AU2009276290B2

AU2009276290B2 - Flower and/or seed active transcriptional control sequences

Info

Publication number: AU2009276290B2
Application number: AU2009276290A
Authority: AU
Inventors: Ainur Ismagul; Nataliya Kovalchuk; Ming Li; Sergiy Lopato
Original assignee: Australian Centre for Plant Functional Genomics Pty Ltd
Current assignee: Australian Centre for Plant Functional Genomics Pty Ltd
Priority date: 2008-07-30
Filing date: 2009-07-30
Publication date: 2015-07-16
Anticipated expiration: 2029-07-30
Also published as: WO2010012034A1; AU2009276290A1

Abstract

The present invention relates generally to transcriptional control sequences tor effecting expression of a nucleotide sequence of interest in a plant. More particularly, the present invention relates to transcriptional control sequences which specifically or preferentially direct expression of an operably connected nucleotide sequence in one or more parts of a plant flower and/or seed.

Description

WO 2010/012034 PCT/AU2009/000968 - 1 FLOWER AND/OR SEED ACTIVE TRANSCRIPTIONAL CONTROL SEQUEN CES PRIORITY CLAIM 5 This application claims priority to Australian provisional patent application 2008904485, filed 30 july 2008, the contents of Which are hereby incorporated by reference. 10 FIELD The present invention relates generally to transcriptional control sequences for effecting expression of a nucleotide sequence of interest in a plant. More particularly, the present invention relates to transcriptional control sequences that direct specific or 15 preferential expression of an operably connected nucleotide sequence of interest in a plant flower and/or seed or one or more particular cell or tissue types therein. BACKGROUND 20 Enhancement of resistance of developing and germinating grain to pathogens reqires identification and characterisation of suitable pathogen responsive (PR) and resistance (R) genes and strategies for modulation of their expression in transgenic plants. Many early attempts to boost disease resistance used constitutive overexpression of defence components but frequently this resulted in poor quality plants. It is now clear that, for 25 many strategies, tissue specific promoters night be the most useful as they limit the gene expression to infection sites. Although substantial effort is currently directed toward the characterisation of PR genes for most agriculturally important cereals, the no less important task of promoter isolation and characterisation has received relatively little attention. 30 Promoters active in the pericarp of cereals would be beneficial for the targeting of transgenes to these tissues. Such promoters, for example, may have application in the mediation of fungal diseases affecting wheat and barley crops, such as infections of Fusarium gramninearum. 5 In light of the above, promoters which can specifically or preferentially direct expression of a gene of interest in at least the pericarp of a plant seed would be desirable. 10 Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country SUMMARY 15 The present invention is predicated, in part on the identification and functional characterization of transcriptional control sequences which specifically or preferentially direct expression of an operably connected nucleotide sequence in one or more parts of a plant flower and/or seed. 20 In a first aspect the present invention provides an isolated nucleic acid comprising: a nucleotide sequence defining a transcriptional control sequence which specifically or preferentially directs expression of an operable connected nucleotide sequence in one or more parts of a plant flower and/or seed, wherein said transcriptional control 25 sequence is derived frorn a thionin gene which comprises the amino acid sequence set forth in SEQ ID NO: 1, or a homolog thereof, wherein the homolog comprises an amino acid sequence which comprises at least 35% identity to SEQ ID NO: 1, In some embodiments, the transcriptional control sequence directs expression of an 30 operably connected nucleotide sequence in at least an ovary or ovule of a flower In WO 2010/012034 PCT/AU2009/000968 - 3 some embodiments, the transcriptional control sequence directs expression of an operably connected nucleotide sequence in at least the pericarp of an immature seed. In a second aspect, the present invention also provides a nucleic acid construct 5 comprising an isolated nucleic acid according to the first aspect of the invention. In a third aspect, the present invention provides a cell comprising a nucleic acid construct according to the second aspect of the invention. 10 The cells contemplated by the third aspect of the invention include any prokaryotic or eukaryotic cell. In some embodiments, the cell is a plant cell. In a fourth aspect, the present invention contemplates a multicellular structure comprising one or more cells according to the third aspect of the invention. 15 In some embodiments, the multicellular structure comprises a plant or a part, organ or tissue thereof. In some embodiments a nucleotide sequence of interest may be operably connected to the transcriptional control sequence or the functionally active fragment or variant thereof, such that the nucleotide sequence of interest is specifically or 20 preferentially expressed in a flower arid/or seed of the plant. In a fifth aspect, the present invention provides a method for specifically or preferentially expressing a nucleotide sequence of interest in one or more parts of a plant flower and/or seed, the method comprising effecting transcription of the 25 nucleotide sequence of interest in a plant under the transcriptional control of a nucleic acid according to the first aspect of the invention. In some embodiments, the nucleotide sequence of interest is expressed in at least an ovary or ovule of a flower. In some embodiments, the nucleotide sequence of interest is 30 expressed in at least the pericarp of an immature seed.

