阅读说明：本技术 用于降低烟草植物的生物碱含量的方法 (Method for reducing alkaloid content of tobacco plants ) 是由 S·本哈立德 F·阿纳斯塔乔德阿布罗埃利马 于 2020-01-23 设计创作，主要内容包括：本发明提供了用于调节(例如降低)植物(例如烟草植物)的生物碱含量的方法,所述方法包括通过调节至少一种编码SOUL血红素-结合蛋白的基因的活性或表达来修饰所述植物。本发明还提供了至少一种编码SOUL血红素-结合蛋白的基因用于调节植物的生物碱含量的用途,以及根据本发明可获得的烟草细胞、植物、植物繁殖材料、收获的叶片、加工的烟草或烟草产品。(The present invention provides methods for modulating (e.g., reducing) alkaloid content in a plant (e.g., a tobacco plant), comprising modifying the plant by modulating the activity or expression of at least one gene encoding a SOUL heme-binding protein. The invention also provides the use of at least one gene encoding a SOUL heme-binding protein for modulating the alkaloid content of a plant, and tobacco cells, plants, plant propagation material, harvested leaves, processed tobacco or tobacco products obtainable according to the invention.)

1. A method of modulating (e.g., reducing) the alkaloid content of a plant or part thereof, or a cell or cell culture, said method comprising modifying said plant or cell culture by modulating the activity or expression of at least one gene encoding a SOUL heme-binding protein.

2. A method of modulating (e.g., reducing) the content of Tobacco Specific Nitrosamines (TSNAs) or TSNA precursors in a tobacco plant or part thereof, said method comprising modifying said plant or cell culture by modulating the activity or expression of at least one gene encoding a SOUL heme-binding protein.

3. Use of at least one gene encoding a SOUL heme-binding protein for modulating the alkaloid content of a cell or plant or part thereof, or a cell or cell culture.

4. A method for producing a plant or part thereof, a cell or cell culture, plant propagation material, leaf, cut harvested leaf, processed leaf or cut and processed leaf having modulated (e.g. reduced) alkaloid content, said method comprising modifying said plant or cell culture to modulate the activity or expression of at least one gene encoding a SOUL heme-binding protein.

5. The method or use of any of the preceding claims, wherein alkaloid content is modulated (e.g., reduced) as compared to a plant or cell culture that has not been modified to modulate the activity or expression of at least one gene encoding a SOUL heme-binding protein.

6. A plant or part thereof, or a cell or cell culture, which is modified to achieve a modulation (e.g. a reduction) in alkaloid content, as compared to an unmodified plant or unmodified cell or cell culture, wherein said modulation is a reduction in activity or expression of at least one gene encoding a SOUL heme-binding protein.

7. Plant propagation material obtainable from a plant or from a cell culture of a plant according to claim 6 or produced by a method according to claim 4 or claim 5.

8. The method or use of any one of claims 1-5, or the plant or part thereof, or the cell or cell culture of claim 6, or the plant propagation material of claim 7, wherein the alkaloid content of said plant is reduced as compared to a plant or cell culture that has not been modified to modulate the activity or expression of at least one gene encoding a SOUL heme-binding protein.

9. The method or use of claim 8, the plant or part thereof, or cell culture of claim 8, or plant propagation material of claim 8, wherein the activity or expression of at least one gene encoding a SOUL heme-binding protein is reduced as compared to a plant or cell culture that has not been modified to modulate the activity or expression of at least one gene encoding a SOUL heme-binding protein.

10. The method or use of any one of claims 1-5, or the plant or part thereof, or the cell or cell culture of claim 6, or the plant propagation material of claim 7, wherein the alkaloid content of said plant or cell culture is increased compared to a plant not modified to modulate the activity or expression of at least one gene encoding a SOUL heme-binding protein.

11. The method or use of claim 10, the plant or part thereof, or cell culture of claim 10, or the plant propagation material of claim 10, wherein the plant is modified to increase the activity or expression of at least one gene encoding a SOUL heme-binding protein and the plant or cell culture exhibits increased alkaloid content as compared to a plant or cell culture not modified to modulate the activity or expression of at least one gene encoding a SOUL heme-binding protein.

12. The method or use according to any one of claims 1 to 5 or 8 to 11, the plant or part thereof, or the cell or cell culture according to claims 6 or 8 to 11, or the plant propagation material according to claims 7 to 11, wherein the total alkaloid content of said plant or cell culture is modulated (e.g. reduced).

13. A method or use according to any one of claims 1 to 5 or 8 to 12, a plant or part thereof, or a cell or cell culture according to claims 6 or 8 to 12, or a plant propagation material according to claims 7 to 12, wherein the content of one or more alkaloids selected from nicotine, nornicotine, PON, neonicotinoid, mugwormin and anatabine is modulated (e.g. reduced), preferably the content of nicotine, nornicotine and/or PON is modulated (e.g. reduced).

14. The method or use according to any one of claims 1-5 or 8-13, the plant or part thereof, or the cell or cell culture according to claims 6 or 8-13, or the plant propagation material according to claims 7-13, wherein said plant or plant cell is from the solanaceae family.

15. The method or use according to any one of claims 1 to 5 or 8 to 14, the plant or part thereof, or the cell or cell culture according to claims 6 or 8 to 14, or the plant propagation material according to claims 7 to 14, wherein the plant or plant cell is from the genus nicotiana.

16. The method or use according to claim 15, the tobacco plant or part thereof or tobacco cell or cell culture according to claim 15, or the plant propagation material according to claim 15, wherein the nicotine content is modulated.

17. The method or use according to claim 16, the tobacco plant or part thereof or tobacco cell or cell culture according to claim 16, or the plant propagation material according to claim 16, wherein the nicotine content is reduced.

18. The method or use according to any one of claims 1 to 5 or 8 to 17, the plant or part thereof, or the cell or cell culture according to claims 6 or 8 to 17, or the plant propagation material according to claims 7 to 17, wherein said at least one gene encoding a SOUL heme-binding protein encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID number 1, 4, 7, 10, 13, 16, 19 or 22, or a functional variant or functional fragment or ortholog thereof, or a sequence having at least 80% identity to SEQ ID number 1, 4, 7, 10, 13, 16, 19 or 22; or

The at least one gene encoding a SOUL heme-binding protein comprises a nucleotide sequence as set forth in SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, or 24, or a functional variant or functional fragment or ortholog thereof, or a sequence having at least 80% identity to SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, or 24.

19. The method or use according to any one of claims 1-5 or 8-18, the plant or part thereof, or the cell or cell culture according to claims 6 or 8-18, or the plant propagation material according to claims 7-18, wherein an additional gene encoding a SOUL heme-binding protein is also modulated, wherein said additional gene encodes a polypeptide comprising an amino acid sequence as set forth in SEQ ID number 1, 4, 7, 10, 13, 16, 19 or 22, or a functional variant or functional fragment or ortholog thereof, or a sequence having at least 80% identity to SEQ ID number 1, 4, 7, 10, 13, 16, 19 or 22; or

Wherein the additional gene comprises a nucleotide sequence as set forth in SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23 or 24 or a functional variant or functional fragment or ortholog thereof, or a sequence having at least 80% identity to SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23 or 24.

20. Use of a plant or part thereof, or a cell or cell culture according to any one of claims 6 or 8-19, or a plant produced by the method of any one of claims 4, 5 or 8-19, for growing plants.

21. Use of a plant or part thereof, or a cell or cell culture according to any one of claims 6 or 8-19, or a plant produced by the method of any one of claims 4, 5 or 8-19, for the production of a product.

22. Use of a plant or part thereof according to any one of claims 6 or 8 to 19, or produced by a method of any one of claims 4, 5 or 8 to 20, for growing a crop.

23. Use of a plant or part thereof according to any one of claims 6 or 8 to 19, or a plant produced by a method of any one of claims 4, 5 or 8 to 19, for the production of leaves.

24. Harvested leaf of a plant according to any one of claims 6 or 8-19, or obtainable from a plant propagated from propagation material according to any one of claims 6-19 or obtainable from a plant obtained by the use according to any one of claims 2 or 20-21 or obtainable from a harvested leaf of a plant produced by the method of any one of claims 4, 5 or 8-19.

25. Harvested leaf of a plant according to claim 24, wherein the harvested leaf of the plant is a cut harvested leaf.

26. A processed lamina, preferably a processed tobacco lamina, preferably a non-viable processed tobacco lamina, which:

obtainable from a plant obtainable from the use according to any one of claims 2 or 20-22;

obtainable by processing a plant according to any one of claims 5 or 7-19;

a plant derivable from propagation of a plant propagation material according to any one of claims 6 to 19; or

Obtainable by processing harvested leaves of a plant according to claim 24 or 25; or

Obtainable from a plant produced by the method of any one of claims 4, 5 or 8-19.

27. The processed leaf of claim 26, wherein the leaf is processed by brewing, fermentation, pasteurization, or a combination thereof.

28. The machined blade of claim 26 or 27, wherein the machined blade is a cut machined blade.

29. A cured tobacco material made from a plant according to any one of claims 15 to 19 or a part or extract thereof.

30. A tobacco blend comprising the cured tobacco material of claim 29.

31. A tobacco industry product prepared from:

a tobacco plant or part thereof according to any one of claims 15-19, or a tobacco cell or cell culture according to any one of claims 15-19;

a tobacco plant or part thereof propagated from tobacco plant propagation material according to claim 15;

harvested leaf of the plant of claim 23 or 24, wherein the plant is tobacco;

a processed leaf according to any one of claims 25 to 27, wherein the plant is tobacco;

or

A plant produced by the method of claim 15.

32. A tobacco industry product according to claim 31, wherein the tobacco product is a combustible smoking article.

33. The tobacco industry product of claim 31, wherein the tobacco product is a smokeless tobacco product.

34. A tobacco product according to claim 31, wherein the tobacco product is a non-combustible aerosol provision system, such as a tobacco heating device or an aerosol generating device.

35. Use of the tobacco cells of claim 15 for modulating alkaloid content in a cell or cell culture.

36. A combustible smoking article, a non-combustible aerosol provision system, a smokeless tobacco product, or a tobacco heating apparatus comprising a plant or part thereof or extract thereof (e.g. a tobacco extract) according to any one of claims 6-19, or a tobacco cell or cell culture according to any one of claims 15-19; or a cured tobacco material according to claim 29; or a tobacco blend according to claim 31.

37. Use of a nucleotide sequence of at least one gene encoding a SOUL heme-binding protein for the selection of plants having modulated (e.g. reduced) alkaloid content and/or modulated (e.g. reduced) Tobacco Specific Nitrosamine (TSNA) or TSNA precursor content, preferably wherein said SOUL heme-binding protein has a sequence selected from SEQ ID number 1, 4, 7, 10, 13, 16, 19 or 22, or a functional variant or functional fragment or ortholog thereof, or a sequence having at least 80% identity to SEQ ID number 1, 4, 7, 10, 13, 16, 19 or 22; or wherein the at least one gene encoding a SOUL heme-binding protein comprises the nucleotide sequence set forth in SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, or 24, or a functional variant or functional fragment or ortholog thereof, or a sequence having at least 80% identity to SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, or 24.

38. A mutant of a plant carrying a genetic mutation in the nucleotide sequence of at least one gene encoding a SOUL heme-binding protein, preferably, wherein the gene is selected from SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23 or 24 or a functional variant or a functional fragment or an orthologue thereof, or a sequence having at least 80% identity to SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23 or 24, wherein the heritable mutation modulates (e.g., reduces) the activity or expression of the at least one gene encoding a SOUL heme-binding protein, and wherein the mutant plant has a modulated (e.g. reduced) alkaloid content and/or a modulated content of Tobacco Specific Nitrosamines (TSNAs) or TSNA precursors relative to a comparable plant not carrying said heritable mutation.

39. Progeny or seed of a mutant plant according to claim 38 carrying a heritable mutation.

40. Harvested leaf, processed leaf or cured tobacco material produced from a plant comprising a modification in the nucleotide sequence of at least one gene encoding a SOUL heme-binding protein, wherein the at least one gene is selected from SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23 or 24 or a functional variant or functional fragment or ortholog thereof, or a sequence having at least 80% identity to SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23 or 24; or a functional variant or a functional fragment or ortholog thereof; wherein the modification modulates (e.g., reduces) the activity or expression of the at least one gene encoding a SOUL heme-binding protein, and wherein the plant has modulated (e.g., reduced) alkaloid content and/or modulated tobacco-specific nitrosamine (TSNA) or TSNA precursor content relative to a comparable plant not carrying the modification in the at least one gene encoding a SOUL heme-binding protein.

41. A method, leaf, plant propagation material, harvested leaf, processed tobacco, tobacco product, use or a combination thereof as described herein with reference to the description and drawings.

Technical Field

The present invention relates to methods of modulating the alkaloid content, e.g., nicotine content, of a plant or portion thereof. The invention also extends to methods of modulating the expression and/or activity of a polypeptide that modulates alkaloid content in a plant. Alternatively, the present invention provides methods of modulating the expression and/or activity of a gene encoding a polypeptide that modulates alkaloid content in a plant. The invention also extends to constructs which may be used to modulate a polypeptide. The invention further relates to plant cells and plants modified to achieve modulation of alkaloid content. The invention also relates to processed and harvested leaf from such regulated plants and their use in tobacco industry products, including combustible smoking articles.

Background

Alkaloids are a group of naturally occurring compounds that contain primarily basic nitrogen atoms and are produced by a large variety of organisms including bacteria, fungi, plants, and animals.

Alkaloids can be classified according to the similarity of carbon backbones such as indole, isoquinoline, and pyridine-like. Pyridine derivatives are a class of monomeric alkaloids; this category includes simple pyridine derivatives, polycyclic condensed and non-condensed pyridine derivatives, and sesquiterpene pyridine derivatives. Examples are nicotine, nornicotine, neonicotinoid, myoxamine and anatabine.

Most of the known biological functions of alkaloids are related to protection. Neuroactive molecules such as caffeine, cocaine, morphine and nicotine act as defense compounds against invading predators. Accumulation of these alkaloids is the result of a signal transduction cascade that monitors gene expression, enzyme activity and alkaloid concentration. Fine-tuning of alkaloid content in plants involves negative feedback loops and degradation pathways.

Nicotine naturally occurs in several plant species, but is found at the highest levels in tobacco plants. Tobacco cultivation produces 2-4% total dry weight of alkaloids. Nicotine in wild and cultivated Nicotiana (Nicotiana) Produced in species and it plays a role in plant defense against herbivores and insectsTo do so (Voelckel et al (2001) Oecology 127(2): 274-280, incorporated herein by reference). Which accounts for 90% of the total alkaloid content. The remaining 10% of the alkaloid pool is mainly composed of the structurally related compounds nornicotine, anatabine, neonicotinoid and pseudooxidized nicotine (PON).

The regulation of alkaloid content in tobacco is complex. Several factors, including genotype, environment, fertilization, and agronomic practices (e.g., topping), affect alkaloid levels in tobacco plants. Some key regulators of nicotine biosynthesis are well characterized, e.g., putrescine N-methyltransferase (PMT), which plays a key role in this pathway, is activated by members of the Ethylene Response Factor (ERF) superfamily, the largest transcription factor family in the tobacco genome (Rushton et al (2008) Plant physiol.147 (1): 280-. Other transcription factors that induce alkaloid biosynthesis belong to the MYC 2-like basic helix-loop-helix (bHLH) family. MYC 2-like bhlhhs directly regulate alkaloid levels through Gbox-mediated binding and activation of alkaloid structural genes, and indirectly regulate alkaloid levels through activation of ERFs.

Modifying the alkaloid content in plants (e.g., tobacco) can have several commercial advantages. For example, reducing the total alkaloid content in a plant can increase the value of the plant as a biomass resource. For example, modifying the alkaloid content can include reducing the alkaloid content, e.g., nicotine content, in a tobacco plant. Tobacco plants and products with reduced nicotine may be desirable in view of "nicotine ceiling", i.e., potential modulation of the average nicotine ceiling in tobacco products. Alternatively, increasing the alkaloid content in plants, such as tobacco plants, can help protect plants against insects and herbivores. There remains a need for plants having modulated alkaloid content, e.g., having modulated nicotine content, with improved commercially desirable traits, and methods of making the same.

During post-harvest leaf conditioning, the reaction between pyridine alkaloids and nitrosating substances results in the formation of tobacco-specific nitrosamines (TSNAs). Nornicotine and PON are precursors to TSNA NNN and NNK, respectively. It is important to reduce the production and accumulation of nornicotine and PON. The CYP82E family of nicotine demethylase genes is one of the major regulators of nicotine conversion to nornicotine, and altering its activity or accumulation may lead to reduced NNN levels. However, to date, no enzymes or genes responsible for PON production have been identified.

As described in the examples, the inventors sought to study the genes responsible for alkaloid synthesis with the aim of modulating alkaloid content in plants, for example reducing nicotine content in tobacco plants.

Summary of The Invention

It has surprisingly been found that by modulating the activity or expression of a gene encoding a SOUL heme-binding protein, the alkaloid content and/or the TSNA content or the TSNA precursor content of a plant can be modulated. SOUL heme-binding proteins as taught herein, e.g., nitab4.5 — 0013616g0010.2, are modulators of nicotine, nornicotine, neonicotinoid, PON, muxosamine, and anatabine in cultivated tobacco. Nitab4.5_0013616g0010.2 encodes a SOUL heme-binding protein according to the present invention. Nitab4.5_0000652g0130.2, nitab4.5_0001140g0220.2, nitab4.5_0003235g0060.2, nitab4.5_0004868g0020.2, nitab4.5_0006614g0010.2, nitab4.5_0006991g0050.2 and nitab4.5_0009023g0010.2 encode homologs of nitab4.5_0013616g0010.2 according to the invention. The SOUL heme-binding proteins according to the present invention contain a conserved three-dimensional structure known as the SOUL motif. The SOUL heme-binding proteins according to the invention bind heme.

According to the present invention, tobacco products can be produced having modulated alkaloid content and commercially desirable traits sought by consumers of the tobacco products. In some cases, consumers may desire products with low levels of alkaloids, such as low levels of TSNA or low nicotine content.

The invention may be particularly useful in the field of plant molecular cultivation, where plants (such as tobacco and other nicotiana species) are used for the production of proteins, peptides and metabolites, for example for the production of therapeutic and pharmaceutical agents such as antibiotics, virus-like particles, or nutraceuticals (neutraceuticals) or small molecules. In a project funded by the EU under the name pharmpont, tobacco has been used to develop HIV-neutralizing antibodies, and Medicago inc.

Thus, plants according to the invention may be used for molecular farming to reduce or eliminate the presence of nicotine, nornicotine, PON and/or other nicotine alkaloids, such as neonicotinoid or anatabine or myoxaline. The use of low nicotine plants or rhizomes (rootstocks) is beneficial in molecular farming and will reduce downstream processing costs associated with purification.

In other instances, it may be desirable to produce plants with high alkaloid levels, such as high levels of nicotine content, so that nicotine may be purified from tobacco plants to produce a pure nicotine product, for example for use in devices utilizing nicotine-containing liquids, such as electronic cigarettes (e-cigarettes), or within tobacco heating devices. For example, the production of plants with leaves containing high levels of nicotine may reduce nicotine extraction costs for producing e-liquid (e-liquids) for e-cigarettes.

The present inventors have surprisingly determined a method of modulating alkaloid content (e.g., nicotine content) in a plant (e.g., a tobacco plant) by modulating the activity or expression of a gene encoding a SOUL heme-binding protein. The alkaloid content (e.g., the content of one or more of nicotine, nornicotine, neonicotinoid, PON, muxosmine, or anatabine) of a plant (e.g., a tobacco plant) can be reduced by reducing the activity or expression of a gene encoding a SOUL heme-binding protein or can be increased by increasing the activity or expression of a gene encoding a SOUL heme-binding protein (e.g., by increasing the heme-binding affinity of a SOUL heme-binding protein). Prior to the present invention, it was not known that modulation of the activity or expression of the gene encoding a SOUL heme-binding protein as described herein could be used to modulate alkaloid levels.

In one aspect, there is provided a method of modulating (e.g., reducing) the alkaloid content of a plant or part thereof, or a cell or cell culture, said method comprising modifying said plant or cell culture by modulating the activity or expression of at least one gene encoding a SOUL heme-binding protein.

In one aspect, there is provided a method of reducing the alkaloid content of a plant or part thereof, or a cell or cell culture, said method comprising modifying said plant or cell culture by reducing the activity or expression of at least one SOUL heme-binding protein.

In another aspect, there is provided a method of modulating (e.g., reducing) the content of Tobacco Specific Nitrosamines (TSNAs) or precursors of TSNAs in a tobacco plant or plant part or cell thereof, the method comprising modifying said plant or cell culture by modulating the activity or expression of at least one gene encoding a SOUL heme-binding protein.

In another aspect, there is provided a method of reducing the content of Tobacco Specific Nitrosamines (TSNAs) or precursors of TSNAs in a tobacco plant or plant part or cell thereof, the method comprising modifying said plant or cell culture by reducing the activity or expression of at least one SOUL heme-binding protein.

In a further aspect, there is provided the use of at least one gene encoding a SOUL heme-binding protein for modulating the alkaloid content of a cell or plant or part thereof, or a cell or cell culture.

In yet another aspect, there is provided a method for producing a plant or part thereof, a cell or cell culture, plant propagation material, leaf, cut harvested leaf, processed leaf or cut and processed leaf having modulated (e.g. reduced) alkaloid content, said method comprising modifying said plant or cell culture to modulate the activity or expression of at least one gene encoding a SOUL heme-binding protein.

Suitably, the alkaloid content may be modulated (e.g., reduced) as compared to a plant or cell culture that has not been modified to modulate the activity or expression of at least one gene encoding a SOUL heme-binding protein.

In a further aspect, there is provided a plant or part thereof, or a cell or cell culture, which is modified to achieve modulation (e.g. reduction) of alkaloid content, as compared to an unmodified plant or unmodified cell or cell culture, wherein said modulation is a reduction in activity or expression of at least one gene encoding a SOUL heme-binding protein.

In a further aspect, there is provided plant propagation material obtainable from a plant according to the invention, or a plant or cell culture produced by a method according to the invention.

In a further aspect, there is provided a method or use according to the invention, or a plant or part thereof, or a cell or cell culture according to the invention, or a plant propagation material according to the invention, wherein the alkaloid content of said plant is reduced compared to a plant or cell culture which has not been modified to modulate the activity or expression of at least one gene encoding a SOUL heme-binding protein.

Suitably, the activity or expression of at least one gene encoding a SOUL heme-binding protein may be reduced as compared to a plant or cell culture that has not been modified to modulate the activity or expression of at least one gene encoding a SOUL heme-binding protein.

Suitably, the alkaloid content of the plant or cell culture may be increased as compared to a plant not modified to modulate the activity or expression of at least one gene encoding a SOUL heme-binding protein.

Suitably, the plant may be modified to increase the activity or expression of at least one gene encoding a SOUL heme-binding protein and the plant or cell culture exhibits increased alkaloid content as compared to a plant or cell culture that has not been modified to modulate the activity or expression of at least one gene encoding a SOUL heme-binding protein.

Suitably, the total alkaloid content of the plant or cell culture may be modulated (e.g. reduced).

Suitably, the content of one or more alkaloids, which may be selected from nicotine, nornicotine, PON, neonicotinoid, mugwormin and anatabine, is adjusted (e.g. reduced), preferably the content of nicotine, nornicotine and/or PON is adjusted (e.g. reduced). In some embodiments, two or more (or three or more) alkaloids selected from nicotine, nornicotine, PON, neonicotinoid, mugwormin, and anatabine may be modulated (e.g., reduced). In some embodiments, the total alkaloid content of the plant or cell is modulated (e.g., reduced).

In one aspect, the plant or plant cell is from the solanaceae family.

Suitably, the plant or plant cell may be from the genus nicotiana.

Suitably, the nicotine content may be adjusted. Suitably, the nicotine content may be reduced.

In one aspect, there is provided a method or use according to the invention, a plant or part thereof, or a cell or cell culture according to the invention, or a plant propagation material according to the invention, wherein said at least one gene encoding a SOUL heme-binding protein encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID number 1, 4, 7, 10, 13, 16, 19 or 22, or a functional variant or fragment or ortholog thereof; or

Wherein the at least one gene encoding a SOUL heme-binding protein encodes a polypeptide comprising an amino acid sequence having at least 80% identity to SEQ ID number 1, 4, 7, 10, 13, 16, 19, or 22; or

Wherein the at least one gene encoding a SOUL heme-binding protein comprises a nucleotide sequence as set forth in SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, or 24 or a functional variant or functional fragment or ortholog thereof; or

Wherein the at least one gene encoding a SOUL heme-binding protein comprises a nucleotide sequence having at least 80% identity to SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, or 24.

