EP-4734754-A1 - MODULATION OF GENES CODING FOR GLUTAMATE DEHYDROGENASE

Abstract

There is disclosed a mutant, non-naturally occurring or transgenic Nicotiana plant leaf or part of the plant leaf having modulated expression or activity of glutamate dehydrogenase (NtGDH), wherein the NtGDH comprises, consists, or consists essentially of at least one of a NtGDH2 polynucleotide or a NtGDH3 polynucleotide or a NtGDH6 polynucleotide or a NtGDH9 polynucleotide or a NtGDH10 polynucleotide or a NtGDH12 polynucleotide or a NtGDH2 polypeptide or a NtGDH3 polypeptide or a NtGDH6 polypeptide or a NtGDH9 polypeptide or a NtGDH10 polypeptide or a NtGDH12 polypeptide, wherein 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 NtGDH2 polypeptide or the NtGDH3 polypeptide or the NtGDH6 polypeptide or the NtGDH9 polypeptide or the NtGDH10 polypeptide or the NtGDH12 polypeptide is modulated as 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 NtGDH2 polypeptide or the NtGDH3 polypeptide or the NtGDH6 polypeptide or the NtGDH9 polypeptide or the NtGDH10 polypeptide or the NtGDH12 polypeptide is not modulated.

Inventors

• BOVET, LUCIEN
• HILFIKER, Aurore Ginette Denise

Assignees

• Philip Morris Products S.A.

Dates

Publication Date

20260506

Application Date

20240625

Claims (1)

