Login| Sign Up| Help| Contact|

Patent Searching and Data


Title:
PLANT PROMOTER
Document Type and Number:
WIPO Patent Application WO/2015/169925
Kind Code:
A1
Abstract:
The present invention relates to an isolated or synthetic nucleic acid promoter capable of directing expression in a plant cell, wherein said nucleic acid promoter comprises a sequence as set forth in SEQ ID NO: 1 or having at least 60% sequence identity thereto, preferably, wherein said plant cell is a tobacco plant cell.

Inventors:
LIEDSCHULTE VERENA (CH)
GOEPFERT SIMON (CH)
BOVET LUCIEN (CH)
SIERRO NICOLAS (CH)
Application Number:
EP2015/060117
Publication Date:
November 12, 2015
Filing Date:
May 07, 2015
Export Citation:
Click for automatic bibliography generation   Help
Assignee:
PHILIP MORRIS PRODUCTS SA (CH)
International Classes:
C12N15/82
Domestic Patent References:
WO2005111217A22005-11-24
WO2010122110A12010-10-28
WO1996026639A11996-09-06
WO2013029800A12013-03-07
WO2013064499A12013-05-10
WO2007044992A22007-04-19
Foreign References:
EP0913469A11999-05-06
US5907082A1999-05-25
Other References:
DIEGO ZAVALLO ET AL: "Isolation and functional characterization of two novel seed-specific promoters from sunflower (Helianthus annuus L.)", PLANT CELL REPORTS, SPRINGER, BERLIN, DE, vol. 29, no. 3, 19 January 2010 (2010-01-19), pages 239 - 248, XP019779612, ISSN: 1432-203X, DOI: 10.1007/S00299-010-0816-X
Attorney, Agent or Firm:
MASCHIO, Antonio (20 Carlton Crescent, Southampton Hampshire SO15 2ET, GB)
Download PDF:
Claims:
CLAIMS

1 . An isolated or synthetic polynucleotide promoter capable of directing expression in a plant cell, wherein said nucleic acid promoter comprises a polynucleotide sequence as set forth in SEQ ID NO: 1 or having at least 60% sequence identity thereto.

2. The isolated or synthetic polynucleotide promoter according to claim 1 , wherein said promoter is capable of driving flower or seed specific expression of an operably linked polynucleotide sequence of interest.

3. A polynucleotide construct comprising a polynucleotide promoter capable of directing expression in a plant cell, wherein said nucleic acid promoter comprises a polynucleotide sequence as set forth in SEQ ID NO: 1 or having at least 60% sequence identity thereto operably linked to a polynucleotide sequence of interest.

4. The polynucleotide construct according to claim 3, comprising, in the 5' to 3' direction, the polynucleotide promoter and the polynucleotide of interest positioned downstream from said promoter and operatively associated therewith.

5. The polynucleotide construct according to claim 4, wherein said polynucleotide of interest encodes a protein that contributes to the flavour profile of tobacco.

6. The polynucleotide construct according to any of claims 3 to 5, wherein said polynucleotide construct is a vector.

7. A plant cell comprising the polynucleotide construct of any of claims 3 to 6.

8. A method of making a plant, comprising regenerating a plant from the plant cell according to claim 7.

9. A plant comprising the plant cell of claim 7.

10. A method for expressing a polynucleotide of interest in a plant cell comprising the use of the polynucleotide construct according to any of claims 3 to 6, preferably, wherein said method comprises the steps of:

(a) transforming a plant cell with the polynucleotide construct of any of claims 3 to 6; (b) growing the transformed plant cell under conditions suitable for the polynucleotide of interest to be expressed by the promoter.

1 1 . A method of modulating the flavour and/or aroma profile of a plant, comprising the steps of:

(a) transforming a plant cell with the polynucleotide construct according to any of claims 3 to 6, wherein the polynucleotide of interest encodes a protein that contributes to the flavour and/or aroma profile of tobacco;

(b) growing a plant containing the plant cell under conditions suitable for the polynucleotide of interest to be expressed;

(c) optionally harvesting material from the plant of step (b); and

(d) determining if the flavour and/or aroma profile of the plant material has been modulated in comparison to control plant material.

12. A method according to claim 1 1 , wherein the plant is a tobacco plant.

13. A method for producing a plant having modified expression of a polynucleotide sequence of interest comprising: a) transforming a plant cell with the polynucleotide construct of any of claims 3 to 6; b) selecting a transformed cell; c) generating a plant from the transformed cell; and d) selecting a plant having modified expression of the polynucleotide sequence of interest.

14. A method according to claim 1 1 or claim 13, wherein the plant is an ornamental plant and the gene is expressed in the flowers of the plant.

15. A method according to any one of claims 1 1 to 13, wherein the plant is intended for human and/or animal consumption.

16. A method according to any one of claims 1 1 to 13, wherein the polynucleotide of interest encodes a chemical, biological or pharmaceutical product which is optionally harvested from the plant.

17. A method of making a transformed plant, comprising transforming a plant cell with the polynucleotide construct of any of claims 3 to 6 to produce a transformed plant cell, and then regenerating a plant from said transformed plant cell.

18. Use of the nucleic acid promoter according to claim 1 or claim 2 or the polynucleotide construct according to any of claims 3 to 6 for expressing a polynucleotide of interest in a plant cell.

19. The use according to claim 18, wherein said polynucleotide of interest encodes a protein that contributes to the flavour and/or aroma profile of the plant.

Description:
PLANT PROMOTER

FIELD OF THE INVENTION

The present invention relates generally to the field of plant molecular biology and the regulation of gene expression in plants. The invention discloses nucleic acid sequences from tobacco containing a promoter region. More specifically, the present invention relates to the regulation of gene expression in plants with specificity to flowers or seeds.

BACKGROUND OF THE INVENTION

In plant molecular biology, it is advantageous to have a choice of a variety of different promoters so as to give the desired effect(s) in the engineered plant. Suitable promoters may be selected for a particular gene construct, for expression in a specific cell type, tissue, plant or environment. Promoters that are useful for plant transgene expression include those that are viral, synthetic, inducible, constitutive, temporally regulated, spatially regulated, tissue-specific, and spatio-temporally regulated. Promoters from bacteria, fungi, viruses and plants have been used to control gene expression in plant cells. It is often desirable to have tissue-specific expression of a gene of interest in a plant. Tissue-specific promoters can promote expression exclusively in one set of tissues without expression throughout the plant. Tissue-preferred promoters can drive expression at a higher level in a subset of tissues with significantly less or even no expression in the other tissues of the plant.

In plant tobacco research, one particular area of interest is modulating the flavour of tobacco to generate a more desirable taste when tobacco is smoked. By way of example, modulation of threonine synthase expression or activity in tobacco plants to modulate methionine concentration and the flavour profile of tobacco is described in WO2013029800. Modulation of the expression or activity of isopropylmalate synthase to alter sucrose ester composition and the flavour profile of tobacco is described in WO2013029799. Modulation of the expression or activity of neoxanthin synthase to modulate the amount of beta- damascenone that is detectable in the aerosol of heated tobacco resulting in new flavour profiles in tobacco is described in WO2013064499.

There is a continuing need in the art for new tobacco plant promoters to obtain those with different expression levels or different specificity of cell-type expression for tissue-specific and tissue-preferred expression, particularly for flower or seed specific expression. These new plant promoters can find a multitude of different uses in plant research - including for use in modulating the chemical profile of tobacco.

SUMMARY OF THE INVENTION Compositions and methods for directing flower or seed specific expression in plants are provided. In particular, a novel nucleic acid molecule isolated from tobacco, that drives expression of genes in a flower or seed specific manner in plants, is provided.

