., NtASN1-S, NtASN1-T, NtASN5-S, and NtASN5-T described in WO2017042162) significantly alters the chemical properties of dried tobacco leaves without affecting biomass. Therefore, modulating the expression and / or activity of ASN and NtGDH combinations may have the potential to reconstitute the chemical properties of dried tobacco leaves (particularly the amino acid chemical properties of Burley or dark tobacco) and thereby alter their functional properties.

[0292] In addition to ASN, other genes and enzymes play a role in the rearrangement of amino acids and / or sugars during leaf yellowing, such as diaminopimelate aminotransferase (DAPAT), which is involved in both the catabolism and anabolism of lysine, and aspartate aminotransferase (AAT), which is expressed during senescence and has the potential to alter the chemical properties of leaves after drying treatment (WO2019 / 185703). Chloroplast sulfate transporters SULTR3, such as NtSULTR3;1A-S, NtSULTR3;1A-T, and NtSULTR3;3-T, play a role in sugar and amino acid metabolism during drying treatment (see WO2021 / 063863). Therefore, further genetic modifications may relate to one or more of DAPAT and / or AAT (such as one or more of NtAATI-S, NtAAT1-T, NtAAT2-S, NtAAT2-T, NtAAT3-S, NtAAT3-T, NtAAT4-S, or NtAAT4-T described in WO2017042162) and / or one or more of NtSULTR3;1A-S, NtSULTR3;1A-T, and NtSULTR3;3-T described in WO2021 / 063863. Modulating the expression and / or activity of the combination of DAPAT and / or AAT and / or SULTR3 and NtGDH may have the potential to reconstitute the chemical properties of dried tobacco leaves, thereby altering sensory properties. Modifications of combinations of NtGDH with one or more, or two or more, or three or 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 ASN and AAT and SULTR3, NtGDH and ASN and DAPAT and AAT and SULTR3.

[0293] One or more traits can be introduced into a mutant, non-natural or transgenic plant from another cultivar, or directly transformed into it.

[0294] Various embodiments provide biomass in which the expression levels of one or more polynucleotides are regulated according to the present disclosure, thereby regulating the levels of the polypeptides encoded thereby, in mutant plants, non-natural plants or transgenic plants.

[0295] The present invention also provides a dried tobacco blend with reduced levels of ammonia. In particular, the tobacco blend can include at least a first dried tobacco plant leaf or a portion thereof according to the present invention and a second dried tobacco plant leaf or a portion thereof having a total amount of ammonia that is less than the total amount of ammonia in at least a second dried tobacco plant leaf or a portion thereof. The blended cigarette typically uses two or three major tobacco types selected from Virginia, Burley, and Oriental. In one embodiment, the second dried tobacco plant leaf or a portion thereof is Burley tobacco or Oriental tobacco or dark tobacco or flue-cured tobacco or a combination of two or more thereof.

[0296] A method is also provided for producing a tobacco blend with reduced ammonia content, the method comprising: (b) providing dried tobacco leaves or portions thereof, wherein the first dried tobacco leaves or portions thereof are from a mutant, non-natural, or transgenic tobacco leaf or portion thereof; and (b) blending the first dried tobacco leaves or portions thereof with at least a second dried tobacco leaf or portion thereof to produce a tobacco blend in which the total amount of ammonia is lower than the total amount of ammonia in at least the second dried tobacco leaf or portion thereof. After the tobaccos have been blended together, the tobacco blend can be finely chopped before being dried to reduce moisture. When the moisture content is at an optimal level, the blend is ready for production.

[0297] Parts of plants described herein, particularly the leaf blades and / or stems and / or midribs of plants, may be incorporated into or used to manufacture a variety of consumer products, including but not limited to aerosol-forming materials, aerosol-forming devices, smoking articles, smokeable articles, smokeless products, pharmaceuticals or cosmetics, intravenous preparations, tablets, powders, and tobacco products. Examples of aerosol-forming materials include tobacco compositions, tobacco, tobacco extracts, shredded tobacco, shredded filler, dried tobacco, expanded tobacco, homogenized tobacco, re-fed tobacco, and pipe tobacco. Smoking articles and smokeable articles are types of aerosol-forming devices. Examples of smoking articles or smokeable articles include cigarettes, cigarillos, and cigars. Examples of smokeless products include chewing tobacco and snuff. In certain aerosol-forming devices, instead of combustion, a tobacco composition or another aerosol-forming material is heated by one or more electric heating elements to produce an aerosol. In other types of heated aerosol-forming devices, aerosols are produced by the transfer of heat to an aerosol-forming material that is physically separated from a combustible fuel element or heat source, and the aerosol-forming material can be located inside, around, or downstream of the heat source. Aerosol-forming materials, including smokeless tobacco products and various tobaccos, can contain tobacco in any form, such as dry particles, shredded, granules, powder, or slurry, and may be deposited, mixed, surrounded, or otherwise combined with other components in any form, such as flakes, films, tabs, foam, or beads. The term “smoke” is used to describe the type of aerosol produced by smoking articles such as cigarettes, or by burning an aerosol-forming material.

[0298] In one embodiment, dried plant material from mutant, transgenic, and non-natural plants described herein is also provided. Processes for drying green tobacco leaves are known to those skilled in the art and include, but are not limited to, air drying, heat drying, fl-curing, and sun drying processes described herein.

[0299] In another embodiment, a tobacco product is described comprising a tobacco-containing aerosol-forming material, which includes plant material from mutant tobacco plants, transgenic tobacco plants, or non-natural tobacco plants described herein, such as leaves, preferably dried leaves. The tobacco product described herein may further be a blended tobacco product containing unmodified tobacco.

[0300] Mutant, non-natural, or transgenic plants may have other uses, for example, in agriculture.

[0301] This disclosure also provides a method for producing seeds, which includes cultivating the mutant plants, non-natural type plants, or transgenic plants described herein, and collecting seeds from the cultivated plants. Seeds from the plants described herein may be prepared in packaging material by means known in the art to form a manufactured article and packed into bags. Packaging materials such as paper and cloth are well known in the art. Seed packaging may have labels, for example, tags or labels attached to the packaging material, or labels printed on the package that describe the properties of the seeds contained therein.

[0302] Compositions, methods, and kits for determining the genotype of plants for identification, selection, or breeding may include means for detecting the presence of NtGDH polynucleotides in a sample of polynucleotides. Accordingly, compositions are described comprising one or more primers for specifically amplifying at least a portion of one or more NtGDH polynucleotides, optionally one or more probes, and optionally one or more reagents for carrying out amplification or detection.