1. CLAIMS 1. A mutant, non-naturally occurring or transgenic Nicotiana plant leaf or part of the plant leaf having modulated expression or activity of glutamate dehydrogenase (NtGDH), wherein the NtGDH comprises, consists, or consists essentially of at least one of a NtGDH2 polynucleotide or a NtGDH3 polynucleotide or a NtGDH6 polynucleotide or a NtGDH9 polynucleotide or a NtGDH10 polynucleotide or a NtGDH12 polynucleotide or a NtGDH2 polypeptide or a NtGDH3 polypeptide or a NtGDH6 polypeptide or a NtGDH9 polypeptide or a NtGDH10 polypeptide or a NtGDH12 polypeptide, wherein (i) the NtGDH2 polynucleotide comprises, consists or consists essentially of a sequence having at least 90% sequence identity to SEQ ID NO: 1; or (ii) the NtGDH2 polypeptide is encoded by the polynucleotide set forth in (i); or (iii) the NtGDH2 polypeptide has at least 94% sequence identity to SEQ ID NO:2; or (iv) the NtGDH3 polynucleotide comprises, consists or consists essentially of a sequence having at least 90% sequence identity to SEQ ID NO: 3; or (v) the NtGDH3 polypeptide is encoded by the polynucleotide set forth in (iv); or (vi) the NtGDH3 polypeptide has at least 94% sequence identity to SEQ ID NO: 4; and (vii) the NtGDH6 polynucleotide comprises, consists or consists essentially of a sequence having at least 90% sequence identity to SEQ ID NO: 7; or (viii) the NtGDH6 polypeptide is encoded by the polynucleotide set forth in (i); or (ix) the NtGDH6 polypeptide has at least 94% sequence identity to SEQ ID NO:8; or (x) the NtGDH9 polynucleotide comprises, consists or consists essentially of a sequence having at least 85% sequence identity to SEQ ID NO: 13; or (xi) the NtGDH9 polypeptide is encoded by the polynucleotide set forth in (i); or (xii) the NtGDH9 polypeptide has at least 91% sequence identity to SEQ ID NO:14; or (xiii) the NtGDH10 polynucleotide comprises, consists or consists essentially of a sequence having at least 89% sequence identity to SEQ ID NO: 15; or (xiv) the NtGDH10 polypeptide is encoded by the polynucleotide set forth in (i); or (xv) the NtGDH10 polypeptide has at least 94% sequence identity to SEQ ID NO:16; or (xix) the NtGDH12 polynucleotide comprises, consists or consists essentially of a sequence having at least 85% sequence identity to SEQ ID NO: 19; or (xx) the NtGDH12 polypeptide is encoded by the polynucleotide set forth in (i); or (xxi) the NtGDH12 polypeptide has at least 91% sequence identity to SEQ ID NO:20, wherein 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 NtGDH2 polypeptide or the NtGDH3 polypeptide or the NtGDH6 polypeptide or the NtGDH9 polypeptide or the NtGDH10 polypeptide or the NtGDH12 polypeptide is modulated as 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 NtGDH2 polypeptide or the NtGDH3 polypeptide or the NtGDH6 polypeptide or the NtGDH9 polypeptide or the NtGDH10 polypeptide or the NtGDH12 polypeptide is not modulated. 2. The mutant, non-naturally occurring or transgenic Nicotiana plant leaf or part of the plant leaf according to claim 1, wherein the expression of the NtGDH6 polynucleotide and the NtGDH10 polynucleotide are modulated or the activity of the NtGDH6 polypeptide and the NtGDH10 polypeptide are modulated; and/or wherein the expression of the NtGDH2 polynucleotide and the NtGDH3 polynucleotide are modulated or the activity of the NtGDH2 polypeptide and the NtGDH3 polypeptide are modulated; and/or wherein the expression of the NtGDH9 polynucleotide and the NtGDH12 polynucleotide are modulated or the activity of the NtGDH9 polypeptide and the NtGDH12 polypeptide are modulated; and/or wherein the expression of each of the NtGDH2 polynucleotide and the NtGDH3 polynucleotide and the NtGDH9 polynucleotide and the NtGDH12 polynucleotide are modulated or the activity of each of the NtGDH2 polypeptide and the NtGDH3 polypeptide and the NtGDH9 polypeptide and the NtGDH12 polypeptide are modulated; and/or wherein the expression of each of the NtGDH2 polynucleotide and the NtGDH3 polynucleotide and the NtGDH6 polynucleotide and the NtGDH9 polynucleotide and the NtGDH10 polynucleotide and the NtGDH12 polynucleotide or the NtGDH2 polypeptide and the NtGDH3 polypeptide and the NtGDH6 polypeptide and the NtGDH9 polypeptide and the NtGDH10 polypeptide and the NtGDH12 polypeptide are modulated. 