In a further aspect, there is provided a method or use according to the invention, a plant or part thereof, or a cell or cell culture according to the invention, or a plant propagation material according to the invention, wherein an additional gene encoding a SOUL heme-binding protein is also modulated, wherein said additional gene encodes a polypeptide comprising the amino acid sequence as set forth in SEQ ID No.1, 4, 7, 10, 13, 16, 19 or 22, or a functional variant or functional fragment or orthologue thereof; or

Wherein the additional gene encodes a polypeptide comprising an amino acid sequence having at least 80% identity to SEQ ID number 1, 4, 7, 10, 13, 16, 19 or 22; or

Wherein the additional gene comprises a nucleotide sequence as set forth in SEQ ID Nos. 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23 or 24 or a functional variant or functional fragment or ortholog thereof; or

Wherein the additional gene comprises a nucleotide sequence having at least 80% identity to SEQ ID Nos. 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23 or 24.

In one aspect, there is provided the use of a plant or part thereof, or a cell or cell culture according to the invention, or a plant produced by a method according to the invention, to cultivate a plant.

In another aspect, there is provided the use of a plant or part thereof, or a cell or cell culture according to the invention, or a plant produced by a method according to the invention, for the production of a product.

In yet another aspect, there is provided the use of a plant according to the invention, or part thereof, or a plant produced by a method according to the invention, for growing a crop.

In another aspect, there is provided the use of a plant according to the invention, or part thereof, or a plant produced by a method according to the invention, to produce a leaf.

In another aspect, harvested leaves of a plant according to the invention are provided, or obtainable from a plant propagated from propagation material according to the invention or obtainable from a plant obtained by use according to the invention or obtainable from a plant produced by a method according to the invention.

Suitably, the harvested leaf may be a cut harvested leaf.

In another aspect, a processed lamina, preferably a processed tobacco lamina, preferably a non-viable processed tobacco lamina is provided which:

obtainable from a plant obtainable from the use according to the invention;

obtainable by processing a plant according to the invention;

obtainable from a plant propagated from a plant propagation material according to the invention; or

Obtainable by processing harvested leaves of a plant according to the invention; or

May be obtained from a plant produced by a method according to the invention.

Suitably, the leaf may be processed by conditioning, fermentation, pasteurisation or a combination thereof.

Suitably, the processing blade may be a cut processing blade.

In another aspect, there is provided a cured tobacco material made from a plant according to the invention or a part thereof or an extract thereof.

In another aspect, a tobacco blend is provided comprising the cured tobacco material according to the present invention.

In another aspect, there is provided a tobacco industry product prepared from:

a tobacco plant or part thereof according to the invention, or a tobacco cell or cell culture according to the invention;

a tobacco plant or part thereof propagated from tobacco plant propagation material according to the invention;

harvested leaf of a plant according to the invention, wherein the plant is tobacco;

processing leaves according to the invention, wherein the plant is tobacco;

or

Plants produced by the method according to the invention.

Suitably, the tobacco product may be a combustible smoking article.

Suitably, the tobacco product may be a smokeless tobacco product.

Suitably, the tobacco product may be a non-combustible aerosol provision system, such as a tobacco heating device or an aerosol generating device.

In one aspect, there is provided the use of a tobacco cell according to the invention for modulating the alkaloid content in a cell culture.

In another aspect, there is provided a combustible smoking article, a non-combustible aerosol supply system, a smokeless tobacco product or a tobacco heating apparatus comprising a plant according to the invention or a part thereof or an extract thereof (e.g. a tobacco extract), or a tobacco cell or cell culture according to the invention; or a reconstituted tobacco material according to the invention; or a tobacco blend according to the invention.

In one aspect, there is provided the use of a nucleotide sequence of at least one gene encoding a SOUL heme-binding protein to select for plants having modulated (e.g. reduced) alkaloid content and/or modulated (e.g. reduced) Tobacco Specific Nitrosamine (TSNA) or TSNA precursor content, preferably wherein the sequence of said SOUL heme-binding protein is selected from SEQ ID number 1, 4, 7, 10, 13, 16, 19 or 22 or a functional variant or functional fragment or ortholog thereof; or wherein the sequence of said SOUL heme-binding protein is a sequence having at least 80% identity to SEQ ID number 1, 4, 7, 10, 13, 16, 19, or 22; or wherein the at least one gene encoding a SOUL heme-binding protein comprises the nucleotide sequence set forth in SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, or 24, or a functional variant or functional fragment or ortholog thereof; or wherein the at least one gene encoding a SOUL heme-binding protein comprises a nucleotide sequence having at least 80% identity to SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, or 24.

In another aspect, there is provided a mutant of a plant carrying a heritable mutation in the nucleotide sequence of at least one gene encoding a SOUL heme-binding protein, preferably wherein said gene is selected from SEQ ID No.2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, or 24 or a functional variant or functional fragment or ortholog thereof; or wherein the gene is selected from a sequence having at least 80% identity to SEQ ID Nos. 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23 or 24; wherein the heritable mutation modulates (e.g., reduces) the activity or expression of the at least one gene encoding a SOUL heme-binding protein, and wherein the mutant plant has a modulated (e.g., reduced) alkaloid content and/or a modulated content of a tobacco-specific nitrosamine (TSNA) or a precursor of a TSNA, relative to a comparable plant not carrying the heritable mutation.

In another aspect, progeny or seeds of the mutant plants carrying the genetic mutations according to the invention are provided.

In another aspect, there is provided harvested leaf, processed leaf or cured tobacco material produced from a plant comprising a modification in the nucleotide sequence of at least one gene encoding a SOUL heme-binding protein, wherein the at least one gene is selected from SEQ ID nos. 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23 or 24 or a functional variant or functional fragment or ortholog thereof; or wherein the at least one gene is selected from a sequence having at least 80% identity to SEQ ID Nos. 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23 or 24; wherein the modification modulates (e.g., reduces) the activity or expression of the at least one gene encoding a SOUL heme-binding protein, and wherein the plant has modulated (e.g., reduced) alkaloid content and/or modulated tobacco-specific nitrosamine (TSNA) or TSNA precursor content relative to a comparable plant not carrying the modification in the at least one gene encoding a SOUL heme-binding protein.

In another aspect, there is provided a method, leaf, plant propagation material, harvested leaf, processed tobacco, tobacco product, use or a combination thereof as described herein with reference to the description and drawings.

Brief Description of Drawings

Embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings, in which:

without wishing to be bound by theory, figure 1 depicts the effect of over-expression of SOUL heme-binding protein on pyridine alkaloid biosynthesis. (A) Following wound or herbivory perception by surface-located receptors, immune signaling is induced via phosphorylation of MAPK and generation of Reactive Oxygen Species (ROS). This activates the Jasmonate (JA) -responsive gene and up-regulates pyridine alkaloid production. Intracellular ROS levels are tightly regulated by Heme Oxygenase (HO) -mediated heme catabolism. (B) SOUL overexpression reduces the available heme that is decomposed by HO. This results in an increase of intracellular ROS, which upregulates the nicotine biosynthesis pathway. PM: plasma membranes.

FIG. 2 shows a vector for transient overexpression of SOUL heme-binding protein in tobacco.

Figure 3 shows the alkaloid (nicotine, nornicotine, neonicotinoid, PON and anatabine) content of 5-week-old TN90 leaf expressing nitab4.5_0013616g0010.2 (SEQ ID number 3). Alkaloid content is expressed relative to control and contains three biological replicates analyzed by t-test. Values are shown as mean ± SEM. Asterisks indicate statistical significance with P-value ≦ 0.001.

Figure 4 shows alkaloid (nicotine, nornicotine, neonicotine, PON, and anatabine) content of 5-week-old TN90 leaves expressing constructs that silenced nitab4.5_0013616g0010.2 (SEQ ID number 3) by virus-induced gene silencing (VIGS). Alkaloid content is expressed relative to control and contains three biological replicates analyzed by t-test. Values are shown as mean ± SEM. Asterisks indicate statistical significance with P-value ≦ 0.001.

FIG. 5 shows the amino acid sequence of Nitab4.5-0013616 g 0010.2-SEQ ID number 1-from tobacco (Nitabu.s.Nicotiana tabacum) according to the inventionNicotiana tabacum) The SOUL heme-binding protein of (a).

FIG. 6 shows the genomic sequence of Nitab4.5-0013616 g 0010.2-SEQ ID number 2-encoding a polypeptide from tobacco (Nitabu) according to the inventionNicotiana tabacum) The SOUL heme-binding protein of (a).

FIG. 7 shows the coding sequence of Nitab4.5-0013616 g 0010.2-SEQ ID number 3-coding sequence from tobacco (Nicotiana tabacum) according to the inventionNicotiana tabacum) The SOUL heme-binding protein of (a).

FIG. 8 shows the amino acid sequence of Nitab4.5-0000652 g 0130.2-SEQ ID number 4-from tobacco (Nicotiana tabacum) according to the inventionNicotiana tabacum) The SOUL heme-binding protein of (a).

FIG. 9 shows the genomic sequence of Nitab4.5-0000652 g 0130.2-SEQ ID number 5-encoding a polypeptide derived from tobacco (Nicotiana tabacum) according to the inventionNicotiana tabacum) The SOUL heme-binding protein of (a).

FIG. 10 shows the coding sequence of Nitab4.5-0000652 g 0130.2-SEQ ID number 6-coding from tobacco (Nicotiana tabacum) according to the inventionNicotiana tabacum) The SOUL heme-binding protein of (a).

FIG. 11 shows the amino acid sequence of Nitab4.5-0001140 g 0220.2-SEQ ID number 7-from tobacco (Nicotiana tabacum) according to the inventionNicotiana tabacum) The SOUL heme-binding protein of (a).

FIG. 12 shows the genomic sequence of Nitab4.5-0001140 g 0220.2-SEQ ID number 8-encoding a polypeptide from tobacco (Nicotiana tabacum) (Nitabacum L.var.Niacinum L.var.Nitabacum L.var.Niacinum L.C.; SEQ ID number 8-according to the inventionNicotiana tabacum) The SOUL heme-binding protein of (a).

FIG. 13 shows the coding sequence of Nitab4.5_0001140g 0220.2-SEQ ID number 9-coding for a polypeptide from tobacco (Nicotiana tabacum) according to the inventionNicotiana tabacum) The SOUL heme-binding protein of (a).

FIG. 14 shows the amino acid sequence of Nitab4.5-0003235 g 0060.2-SEQ ID number 10-from tobacco (Nicotiana tabacum) according to the inventionNicotiana tabacum) The SOUL heme-binding protein of (a).

FIG. 15 shows the genomic sequence of Nitab4.5-0003235 g 0060.2-SEQ ID number 11-encoding a polypeptide from tobacco (Nicotiana tabacum) (Nitabacum) according to the inventionNicotiana tabacum) The SOUL heme-binding protein of (a).

FIG. 16 shows the coding sequence of Nitab4.5-0003235 g 0060.2-SEQ ID number 12-coding for a polypeptide from tobacco (Nicotiana tabacum) according to the inventionNicotiana tabacum) The SOUL heme-binding protein of (a).

FIG. 17 shows the amino acid sequence of Nitab4.5-0004868 g 0020.2-SEQ ID number 13-from tobacco (Nicotiana tabacum) according to the inventionNicotiana tabacum) The SOUL heme-binding protein of (a).

FIG. 18 shows the genomic sequence of Nitab4.5-0004868 g 0020.2-SEQ ID number 14-encoding a polypeptide from tobacco (Nicotiana tabacum) (Nitabacum) according to the inventionNicotiana tabacum) The SOUL heme-binding protein of (a).

FIG. 19 shows the coding sequence of Nitab4.5_0004868g 0020.2-SEQ ID number 15-coding for a polypeptide from tobacco (Nicotiana tabacum) according to the inventionNicotiana tabacum) The SOUL heme-binding protein of (a).

FIG. 20 shows the amino acid sequence of Nitab4.5-0006614 g 0010.2-SEQ ID number 16-from tobacco (Nitabu.s.Nicotiana tabacum) according to the inventionNicotiana tabacum) The SOUL heme-binding protein of (a).

FIG. 21 shows the genomic sequence of Nitab4.5-0006614 g 0010.2-SEQ ID number 17-encoding a polypeptide from tobacco (Nitabu) according to the inventionNicotiana tabacum) The SOUL heme-binding protein of (a).

FIG. 22 shows the coding sequence of Nitab4.5-0006614 g 0010.2-SEQ ID number 18-coding sequence from tobacco (Nicotiana tabacum) according to the inventionNicotiana tabacum) The SOUL heme-binding protein of (a).

FIG. 23 shows the amino acid sequence of Nitab4.5-0006991 g 0050.2-SEQ ID number 19-from tobacco (Nicotiana tabacum) according to the inventionNicotiana tabacum) The SOUL heme-binding protein of (a).

FIG. 24 shows the genomic sequence of Nitab4.5-0006991 g 0050.2-SEQ ID number 20-encoding a polypeptide from tobacco (Nicotiana tabacum) (Nitabacum L.var.Nicotiana tabacum) The SOUL heme-binding protein of (a).

FIG. 25 shows the coding sequence of Nitab4.5_0006991g 0050.2-SEQ ID number 21-coding for a polypeptide from tobacco (Nicotiana tabacum) (Nitabacum) according to the inventionNicotiana tabacum) The SOUL heme-binding protein of (a).

FIG. 26 shows the amino acid sequence of Nitab4.5-0009023 g 0010.2-SEQ ID number 22-from tobacco (Nitabacum) according to the inventionNicotiana tabacum) The SOUL heme-binding protein of (a).

FIG. 27 shows the genomic sequence of Nitab4.5-0009023 g 0010.2-SEQ ID number 23-encoding a polypeptide from tobacco (Nitabacum) according to the inventionNicotiana tabacum) The SOUL heme-binding protein of (a).

FIG. 28 shows the coding sequence of Nitab4.5_0009023g 0010.2-SEQ ID number 24-code from tobacco (A) according to the inventionNicotiana tabacum) The SOUL heme-binding protein of (a).

FIG. 29 shows the 300-nucleotide cDNA fragment used in example 2, SEQ ID number 25.

FIG. 30 shows SEQ ID number 26, the sequence TRV1 used in example 2.

FIG. 31 shows SEQ ID number 27, the sequence TRV2 used in example 2.

Figure 32 shows the nornicotine content of 5-week-old TN90 leaves transiently expressing the indicated constructs: OE = expressing nitab4.5_0013616g 0010.2; or As = antisense construct silencing nitab4.5_0013616g 0010.2. Levels are expressed relative to controls and contain three biological replicates analyzed by multiple comparative post-hoc tests of one-way ANOVA and Tukey. Values are shown as mean ± SEM. Asterisks indicate statistical significance with P-value ≦ 0.001.

FIG. 33 shows the nornicotine content of 5-week-old TN90 leaf transiently expressing constructs encoding the homologous sequences shown (see example 3). The content is expressed as a percentage relative to the control (100%) and contains two biological replicates analyzed by t-test. Values are shown as mean ± SEM.

Figure 34 shows the nornicotine content of greenhouse grown TN90 plants at 12 leaf stage or control plants overexpressing nitab4.5_0013616g 0010.2. (n = 24).

FIG. 35 shows in panel (A) the reduced nicotine levels of field grown control (wild type WT) TN90 leaf and Nitab4.5_0013616g0010.2 Overexpressors (OX). The amounts are expressed relative to the control. The results were analyzed by t-test. Values are shown as mean ± SEM. Asterisks indicate statistical significance with P-value ≦ 0.001. In panel (B), NNN content of field grown control (WT) TN90 leaf and Nitab4.5_0013616g0010.2 overexpressors. The amounts are expressed relative to the control. The results were analyzed by t-test. Values are shown as mean ± SEM. Asterisks indicate statistical significance with P-value ≦ 0.01.

Sequence listing

A summary of sequence identifiers and corresponding sequence listing is provided for use throughout the subject specification, wherein:

SEQ ID No.1 corresponds to the amino acid sequence of Nitab4.5-0013616 g 0010.2.

SEQ ID No.2 corresponds to the genomic sequence of Nitab4.5-0013616 g 0010.2.

SEQ ID No.3 corresponds to the coding sequence of Nitab4.5_0013616g 0010.2.

SEQ ID No.4 corresponds to the amino acid sequence of Nitab4.5-0000652 g 0130.2.

SEQ ID No.5 corresponds to the genomic sequence of Nitab4.5_0000652g 0130.2.

SEQ ID No.6 corresponds to the coding sequence of Nitab4.5_0000652g 0130.2.

SEQ ID No.7 corresponds to the amino acid sequence of Nitab4.5-0001140 g 0220.2.

SEQ ID No.8 corresponds to the genomic sequence of Nitab4.5_0001140g 0220.2.

SEQ ID No.9 corresponds to the coding sequence of Nitab4.5_0001140g 0220.2.

SEQ ID No.10 corresponds to the amino acid sequence of Nitab4.5-0003235 g 0060.2.

SEQ ID No.11 corresponds to the genomic sequence of Nitab4.5_0003235g 0060.2.

SEQ ID No.12 corresponds to the coding sequence of Nitab4.5_0003235g 0060.2.

SEQ ID No.13 corresponds to the amino acid sequence of Nitab4.5-0004868 g 0020.2.

SEQ ID No.14 corresponds to the genomic sequence of Nitab4.5_0004868g 0020.2.

SEQ ID No.15 corresponds to the coding sequence of Nitab4.5_0004868g 0020.2.

SEQ ID No.16 corresponds to the amino acid sequence of Nitab4.5-0006614 g 0010.2.

SEQ ID No.17 corresponds to the genomic sequence of Nitab4.5-0006614 g 0010.2.

SEQ ID No.18 corresponds to the coding sequence of Nitab4.5_0006614g 0010.2.

SEQ ID No.19 corresponds to the amino acid sequence of Nitab4.5-0006991 g 0050.2.

SEQ ID No.20 corresponds to the genomic sequence of Nitab4.5_0006991g 0050.2.

SEQ ID No.21 corresponds to the coding sequence of Nitab4.5_0006991g 0050.2.

SEQ ID No.22 corresponds to the amino acid sequence of Nitab4.5-0009023 g 0010.2.

SEQ ID No.23 corresponds to the genomic sequence of Nitab4.5_0009023g 0010.2.

SEQ ID No.24 corresponds to the coding sequence of Nitab4.5_0009023g 0010.2.

SEQ ID number 25 is the 300-nucleotide cDNA fragment used in example 2.

SEQ ID number 26 is the sequence TRV1 used in example 2.

SEQ ID number 27 is the sequence TRV2 used in example 2.

Some of the sequences disclosed herein contain an "X" or "N" in the nucleotide sequence. "X" or "N" may be a deletion or insertion of any nucleotide or one or more nucleotides. For example, in some cases, a string of "X" or "N" is displayed. The number of "X" or "N" does not necessarily correlate with the actual number of nucleotides at that position. More or fewer nucleotides may be present in the sequence than indicated by "X" or "N".

Detailed description of the invention

The present inventors have shown for the first time that alkaloid and/or TSNA content of a plant (or processed plant) can be modulated by modulating the activity or expression of at least one gene encoding a SOUL heme-binding protein in the plant (e.g., a tobacco plant).

The present invention provides a method of modulating (e.g., reducing) alkaloid content in a plant or part thereof, said method comprising modifying said plant by modulating (e.g., reducing) the activity or expression of at least one gene encoding a SOUL heme-binding protein.

Also provided are methods of modulating (e.g., reducing) the content of Tobacco Specific Nitrosamines (TSNAs) or precursors of TSNAs in a tobacco plant or plant part thereof, comprising modifying the plant by modulating (e.g., reducing) the activity or expression of at least one gene encoding a SOUL heme-binding protein.

The at least one gene encoding a SOUL heme-binding protein may be selected from at least one gene encoding a SOUL heme-binding protein comprising an amino acid sequence as set forth in SEQ ID number 1, 4, 7, 10, 13, 16, 19, or 22, or a functional variant or functional fragment or ortholog thereof, or a sequence having at least 80% identity to SEQ ID number 1, 4, 7, 10, 13, 16, 19, or 22; or wherein the at least one gene encoding a SOUL heme-binding protein comprises a nucleotide sequence set forth in SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, or 24, or a functional variant or fragment or ortholog of SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, or 24, or a nucleic acid sequence having at least 80% identity to SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, or 24.

Suitably, the SOUL heme-binding protein may comprise a SOUL motif.

Suitably, more than one SOUL heme-binding protein may be modified. In one embodiment, at least one SOUL heme-binding protein gene is modified selected from the group consisting of a gene encoding a polypeptide comprising an amino acid sequence set forth in SEQ ID number 1, 4, 7, 10, 13, 16, 19, or 22, or a functional variant or functional fragment or ortholog thereof, or a sequence having at least 80% identity to SEQ ID number 1, 4, 7, 10, 13, 16, 19, or 22; or wherein the at least one gene encoding a SOUL heme-binding protein comprises a nucleotide sequence set forth in SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, or 24, or a functional variant or fragment or ortholog of SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, or 24, or a nucleic acid sequence having at least 80% identity to SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, or 24.

In one embodiment, at least two SOUL heme-binding protein genes are modified selected from the group consisting of a gene encoding a polypeptide comprising an amino acid sequence set forth in SEQ ID number 1, 4, 7, 10, 13, 16, 19, or 22, or a functional variant or functional fragment or ortholog thereof, or a sequence having at least 80% identity to SEQ ID number 1, 4, 7, 10, 13, 16, 19, or 22; or wherein the at least one gene encoding a SOUL heme-binding protein comprises a nucleotide sequence set forth in SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, or 24, or a functional variant or fragment or ortholog of SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, or 24, or a nucleic acid sequence having at least 80% identity to SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, or 24.

In one embodiment, at least three SOUL heme-binding protein genes are modified selected from the group consisting of a gene encoding a polypeptide comprising an amino acid sequence set forth in SEQ ID number 1, 4, 7, 10, 13, 16, 19, or 22, or a functional variant or functional fragment or ortholog thereof, or a sequence having at least 80% identity to SEQ ID number 1, 4, 7, 10, 13, 16, 19, or 22; or wherein the at least one gene encoding a SOUL heme-binding protein comprises a nucleotide sequence set forth in SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, or 24, or a functional variant or fragment or ortholog of SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, or 24, or a nucleic acid sequence having at least 80% identity to SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, or 24.

In one embodiment, at least four SOUL heme-binding protein genes are modified selected from the group consisting of a gene encoding a polypeptide comprising an amino acid sequence set forth in SEQ ID number 1, 4, 7, 10, 13, 16, 19, or 22, or a functional variant or functional fragment or ortholog thereof, or a sequence having at least 80% identity to SEQ ID number 1, 4, 7, 10, 13, 16, 19, or 22; or wherein the at least one gene encoding a SOUL heme-binding protein comprises a nucleotide sequence set forth in SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, or 24, or a functional variant or fragment or ortholog of SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, or 24, or a nucleic acid sequence having at least 80% identity to SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, or 24.

In one embodiment, at least five of the SOUL heme-binding protein genes are modified selected from the group consisting of a gene encoding a polypeptide comprising an amino acid sequence set forth in SEQ ID number 1, 4, 7, 10, 13, 16, 19, or 22, or a functional variant or functional fragment or ortholog thereof, or a sequence having at least 80% identity to SEQ ID number 1, 4, 7, 10, 13, 16, 19, or 22; or wherein the at least one gene encoding a SOUL heme-binding protein comprises a nucleotide sequence set forth in SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, or 24, or a functional variant or fragment or ortholog of SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, or 24, or a nucleic acid sequence having at least 80% identity to SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, or 24.

In one embodiment, at least six SOUL heme-binding protein genes are modified selected from the group consisting of a gene encoding a polypeptide comprising an amino acid sequence set forth in SEQ ID number 1, 4, 7, 10, 13, 16, 19, or 22, or a functional variant or functional fragment or ortholog thereof, or a sequence having at least 80% identity to SEQ ID number 1, 4, 7, 10, 13, 16, 19, or 22; or wherein the at least one gene encoding a SOUL heme-binding protein comprises a nucleotide sequence set forth in SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, or 24, or a functional variant or fragment or ortholog of SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, or 24, or a nucleic acid sequence having at least 80% identity to SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, or 24.