ASPECTS AND EMBODIMENTS OF THE INVENTION

Aspects and embodiments of the present invention are set forth in the accompanying claims. In a first aspect, there is provided an isolated or synthetic polynucleotide promoter capable of directing expression in a plant cell, wherein said nucleic acid promoter comprises a polynucleotide sequence as set forth in SEQ ID NO: 1 or having at least 60% sequence identity thereto.

In one embodiment, said promoter is capable of driving flower or seed specific expression of an operably linked polynucleotide sequence of interest.

In a further aspect, there is provided a polynucleotide construct comprising a polynucleotide promoter capable of directing expression in a plant cell, wherein said nucleic acid promoter comprises a polynucleotide sequence as set forth in SEQ ID NO: 1 or having at least 60% sequence identity thereto operably linked to a polynucleotide sequence of interest.

In one embodiment, the polynucleotide construct comprises, in the 5' to 3' direction, the polynucleotide promoter and the polynucleotide of interest positioned downstream from said promoter and operatively associated therewith.

In one embodiment, said polynucleotide of interest encodes a protein that contributes to the flavour profile of tobacco.

In one embodiment, said polynucleotide construct is a vector.

In a further aspect, there is provided a plant cell comprising the polynucleotide construct. In a further aspect, there is provided a method of making a plant, comprising regenerating a plant from the plant cell.

In a further aspect, there is provided a plant comprising the plant cell.

In a further aspect, there is provided a method for expressing a polynucleotide of interest in a plant cell comprising the use of the polynucleotide construct, preferably, wherein said method comprises the steps of: (a) transforming a plant cell with the polynucleotide construct; and (b) growing the transformed plant cell under conditions suitable for the polynucleotide of interest to be expressed by the promoter.

In a further aspect, there is provided a method of modulating the flavour profile of tobacco comprising the steps of: (a) transforming a tobacco plant cell with the polynucleotide construct, wherein the polynucleotide of interest encodes a protein that contributes to the flavour profile of tobacco; (b) growing a tobacco plant containing the plant cell under conditions suitable for the polynucleotide of interest to be expressed; (c) harvesting and optionally curing tobacco plant material from the tobacco plant of step (b); and (d) determining if the flavour profile of the tobacco plant material has been modulated in comparison to control tobacco plant material.

In a further aspect, there is provided a method for producing a plant having modified expression of a polynucleotide sequence of interest comprising: (a) transforming a plant cell with the polynucleotide construct; (b) selecting a transformed cell; (c) generating a plant from the transformed cell; and (d) selecting a plant having modified expression of the polynucleotide sequence of interest.

In a further aspect, there is provided a method of making a transformed plant, comprising transforming a plant cell with the polynucleotide construct to produce a transformed plant cell, and then regenerating a plant from said transformed plant cell.

In a further aspect, there is provided the use of the nucleic acid promoter or the polynucleotide construct for expressing a polynucleotide of interest in a plant cell.

In one embodiment, said polynucleotide of interest encodes a protein that contributes to the flavour profile of tobacco.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the relative expression levels of NND3 and related functional CYP82E genes in different plant tissues. Bars indicate mean ± SD of three biological replicates taken from three greenhouse grown mature N. tabacum var. TN90 plants.

Figure 2 shows relative NND3 expression levels in different parts of flowers.

Figure 3 shows relative expression levels of NND3 and related functional CYP82E genes in N. tabacum var. Stella leaves at different curing time points. Samples are taken from pools of several leaves. Two pools are analyzed as biological replicates - (a) replicate 1 and (b) replicate 2. Bars indicate mean ± SD of three technical replicates.

DEFINITIONS

The technical terms and expressions used within the scope of this application are generally to be given the meaning commonly applied to them in the pertinent art of plant and molecular biology. All of the following term definitions apply to the complete content of this application. The word "comprising" does not exclude other elements or steps, and the indefinite article "a" or "an" does not exclude a plurality. A single step may fulfil the functions of several features recited in the claims. The terms "about", "essentially" and "approximately" in the context of a given numerate value or range refers to a value or range that is within 20%, within 10%, or within 5%, 4%, 3%, 2% or 1 % of the given value or range.

The term "isolated" refers to any entity that is taken from its natural milieu, but the term does not connote any degree of purification.

An "expression vector" is a nucleic acid vehicle that comprises a combination of nucleic acid components for enabling the expression of nucleic acid. Suitable expression vectors include episomes capable of extra-chromosomal replication such as circular, double-stranded nucleic acid plasmids; linearized double-stranded nucleic acid plasmids; and other functionally equivalent expression vectors of any origin. An expression vector comprises at least a promoter positioned upstream and operably-linked to a nucleic acid, nucleic acid constructs or nucleic acid conjugate.

The term "construct" refers to a double-stranded, recombinant nucleic acid fragment comprising one or more polynucleotides. The construct comprises a "template strand" base- paired with a complementary "sense or coding strand." A given construct can be inserted into a vector in two possible orientations, either in the same (or sense) orientation or in the reverse (or anti-sense) orientation with respect to the orientation of a promoter positioned within a vector - such as an expression vector.

A "vector" refers to a nucleic acid vehicle that comprises a combination of nucleic acid components for enabling the transport of nucleic acid, nucleic acid constructs and nucleic acid conjugates and the like. Suitable vectors include episomes capable of extra- chromosomal replication such as circular, double-stranded nucleic acid plasmids; linearized double-stranded nucleic acid plasmids; and other vectors of any origin.

A "promoter" is an untranslated DNA sequence typically upstream of a coding region that contains the binding site for RNA polymerase and initiates transcription of the DNA. The promoter region may include other elements - such as promoter regulatory sequences - that act as regulators of gene expression. Promoter regulatory sequences can contain proximal and/or distal upstream elements, the latter elements often being referred to as enhancers. An enhancer is a DNA sequence that can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue specificity of the promoter. It is generally capable of operating in both orientations and is capable of functioning even when moved either upstream or downstream from the promoter. "Operably-linked" refers to the association of polynucleotide sequences on a single nucleic acid fragment or construct so that the function of one polynucleotide sequence is affected by the other polynucleotide sequence. For example, a promoter is operably-linked with a coding sequence when it is capable of affecting the expression of that coding sequence. The coding sequence is therefore under the transcriptional control of the promoter.

The terms "homology, identity or similarity" refer to the degree of sequence similarity between two polypeptides or between two nucleic acid molecules compared by sequence alignment. The degree of homology between two discrete nucleic acid sequences being compared is a function of the number of identical, or matching, nucleotides at comparable positions. The percent identity may be determined by visual inspection and mathematical calculation. Alternatively, the percent identity of two nucleic acid sequences may be determined by comparing sequence information using a computer program such as - ClustalW, BLAST, FASTA or Smith-Waterman.

A "variant" means a substantially similar sequence. A variant can have a similar function or substantially similar function as a wild-type sequence. For a promoter, a similar function is at least about 50%, 60%, 70%, 80% or 90% of wild-type promoter activity under the same conditions. For a promoter, a substantially similar function is at least about 90%, 95%, 96%, 97%, 98% or 99% of wild-type promoter function under the same conditions.

The term "plant" refers to any plant or plant part at any stage of its life cycle or development, and its progenies. In one embodiment, the plant is a "tobacco plant", which refers to a plant belonging to the genus Nicotiana. Preferred species of tobacco plant are described herein. "Plant parts" include plant cells, plant protoplasts, plant cell tissue cultures from which a whole plant can be regenerated, plant calli, plant clumps and plant cells that are intact in plants or parts of plants such as embryos, pollen, anthers, ovules, seeds, leaves, flowers, stems, branches, fruit, roots, root tips and the like. Progeny, variants and mutants of regenerated plants are also included within the scope of the disclosure, provided that they comprise the introduced polynucleotides described herein.