3. The mutant, non-naturally occurring or transgenic Nicotiana plant leaf or part of the plant leaf according to claim 1 or claim 2, wherein the expression of one or more of a NtGDH4 polynucleotide or a NtGDH7 polynucleotide or a NtGDH8 polynucleotide or a NtGDH11 polynucleotide or a NtGDH4 polypeptide or a NtGDH7 polypeptide or a NtGDH8 polypeptide or a NtGDH11 polypeptide is not modulated, wherein (i) the NtGDH4 polynucleotide comprises, consists or consists essentially of a sequence having at least 88% sequence identity to SEQ ID NO: 5; or (ii) the NtGDH4 polypeptide is encoded by the polynucleotide set forth in (i); or (iii) the NtGDH4 polypeptide has at least 92% sequence identity to SEQ ID NO:6; or (iv) the NtGDH7 polynucleotide comprises, consists or consists essentially of a sequence having at least 92% sequence identity to SEQ ID NO: 9; or (v) the NtGDH7 polypeptide is encoded by the polynucleotide set forth in (iv); or (vi) the NtGDH7 polypeptide has at least 96% sequence identity to SEQ ID NO: 10; or (vii) the NtGDH8 polynucleotide comprises, consists or consists essentially of a sequence having at least 92% sequence identity to SEQ ID NO: 11; or (viii) the NtGDH8 polypeptide is encoded by the polynucleotide set forth in (i); or (ix) the NtGDH8 polypeptide has at least 95% sequence identity to SEQ ID NO:12; or (x) the NtGDH11 polynucleotide comprises, consists or consists essentially of a sequence having at least 87% sequence identity to SEQ ID NO: 17; or (xi) the NtGDH11 polypeptide is encoded by the polynucleotide set forth in (i); or (xii) the NtGDH11 polypeptide has at least 92% sequence identity to SEQ ID NO:18. 4. The mutant, non-naturally occurring or transgenic Nicotiana plant leaf or part of the plant leaf according to any of the preceding claims, wherein the plant leaf or part of the plant leaf comprises at least one genetic alteration that modulates the expression or activity of the at least one NtGDH polynucleotide or the at least one NtGDH polypeptide; or wherein the plant leaf or part of the plant leaf comprises one or more of exogenous DNA or exogenous RNA that modulates the expression or activity of the at least one NtGDH polynucleotide or the at least one NtGDH polypeptide; or wherein the plant leaf or part of the plant leaf comprises one or more of a vector or a viral vector or an Agrobacterium vector or a CRISPR vector that modulates the expression or activity of the at least one NtGDH polynucleotide or the at least one NtGDH polypeptide; or wherein the plant leaf or part of the plant leaf comprises at least one modification that is capable of driving one or more of RNA interference or transcriptional gene silencing or virus induced gene silencing that modulates the expression or activity of the at least one NtGDH polynucleotide or the at least one NtGDH polypeptide; or wherein the plant leaf or part of the plant leaf comprises one or more of exogenous double stranded RNA (dsRNA) or exogenous hairpin RNA (hpRNA) or exogenous small interfering RNA that modulates 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 thereof. 5. The mutant, non-naturally occurring or transgenic Nicotiana plant leaf or part of the plant leaf according to any of the preceding claims, wherein the modulated expression or activity of the at least one NtGDH polynucleotide or NtGDH polypeptide modulates the amount of ammonia and amino acids and sugar and total alkaloids in the plant leaf or part of the plant leaf when cured; suitably, wherein the amino acids are proline, aspartate, serine, threonine and arginine. 6. The mutant, non-naturally occurring or transgenic Nicotiana plant leaf or part of the plant leaf according to any of the preceding claims, wherein the plant leaf or part thereof is air cured, suitably, wherein the air cured leaf or part thereof is sun cured or fire cured; or wherein the plant leaf or part thereof is air dried, suitably, wherein the air dried leaf or part thereof is sun dried or fire dried; and/or wherein the Nicotiana plant leaf or part of the plant leaf is a Nicotiana tabacum plant leaf or part of the plant leaf. 7. A method of preparing a cured Nicotiana plant leaf or a part of the plant leaf with modulated levels of ammonia and amino acids and sugar and total alkaloids as compared to a Nicotiana plant leaf or a part of the plant leaf from a control cured Nicotiana plant, said method comprising the steps of: (a) providing a Nicotiana plant comprising a NtGDH that comprises, consists, or consists essentially of at least one of a NtGDH2 polynucleotide or a NtGDH3 polynucleotide or a NtGDH6 polynucleotide or a NtGDH9 polynucleotide or a NtGDH10 polynucleotide or a NtGDH12 polynucleotide