WO 2010/012034 PCT/AU2009/000968 -4 Nucleotide and amino acid sequences are referred to herein by a sequence identifier number (SEQ ID NO:). A summary of the sequence identifiers is provided in Table 1. A sequence listing is provided at the end of the specification. 5 Table I - Summary of Sequence Identifiers SEQ ID NO 1 TdPRPI-10 protein anino acid sequence SEQ ID NO:2 TdPRPI-10 cDNA nucleotide sequence SEQ ID NO: 3 TdPRPI-10 promoter nuleotide sequence SEQ ID NO: 4 TdPRPI-10 gene nucleotide sequence SEQ ID NO: 5 TdPRPI-10 promoter and gene nucleotide sequence SEQ ID NO: 6 TaPRPI protein amino acid sequence SEQ ID NO: 7 TaPRPI cDNA nucleotide sequence SEQ ID NO: 8 OsPRPI protein anino acid sequence SEQ ID NO: 9 OsPRPI cDNA nucleotide sequence SEQ ID NO: 10 OsPRPI promoter nucleotide sequence SEQ ID NO: 11 OsPRPI gene nucleotice sequence SEQ ID NO: 12 OsPRPI promoter1an gene nucleotide sequence SEQ ID NO: 13 TdPRPI- I protein anino acid sequence SEQ ID NO: 14 TdPRPI-1l cDNA nucleotide sequence SEQ ID NO: 15 TdPRPI-1 1 promoter n uceotide sequence SEQ ID NO: 16 TdPRPI-11 gene nucleoide sequence SEQ ID NO: 17 TdPRPI-11 promoter and gene nucleoide sequence SEQ ID NO: 18 TdPRPI-1 protein minor acid sequence SEQ ID NO: 19 TdPRPI-1 cDNA nucleotide sequence SEQ ID NO: 20 TdPRPI-1 promoter nucleotide sequence SEQ ID NO: 21 TdPRPI-1 gene nucleotide sequence SEQ ID NO: 22 TdPRPI-1I promoter and gene n ucleotide sequence SEQ ID NO: 23 TdPRPI-5 protein amino acid sequence SEQ ID NO: 24 TdPRPI-5 cDNA nucleotide sequence WO 2010/012034 PCT/AU2009/000968 -5 SEQ ID NO: 25 TdPRPI-5 promoter nucleotide sequence SEQ ID NO: 26 TdPRPI-5 gene nucleotide sequence SEQ ID NO: 27 TdPRPI-5 promoter and gene nucleotide sequence SEQ ID NO: 28 TdPRPI-7 protein amino acid sequence SEQ ID NO: 29 TdPRPI-7 cDNA nucleotide sequence SEQ ID NO: 30 TdPRPI-7 promoter nucleotide sequence SEQ ID NO: 31 TdPRPI-7 gene nucleotide sequence SEQ ID NO: 32 TdPRPI-7 promoter and gene nucleotide sequence SEQ ID NO: 33 TdPRPI-8 protein amino acid sequence SEQ ID NO: 34 TdPRPI-8 cDNA nucleotide sequence SEQ ID NO: 35 TdPRPI-8 promoter nucleotide sequence SEQ ID NO: 36 TdPRPI-8 gene nucleotide sequence SEQ ID NO: 37 TdPRPI-8 promoter and gene nucleotide sequence DESCRIPTION OF EXEMPLARY EMBODIMENTS It is to be understood that the following description is for the purpose of describing 5 particular embodiments only and is not intended to be limiting with respect to the above description. The present invention is predicated, in part, on the identification and functional characterisation of transcriptional control sequences which specifically or preferentially 10 direct expression of an operably connected nucleotide sequence in one or more parts of a plant flower and/or seed. In some embodiments, the present invention is predicated, in part, on the cloning of 'y thionin like defensins and their associated promoters from wheat and rice. 15 As used herein, the term "transcriptional control sequence" should be understood as a nucleotide sequence that modulates at least the transcription of an operably connected nucleotide sequence. As such, the transcriptional control sequences of the present WO 2010/012034 PCT/AU2009/000968 -6 invention may comprise any one or more of, for example, a leader, promoter, enhancer or upstream activating sequence. As referred to herein, the term "transcriptional control sequence" preferably at least includes a promoter. A "promoter" as referred to herein, encompasses any nucleic acid that confers, activates or enhances expression of 5 an operably connected nucleotide sequence in a cell. As used herein, the term "operably connected" refers to the connection of a transcriptional control sequence, such as a promoter, and a nucleotide sequence of interest in such a way as to bring the nucleotide sequence of interest under the 10 transcriptional control of the transcriptional control sequence. For example, promoters are generally positioned 5' (upstream) of a nucleotide sequence to be operably connected to the promoter. In the construction of heterologous transcriptional control sequence/nucleotide sequence of interest combinations, it is generally preferred to position the promoter at a distance from the transcription start site that is 15 approximately the same as the distance between that promoter and the gene it controls in its natural setting, i.e. the gene from which the promoter is derived. As is known in the art, some variation in this distance can be accommodated without loss of promoter function. 