In one embodiment, at least seven of the SOUL heme-binding protein genes are modified selected from the group consisting of a gene encoding a polypeptide comprising an amino acid sequence set forth in SEQ ID number 1, 4, 7, 10, 13, 16, 19, or 22, or a functional variant or functional fragment or ortholog thereof, or a sequence having at least 80% identity to SEQ ID number 1, 4, 7, 10, 13, 16, 19, or 22; or wherein the at least one gene encoding a SOUL heme-binding protein comprises a nucleotide sequence set forth in SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, or 24, or a functional variant or fragment or ortholog of SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, or 24, or a nucleic acid sequence having at least 80% identity to SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, or 24.

In one embodiment, at least eight SOUL heme-binding protein genes are modified selected from the group consisting of a gene encoding a polypeptide comprising an amino acid sequence set forth in SEQ ID number 1, 4, 7, 10, 13, 16, 19, or 22, or a functional variant or functional fragment or ortholog thereof, or a sequence having at least 80% identity to SEQ ID number 1, 4, 7, 10, 13, 16, 19, or 22; or wherein the at least one gene encoding a SOUL heme-binding protein comprises a nucleotide sequence set forth in SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, or 24, or a functional variant or fragment or ortholog of SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, or 24, or a nucleic acid sequence having at least 80% identity to SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, or 24.

Suitably, a SOUL heme-binding protein of the invention may comprise a SOUL motif.

In one aspect, the at least one SOUL heme-binding protein gene encodes a polypeptide comprising an amino acid sequence as set forth in SEQ ID number 1, or a functional variant or functional fragment or ortholog thereof, or a sequence having at least 80% identity to SEQ ID number 1; or wherein the at least one gene encoding a SOUL heme-binding protein comprises a nucleotide sequence as set forth in SEQ ID number 2 or 3, or a functional variant or functional fragment or ortholog of SEQ ID number 2 or 3, or a nucleic acid sequence having at least 80% identity to SEQ ID number 2 or 3.

Suitably, the SOUL heme-binding protein may comprise a SOUL motif.

In one aspect, the activity or expression of at least one further gene is modulated. Suitably, at least two (or at least three or at least four or at least five or at least six or at least seven or at least eight or at least nine) further genes selected from SEQ ID number 2,3, 5, 6, 8, 14, 15, 17, 18, 20, 21, 23 or 24, or functional variants or functional fragments or orthologs of SEQ ID number 2,3, 5, 6, 8, 9, 12, 15, 17, 18, 20, 21, 23 or 24, or nucleic acid sequences having at least 80% identity to SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23 or 24, or further genes of the nucleic acid sequences having at least 80% identity to SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23 or 24, may also be modulated. Suitably, the SOUL heme-binding protein may comprise a SOUL motif.

The "activity" of a gene encoding a SOUL heme-binding protein may refer to the ability of the SOUL heme-binding protein produced by the gene to bind other molecules, such as other proteins. Suitably, the "activity" of a gene encoding a SOUL heme-binding protein refers to the ability of the SOUL heme-binding protein produced by the gene to bind heme.

"expression" of a gene encoding a SOUL heme-binding protein may refer to the level of transcription, translation, i.e., protein expression.

Measurement of the level or amount of the gene product can be performed by any suitable method, such as comparison of mRNA transcript levels, protein or peptide levels, and/or plant phenotype between a modified plant and a comparable plant that has not been modified according to the present invention.

The term "comparable product" as defined herein will be a product derived from a plant (e.g. a tobacco plant) which has not been modified according to the present invention, but wherein all other relevant features are the same (e.g. plant species, growth conditions, methods of processing plants such as tobacco, etc.). Comparable products according to the invention may mean a plant (e.g. a tobacco plant) or a part thereof, such as a leaf (e.g. a tobacco leaf), a harvested leaf (e.g. a harvested tobacco leaf), a cut harvested leaf (e.g. a cut harvested tobacco leaf), a processed leaf (e.g. a processed tobacco leaf), or a plant propagation material (e.g. a tobacco plant propagation material), or a product comprising said plant or a part thereof, e.g. a tobacco product or a combination thereof, which may be obtained or obtained from a plant which has not been modified according to the invention, e.g. to modulate the activity or expression of a gene encoding a SOUL heme-binding protein. In one embodiment, a comparable product is a product that does not comprise a gene encoding a SOUL heme-binding protein whose activity or expression has been modulated.

The term "modified" or "modified" as used herein means a plant (e.g., a tobacco plant) or nucleic acid sequence that has been altered or changed. The present invention encompasses plant modification using techniques for genetic modification of plants or non-genetic modification of plants. Such methods are well known in the art, and examples of genetic modification techniques include transformation, transgenics, homologous transgenics (cisgenetics), and gene editing methods. Examples of non-genetic modification techniques include fast neutron mutagenesis, chemical mutagenesis such as Ethyl Methanesulfonate (EMS) mutagenesis, and modern population analysis methods.

The term "unmodified plant" as defined herein shall be a plant (e.g. a tobacco plant) which has not been modified according to the present invention, e.g. to modulate the activity or expression of a gene encoding a SOUL heme-binding protein or to modify the nucleic acid sequence of at least one gene encoding a SOUL heme-binding protein; and wherein all other relevant characteristics are the same (e.g., plant species, growth conditions, methods of processing tobacco, etc.). In one embodiment, the unmodified plant is a plant that does not comprise a gene encoding a SOUL heme-binding protein whose activity or expression has been modulated. In one embodiment, an unmodified plant is a plant that does not comprise a modified nucleic acid sequence encoding at least one gene encoding a SOUL heme-binding protein.

SOUL heme-binding proteins

As used herein, "SOUL heme-binding protein" has its usual meaning in the art and refers to a protein that comprises a SOUL motif, which is a hydrophobic cleft flanked by an alpha-helix and a beta-loop.

SOUL heme-binding proteins are present in bacteria, plants, and animals. SOUL heme-binding proteins bind tetrapyrroles such as heme (Takahashi et al (2008) Photochem. Photobiol. Sci. 7, 1216-1224). It has been suggested that the SOUL proteins in plants may be involved in the transport of heme to other heme-containing proteins such as cytochrome P450 and in the binding of free heme (Freere et al (2009) Acta Crystal. F65, 723-.

The three-dimensional structure of the mouse-derived SOUL heme-binding protein has been determined. The murine SOUL structure consists of nine twisted β -barrels, two α -helices and one hydrophobic cleft. The hydrophobic cleft is flanked by one of the alpha helices and the beta 8-9 loop, forming a conserved "SOUL motif" which is critical for heme-binding (Dias et al (2006) j. biol. chem. 276, 18161-. Similar structures were predicted for Arabidopsis thaliana SOUL heme-binding proteins (Takahashi et al (2008) Photochem. Photobiol. Sci. 7, 1216-1224).

In one embodiment, the SOUL heme-binding protein comprises a SOUL motif. As used herein, the term "SOUL motif refers to a conserved structural motif. The SOUL heme-binding protein and the SOUL motif can be identified by comparing predicted protein structures to known protein structures. For example, a SOUL heme-binding protein and a SOUL motif can be identified by comparing predicted protein structures against the predicted structure of SEQ ID No.1, wherein the presence of a hydrophobic cleft flanked by an alpha-helix and a beta-loop identifies the protein as a SOUL heme-binding protein.

In one embodiment, the SOUL motif is a region of the protein corresponding to a hydrophobic cleft flanked by an alpha-helix and a beta-loop. The SOUL motif may be involved in heme-binding.

Without wishing to be bound by theory, it is hypothesized that increasing the content of SOUL heme-binding protein in plant cells or increasing the activity of SOUL heme-binding proteins in plants, such as heme-binding activity, results in increased binding of free heme. A decrease in free heme levels would mean a decrease in heme degradation by heme oxygenase, resulting in a decrease in the clearance of Reactive Oxygen Species (ROS). ROS stimulate the activation of nicotine biosynthesis genes. Increased activity or expression of the SOUL heme-binding protein thereby results in increased levels of pyridine alkaloids. Conversely, according to this hypothesis, a decrease in the level or activity of the SOUL heme-binding protein (such as heme-binding activity) in a plant cell increases the amount of free heme, resulting in increased ROS clearance and thus decreased activation of nicotine biosynthesis genes, thereby decreasing the level of pyridine alkaloids in the cell. This hypothetical illustration is depicted in fig. 1.

In one embodiment, the SOUL heme-binding protein comprises the amino acid sequence set forth as SEQ ID number 1 or a sequence having at least 80% identity thereto, or a homologue thereof. Suitably, the SOUL heme-binding protein may comprise a SOUL motif. Suitably, homologues of SEQ ID number 1 may be selected from: SEQ ID number 4, 7, 10, 13, 16, 19 or 22, or a sequence having at least 80% identity thereto. Suitably, homologues of SEQ ID number 1 may be selected from: SEQ ID No.4, 7, 10, 13, 16, 19 or 22, wherein said sequence comprises a SOUL motif or a sequence having at least 80% identity to SEQ ID No.4, 7, 10, 13, 16, 19 or 22 and comprising a SOUL motif.

In one embodiment, the SOUL heme-binding protein comprises an amino acid sequence as set forth in SEQ ID number 1, 4, 7, 10, 13, 16, 19, or 22, or a sequence at least 80% identical thereto (preferably at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical thereto). In one embodiment, a SOUL heme-binding protein comprises an amino acid sequence as set forth in SEQ ID number 1, 4, 7, 10, 13, 16, 19, or 22, or a sequence at least 80% identical thereto (preferably at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical thereto) and comprising a SOUL motif.

Suitably, a SOUL heme-binding protein according to the invention may comprise an amino acid sequence as set forth in SEQ ID number 1, or a sequence having at least 80% identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 97%, or at least 99% identity thereto). Suitably, the SOUL heme-binding protein may comprise a SOUL motif.

Suitably, a SOUL heme-binding protein according to the invention may comprise an amino acid sequence as shown in SEQ ID number 4, or a sequence having at least 80% identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 97% or at least 99% identity thereto). Suitably, the SOUL heme-binding protein may comprise a SOUL motif.

Suitably, a SOUL heme-binding protein according to the invention may comprise an amino acid sequence as shown in SEQ ID number 7, or a sequence having at least 80% identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 97% or at least 99% identity thereto). Suitably, the SOUL heme-binding protein may comprise a SOUL motif.

Suitably, a SOUL heme-binding protein according to the invention may comprise the amino acid sequence as shown in SEQ ID number 10, or a sequence having at least 80% identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 97% or at least 99% identity thereto). Suitably, the SOUL heme-binding protein may comprise a SOUL motif.

Suitably, a SOUL heme-binding protein according to the invention may comprise the amino acid sequence as shown in SEQ ID number 13, or a sequence having at least 80% identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 97% or at least 99% identity thereto). Suitably, the SOUL heme-binding protein may comprise a SOUL motif.

Suitably, a SOUL heme-binding protein according to the invention may comprise an amino acid sequence as shown in SEQ ID number 16, or a sequence having at least 80% identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 97% or at least 99% identity thereto). Suitably, the SOUL heme-binding protein may comprise a SOUL motif.

Suitably, a SOUL heme-binding protein according to the invention may comprise the amino acid sequence as shown in SEQ ID number 19, or a sequence having at least 80% identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 97% or at least 99% identity thereto). Suitably, the SOUL heme-binding protein may comprise a SOUL motif.

Suitably, a SOUL heme-binding protein according to the invention may comprise the amino acid sequence as shown in SEQ ID number 22, or a sequence having at least 80% identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 97% or at least 99% identity thereto). Suitably, the SOUL heme-binding protein may comprise a SOUL motif.

In one embodiment, the SOUL heme-binding protein according to the invention comprises or consists of an amino acid sequence selected from the group consisting of: SEQ ID number 1, 4, 7, 10, 13, 16, 19 or 22.

Suitably, the protein may be from tobacco.

In one embodiment, the SOUL heme-binding protein is encoded by a polynucleotide sequence, wherein a gene (prior to mutation) comprises a sequence as set forth in SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, or 24; or a sequence having at least 80% sequence identity thereto. Suitably, the SOUL heme-binding protein may comprise a SOUL motif.

Suitably, the SOUL heme-binding protein for use according to the invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises a sequence as shown in SEQ ID number 2, or a sequence having at least 80% sequence identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 97% or at least 99% identity thereto). Suitably, the SOUL heme-binding protein may comprise a SOUL motif.

Suitably, the SOUL heme-binding protein for use according to the invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises a sequence as shown in SEQ ID number 3, or a sequence having at least 80% sequence identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 97% or at least 99% identity thereto). Suitably, the SOUL heme-binding protein may comprise a SOUL motif.

Suitably, the SOUL heme-binding protein for use according to the invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises a sequence as shown in SEQ ID number 5, or a sequence having at least 80% sequence identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 97% or at least 99% identity thereto). Suitably, the SOUL heme-binding protein may comprise a SOUL motif.

Suitably, the SOUL heme-binding protein for use according to the invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises a sequence as shown in SEQ ID number 6, or a sequence having at least 80% sequence identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 97% or at least 99% identity thereto). Suitably, the SOUL heme-binding protein may comprise a SOUL motif.

Suitably, the SOUL heme-binding protein for use according to the invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises a sequence as shown in SEQ ID number 8, or a sequence having at least 80% sequence identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 97% or at least 99% identity thereto). Suitably, the SOUL heme-binding protein may comprise a SOUL motif.

Suitably, the SOUL heme-binding protein for use according to the invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises a sequence as shown in SEQ ID number 9, or a sequence having at least 80% sequence identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 97% or at least 99% identity thereto). Suitably, the SOUL heme-binding protein may comprise a SOUL motif.

Suitably, the SOUL heme-binding protein for use according to the invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises the sequence as shown in SEQ ID number 11, or a sequence having at least 80% sequence identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 97% or at least 99% identity thereto). Suitably, the SOUL heme-binding protein may comprise a SOUL motif.

Suitably, the SOUL heme-binding protein for use according to the invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises a sequence as shown in SEQ ID number 12, or a sequence having at least 80% sequence identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 97% or at least 99% identity thereto). Suitably, the SOUL heme-binding protein may comprise a SOUL motif.

Suitably, the SOUL heme-binding protein for use according to the invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises a sequence as shown in SEQ ID number 14, or a sequence having at least 80% sequence identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 97% or at least 99% identity thereto). Suitably, the SOUL heme-binding protein may comprise a SOUL motif.

Suitably, the SOUL heme-binding protein for use according to the invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises a sequence as shown in SEQ ID number 15, or a sequence having at least 80% sequence identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 97% or at least 99% identity thereto). Suitably, the SOUL heme-binding protein may comprise a SOUL motif.

Suitably, the SOUL heme-binding protein for use according to the invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises a sequence as shown in SEQ ID number 17, or a sequence having at least 80% sequence identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 97% or at least 99% identity thereto). Suitably, the SOUL heme-binding protein may comprise a SOUL motif.

Suitably, the SOUL heme-binding protein for use according to the invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises a sequence as shown in SEQ ID number 18, or a sequence having at least 80% sequence identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 97% or at least 99% identity thereto). Suitably, the SOUL heme-binding protein may comprise a SOUL motif.

Suitably, the SOUL heme-binding protein for use according to the invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises a sequence as shown in SEQ ID number 20, or a sequence having at least 80% sequence identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 97% or at least 99% identity thereto). Suitably, the SOUL heme-binding protein may comprise a SOUL motif.

Suitably, the SOUL heme-binding protein for use according to the invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises a sequence as shown in SEQ ID number 21, or a sequence having at least 80% sequence identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 97% or at least 99% identity thereto). Suitably, the SOUL heme-binding protein may comprise a SOUL motif.

Suitably, the SOUL heme-binding protein for use according to the invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises a sequence as shown in SEQ ID number 23, or a sequence having at least 80% sequence identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 97% or at least 99% identity thereto). Suitably, the SOUL heme-binding protein may comprise a SOUL motif.

Suitably, the SOUL heme-binding protein for use according to the invention may be encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises a sequence as shown in SEQ ID number 24, or a sequence having at least 80% sequence identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 97% or at least 99% identity thereto). Suitably, the SOUL heme-binding protein may comprise a SOUL motif.

In one embodiment, the SOUL heme-binding protein is encoded by a polynucleotide sequence, wherein the gene (prior to mutation) is selected from the group consisting of: SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23 or 24.

Suitably, the protein for use according to the invention may be encoded by a polynucleotide sequence from tobacco.

In one aspect, the present invention provides a method of reducing the alkaloid content of a plant or part or cell thereof (e.g., a plant cell), said method comprising modifying said plant by reducing or inhibiting the activity or expression of at least one gene encoding a SOUL heme-binding protein.

In one aspect, the invention provides a method of reducing alkaloid content in a plant or part or plant cell thereof, said method comprising modifying said plant by reducing or inhibiting the activity or expression of at least one gene encoding a SOUL heme-binding protein comprising the amino acid sequence set forth as SEQ ID number 1, 4, 7, 10, 13, 16, 19 or 22 or a sequence having at least 80% identity thereto, or wherein said at least one gene encoding a SOUL heme-binding protein comprises the nucleotide sequence set forth as SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23 or 24, or a functional variant or functional fragment or ortholog of SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23 or 24, Or a nucleic acid sequence having at least 80% identity to SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23 or 24. Suitably, the SOUL heme-binding protein may comprise a SOUL motif.

In one aspect, the present invention provides a method of reducing the content of Tobacco Specific Nitrosamines (TSNAs) or TSNA precursors in a plant or part thereof (e.g. leaf), comprising modifying said plant by reducing or inhibiting the activity or expression of at least one gene encoding a SOUL heme-binding protein.

In one aspect, the invention provides a method of reducing the content of Tobacco Specific Nitrosamines (TSNAs) or TSNA precursors in a plant or part thereof (e.g. leaf), the method comprising modifying said plant by reducing or inhibiting the activity or expression of at least one gene encoding a SOUL heme-binding protein comprising the amino acid sequence shown as SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23 or 24 or a sequence having at least 80% identity thereto, or wherein said at least one gene encoding a SOUL heme-binding protein comprises the nucleotide sequence shown as SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23 or 24, or SEQ ID number 2, 2, 3. 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23 or 24 or a functional variant or functional fragment or ortholog thereof, or a nucleic acid sequence having at least 80% identity to SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23 or 24.

The term "reduce" or "inhibit" (e.g., inhibiting the activity or expression of a gene encoding a SOUL heme-binding repeat protein) as used herein means that the activity or expression of the gene encoding the SOUL heme-binding repeat protein is lower or reduced as compared to the activity or expression of the gene in a comparable product.

In one aspect, the present invention provides a method of increasing the alkaloid content of a plant or part or cell thereof (e.g., a plant cell), said method comprising modifying said plant by increasing or enhancing the activity or expression of at least one gene encoding a SOUL heme-binding protein.

In one aspect, the invention provides a method of increasing the alkaloid content of a plant or part or plant cell thereof, said method comprising modifying said plant by increasing or enhancing the activity or expression of at least one gene encoding a SOUL heme-binding protein comprising the amino acid sequence as set forth in SEQ ID number 1, 4, 7, 10, 13, 16, 19 or 22 or a sequence having at least 80% identity thereto, or wherein said at least one gene encoding a SOUL heme-binding protein comprises the nucleotide sequence as set forth in SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23 or 24, or a functional variant or functional fragment or ortholog of SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23 or 24, Or a nucleic acid sequence having at least 80% identity to SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23 or 24. Suitably, the SOUL heme-binding protein may comprise a SOUL motif.

In one aspect, the present invention provides a method of increasing the content of Tobacco Specific Nitrosamines (TSNAs) or TSNA precursors in a plant or part thereof (e.g. leaf), said method comprising modifying said plant by increasing or enhancing the activity or expression of at least one gene encoding a SOUL heme-binding protein.

In one aspect, the present invention provides a method of increasing the content of Tobacco Specific Nitrosamines (TSNAs) or TSNA precursors in a plant or part thereof (e.g. leaves), the method comprising modifying said plant by increasing or enhancing the activity or expression of at least one gene encoding a SOUL heme-binding protein comprising the amino acid sequence as set forth in SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23 or 24 or a sequence having at least 80% identity thereto, or wherein said at least one gene encoding a SOUL heme-binding protein comprises the nucleotide sequence as set forth in SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23 or 24, or SEQ ID number 2, 2, 3. 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23 or 24 or a functional variant or functional fragment or ortholog thereof, or a nucleic acid sequence having at least 80% identity to SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23 or 24.

The term "increase" or "enhancing" (e.g., increasing the activity or expression of a gene encoding a SOUL heme-binding repeat protein) as used herein means that the activity or expression of the gene encoding the SOUL heme-binding repeat protein is higher or increased as compared to the activity or expression of the gene in a comparable product.

According to the present invention, the activity or expression of a gene encoding a SOUL heme-binding protein is modulated.

In one aspect, the present invention provides a method of modulating (i.e., increasing or decreasing) the alkaloid content of a plant or part or cell thereof (e.g., a plant cell), said method comprising modifying said plant by modulating (i.e., increasing or decreasing) the activity of at least one gene encoding a SOUL heme-binding protein.

The term "activity" refers to any function of the SOUL heme-binding protein encoded by at least one gene. Examples of activity include enzymatic activity or localization of the SOUL heme-binding protein. Suitably, the activity is the ability of the SOUL heme-binding protein to interact with another molecule or molecules. In some embodiments, the present invention provides methods of modulating (i.e., increasing or decreasing) the alkaloid content of a plant or part or cell thereof (e.g., a plant cell), comprising modifying said plant by modulating (i.e., increasing or decreasing) the ability of a SOUL heme-binding protein to interact with another molecule.

Suitably, the ability of the SOUL heme-binding protein to interact with other molecules is the ability to bind other molecules. The other molecule may be a protein. Other molecules may be tetrapyrroles. The other molecule may be heme. In some embodiments, the present invention provides methods of modulating (i.e., increasing or decreasing) the alkaloid content of a plant or part or cell thereof (e.g., a plant cell), comprising modifying said plant by modulating (i.e., increasing or decreasing) the ability of a SOUL heme-binding protein to bind heme.

Suitably, the other molecule is more than one molecule, such as one or more molecules, such as two or more molecules, such as three or more molecules. Where the other molecule is more than one molecule, the other molecules may be the same molecule or may be different molecules. Where the other molecule is more than one molecule, at least one of the other molecules may be heme.

Modulation of the activity of a gene encoding a SOUL heme-binding protein may require an increase or decrease in the activity of the SOUL heme-binding protein.

Increasing the activity of a SOUL heme-binding protein refers to enhancing or improving the ability of a SOUL heme-binding protein to perform a specific function as compared to a SOUL heme-binding protein in a plant that has not been modified according to the present invention.

Reducing the activity of a SOUL heme-binding protein refers to reducing, inhibiting, or disrupting the ability of a SOUL heme-binding protein to perform a particular function as compared to a SOUL heme-binding protein in a plant that has not been modified according to the present invention. The activity of the SOUL heme-binding protein may be reduced to such an extent that the activity is prevented or eliminated.

In some embodiments, the activity of a SOUL heme-binding protein may be modulated (i.e., increased or decreased) by at least about 10%, 20%, 30%, or 40%, suitably at least about 50%, 60%, 70%, more suitably at least about 80%, 90%, 95%, or 100% as compared to the activity of a gene encoding a SOUL heme-binding protein in a plant (e.g., a tobacco plant) that is not modified according to the present invention.

In some embodiments, a modulated SOUL heme-binding protein exhibits increased or decreased activity as compared to an unmodified SOUL heme-binding protein. A modulated SOUL heme-binding protein may exhibit at least about 1%, at least about 3%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% increased or decreased activity as compared to an unmodified SOUL heme-binding protein.

Suitably, the modulation of activity is an increase or decrease in the ability of the SOUL heme-binding protein to interact with (such as bind to) another molecule or molecules (such as heme).

In some embodiments, the present invention provides methods of modulating the alkaloid content of a plant or part or cell thereof, comprising modifying said plant by modulating the ability of a SOUL heme-binding protein to bind heme. Suitably, the ability of the SOUL heme-binding protein to bind heme is increased. Suitably, the ability of the SOUL heme-binding protein to bind heme is reduced. An increase or decrease in the ability of a SOUL heme-binding protein to bind another molecule can be expressed as an increase or decrease in the binding affinity of the SOUL heme-binding protein. In some embodiments, the present invention provides methods of modulating the alkaloid content of a plant or part or cell thereof, comprising modifying said plant by modulating the binding affinity of a SOUL heme-binding protein for heme.