or a NtGDH2 polypeptide or a NtGDH3 polypeptide or a NtGDH6 polypeptide or a NtGDH9 polypeptide or a NtGDH10 polypeptide or a NtGDH12 polypeptide, wherein: (i) the NtGDH2 polynucleotide comprises, consists or consists essentially of a sequence having at least 90% sequence identity to SEQ ID NO: 1; or (ii) the NtGDH2 polypeptide is encoded by the polynucleotide set forth in (i); or (iii) the NtGDH2 polypeptide has at least 94% sequence identity to SEQ ID NO:2; or (iv) the NtGDH3 polynucleotide comprises, consists or consists essentially of a sequence having at least 90% sequence identity to SEQ ID NO: 3; or (v) the NtGDH3 polypeptide is encoded by the polynucleotide set forth in (iv); or (vi) the NtGDH3 polypeptide has at least 94% sequence identity to SEQ ID NO: 4; and (vii) the NtGDH6 polynucleotide comprises, consists or consists essentially of a sequence having at least 90% sequence identity to SEQ ID NO: 7; or (viii) the NtGDH6 polypeptide is encoded by the polynucleotide set forth in (i); or (ix) the NtGDH6 polypeptide has at least 94% sequence identity to SEQ ID NO:8; or (x) the NtGDH9 polynucleotide comprises, consists or consists essentially of a sequence having at least 85% sequence identity to SEQ ID NO: 13; or (xi) the NtGDH9 polypeptide is encoded by the polynucleotide set forth in (i); or (xii) the NtGDH9 polypeptide has at least 91% sequence identity to SEQ ID NO:14; or (xiii) the NtGDH10 polynucleotide comprises, consists or consists essentially of a sequence having at least 89% sequence identity to SEQ ID NO: 15; or (xiv) the NtGDH10 polypeptide is encoded by the polynucleotide set forth in (i); or (xv) the NtGDH10 polypeptide has at least 94% sequence identity to SEQ ID NO:16; or (xix) the NtGDH12 polynucleotide comprises, consists or consists essentially of a sequence having at least 85% sequence identity to SEQ ID NO: 19; or (xx) the NtGDH12 polypeptide is encoded by the polynucleotide set forth in (i); or (xxi) the NtGDH12 polypeptide has at least 91% sequence identity to SEQ ID NO:20; (b) modulating the expression of the at least one 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 in the Nicotiana plant leaf or part of the plant leaf; (c) harvesting the plant leaf or part of the plant leaf from the Nicotiana plant; (d) curing the plant leaf or part of the plant leaf; (e) optionally, measuring the levels of ammonia and one or more of amino acids and sugars and total alkaloids in the cured Nicotiana plant leaf or part of the cured plant leaf; and (f) obtaining a cured Nicotiana plant leaf or part of the plant leaf with modulated levels of ammonia and amino acids and sugars and total alkaloids as 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 modulated. 8. The method according to claim 7, wherein in step (b) the expression of the NtGDH6 polynucleotide and the NtGDH10 polynucleotide are modulated or the activity of the NtGDH6 polypeptide and the NtGDH10 polypeptide are modulated; and/or wherein the expression of the NtGDH2 polynucleotide and the NtGDH3 polynucleotide are modulated or the activity of both the NtGDH2 polypeptide and the NtGDH3 polypeptide are modulated; and/or wherein the expression of the NtGDH9 polynucleotide and the NtGDH12 polynucleotide are modulated or the activity of the NtGDH9 polypeptide and the NtGDH12 polypeptide are modulated; and/or wherein the expression of each of the NtGDH2 polynucleotide and the NtGDH3 polynucleotide and the NtGDH9 polynucleotide and the NtGDH12 polynucleotide are modulated or the activity of each of the NtGDH2 polypeptide and the NtGDH3 polypeptide and the NtGDH9 polypeptide and the NtGDH12 polypeptide are modulated; and/or wherein the expression of each of the NtGDH2 polynucleotide and the NtGDH3 polynucleotide and the NtGDH6 polynucleotide and the NtGDH9 polynucleotide and the NtGDH10 polynucleotide and the NtGDH12 polynucleotide or the NtGDH2 polypeptide and the NtGDH3 polypeptide and the NtGDH6 polypeptide and the NtGDH9 polypeptide and the NtGDH10 polypeptide and the NtGDH12 polypeptide are modulated. 