[0303] Accordingly, gene-specific oligonucleotide primers or probes comprising about 10 or more consecutive polynucleotides corresponding to the NtGDH polynucleotides described herein are disclosed. The primers or probes may comprise or consist of about 15, 20, 25, 30, 40, 45, or 50 or more consecutive polynucleotides that hybridize (e.g., specifically hybridize) to the NtGDH polynucleotides described herein. In some embodiments, the primers or probes may comprise or consist of about 10 to 50 consecutive nucleotides, about 10 to 40 consecutive nucleotides, about 10 to 30 consecutive nucleotides, or about 15 to 30 consecutive nucleotides that can be used in sequence-dependent gene identification methods (e.g., Southern hybridization) or isolation (e.g., in situ hybridization of bacterial colonies or bacteriophage plaques) or gene detection (e.g., as one or more amplification primers in amplification or detection). One or more specific primers or probes may be designed and used to amplify or detect some or all of the polynucleotides. As a specific example, a polynucleotide fragment can be amplified using two primers in a PCR protocol. PCR can also be performed using one primer derived from the polynucleotide sequence and a second primer that hybridizes to an upstream or downstream sequence of the polynucleotide sequence (such as a promoter sequence, the 3' end of the mRNA precursor, or a sequence derived from a vector). Examples of thermal and isothermal techniques useful for in vitro amplification of polynucleotides are well known in the art. The sample may be a plant, plant cell or plant material as described herein, or a tobacco product made from or derived from a plant, plant cell or plant material, or derived therefrom.

[0304] In a further embodiment, a method is also provided for detecting NtGDH polynucleotides (or any combination thereof) described herein in a sample, the method comprising: (a) providing a sample containing or suspected to contain polynucleotides; (b) contacting the sample with one or more primers or one or more probes for the specific detection of at least a portion of NtGDH polynucleotides; and (c) detecting the presence of an amplification product, the presence of which indicates the presence of NtGDH polynucleotides in the sample. In a further embodiment, the use of one or more primers or probes for the specific detection of at least a portion of NtGDH polynucleotides is also provided. A kit for detecting at least a portion of NtGDH polynucleotides is also provided, which comprises one or more primers or probes for the specific detection of at least a portion of NtGDH polynucleotides. The kit may include reagents for polynucleotide amplification, such as PCR, or reagents for probe hybridization detection techniques, such as Southern blotting, Northern blotting, in-situ hybridization, or microarrays. The kit may include reagents for antibody-conjugated detection techniques such as Western blotting, ELISA, SELDI mass spectrometry, or test strips. The kit may also include reagents for DNA sequencing. Finally, the kit may include reagents and instructions for using the kit.

[0305] In some embodiments, the kit may include instructions for one or more of the described methods. The described kit may be useful for determining genetic identity, phylogenetic studies, genotyping, haplotype determination, pedigree analysis, or plant breeding, particularly codominance scoring.

[0306] This disclosure also provides methods for genotyping plants, plant cells, or plant materials containing the NtGDH polynucleotides described herein. Genotyping provides a means for distinguishing chromosome homologs and can be used to distinguish segregates in plant populations. Molecular marker methods can be used for phylogenetic studies, characterization of genetic relationships between crop varieties, identification of hybrids or somatic cell hybrids, localization of chromosome segments affecting single gene traits, map-based cloning, and quantitative genetic studies. Specific methods of genotyping can employ any number of molecular marker analysis techniques, including amplified fragment length polymorphism (AFLP). AFLP is the product of allelic differences between amplified fragments caused by polynucleotide mutations. Thus, this disclosure further provides means for tracking the segregation of one or more genes or polynucleotides, as well as chromosomal sequences genetically linked to these genes or polynucleotides, using techniques such as AFLP analysis.

[0307] A method for producing a liquid tobacco extract, and a liquid tobacco extract produced by this method are also disclosed herein.

[0308] A specific extraction temperature is selected for the tobacco starting material. The extraction temperature is typically selected from a range of about 100°C to about 160°C. The duration of the heating step can optionally be controlled to provide some control over the composition of the extract obtained from the tobacco starting material. Preferably, the tobacco starting material is heated at the extraction temperature for at least about 90 minutes, more preferably at least about 120 minutes. The heating step is typically carried out in an inert atmosphere. Preferably, a flow of an inert gas, such as nitrogen, is passed through the starting tobacco material during the heating step. Volatile tobacco compounds are released into the flow of inert gas during the heating step, and as a result, the inert gas acts as a carrier for the volatile components. The flow of inert gas may be at least about 25 L / min, more preferably at least about 30 L / min. Relatively high flow rates of inert gas can advantageously improve the efficiency of extraction from the tobacco starting material. Optionally, the heating step may be carried out in a vacuum. Suitable heating methods for heating tobacco starting materials are known to those skilled in the art and include dry distillation, steam distillation, vacuum distillation, flash distillation, and thin-film steam distillation.

[0309] When volatile compounds are collected by absorption into a liquid solvent, the step of forming a liquid tobacco extract may include drying the solution of volatile compounds in the liquid solvent to concentrate the solution. Drying can be carried out using any suitable means, including but not limited to drying, molecular sieving, freeze-drying, phase separation, distillation, membrane permeation, controlled crystallization and filtration of water, reverse hygroscopicity, ultracentrifugation, liquid chromatography, reverse osmosis, or chemical drying.

[0310] Liquid tobacco extracts are particularly suitable for producing compositions, formulations, or gel compositions for use in aerosol generating systems. An aerosol generating system comprising a composition, formulation, or gel composition is disclosed. In such an aerosol generating system, the composition, formulation, or gel is typically heated within an aerosol generating device, such as a device including a heater element that interacts with the composition, formulation, or gel incorporating the liquid tobacco extract to produce an aerosol. During use, volatile compounds are released by heat transfer and entrained in the air drawn in through the aerosol generating device. As the released compounds cool, they condense to form an aerosol that is inhaled by the consumer.

[0311] The present invention is further described in the following embodiments provided to illustrate the present invention in more detail. These embodiments, which describe preferred modes currently intended for carrying out the present invention, are intended to illustrate the present invention and not to limit it. [Examples]

[0312] Example 1 - Materials and Methods Plant materials and cultivation conditions Before germination, the seeds are sterilized with chlorine gas. A 5% final chlorine solution is placed in a bell-shaped container along with the glass tubes containing the seeds. Then, hydrochloric acid (37%) is added to the solution, and the seeds are incubated for 2 hours. The seeds are then placed in Murashige & Skoog propagation medium (Murashige & Skoog (1962) Physiologia Plantarum 15, 3, 473-497) and transferred to a plant growth chamber (24°C, 16 hours light / 20°C, 8 hours dark) for 4 weeks. Well-developed seedlings are transferred to a greenhouse and grown in 10L pots until fully grown. Artificial light is applied for 16 hours daily. All plants are grown at the same spacing throughout all stages of cultivation to avoid any impact on the leaves.

[0313] Method for determining amino acid content The amino acid content is measured using Method MP 1471 rev 5 2011, Resana, Italy: Chelab Silliker S.r.l, Merieux NutriSciences Company. For the determination of amino acids in dried plant leaves, after removing the midrib, if necessary, the dried leaf blade is dried at 40°C for 2 - 3 days. Then, the tobacco material is ground into fine powder (-100uM) before analysis of the amino acid content. Alternatively, the 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 analyzer or an HPLC device.