20 Thus, in a first aspect, the present invention provides an isolated nucleic acid comprising: (i) a nucleotide sequence defining a transcriptional control sequence w which specifically or preferentially directs expression of an operably connected nucleotide sequence in one or more parts of a plant flower and/or seed, wherein said 25 transcriptional control sequence is derived from a gene which encodes a y-thionin; or (ii) a nucleotide sequence defining a functionally active fragment or variant of the nucleotide sequence defined at (i). In the present invention, "isolated" refers to material removed from its original 30 environment (e.g. the natural environment if it is naturally occurring), and thus is WO 2010/012034 PCT/AU2009/000968 -7 altered "by the hand of man" from its natural state. For example, an isolated nucleic acid could be part of a vector or a composition of matter, or could be contained within a cell, and still be "isolated" because that vector, composition of matter, or particular cell is not the original environment of the nucleic acid. An "isolated" nucleic acid 5 molecule should also be understood to include a synthetic nucleic acid molecule, including those produced by chemical synthesis using known methods in the art or by in-vitro amplification (e.g. polymerase chain reaction and the like). The isolated nucleic acid of the present invention may comprise any 10 polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. For example, the isolated nucleic acid molecules of the invention may comprise single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions., hybrid molecules comprising 15 DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, the isolated nucleic acid molecules may comprise triple-stranded regions comprising RNA or DNA or both RNA and DNA. The isolated nucleic acid molecules may also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. 20 "Modified" bases include, for example., tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus the term "nucleic acid" also embraces chemically, enzymatically, or metabolically modified forms of DNA and RNA. 25 As set out above, the method of the present invention contemplates a transcriptional control sequence which specifically or preferentially directs expression of an operably connected nucleotide sequence in one or more parts of a plant flower and/or seed. The term plant "flower" would be readily understood by a person skilled in the art. 30 However, for clarity, flowers generally include at least some of the main structures as WO 2010/012034 PCT/AU2009/000968 -8 follows (although not all flowers within the meaning of the term as used herein will include all of the structures noted below): Calyx: the outer whorl of sepals; typically these are green, but are petal--like in some species. 5 Corolla: the whorl of petals, which are usually thin, soft and colored to attract insects that help the process of pollination. Androeciun: one or two whorls of stamens, each a filament topped by an anther where pollen is produced. Pollen contains the male gametes. Gynocciuin: one or more pistils. The female reproductive organ is the carpel: this 10 contains an ovary with ovules (which contain female gametes). A pistil may comprise a number of carpets merged together, in which case there is only one pistil to each flower, or of a single individual carpel (the flower is then called apocarpous). The sticky tip of the pistil, the stigma, is the receptor of pollen. The supportive stalk, the style becomes the pathway for pollen tubes to grow from pollen grains adhering to the 15 stigma, to the ovules, carrying the reproductive material. Although the floral structure described above is considered the "typical" structural plan, plant species show a wide variety of modifications from this plan. For example, the two subclasses of flowering plants may be distinguished by the number of floral 20 organs in each whorl: dicotyledons typically having 4 or 5 organs (or a multiple of 4 or 5) in each whorl and monocotyledons having three or some multiple of three. In the majority of species individual flowers have both pistils and stamens as described above. However, in some species of plants the flowers have only either male (stamens) 25 or female (pistil) parts. The term "flower" should also be understood to include immature flowers prior to anthesis, mature flowers after anthesis as well as fertilised flowers. Furthermore, the term "flower" should