A "plant cell" refers to a structural and physiological unit of a plant. The plant cell may be in the form of a protoplast without a cell wall, an isolated single cell or a cultured cell, or as a part of higher organized unit such as but not limited to, plant tissue, a plant organ, or a whole plant.

The term "plant material" refers to any solid, liquid or gaseous composition, or a combination thereof, obtainable from a plant, including biomass, leaves, stems, roots, flowers or flower parts, fruits, pollen, egg cells, zygotes, seeds, cuttings, secretions, extracts, cell or tissue cultures, or any other parts or products of a plant. In one embodiment, the plant material comprises or consists of biomass, stem, seed or leaves. In another embodiment, the plant material comprises or consists of leaves.

The term "variety" refers to a population of plants that share constant characteristics which separate them from other plants of the same species. While possessing one or more distinctive traits, a variety is further characterized by a very small overall variation between individuals within that variety. A variety is often sold commercially.

The term "modulating" may refer to reducing, inhibiting, increasing or otherwise affecting, for example, polynucleotide expression or transcriptional activity.

The term "reduce" or "reduced" as used herein, refers to a reduction of from about 10% to about 99%, or a reduction of at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% or more of a quantity or an activity - such as polynucleotide expression or transcriptional activity. The term "inhibit" or "inhibited" as used herein, refers to a reduction of from about 98% to about 100%, or a reduction of at least 98%, at least 99%, but particularly of 100%, of a quantity or an activity - such as polynucleotide expression or transcriptional activity

The term "increase" or "increased" as used herein, refers to an increase of from about 5% to about 99%, or an increase of at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% or more of a quantity or an activity - such as polynucleotide expression or transcriptional activity.

The term "control" in the context of a control plant cell means a plant cell in which the expression of a polynucleotide has not been modified (for example, increased or reduced) and so it can provide a comparison with a plant cell in which the expression of the polynucleotide has been modified. The control plant may comprise an empty vector. The control plant cell may correspond to a wild-type plant cell. For example, the control plant cell can be the same genotype as the starting material for the genetic alteration that resulted in the subject plant cell. In all such cases, the subject plant cell and the control plant cell are cultured and harvested using the same protocols for comparative purposes. Changes in levels, ratios, activity, or distribution of the polynucleotides or genes can be measured by comparing a subject plant cell to the control plant cell, where the subject plant cell and the control plant cell have been cultured and/or harvested using the same protocols.

DETAILED DESCRIPTION

In one embodiment, there is provided a promoter polynucleotide. The promoter polynucleotide can be an isolated or an artificial or a synthetic promoter polynucleotide. The promoter polynucleotide can comprise, consist or consist essentially of a polynucleotide sequence having at least 60% sequence identity to any of the sequences described herein, including any of polynucleotides shown in the sequence listing. The sequence of the full- length complement, the reverse full-length complement, and the reverse sequence thereof are also disclosed.

Suitably, the isolated promoter polynucleotide comprises, consists or consists essentially of a sequence having at least 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, 80%, 85%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99% or 100% sequence identity thereto.

In another embodiment, there is provided an isolated promoter polynucleotide comprising, consisting or consisting essentially of a polynucleotide sequence having at least 60% sequence identity to SEQ ID NO:1. Suitably, the isolated promoter polynucleotide comprises, consists or consist essentially of a sequence having at least about 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, 80%, 85%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, 99.1 %, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to SEQ ID NO:1.

In another embodiment, there is provided a promoter polynucleotide comprising, consisting or consisting essentially of a polynucleotide with substantial homology (that is, sequence similarity) or substantial identity to SEQ ID NO:1 .

In another embodiment, there is provided a promoter polynucleotide variant that has at least about 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, 80%, 85%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, 99.1 %, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity to the sequence of SEQ ID NO:1 .

In another embodiment, there is provided fragments of SEQ ID NO:1 with substantial homology (that is, sequence similarity) or substantial identity thereto that have at least about 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, 80%, 85%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, 99.1 %, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to the corresponding fragments of SEQ ID NO:1.

In another embodiment, there is provided a promoter polynucleotide comprising a sufficient or substantial degree of identity or similarity to SEQ ID NO:1.

Fragments of SEQ ID NO:1 that function as a promoter are also disclosed. The fragments include those that can be assembled within recombinant constructs. Fragments of the polynucleotide sequence may range from at least about 100 nucleotides up to the full-length polynucleotide.

The polynucleotides described herein include a polymer of nucleotides comprising or consisting of deoxyribonucleic acid (DNA). Although the polynucleotide sequences described herein are shown as DNA sequences, the sequences include corresponding RNA sequences, and their complementary (for example, completely complementary) DNA or RNA sequences, including the reverse complements thereof. The polynucleotides will generally contain phosphodiester bonds, although in some cases, polynucleotide analogues are included that may have alternate backbones, comprising, for example, phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphophoroamidite linkages; and peptide polynucleotide backbones and linkages. Other analogue polynucleotides include those with positive backbones; non-ionic backbones, and non-ribose backbones. Modifications of the ribose-phosphate backbone may be done for a variety of reasons, for example, to increase the stability and half-life of such molecules in physiological environments or as probes on a biochip. Mixtures of naturally occurring polynucleotides and analogues can be made; alternatively, mixtures of different polynucleotide analogues, and mixtures of naturally occurring polynucleotides and analogues may be made. A variety of polynucleotide analogues are known, including, for example, phosphoramidate, phosphorothioate, phosphorodithioate, O-methylphophoroamidite linkages and peptide polynucleotide backbones and linkages. Other analogue polynucleotides include those with positive backbones, non-ionic backbones and non-ribose backbones. Polynucleotides containing one or more carbocyclic sugars are also included. Other analogues include peptide polynucleotides which are peptide polynucleotide analogues. These backbones are substantially non-ionic under neutral conditions, in contrast to the highly charged phosphodiester backbone of naturally occurring polynucleotides. This may result in advantages. First, the peptide polynucleotide backbone may exhibit improved hybridization kinetics. Peptide polynucleotides have larger changes in the melting temperature for mismatched versus perfectly matched base pairs. DNA and RNA typically exhibit a 2-4 °C drop in melting temperature for an internal mismatch. With the non-ionic peptide polynucleotide backbone, the drop is closer to 7-9 °C. Similarly, due to their non-ionic nature, hybridization of the bases attached to these backbones is relatively insensitive to salt concentration. In addition, peptide polynucleotides may not be degraded or degraded to a lesser extent by cellular enzymes, and thus may be more stable.