[0314] Method for determining sugar content The sugar content is adapted by Skalar Instrument Co (West Chester, PA) and measured using the segmented flow colorimetry developed for the analysis of tobacco samples as described in Tobacco Science 20:139 - 144 (1976). The measurement of the sugar content is also described in Coresta Recommended Method No. 38, CRM38, CRM and ISO 15154:2003. For the determination of sugars in dried leaves, after removing the midrib, if necessary, the dried leaf blade is dried at 40°C for 2 - 3 days. Then, the tobacco material is ground into fine powder (-100uM) before analysis of the sugars. Alternatively, the sugar content is measured according to ISO 15154:2003 which defines a method for the determination of the content of reducing carbohydrates in tobacco by continuous flow analysis. <00009​​​Ammonia is measured using Coresta Recommended Method No. 79 (2018), Determination of ammonia in tobacco and tobacco products by ion chromatographic analysis. In short, the ammonia content is determined by extraction of the tobacco sample into a sulfuric acid solution. Ion chromatography is used to separate ammonium ions from other cation species. The response of ammonium ions is measured using a conductivity detector and quantified against an external standard calibration.

[0316] Method for determining alkaloid content Alkaloids are measured according to Coresta Recommendation Method 35 (2010), Determination of total alkaloids (as nicotine) in tobacco by continuous flow analysis. In short, an aqueous extract of tobacco is prepared, and the total alkaloid (as nicotine) content of the extract is determined by the reaction of sulfanilic acid and cyanogen chloride. Cyanogen chloride is produced in situ by the reaction of potassium cyanide with chloramine T. The resulting color is measured at 460 nm.

[0317] Method for determining nitrate content Nitrates are measured using Coresta Recommended Method 36 (2015), Determination of nitrate in tobacco and smokeless tobacco products by reduction to nitrite and continuous flow analysis. In short, aqueous extracts of tobacco or smokeless tobacco products are prepared, and the nitrate content of the extract is determined by reduction to nitrite using hydrazinosulfate in the presence of a copper catalyst, which is then reacted with sulfanilamide to form a diazo compound. This compound combines with N-1-naphthylethylenediamine dihydrochloride to form a colored complex, the absorbance of which is measured at 520 nm.

[0318] dry weight basis Data can be reported in %DWB±SD. The dry weight basis is calculated after drying at 105°C until a certain mass is reached, which is essentially 100 percent solids.

[0319] Gene expression analysis The generated sequencing data is demultiplexed using Illumina BaseSpace® Clarity LIMS (Illumina, Inc.) and then imported into Qiagen CLC Genomics Workbench version 12.0.1 (CLC bio, a QIAGEN Company). Transcriptome reads are mapped to an updated version of the N. tabacum reference genome (Sierro et al., (2014) Nat Commun 5, 3833) using the RNA-Seq Analysis 2.16 tool with similarity 0.8 (S=0.8) and fraction length 0.8 (L=0.8) as mapping criteria. Mismatch cost is set to 2, insertion cost to 3, and deletion cost to 3. Global alignment is not performed, while pair distances are automatically detected. The maximum number of read hits is set to 10, and paired reads are counted as one. Gene expression-FPKM values ​​are obtained for each gene in the reference genome, as well as for genes for which there are no transcript models.

[0320] RNAi procedure DNA fragment sequence number 21 was selected to repress NtGDH expression and cloned between a potent constitutive MMV promoter and the 3' nos terminator sequence of the Agrobacterium tumefaciens nopalin synthase gene (Cheng et al. (1997) Plant Physiol. 115(3):971-980). Nicotiana tabacum was transformed using a standard Agrobacterium-mediated transformation protocol (Horsch et al. (1985) Science, 227, 1229-1232). Seeds were harvested from independent T0 lines exhibiting the strongest NtGDH silencing. T1 plants from these T0 lines were grown in a greenhouse under standard agricultural practices and selected by RT-qPCR experiments to assess NtGDH gene expression levels.

[0321] Example 2 - Identification of tobacco genes that cause ammonia accumulation To identify genes that may cause ammonia accumulation in Burley leaves during the first 15 days of drying, several gene targets are tested, primarily based on their expression during the yellowing stage and their potential association with amino acid catabolism. One family of tobacco genes that alters the amino acid content after drying is a cluster of aspartate aminotransferase genes. While such genes affect the amino acid content after drying, manipulating these genes does not result in significant changes in ammonia. Therefore, GDH may be responsible for the potential NH4 during the initial drying stage (leaf cellular senescence). + Assuming they could be (ammonia) donors, tobacco gene orthologues for Arabidopsis thaliana glutamate dehydrogenase (NAD(H)-dependent GDH) encoding AtGDH1 (At5g18170), AtGDH2 (At5g07440), and AtGDH3 (At3g03910) were tested.

[0322] In the first step, all tobacco sequences related to GDH were identified. It was found that tobacco has two main clusters: one containing six proteins from tobacco (NtGDH2, NtGDH3, NtGDH7, NtGDH8, NtGDH4, and NtGDH11), and the other containing four proteins from tobacco (NtGDH6, NtGDH10, NtGDH9, and NtGDH12). In total, 10 tobacco gene products were found, five of which originated from Nicotiana sylvestris (NtGDH2, NtGDH8, NtGDH4, NtGDH6, and NtGDH12), and five of which originated from Nicotiana tomentosiformis (NtGDH3, NtGDH7, NtGDH11, NtGDH10, and NtGDH9).

[0323] To identify NtGDH genes particularly expressed during the initial desiccation stage, transcript data from RNA-seq data (Fragments Per Kilobase of transcript per Million mapped reads) are plotted (see Figure 1). For control, SAG12 and SGR1, typical markers of cellular senescence, are strongly expressed at this stage, showing peak expression at approximately 96 hours of desiccation. Several NtGDH genes, including NtGDH7, NtGDH8, NtGDH4, and NtGDH11, are not expressed during the initial desiccation stage. On the other hand, six other NtGDH genes are expressed during the initial desiccation stage, namely the pairs NtGDH6-10, NtGDH2-3, and NtGDH9-12. Interestingly, NtGDH6 and NtGDH10 are primarily expressed after 24 hours of desiccation, while the NtGDH2-3 and NtGDH9-12 pairs are more induced in the later stages, from 48 to 192 hours of desiccation. Both NtGDH2-3 and NtGDH9-12 follow similar induction profiles, but the expression of NtGDH2 and NtGDH3 continuously increases during the drying process, reaching peak FPKM values ​​after 192 hours, whereas this is not the case for NtGDH9-12.

[0324] Based on the expression data presented in Figure 1, NtGDH2 and NtGDH3 are silenced using an RNAi approach to determine their effect on ammonia accumulation in dried leaves.

[0325] Example 3 - RNAi silencing of NtGDH2-3 An RNAi approach was used to silence the expression of the NtGDH2-3 cluster. The sequence inserted into the GATEWAY vector to specifically generate anti-GDH2-3 plants is presented below as Sequence ID No. 21. Following plant transformation (T0), three plants E459-2, E459-3, and E459-5 were selected, showing a positive decrease in NtGDH2-3 expression. The strongest silencing effect was found in plants E459-3 and E459-5 48 hours after leaf drying treatment compared to control plant leaves (C1-3), while the E459-2 line was only slightly weakly silenced (see Figure 2).