also be understood to include flowers which may comprise a 30 mature or immature seed and associated structures. Such flowers generally include WO 2010/012034 PCT/AU2009/000968 -9 fertilised flowers, but may also include unfertilised flowers in which a seed develops, such as by the process of apomixis. As would be appreciated, the term "seed" encompasses whole seeds as well as the 5 various cells and tissues that make up a mature or immature seed. For example, seeds may include tissue types such as the embryo, embryo surrounding region, endosperm transfer layer, endosperm, aleurone layer, pericarp and the like. Meanwhile, immature seeds may include, for example, fertilised egg cells, zygotes, fertilised central cells, embryos, the endosperm coenocyte, the endosperm syncytium and the like. 10 The term "seed" as used herein may also include one or more immature plant structures associated with a germinating seed, including, for example, the embryo, scutellum or cotyledon(s), radicle, coleorhiza, coleoptile, hypocotyl and epicotyl. 15 As set out later, in some specific embodiments, the present invention contemplates plants of the grass family (Poaceae) and cereal crop plants. In these embodiments, the term "flower" should be understood to include a floret of such plants, which may include support structures such as the lemma and palea. Furthermore, the term flower or floret should also be understood to include florets in the grass family which may 20 include a seed, grain or caryopsis. It should be understood that reference herein to expression in a plant flower and/or seed refers to the transcription and/or translation of a nucleotide sequence in one or more cells or tissues of a plant flower and/or seed and/or at one or more 25 developmental stages of the plant flower and/or seed. Thiis in no way implies that expression of the nucleotide sequence must occur in all cells of the plant flower and/or seed or at all developmental stages of the flower andl/or seed. As set out later, the nucleic acids of the present invention may direct expression in particular parts of a flower and/or seed and/or at particular developmental stages of a flower and/or seed. 30 WO 2010/012034 PCT/AU2009/000968 -10 As set out above, the transcriptional control sequences contemplated by the present invention "specifically or preferentially" direct expression of an operably connected nucleotide sequence in a plant flower and/or seed. As used herein, "specifically expressing" means that the nucleotide sequence of interest is expressed substantially 5 only in a plant flower and/or seed (or a particular tissue or cell type therein). "Preferentially expressing" should be understood to mean that the nucleotide sequence of interest is expressed at a higher level in a plant flower and/or seed (or tissue or cell type therein) than in one or more other tissues of the plant, e.g. mature leaf or stem tissue. In some embodiments "preferential" expression in a plant flower and/or seed 10 includes expression of a nucleotide sequence of interest in a plant flower and/or seed (or a tissue or cell type therein) at a level of, for example, at least twice, at least 5 times or at least 10 times the level of expression seen in at least one other non-flower tissue and/or non-seed tissue of the plant. 15 The transcriptional control sequence or functionally active fragment or variant thereof may effect specific or preferential expression in a flower and/or seed from at least one flowering plant species, including monocotyledonous angiosperm plants ("monocots") or dicotyledonous angiosperm plants ("dicots"). For clarity, this should be understood as the transcriptional control sequence or functionally active fragment or variant 20 thereof being able to effect specific or preferential expression in a flower and/or seed in at least one plant species. The transcriptional control sequence may or may not effect expression in one or more other plant species, and this expression may or may not be specific or preferential to the flower and/or seed. Thus, the transcriptional control sequences of the present invention need not be active in all plant species, and need not 25 necessarily direct specific or preferential expression in the flower and/or seed in all plants in which they are active. In some embodiments, the transcriptional control sequence specifically or preferentially directs expression of an operably connected nucleotide sequence in a 30 flower and/or seed of a mronocotyledonous plant.