Techniques for measuring protein activity are known in the art. For example, assays for measuring enzymatic activity of proteins are known, and microscopic techniques can be used to identify the localization of proteins.

In particular, the ability of a SOUL heme-binding protein to bind another molecule can be measured using techniques known in the art. Examples of such techniques include immunoprecipitation, isothermal calorimetry, surface plasmon resonance, and microlayer thermophoresis. For example, the ability of a modulated or mutated SOUL heme-binding protein to bind to other molecules can be determined by co-immunoprecipitation experiments using a modulated or mutated SOUL heme-binding protein and a corresponding unmodified or unmutated SOUL heme-binding protein. If modulation or mutation of a SOUL heme-binding protein reduces, inhibits, or eliminates the ability of the SOUL heme-binding protein to bind other molecules, then the co-immunoprecipitation will show that the modulated or mutated SOUL heme-binding protein binds less of the other molecules.

According to the present invention, the activity or expression of a SOUL heme-binding protein is modulated.

In one aspect, the present invention provides a method of modulating (i.e., increasing or decreasing) the alkaloid content of a plant or part or cell thereof (e.g., a plant cell), said method comprising modifying said plant by modulating (i.e., increasing or decreasing) the expression of at least one gene encoding a SOUL heme-binding protein.

"expression" of a gene refers to the degree to which the information encoded in the gene is converted into function. The expression level of a gene may be equivalent to the amount of the product of the gene present in the cell or organism. A modification that modulates (i.e., increases or decreases) gene expression is a modification that increases the amount of the gene product in a plant or cell as compared to an unmodified plant or cell.

In some embodiments, expression of a SOUL heme-binding protein is modulated (i.e., increased or decreased) as compared to the expression of a gene encoding a SOUL heme-binding protein in a plant that is not modified according to the present invention (e.g., a tobacco plant).

In some embodiments, the expression of a SOUL heme-binding protein may be modulated (i.e., increased or decreased) by at least about 10%, 20%, 30%, or 40%, suitably at least about 50%, 60%, 70%, more suitably at least about 80%, 90%, 95%, or 100% as compared to the expression of a gene encoding a SOUL heme-binding protein in a plant (e.g., a tobacco plant) that is not modified according to the present invention.

In some embodiments, a modulated SOUL heme-binding protein exhibits increased or decreased expression compared to an unmodified SOUL heme-binding protein. A modulated SOUL heme-binding protein may exhibit at least about 1%, at least about 3%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% increased or decreased expression as compared to an unmodified SOUL heme-binding protein.

Typically, a gene is transcribed into mRNA, which is translated into a protein, the final gene product. Proteins can be sequestered in cellular storage and/or degraded. The expression of a gene can be modulated by modulating any or all of these steps. Thus, in some embodiments, the modification modulates the expression of at least one SOUL heme-binding protein gene in one of the following ways:

modulating transcription from at least one SOUL heme-binding protein gene;

modulating translation of mRNA from at least one SOUL heme-binding protein gene;

modulating release of SOUL heme-binding protein from intracellular storage; and/or

Modulating the degradation rate of the SOUL heme-binding protein.

Expression of a particular gene encoding a SOUL heme-binding protein can be measured by measuring transcription and/or translation of the gene. Methods for measuring transcription are well known in the art and include, inter alia, northern blotting, RNA-Seq, in situ hybridization, DNA microarrays, and RT-PCR. Alternatively, expression of a gene can be measured indirectly by measuring the level of a gene product, such as a protein encoded by the gene. For example, expression of a SOUL heme-binding protein can be determined by measuring the presence of the protein by western blot using an antibody specific for a SOUL heme-binding protein (e.g., an antibody specific for a SOUL motif).

Decoration

The term "modification" as used herein refers to an alteration of the genetic material of a plant or cell. The plant or cell may be modified in any manner that modulates the activity or expression of at least one gene encoding a SOUL heme-binding protein. The types of modifications to plants and cells that modulate the activity or expression of genes, and the techniques to achieve these modifications, are known in the art.

In some embodiments, the present invention provides methods of reducing the alkaloid content of a plant or a part or cell thereof (e.g., a plant cell), comprising modifying said plant by reducing or inhibiting the activity or expression of at least one gene encoding a SOUL heme-binding protein.

In some embodiments, the present invention provides a method of reducing the content of Tobacco Specific Nitrosamines (TSNAs) or precursors of TSNAs in a tobacco plant or plant part thereof, the method comprising modifying said plant or cell culture by reducing the activity or expression of at least one gene encoding a SOUL heme-binding protein.

Any method known in the art for reducing or inhibiting the activity or expression of a gene may be used in the method according to the invention.

Suitably, the activity or expression of a SOUL heme-binding protein gene may be reduced, partially inactivated, inhibited, eliminated, knocked out, or lost such that the protein activity, expression, or function of the SOUL heme-binding protein gene is undetectable.

In one aspect, at least one of the SOUL heme-binding protein genes is knocked out. In other words, the SOUL heme-binding protein gene has been rendered completely ineffective.

For example, the method may comprise:

● providing a mutation in a nucleic acid sequence encoding a protein comprising an amino acid sequence as set forth in SEQ ID No.1, 4, 7, 10, 13, 16, 19 or 22, or an amino acid sequence having at least 80% sequence identity thereto;

● provides mutations in regulatory regions (e.g., promoters or enhancers) that contribute to the control of the expression of a protein comprising an amino acid sequence as set forth in SEQ ID No.1, 4, 7, 10, 13, 16, 19 or 22, or an amino acid sequence having at least 80% sequence identity thereto;

● provides an antisense RNA, siRNA or miRNA that reduces the level of a nucleic acid sequence encoding a protein comprising an amino acid sequence set forth as SEQ ID No.1, 4, 7, 10, 13, 16, 19 or 22, or an amino acid sequence having at least 80% sequence identity thereto.

Each of the above methods results in a reduction or prevention of the following activities or expressions: a protein comprising the amino acid sequence set forth as SEQ ID number 1, 4, 7, 10, 13, 16, 19 or 22, or an amino acid sequence having at least 80% sequence identity thereto, or wherein at least one of the genes encoding a SOUL heme-binding protein comprises the nucleotide sequence set forth as SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23 or 24, or a functional variant or functional fragment or ortholog of SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23 or 24, or a nucleic acid sequence having at least 80% identity to SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23 or 24.

As used herein, the term "mutation" includes natural genetic variants or engineered variants. In particular, the term "mutation" refers to a variation in the nucleotide sequence or amino acid sequence encoding an amino acid sequence compared to the sequence as set forth in SEQ ID number 1, 4, 7, 10, 13, 16, 19 or 22, or to an amino acid sequence having at least 80% (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, preferably at least 98%, preferably at least 99%) sequence identity thereto.

In one embodiment, the mutation reduces the alkaloid content of the plant. In another embodiment, the mutation reduces the level of at least one TSNA or TSNA precursor in tobacco.

In one embodiment, a method according to the present invention may comprise providing a plant or a part thereof or a plant cell with a nucleic acid sequence, wherein said nucleic acid results in a reduction or elimination of the activity or expression of at least one gene encoding a SOUL heme-binding protein.

In one embodiment, the method according to the invention may comprise providing a plant or a part thereof or a plant cell with a nucleic acid sequence, wherein said nucleic acid results in a modification of the nucleic acid sequence of at least one gene encoding a SOUL heme-binding protein.

Suitably, the nucleic acid sequence may be introduced into a plant or part or cell thereof. Suitably, an endogenous nucleic acid sequence in a plant or part or cell thereof may be modified to encode a polypeptide according to the invention (e.g. by gene editing). For example, the endogenous nucleotide sequence may be modified to reduce the activity or expression of at least one gene encoding a SOUL heme-binding protein.

In a preferred embodiment, each copy of a nucleic acid sequence encoding a protein comprising the sequence shown as SEQ ID number 1, 4, 7, 10, 13, 16, 19 or 22 or a sequence having at least 80% sequence identity thereto or wherein the at least one gene encoding a SOUL heme-binding protein comprises the nucleotide sequence shown in SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23 or 24 or a functional variant or functional fragment or ortholog of SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23 or 24 or a nucleic acid sequence having at least 80% identity to SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23 or 24 (which is present in plants) is modified, for example, a mutation as defined herein (e.g., every genomic copy of the gene encoding the protein in the plant is mutated). For example, each copy of a gene in the tobacco's allotetraploid genome may be mutated.

In a preferred embodiment, some or all of the homologs of the SOUL heme-binding proteins described herein are modified, e.g., inhibited or mutated. Suitably, some or all of SEQ ID number 1, 4, 7, 10, 13, 16, 19 or 22 or the corresponding sequence having at least 80% sequence identity thereto is modified, e.g. inhibited or mutated.

In some embodiments, the plant or plant cell according to the invention is homozygous. Suitably, the plant or plant cell may be homozygous for a modification, such as inhibition or mutation.

In some embodiments, the plant or plant cell according to the invention expresses only modified, e.g. mutated, nucleic acids encoding a SOUL heme-binding protein. In other words, in some embodiments, no endogenous (or endogenous and functional protein) is present in the plant according to the invention. In other words, if any endogenous protein is present, it is preferably in an inactive form.

In one embodiment, the method may comprise providing a mutation in a nucleic acid sequence as set forth in SEQ ID No.2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23 or 24 or a nucleic acid sequence having at least 80% identity thereto.

The mutation may alter the plant genome such that a nucleic acid sequence encoding a protein comprising the amino acid sequence shown as SEQ ID No.1, 4, 7, 10, 13, 16, 19 or 22 or an amino acid sequence having at least 80% sequence identity thereto is deleted or otherwise modified, in whole or in part, to inhibit or eliminate the ability of the SOUL heme-binding protein to interact with other molecules (such as to bind other molecules) compared to a protein shown as SEQ ID No.1, 4, 7, 10, 13, 16, 19 or 22 or a sequence having at least 80% sequence identity thereto. In some embodiments, the mutation does not alter the level or expression of the protein, but reduces, inhibits or eliminates the ability of the SOUL heme-binding protein to interact with other molecules (such as to bind other molecules), as compared to a protein as set forth in SEQ ID nos. 1, 4, 7, 10, 13, 16, 19, or 22, or a sequence having at least 80% sequence identity thereto. Suitably, the mutation inhibits or abrogates the ability of the SOUL heme-binding protein to interact with other proteins. Suitably, the mutation inhibits or abrogates the ability of the SOUL heme-binding protein to bind to other molecules. Suitably, the mutation inhibits or abrogates the ability of the SOUL heme-binding protein to bind to other proteins. Suitably, the mutation inhibits or abrogates the ability of the SOUL heme-binding protein to bind tetrapyrroles or tetrapyrroles. Suitably, the mutation inhibits or abolishes the ability of the SOUL heme-binding protein to bind heme. The expression "inhibit or eliminate" means to reduce the interaction between the SOUL heme-binding protein and the other molecule, suitably to the extent that the interaction is prevented (i.e. the SOUL heme-binding protein does not interact at all with the other molecule). Suitably, inhibition or abolishing of binding of a SOUL heme-binding protein to another molecule means that the binding affinity of the SOUL heme-binding protein to the other molecule is reduced. In some embodiments, the mutation reduces the binding affinity of the SOUL heme-binding protein for heme.

Suitably, the mutation may be in a domain of the SOUL heme-binding protein that mediates interaction with other molecules. The mutation may be in the domain of the SOUL heme-binding protein that mediates binding to heme. The mutation may be in the SOUL motif of the SOUL heme-binding protein. In some embodiments, the SOUL motif can be mutated, thereby altering the ability of the SOUL heme-binding protein to interact with other molecules. In other words, the SOUL motif can be mutated, resulting in an alteration of the binding affinity of the SOUL heme-binding protein for other molecules. Suitably, the SOUL motif is mutated such that the binding affinity of the SOUL heme-binding protein to other molecules is increased. Suitably, the SOUL motif is mutated such that the binding affinity of the SOUL heme-binding protein to other molecules is reduced.

The mutation may disrupt the nucleic acid sequence encoding the protein comprising an amino acid sequence as set forth in SEQ ID number 1, 4, 7, 10, 13, 16, 19, or 22, or an amino acid sequence having at least 80% sequence identity thereto.

The disruption may result in the nucleic acid sequence not being transcribed and/or translated.

The nucleic acid sequence may be interrupted, for example, by deleting or otherwise modifying the ATG start codon of the nucleic acid sequence, such that translation of the protein is reduced or prevented.

The nucleic acid sequence may comprise one or more nucleotide changes that reduce or prevent protein expression or affect protein trafficking. For example, expression of a protein may be reduced or prevented by introducing one or more premature stop codons, frame shifts, splicing mutations, or non-tolerant amino acid substitutions in the open reading frame.

A premature stop codon refers to a mutation that introduces a stop codon into the open reading frame and prevents translation of the entire amino acid sequence. The premature stop codon can be a TAG ("amber"), TAA ("ochre") or TGA ("opal" or "umber") codon.

Frame-shifting mutations (also known as reading frame errors (frameshift) or reading frame shifts) are mutations that result from insertions/deletions (insertions or deletions) of multiple nucleotides in a nucleic acid sequence that are not divisible by three. Due to the triplet nature of gene expression by codons, insertions or deletions can alter the reading frame, resulting in translation that is completely different from the original. Frame-shifting mutations will often result in post-mutation codon reads to encode different amino acids. Frame-shifting mutations will typically result in the introduction of a premature stop codon.

Splicing mutations insert, delete or alter many nucleotides at specific sites where splicing occurs during processing of precursor messenger RNA into mature messenger RNA. Deletion of the splice site results in retention of one or more introns in the mature mRNA and may lead to production of abnormal proteins.

Non-tolerant amino acid substitutions refer to mutations that result in non-synonymous amino acid substitutions in a protein that result in the function of reduction or removal of the protein.

Any method known in the art for providing mutations in nucleic acid sequences may be used in the method according to the invention. For example, homologous recombination can be used, wherein a vector is generated in which one or more related nucleic acid sequences are mutated, and used to transform a plant or plant cell. Recombinant plants or plant cells expressing the mutated sequences can then be selected.

In one embodiment, the mutation introduces a non-tolerant amino acid substitution in a protein comprising the amino acid sequence set forth as SEQ ID number 1, 4, 7, 10, 13, 16, 19, or 22, or a sequence having at least 80% sequence identity thereto.

In some embodiments, the SOUL motif may contain a mutation that reduces the expression of at least one gene encoding a SOUL heme-binding protein.

The mutation may be a deletion, a splice mutant or a codon encoding a non-tolerant amino acid substitution.

In one embodiment, the nucleic acid sequence encoding the SOUL heme-binding protein may be deleted in whole or in part. The deletion may be contiguous or may comprise multiple segments of the sequence. Deletions preferably remove a sufficient amount of the nucleotide sequence such that the nucleic acid sequence no longer encodes a functional SOUL heme-binding protein. The deletion may be complete when compared to the corresponding genome of a comparable unmodified plant, in which case 100% of the coding part of the nucleic acid sequence is absent. Deletions may, for example, remove at least 50, 60, 70, 80, or 90% of the coding portion of the nucleic acid sequence. Suitably, at least a portion of the protein may be deleted. The deletion may, for example, remove at least 10, 20, 30, 40, 50, 60, 70, 80, or 90% of the coding portion of the protein.

Deletions may remove at least a portion of the SOUL motif.

Deletions may, for example, remove at least 10, 20, 30, 40, 50, 60, 70, 80, or 90% of the SOUL motif.

Suitably, a deletion can remove at least 5 amino acids, at least 10 amino acids, at least 15, at least 20, at least 25, at least 30 amino acids from the SOUL motif. Suitably, a deletion can remove at least 5 amino acids, at least 10 amino acids, at least 15, at least 20, at least 25, at least 30 amino acids from the SOUL motif.

In one embodiment, a deletion can remove at least 100 amino acids, at least 150, at least 200, at least 250, at least 300, at least 350 amino acids from the C-terminus of a SOUL heme-binding protein.

Suitably, the mutated protein may be a truncated protein lacking at least about 100 amino acids, at least about 150 amino acids, or a sequence having at least 80% (preferably at least 85%, at least 90%, at least 95%, at least 98%) sequence identity to a truncated protein lacking at least about 100 amino acids, at least about 150 amino acids, corresponding to the amino acid from the C-terminus of SEQ ID number 1.

Methods for deleting nucleic acid sequences in plants are known in the art. For example, homologous recombination can be used, wherein a vector is produced in which one or more related nucleic acid sequences are lost, and used to transform a plant or plant cell. Recombinant plants or plant cells expressing the new sequence portions can then be selected.

Plant cells transformed with the vectors as described herein can be grown and maintained according to well known tissue culture methods, such as by culturing the cells in a suitable medium supplied with essential growth factors such as amino acids, plant hormones, vitamins, and the like.

Modification of nucleic acid sequences can be performed using targeted mutagenesis methods (also known as Targeted Nucleotide Exchange (TNE) or oligonucleotide-directed mutagenesis (ODM)). Targeted mutagenesis methods include, but are not limited to, those employing zinc finger nucleases, TALENs (see WO2011/072246 and WO2010/079430), Cas 9-like, Cas9/crRNA/tracrRNA, Cas9/gRNA, or other CRISPR systems (see WO 2014/071006 and WO2014/093622), meganucleases (see WO2007/047859 and WO2009/059195), or targeted mutagenesis methods employing mutagenic oligonucleotides that may contain chemically modified nucleotides with sequence complementarity to the gene for enhanced mutagenesis (e.g., KeyBase @, or TALENs) that enter into plant protoplasts.

Alternatively, mutagenesis systems such as TILLING (Targeted Induced Local mutations IN Genomics); McCallum et al (2000) nat. Biotech. 18:455, and McCallum et al (2000) Plant Physiol. 123, 439-442, both incorporated herein by reference), can be used to generate Plant lines comprising genes encoding proteins having mutations. TILLING uses traditional chemical mutagenesis (e.g. Ethyl Methane Sulfonate (EMS) mutagenesis to generate random mutations) followed by high throughput screening of the mutations. Thus, plants, seeds, cells and tissues comprising the gene with the desired mutation can be obtained.

The method may comprise the steps of: mutagenized plant seeds (e.g., EMS mutagenesis), pooling of plant individuals or DNA, PCR amplification of target regions, heteroduplex formation and high throughput detection, identification of mutated plants, sequencing of mutated PCR products. It is understood that other mutagenesis and selection methods may be equally employed to generate such modified plants. The seeds may be, for example, irradiated or chemically treated, and the plants may be screened for the modified phenotype.

Fast neutron deletion mutagenesis can be used in a reverse genetics sense (i.e., using PCR) to identify plant lines that carry deletions in endogenous genes. See, e.g., Ohshima et al (1998) Virology 213: 472-; okubara et al (1994) Genetics 137: 867-; and Quesada et al (2000) Genetics 154: 421-.

In another approach, dominant mutants can be used to trigger RNA silencing due to gene inversion and recombination of repeated loci. See, e.g., Kusaba et al (2003) Plant Cell 15:1455-1467 (incorporated herein by reference).

Modified plants can be distinguished from unmodified plants, i.e. wild-type plants, by molecular methods such as one or more mutations present in the DNA, and by modified phenotypic characteristics. The modified plant may be homozygous or heterozygous for the modification. Preferably, the modified plant is homozygous for the modification.

In one embodiment, the method of reducing or preventing the activity or expression of a protein comprising an amino acid sequence as set forth in SEQ ID number 1, 4, 7, 10, 13, 16, 19, or 22, or an amino acid sequence having at least 80% sequence identity thereto, does not comprise treating the plant with a chemical (e.g., an agrochemical).

Other ways of reducing or preventing expression will be apparent to those skilled in the art and include the use of virus-Induced Gene Silencing (VIGs), microrna silencing, RNAi, antisense, tDNA insertion, or dominant negative constructs (or antisense allelic mutations).

In one embodiment, expression of a protein comprising an amino acid sequence as set forth in SEQ ID number 1, 4, 7, 10, 13, 16, 19, or 22, or an amino acid sequence having at least 80% sequence identity thereto, can be reduced or eliminated by virus-induced gene silencing.

In one embodiment, expression of a protein comprising an amino acid sequence as set forth in SEQ ID number 1, 4, 7, 10, 13, 16, 19 or 22, or an amino acid sequence having at least 80% sequence identity thereto, can be reduced or eliminated by means of micro RNAs.

In one embodiment, expression of a protein comprising an amino acid sequence as set forth in SEQ ID number 1, 4, 7, 10, 13, 16, 19 or 22, or an amino acid sequence having at least 80% sequence identity thereto, can be reduced or eliminated by RNAi.

In one embodiment, expression of a protein comprising an amino acid sequence as set forth in SEQ ID number 1, 4, 7, 10, 13, 16, 19 or 22, or an amino acid sequence having at least 80% sequence identity thereto, can be reduced or eliminated by antisense suppression.

In one embodiment, expression of a protein comprising an amino acid sequence as set forth in SEQ ID number 1, 4, 7, 10, 13, 16, 19, or 22, or an amino acid sequence having at least 80% sequence identity thereto, can be reduced or eliminated by sense suppression.

In one embodiment, expression of a protein comprising an amino acid sequence as set forth in SEQ ID number 1, 4, 7, 10, 13, 16, 19 or 22, or an amino acid sequence having at least 80% sequence identity thereto, can be reduced or eliminated by a tDNA insertion.

In one embodiment, expression of a protein comprising an amino acid sequence as set forth in SEQ ID number 1, 4, 7, 10, 13, 16, 19, or 22, or an amino acid sequence having at least 80% sequence identity thereto, can be reduced or eliminated by a dominant negative construct (or a negative allelic mutation).

In one embodiment, expression of a protein comprising an amino acid sequence as set forth in SEQ ID number 1, 4, 7, 10, 13, 16, 19 or 22, or an amino acid sequence having at least 80% sequence identity thereto, can be reduced or eliminated by a targeted mutagenesis based system.

In one embodiment, expression of a protein comprising an amino acid sequence as set forth in SEQ ID number 1, 4, 7, 10, 13, 16, 19, or 22, or an amino acid sequence having at least 80% sequence identity thereto, can be reduced or eliminated by a CRISPR-based system.

In one embodiment, expression of a protein comprising an amino acid sequence as set forth in SEQ ID number 1, 4, 7, 10, 13, 16, 19 or 22, or an amino acid sequence having at least 80% sequence identity thereto, can be reduced or eliminated by a zinc finger nuclease, TALENs, meganucleases, mutagenic oligonucleotides or TILLING.

In some embodiments, the present invention provides methods of increasing the alkaloid content of a plant or a part or cell thereof (e.g., a plant cell), comprising modifying said plant by increasing or enhancing the activity or expression of at least one gene encoding a SOUL heme-binding protein.

Any method known in the art for increasing or enhancing the activity or expression of a gene may be used in the method according to the invention.

In some embodiments, the method may comprise overexpressing at least one gene encoding a SOUL heme-binding protein. Suitably, the method may comprise expressing in the plant or cell one or more additional copies of at least one gene encoding a SOUL heme-binding protein. Suitably, the method may comprise modifying the endogenous copy of at least one gene encoding a SOUL heme-binding protein such that its expression is increased. The methods can include mutating the coding sequence of at least one gene encoding a SOUL heme-binding protein. The methods can include mutating a regulatory sequence that regulates the expression of at least one gene encoding a SOUL heme-binding protein.

Suitably, the method may comprise transforming cells of a plant (e.g. a tobacco plant) with a genetic construct encoding at least one SOUL heme-binding protein comprising an amino acid sequence as set forth in SEQ ID number 1, 4, 7, 10, 13, 16, 19, or 22, or a functional variant or functional fragment or ortholog thereof, or a sequence having at least 80% identity to SEQ ID number 1, 4, 7, 10, 13, 16, 19, or 22; or wherein the at least one gene encoding a SOUL heme-binding protein comprises a nucleotide sequence as set forth in SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, or 24, or a functional variant or fragment or ortholog of SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, or 24, or a nucleic acid sequence having at least 80% identity to SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, or 24; or which comprises a nucleotide sequence encoding a protein capable of promoting or enhancing at least one endogenous SOUL heme-binding protein gene. It will be appreciated that each of these options will result in increased activity and expression of the polypeptide encoded by at least one SOUL heme-binding protein gene. The method may comprise regenerating a plant from the transformed cell. There is provided the use of a genetic construct capable of increasing the activity and/or expression of a polypeptide encoded by at least one SOUL heme-binding protein gene for increasing the alkaloid content (e.g. nicotine content) and/or TSNA content (or precursors thereof) in a plant, or part or cell thereof, transformed with said construct.