[0326] Table 1 shows the chemical effects of NtGDH2-3 silencing on air-dried leaves. The original target, ammonia content, is reduced by approximately 50% (see Figure 2 when considering the two lines E459-3 and E459-5 that were effectively silenced for NtGDH2-3). As expected, the ammonia content in line E459-2 is also lower than the control, but to a smaller degree compared to E459-3 and E459-5. Nevertheless, the ammonia reduction is still statistically significant across all three lines: E459-2, E459-3, and E459-5. Surprisingly, the total alkaloid content is also significantly reduced by approximately 30%, showing a negative correlation with a 30% increase in total amino acids (when considering the two lines E459-3 and E459-5 that were effectively silenced for NtGDH2-3). In dried leaves of NtGDH2-3 silencing lines, the amino acids that are statistically most significantly increased compared to the wild type are proline (4.6 times more when considering the two lines E459-3 and E459-5 that effectively silencing NtGDH2-3), aspartic acid (1.8 times more when considering the two lines E459-3 and E459-5 that effectively silencing NtGDH2-3), as well as serine, threonine, and arginine (1.4 times, 1.8 times, and 2.4 times more, respectively, when considering the two lines E459-3 and E459-5 that effectively silencing NtGDH2-3). It is noteworthy that the amino acids aspartic acid and arginine, which are increased in anti-NtGDH lines, have also been reported to be substrates for nicotine synthesis in the roots.

[0327] Figure 3 shows box plots of the major metabolic changes to ammonia, sugar, aspartic acid, and proline induced by NtGDH2-3 silencing. All of these modifications are significant compared to the control (WT). The most striking effect is on ammonia, reaching a 50% reduction in dried tobacco.

[0328] Inactivation of NtGDH2-3 not only limits the increase in ammonia but also alters the chemical properties of dried leaves by changing the amino acid and sugar content. Knocking out additional NtGDH genes such as NtGDH9-12 is believed to result in a stronger reduction in ammonia, and consequently, a reduction in cytotoxicity caused by ammonium during the cellular senescence process. However, additional side effects on the chemical properties of tobacco, including alkaloids, amino acids, and sugars, are also expected.

[0329] Example 4 - Effects of silencing on biomass and plant height Silencing NtGDH2 and NtGDH3 does not affect plant biomass and height (see Figure 4). This suggests that the presence or absence of active NtGDH2 and NtGDH3 proteins does not affect plant growth and development. This indirectly confirms a specific function in leaves for reusing nitrogen from amine groups. This may play a crucial role in inducing the synthesis of novel amino acids, such as proline, under stress or cellular senescence conditions, for example, under drought stress.

[0330] Example 5 - Sensory Analysis The control tobacco (TN90) and leaf material (upper and middle leaves) of the NtGDH-RNAi strain described above were pooled and subjected to RRP sensory testing using IQOS. The sticks were prepared in cast-leaf mode corresponding to a 60% test tobacco supplemented with 40% full-cured background tobacco. Four trained panelists tested the samples, and the results are shown in Table 2.

[0331] In summary, TN90 unfolds with a dark, leathery, and animalistic fresh scent with some stimulating notes. Interestingly, the absence of NtGDH2-3 dramatically alters the sensory perception, offering some distinct differentiation. The dark scent perceived in the mainstream aerosol is reduced, replaced by additional complex aromatic notes, resulting in a good balance with some warmth and less stimulating notes.

[0332] Any publications cited or referenced herein provide relevant information disclosed prior to the filing date of this application. Statements herein should not be construed as admitting that the inventor has no right to retroactively access such disclosures. All publications referenced herein are incorporated herein by reference. Various modifications and variations of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the present invention is described in relation to certain preferred embodiments, the claimed invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the described form for carrying out the present invention, which will be apparent to those skilled in the art in cell, molecular and plant biology or related fields, are intended to be included within the scope of the following claims. [Table 1] [Table 2]

[0333] array Sequence ID 1: Polynucleotide coding sequence of NtGDH2

[0334] Sequence ID 2: polypeptide sequence of NtGDH2 MNALAATNRNFRQAARILGLDSKLEKSLLIPFREIKVECTIPKDDGTLVSYVGFRVQHDNARGPMKGGIRYHPEVDLDEVNALAQLMTWKTAVVDIPYGGAKGGIGCKPKDLSKSELERLTRVFTQKIHDLIGINTDVPAPDMGTNAQTMAWILDEYSKFHGHSPAIVTGKPIDLGGSLGREAATGRGVVYATEALLAEYGKNIK DLTFAIQGFGNVGAWAAKLIHERGGKVIAVSDITGAVKNPNGLDIPALLNHKEATGKLIDFSGGDVMNSDEVLTHECDVLIPCALGGVLNRENADNVKAKFIIEAANHPTDPEADEILSKKGIVILPDIYANAGGVTVSYFEWVQNIQGFMWDEEKVNRELKKYMTKAFHKLKNMCQSHDCNLRMGAFTLGVNRVARATTLRGWEA

[0335] Sequence ID 3: Polynucleotide coding sequence of NtGDH3

[0336] Sequence ID 4: Polypeptide sequence of NtGDH3 MNALAATNRNFRQAARILGLDSKLEKSLLIPFREIKVECTIPKDDGTLVSYVGFRVQHDNARGPMKGGIRYHPEVDLDEVNALAQLMTWKTAVADIPYGGAKGGIGCKPKDLSKSELERLTRVFTQKIHDLIGINTDVPAPDMGTNAQTMAWILDEYSKFHGHSPAIVTGKPIDLGGSLGREAATGRGVVYATEALLAEYGKNIK DLTFAIQGFGNVGAWAAKLIHERGGKVIAVSDITGAVKNPNGLDIPALLNHKEATGKLIDFCGGDVMNSDEVLTHECDVLIPCALGGVLNRENADNVKAKFII EAANHPTDPEADEILSKKGIVILPDIYANAGGVTVSYFEWVQNIQGFMWDEEKVNRELRKYMTKAFHNLKNMCQLHNCNLRMGAFTLGVNRVARATTLRGWEA

[0337] Sequence ID 5: Polynucleotide sequence of NtGDH4

[0338] Sequence ID 6: Polypeptide sequence of NtGDH4 MNALAATNRNFRQAARILGLDSKLEKSLLIPFREIKVECTIPKDDGTLVSYVGFRVQHDNARGPMKGGIRYHPEVDLDEVNALAQLMTWKTAVVDIPYGGAKGGIGCVPKELSKSELERLTRVFTQKIHDLIGINTDVPAPDMGTNAQTMAWILDEYSKFHGHSLAIVTGKPVDLGGSLGREAATGRGVVYATEALLAEYGKHIK DMTFAIQGFGNVGAWAARIIHERGGKVVAVSDITGAVKNQNGLDIPALLNHKEATGTLAGFSGGDAMSSDELLTHDCDVLIPCALGGVLNRENADSVKAKYIVEAANHPTDPDADEILSKKGVVILPDIYANAGGVTVSYFEWVQNIQGFMWDEEHVNRELKKYMTRAFHNLKNMCKSHNCNLRMGAFTLGVNRVARATQLRGWEA