WO 2010/012034 PCT/AU2009/000968 -11 In some embodiments, the transcriptional control sequence specifically or preferentially directs expression of an operably connected nucleotide sequence in a flower and/or seed of a plant in the family Poaceae. 5 In some embodiments, the transcriptional control sequence specifically or preferentially directs expression of an operably connected nucleotide sequence in a flower and/or seed of a cereal crop plant. 10 As used herein, the term "cereal crop plant" may be a member of the Poaceae (grass family) that produces grain. Examples of Poaceae cereal crop plants include wheat, rice, maize, millets, sorghum, rye, triticale, oats, barley, teff, wild rice, spelt and the like. The term cereal crop plant should also be understood to include a number of non Poaceae plant species that also produce edible grain, which are known as the 15 pseudocereals and include, for example, amaranth, buckwheat and quinoa. In some embodiments, the transcriptional control sequence specifically or preferentially directs expression of an operably connected nucleotide sequence in a flower and/or seed of a wheat plant. 20 As referred to herein, "wheat" should be understood as a plant of the genus Triticum. Thus, the term "wheat" encompasses diploid wheat, tetraploid wheat and hexaploid wheat. In some embodiments, the wheat plant may be a cultivated species of wheat including, for example, T. aestivum, T. durum, T'. nococcum or T. spelta. In some 25 embodiments, the term "wheat" refers to wheat of the species Triticum aestivum. In some embodiments, the transcriptional control sequence specifically or preferentially directs expression of an operably connected nucleotide sequence in a flower and/or seed of a rice plant. 30 WO 2010/012034 PCT/AU2009/000968 -12 As referred to herein, "rice" includes several members of the genus Oryza including the species Oryza sativa and Oryza giaberrima. The term "rice" thus encompasses rice cultivars such as japonica or sinica varieties, indica varieties and javonica varieties. In some embodiments, the term "rice" refers to rice of the species Oryza sativa. 5 As set out above, the nucleic acid of the first aspect of the present invention may also specifically or preferentially direct expression in a particular cell or tissue of a plant flower and/or seed and/or specifically or preferentially direct expression at a particular developmental stage of a plant flower and/or seed. 10 In some embodiments, the nucleic acid according to the first aspect of the invention comprises a transcriptional control sequence which directs expression of an operably connected nucleotide sequence in one or more parts of a plant flower at one or more developmental stages selected from before anthesis, after anthesis or after fertilisation. 15 In some embodiments, the transcriptional control sequence directs expression of an operably connected nucleotide sequence in one or more parts of a plant flower selected from: an ovary or ovule, the lemma and/or palea (in flowers having such structures) or an anther. In some embodiments, the transcriptional control sequence directs 20 expression of an operably connected nucleotide sequence in at least an ovary or ovule of a flower. In some embodiments, the nucleic acid according to the first aspect of the invention comprises a transcriptional control sequence which directs expression of an operably 25 connected nucleotide sequence in one or more parts of a plant seed at one or more developmental stages selected from: immature seed or germinating seed. In some embodiments, the transcriptional control sequence directs expression of an operably connected nucleotide sequence in one or more parts of a plant seed selected 30 from: an imnmature seed in a flower, one or both poles of an immature seed, the WO 2010/012034 PCT/AU2009/000968 -13 pericarp of a seed, an embryo in a seed, vascular tissue in a seed, or one or more structures associated with a germinating seed (including, for example, the embryo, scutelium or cotyledon(s), radicle, coleorhiza, coleoptile, hypocotyl and epicotyl). In some embodiments, the transcriptional control sequence directs expression of an 5 operable connected nucleotide sequence in at least the pericarp of an immature seed. As referred to herein, the "pericarp" of a seed should be understood to encompass... The pericarp is the tissue that develops from the ovary wall of the flower and 10 surrounds the seeds of a plant. The pericarp itself is typically made up of three distinct layers: the exocarp, which is the most-outside layer, the mesocarp, which is the middle layer, and the endocarp, which is the inner layer surrounding the ovary or the seeds. The grains of grasses are single-seed simple fruits