9. The method according to claim 7 or 8, wherein the expression of one or more of a NtGDH4 polynucleotide or a NtGDH7 polynucleotide or a NtGDH8 polynucleotide or a NtGDH11 polynucleotide or the activity of a NtGDH4 polypeptide or a NtGDH7 polypeptide or a NtGDH8 polypeptide or a NtGDH11 polypeptide is not modulated, wherein (i) the NtGDH4 polynucleotide comprises, consists or consists essentially of a sequence having at least 88% sequence identity to SEQ ID NO: 5; or (ii) the NtGDH4 polypeptide is encoded by the polynucleotide set forth in (i); or (iii) the NtGDH4 polypeptide has at least 92% sequence identity to SEQ ID NO:6; or (iv) the NtGDH7 polynucleotide comprises, consists or consists essentially of a sequence having at least 92% sequence identity to SEQ ID NO: 9; or (v) the NtGDH7 polypeptide is encoded by the polynucleotide set forth in (iv); or (vi) the NtGDH7 polypeptide has at least 96% sequence identity to SEQ ID NO: 10; or (vii) the NtGDH8 polynucleotide comprises, consists or consists essentially of a sequence having at least 92% sequence identity to SEQ ID NO: 11; or (viii) the NtGDH8 polypeptide is encoded by the polynucleotide set forth in (i); or (ix) the NtGDH8 polypeptide has at least 95% sequence identity to SEQ ID NO:12; or (x) the NtGDH11 polynucleotide comprises, consists or consists essentially of a sequence having at least 87% sequence identity to SEQ ID NO: 17; or (xi) the NtGDH11 polypeptide is encoded by the polynucleotide set forth in (i); or (xii) the NtGDH11 polypeptide has at least 92% sequence identity to SEQ ID NO:18. 10. The method according to any of claims 7 to 9, wherein the Nicotiana plant leaf or part of the plant leaf is a Nicotiana tabacum plant leaf or part of the plant leaf; and/or wherein in step (b) expression or activity is modulated by genome editing; suitably, wherein the genome editing is selected from CRISPR-mediated genome editing, mutagenesis, zinc finger nuclease-mediated mutagenesis, chemical or radiation mutagenesis, homologous recombination, oligonucleotide-directed mutagenesis and meganuclease- mediated mutagenesis; or wherein in step (b) expression or activity is modulated using an interference polynucleotide. 11. A cured mutant, non-naturally occurring or transgenic Nicotiana tabacum plant leaf or a part thereof obtained or obtainable by the method of any of claims 7 to 10. 12. Cured Nicotiana plant leaf or a part of the cured plant leaf, wherein: (i) the ammonia content is between about 0.16 ± 0.04 % DWB to 0.110.16 ± 0.03 % Dry Weight Basis (DWB); and (ii) the glucose, fructose and sucrose content is from 0.51 ± 0.58 % DWB to 1.55 ± 1.10 % DWB; and (iii) the total free amino acid content is from 51.0 ± 6.60 mg/g DWB to 60.1 ± 4.58 mg/g DWB; and (iv) the total alkaloid content is from 2.24 ± 0.8 % DWB to 4.20 ± 0.39 %. 13. A cured tobacco blend comprising at least two different types of cured tobacco, wherein at least one of the cured tobaccos is cured tobacco from the mutant, non-naturally occurring or transgenic Nicotiana plant leaf or part of the plant leaf according to any of claims 1 to 6 or the cured mutant, non-naturally occurring or transgenic Nicotiana tabacum plant leaf or a part thereof according to claim 11 or the cured Nicotiana plant leaf or a part of the cured plant leaf according to claim 12; suitably, wherein the at least one other cured tobacco is a Burley tobacco or an Oriental tobacco or a Dark tobacco or a flue cured tobacco or a combination of two or more thereof. 14. A method for producing a tobacco blend having a reduced amount of ammonia comprising: (a) providing a cured Nicotiana plant leaf or a part thereof, wherein the first cured Nicotiana plant leaf or a part thereof is from the mutant, non-naturally occurring or transgenic Nicotiana plant leaf or part of the plant leaf according to any of claims 1 to 6 or the cured mutant, non-naturally occurring or transgenic Nicotiana tabacum plant leaf or a part thereof according to claim 11 or the cured Nicotiana plant leaf or a part of the cured plant leaf according to claim 12; and (b) blending the first cured Nicotiana plant leaf or a part thereof with at least one second cured Nicotiana plant leaf or a part thereof to produce a tobacco blend in which the sum of ammonia is lower than the sum of ammonia in the at least one second cured Nicotiana plant leaf or a part thereof. 15. A cured tobacco blend obtained or obtainable by the method of claim 14; or a tobacco product or a smoking article comprising a cured form of the mutant, non- naturally occurring or transgenic Nicotiana plant leaf or part of the plant leaf according to any of claims 1 to 6 or the cured mutant, non-naturally occurring or transgenic Nicotiana tabacum plant leaf or a part thereof according to claim 11 or the cured Nicotiana plant leaf or a part of the cured plant leaf according to claim 12 or the cured tobacco blend obtained or obtainable by the method of claim 14.