[0339] Sequence ID 7: Polynucleotide sequence of NtGDH6

[0340] Sequence ID 8: polypeptide sequence of NtGDH6 MNALAATNRNFKLAARLLGLDSKLEKSLLIPFREIKVECTIPKDDGSLASFVGFRVQHDNARGPMKGGIRYHPEVDPDEVNALAQLMTWKTAVANIPYGGAKGGIGCSPSDLSNSELERLTRVFTQKIHDLIGIHTDVPAPDMGTNPQTMAWILDEYSKFHGYSPAVVTGKPIDLGGSLGRDAATGRGVLFATEALLKEHGKSIA GQRFVIQGFGNVGSWAAKLINEQGGKIVAVSDITGAIKNENGLNIASLLKHVKENRGVKGFNDARPIDPHSILVEDCDVLIPAALGGVINRDNANDIKAKYIIEAANHPTDPEADEILAKKGVVILPDIYANSGGVTVSYFEWVQNIQGFMWDEDKVNAELKTYMTRGFKDVKDMCKTHNCDLRMGAFTLGVNRVARATVLRGWEA

[0341] Sequence ID 9: Polynucleotide sequence of NtGDH7

[0342] Sequence ID 10: Polypeptide sequence of NtGDH7 MNALAATNRNFRQAARILGLDSKIEKSLLIPFREIKVECTIPKDDGTLVSYVGFRVQHDNARGPMKGGIRYHHEVELDEVNALAQLMTWKTAVANIPYGGAKGGIGCTPKDLSVSELERLTRVFTQKIHDLIGINTDVPAPDMGTNAQTMAWILDEYSKFHGHSPAIVTGKPIDLGGSLGREAATGRGAVYATEALLAEYGKNIK DLTFAIQGFGNVGAWAGKIIHERGGKVIAVSDITGAIKNPNGLDIPALLSHREKTGKLTDFTGGDVMNSDELLTHECDVLIPCALGGVLNRENADHVKAKFII EAANHPTDPDADEILSKKGVVILPDIYANAGGVTVSYFEWVQNIQGFMWDEEKVNAELKKYMTRAFHNLKSMCHSHNCNLRMGAFTLGVNRVARATQLRGWEA

[0343] Sequence ID 11: Polynucleotide sequence of NtGDH8

[0344] Sequence ID 12: Polypeptide sequence of NtGDH8 MNALAATNRNFRQAARILGLDSKIEKSLLIPFREIKVECTIPKDDGTLVSYIGFRVQHDNARGPMKGGIRYHHEVELDEVNALAQLMTWKTAVANIPYGGAKGGIGCTPKDLSLSELERLTRVFTQKIHDLIGINTDVPAPDMGTNAQTMAWILDEYSKFHGHSPAIVTGKPIDLGGSLGREAATGRGAVYATEALLAEYGMNIK DLTFAIQGFGNVGAWAGKIIHERGGKVIAVSDITGAIKNPNGLDIPALLSHREKTGKLTDFAGGDVMNSDELLTHECDVLIPCALGGVLNRENADHVKAKFII EAANHPTDPDADEILSKKGVVILPDIYANAGGVTVSYFEWVQNIQGFMWDEEKVNAELKKYMTRAFHNLKSMCHSHNCNLRMGAFTLGVNRVARATQLRGWEA

[0345] Sequence ID 13: Polynucleotide sequence of NtGDH9

[0346] Sequence ID 14: Polypeptide sequence of NtGDH9 MNALAATNRNFKLASRLLGLDSKLEQCLLIPFREIKVECTIPKDDGSLATFIGFRVQHDNARGPMKGGIRYHPEVDPDEVNALAQLMTWKTAVANIPYGGAKGGIGCSPSDLSISELERLTRVFTQKIHDLIGIHTDVPAPDMGTNPQTMAWILDEYSKFHGYSPAVVTGKPIDLGGSLGRDAATGRGVLFAAEALLRDHGKSIA GQRFVVQGFGNVGSWAAQLITEQGGKIVAVSDITGAIKNKNGIDIASLLKHVKENRGVKGFHGADSIDPNSILVEDCDVLIPAALGGVINRDNAKDIKAKFIV EAANHPTDPEADEILAKKGVVILPDIYANSGGVTVSYFEWVQNIQGFMWDEERVNTELKAYMNRGFKDVKDMCKTHNCDLRMGAFTLGVNRVARATTLRGWEA

[0347] Sequence ID 15: Polynucleotide sequence of NtGDH10

[0348] Sequence ID 16: Polypeptide sequence of NtGDH10 MNALAATNRNFKLAARLLGLDSKLEKSLLIPFREIKVECTIPKDDGSLASFVGFRVQHDNARGPMKGGIRYHPEVDPDEVNALAQLMTWKTAVANIPYGGAKGGIGCSPSDLSNSELERLTRVFTQKIHDLIGIHTDVPAPDMGTNPQTMAWILDEYSKFHGYSPAVVTGKPIDLGGSLGRDAATGRGVLFATEALLKEHGKSIA GQCFVIQGFGNVGSWAAKLINEQGGKIVAVSDITGAIKNKNGLDIASLLKHVKENRGVKGFNDARSIDPDSILVEDCDVLIPAALGGVINRDNANNIKAKYIIEAANHPTDPEADEILAKKGVVILPDIYANSGGVTVSYFEWVQNIQGFMWDEDKVNAELKTYMTRGFKDVKDMCKTHNCDLRMGAFTLGVNRVARATALRGWEA

[0349] Sequence ID 17: Polynucleotide sequence of NtGDH11

[0350] Sequence ID 18: Polypeptide sequence of NtGDH11 MNALAATNRNFRQAARILGLDSKLEKSLLIPFREIKVECTIPKDDGTLVSYVGFRVQHDNARGPMKGGIRYHPEVDLDEVNALAQLMTWKTAVVDIPYGGAKGGIGCVPKELSKSELERLTRVFTQKIHDLIGINTDVPAPDMGTNAQTMAWILDEYSKFHGHSLAIVTGKPVDLGGSLGREAATGRGVVYATEALLAEYGKHIK DMTFAIQGFGNVGAWAARIIHERGGKVVAVSDITGAVKNQNGLDIPALLNHKEATGTLAGFSGGDAMSSDELLTQECDVLIPCALGGVLNRENADSVKAKYIVEAANHPTDPDADEILSKKGVVILPDIYANAGGVTVSYFEWVQNIQGFMWDEEQVNRELKKYMTRAFHNLKNMCKSHNCNLRMGAFTLGVNRVARATQLRGWEA