wherein the pericarp (ovary wall) and seed coat are fused into one layer. This type of fruit may be referred to as a 15 caryopsis. Examples include cereal grains, such as wheat, barley, and rice. As would be appreciated, expression in one or more particular parts of the flower and/or seed may occur at one or more specific developmental stages of the flower and/or seed, including, for example, the developmental stages of the flower and/or 20 seed during which the particular part exists. For example, expression in a seed or caryopsis of the flower would be restricted to a developmental stage of the flower when a seed or caryopsis is present in the flower. As set out above, the present invention contemplates transcriptional control sequences 25 which specifically or preferentially direct expression of an operably connected nucleotide sequence in one or more parts of a plant flower and/or seed, wherein said transcriptional control sequence is derived from a gene which encodes a y-thionin The term "derived from", as used herein, refers to a source or origin for the 30 transcriptional control sequence. For example, a transcriptional control sequence WO 2010/012034 PCT/AU2009/000968 -14 "derived from a gene which encodes a y-thionin" refers to a transcriptional control sequence which, in its native state, exerts at least some transcriptional control over a gene which encodes a w-thionin in a plant. The term derived from should also be understood to refer to the source of the sequence information for a transcriptional 5 control sequence and not be limited to the source of a nucleic acid itself. Thus, a transcriptional control sequence derived from a gene which encodes a y-thionin, need not necessarily be directly isolated from the gene. For example, a synthetic nucleic acid having a sequence that is determined with reference to a transcriptional control sequence which, in its native state, exerts at least some transcriptional control over a 10 gene which encodes a y-thionin, should be considered derived from a gene which encodes a y-thionin. Gamma-thionins (y-thionins) are small evolutionarily related proteins of plants that function as defensive proteins from pathogens and/or parasites. In their mature form, 15 y-thionins generally consist of about 45 to 85 amino-acid residues. However, y-thionins of larger or smaller sizes may also fall within the scope of the term y-thionins as referred to herein. Plant y-thionins have three dimensional structures similar to defensins from insects. 20 Thus, they may also be referred to as "plant defensins" due to structural and functional similarities to animal defensins. Defensins demonstrate anti-bacterial and or anti fungal activity as a result of pathogen membrane permeabilization. In high concentrations plant defensins can be potentially toxic to insects because of their ability to inhibit animal a-amylases and proteinases. 25 The folded structure of gamma-purothionin is characterised by a well-defined 3 stranded anti-parallel beta-sheet and a short alpha-helix. Three disulfide bridges are located in the hydrophobic core between the helix and sheet, forming a cysteine stabilised alpha-helical motif. This structure differs from that of the plant alpha-- and 30 beta-thionins, but is analogous to scorpion toxins and insect defensins.

WO 2010/012034 PCT/AU2009/000968 -15 Examples of known y-thionins include: Gamma-thionins from Trfiticum aestivum endosperm (gamma-purothionins) and gamma-hordothionins from Hordeum culgare which are toxic to animal cells and inhibit 5 protein synthesis in cell free systems; A flower-specific thionin (FS'T) from Nicotiana tabacuim; Antifungal proteins (AFP) from the seeds of Brassicaceae species such as radish, mustard, turnip and Arabidopsis thaliana; Inhibitors of insect alpha-amylases from sorghum; 10 Probable protease inhibitor P322 from Solanum tuberosum; A germination-related protein from Vigna unguiculata; Anther-specific protein SF18 from sunflower, which contains a ganmma-thionin domain at its N-terminus and a proline-rich C-terminal domain; Glycine max sulfur-rich protein SE60; and 15 Viciafaba antibacterial peptides fabatin-I and -2. In some embodiments, the y-thionin contemplated in accordance with the present invention comprises the amino acid sequence set forth in SEQ ID NO: I or a homolog thereof. 