The genetic construct may encode a polypeptide comprising amino acids SEQ ID number 1, 4, 7, 10, 13, 16, 19 or 22, or a functional variant or functional fragment or ortholog thereof, or a sequence having at least 80% identity to SEQ ID number 1, 4, 7, 10, 13, 16, 19 or 22; or wherein the at least one gene encoding a SOUL heme-binding protein comprises a nucleotide sequence set forth in SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, or 24, or a functional variant or fragment or ortholog of SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, or 24, or a nucleic acid sequence having at least 80% identity to SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, or 24.

In another embodiment, the present invention relates to a method of increasing the alkaloid content of a plant or part thereof and/or the TSNA content (or a precursor thereof) in a plant or plant part thereof, comprising modifying said plant by increasing the activity of at least one gene encoding a SOUL heme-binding protein.

In one embodiment, the activity of at least one gene encoding a SOUL heme-binding protein may be increased by introducing (or providing) a mutation into at least one gene encoding a SOUL motif.

Suitably, the activity of at least one gene encoding a SOUL heme-binding protein comprising an amino acid sequence as set forth in SEQ ID number 1, 4, 7, 10, 13, 16, 19, or 22 may be increased by introducing a mutation into the at least one gene encoding a SOUL heme-binding protein; or a functional variant or a functional fragment or ortholog thereof, or a sequence having at least 80% identity to SEQ ID number 1, 4, 7, 10, 13, 16, 19 or 22; or wherein the at least one gene encoding a SOUL heme-binding protein comprises a nucleotide sequence set forth in SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, or 24, or a functional variant or fragment or ortholog of SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, or 24, or a nucleic acid sequence having at least 80% identity to SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, or 24.

In some embodiments, the modification that increases the activity or expression of at least one SOUL heme-binding protein gene and thereby increases alkaloid content and/or TSNA content (or precursors thereof) is by one of:

modulating transcription from at least one SOUL heme-binding protein gene;

modulating translation of mRNA from at least one SOUL heme-binding protein gene;

modulating release of SOUL heme-binding protein from intracellular storage; and/or

Modulating the degradation rate of the SOUL heme-binding protein.

Alkaloid content

In one embodiment, the present invention provides a method of modulating the alkaloid content of a plant (e.g., a tobacco plant) or part thereof, comprising modifying said plant by modulating the activity or expression of at least one gene encoding a SOUL heme-binding protein.

The term "modulate" is used herein to mean increase or decrease.

The term "increasing the alkaloid content" is used herein to mean that the alkaloid content is higher in a product of the invention (e.g. a plant, part thereof (e.g. leaf), processed leaf or a product prepared from a plant (e.g. a tobacco product)) compared to a comparable product which has not been modified according to the invention.

The term "reduced alkaloid content" is used herein to mean a lower alkaloid content in a product of the invention (e.g., a plant, part thereof (e.g., leaf), processed leaf, or product made from a plant (e.g., a tobacco product)) as compared to a comparable product that has not been modified according to the invention.

In some embodiments, modulation of alkaloid content refers to an increase in alkaloid content, wherein the activity or expression of at least one SOUL heme-binding protein gene is increased (or in other words, the protein is overexpressed).

In some embodiments, modulation of alkaloid content refers to a reduction in alkaloid content, wherein expression of at least one gene encoding a SOUL heme-binding protein is reduced or inhibited or eliminated.

In a further aspect, the alkaloid content is measured from the leaf. In one aspect, alkaloid content is measured from green leaves. In a further aspect, the alkaloid content is measured from conditioned leaves, such as air-cured, flue-cured, fire-cured, or sun-cured leaves. In a further aspect, the alkaloid content is measured from flue roasted leaves. In a further aspect, the alkaloid content is measured from air-cured leaves.

The term "alkaloid content" is used herein to mean the concentration and/or total amount of the entire group of compounds classified as alkaloids or the concentration and/or total amount of one or more compounds classified as alkaloids. Alkaloids commonly found in tobacco include nicotine, nornicotine, PON, anatabine, neonicotinoid, and myoxamine. In some embodiments, the content of one or more alkaloids selected from nicotine, nornicotine, PON, anatabine, neonicotinoid, and myosamine, such as two or more alkaloids, such as three or more alkaloids, such as four or more alkaloids, such as five or more alkaloids, such as all six alkaloids, is adjusted. In some embodiments, the content of one or more alkaloids selected from nicotine, nornicotine, PON, anatabine, neonicotinoid, and myosamine, such as two or more alkaloids, such as three or more alkaloids, such as four or more alkaloids, such as five or more alkaloids, such as all six alkaloids, is increased. In some embodiments, the content of one or more alkaloids selected from nicotine, nornicotine, PON, anatabine, neonicotinoid, and myosamine, such as two or more alkaloids, such as three or more alkaloids, such as four or more alkaloids, such as five or more alkaloids, such as all six alkaloids, is reduced. In some embodiments, the total alkaloid content of the plant or cell is modulated. In some embodiments, the total alkaloid content is increased. In some embodiments, the total alkaloid content is increased.

Suitably, the nicotine content may be adjusted. In one embodiment, the nicotine content is reduced. In another embodiment, the nicotine content is not modulated, but the content of one or more alkaloids selected from nornicotine, PON, anatabine, neonicotinoid, and myoxaline is modulated. In another embodiment, the nicotine content is not modulated, but the content of one or more alkaloids selected from nornicotine, PON, anatabine, and anabasine is modulated.

Any method known in the art for determining the concentration and/or total amount of alkaloids can be used. One preferred method for analyzing alkaloid content involves analysis by gas chromatography-flame ionization detection method (GC-FID).

In one embodiment, a process for producing: a plant (e.g., a tobacco plant) or a part thereof, a plant propagation material (e.g., a tobacco plant propagation material), a cell (e.g., a tobacco cell), a leaf (e.g., a tobacco leaf), a harvested leaf (e.g., a harvested tobacco leaf), a cut harvested leaf (e.g., a cut harvested tobacco leaf), a processed leaf (e.g., a processed tobacco leaf), a cut and processed leaf (e.g., a cut and processed tobacco leaf), a product (e.g., a tobacco product) comprising said plant or a part thereof obtainable or obtained by a plant of the invention having modulated alkaloid content, or a combination thereof, comprising modifying said plant to modulate the activity or expression of a gene encoding a SOUL heme-binding protein. The adjusted alkaloid content can be determined by comparing the alkaloid content in: a plant (e.g., a tobacco plant) or a part thereof, a plant propagation material (e.g., a tobacco plant propagation material), a cell (e.g., a tobacco cell), a leaf (e.g., a tobacco leaf), a harvested leaf (e.g., a harvested tobacco leaf), a cut harvested leaf (e.g., a cut harvested tobacco leaf), a processed leaf (e.g., a processed tobacco leaf), a cut and processed leaf (e.g., a cut and processed tobacco leaf), a product comprising a plant or a part thereof of the invention, e.g., a tobacco product, or a combination thereof.

Suitably, the alkaloid content may be modulated in a plant, e.g. a tobacco plant, e.g. a modified tobacco plant. Suitably, the alkaloid content may be modulated in lamina (e.g. tobacco lamina, e.g. from a modified tobacco plant). Suitably, the alkaloid content may be modulated in harvested leaf (e.g. harvested tobacco leaf from a modified tobacco plant). Suitably, the alkaloid content may be adjusted in cut harvested leaf (e.g. cut harvested tobacco leaf from a modified tobacco plant). Suitably, the alkaloid content may be modulated in a processed leaf (e.g. a processed tobacco leaf, such as a processed tobacco leaf from a modified tobacco plant). Suitably, the alkaloid content may be adjusted in cut and processed lamina (e.g. cut and processed tobacco lamina, such as cut and processed tobacco lamina from a modified tobacco plant). Suitably, the alkaloid content may be modulated in a cured leaf (e.g. cured tobacco leaf from a modified tobacco plant). Suitably, the alkaloid content may be modulated in an extract of a green leaf (e.g. a green tobacco leaf from a modified tobacco plant). Suitably, the alkaloid content may be modulated in a product comprising a plant of the invention or part thereof (e.g. a tobacco product, such as a tobacco product produced from a modified tobacco plant or part thereof). Suitably, the alkaloid content may be adjusted in any one of the above products or combinations thereof. Suitably, the above-mentioned adjustment of the alkaloid content may be an increase in the alkaloid content. Suitably, the modulation of the alkaloid content may be a reduction in alkaloid content (e.g. a reduction in nicotine content).

In one embodiment, the content of one or more alkaloids selected from nicotine, nornicotine, PON, anatabine, neonicotinoid and myoxamine is reduced. In one embodiment, the content of one or more alkaloids selected from nicotine, nornicotine, PON, anatabine and anabasine is reduced.

Suitably, the above-mentioned adjustment of the alkaloid content may be a reduction of the nicotine content. Suitably, the modulation of the alkaloid content may be a reduction in the content of one or more alkaloids selected from nornicotine, PON, anatabine, neonicotinoid and myoxamine, rather than a reduction in the content of nicotine. Suitably, the modulation of the alkaloid content may be a reduction in the content of one or more alkaloids selected from nornicotine, PON, anatabine and anabasine, rather than a reduction in the nicotine content.

In one embodiment, the nicotine content of a modified plant (e.g., a tobacco plant), a plant propagation material (e.g., a tobacco plant propagation material), a lamina (e.g., a tobacco lamina), a harvested lamina (e.g., a harvested tobacco lamina), a cut harvested lamina (e.g., a cut harvested tobacco lamina), a processed lamina (e.g., a processed tobacco lamina), a cut and processed lamina (e.g., a cut and processed tobacco lamina), or a tobacco product from a modified tobacco plant is reduced.

In one embodiment, the alkaloid content of a plant (e.g., a tobacco plant) or a part thereof may be modulated to at least 0.5, 1.5, or 2 fold when compared to the alkaloid content of a plant (e.g., a tobacco plant) or a part thereof, respectively, that has not been modified to modulate the activity or expression of at least one gene encoding a SOUL heme-binding protein and that has been grown under similar growth conditions. Suitably, the alkaloid content may be adjusted to about 0.5-fold to about 2-fold. Suitably, the modification may be an increase or decrease in alkaloid content. Suitably, the modulation may be modulation of one or more alkaloids selected from nicotine, nornicotine, PON, anatabine, anabasine and myoxaline. Suitably, the modulation may be modulation of one or more alkaloids selected from nicotine, nornicotine, PON, anatabine and anabasine. Suitably, the nicotine content is regulated.

In one embodiment of the invention, the alkaloid content of a plant (e.g. a tobacco plant) or a part thereof may be modulated by at least 1%, 2%, 5%, 8%, 10%, 12%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% compared to a plant (e.g. a tobacco plant) or a part thereof which has not been modified according to the invention. In one embodiment, the alkaloid content may be adjusted by at least 30% compared to an unmodified plant or part thereof. In one embodiment, the alkaloid content may be adjusted by at least 40% compared to an unmodified plant or part thereof. In one embodiment, the alkaloid content may be adjusted by at least 50% compared to an unmodified plant or part thereof. In one embodiment, the alkaloid content may be adjusted by at least 60% compared to an unmodified plant or part thereof. The modulation may be an increase or decrease in alkaloid content when compared to an unmodified plant (e.g., a tobacco plant) or portion thereof. Suitably, the adjustment may be of total alkaloid content. Suitably, the modulation may be modulation of one or more alkaloids selected from nicotine, nornicotine, PON, anatabine, anabasine and myoxaline. Suitably, the modulation may be modulation of one or more alkaloids selected from nicotine, nornicotine, PON, anatabine and anabasine. Suitably, the modulation is a modulation of nicotine content, such as a reduction in nicotine content. Suitably, the modulation is a modulation of nornicotine content, such as a reduction in nornicotine content. Suitably, the modulation is a modulation of the neonicotinoid content, such as a reduction of the neonicotinoid content. Suitably, the adjustment is an adjustment of PON content, such as a reduction in PON content. Suitably, the adjustment is an adjustment of anatabine content, such as a reduction in anatabine content. Suitably, the modulation is modulation of more than one alkaloid selected from nicotine, nornicotine, PON, anatabine, neonicotinoid and myosamine, such as two or more alkaloids, such as three or more alkaloids, such as four or more alkaloids, such as five or more alkaloids, such as all six alkaloids.

In some embodiments, the alkaloid content of a plant may be adjusted from about 5% to about 100%, from about 10% to about 90%, from about 20% to about 80%, from about 30% to about 70%, from about 40% to 60%, or from about 40% to 50%.

In one embodiment, the alkaloid content of the plant may be adjusted from about 50% to about 100%, preferably from about 60% to 90%, preferably 80% or more.

Tobacco Specific Nitrosamine (TSNA) content

In one embodiment, the present invention provides a method of modulating (i.e., increasing or decreasing) the tobacco-specific nitrosamine (TSNA) or TSNA precursor content in a plant (e.g., a tobacco plant) or portion thereof. Suitably, the method may comprise modifying said plant by modulating activity or expression of at least one gene encoding a SOUL heme-binding protein. Suitably, the content of TSNA or TSNA precursor may be reduced. Suitably, the total TSNA content of the plant or part thereof may be modulated.

TSNAs can be measured in processed tobacco, such as cured tobacco or reconstituted tobacco. In one embodiment, the TSNA content is measured and/or modified (e.g., reduced) in a cured tobacco plant or portion thereof (e.g., cured tobacco lamina).

The term "tobacco specific nitrosamines" or "TSNAs" as used herein has its usual meaning in the art, i.e. nitrosamines found only in tobacco products or other nicotine-containing products. Suitably, the at least one tobacco-specific nitrosamine may be 4- (methylnitrosamine) -1- (3-pyridyl) -1-butanone (NNK), N ' -nitrosonornicotine (NNN), N ' -Nitrosoanatabine (NAT) or N ' -Nitrosoanabasine (NAB).

The term "precursor thereof, when used in relation to at least one tobacco-specific nitrosamine, refers to one or more chemicals or compounds of the tobacco plant that cause the formation of tobacco-specific nitrosamines or are associated with nitrosation reactions that lead to the production of tobacco-specific nitrosamines. Suitably, the term "precursor thereof may refer to nitrate, nitrite or nitric oxide.

The term "modulate" is used herein to mean increase or decrease.

In one embodiment, the TSNA is N' nitrosonornicotine (NNN) and/or the precursor is nornicotine.

In one embodiment, the TSNA may be one or more selected from the group of: n ' -nitrosonornicotine (NNN), N ' -Nitrosonornicotine (NAT), N ' -Nitrosoneonicotine (NAB), and 4- (methylnitrosamino) -1- (3-pyridyl) -1-butanone (NNK). Suitably, the at least one tobacco-specific nitrosamine may be NNK or NNN. In one embodiment, the tobacco specific nitrosamine is NNN.

In one embodiment, the precursor of the TSNA is one or more selected from the group of nornicotine, neonicotine, anatabine, and oxidized derivatives of nicotine such as pseudooxidized nicotine (PON).

In a preferred embodiment, the precursor of the TSNA is nornicotine.

In one embodiment, the precursor of the TSNA may be PON. Precursors of TSNAs (e.g., NNN, NNK, NAB and/or NAT) can be measured in green tobacco lamina, e.g., prior to processing, e.g., prior to modulation. In one embodiment, precursors of TSNAs (e.g., NNN, NNK, NAB and/or NAT) are measured and/or modified (e.g., reduced) in green tobacco lamina, e.g., prior to processing, e.g., prior to modulation.

In one embodiment, performing the method and or use of the invention results in a reduction of at least one TSNA or precursor thereof in the modified tobacco plant (or part thereof) when compared to a tobacco plant (or part thereof) which has not been modified according to the invention.

The term "reducing at least one TSNA or precursor thereof" or "reduction of at least one TSNA or precursor thereof" is used herein to mean that the concentration and/or total content of at least one TSNA or precursor thereof in a product, method or use of the invention is lower relative to a comparable product, method or use. For example, a comparable tobacco industry product would be derived from a tobacco plant that has not been modified according to the present invention, but where all other relevant characteristics are the same (e.g., plant species, growth conditions, methods of processing tobacco, etc.).

Any method known in the art for determining the concentration and/or level of at least one TSNA or precursor thereof may be used. In particular, such methods may be used, which may include addition of deuterium labeled internal standards, water extraction and filtration, followed by analysis using reverse phase high performance liquid chromatography with tandem mass spectrometry (LC-MS/MS). Other examples for determining the concentration and/or level of tobacco-specific nitrosamine precursors include methods, such as the CORESTA recommended method CRM-72: determination of Tobacco-Specific Nitrosamines in Tobacco and Tobacco Products by LC-MS/MS (CORESTA reconstructed method CRM-72: Determination of Tobacco Specific Nitrosamines in Tobacco and Tobacco Products by LC-MS/MS); the methods detailed in CRM or Wagner et al (2005) Analytical Chemistry, 77(4), 1001-1006, being developed as ISO/DIS 21766, are incorporated herein by reference in their entirety.

Suitably, the concentration and/or total content of at least one tobacco-specific nitrosamine or a precursor thereof may be reduced by carrying out the method and/or use of the present invention. Suitably, the concentration and/or level of at least one tobacco-specific nitrosamine or a precursor thereof may be reduced in a tobacco plant of the invention (e.g. obtainable or obtained by a method and/or use of the invention) when compared to the concentration and/or level of at least one tobacco-specific nitrosamine or a precursor thereof in a tobacco plant that has not been modified according to the invention.

The concentration and/or total content of the at least one tobacco-specific nitrosamine or precursor thereof may be reduced in tobacco lamina, harvested lamina, processed tobacco lamina, tobacco industry products or combinations thereof obtainable from or obtained from a tobacco plant (or part of a tobacco plant or a tobacco cell or cell culture) of the present invention when compared to tobacco lamina, harvested lamina, processed tobacco lamina, tobacco industry products or combinations thereof obtainable from or obtained from a tobacco plant (or part of a tobacco plant or a tobacco cell or cell culture) that is not modified according to the present invention.

Suitably, the concentration and/or total content of the at least one tobacco-specific nitrosamine or precursor thereof may be reduced in processing tobacco lamina.

Suitably, the concentration and/or level of the at least one tobacco-specific nitrosamine or precursor thereof may be reduced in a tobacco industry product.

In one embodiment, the at least one tobacco-specific nitrosamine or precursor thereof may be reduced by at least about 1%, at least about 3%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50%. In some embodiments, the at least one tobacco-specific nitrosamine or precursor thereof may be reduced by about 5% to about 50%, about 10% to about 50%, about 20% to about 50%, about 30% to about 50%, or about 40% to 50%.

With respect to processing (e.g., conditioned) tobacco lamina (e.g., conditioned or reconstituted), the at least one tobacco-specific nitrosamine or precursor thereof may be reduced by about 5000 ng/g to about 50 ng/g, about 4000 ng/g to about 100 ng/g, about 3000 ng/g to 500 ng/g, or 2000 ng/g to 1000 ng/g. In some embodiments, at least one tobacco-specific nitrosamine or precursor thereof may be reduced by at least about 5000 ng/g, at least about 4000 ng/g, at least about 3000 ng/g, at least about 2000 ng/g, at least about 1000 ng/g, at least about 500 ng/g, at least about 100 ng/g, or at least about 50 ng/g.

Biomass production

In one aspect, the present invention provides a method of producing biomass comprising:

growing cells under conditions that produce biomass, the cells having been engineered to modulate (e.g., reduce) the activity or expression of a gene encoding a SOUL heme-binding protein.

In one embodiment, the invention provides a method of producing biomass having an altered (e.g., reduced) concentration and/or total content of nicotine, comprising growing a cell that has been engineered to reduce the activity or expression of at least one gene encoding a SOUL heme-binding protein comprising an amino acid sequence as set forth in SEQ ID number 1, 4, 7, 10, 13, 16, 19, or 22, or a functional variant or functional fragment or ortholog thereof, or a sequence having at least 80% identity to SEQ ID number 1, 4, 7, 10, 13, 16, 19, or 22; or

Wherein the at least one gene encoding a SOUL heme-binding protein comprises a nucleotide sequence set forth in SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, or 24, or a functional variant or fragment or ortholog of SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, or 24, or a nucleic acid sequence having at least 80% identity to SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, or 24.

Cells can be engineered by any method known in the art to alter the activity or expression of at least one gene encoding a SOUL heme-binding protein. Suitably, the cell may be engineered to express a foreign gene encoding a SOUL heme-binding protein. Suitably, the cell may be engineered to overexpress a gene encoding a SOUL heme-binding protein. Suitably, the cell may be engineered to reduce the activity or expression of a gene encoding a SOUL heme-binding protein.

Suitably, the biomass may contain a lower concentration and/or total content of nicotine than biomass produced from a comparable cell that has not been modified according to the invention.

Suitably, the cell used for biomass production may be a plant cell, such as a tobacco cell.

Suitably, the cells used for biomass production may be yeast cells.

In one embodiment, the cell (e.g., yeast cell) can be further modified to comprise one or more sequences that increase nicotine alkaloid biosynthesis. Suitably, these one or more sequences may be incorporated into a nucleic acid construct suitable for transformation of a cell (e.g., a yeast cell). One or more sequences may be overexpressed in a cell (e.g., a yeast cell). The sequence may be selected from one or more of the following genes: MPO (or methyl putrescine oxidase or MPO1 or MPO 2); a622 (or isoflavone reductase-like protein or isoflavone reductase homolog (homolog) or isoflavone reductase-like protein); BBL (or berberine bridge enzyme-like or BBE or NBB 1); PMT (or putrescine N-methyltransferase or putrescine methyltransferase or S-adenosyl-L-methionine: putrescine N-methyltransferase or PMT1 or PMT2 or PMT3 or PMT4) and QPT (or quinolinate phosphoribosyltransferase). In one embodiment, the sequence may be selected from one or more of the following genes: BBL, A622, PMT and MPO (MPO1 or MPO 2). Genes suitable for modification in this manner can be taught, for example, in US2016032299, which is incorporated herein by reference.

Commercially desirable traits

In one embodiment, the plants of the invention have a modified (i.e. increased or decreased) total alkaloid content, and/or a modified (i.e. increased or decreased) content of one or more alkaloids selected from nicotine, nornicotine, neonicotinoid, muoxinamine and anatabine and/or decreased nicotine, while the flavor profile and/or other commercially desirable traits are at least maintained. In one embodiment, the plant of the invention produces leaves of similar grade and/or quality as plants not modified according to the invention.

In one embodiment, a plant of the invention has reduced nicotine content (e.g., as compared to the same plant not modified according to the invention) without a significant change in the flavor profile of the plant.

In one embodiment, a plant of the invention has modified (i.e., increased or decreased) alkaloid and/or TSNA content (e.g., as compared to the same plant not modified according to the invention) in the absence of a significant change (e.g., decrease) in other commercially desirable traits of the plant. In particular, the yield of the modified plant is preferably not reduced compared to the same plant not modified according to the present invention.

Thus, in one embodiment, the methods and uses of the present invention involve modifying (i.e. increasing or decreasing) the total alkaloid content, and/or modifying (i.e. increasing or decreasing) one or more alkaloids selected from nicotine, nornicotine, neonicotinoid and anatabine, and/or modifying (i.e. increasing or decreasing) the nicotine content and/or the TSNA content, while maintaining the flavour profile and/or other commercially desirable traits (e.g. yield).

The term "commercially desirable trait" as used herein shall include traits such as yield, mature plant height, harvestable leaf number, average node length, waist leaf (cutter leaf) length, waist leaf width, mass (e.g., leaf mass, suitably modulated leaf mass), abiotic (e.g., drought) stress tolerance, herbicide tolerance, and/or biotic (e.g., insect, bacterial or fungal) stress tolerance.

The leaf quality may be measured based on the color, texture and aroma of the modulated leaves, for example, according to the United States Department of Agriculture (USDA) grades and standards.

Tobacco grade is evaluated based on factors including, but not limited to: petiole position, leaf size, leaf color, leaf uniformity and integrity, maturity, texture, elasticity, luster (related to leaf color strength and depth, and shine), hygroscopicity (the ability of tobacco leaves to absorb and retain environmental moisture), and green nuance or trait (cast).