[0351] Sequence ID 19: Polynucleotide sequence of NtGDH12

[0352] Sequence ID 20: Polypeptide sequence of NtGDH12 MNALAATNRNFKLASRLLGLDSKLEQCLLIPFREIKVECTIPKDDGSLATFIGFRVQHDNARGPMKGGIRYHPEVDPDEVNALAQLMTWKTAVANIPYGGAKGGIGCSPSDLSISELERLTRVFTQKIHDLIGIHTDVPAPDMGTNPQTMAWILDEYSKFHGYSPAVVTGKPIDLGGSLGRDAATGRGVLFAAEALLRDHGKSIA GQHFVVQGFGNVGSWAAQLITEQGGKIVAVSDITGAIKNKNGIDIASLLKHVKENRGVKGFHGADSIDPNSILVEDCDVLIPAALGGVINRDNAKDIKAKFIV EAANHPTDPEADEILAKKGVVILPDIYANSGGVTVSYFEWVQNIQGFMWDEERVNTELKAYMNRGFKDVKDMCKTHNCDLRMGAFTLGVNRVARATTLRGWEA

[0353] Sequence ID 21: Polynucleotide sequence used for silencing tgacttttgcaattcagggttttggaaacgtgggagcatgggcagcaaagcttatt

Claims

1. A mutant, non-natural, or transgenic tobacco plant leaf or a portion of that plant leaf in which the expression or activity of glutamate dehydrogenase (NtGDH) is regulated, wherein the NtGDH comprises or essentially consists of at least one of the following: NtGDH2 polynucleotide, or NtGDH3 polynucleotide, or NtGDH6 polynucleotide, or NtGDH9 polynucleotide, or NtGDH10 polynucleotide, or NtGDH12 polynucleotide, or NtGDH2 polypeptide, or NtGDH3 polypeptide, or NtGDH6 polypeptide, or NtGDH9 polypeptide, or NtGDH10 polypeptide, or NtGDH12 polypeptide. (i) The NtGDH2 polynucleotide comprises, or essentially comprises, a sequence having at least 90% sequence identity with SEQ ID NO: 1, or (ii) The NtGDH2 polypeptide is encoded by the polynucleotide described in (i), or (iii) The NtGDH2 polypeptide has at least 94% sequence identity with respect to SEQ ID NO: 2, or (iv) The NtGDH3 polynucleotide comprises, or essentially comprises, a sequence having at least 90% sequence identity with SEQ ID NO: 3, or (v) The NtGDH3 polypeptide is encoded by the polynucleotide described in (iv), or (vi) The NtGDH3 polypeptide has at least 94% sequence identity with respect to SEQ ID NO: 4, and (vii) The NtGDH6 polynucleotide comprises, or essentially comprises, a sequence having at least 90% sequence identity with SEQ ID NO: 7, or (viiii) The NtGDH6 polypeptide is encoded by the polynucleotide described in (i), or (ix) The NtGDH6 polypeptide has at least 94% sequence identity with respect to SEQ ID NO: 8, or (x) The NtGDH9 polynucleotide comprises, or essentially comprises, a sequence having at least 85% sequence identity with SEQ ID NO: 13, or (xi) The NtGDH9 polypeptide is encoded by the polynucleotide described in (i), or (xi) The NtGDH9 polypeptide has at least 91% sequence identity with respect to SEQ ID NO: 14, or (xiiii) The NtGDH10 polynucleotide comprises, or essentially comprises, a sequence having at least 89% sequence identity with SEQ ID NO: 15, or (xiv) The NtGDH10 polypeptide is encoded by the polynucleotide described in (i), or (xv) The NtGDH10 polypeptide has at least 94% sequence identity with respect to SEQ ID NO: 16, or (xix) The NtGDH12 polynucleotide comprises, or essentially comprises, a sequence having at least 85% sequence identity with SEQ ID NO:

19. (xx) The NtGDH12 polypeptide is encoded by the polynucleotide described in (i), or (xxi) The NtGDH12 polypeptide has at least 91% sequence identity with respect to SEQ ID NO: 20, Expression of the NtGDH2 polynucleotide, or the NtGDH3 polynucleotide, or the NtGDH6 polynucleotide, or the NtGDH9 polynucleotide, or the NtGDH10 polynucleotide, or the NtGDH12 polynucleotide, or the NtGDH2 polypeptide, or the NtGDH3 polypeptide, or the NtGDH6 polypeptide, or the NtGDH9 polypeptide, or the NtGDH10 polypeptide, or the NtGDH12 polypeptide is performed by the NtGDH2 polynucleotide, or the NtGDH3 polynucleotide Mutant, non-natural, or transgenic tobacco plant leaves or portions of its leaves in which the expression of creotide, or the NtGDH6 polynucleotide, or the NtGDH9 polynucleotide, or the NtGDH10 polynucleotide, or the NtGDH12 polynucleotide, or the NtGDH2 polypeptide, or the NtGDH3 polypeptide, or the NtGDH6 polypeptide, or the NtGDH9 polypeptide, or the NtGDH10 polypeptide, or the NtGDH12 polypeptide is regulated compared to a control plant in which the expression is regulated.

2. The expression of the NtGDH6 polynucleotide and the NtGDH10 polynucleotide is regulated, or the activity of the NtGDH6 polypeptide and the NtGDH10 polypeptide is regulated, and / or The expression of the NtGDH2 polynucleotide and the NtGDH3 polynucleotide is regulated, or the activity of the NtGDH2 polypeptide and the NtGDH3 polypeptide is regulated, and / or The expression of the NtGDH9 polynucleotide and the NtGDH12 polynucleotide is regulated, or the activity of the NtGDH9 polypeptide and the NtGDH12 polypeptide is regulated, and / or The expression of the NtGDH2 polynucleotide, the NtGDH3 polynucleotide, the NtGDH9 polynucleotide, and the NtGDH12 polynucleotide is regulated, or the activity of the NtGDH2 polypeptide, the NtGDH3 polypeptide, the NtGDH9 polypeptide, and the NtGDH12 polypeptide is regulated, and / or A mutant, non-natural or transgenic tobacco plant leaf or a portion of a tobacco plant leaf according to claim 1, wherein the expression of each of the NtGDH2 polynucleotide, the NtGDH3 polynucleotide, the NtGDH6 polynucleotide, the NtGDH9 polynucleotide, the NtGDH10 polynucleotide and the NtGDH12 polynucleotide, or the NtGDH2 polypeptide, the NtGDH3 polypeptide, the NtGDH6 polypeptide, the NtGDH9 polypeptide, the NtGDH10 polypeptide and the NtGDH12 polypeptide is regulated.