20 The term "homolog", as used herein with reference to homologs of polypeptides comprising the amino acid sequence set forth in SEQ ID NO: 1, should be understood to include, for example, homologs, orthologs, paralogs, mutants and variants of polypeptides comprising the amino acid sequence set forth in SEQ ID NO: 1. In some 25 embodiments, the homolog, ortholog, paralog, mutant or variant of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 comprises an amino acid sequence which comprises at least 35% sequence identity, at least 40% sequence identity, at least 45% sequence identity, at least 50% sequence identity, at least 55% sequence identity, at least 60% sequence identity, at least 65% sequence identity, at 30 least 70% sequence identity, at least 75% sequence identity, at least 80% sequence WO 2010/012034 PCT/AU2009/000968 -16 identity, at least 85% sequence identity, at least 90% sequence identity or at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. When comparing amino acid sequences to calculate a percentage identity, the 5 compared sequences should be compared over a comparison window of at least 20 amino acid residues, at least 40 amino acid residues, at least 60 amino acid residues, at least 80 amino acid residues, or over the full length of SEQ ID NO: 1. The comparison window may comprise additions or deletions (i.e. gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) 10 for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerised implementations of algorithms such the BLAST family of programs as, for example, disclosed by Altschul et al. (Nucl. Acids Rcs. 25: 3389-3402, 1997). A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al. (Current Protocols in Molecular 15 Biology, John Wiley & Sorts Inc, 1994-1998, Chapter 15, 1998). Examples of homologs of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 include for example: a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 6, 20 which is derived from Triticunm aestivum. a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 8, which is derived from Oryza sativa; and a polypeptide comprising an amino acid sequence selected from the list consisting of SEQ ID NO: 13, SEQ ID NO: 18, SEQ ID NO: 23, SEQ ID NO: 28 or SEQ 25 ID NO: 33, each of which are derived from Triticum durum. The transcriptional control sequences of the present invention may be derived from any source, including isolated from any suitable organism or they may be synthetic nucleic acid molecules. 30 WO 2010/012034 PCT/AU2009/000968 -17 In some embodiments, however, the transcriptional control sequences contemplated herein are derived from a plant. In some embodiments, the transcriptional control sequences of the present invention are derived from a monocot plant species. In some embodiments the transcriptional control sequences of the present invention are derived 5 from a plant in the family Poaceae. In some embodiments, the transcriptional control sequences of the present invention are derived from a cereal crop plant species. In one specific embodiment, the transcriptional control sequence is derived from a Triticum species (for example T. aestivum, T. durum, T. monococcum, T. dicoccon, T. spelta 10 or T. polonicum). In some embodiments, the transcriptional control sequence is derived from a tetraploid wheat (for example T. durum, T. dicoccon, or T. polonicum). In some embodiments, the transcriptional control sequence is derived from a durum wheat, and in some embodiments, the transcriptional control sequence is derived from Triticum durum. 15 In some embodiments, the transcriptional control sequence is derived from a gene which comprises an open reading frame comprising the nucleotide sequence set forth in SEQ ID NO: 2 or a homology thereof. 20 One example of a gene which comprises an open reading frame comprising the nucleotide sequence set forth in SEQ ID NO: 2 is a gene comprising the nucleotide sequence set forth in SEQ ID NO: 4. The term "homolog", as used herein with reference to homologs of genes comprising 25 an open reading frame comprising the nucleotide sequence set forth in SEQ ID NO: 2, should be understood to include, for example, homologs, orthologs, paralogs, mutants and variants of genes comprising an open reading frame which comprises the nucleotide sequence set forth in SEQ ID NO: 2. In some embodiments, the homolog, ortholog, paralog, mutant or variant of a polypeptide comprising an open reading 30 frame which comprises the nucleotide sequence set forth in SEQ ID NO: 2 comprises a WO 2010/012034 PCT/AU2009/000968 -18 nucleotide sequence which comprises at least 35% sequence identity, at least 40% sequence identity, at least 45% sequence identity, at least 50% sequence identity, at least 55% sequence identity, at least 60% sequence identity, at least 65% sequence identity, at least 70% sequence identity, at least 75% sequence identity, at least 80% 5 sequence identity, at least 85% sequence identity, at least 90% sequence identity or at least 95% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 2. When comparing nucleotide sequences to calculate a percentage identity, the compared sequences should be compared