The blade grade may be determined using Standard methods known in the art, for example, using the Official Standard Grades (7 U.S. C. 511) published by the U.S. Department of Agriculture product Marketing Service of the US Department of Agriculture. See, for example, official standard ratings (55 f.r. 40645) for Burley tobaco (american type 31 and foreign type 93) that were in effect at 11/5 days 1990; official standard grades (54 f.r. 7925) of flue-cured tobacco (us types 11, 12, 13, 14 and foreign type 92) in effect at 27/3/1989; official standard grade of broadleaf holly leaf (Pennsylvania Seedleaf Tobacco) (U.S. type 41) effective on 8 days 1 month 1965 (29 F.R. 16854); official standard grades (28 f.r. 11719 and 28 f.r. 11926) of Cigar Leaf Tobacco (Ohio Cigar-Leaf tobaco), usa types 42, 43 and 44, effective 12, 8 days 1963; official standard grades of Wisconsin Cigar-Binder Tobacco (U.S. model 54 and 55) for Cigar wrappers, Wisconsin, effective at 20/11/1969 (34 F.R. 17061); official standard ratings (34 f.r. 17061) of cigar inner wrappers of wisconsin (us types 54 and 55) that were in effect at 11/20/1969; official standard grades of Cigar overwrap Tobacco (ShadeGrown Cigar-writer Tobacco) (U.S. model 62) were cultivated in shade in Georgia and Florida, which became effective in 4 months 1971. The USDA grade index value may be determined according to industry recognized grade indices. See, e.g., Bowman et al (1988) Tobacco Science, 32: 39-40; the Library of Tobacco heritage literature (Legacy Tobacco Document Library) (Bates Document #523267826 and 523267833, 1.7.1988, the Burley Grade Index Memorandum (Melandrum on the deployed Burley Tobacco Grade Index)); and Miller et al (1990) Tobacco Intern, 192:55-57 (all of the aforementioned references are incorporated herein in their entirety).

In one aspect, the USDA grade index is a 0-100 numerical representation of the received federal grade and is a weighted average of all handle positions. A higher ranking index indicates a higher quality. Alternatively, blade grade may be determined via hyper-spectral imaging. See, for example, WO 2011/027315 (which is incorporated herein by reference).

In one embodiment, the tobacco plant of the present invention provides a commercially acceptable grade of tobacco.

Suitably, the tobacco plant of the present invention provides a commercially acceptable grade of cured tobacco.

In one embodiment, the tobacco plants of the present invention are capable of producing lamina having a USDA grade index value that is at least about 70% of the USDA grade index value of a comparable plant lamina when grown under similar growth conditions. Suitably, the tobacco plants disclosed herein may be capable of producing lamina having a USDA grade index value that is at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 98% of the USDA grade index value of the control plant when grown under similar growth conditions. Suitably, a tobacco plant disclosed herein may be capable of producing lamina having a USDA grade index value of 65% to 130%, 70% to 130%, 75% to 130%, 80% to 130%, 85% to 130%, 90% to 130%, 95% to 130%, 100% to 130%, 105% to 130%, 110% to 130%, 115% to 130%, or 120% to 130% of the USDA grade index value of a comparable plant.

In one aspect, the tobacco plants of the invention are capable of producing lamina having a USDA grade index value of at least 50. Suitably, the tobacco plants disclosed herein may be capable of producing lamina having a USDA grade index value of 55 or higher, 60 or higher, 65 or higher, 70 or higher, 75 or higher, 80 or higher, 85 or higher, 90 or higher, and 95 or higher.

Unless otherwise indicated, tobacco yield as used herein refers to cured leaf yield, which is calculated based on cured tobacco leaf weight per acre under standard field conditions, following standard agronomic and curing practices.

In one aspect, a plant of the invention (e.g., a tobacco plant) has a yield that is 50% to 150%, 55% to 145%, 60% to 140%, 65% to 135%, 70% to 130%, 75% to 125%, 80% to 120%, 85% to 115%, 90% to 110%, 95% to 105%, 50% to 100%, 55% to 100%, 60% to 100%, 65% to 100%, 70% to 100%, 75% to 100%, 80% to 100%, 85% to 100%, 90% to 100%, 95% to 100%, 100% to 150%, 105% to 150%, 110% to 150%, 115% to 150%, 120% to 150%, 125% to 150%, 130% to 150%, 135% to 150%, 140% to 150%, or 145% to 150% of the yield of a comparable plant when grown under similar field conditions.

In another aspect, a plant of the invention (e.g., a tobacco plant) has a yield that is about 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 times the yield of a comparable plant when grown under similar field conditions.

In another aspect, the yield of tobacco plants of the invention is comparable to the yield of comparable flue-cured plants when grown under similar field conditions.

In one aspect, the tobacco plants of the invention provide a yield selected from the group consisting of: about 1200 to 3500, 1300 to 3400, 1400 to 3300, 1500 to 3200, 1600 to 3100, 1700 to 3000, 1800 to 2900, 1900 to 2800, 2000 to 2700, 2100 to 2600, 2200 to 2500, and 2300 to 2400 pounds per acre.

In another aspect, the tobacco plants of the invention provide a yield selected from the group consisting of: about 1200 to 3500, 1300 to 3500, 1400 to 3500, 1500 to 3500, 1600 to 3500, 1700 to 3500, 1800 to 3500, 1900 to 3500, 2000 to 3500, 2100 to 3500, 2200 to 3500, 2300 to 3500, 2400 to 3500, 2500 to 3500, 2600 to 3500, 2700 to 3500, 2800 to 3500, 2900 to 3500, 3000 to 3500 and 3100 to 3500 pounds per acre.

In a further aspect, the tobacco plants of the invention provide a yield selected from the group consisting of: about 1200 to 3500, 1200 to 3400, 1200 to 3300, 1200 to 3200, 1200 to 3100, 1200 to 3000, 1200 to 2900, 1200 to 2800, 1200 to 2700, 1200 to 2600, 1200 to 2500, 1200 to 2400, 1200 to 2300, 1200 to 2200, 1200 to 2100, 1200 to 2000, 1200 to 1900, 1200 to 1800, 1200 to 1700, 1200 to 1600, 1200 to 1500, and 1200 to 1400 pounds per acre.

Plant breeding

In one embodiment, the present invention provides a method of producing a plant having a modified alkaloid content and/or a modified Tobacco Specific Nitrosamine (TSNA) or TSNA precursor content comprising:

a. crossing a donor plant with a recipient tobacco plant, the donor plant having a modified nicotine content and/or a modified tobacco-specific nitrosamine (TSNA) or TSNA precursor content and wherein the activity or expression of at least one gene encoding a SOUL heme-binding protein according to the invention has been modulated according to the invention in the donor plant, the recipient tobacco plant not having a modified nicotine content or a modified tobacco-specific nitrosamine (TSNA) or TSNA precursor content and having a commercially desirable trait;

b. isolating genetic material from progeny of the donor plant that is crossed with the recipient plant; and

c. performing molecular marker assisted selection with a molecular marker, comprising:

i. identifying a region of the introgression comprising a mutation in the polynucleotide sequence encoding the protein defined in a.

Suitably, the activity or expression of a protein comprising an amino acid sequence as set forth in SEQ ID number 1, 4, 7, 10, 13, 16, 19 or 22, or a functional variant or functional fragment or ortholog thereof, or a sequence having at least 80% identity to SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23 or 24 is modulated in a donor plant when compared to a comparable plant; or the protein is encoded by: a nucleotide sequence as set forth in SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23 or 24, or a functional variant or fragment or ortholog of SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23 or 24, or a nucleic acid sequence having at least 80% identity to SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23 or 24.

Molecular marker assisted selection can include performing PCR to identify introgressed nucleic acid sequences comprising mutations that modulate the activity or expression of proteins comprising an amino acid sequence as set forth in SEQ ID number 1, 4, 7, 10, 13, 16, 19, or 22 or an amino acid sequence having at least 80% sequence identity thereto.

Plant and method for producing the same

Suitable plants according to the invention include plants of the solanaceae family, which include, for example, datura, eggplant, mandragora, lethal solanum (belladonna), capsicum (paprika, capsicum), potato and tobacco.

In one embodiment, a suitable genus of the Solanaceae family is the genus Nicotiana (Nicotiana tabacum: (Nicotiana tabacum) ((Nicotiana tabacum)Nicotiana) For example tobacco or yellow tobacco: (Nicotiana rustica)。

A suitable species of nicotiana can be tobacco. The species of the genus nicotiana may be referred to herein as a tobacco plant, or simply tobacco.

Tobacco plants

The invention provides methods, uses and cells (e.g., tobacco cells), plants (e.g., tobacco plants) and plant propagation material directed against plants (e.g., tobacco plants).

The term "tobacco plant" as used herein refers to a plant of the genus nicotiana used in the production of tobacco products. Non-limiting examples of suitable "tobacco" plants include tobacco and yellow tobacco (e.g., tobacco: (i.e.,), (ii)N. tabacumL.), LA B21, LN KY171, Tl 1406, Basma, Galpao, Perique, Beninhart 1000-1, and Pelico).

Tobacco material may be derived or obtained from tobacco type varieties, commonly known as the burley variety, the flue-cured (tobacco) or bright (bright) and dark (dark) varieties. In some embodiments, the tobacco material is derived from burley tobacco, Virginia (Virginia), or dark tobacco plants. The tobacco plant may be selected from burley tobacco, rare tobacco, specialty tobaccos (specialty tobaccos), expanded tobacco, and the like.

Also contemplated herein is the use of tobacco cultivars and elite (elite) tobacco cultivars. Thus, a tobacco plant for use herein can be a tobacco variety or an elite tobacco cultivar. Particularly useful tobacco varieties include flue-cured virginia, burley and Oriental types.

In some embodiments, the tobacco plant may be selected, for example, from one or more of the following varieties: l. cultivars T.I. 1068, AA 37-1, B13P, Xanthi (Mitchell-Mor), KT D #3 Hybrid (Hybrid)107, Bel-W3, 79-615, Samsun Holmes NN, F4 from Hybrid BU21 x Hoja paramo, line 97, KTRDC #2 Hybrid 49, KTRDC #4 Hybrid 110, Burley 21, PM016, KTRDC #5 KY 160 SI, KTRDC #7 FCA, KTRDC #6 TN86 SI, PM021, K149, K326, K346, K358, K394, K399, K730, KY10, KY14, KY 160, KY17, KY 8959, KY9, MKY 907, MD 609, Nair 373, NC 2000, PG 01, PG 04, P01, P02, P7, RG 17, RG 11, KY 3586, BAS 44, DRAMIG 31, RG # 11, RG 31, RG # 26 x, K35, K31, K35, K44, K31, K21 x III K35, K21, K11, K35, K23, K11, K23, K35, K3, K23, K3, K11, K3, K11, K23, K11, K3, K23, K3, K11, K3, K23, K3, K23, K3, K11, K3, K23, K11, K23, K3, K11, K3, basma I Zichna ZP4/B, Basma Xanthi BX 2A, Batek, Besuki Jember, C104, Coker 319, Coker 347, Croollo Misionero, PM092, Delturn, Djebel 81, DVH 405, Galpao Commum, HB04P, Hicks Broadleaf, Kabakulak Elassona, PM102, Kutsage E1, KY14, KY171, LA BU21, McNairr 944, 232NC 6, NC 71, NC 297, NC 3, PVH 40203, PVH09, PVH19, PVH 2110, Red Russian, Samsun, Saplak, SimlabGR, Talgar 28, PM132, Wislidica, Yayaagle, NC 4, Madola, Priplex-149, Priplex 23, Priplex 3526, Priplex P3526, TK 42-368, TK K3646, TK 26, TK-K26, TK-3646, TK K26, TK-368, TK 46-K3648, TK 46-K3526, TK 26, TK-K3646, TK 46, TK-K1068, TK 3, ZK 3, and K3, TK III, GR153 and Petit Havana.

Non-limiting examples of varieties or cultivars are: BD 64, CC 101, CC 200, CC 27, CC 301, CC 400, CC 500, CC 600, CC 700, CC 800, CC 900, Coker 176, Coker 319, Coker 371 Gold, Coker 48, CD 263, DF 911, DT 538 LC, Galpao tobacco, GL 26H, GL 350, GL 600, GL 737, GL 939, GL 973, HB04P, HB04P LC, HB3307PLC, hybrid 403LC, hybrid 404LC, hybrid 501 LC, K149, K326, K346, K358, K394, K399, K730, KDH 959, KT 200, KT204LC, KY10, KY14, KY 160, KY17, KY171, KY907LC, KTY14xL8 LC, Littlenden, Nair 373, Nair 944, Narr 35L 8, Narr 3543, Madur 33, Nrad N737N NC 1 LC, Ndol NC 1-L7798, Narr LC, NC 102, NC 2000, NC 291, NC 297, NC 299, NC 3, NC 4, NC 5, NC 6, NC7, NC 606, NC 71, NC 72, NC 810, NC BH 129, NC 2002, Neal Smith Madole, OXFORD 207, PD 7302 LC, PD 7309 LC, PD 7312 LC ' Periq ' e ' tobacco, PVH03, PVH09, PVH19, PVH50, PVH51, R610, R630, R7-11, R7-12, RG 17, RG 81, RGH 51, 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, Speight H179, Speight TN 6, Speight TN86, Speight TN 35, Speight LC, Speight 95, Speight TN 35, and T16, VA 309, VA359, AA 37-1, B13P, Xanthi (Mitchell-Mor), Bel-W3, 79-615, Samsun Holmes NN, KTRDC No.2 hybrid 49, Burley 21, KY 8959, KY9, MD 609, PG 01, PG 04, P01, P02, P03, RG 11, RG 8, VA 509, AS44, Bank A1, Basma Drama B84/31, Basma I Zichna ZP4/B, Basma Xanthi BX 2, Besuki Jember, C104, Coker, Criollo Misionero, Delcorest, Djebel 81, DVH 405, Galpao Com, HB04P, Hicks Broadleaf, Kabakukukukukukukutaksonka, E25, Saistsa 2326, Yakulslag 33, Prilep 32, Priple-P9634, Priple NC-3648, Pillka NC-9634, Pillcap 6, Priple NC-9, Prakura N6, Pillyama C9, Pillyamsp 9685, Pillka, TI-1068, KDH-960, Tl-1070, TW136, Basma, TKF 4028, L8, TKF 2002, GR141, Basma xanthhi, GR149, GR153, Petit Havana. Even if not specifically identified herein, the Low transformant subvariates described above (Low converter subvariates) are also contemplated.

The tobacco plant may be burley, flue-cured virginia or oriental.

In one embodiment, the plant propagation material may be obtainable from a plant of the invention (e.g. a tobacco plant).

As used herein, "plant propagation material" refers to any plant matter taken from a plant from which further plants can be produced. Suitably, the plant propagation material may be selected from seeds, plant calli and plant pieces. Suitably, the plant propagation material may be a seed. Suitably, the plant propagation material may be plant callus. Suitably, the plant propagation material may be a plant mass.

In one embodiment, the cell (e.g. a tobacco cell), tobacco plant and/or plant propagation material may be obtainable (e.g. obtained) by a method according to the invention.

Suitably, a tobacco plant according to the invention may have modulated (e.g. reduced) nicotine content when compared to an unmodified tobacco plant, wherein the tobacco plant has been modified to modulate (e.g. reduce) the activity or expression of at least one gene encoding a SOUL heme-binding protein.

Suitably, a tobacco plant according to the invention may have a modulated (e.g. reduced) Tobacco Specific Nitrosamine (TSNA) or TSNA precursor content when compared to an unmodified tobacco plant, wherein said tobacco plant has been modified to modulate (e.g. increase) the activity or expression of at least one gene encoding a SOUL heme-binding protein.

In one embodiment, a tobacco plant according to the invention comprises a tobacco cell of the invention.

In another embodiment, the plant propagation material may be obtainable (e.g. obtained) from a tobacco plant of the invention.

In one embodiment, there is provided the use of a tobacco plant breeding tobacco plant as described herein.

In another embodiment, the present invention also provides the use of a tobacco plant of the preceding embodiment for the production of a tobacco industry product.

In another embodiment, there is provided the use of a tobacco plant of the invention for growing a crop.

In one embodiment, there is provided the use of a cell as provided in the preceding embodiments for the production of a tobacco industry product.

In one embodiment, the invention provides a plant cell or cell culture (e.g., an in vitro culture).

The tobacco cell culture may be a cell suspension culture. These in vitro cultured cells can be incorporated into tobacco industry products, for example, as substitutes for conventional tobacco particles, cut tobacco (shreds), fine or long cut tobacco sheets, as additive components, or as both substitutes and additives. Suitably, the cell culture may produce nicotine.

In one embodiment, there is provided the use of a cell culture, e.g. a harvested and/or processed cell culture according to the invention, for the production of a tobacco industry product.

Tobacco cells harvested from in vitro cultures may be dried, e.g., freeze dried, e.g., to produce a powder.

In one embodiment, the plant cell is a tobacco plant cell.

In one embodiment, the cell culture is a tobacco cell culture. The skilled person will be aware of known methods for establishing in vitro cultures of tobacco cells. By way of example only, the following methods may be used: the method includes the steps of collecting seeds from a tobacco plant of interest and sterilizing the exterior thereof to eliminate unwanted organisms, planting the seeds to grow the tobacco plant of interest, removing tissue from the tobacco plant (e.g., from tobacco stems) for use as an explant, establishing a callus culture from the tobacco explant, establishing a cell suspension culture from the callus culture, and harvesting culture material (e.g., including tobacco cells) to produce a tobacco cell culture.

Tobacco cells can be harvested by various methods, including filtration, such as vacuum filtration. The sample can be washed in the filter by adding water and the remaining liquid removed by filtration, e.g. vacuum filtration.

The harvested tobacco cells or cell cultures may be further processed, for example dried, such as air dried and/or freeze dried. Harvested tobacco cells or cell cultures or dried harvested tobacco cells or cell cultures or extracts thereof may be incorporated into tobacco industry products according to the invention.

In one embodiment, the invention provides a plant (e.g., a tobacco plant) or portion thereof for molecular planting. Suitably, the plants or parts thereof modified according to the invention may be used for the manufacture of proteins, such as therapeutic agents, e.g. antibiotics, virus-like particles, nutraceuticals or small molecules.

In one embodiment, the present invention provides a method for producing a protein (e.g., a therapeutic protein), the method comprising modifying a plant or portion thereof capable of producing the protein (e.g., a therapeutic protein) by modulating the activity or expression of at least one SOUL heme-binding protein gene encoding an amino acid sequence set forth in SEQ ID number 1, 4, 7, 10, 13, 16, 19, or 22, or a functional variant or functional fragment or ortholog thereof, or a sequence having at least 80% identity to SEQ ID number 1, 4, 7, 10, 13, 16, 19, or 22; or wherein at least one of the genes encoding a SOUL heme-binding protein comprises a nucleotide sequence set forth in SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, or 24, or a functional variant or fragment or ortholog of SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, or 24, or a nucleic acid sequence having at least 80% identity to SEQ ID number 2,3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, or 24; and culturing the plant under conditions sufficient to allow production of the protein (e.g., a therapeutic protein).

Product(s)

The invention also provides products obtainable from or obtained from the plants according to the invention. Products obtainable or obtained from plants in which the activity or expression of a gene encoding a SOUL heme-binding protein has been modulated are provided.

In one embodiment, the product may comprise a construct of the invention that modulates the activity or expression of at least one gene encoding a SOUL heme-binding protein as defined herein. In one embodiment, the product may comprise a construct of the invention that modifies the nucleic acid sequence of at least one gene encoding a SOUL heme-binding protein as defined herein.

The invention also provides products obtainable or obtained from the tobacco according to the invention.

In one embodiment, there is provided the use of a tobacco plant of the invention to produce tobacco lamina.

Suitably, the tobacco lamina may be subjected to downstream applications, such as processing.

Thus, in one embodiment, the use of the foregoing embodiments can provide a processed tobacco lamina. Suitably, the tobacco lamina may be subjected to conditioning, fermentation, pasteurization or a combination thereof. In another embodiment, tobacco lamina may be cut. In some embodiments, the tobacco lamina may be cut before or after being subjected to conditioning, fermentation, pasteurization, or combinations thereof.

In one embodiment, the invention provides harvested leaf of the tobacco plant of the invention.

In a further embodiment, the harvested leaf may be obtainable (e.g. obtained) from a tobacco plant propagated from the propagation material of the invention.

In another embodiment, harvested leaves obtainable from the method or use of the invention are provided.

Suitably, the harvested leaf may be a cut harvested leaf.

In some embodiments, the harvested leaf may comprise live tobacco cells. In other embodiments, the harvested leaves may be subjected to further processing.

Processed tobacco lamina is also provided.

The processed tobacco leaf may be obtainable from a tobacco plant of the invention. Suitably, the processed tobacco leaf may be obtainable from a tobacco plant obtained according to any method and/or use of the present invention.

In another embodiment, the processed tobacco leaf may be obtainable from a tobacco plant propagated from a tobacco plant propagation material according to the invention.

The processed tobacco lamina of the invention may be obtainable by processing the harvested lamina of the invention.

The term "processed tobacco lamina" as used herein refers to a tobacco lamina that has been subjected to one or more processing steps to which tobacco in the art has been subjected. "processed tobacco leaf" contains no or substantially no living cells.

The term "living cell" refers to a cell that is capable of growing and/or has metabolic activity. Thus, if a cell is said to be non-viable, also referred to as "non-viable," the cell does not exhibit the characteristics of a viable cell.

The term "substantially free of viable cells" means that less than about 5% of the total cells are viable. Preferably less than about 3%, more preferably less than about 1%, and even more preferably less than about 0.1% of the total cells are viable.

In one embodiment, processing tobacco lamina may be performed by one or more of the following: brewing, fermentation and/or pasteurization.

Suitably, the processed tobacco lamina may be processed by modulation.

The tobacco lamina may be cured by any method known in the art. In one embodiment, the tobacco lamina may be cured by one or more curing methods selected from the group consisting of: air-curing, open fire-curing, flue-curing and sun-curing.

Suitably, the tobacco lamina may be air cured.

Typically, air curing is accomplished by hanging tobacco lamina in a well-ventilated curing barn and allowing for drying. This is typically done during a period of four to eight weeks. The air-curing is particularly suitable for burley tobacco.

Suitably, the tobacco lamina may be cured by an open flame. Open fire curing is typically achieved by suspending the tobacco lamina in a large curing barn where the hardwood fire is left to fire continuously or intermittently with low smoldering at all times, and depending on the process and tobacco, typically takes three to ten weeks.

In another embodiment, the tobacco lamina may be flue cured. Flue-curing may include stringing tobacco leaves onto tobacco rods and hanging them on tobacco hanging rods (tier-poles) in a curing barn. Baking houses typically have flues that run in externally supplied fire boxes. Typically, this results in tobacco that has been thermally conditioned without exposure to smoke. Typically, the temperature is slowly increased during the course of the modulation, while the entire course takes about 1 week.

Suitably, the tobacco lamina may be cured by curing. Such methods typically involve exposing the uncovered tobacco to the sun.

Suitably, the processed tobacco leaf may be processed by fermentation.

Fermentation may be carried out in any manner known in the art. Typically, during fermentation, tobacco lamina is stacked into stacks of cured tobacco covered in, for example, burlap bags to retain moisture. The combination of the remaining water inside the lamina and the weight of the tobacco generates natural heat to mature the tobacco. The temperature in the center of the stack was monitored daily. In some methods, the entire stack is opened weekly. The blades are then removed to shake and wet, and the stack is rotated so that the inner blades are out and the bottom blades are placed on top of the stack. This ensures uniform fermentation throughout the pile. The additional moisture on the lamina, coupled with the actual rotation of the lamina itself, generates heat, thereby releasing the natural ammonia of the tobacco and reducing nicotine, while also darkening color and improving the aroma of the tobacco. Typically, the fermentation process extends for up to 6 months, depending on the variety of tobacco, the position of the stalk on the lamina, the thickness of the lamina and the intended use.

Suitably, the processed tobacco lamina may be processed by pasteurisation. Pasteurization may be particularly preferred when the tobacco lamina is to be used in the preparation of smokeless tobacco industry products, most preferably snus (snus).

Tobacco lamina pasteurization may be carried out by any method known in the art. For example, pasteurization can be performed with, for example, J cultures, L Ramstrom, M Burke, K growth. Effect of microbial on microbial and microbial health in Sweden. Tobacco Control (2003)12349-359 (the teachings of which are incorporated herein by reference) is described in detail.

In buccal cigarette production, pasteurization is typically carried out by a process in which the tobacco is heat treated with steam for 24-36 hours (to a temperature of about 100 ℃). This results in a nearly sterile product and without wishing to be bound by theory, one of the consequences of this is believed to limit further TSNA formation.

In one embodiment, the pasteurization process may be steam pasteurization.

In some embodiments, the processed tobacco lamina may be cut. The processed tobacco lamina may be cut before or after processing. Suitably, the fabricated tobacco lamina may be cut after processing.