3. If the expression of one or more NtGDH4 polynucleotides, or NtGDH7 polynucleotides, or NtGDH8 polynucleotides, or NtGDH11 polynucleotides, or NtGDH4 polypeptides, NtGDH7 polypeptides, or NtGDH8 polypeptides, or NtGDH11 polypeptides is not regulated, (i) The NtGDH4 polynucleotide comprises, or essentially comprises, a sequence having at least 88% sequence identity with SEQ ID NO: 5, or (ii) The NtGDH4 polypeptide is encoded by the polynucleotide described in (i), or (iii) The NtGDH4 polypeptide has at least 92% sequence identity with respect to SEQ ID NO: 6, or (iv) The NtGDH7 polynucleotide comprises, or essentially comprises, a sequence having at least 92% sequence identity with SEQ ID NO: 9, or (v) The NtGDH7 polypeptide is encoded by the polynucleotide described in (iv), or (vi) The NtGDH7 polypeptide has at least 96% sequence identity with SEQ ID NO: 10, or (vii) The NtGDH8 polynucleotide comprises, or essentially comprises, a sequence having at least 92% sequence identity with SEQ ID NO: 11, or (viiii) The NtGDH8 polypeptide is encoded by the polynucleotide described in (i), or (ix) The NtGDH8 polypeptide has at least 95% sequence identity with SEQ ID NO: 12, or (x) The NtGDH11 polynucleotide comprises, or essentially comprises, a sequence having at least 87% sequence identity with SEQ ID NO: 17, or (xi) The NtGDH11 polypeptide is encoded by the polynucleotide described in (i), or (xi) The mutant, non-natural, or transgenic tobacco plant leaf or part of a plant leaf according to claim 1 or 2, wherein the NtGDH11 polypeptide has at least 92% sequence identity with SEQ ID NO:

18.

4. The plant leaf or a portion of the plant leaf contains at least one genetic modification that modulates the expression or activity of the at least one NtGDH polynucleotide or the at least one NtGDH polypeptide, or The plant leaf or a portion of the plant leaf contains one or more exogenous DNAs or exogenous RNAs that regulate the expression or activity of the at least one NtGDH polynucleotide or the at least one NtGDH polypeptide, or The plant leaf or a portion of the plant leaf contains one or more vectors that regulate the expression or activity of the at least one NtGDH polynucleotide or the at least one NtGDH polypeptide, or a viral vector, or an Agrobacterium vector or a CRISPR vector, or The plant leaf or a portion of the plant leaf contains at least one modification that can induce one or more RNA interferences that regulate the expression or activity of the at least one NtGDH polynucleotide or the at least one NtGDH polypeptide, or one or more transcription gene silencing or virus-induced gene silencing, The plant leaf or a portion of the plant leaf contains one or more exogenous double-stranded RNAs (dsRNAs), exogenous hairpin RNAs (hpRNAs), or exogenous small interfering RNAs that regulate the expression or activity of the at least one NtGDH polynucleotide or the at least one NtGDH polypeptide, or A combination of two or more of these, a mutant, non-natural, or transgenic tobacco plant leaf or a part of a plant leaf according to any one of claims 1 to 3.

5. The regulated expression or activity of at least one NtGDH polynucleotide or NtGDH polypeptide, when subjected to drying, regulates the amounts of ammonia, amino acids, sugars, and total alkaloids in the plant leaves or a portion of the plant leaves, preferably, A mutant, non-natural, or transgenic tobacco plant leaf or a part of a plant leaf according to any one of claims 1 to 4, wherein the amino acids are proline, aspartic acid, serine, threonine, and arginine.

6. The plant leaves or a part thereof are air-dried, preferably the air-dried leaves or a part thereof are sun-dried or heat-dried, or the plant leaves or a part thereof are air-dried, preferably the air-dried leaves or a part thereof are sun-dried or heat-dried, and / or The mutant, non-natural type, or transgenic tobacco leaf or part of the leaf according to any one of claims 1 to 5, wherein the tobacco leaf or a part of the leaf is a Nicotiana tabacum leaf or a part of the leaf.

7. A method for preparing dried tobacco leaves or a portion of tobacco leaves having controlled levels of ammonia, amino acids, sugars, and total alkaloids compared to tobacco leaves or a portion of tobacco leaves from a control dried tobacco plant, wherein the method is: (a) A step of providing a tobacco plant comprising, or essentially comprising, an NtGDH plant comprising at least one of the following: NtGDH2 polynucleotide, or NtGDH3 polynucleotide, or NtGDH6 polynucleotide, or NtGDH9 polynucleotide, or NtGDH10 polynucleotide, or NtGDH12 polynucleotide, or NtGDH2 polypeptide, or NtGDH3 polypeptide, or NtGDH6 polypeptide, or NtGDH9 polypeptide, or NtGDH10 polypeptide, or NtGDH12 polypeptide, (i) The NtGDH2 polynucleotide comprises, or essentially comprises, a sequence having at least 90% sequence identity with SEQ ID NO: 1, or (ii) The NtGDH2 polypeptide is encoded by the polynucleotide described in (i), or (iii) The NtGDH2 polypeptide has at least 94% sequence identity with respect to SEQ ID NO: 2, or (iv) The NtGDH3 polynucleotide comprises, or essentially comprises, a sequence having at least 90% sequence identity with SEQ ID NO: 3, or (v) The NtGDH3 polypeptide is encoded by the polynucleotide described in (iv), or (vi) The NtGDH3 polypeptide has at least 94% sequence identity with respect to SEQ ID NO: 4, and (vii) The NtGDH6 polynucleotide comprises, or essentially comprises, a sequence having at least 90% sequence identity with SEQ ID NO: 7, or (viiii) The NtGDH6 polypeptide is encoded by the polynucleotide described in (i), or (ix) The NtGDH6 polypeptide has at least 94% sequence identity with respect to SEQ ID NO: 8, or (x) The NtGDH9 polynucleotide comprises, or essentially comprises, a sequence having at least 85% sequence identity with SEQ ID NO: 13, or (xi) The NtGDH9 polypeptide is encoded by the polynucleotide described in (i), or (xi) The NtGDH9 polypeptide has at least 91% sequence identity with respect to SEQ ID NO: 14, or (xiiii) The NtGDH10 polynucleotide comprises, or essentially comprises, a sequence having at least 89% sequence identity with SEQ ID NO: 15, or (xiv) The NtGDH10 polypeptide is encoded by the polynucleotide described in (i), or (xv) The NtGDH10 polypeptide has at least 94% sequence identity with respect to SEQ ID NO: 16, or (xix) The NtGDH12 polynucleotide comprises, or essentially comprises, a sequence having at least 85% sequence identity with SEQ ID NO:

19. (xx) The NtGDH12 polypeptide is encoded by the polynucleotide described in (i), or (xxi) The NtGDH12 polypeptide has at least 91% sequence identity with respect to SEQ ID NO: 20, in the process of providing (b) A step of adjusting the expression of at least one of the NtGDH2 polynucleotides, or the NtGDH3 polynucleotide, or the NtGDH6 polynucleotide, or the NtGDH9 polynucleotide, or the NtGDH10 polynucleotide, or the NtGDH12 polynucleotide, or the activity of the NtGDH2 polypeptide, or the NtGDH3 polypeptide, or the NtGDH6 polypeptide, or the NtGDH9 polypeptide, or the NtGDH10 polypeptide, or the NtGDH12 polypeptide, in the tobacco plant leaf or a part of the plant leaf. (c) A step of harvesting plant leaves or a portion of the plant leaves from the tobacco plant, (d) A step of drying the plant leaves or a part of the plant leaves, (e) Optionally, a step of measuring the levels of ammonia, amino acids, sugars, and total alkaloids in the dried tobacco plant leaves or a portion of the dried plant leaves, (f) A method comprising the step of obtaining a dried tobacco plant leaf or a portion of the plant leaf having regulated levels of ammonia, amino acids, sugars, and total alkaloids compared to a control plant in which the expression of the NtGDH2 polynucleotide, or the NtGDH3 polynucleotide, or the NtGDH6 polynucleotide, or the NtGDH9 polynucleotide, or the NtGDH10 polynucleotide, or the NtGDH12 polynucleotide, or the activity of the NtGDH2 polypeptide, or the NtGDH3 polypeptide, or the NtGDH6 polypeptide, or the NtGDH9 polypeptide, or the NtGDH10 polypeptide, or the NtGDH12 polypeptide is not regulated.