Also of use are polynucleotides that hybridize under stringent conditions, typically moderately stringent conditions, and commonly highly stringent conditions, to the polynucleotide promoter described herein. The basic parameters affecting the choice of hybridization conditions and guidance for devising suitable conditions are described in Sambrook, J., E. F. Fritsch, and T. Maniatis (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) and can be readily determined by those having ordinary skill in the art based on, for example, the length or base composition of the polynucleotide. One way of achieving moderately stringent conditions involves the use of a prewashing solution containing 5x Standard Sodium Citrate, 0.5% Sodium Dodecyl Sulphate, 1 .0 mM Ethylenediaminetetraacetic acid (pH 8.0), hybridization buffer of about 50% formamide, 6x Standard Sodium Citrate, and a hybridization temperature of about 55 °C (or other similar hybridization solutions, such as one containing about 50% formamide, with a hybridization temperature of about 42 °C), and washing conditions of about 60 °C, in 0.5x Standard Sodium Citrate, 0.1 % Sodium Dodecyl Sulphate. Generally, highly stringent conditions are defined as hybridization conditions as above, but with washing at approximately 68 °C, 0.2 x Standard Sodium Citrate, 0.1 % Sodium Dodecyl Sulphate. SSPE (1 x SSPE is 0.15M sodium chloride, 10 mM sodium phosphate, and 1.25 mM Ethylenediaminetetraacetic acid, pH 7.4) can be substituted for Standard Sodium Citrate (1 x Standard Sodium Citrate is 0.15M sodium chloride and 15 mM sodium citrate) in the hybridization and wash buffers; washes are performed for 15 minutes after hybridization is complete. It should be understood that the wash temperature and wash salt concentration can be adjusted as necessary to achieve a desired degree of stringency by applying the basic principles that govern hybridization reactions and duplex stability, as known to those skilled in the art and described further below (see, for example, Sambrook, J., E. F. Fritsch, and T. Maniatis (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). When hybridising a polynucleotide to a target polynucleotide of unknown sequence, the hybrid length is assumed to be that of the hybridising polynucleotide. When polynucleotides of known sequence are hybridised, the hybrid length can be determined by aligning the sequences of the polynucleotides and identifying the region(s) of optimal sequence complementarity. The hybridisation temperature for hybrids anticipated to be less than 50 base pairs in length should be about 5 to 10 °C less than the melting temperature (T m ) of the hybrid, where T m is determined according to the following equations. For hybrids less than 18 base pairs in length, T m (°C) = 2(number of A+T bases) + 4(number of G+C bases). For hybrids above 18 base pairs in length, T m (°C) = 81.5+16.6 (Iog10 [Na+]) + 0.41 (% G+C) - (600/N), where N is the number of bases in the hybrid, and [Na+] is the concentration of sodium ions in the hybridisation buffer ([Na+] for 1x Standard Sodium Citrate=0.165M).

As will be understood by the person skilled in the art, a linear DNA molecule has two possible orientations: the 5'-to-3' direction and the 3'-to-5' direction. For example, if a reference sequence is positioned in the 5'-to-3' direction, and if a second sequence is positioned in the 5'-to-3' direction within the same polynucleotide molecule/strand, then the reference sequence and the second sequence are orientated in the same direction, or have the same orientation. Typically, the promoter sequence as described herein and a polynucleotide (gene) of interest under the regulation of the given promoter are positioned in the same orientation. However, with respect to the reference sequence positioned in the 5'- to-3' direction, if a second sequence is positioned in the 3'-to-5' direction within the same polynucleotide molecule/strand, then the reference sequence and the second sequence are orientated in anti-sense direction, or have anti-sense orientation. Two sequences having anti-sense orientations with respect to each other can be alternatively described as having the same orientation, if the reference sequence (5'-to-3' direction) and the reverse complementary sequence of the reference sequence (reference sequence positioned in the 5'-to-3') are positioned within the same polynucleotide molecule/strand. The sequences set forth herein are shown in the 5'-to-3' direction.

The promoter described herein can be used to express of one or more polynucleotides of interest in a host cell. The promoter described herein can be used to express of one or more polynucleotides of interest in a plant cell - such as a tobacco plant cell. By way of example, a construct (which can be, for example, a vector or an expression vector or a plasmid and the like) that is compatible with the cell to be transformed can be prepared which comprises one or more polynucleotides of interest together with the promoter described herein positioned upstream to express (suitably, overexpress) the polynucleotide(s) in the cell. The coding sequence of the polynucleotide(s) of interest can be cloned between the promoter described herein and an optional transcriptional terminator whereby the coding sequence is operatively linked to the promoter and the transcriptional terminator is operatively linked to the coding sequence. The construct can optionally include a selectable marker coding sequence. Examples of visible markers include, but are not limited to, .beta. -glucuronidase (GUS), Chloramphenicol Acetyl Transferase (CAT), Luciferase (LUC) and proteins with fluorescent properties - such as Green Fluorescent Protein (GFP) from Aequora victoria. Following transformation and when grown under suitable conditions, the promoter can drive expression of the polynucleotide(s) of interest to produce the encoded protein in the cell. The polypeptide(s) of interest encoded by the recombinant polynucleotide(s) of interest can be a native polypeptide, or can be heterologous to the cell. In one exemplary embodiment, a construct carrying one or more polynucleotides of interest is generated to express a polynucleotide(s) of interest in a plant cell. The construct carries the promoter described herein upstream of the polynucleotide(s) driving its expression in the plant cell. The construct can optionally carry an antibiotic resistance gene to confer selection of the transformed cell. Additionally, one or more targeting sequences may be employed to target the polypeptide of interest to an intracellular compartment within cells or to the extracellular environment. For example, a DNA sequence encoding a transit or signal peptide sequence may be operably linked to a sequence encoding a desired polypeptide of interest such that, when translated, the transit or signal peptide can transport the polypeptide of interest to a particular intracellular or extracellular destination, respectively, and can then be post- translationally removed. Transit or signal peptides act by facilitating the transport of proteins through intracellular membranes, e.g., vacuole, vesicle, plastid and mitochondrial membranes, whereas signal peptides direct proteins through the extracellular membrane. For example, the transit or signal peptide can direct a desired protein to a particular organelle - such as a plastid, rather than to the cytoplasm.

The polynucleotide of interest may be identified in genomic DNA sequences - such as genomic DNA sequences of plants - for which genome sequence information is available to the public or isolated from polynucleotide libraries. The polynucleotides may be artificial or synthetic polynucleotides. Such artificial or synthetic polynucleotides can be synthesised using known state-of-the-art techniques. The polynucleotides may be synthesised using automated oligonucleotide synthesizers (for example, the Beckman DNA OLIGO 1000M synthesiser) so as to obtain polynucleotide fragments of desired length. A multitude of these polynucleotide fragments may then be linked using known DNA manipulation techniques. A recombinant polynucleotide construct for use in the present disclosure can comprise one or more polynucleotides of interest encoding one or more polypeptides of interest, operably linked to the promoter described herein for expressing the polynucleotides in a plant cell. Plant cells in which protein expression occurs can be non-naturally occurring plant cells, transgenic plant cells, man-made plant cells, genetically engineered plant cells or mutant plant cells. Plants in which protein expression occurs can be non-naturally occurring plants, transgenic plants, man-made plants, genetically engineered plants or mutant plants. Expression may occur in certain cells or in certain parts of a plant. Expression may occur in certain tissues of a plant. Expression may occur in specific tissues of a plant. Expression may occur in a specific tissue of a plant. Advantageously, the promoter of the present invention is capable of expressing polynucleotides in flowers or seeds.

The non-naturally occurring, transgenic, man-made, genetically engineered or mutant plant cell or plant can comprise a genome that has been altered by the integration (suitably, the stable integration) of recombinant DNA therein. Recombinant DNA includes DNA which has been genetically engineered and constructed outside of a cell and includes DNA containing naturally occurring DNA or cDNA or synthetic DNA. A transgenic, man-made, genetically engineered or mutant plant cell can include a plant or plant cell regenerated from an originally-transformed plant or plant cell and progeny transgenic plants or plant cells from later generations or crosses of a transformed plant or plant cell.

A plant cell can be transformed by having the recombinant polynucleotide or a construct comprising same integrated into its genome to become stably transformed. The plant cell can be stably transformed. Stably transformed cells typically retain the introduced polynucleotide(s) with each cell division. A plant cell can be transiently transformed such that the recombinant polynucleotide or a construct comprising same is not integrated into its genome. Transiently transformed cells typically lose all or some portion of the introduced recombinant polynucleotide or construct comprising same with each cell division such that the introduced recombinant polynucleotide cannot be detected in daughter cells after a sufficient number of cell divisions. Suitably, the plant cell is stably transformed.