Description

MODULATION OF GENES CODING FOR GLUTAMATE DEHYDROGENASE FIELD OF THE INVENTION The present invention relates in general to plants having modulated expression or activity of glutamate dehydrogenase. BACKGROUND OF THE INVENTION Heated tobacco products, which heat real tobacco instead of burning it, do not emit side- stream smoke, but smokers exhale aerosols into the atmosphere. The inhalation of ammonia may cause nasopharyngeal and tracheal burns, bronchiolar and alveolar oedema, and airway destruction, resulting in respiratory distress or failure. Yamamoto et al. (2022) Toxics Oct 6;10(10):592 showed that the total emission amount of ammonia increased with an increase in heating temperature regardless of the tobacco heating device used. They concluded that ammonia in the mainstream aerosols was emitted from a common thermal process, probably thermal extraction in water vapor from a tobacco leaf. The ammonia released in the aerosol is correlated with the ammonia (ammonium ions) present in the tobacco material before the heating experience. These data also show that the type of curing will be determinant in the accumulation of ammonia within the tobacco matrix, both tobacco and aerosolized ammonia being predominant when the tobacco is air-cured. The presence of ammonia in tobacco material is not only linked to the type of curing used, but also to the tobacco type. For example, it is known that Burley tobacco generates significantly more ammonia than Virginia, as shown by Lefingwell in Chap.8, Production, Chemistry, And Technology, D. Layten Davis and Mark T. Nielson, Eds., Blackwell Science (Pub.), 1999. Figure 2 of this publication shows that when Burley fresh leaves are hung in an air-cured barn just after harvest, ammonia content (as a toxic compound for green leaf cells) is extremely low in the green leaves reaching a maximum value only after about 15 days (end of the so-called yellowing process which is associated to an induced leaf-senescence process). Very similar data were already described by Burton et al. (1983) Rec. Adc. Tob. Sci., 9, 91-153. These data also suggest that ammonia is a by-product of the senescence process, likely resulting from an active proteolysis and the 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 a catabolic process leading to the production of ammonia in cured tobacco has not been identified. There remains a need in the art to reduce the levels of ammonia in tobacco material. The present invention seeks to address this and other needs. SUMMARY OF THE INVENTION Certain GDH genes from Nicotiana tabacum that are referred to herein as NtGDH are disclosed that are involved in the accumulation of ammonia in leaf during curing. NtGDH genes were investigated to identify those that are particularly expressed during the early curing phase. Unexpectedly, certain NtGDH genes were found not to be expressed during the early curing phase – such as NtGDH4, NtGDH7, NtGDH8 and NtGDH11 – whereas other NtGDH genes were expressed during the early curing phase. In particular, 6 NtGDH genes were expressed during the early curing phase, namely NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10 and NtGDH12. Interestingly, NtGDH6 and NtGDH10 were found to be mainly expressed after 24 hours curing, whereas NtGDH2, NtGDH3, NtGDH9 and NtGDH12 were more induced in the late phase of curing from 48 hours to 192 hours. NtGDH2, NtGDH3, NtGDH9 and NtGDH12 followed similar induction profiles, however, the expression of NtGDH2 and NtGDH3 constantly increased during the curing process and reached the highest expression values (based on Fragments Per Kilobase of transcript per Million mapped reads (FPKM values) after 192 hours, which was not the case for NtGDH9 or NtGDH12. The effect of downregulating (for example, silencing) certain NtGDH genes on air-cured leaves showed that ammonia content is reduced. Suprisingly, the content of total alkaloids was also reduced, which anti-correlates with an increase in total amino acids. The amino acids mainly increasing in the cured leaves were proline, aspartate, serine, threonine and arginine. Sugars also increased. In summary therefore, inactivating or reducing the expression of certain NtGDH genes – such as NtGDH2, NtGDH3, NtGDH6, NtGDH9, NtGDH10 and NtGDH12 – can be used change the chemistry of cured leaves not only by limiting the increase of ammonia, but also by changing amino acid content - including proline, aspartate, serine, threonine and arginine content - and sugar content – such as glucose, fructose and lactose. Conversly, it is expected that upregulating (for example, overexpressing) certain NtGDH genes would also change the chemistry of cured leaves by increasing ammonia, increasing total alkaloids, decreasing amino acids – including proline, aspartate, serine, threonine and arginine – and decreasing sugars - such as glu