8. In step (b), the expression of the NtGDH6 polynucleotide and the NtGDH10 polynucleotide is regulated, or the activity of the NtGDH6 polypeptide and the NtGDH10 polypeptide is regulated, and / or The expression of the NtGDH2 polynucleotide and the NtGDH3 polynucleotide is regulated, or the activity of both the NtGDH2 polypeptide and the NtGDH3 polypeptide is regulated, and / or The expression of the NtGDH9 polynucleotide and the NtGDH12 polynucleotide is regulated, or the activity of the NtGDH9 polypeptide and the NtGDH12 polypeptide is regulated, and / or The expression of the NtGDH2 polynucleotide, the NtGDH3 polynucleotide, the NtGDH9 polynucleotide, and the NtGDH12 polynucleotide is regulated, or the activity of the NtGDH2 polypeptide, the NtGDH3 polypeptide, the NtGDH9 polypeptide, and the NtGDH12 polypeptide is regulated, and / or The method according to claim 7, wherein the expression of each of the NtGDH2 polynucleotide, the NtGDH3 polynucleotide, the NtGDH6 polynucleotide, the NtGDH9 polynucleotide, the NtGDH10 polynucleotide and the NtGDH12 polynucleotide, or the NtGDH2 polypeptide, the NtGDH3 polypeptide, the NtGDH6 polypeptide, the NtGDH9 polypeptide, the NtGDH10 polypeptide and the NtGDH12 polypeptide is controlled.

9. Expression of one or more NtGDH4 polynucleotides, NtGDH7 polynucleotides, NtGDH8 polynucleotides, or NtGDH11 polynucleotides, or the activity of NtGDH4 polypeptides, NtGDH7 polypeptides, NtGDH8 polypeptides, or NtGDH11 polypeptides is not regulated. (i) The NtGDH4 polynucleotide comprises, or essentially comprises, a sequence having at least 88% sequence identity with SEQ ID NO: 5, or (ii) The NtGDH4 polypeptide is encoded by the polynucleotide described in (i), or (iii) The NtGDH4 polypeptide has at least 92% sequence identity with respect to SEQ ID NO: 6, or (iv) The NtGDH7 polynucleotide comprises, or essentially comprises, a sequence having at least 92% sequence identity with SEQ ID NO: 9, or (v) The NtGDH7 polypeptide is encoded by the polynucleotide described in (iv), or (vi) The NtGDH7 polypeptide has at least 96% sequence identity with SEQ ID NO: 10, or (vii) The NtGDH8 polynucleotide comprises, or essentially comprises, a sequence having at least 92% sequence identity with SEQ ID NO: 11, or (viiii) The NtGDH8 polypeptide is encoded by the polynucleotide described in (i), or (ix) The NtGDH8 polypeptide has at least 95% sequence identity with SEQ ID NO: 12, or (x) The NtGDH11 polynucleotide comprises, or essentially comprises, a sequence having at least 87% sequence identity with SEQ ID NO: 17, or (xi) The NtGDH11 polypeptide is encoded by the polynucleotide described in (i), or (xi) The method according to claim 7 or 8, wherein the NtGDH11 polypeptide has at least 92% sequence identity with respect to SEQ ID NO:

18.

10. The tobacco plant leaf or a part thereof is a Nicotiana tabacum plant leaf or a part thereof, and / or In step (b), expression or activity is regulated by genome editing, preferably, The genome editing is selected from CRISPR-mediated genome editing, mutagenesis, zinc finger nuclease-mediated mutagenesis, chemical or radioactive mutagenesis, homologous recombination, oligonucleotide-directed mutagenesis, and meganuclease-mediated mutagenesis, or The method according to any one of claims 7 to 9, wherein in step (b), expression or activity is regulated using interfering polynucleotides.

11. Dried mutant, non-natural type, or transgenic Nicotiana tabacum plant leaf or part thereof obtained or obtainable by the method of any one of claims 7 to 10.

12. (i) Ammonia content is approximately 0.16 ± 0.04% DWB to 0.11 ± 0.16 ± 0.03% on a dry weight basis (DWB), and (ii) Glucose, fructose, and sucrose content is 0.51 ± 0.58% DWB to 1.55 ± 1.10% DWB, and (iii) Total free amino acid content is 51.0 ± 6.60 mg / g DWB to 60.1 ± 4.58 mg / g DWB, and (iv) Total alkaloid content is 2.24 ± 0.8% DWB to 4.20 ± 0.39%, dried tobacco plant leaves or a portion of the said dried plant leaves.

13. A dried tobacco blend comprising at least two different dried tobaccos, wherein at least one of the dried tobaccos is a dried tobacco derived from a mutant, non-natural, or transgenic tobacco plant leaf or a portion thereof as described in any of claims 1 to 6, or a dried mutant, non-natural, or transgenic Nicotiana tabacum plant leaf or a portion thereof as described in claim 11, or a dried tobacco plant leaf or a portion thereof as described in claim 12, preferably, A dried tobacco blend in which at least one other dried tobacco is Burley tobacco, or Oriental tobacco, or Dark tobacco, or Flu-cured tobacco, or a combination of two or more of these.

14. A method for producing a tobacco blend with reduced ammonia content, (a) To provide dried tobacco leaves or a portion thereof, wherein the first dried tobacco leaves or a portion thereof are derived from a mutant, non-natural, or transgenic tobacco leaf or a portion thereof as described in any of claims 1 to 6, or from a dried mutant, non-natural, or transgenic Nicotiana tabacum leaf or a portion thereof as described in claim 11, or from a dried tobacco leaf or a portion thereof as described in claim 12, and (b) A method comprising blending the first dried tobacco leaves or a portion thereof with at least one second dried tobacco leaves or a portion thereof to produce a tobacco blend in which the total amount of ammonia is less than the total amount of ammonia in the at least one second dried tobacco leaves or a portion thereof.

15. A dried tobacco blend obtained or obtainable by the method of claim 14, or A tobacco product or smoking article comprising a dried form of a mutant, non-natural, or transgenic tobacco plant leaf or a portion thereof according to any one of claims 1 to 6, or a dried mutant, non-natural, or transgenic Nicotiana tabacum plant leaf or a portion thereof according to claim 11, or a dried tobacco plant leaf or a portion thereof according to claim 12, or a dried tobacco blend obtained or obtainable by the method of claim 14.

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