The constructs, vectors and the like may be introduced into a plant genome by a variety of conventional techniques. For example, A. tumefaciens mediated transformation, electroporation, protoplast fusion, injection in reproductive organs, injection in immature embryos; microinjection of plant cell protoplasts; use of ballistic methods - such as DNA coated particle bombardment. The choice of technique will depend on the plant type to be transformed. For example, dicot plants and some monocots and gymnospermae may be transformed using Agrobacterium Ti plasmid technology. The constructs, vectors and the like may be combined with appropriate T-DNA flanking regions and introduced into the conventional A. tumefaciens host vector. The virulence factor of the A. tumefaciens host will conduct the insertion of the genetic constructs and adjacent marker into the DNA of the plant cell when the cell is infected by the bacteria. Microinjection techniques, the use of polyethylene glycol precipitations, electroporation techniques and ballistic transformation techniques are all well known in the art. Tissues, such as leaf tissues, dissociated cells, protoplasts, seeds, embryos, meristemic regions, cotyledons, hypocotyledons and others can be transformed.

Vectors containing the recombinant polynucleotide constructs are also provided. Suitable vector backbones include, for example, those routinely used in the art such as plasmids, viruses, artificial chromosomes, bacterial artificial chromosomes, yeast artificial chromosomes, or bacteriophage artificial chromosomes. Suitable expression vectors include, without limitation, plasmids and viral vectors derived from, for example, bacteriophage, baculoviruses, and retroviruses. Numerous vectors and expression systems are commercially available. The vectors can include, for example, origins of replication, scaffold attachment regions or markers. A marker gene can confer a selectable phenotype on a plant cell. For example, a marker can confer biocide resistance, such as resistance to an antibiotic (for example, kanamycin, G418, bleomycin, or hygromycin), or an herbicide (for example, glyphosate, chlorsulfuron or phosphinothricin). In addition, an expression vector can include a tag sequence designed to facilitate manipulation or detection (for example, purification or localization) of the expressed polypeptide. Tag sequences, such as luciferase, beta-glucuronidase, green fluorescent protein, glutathione S-transferase, polyhistidine, c- myc or hemagglutinin sequences typically are expressed as a fusion with the encoded polypeptide. Such tags can be inserted anywhere within the polypeptide, including at either the carboxyl or amino terminus.

Numerous sequences have been found to enhance expression from within the transcriptional unit and these sequences can be used in conjunction with the promoter described herein to increase expression. For example, various intron sequences have been shown to enhance expression. A number of non-translated leader sequences derived from viruses are also known to enhance expression. Specifically, leader sequences from Tobacco Mosaic Virus (TMV, the "W-sequence"), Maize Chlorotic Mottle Virus (MCMV), and Alfalfa Mosaic Virus (AMV) have been shown to be effective in enhancing expression. Other leader sequences known in the art include but are not limited to: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5' noncoding region), potyvirus leaders, for example, TEV leader (Tobacco Etch Virus), MDMV leader (Maize Dwarf Mosaic Virus), human immunoglobulin heavy-chain binding protein (BiP) leader, untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4) and tobacco mosaic virus leader (TMV). Plants suitable for use in the present disclosure include, but are not limited to, monocotyledonous and dicotyledonous plants and plant cell systems, including species from one of the following families: Acanthaceae, Alliaceae, Alstroemeriaceae, Amaryllidaceae, Apocynaceae, Arecaceae, Asteraceae, Berberidaceae, Bixaceae, Brassicaceae, Bromeliaceae, Cannabaceae, Caryophyllaceae, Cephalotaxaceae, Chenopodiaceae, Colchicaceae, Cucurbitaceae, Dioscoreaceae, Ephedraceae, Erythroxylaceae, Euphorbiaceae, Fabaceae, Lamiaceae, Linaceae, Lycopodiaceae, Malvaceae, Melanthiaceae, Musaceae, Myrtaceae, Nyssaceae, Papaveraceae, Pinaceae, Plantaginaceae, Poaceae, Rosaceae, Rubiaceae, Salicaceae, Sapindaceae, Solanaceae, Taxaceae, Theaceae, or Vitaceae.

Suitable species may include members of the genera Abelmoschus, Abies, Acer, Agrostis, Allium, Alstroemeria, Ananas, Andrographis, Andropogon, Artemisia, Arundo, Atropa, Berberis, Beta, Bixa, Brassica, Calendula, Camellia, Camptotheca, Cannabis, Capsicum, Carthamus, Catharanthus, Cephalotaxus, Chrysanthemum, Cinchona, Citrullus, Coffea, Colchicum, Coleus, Cucumis, Cucurbita, Cynodon, Datura, Dianthus, Digitalis, Dioscorea, Elaeis, Ephedra, Erianthus, Erythroxylum, Eucalyptus, Festuca, Fragaria, Galanthus, Glycine, Gossypium, Helianthus, Hevea, Hordeum, Hyoscyamus, Jatropha, Lactuca, Linum, Lolium, Lupinus, Lycopersicon, Lycopodium, Manihot, Medicago, Mentha, Miscanthus, Musa, Nicotiana, Oryza, Panicum, Papaver, Parthenium, Pennisetum, Petunia, Phalaris, Phleum, Pinus, Poa, Poinsettia, Populus, Rauwolfia, Ricinus, Rosa, Saccharum, Salix, Sanguinaria, Scopolia, Secale, Solanum, Sorghum, Spartina, Spinacea, Tanacetum, Taxus, Theobroma, Triticosecale, Triticum, Uniola, Veratrum, Vinca, Vitis, and Zea. Suitable species may include Panicum spp., Sorghum spp., Miscanthus spp., Saccharum spp., Erianthus spp., Populus spp., Andropogon gerardii (big bluestem), Pennisetum purpureum (elephant grass), Phalaris arundinacea (reed canarygrass), Cynodon dactylon (bermudagrass), Festuca arundinacea (tall fescue), Spartina pectinata (prairie cord-grass), Medicago sativa (alfalfa), Arundo donax (giant reed), Secale cereale (rye), Salix spp. (willow), Eucalyptus spp. (eucalyptus), Triticosecale (tritic wheat times rye), bamboo, Helianthus annuus (sunflower), Carthamus tinctorius (safflower), Jatropha curcas (jatropha), Ricinus communis (castor), Elaeis guineensis (palm), Linum usitatissimum (flax), Brassica juncea, Beta vulgaris (sugarbeet), Manihot esculenta (cassaya), Lycopersicon esculentum (tomato), Lactuca sativa (lettuce), Musyclise alca (banana), Solanum tuberosum (potato), Brassica oleracea (broccoli, cauliflower, Brussels sprouts), Camellia sinensis (tea), Fragaria ananassa (strawberry), Theobroma cacao (cocoa), Coffea arabica (coffee), Vitis vinifera (grape), Ananas comosus (pineapple), Capsicum annum (hot & sweet pepper), Allium cepa (onion), Cucumis melo (melon), Cucumis sativus (cucumber), Cucurbita maxima (squash), Cucurbita moschata (squash), Spinacea oleracea (spinach), Citrullus lanatus (watermelon), Abelmoschus esculentus (okra), Solanum melongena (eggplant), Rosa spp. (rose), Dianthus caryophyllus (carnation), Petunia spp. (petunia), Poinsettia pulcherrima (poinsettia), Lupinus albus (lupin), Uniola paniculata (oats), bentgrass (Agrostis spp.), Populus tremuloides (aspen), Pinus spp. (pine), Abies spp. (fir), Acer spp. (maple), Hordeum vulgare (barley), Poa pratensis (bluegrass), Lolium spp. (ryegrass) and Phleum pratense (timothy), Panicum virgatum (switchgrass), Sorghum bicolor (sorghum, sudangrass), Miscanthus giganteus (miscanthus), Saccharum sp. (energycane), Populus balsamifera (poplar), Zea mays (corn), Glycine max (soybean), Brassica napus (canola), Triticum aestivum (wheat), Gossypium hirsutum (cotton), Oryza sativa (rice), Helianthus annuus (sunflower), Medicago sativa (alfalfa), Beta vulgaris (sugarbeet), or Pennisetum glaucum (pearl millet).