In one embodiment, the use of the foregoing embodiments can provide reconstituted tobacco.

In one embodiment, reconstituted tobacco is provided.

"reconstituted" as used herein may also be referred to as reconstituted (recon), recycled or homogenized sheet tobacco, and refers to tobacco material produced from processed tobacco lamina residue. Reconstituted tobacco allows for the production of consistent, high quality blends and allows for the adjustment of the ratios of the various components.

Reconstituted tobacco may be either nanofibrous (nanofiber) reconstituted (nanofibers can be extracted in solid or liquid form), paper reconstituted (which uses stems, scraps and midribs as raw materials), or slurry type reconstituted (which uses a mixture of finely divided material and tobacco stems pulverized into powder, mixed with water and plant binders; flaking the soluble residues by extracting water).

Any method known in the art may be used to prepare reconstituted tobacco, see, for example, CORESTA consistency, Sapporo, 2012, cook Science/Product Technology Groups, SSPT 12 (incorporated herein by reference).

In some embodiments, tobacco plants, harvested leaves of tobacco plants, and/or processed tobacco leaves may be used to extract nicotine. Extraction of nicotine can be accomplished using any method known in the art. For example, a process for extracting nicotine from tobacco is taught in US2,162,738, which is incorporated herein by reference.

In one aspect, the invention provides a cured tobacco material made from a tobacco plant according to the invention or a part thereof.

In another aspect, the invention provides a tobacco blend comprising a tobacco material made from a tobacco plant or part thereof according to the invention, or a tobacco cell or cell culture according to the invention. In one aspect, the present invention provides a tobacco blend comprising a cured tobacco material according to the present invention.

Suitably, the tobacco blend according to the invention may comprise about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% tobacco from a tobacco plant or part thereof according to the invention or from a tobacco cell or cell culture according to the invention. Suitably, the tobacco blend may comprise about 10% tobacco from a tobacco plant or part thereof according to the invention or from a tobacco cell or cell culture according to the invention. Suitably, the tobacco blend may comprise about 20% tobacco from a tobacco plant or part thereof according to the invention or from a tobacco cell or cell culture according to the invention. Suitably, the tobacco blend may comprise about 30% tobacco from a tobacco plant or part thereof according to the invention or from a tobacco cell or cell culture according to the invention. Suitably, the tobacco blend may comprise about 40% tobacco from a tobacco plant or part thereof according to the invention or from a tobacco cell or cell culture according to the invention. Suitably, the tobacco blend may comprise about 50% tobacco from a tobacco plant or part thereof according to the invention or from a tobacco cell or cell culture according to the invention. Suitably, the tobacco blend may comprise about 60% tobacco from a tobacco plant or part thereof according to the invention or from a tobacco cell or cell culture according to the invention. Suitably, the tobacco blend may comprise about 70% tobacco from a tobacco plant or part thereof according to the invention or from a tobacco cell or cell culture according to the invention. Suitably, the tobacco blend may comprise about 80% tobacco from a tobacco plant or part thereof according to the invention or from a tobacco cell or cell culture according to the invention. Suitably, the tobacco blend may comprise about 90% tobacco from a tobacco plant or part thereof according to the invention or from a tobacco cell or cell culture according to the invention.

In one aspect, the tobacco blend product of the present invention comprises at least about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95 percent by dry weight of tobacco cured from a tobacco plant or portion thereof according to the present invention or a tobacco cell or cell culture according to the present invention.

Suitably, the cured tobacco material may be air cured. Suitably, the cured tobacco material may be flue cured. Suitably, the cured tobacco material may be cured by curing. Suitably, the cured tobacco material may be cured by an open flame.

Tobacco industry products or smoking articles according to the invention may comprise tobacco material (e.g. cured tobacco material or reconstituted tobacco material) according to the invention.

In another aspect, the invention provides a tobacco industry product.

In one embodiment, the tobacco industry product according to the invention may be a blended tobacco industry product. Suitably, the tobacco blend may comprise a cured tobacco material according to the invention.

In one embodiment, tobacco industry products can be prepared from the tobacco plants of the present invention or parts thereof.

Suitably, a tobacco plant or part thereof may be propagated from a tobacco plant propagation material according to the present invention.

The term "part thereof" as used herein in the context of a tobacco plant refers to a portion of a tobacco plant. Suitably, a "part thereof" may be a leaf, root or stem or flower of a tobacco plant. Suitably, a "part thereof" may be a leaf, root or stem of a tobacco plant.

Tobacco industry products

As used herein, the term "tobacco industry product" is intended to include combustible smoking articles, such as cigarettes, cigarillos, cigars, tobacco for pipes or cigarettes (whether based on tobacco, tobacco derivatives, expanded tobacco, reconstituted tobacco, tobacco substitutes or other smokable materials), non-combustible aerosol provision systems, such as heating products that release compounds from a matrix material without combustion, such as e-cigarettes, tobacco heating products, and hybrid systems that generate aerosols from combinations of matrix materials, e.g. hybrid systems containing a liquid or gel or solid matrix, and aerosolizable matrix materials for use within these aerosol provision systems; and aerosol-free delivery articles, such as lozenges, chewing gums, patches, articles containing powders that can be inhaled, and smokeless tobacco industry products such as snus and snuff, which may or may not deliver nicotine.

In one embodiment, a tobacco industry product can be prepared from (e.g., can comprise) a tobacco plant of the invention or a portion thereof.

Suitably, a tobacco plant or part thereof may be propagated from a tobacco plant propagation material according to the present invention.

As used herein in the context of a tobacco plant, the term "a portion thereof" refers to a portion of a tobacco plant. Preferably, the "part thereof" is a lamina of a tobacco plant.

In another embodiment, tobacco industry products may be prepared from harvested leaf blades of the present invention.

In a further embodiment, tobacco industry products may be prepared from the processed tobacco lamina of the present invention.

Suitably, the tobacco industry product may be prepared from tobacco lamina processed by one or more of the following: brewing, fermentation and/or pasteurization.

Suitably, the tobacco industry product may comprise cut tobacco lamina, optionally processed according to the previous embodiments.

In another embodiment, a tobacco industry product may be prepared from a tobacco cell or cell culture according to the invention.

In another embodiment, a tobacco industry product may be prepared from (e.g., may comprise) a cured tobacco material according to the present invention.

In another embodiment, a tobacco industry product may be prepared from (e.g., may comprise) a tobacco blend according to the present invention.

In one embodiment, the tobacco industry product may be a smoking article.

As used herein, the term "smoking article" may include smokeable products, such as cigarettes, cigars and cigarillos, whether based on tobacco, tobacco derivatives, expanded tobacco, reconstituted tobacco or tobacco substitutes.

In another embodiment, the tobacco industry product can be a smokeless tobacco industry product.

The term "smokeless tobacco industry product" as used herein refers to tobacco industry products that are not intended to be smoked and/or subjected to combustion.

Smokeless tobacco industry products, including heat-not-burn materials, may contain tobacco in any form, including dry granules, cut filler, granules, powders, or slurries, deposited on, mixed in, surrounded by, or combined with other ingredients, in any form, such as sheets, films, tabs, foams, or beads.

In one embodiment, the smokeless tobacco industry products can include mouth tobacco, snuff, chewing tobacco, and the like.

In one embodiment, the tobacco industry product is a combustible smoking article selected from cigarettes, cigarillos and cigars.

In one embodiment, the tobacco industry product comprises one or more components of a combustible smoking article, such as a filter, a filter rod (filter rod), a filter rod segment (filter rod segment), tobacco, a tobacco rod segment, a plug (spill), an additive releasing component such as a capsule, a thread, beads, paper such as plug wrap (plug wrap), tipping paper or cigarette paper.

In one embodiment, the tobacco industry product is a non-combustible aerosol provision system.

In one embodiment, the tobacco industry product comprises one or more components of a non-combustible aerosol provision system, such as a heater and an aerosolizable substrate.

In one embodiment, the aerosol provision system is an electronic cigarette, also known as a vaping device (vaping device).

In one embodiment, the electronic cigarette includes a heater, a power source capable of powering the heater, an aerosolizable matrix such as a liquid or gel, a shell, and an optional interface.

In one embodiment, the aerosolizable substrate is contained in a substrate container. In one embodiment, the substrate container is combined with or includes a heater.

In one embodiment, the tobacco industry product is a heating product that releases one or more compounds by heating rather than burning the matrix material. The matrix material is an aerosolizable material, which may be, for example, tobacco or other non-tobacco products, which may or may not contain nicotine. In one embodiment, the heating product is a tobacco heating product.

In one embodiment, the heating product is an electronic device.

In one embodiment, the tobacco heating product comprises a heater, a power source capable of powering the heater, an aerosolizable matrix such as a solid or gel material.

In one embodiment, the heating product is a non-electronic article.

In one embodiment, the heating product comprises an aerosolizable matrix, such as a solid or gel material, and a heat source capable of supplying thermal energy to the aerosolizable matrix without any electronic means, such as by burning a combustion material, such as charcoal.

In one embodiment, the heating product further comprises a filter capable of filtering an aerosol generated by heating the aerosolizable substrate.

In some embodiments, the aerosolizable matrix material can comprise a vapor or aerosol generating agent or humectant, such as glycerin, propylene glycol, glyceryl triacetate, or diethylene glycol.

In one embodiment, the tobacco industry product is a hybrid system to generate an aerosol by heating without combusting a combination of matrix materials. The matrix material may comprise, for example, a solid, liquid or gel, which may or may not contain nicotine. In one embodiment, the hybrid system includes a liquid or gel matrix and a solid matrix. The solid substrate may be, for example, tobacco or other non-tobacco products, which may or may not contain nicotine. In one embodiment, the hybrid system comprises a liquid or gel matrix and tobacco.

In further embodiments, the tobacco industry product may be a tobacco heating device or a hybrid device or an electronic cigarette or the like.

Typically in tobacco heating or hybrid devices, aerosols are generated by transferring heat from a heat source to a physically separate aerosol-forming substrate or material, which may be located within, around or downstream of the heat source. During smoking, volatile compounds are released from the aerosol-forming substrate by heat transfer from the heat source and become entrained in the air drawn through the smoking article. As the released compounds cool, they condense to form an aerosol which is inhaled by the user.

Aerosol generating articles and devices for consuming or smoking tobacco heating devices are known in the art. They may comprise, for example, electrically heated aerosol generating devices in which the aerosol is generated by transferring heat from one or more electrical heating elements of the aerosol generating device to an aerosol-forming substrate of the tobacco heating device.

Suitably, the tobacco heating apparatus may be an aerosol generating apparatus.

Preferably, the tobacco heating apparatus may be a heat non-combustion apparatus. Heat non-combustion devices are known in the art and release compounds by heating rather than burning tobacco.

An example of a suitable, heat non-combustible apparatus may be that taught in WO2013/034459 or GB2515502, which are incorporated herein by reference.

In one embodiment, the aerosol-forming substrate of the tobacco heating apparatus may be a tobacco industry product according to the invention.

In one embodiment, the tobacco heating apparatus may be a hybrid apparatus.

Polynucleotide/polypeptide/construct

In certain embodiments of the present invention, constructs that modulate the activity or expression of at least one gene encoding a SOUL heme-binding protein may be transformed into plant cells, suitably under the direction of a promoter.

In certain embodiments of the invention, a construct that reduces (i.e., inhibits) the activity or expression of at least one gene encoding a SOUL heme-binding protein under the direction of a promoter may be transformed into a plant cell. For example, the genetic construct may be a gene editing construct or may comprise an RNAi molecule, which may comprise a small interfering rna (sirna) molecule or a short hairpin loop (shRNA) molecule.

In certain embodiments of the invention, a construct that increases the activity or expression of a gene encoding a SOUL heme-binding protein, e.g., a construct of a gene encoding a SOUL heme-binding protein, such as an endogenous SOUL heme-binding protein, may be transformed into a plant cell, suitably under the direction of a promoter.

The constructs may be introduced into the plants according to the invention with the aid of suitable vectors, for example plant transformation vectors. The plant transformation vector may comprise an expression cassette comprising, in the direction of transcription 5' -3', a promoter sequence, a construct sequence targeting a gene encoding a SOUL heme-binding protein and optionally a 3' untranslated terminator sequence, including a termination signal for RNA polymerase and a polyadenylation signal for polyadenylase. The promoter sequence may be present in one or more copies, and such copies may be the same or variants of the promoter sequence as described above. The terminator sequence may be derived from a plant, bacterial or viral gene. Suitable terminator sequences are, for example, pearbcS E9Terminator sequence derived from Agrobacterium tumefaciens (A)Agrobacterium tumefaciens) Of the nopaline synthase genenosTerminator sequences, and from cauliflower mosaic virus35SA terminator sequence. Other suitable terminator sequences will be readily known to those skilled in the art.

The constructs of the invention may also comprise gene expression enhancing mechanisms to increase the strength of the promoter. An example of such an enhancer element is an enhancer element derived from part of the promoter of the pea plastocyanin gene and is the subject of international patent application No. WO 97/20056, which is incorporated herein by reference. Suitable enhancer elements may be, for example, the nopaline synthase gene derived from Agrobacterium tumefaciensnosEnhancer element, and from cauliflower mosaic virus35SAn enhancer element.

These regulatory regions may be derived from the same gene as the promoter DNA sequence, or may be derived from tobaccoGrasses or other organisms, e.g. from plants of the Solanaceae family or from the subfamily, the Asclepiadaceae (Cestroideae) Of (2) or (ii) a different gene of (c). All regulatory regions should be able to function in the cells of the tissue to be transformed.

The promoter DNA sequence may be derived from the same gene as the gene of interest, e.g. the gene the promoter is intended to direct, e.g. the gene encoding the SOUL heme-binding protein according to the invention, the coding sequence used in the invention, or may be derived from a different gene from tobacco or another organism, e.g. from a plant of the solanaceae family or from the subfamily amygdalina subfamily.

The expression cassette can be incorporated into a base plant transformation vector, such aspBIN 19 Plus、pBI 101pKYLX71:35S2, pCAMBIA2300, or other suitable plant transformation vectors known in the art. In addition to the expression cassette, the plant transformation vector will contain such sequences as are necessary for the transformation process. These may include Agrobacterium (A) or (B)Agrobacterium)virGenes, one or more T-DNA border sequences (border sequences), and selectable markers or other means of identifying transgenic plant cells.

The term "expression vector or plant transformation vector" means a construct capable of expression in vivo or in vitro. Preferably, the expression vector is incorporated into the genome of the organism. In one embodiment, the vectors of the invention express a protein, such as a SOUL heme-binding protein as described herein. The term "incorporated" preferably includes stable incorporation into the genome.

Techniques for transforming plants are well known in the art and include, for example, Agrobacterium-mediated transformation. The rationale in the construction of genetically modified plants is to insert genetic information into the plant genome in order to obtain a stable maintenance of the inserted genetic material. An overview of the general technology may be found in the synthesis of peptides by Potrykus (A), (B), (C), (Annu Rev Plant Physiol Plant Mol Biol [1991]42: 205-.

Generally, in Agrobacterium-mediated transformation, the transformation is carried out by Agrobacterium with a vector from a target plantCo-cultivation of the explants, a binary vector (construct according to the invention) carrying the foreign DNA of interest, is transferred from the appropriate agrobacterium strain to the target plant. Transformed plant tissue is then regenerated on a selection medium comprising a selectable marker and a plant growth hormone. An alternative is the floral dip (Clough)&Bent, 1998 Plant J.1998 month 12; 735-43, incorporated herein by reference), whereby flower buds of intact plants are contacted with a suspension of an agrobacterium strain containing the chimeric gene, and after setting, transformed individuals are germinated and identified by growth on a selection medium. Direct infection of plant tissue by Agrobacterium is a simple technique which has been widely adopted and is described in Butcher et al, (1980),Tissue Culture Methods for Plant PathologistsD.S. implants and J.P. Helgeson, 203-208, incorporated herein by reference.

Further suitable transformation methods include, for example, direct gene transfer into protoplasts using polyethylene glycol or electroporation techniques, particle bombardment, microinjection, and the use of silicon carbide fibers. Transformation of plants using impact transformation (balistic transformation) and production of fertile transgenic maize plants by silicon carbide whisker-mediated transformation is taught in Frame et al (1994) The Plant Journal 6(6): 941-948 (which is incorporated herein by reference), and viral transformation techniques are taught, for example, in Meyer et al (1992) mol. Gen. Gene. 231(3): 345-352 (which is incorporated herein by reference). The use of cassava mosaic virus as a vector system for plants is taught in Meyer et al (1992) Gene 110: 213-217, which is incorporated herein by reference. Further teachings on plant transformation can be found in EP-A-0449375, which is incorporated herein by reference.

In a further aspect, the invention relates to a vector system which carries the construct and introduces it into the genome of an organism, such as a plant, suitably a tobacco plant. The vector system may comprise one vector, but it may comprise two vectors. In the case of two vectors, the vector system is usually referred to as dualA meta-vector system. Binary vector systems are described in Gynheung et al, (1980), Binary Vectors,Plant Molecular Biology Manual a3, 1-19, which references are incorporated herein by reference.

A widely used system for transforming plant cells uses Ti plasmids from Agrobacterium tumefaciens or from Agrobacterium rhizogenes (R) ((R))Agrobacterium rhizogenes) The Ri plasmid of (1), which was obtained by An et al (1986) Plant Physiol.81, 301-305 and Butcher et al (1980)Tissue Culture Methods for Plant PathologistsD.S. Ingrams and J.P. Helgeson, 203-208, which are incorporated herein by reference. After each method of introduction of the desired foreign gene according to the invention in plants, the presence and/or insertion of further DNA sequences may be necessary. The use of T-DNA for Plant cell transformation has been well studied and is described in EP-A-120516, HoekemｃA (1985) The Binary Plant Vector System, Offset-drukkerij Kanters B.B., Amsterdam Chapter V, Fraley et al Crit. Rev. Plant Sci.4:1-46, and An et al (1985) EMBO J4: 277-284, which is incorporated herein by reference.

Plant cells transformed with one or more constructs that modulate the activity or expression of at least one gene encoding a SOUL heme-binding protein may be grown and maintained according to well known tissue culture methods, such as by culturing the cells in a suitable medium supplied with essential growth factors such as amino acids, plant hormones, vitamins, and the like.

The term "transgenic plant" in connection with the present invention includes any plant comprising a construct which modulates the activity or expression of at least one gene encoding a SOUL heme-binding protein according to the present invention. Accordingly, a transgenic plant is a plant which has been transformed with a construct according to the invention. Preferably, according to the present invention, the transgenic plant exhibits a modulated alkaloid content and/or a modulated TSNA content (or precursors thereof). The term "transgenic plant" does not include a native nucleotide coding sequence that is in its natural environment when under the control of its native promoter (which is also in its natural environment).

In one aspect, a gene, construct, plant transformation vector or plant cell encoding a SOUL heme-binding protein according to the invention is in isolated form. The term "isolated" means that the sequence is at least substantially free of at least one other component with which the sequence is naturally associated in nature and as found in nature.

In one aspect, a gene, construct, plant transformation vector or plant cell encoding a SOUL heme-binding protein according to the invention is in a purified form. The term "purified" means in a relatively pure state, e.g., at least about 90% pure, or at least about 95% pure or at least about 98% pure.

The term "nucleotide sequence" as used herein refers to an oligonucleotide sequence or a polynucleotide sequence, as well as variants, homologues, fragments and derivatives thereof (such as portions thereof). The nucleotide sequence may be of genomic or synthetic or recombinant origin, which may be double-stranded or single-stranded, whether representing the sense or antisense strand.

The term "nucleotide sequence" in connection with the present invention includes genomic DNA, cDNA, synthetic DNA and RNA. Preferably, it means a DNA, more preferably a cDNA sequence, encoding the present invention.

In a preferred embodiment, the nucleotide sequence, i.e. the gene encoding a SOUL heme-binding protein, when relevant to and encompassed by the scope of the invention itself includes the native nucleotide sequence when in its natural environment and when it is linked to one or more of its naturally associated sequences also in its natural environment. For ease of reference, we shall refer to this preferred embodiment as the "native nucleotide sequence". In this respect, the term "native nucleotide sequence" means the entire nucleotide sequence that is in its natural environment and, when operably linked to the entire promoter with which it is naturally associated, the promoter is also in its natural environment.

The nucleotide sequence for use in the present invention may be present in a vector, wherein the nucleotide sequence is operably linked to a regulatory sequence capable of providing for expression of the nucleotide sequence by a suitable host organism. Constructs for use in the present invention may be transformed into a suitable host cell as described herein to provide for expression of a polypeptide of the present invention. The choice of a vector, such as a plasmid, cosmid, or phage vector, often depends on the host cell into which it is to be introduced. The vector may be used in vitro, for example, for the production of RNA, or for transfection, transformation, transduction, or infection of a host cell.

In some applications, the nucleotide sequence for use in the present invention is operably linked to a regulatory sequence capable of providing for expression of the nucleotide sequence, e.g., by a selected host cell. For example, the invention includes a vector comprising the nucleotide sequence of a gene encoding a SOUL heme-binding protein as described herein operably linked to such regulatory sequences, i.e., the vector is an expression vector.

The term "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A regulatory sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.

The term "regulatory sequence" includes promoters and enhancers and other expression regulatory signals. The term "promoter" is used in the usual sense in the art, e.g., an RNA polymerase binding site. The nucleotide sequence within the construct encoding the gene encoding the SOUL heme-binding protein may be operably linked to at least one promoter.

The term "construct" synonymous with terms such as "cassette" or "vector" includes a nucleotide sequence for use according to the present invention attached directly or indirectly to a promoter.

An example of indirect attachment is the provision of a suitable spacer group, such as an intron sequence, such as the Sh 1-intron or the ADH intron, intermediate to the promoter and the nucleotide sequence of the present invention. The same is true for the term "fused" in connection with the present invention, which includes direct or indirect attachment. In some cases, the term does not include the natural combination of nucleotide sequences encoding proteins normally associated with wild-type gene promoters, as well as when they are both in their natural environment. The construct may even contain or express a marker that allows for selection of the genetic construct.

In some embodiments, the promoter may be operably linked to a nucleotide sequence in a construct or vector for modulating the concentration and/or total content of nicotine in a cell or cell culture or tobacco plant or portion thereof.

In some embodiments, the promoter may be selected from: constitutive promoters, tissue specific promoters, developmentally regulated promoters, and inducible promoters.

In one embodiment, the promoter may be a constitutive promoter.

Constitutive promoters direct the expression of genes continuously throughout various parts of a plant during plant development, although genes may not be expressed at the same level in all cell types. Examples of known constitutive promoters include those associated with: cauliflower mosaic virus 35S transcript (Odell JT, Nagy F, Chua NH. (1985), Identification of DNA sequences required for Activity of the cauliflower mosaic virus 35S promoter, Nature 313810-2), rice actin 1 gene (Zhang W, McElroy D, Wu R. (1991), Analysis of rice Act 15' region Activity in transgenic Plant Cell 31155-65) and maize ubiquitin 1 gene (Cornejo MJ, Luth D, Blakeship KM, Anderson OD, Blechll et al (1993) AE video of a maize ubiquitin promoter in promoter 23567). Constitutive promoters, such as The carnation corrosion ring virus (CERV) promoter (Hull R, Sadler J, Longstaff M (1986) (CaMV/35S), The figwort mosaic virus (figwort mosaic virus) 35S promoter The sequence of verified virus DNA, complex with a cauliflower mosaic virus and retroviruses, EMBO Journal, 5(2):3083 and 3090).

The constitutive promoter may be selected from: a carnation ringworm virus (CERV) promoter, a cauliflower mosaic virus (CaMV 35S promoter), a promoter from the rice actin 1 gene or the maize ubiquitin 1 gene.

The promoter may be a tissue specific promoter. Tissue-specific promoters are promoters that direct the expression of genes in one (or several) parts of a plant, usually throughout the life of those plant parts. The class of tissue-specific promoters also typically includes promoters whose specificity is not absolute, i.e., they may also direct expression at lower levels in tissues other than the preferred tissue. Tissue-specific promoters include phaseolin promoter, legumin B4-promoter, usp-promoter, sbp-promoter, ST-LS1 promoter, B33 (patatin) class I promoter).

In another embodiment, the promoter may be a developmentally regulated promoter.

Developmentally regulated promoters direct changes in the expression of a gene in one or more parts of a plant at specific times during plant development. Genes may be expressed at different (usually lower) levels in the plant part at other times, and may also be expressed in other plant parts.