The disclosure can be applied to any species of the genus Nicotiana, including N. rustica and N. tabacum (for example, LA B21 , LN KY171 , Tl 1406, Basma, Galpao, Perique, Beinhart 1000-1 , and Petico). Other species include N. acaulis, N. acuminata, N. africana, N. alata, N. ameghinoi, N. amplexicaulis, N. arentsii, N. attenuata, N. azambujae, N. benavidesii, N. benthamiana, N. bigelovii, N. bonariensis, N. cavicola, N. clevelandii, N. cordifolia, N. corymbosa, N. debneyi, N. excelsior, N. forgetiana, N. fragrans, N. glauca, N. glutinosa, N. goodspeedii, N. gossei, N. hybrid, N. ingulba, N. kawakamii, N. knightiana, N. langsdorffii, N. linearis, N. longiflora, N. maritima, N. megalosiphon, N. miersii, N. noctiflora, N. nudicaulis, N. obtusifolia, N. occidentalis, N. occidentalis subsp. hesperis, N. otophora, N. paniculata, N. pauciflora, N. petunioides, N. plumbaginifolia, N. quadrivalvis, N. raimondii, N. repanda, N. rosulata, N. rosulata subsp. ingulba, N. rotundifolia, N. setchellii, N. simulans, N. solanifolia, N. spegazzinii, N. Stockton ii, N. suaveolens, N. sylvestris, N. thyrsiflora, N. tomentosa, N. tomentosiformis, N. trigonophylla, N. umbratica, N. undulata, N. velutina, N. wigandioides, and N. x sanderae.

Particularly useful Nicotiana tabacum varieties include Burley type, dark type, flue-cured type, and Oriental type tobaccos. Non-limiting examples of varieties or cultivars are: BU 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, DF91 1 , DT 538 LC Galpao tobacco, GL 26H, GL 350, GL 600, GL 737, GL 939, GL 973, HB 04P, HB 04P LC, HB3307PLC, Hybrid 403LC, Hybrid 404LC, Hybrid 501 LC, K 149, K 326, K 346, K 358, K394, K 399, K 730, KDH 959, KT 200, KT204LC, KY10, KY14, KY 160, KY 17, KY 171 , KY 907, KY907LC, KTY14xL8 LC, Little Crittenden, McNair 373, McNair 944, msKY 14xL8, Narrow Leaf Madole, Narrow Leaf Madole LC, NBH 98, N-126, N-777LC, N-7371 LC, NC 100, NC 102, NC 2000, NC 291 , NC 297, NC 299, NC 3, NC 4, NC 5, NC 6, NC7, NC 606, NC 71 , NC 72, NC 810, NC BH 129, NC 2002, Neal Smith Madole, OXFORD 207, PD 7302 LC, PD 7309 LC, PD 7312 LC 'Perique' tobacco, PVH03, PVH09, PVH19, PVH50, PVH51 , R 610, R 630, R 7-1 1 , R 7-12, RG 17, RG 81 , RG H51 , RGH 4, RGH 51 , RS 1410, Speight 168, Speight 172, Speight 179, Speight 210, Speight 220, Speight 225, Speight 227, Speight 234, Speight G-28, Speight G-70, Speight H-6, Speight H20, Speight NF3, Tl 1406, Tl 1269, TN 86, TN86LC, TN 90, TN 97, TN97LC, TN D94, TN D950, TR (Tom Rosson) Madole, VA 309, VA359, AA 37-1 , B13P, Xanthi (Mitchell-Mor), Bel-W3, 79-615, Samsun Holmes NN, KTRDC number 2 Hybrid 49, Burley 21 , KY8959, KY9, MD 609, PG01 , PG04, P01 , P02, P03, RG1 1 , RG 8, VA509, AS44, Banket A1 , Basma Drama B84/31 , Basma I Zichna ZP4/B, Basma Xanthi BX 2A, Batek, Besuki Jember, C104, Coker 347, Criollo Misionero, Delcrest, Djebel 81 , DVH 405, Galpao Comum, HB04P, Hicks Broadleaf, Kabakulak Elassona, Kutsaga E1 , LA BU 21 , NC 2326, NC 297, PVH 21 10, Red Russian, Samsun, Saplak, Simmaba, Talgar 28, Wislica, Yayaldag, Prilep HC-72, Prilep P23, Prilep PB 156/1 , Prilep P12-2/1 , Yaka JK-48, Yaka JB 125/3, TI-1068, KDH-960, TI-1070, TW136, Basma, TKF 4028, L8, TKF 2002, GR141 , Basma xanthi, GR149, GR153, Petite Havana. Low converter subvarieties of the above, even if not specifically identified herein, are also contemplated. A further aspect relates to a seed of a plant described herein. Suitably, the seed is a tobacco seed of a tobacco plant. A further aspect relates to pollen or an ovule of the plant. In addition, there is provided a plant as described herein which further comprises a nucleic acid conferring male sterility. Also provided is a tissue culture of regenerable cells of the plant or a part thereof, which culture regenerates plants capable of expressing all the morphological and physiological characteristics of the parent. The regenerable cells include but are not limited to cells from leaves, pollen, embryos, cotyledons, hypocotyls, roots, root tips, anthers, flowers and a part thereof, ovules, shoots, stems, stalks, pith and capsules or callus or protoplasts derived therefrom.

The promoter described here may increase the level of expression of one or more polynucleotides of interest. An increase in expression as compared to a control may be from about 5 % to about 100 %, or an increase of at least 10 %, at least 20 %, at least 25 %, at least 30 %, at least 40 %, at least 50 %, at least 60 %, at least 70 %, at least 75 %, at least 80 %, at least 90 %, at least 95 %, at least 98 %, or 100 % or more - such as 200% or 300% or more, which includes an increase in transcriptional activity.

The promoter described here may reduce the level of expression of one or more polynucleotides of interest. A reduction in expression as compared to a control may be from about 5 % to about 100 %, or a reduction of at least 10 %, at least 20 %, at least 25 %, at least 30 %, at least 40 %, at least 50 %, at least 60 %, at least 70 %, at least 75 %, at least 80 %, at least 90 %, at least 95 %, at least 98 %, or 100 %, which includes a reduction in transcriptional activity.

Several methods are available to assess promoter activity. Expression cassettes can be constructed with a visible marker. Transient transformation methods can be used to assess promoter activity. Using transformation methods - such as microprojectile bombardment, Agrobacterium transformation or protoplast transformation, expression cassettes can be delivered to plant cells or tissues. Reporter gene activity - such as .beta. -glucuronidase activity, luciferase activity or GFP fluorescence - can be monitored after transformation over time after DNA delivery using methods well known in the art. Reporter gene activity can be monitored by enzymatic activity, by staining cells or tissue with substrate for the enzyme encoded by the reporter gene or by direct visualisation under an appropriate wavelength of light. Full-length promoter sequences, deletions and mutations of the promoter sequence may be assayed and their expression levels compared. Additionally, RNA levels may be measured using methods well known in the art - such as Northern blotting, competitive reverse transcriptase PCR and RNAse protection assays. These assays can be used to measure the level of expression of a promoter. Further confirmation of promoter activity can be obtained by stable transformation of the promoter in an expression cassette comprising a visible marker or polynucleotide of interest into a plant. Using various methods - such as enzymatic activity assays, RNA analysis and protein assays - promoter activity can be monitored over development, and additionally by monitoring expression in different tissues in the primary transformants and through subsequent generations of transgenic plants.

Of particular interest in the present disclosure is plants in which the specific and relative concentrations of flavour and/or non-flavour compounds stored have been artificially manipulated. Desirably, such plants can be manipulated to synthesise and store flavour and non-flavour compounds at desired concentrations. Flavour and non-flavour compounds synthesised in plant cells can be so synthesised from precursor compounds by the activity of enzymes. The promoter of the present invention is useful for this purpose as it can be used to express one or more genes at modulated levels or in certain locations. Examples of such polynucleotides of interest (genes) that can be expressed using the promoter of the present invention include, but are not limited to polynucleotides selected from the group consisting of threonine synthase (see WO2013/029800), isopropylmalate synthase (WO2013/029799) or neoxanthin synthase (see WO2013/064499) or a combination of two or more thereof.

In embodiments, the promoter described here can be used to produce a polynucleotide either having a direct influence or encoding a polypetide having and influence on the reproductive function of the plant. Therefore, the promoter can influence the expression of a ribonucleic acid or a polypeptide which can promote sterility, induce mutations and/or DNA strand breaks or facilitate chromosomal crossings.

For example, genes can be introduced and specifically expressed in seeds and flowers, which are responsible for induction of sterility in the plant. Exemplary genes include the atp9 mitochondrial gene; see Hernould, et al., Proc Natl Acad Sci U S A. 1993 Mar 15;90(6):2370-4. Moreover, ribonucleases, antisense RNA or RNAi mediators can be encoded, which can suppress the expression of genes specifically in the seeds or flowers of the plant (see Mariani et al., 1990 Nature 347:737-741 ; AN et al., 2013 PLOS ONE vol. 8, e6816).

The promoter described herein can also be used to express genes in plant flowers, modifying the scent of flowers for commercial ends. For example, see Luecker et al., 2004 Plant Pysiol 134:510; El Tamer et al., 2003 J. Biotechnol 106:15; Luecker et al., 2004 Plant J. 39:135). Flower scents can be modified in ornamental species, as well as comestible species and smoking tobacco.

Cured plant material from the tobacco plants described herein is also provided. Processes of curing green tobacco leaves are known by those having skills in the art and include without limitation air-curing, fire-curing, flue-curing and sun-curing. The process of curing green tobacco leaves depends on the type of tobacco harvested. For example, Virginia flue (bright) tobacco is typically flue-cured, Burley and certain dark strains are usually air-cured, and pipe tobacco, chewing tobacco, and snuff are usually fire-cured.

Dried plant material from the tobacco plants described herein is also provided. Processes of drying tobacco flowers or seeds are known by those having skills in the art and include without limitation freeze drying, air drying, or sun curing.

There is contemplated the use of flower and/or seed materials which have been modified with a promoter as described herein. For example, such materials can be used for inclusion in tobacco products, for animal feeds, for the production of commercially desirable products including chemicals, pharmaceuticals and nutraceuticals, for the production of human and/or animal feedstuffs and as biological reactors. For example, flower material which has a modified scent can be included in tobacco products.

In another embodiment, there is described tobacco products including tobacco-containing aerosol forming materials comprising plant material - such as leaves, preferably cured leaves - from the plants described herein. The tobacco products described herein can be a blended tobacco product which may further comprise unmodified tobacco.

Methods for producing seeds are also described comprising cultivating the plant described herein, and collecting seeds from the cultivated plants. Seeds from the plants can be conditioned and bagged in packaging material by means known in the art to form an article of manufacture. Packaging material such as paper and cloth are well known in the art. A package of seed can have a label, for example, a tag or label secured to the packaging material, a label printed on the package that describes the nature of the seeds therein.

The invention is further described in the Examples below, which are provided to describe the invention in further detail. These examples, which set forth a preferred mode presently contemplated for carrying out the invention, are intended to illustrate and not to limit the invention. EXAMPLES

The following examples are provided as an illustration and not as a limitation. Unless otherwise indicated, conventional techniques and methods of molecular biology and plant biology are employed.

Example 1

Analysis of the NND3 promoter expression in different tissues

To determine the tissue specificity of the newly identified NND3 promoter sequence, different tissues of greenhouse grown N. tabacum var. TN90 are harvested and analysed for NND3 gene expression via quantitative PCR.

RNA extraction is performed using the RNeasy Mini kit (Qiagen) according to the manufacturer's instructions. RNA samples are diluted in water to obtain 1 μg RNA in 10 μΙ final volume. Then DNase digestion is performed with RQ1 RNase-Free DNase (Promega). The DNase reaction is stopped using RQ1 DNase stop solution (Promega). Immediately, the reverse transcriptase (RT) reaction is performed to convert RNA into complementary DNA (cDNA). For RT reactions, M-MLV Reverse Transcriptase, RNase H Minus, Point Mutant (Promega) was used in combination with oligo(dT)15 primers.

Quantitative real-time PCR is performed using the Stratagene Mx3005P and the corresponding software. For each target, different primer pairs are designed according to the guidelines of the Mx3005P user handbook. The primer pairs are tested for primer dimer formation and their performance in a qPCR run. Their efficiency is tested using a standard curve with a five-fold dilution of cDNA. The PCR products are sequenced in order to verify that the primers specifically amplify their target sequence. As the different CYP82E genes are close in sequence, primer design is complex and the chosen primers do not always show optimal efficiency.

The discrepancies in efficiency mean that the qPCR experiment values are gene specific and do not represent an absolute expression value. Therefore, the comparison in expression values is valid for each gene between the various tissues but not between genes. Employed primer pairs and their efficiency are listed in Table 1 . ABsolute Blue QPCR SYBR Green low ROX Mix (Thermo Scientific) is used with primer concentrations of 300 nM. In a qPCR run a denaturation temperature of 95°C is employed, initially for 15 minutes and then in each cycle for 15 seconds, 15 seconds at 60°C for annealing and 25 seconds at 72°C for elongation, for 50 cycles. All samples are run in triplicates. Furthermore, biological triplicates are employed. The expression of the actin9 gene (house-keeping gene) is used for all samples as normaliser. The results are shown in Figure 1 and confirm the expression of CYP82E5 and CYP82E10 in green leaves. These genes are also highly expressed in all other tissues that were tested. They show a very similar expression pattern. Whilst CYP82E4 and NND3 each show expression in flowers and very minor expression in roots, NND3 is not expressed at detectable levels in leaves whereas CYP82E4 shows expression exclusively in senescent leaves. For primer specificity verification, the PCR products have been purified and sequenced. The low signal observed for NND3 in roots contained mixed products of NND3 and CYP82E4 amplification whereas products from flower material contained NND3 amplification product only.

The tissue specificity of NND3 expression in flowers was further analyzed. Flowers were dissected into different tissues, RNA was extracted and NND3 expression analysis was performed (using primers NND3_F6 and NND3_R7). The results are shown in Figure 2 and demonstrate specific expression of NND3 in ovary tissue. More than 10-times reduced expression can be observed in other reproductive tissues (stigma, filaments, and anthers). However, in the leaves surrounding the reproductive organs, only very low NND3 expression was observed (100-fold and more than 1000-fold reduced expression in petals and sepals, respectively, compared to the ovaries).