In one embodiment, the promoter may be an inducible promoter.

Inducible promoters are capable of directing the expression of a gene in response to an inducer. In the absence of an inducer, the gene will not be expressed. The inducer may act directly on the promoter sequence or may act by counteracting the effect of the repressor molecule. The inducer may be a chemical agent such as a metabolite, a protein, a growth regulator (such as auxin and salicylic acid that activates the OCS promoter) or a toxic element, a physiological stress such as heat, light (such as the soybean SSU promoter), injury (e.g. nos, nopaline synthase promoter), or osmotic pressure, or an indirect consequence of the action of a pathogen or pest. Developmentally regulated promoters can be described as specific types of inducible promoters that respond to endogenous inducers produced by a plant or environmental stimuli at specific times in the plant's life history. Examples of known inducible promoters include those associated with wound responses such as described by Warner SA, Scott R, Draper J. ((1993) Plant J. 3191-201), temperature responses such as disclosed by Benfey & Chua (1989) (Benfey, P.N., and Chua, N-H. ((1989) Science 244174-181), and chemical induction such as described by Gatz ((1995) Methods in Cell biol. 50411-424).

A nucleotide sequence encoding a protein having the specific properties of a gene encoding a SOUL heme-binding protein as defined herein or a protein suitable for modification may be identified and/or isolated and/or purified from any cell or organism producing said protein. Various methods for the identification and/or isolation and/or purification of nucleotide sequences are well known in the art. For example, once the appropriate sequence has been identified and/or isolated and/or purified, PCR amplification techniques can be used to prepare further sequences.

In a still further alternative, the nucleotide sequence encoding the SOUL heme-binding protein may be prepared synthetically by established standard methods, e.g., by Beucage et al (1981)Tetrahedron Letters22, 1859-1869 (incorporated herein by reference) or by Matthes et al (1984)EMBO J3, 801-805 (which is incorporated herein by reference). In the phosphoramidite method, oligonucleotides are synthesized, purified, annealed, ligated and cloned in appropriate vectors, for example in an automated DNA synthesizer.

As used herein, the term "amino acid sequence" is synonymous with the term "polypeptide" and/or the term "protein". In some instances, the term "amino acid sequence" is synonymous with the term "peptide". In some instances, the term "amino acid sequence" is synonymous with the term "enzyme".

The present invention also encompasses the use of sequences having a degree of sequence identity or sequence homology (hereinafter referred to as "homologous sequence(s)") to one or more amino acid sequences of a polypeptide having the specific properties defined herein, or any nucleotide sequence, i.e., the SOUL heme-binding protein gene encoding such a polypeptide. Herein, the term "homologue" means an entity having a certain homology with the subject amino acid sequence and the subject nucleotide sequence. Here, the term "homology" may be equivalent to "identity".

The homologous amino acid sequences and/or nucleotide sequences and/or fragments should provide and/or encode polypeptides that retain the functional activity of the SOUL heme-binding protein gene and/or enhance the activity of the SOUL heme-binding protein gene. Typically, homologous sequences will comprise, for example, the same active site or the like as the subject amino acid sequence, or will encode the same active site. Although homology may also be considered in terms of similarity (i.e. amino acid residues with similar chemical properties/functions), in the context of the present invention it preferably denotes homology in terms of sequence identity. Homologous sequences typically retain a functional domain or motif. Suitably, a homologue of the SOUL heme-binding protein may contain a heme-binding site.

In one embodiment, homologous sequences are employed to include amino acid sequences or nucleotide sequences having one, two or several additions, deletions and/or substitutions as compared to the subject sequence.

Sequence identity

Sequence identity comparisons can be performed by eye or, more commonly, by means of readily available sequence comparison programs. These commercially available computer programs can calculate% homology between two or more sequences. % homology or% identity can be calculated over contiguous sequences, i.e., one sequence is aligned with the other and each amino acid in one sequence is directly compared to the corresponding amino acid in the other sequence, one residue at a time. This is called an "unnotched" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.

Although this is a very simple and consistent method, it fails to take into account, for example, that in an otherwise equivalent pair of sequences, an insertion or deletion will result in the following amino acid residues not being aligned, thus potentially resulting in a substantial reduction in% homology when making an overall alignment. Thus, most sequence comparison methods are designed to produce optimal alignments that take into account possible insertions and deletions without unduly penalizing the overall homology score. This is achieved by inserting "gaps" in the sequence alignment in an attempt to maximize local homology.

However, these more complex methods assign a "gap penalty" to each gap that occurs in the alignment, such that for the same number of identical amino acids, an alignment of sequences with as few gaps as possible-reflecting a higher correlation between the two compared sequences-will result in a higher score than an alignment with many gaps. An "Affine gap cost" (Affine gap cost) is typically used, which imposes a relatively high cost for the presence of a gap, while imposing a small penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties will of course result in an optimized alignment with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, when using such software for sequence comparison, it is preferred to use default values.

Therefore, the calculation of maximum% homology first requires the generation of an optimal alignment taking into account gap penalties. A suitable computer program for performing this alignment is Vector NTI (Invitrogen Corp.). Examples of software that can be compared include, but are not limited to, for example, the BLAST package (see Ausubel et al 1999 Short Protocols in Molecular Biology, 4 th edition-18 th chapter), BLAST 2 (see FEMS Microbiol Lett 1999174 (2): 247-50; FEMS Microbiol Lett 1999177 (1): 187-8 and[email protected] ncbi.nlm.nih.gov) FASTA (Altschul et al 1990J. mol. biol. 403-. At least BLAST, BLAST 2, and FASTA are available for offline and online searches (see Ausubel et al 1999, pages 7-58 to 7-60).

Although the final% homology can be measured in terms of identity, the alignment process itself is generally not based on an all or no pairwise comparison. Instead, a scaled similarity score matrix is typically used that assigns a score to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix that is commonly used is the BLOSUM62 matrix-the default matrix of the BLAST suite of programs. The Vector NTI program typically uses public default values or convention symbol comparison tables (if provided) (see user manual for further details). For some applications, it is preferable to use the default values of the Vector NTI package.

Alternatively, multiple alignment features in Vector NTI (Invitrogen Corp.) can be used for percent homology calculation based on an algorithm similar to CLUSTAL (Higgins DG & Sharp PM (1988), Gene 73(1), 237- & 244). Once the software has produced an optimal alignment, it is possible to calculate% homology, preferably% sequence identity. Software typically performs this as part of a sequence comparison and generates numerical results.

If gap penalties should be used when determining sequence identity, the following parameters are preferably used for pairwise alignments:

for BLAST
		GAP OPEN (GAP OPEN)	0
GAP EXTENSION (GAP EXTENSION)	0

For CLUSTAL	DNA	Protein
				Word length	2	1	K triad
Gap penalties	15	10
				Extension of the notch	6.66	0.1

In one embodiment, CLUSTAL can be used with the gap penalty and gap extension set as defined above. In some embodiments, the gap penalties for BLAST or CLUSTAL alignments may be different from those detailed above. The skilled artisan will appreciate that the standard parameters used to perform BLAST and CLUSTAL alignments may be changed periodically, and will be able to select the appropriate parameters based on the standard parameters detailed for the BLAST or CLUSTAL alignment algorithms at the time.

Suitably, the nucleotide sequence is determined over at least 50 contiguous nucleotides, preferably over at least 60 contiguous nucleotides, preferably over at least 70 contiguous nucleotides, preferably over at least 80 contiguous nucleotides, preferably over at least 90 contiguous nucleotides, preferably over at least 100 contiguous nucleotides, preferably over at least 150 contiguous nucleotides, preferably over at least 200 contiguous nucleotides, preferably over at least 250 contiguous nucleotides, preferably over at least 300 contiguous nucleotides, preferably over at least 350 contiguous nucleotides, preferably over at least 400 contiguous nucleotides, preferably over at least 450 contiguous nucleotides, preferably over at least 500 contiguous nucleotides, preferably over at least 550 contiguous nucleotides, preferably over at least 600 contiguous nucleotides, preferably over at least 650 contiguous nucleotides, or preferably over at least 700 contiguous nucleotides, the degree of identity with respect to the nucleotide sequence is determined.

Suitably, the degree of identity with respect to a nucleotide, cDNA, cds or amino acid sequence may be determined over the entire sequence.

The sequences may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent substance. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues, so long as the secondary binding activity of the substance is retained. 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 hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.

Conservative substitutions may be made, for example, according to the following table. Amino acids in the same group in the second column, and preferably in the same row in the third column, may be substituted for each other:

。

the present invention also includes homologous substitutions (both substitutions and replacements are used herein to mean the interchange of existing amino acid residues with substitute residues), i.e., homologous (like-for-like) substitutions, such as basic for basic, acidic for acidic, polar for polar, and the like, that may occur. Nonhomologous substitutions may also occur, i.e., from one class of residue to another, or on the other hand involve the inclusion of unnatural amino acids such as ornithine (hereinafter referred to as Z), diaminobutyric acid ornithine (hereinafter referred to as B), norleucine ornithine (hereinafter referred to as O), pyridylalanine, thienylalanine, naphthylalanine and phenylglycine.

Substitutions may also be made with unnatural amino acids, including; alpha and alpha-disubstituted amino acids, N-alkyl amino acids, lactic acid, halide derivatives of natural amino acids such as trifluorotyrosine, p-Cl-phenylalanine, p-Br-phenylalanine, p-I-phenylalanine, L-allyl-glycine, beta-alanine, L-alpha-aminobutyric acid, L-gamma-aminobutyric acid, L-alpha-aminoisobutyric acid, L-epsilon-aminocaproic acid^#7-aminoheptanoic acid, L-methionine sulfone^#L-norleucine, L-norvaline, p-nitro-L-phenylalanine, L-hydroxyproline^#L-Thioproline, methyl derivatives of phenylalanine (Phe) such as 4-methyl-Phe, pentamethyl-Phe, L-Phe (4-amino)^#L-Tyr (methyl), L-Phe (4-isopropyl), L-Tic (1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid), L-diaminopropionic acid^#And L-Phe (4-benzyl). The notation, # has been used for the purposes discussed above (in relation to homologous or non-homologous substitutions) to indicate the hydrophobic character of the derivative, while # has been used to indicate the hydrophilic character of the derivative, # indicates the amphipathic character.

In addition to amino acid spacers such as glycine or β -alanine residues, variant amino acid sequences can include suitable spacers, which can be inserted between any two amino acid residues of the sequence, including alkyl groups such as methyl, ethyl, or propyl. Further variations relate to the presence of one or more amino acid residues in the peptidomimetic form, as will be well understood by those skilled in the art. For the avoidance of doubt, "peptidomimetic form" is used to refer to variant amino acid residues in which the alpha-carbon substituent is located on the nitrogen atom of the residue rather than on the alpha-carbon. Methods for preparing peptides in peptidomimetic form are known in the art, for example, Simon et al (1992) PNAS 89(20), 9367-.

The nucleotide sequence used in the present invention may include a synthetic or modified nucleotide therein. Many different types of modifications to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones and/or acridine or polylysine chain additions at the 3 'and/or 5' ends of the molecule. For the purposes of the present invention, it is understood that the nucleotide sequences described herein may be modified by any method available in the art. Such modifications may be made to enhance the in vivo activity or longevity of the nucleotide sequences of the present invention.

The invention also includes sequences that are complementary to the nucleic acid sequences of the invention, or sequences that are capable of hybridizing to the sequences of the invention or to sequences complementary thereto. The term "hybridization" as used herein shall include "the process of joining with a complementary strand through base pairing of its nucleic acid strand" as well as the process of amplification as carried out in Polymerase Chain Reaction (PCR) techniques.

The invention also relates to nucleotide sequences that can hybridize to the nucleotide sequences of the invention (including the complements of those presented herein). Preferably, hybridization is determined under stringent conditions (e.g., 50 ℃ and 0.2xSSC {1xSSC = 0.15M NaCl, 0.015M sodium citrate, pH 7.0 }). More preferably, hybridization is determined under high stringency conditions (e.g., 65 ℃ and 0.1xSSC {1xSSC = 0.15M NaCl, 0.015M sodium citrate, pH 7.0 }).

An overview of the general techniques used to transform plants can be found in papers such as Potrykus et al (1991) Annu Rev Plant physiol. Plant mol. biol. 42:205-225 and Christou et al (1994) Agro-Food-Industry Hi-Tech March/April 17-27, which are incorporated herein by reference. Further teachings on plant transformation can be found in EP-A-0449375, which is incorporated herein by reference.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Singleton et al, DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20 ED., John Wiley AND Sons, New York (1994), AND Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide the skilled artisan with a general DICTIONARY OF many OF the terms used in this disclosure.

The present disclosure is not limited to the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present disclosure. Numerical ranges include the numbers defining the range. Unless otherwise indicated, any nucleic acid sequence is written left to right in a 5 'to 3' orientation, respectively; amino acid sequences are written from left to right in the amino to carboxyl orientation.

The headings provided herein are not limitations of the various aspects or embodiments of the disclosure which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

Amino acids are referred to herein using the name of the amino acid, a three-letter abbreviation, or a single letter abbreviation. As used herein, the term "protein" includes proteins, polypeptides and peptides. As used herein, the term "amino acid sequence" is synonymous with the term "polypeptide" and/or the term "protein". In some instances, the term "amino acid sequence" is synonymous with the term "peptide". In some instances, the term "amino acid sequence" is synonymous with the term "enzyme".

In the present disclosure and claims, the conventional single and three letter codes for amino acid residues may be used. The 3-letter code of an amino acid is as defined in accordance with the IUPACIUB Joint Commission on Biochemical Nomenclature (JCBN). It is also understood that due to the degeneracy of the genetic code, a polypeptide may be encoded by more than one nucleotide sequence.

Other definitions of terms may appear throughout the specification. Before the exemplary embodiments are described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in or excluded from the range, depending on the particular exclusion limit in the stated range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the disclosure. Where a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

It must be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an enzyme" or "nitrate reductase" includes a variety of such candidate agents and equivalents thereof known to those skilled in the art, and so forth.

Advantages of the invention

It has been surprisingly found that by modulating the activity or expression of at least one gene encoding a SOUL heme-binding protein which acts as a positive regulator of nicotine in tobacco as taught herein, alkaloid content (e.g., nicotine and/or nornicotine and/or PON and/or anatabine and/or neonicotinoid and/or oxylamine content or total alkaloid content) and/or TSNA content of a plant can be modulated. Thus, tobacco products can be produced having modulated alkaloid (e.g., nicotine and/or nornicotine and/or PON and/or anatabine and/or anabasine and/or myodoxine content or total alkaloid content) and/or TSNA content, as well as commercially desirable traits sought by tobacco product consumers.

The present inventors have surprisingly determined a method of modulating alkaloid content (e.g. nicotine and/or nornicotine and/or PON and/or anatabine and/or anabasine and/or mugwort content or total alkaloid content) and/or TSNA content in a plant (e.g. a tobacco plant) by modulating the activity or expression of a gene encoding a SOUL heme-binding protein. Alkaloid (e.g., nicotine and/or nornicotine and/or PON and/or anatabine and/or anabasine and/or oxylamine content or total alkaloid content) and/or TSNA content of a plant (e.g., a tobacco plant) can be reduced by reducing or inhibiting the activity or expression of a gene encoding a SOUL heme-binding protein. Alkaloid (e.g., nornicotine or nicotine content) or TSNA content of a plant (e.g., a tobacco plant) can be increased by increasing the activity or expression of a gene encoding a SOUL heme-binding protein. Prior to the present invention, it was not known that modulation of the activity or expression of a gene encoding a SOUL heme-binding protein as described herein could be used to modulate alkaloid (e.g., nicotine and/or nornicotine and/or PON and/or anatabine and/or anabasine and/or mugwort content or total alkaloid content) and/or TSNA content in a plant (e.g., a tobacco plant).

The present inventors have determined that inhibition of the activity or expression of a gene encoding a SOUL heme-binding protein can reduce the alkaloid content (e.g., nicotine and/or nornicotine and/or PON and/or anatabine and/or neonicotine and/or myoxine content or total alkaloid content) of modified plants to surprisingly low levels.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that such publication forms the prior art of the claims appended hereto.

Examples

Example 1 transient overexpression of SOUL heme-binding protein increases alkaloid content in leaves

Method and material

Cloning

SOUL heme-binding protein expression vector

The SOUL heme-binding protein gene sequence (SEQ ID number 3) was amplified from a Gateway-compatible cDNA library using primers located outside the restriction sites flanking the gene sequence. The gene sequence was then transferred to an expression vector, depicted in fig. 2.

The resulting plasmid was sequenced and transformed into Agrobacterium tumefaciens GV3101pMP90 by heat shock and transiently expressed in TN90 leaves.

Transient gene expression

Agrobacterium tumefaciens GV3101 strain carrying the construct of interest was grown overnight in Luria-Bertani (LB) medium supplemented with the appropriate antibiotics. The cultures were rapidly centrifuged (spin down) and resuspended to OD600=0.6 in buffer containing 10 mM MgCl2, 10 mM MES pH 5.6 and 100 μ M acetosyringone and incubated for 1 hour at room temperature. Infiltration into the TN90 leaf was performed with a needleless syringe. Samples were obtained 5 days after infiltration.

The test was performed in three biological replicates.

Alkaloid measurement

The relative content of pyridine alkaloids was determined by reversed phase high performance liquid chromatography with tandem mass spectrometry (LC-MS/MS). The chromatographic separation was achieved using a Gemini-NX column (100 mM. times.3.0 mM, particle size 3 μm, Phenomenex) and gradient chromatography using 6.5 mM ammonium acetate buffer (aq) (pH10) and methanol.

The mass spectrometer was operated in Electrospray (ESI) positive mode using a predetermined MRM data acquisition. Two MRM transitions were monitored for each analyte and once for an isotopically labeled internal standard.

Analyte	Precursor ion	Ionic ion (amount/confirmation)
			Nicotine	163.1	130/106
Nicotine d4	167.1	134.1
			New nicotinoids	163.1	80/120
Anatabine	161.1	144/80
			Nicotine reduction	149.1	80/130
Nornicotine d4	153.1	84.1
			PON	176.1	106.0/148
PON d4	183.1	110.0

。

Statistical analysis

Statistical significance based on one-way ANOVA analysis was performed with Prism 5.01 Software (GraphPad Software).

Results

Alkaloid content of 5-week-old TN90 leaves expressing the nitab4.5_0013616g0010.2 construct is shown in figure 3. Nicotine, nornicotine, and PON levels are expressed relative to controls and include three biological replicates analyzed by one-way ANOVA and Tukey's multiple comparisons post-test. Values are shown as mean ± SEM. Asterisks indicate statistical significance with P-value ≦ 0.001.

Overexpression of nitab4.5_0013616g0010.2 resulted in a significant increase in nornicotine content in the leaf.

Conclusion

Nitab4.5_0013616g0010.2 is a positive regulator of alkaloid content, particularly nornicotine content, in lamina and is a regulator of pyridine alkaloids in tobacco.

Example 2 viral-induced Gene silencing of SOUL heme-binding protein (VIGS) reduction of organisms in leaves Alkali content

Virus Induced Gene Silencing (VIGS)

For virus-induced gene silencing, a 300-nucleotide cDNA fragment (SEQ ID number 25) was synthesized and cloned into pTV00 using the In-Fusion cloning kit. The plasmid was then transformed into agrobacterium tumefaciens GV 3101.

TRV vectors comprising both TRV RNA1 (SEQ ID number 26) and TRV RNA2 (SEQ ID number 27) containing the targeted nucleotide sequences were amplified separately in agrobacterium tumefaciens. These cultures were mixed (1:1) and infiltrated with a syringe into 2-week-old TN90 plants. Silencing effects five weeks after viral infection were assessed by assessing the expression level of the target gene.

Silencing

The VIGS assay was performed as previously described (Ratcliff et al (2001) The Plant Journal 25: 237-. Briefly, independent cultures of Agrobacterium tumefaciens GV3101 harboring plasmids TRV2 and TRV1 were propagated overnight in LB medium supplemented with appropriate antibiotics. Cultures were plated in VIGS buffer (10 mM morpholine ethanesulfonic acid pH 5.6, 10 mM MgCL)₂And 100 μ M acetosyringone), the optical density was adjusted to OD₆₀₀=1, and incubated overnight in the dark at room temperature. These cultures were mixed (1:1),and infiltrated into 2-week old TN90 plants with a syringe. The silencing effect two weeks after viral infection was assessed by assessing the expression level of the target gene. TRV-luciferase was used as a negative control and TRV-PDS (reduced chlorophyll content of silenced leaves) was used as a phenotypically silenced control.

Results

Nicotine, nornicotine, neonicotinoid, PON, and anatabine contents of 5-week-old TN90 leaves expressing the indicated construct silencing nitab4.5_0013616g0010.2 are shown in figure 4. The content is expressed relative to the control and includes three biological replicates analyzed by t-test. Values are shown as mean ± SEM. Asterisks indicate statistical significance with P-value ≦ 0.001.

Silencing nitab4.5_0013616g0010.2 resulted in a reduction in nicotine, nornicotine, neonicotine, PON and anatabine content in the leaf.

Conclusion

Nitab4.5_0013616g0010.2 is a positive regulator of alkaloid content in leaves, in particular nicotine, nornicotine, neonicotinoid, PON and anatabine content, and is a regulator of pyridine alkaloid in tobacco.

Example 3 homolog test

The effect of homologues of SEQ ID No.1, i.e. SEQ ID nos 4, 7, 10, 13, 16, 19 or 22, was tested in a transient assay as described in example 2.

Transient expression of the SOUL heme-binding protein expression vectors of homologs SEQ ID Nos 4, 7, 10, 13, 16, 19, and 22 is shown in FIG. 33.

Transient overexpression of all tested homologues resulted in a significant increase in nornicotine content.

Example 4-Nitab4.5-0013616 g0010.2 antisense results in a significant reduction in nornicotine levels

Nitab4.5_0013616g0010.2_ AS expression vector

The Nitab4.5_0013616g0010.2_ AS (antisense) construct was generated by two-step amplification and Gateway-compatible primer cloning using the Nitab4.5_0013616g0010.2 expression vector, the first set of gene-specific primers and the second set of Gateway-compatible primers AS templates. The amplification product was inserted into a Gateway ™ pDONR ™ cassette/Zeo vector (ThermoFisher Scientific). The sequence was then transferred to an expression vector.

The resulting plasmids were sequenced and transformed into Agrobacterium tumefaciens GV3101pMP90 by heat shock and transiently expressed in TN90 leaf discs.

Results

The nornicotine content of 5-week-old TN90 leaves expressing the indicated constructs silencing nitab4.5_0013616g0010.2 is shown in figure 32. Levels are expressed relative to controls and include three biological replicates analyzed by one-way ANOVA and Tukey's multiple comparisons post-test. Values are shown as mean ± SEM. Asterisks indicate statistical significance with P-value ≦ 0.001.

Conclusion

Nitab 4.5-0013616 g0010.2 is a positive regulator of nornicotine content in leaves.

Example 5 Stable T1 lines of the greenhouse overexpressing Nitab4.5-0013616 g0010.2

We also verified the function of nitab4.5 — 0013616g0010.2 with a stable transformation assay.

24 TN 90T 1 plants overexpressing Nitab4.5-0013616 g0010.2 were grown in the greenhouse. At the 12-leaf stage, nornicotine content was measured as described above.

Results

The nornicotine content of greenhouse grown 12-leaf stage TN90 plants overexpressing nitab4.5_001316g0010.2 is shown in fig. 34.

Example 6 field grown T1 plants overexpressing Nitab4.5-0013616 g0010.2Plant strain

24 TN 90T 1 plants overexpressing Nitab4.5-0013616 g0010.2 were grown in the greenhouse. At the 12-leaf stage, nornicotine content was measured as described above.

Results

The reduced nicotine content of field grown leaves of TN90 overexpressing nitab4.5_0013616g0010.2 is shown in fig. 35A. The amounts are expressed relative to the control. The results were analyzed by t-test. Values are shown as mean ± SEM. Asterisks indicate statistical significance with P-value ≦ 0.001.

The NNN content of leaves of TN90 leaves overexpressing nitab4.5_0013616g0010.2 grown in the field is shown in fig. 35B. The amounts are expressed relative to the control. The results were analyzed by t-test. Values are shown as mean ± SEM. Asterisks indicate statistical significance with P-value ≦ 0.01.

Conclusion

Overexpression of nitab4.5_0013616g0010.2 in T1 plants increased nornicotine and NNN levels.

Nitab 4.5-0013616 g0010.2 is a positive regulator of nornicotine content in leaves.