TABLE 1

Primers used for expression analysis of NND3 and related functional CYP82E genes

Primer name Target gene Sequence (5'-3') Efficency (%)

NND3_F1 NND3 TTGATCCAGGGTTTCAATTACAGC 102.7 NND3 R2 NND3 AACGTACCAAATTAGAAAAACGTGTACC

E4_F1 CYP82E4 TTTTCAGAATTGGTTAGAGGAACATATTAAT 80.7 E4 R1 CYP82E4 TGTGTCTATCTCTTCTTGTGCTTTCG

E5_F1 CYP82E5 AGAGATTCTTCGCTGATGATATTGACTAC 101 .3 E5 R1 CYP82E5 CCGTAATTGTCACTTCTACAGGA I I I ACT

E10_F2 CYP82E10 GCTGATATTGACTTTCGTGGTCAA 87.3 E10 R2 CYP82E10 GCGAGGCGTAATTACCACTTCTAT

PQ0014_F Actin9 CTATTCTCCG CTTTG G ACTTGG CA

PQ0014 R Actin9 AGGACCTCAGGACAACGGAAACG

NND3 F6 NND3 AATTTTG GTCTCATCGTG AAG ATG ATA

NND3 R7 NND3 TCACTCTCTTCTACCCATCTATCCTTG Example 2

Relative expression levels of NND3 in N. tabacum var. Stella leaves at different curing time points

Relative expression levels of NND3 and related functional CYP82E genes in N. tabacum var. Stella leaves at different curing time points is analysed. Samples are taken in "green" leaves (upper stalk position) and in "ripe" leaves (lower stalk position) at harvest time. The leaves are transported to an air curing barn and a sample is taken when curing starts ("0 h") and then again after 12 hours ("12 h"), 24 hours ( "24 h") and 48 hours ("48 h"). Samples are taken from pools of several leaves. Two pools are analyzed as biological replicates - replicate 1 and 2. The results are shown in Figure 3. Bars indicate mean ± SD of three technical replicates. Expression levels of the different CYP82E genes can only be considered independently and not by direct comparison due to different PCR efficiencies. CYP82E5 and CYP82E10 are expressed to a similar extent in all leaf samples. Expression of CYP82E4 is activated in senescent leaves and increases under curing conditions. NND3 is not expressed in leaves under all conditions analyzed. Surprisingly NND3 shows a flower specific expression pattern with significant expression in flowers and flower derived material (capsules: i.e. dry fruit formed from fower tissue containing seeds).

Any publication cited or described herein provides relevant information disclosed prior to the filing date of the present application. Statements herein are not to be construed as an admission that the inventors are not entitled to antedate such disclosures. All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in cellular, molecular and plant biology or related fields are intended to be within the scope of the following claims.

SEQUENCES

SEQ ID NO: 1 AAAAAATAAAAATTTAAAGTTGAATTGTTTGGCCAAACTTTTGGAGTAAAAAAAGTGTTT T

G AAG AG AAG CAG AAG CAGTTTTG G AG AAG AAG AAAAAAATAGTTTCTCTC CAAAAGTAC

TTTTGAGAAAAATATACTTAGAACCAATTTTTAAAAATTTGATCAAATACTAATTAT TGCC

CATAAGTGATTTTCAAATTAATTAGTGAAACAAGCCAAACAAGTTATAACTTCAACC TTCA

ATATGTCCCTATTATCTTTAAACGGTAAAAAATCTCCTCTCTGTTTAGAACATTATC TCCA

TCTCGTGTCCTGTGCCCTTAAAAATGGCCGGCCATGTGAAGGCTCTGTTATCTGATA AA

TCTATTAATTAGACTTTACTTCCATTAATTTTTTTTTTTTTTTTTTTTTTTTTTTTT TTTTTTTT

TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGCTATGGTGTTAATTTTAG AATGACA

TTTCCTAAGAATCAAGATTTACACGTAGCTTAAAGAGTAACTCAATATTTTATTCTC AAGT

TGTATGTAGACATAAATTTTCTGTCGCTTTTGTCTTGATAATTCAAAATGCAAAATC TTAT

CTCTACTTTCTATCCTGTCTGTGATTGACCGGATCCGGTACTTATCTCACACTTATG CTA

GTGCACGTATTGTGTCGATATAGGTGCGGCACCACACATAAAGAGTTTGTACATCTT AG

ATCAGTGACGGGTTTACAATGTAACCTATGAATTTATGGAAATCTACCATAATAACA ATT

ATAATTGAGTCTTGGGTGGTTCCACGATTTGAATCCGTAACCTAAGTCACAAAAAAA CA

GATTTACTGTTTTAACGCCCAGCAAATTCATGATAAAAACAGTTCTAACATGACCAT CAA

AATGAAAATGATTGAAAAAAAGATGACTTTAAACAGAAAAAAACAGTTACCGCTGTG GTT

G AAAAG GG ATTGTG G CG CTGCTATTTCTG AAATGG AAG AC AG G GAACTTATCAAGTG A

GCTAATATATATTTCGATATTATTTTTTTAATATCAAATATTATATATCTAATATCA ATTTTA

TTTTAAAAAG G C C C AAATAC ATAAATAAAAAAAAAAAAAC CTTAATGTTG G CAG C AAAAT

CCATTTAAATACCTCTAACTAACTCTTGTACCTATTAGACATTCAAACACTCACTGA AGT

GTACCTATTGAACCCTTCATGCACATGTGGCACAAAGAGTGAGTTTCACTTCAGGTC CC

GCGCGTGAATGAATAACTAAAATATAATTTTATTTATTATCTTTGTTTTTCTTCACT CTCCT

TGGTCGCCTGAATCAGTCATCTTCATATTTTCAAGCTTCTCACCATTTTTCTTCTTA AATC

TTCTTGATAACATTGTTCTTCACAAAAATTATAAAATTTACACAACTCAAAATTACA CACA

ATCAAAAATAATATCTTGAAATTGATAACAACATCACTCTCTCCGCCACCACCAACC ACC

CTTGCTTCTTCCTCACTCCACCCTCACCAAAAATTTCATCAATGTTTGCTCCATCCG GCA

AGAACTCATATGGCAAGAACTCAGAAGCGAAAATGTTGGACTATTTCTAATTAAGTC CAA

AACTAAGTGCAACATATTTAAAAAATGTCAATTGACCCAATTAAGTTTTAAAATTCA ACAC

AACCCACTTCAAATCAAGCGTTATTTTCTTTAATTTTCTTGTCTTCAAGGAGTTTGG TGTT

TG ATTTG G AACAATAAATTTATTG G AAAGCAAGTTG AG AC GAT AAG CTTTGTTACG GG A

GGAATCCAAGGAGTCATCGGTATCGATTTTTATCGCTGGTTTCCTTCATTTTGTCGA GG

CACTATTATAGTTGTCATTTGTTTAGGCGTGGCAAAATAAGAACTTGGTTGTATAAT ATG

AATTCTAGCATAATTTGGTCTGAAAATGATGAATTCTAGCATAATTTGGTCTGAAAA GCA

AAACAAGAACACTAGCCAAATGGCTCATATCCCTTGCTAACTTGAATATGTTCGTCT TTC

TTTAGAGTTTAGATGAAGAAGAAGAAAAAACAACAGAAAAGCAACAAAAAAAAGAGA GA

AACAATGGAGAAGAAAAGGGGATAATCAGAAAGGAACGACGTCGGACATGAAGGAAG G

AAAGGGGATAATCAGAAAGGAAGTTTTTTCTTTTTTTGGCTTAAAGTCTTTTGCTTT TCCT TTTAAGTTTTATTTTATTCTTTTGTATTTTACATGTGTCACCCATTAATTGGTCCCTTTT CA CATCAGCATATAATAAAAACACGCTCTCCTTTTTGACAAAAATTGTGACTCAAGTGTTTA ATAGGTATACTTGAGTGATGTTGAAGTGTCTGATGGGTGCAAGGTGAGCTGAGGTATCT AAATGAATTTTGGTATCAACTTTAAGGAGTTGAGGTTAAAGTTAAGGTGTCTAAATAAAT TTTG ATATCAACTTTAAG G GTTTGTCTATTTATTTG G CCTTTTAAAAACTTAAACCAGTTA CCCTGTCAAAACCAAAAAACTGAATATAAAATATCAATATTTTTTATTTCGGGTACGATA A TTGTATTAAC CACCTTACAATTATATATAAAAAG GAAGTTG GTGATAG CTTG ATTC CCAA GTTCTTTTCTAAAAATCCATA