over a comparison window of at least 20 10 nucleotide residues, at least 50 nucleotide residues, at least 100 nucleotide residues, at least 150 nucleotide residues, at least 200 nucleotide residues or over the full length of SEQ ID NO: 2. The comparison window may comprise additions or deletions (i.e. gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal 15 alignment of sequences for aligning a comparison window may be conducted by computerised implementations of algorithms such the BLAST family of programs as, for example, disclosed by Altschul et al. (Nucl. Acids Res. 25: 3389-3402, 1997). A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et a!. (Current Protocols in Aolecular Biology, John Wiley & Sons Inc, 1994-1998, Chapter 15, 20 1998). One example of a gene comprising a homolog of an open reading frame comprising the nucleotide sequence set forth in SEQ ID NO: 2 includes a gene comprising an open reading frame comprising the nucleotide sequence set forth in SEQ ID NO: 7. 25 Further examples of genes comprising an open reading frame homologous to the nucleotide sequence set forth in SEQ ID NO: 2 include: a gene comprising an open reading frame comprising the nucleotide sequence set forth in SEQ ID NO: 9; and/or a gene which comprises the nucleotide sequence set 30 forth in SEQ ID NO: 11; WO 2010/012034 PCT/AU2009/000968 -19 a gene comprising an open reading frame comprising the nucleotide sequence set forth in SEQ ID NO: 14; and/or a gene which comprises the nucleotide sequence set forth in SEQ ID NO: 16; a gene comprising an open reading frame comprising the nucleotide sequence 5 set forth in SEQ ID NO: 19; and/or a gene which comprises the nucleotide sequence set forth in SEQ ID NO: 21; a gene comprising an open reading frame comprising the nucleotide sequence set forth in SEQ ID NO: 24; and/or a gene which comprises the nucleotide sequence set forth in SEQ ID NO: 26; 10 a gene comprising an open reading frame comprising the nucleotide sequence set forth in SEQ ID NO: 29; and/or a gene which comprises the nucleotide sequence set forth in SEQ ID NO: 31; and a gene comprising an open reading frame comprising the nucleotide sequence set forth in SEQ ID NO: 34; and/or a gene which comprises the nucleotide sequence set 15 forth in SEQ ID NO: 36. In some embodiments, the transcriptional control sequence contemplated by the first aspect of the invention comprises the nucleotide sequence set forth in SEQ ID NO: 3 or a functionally active fragment or variant thereof. 20 In some embodiments, the transcriptional control sequence contemplated by the first aspect of the invention comprises the nucleotide sequence set forth in SEQ ID NO: 10 or a functionally active fragment or variant thereof. 25 In some embodiments, the transcriptional control sequence contemplated by the first aspect of the invention comprises the nucleotide sequence set forth in SEQ ID NO: 15 or a functionally active fragment or variant thereof. In some embodiments, the transcriptional control sequence contemplated by the first 30 aspect of the invention comprises the nucleotide sequence set forth in SEQ ID NO: 20 WO 2010/012034 PCT/AU2009/000968 -20 or a functionally active fragment or variant thereof. In some embodiments, the transcriptional control sequence contemplated by the first aspect of the invention comprises the nucleotide sequence set forth in SEQ ID NO: 25 5 or a functionally active fragment or variant thereof. In some embodiments, the transcriptional control sequence contemplated by the first aspect of the invention comprises the nucleotide sequence set forth in SEQ ID NO: 30 or a functionally active fragment or variant thereof. 10 In some embodiments, the transcriptional control sequence contemplated by the first aspect of the invention comprises the nucleotide sequence set forth in SEQ ID NO: 35 or a functionally active fragment or variant thereof. 15 As set out above, the present invention also contemplates "functionally active fragments or variants" of the transcriptional control sequences of the present invention, including (but not limited to) functionally active fragments or variants of a transcriptional control sequence comprising the nucleotide sequence set forth in any of SEQ ID NO: 3, SEQ ID NO: 10, SEQ ID NO: 15, SEQ ID NO: 20, SEQ ID NO: 25, SEQ ID 20 NO: 30 or SEQ ID NO: 35. "Functionally active fragments" of the transcriptional control sequence of the invention include fragments of a transcriptional control sequence which retain the capability to specifically or preferentially direct expression of an operably connected nucleotide 25 sequence in a plant flower and/or seed (or a particular cell or tissue type thereof as hereinbefore described) in at least one plant type. In some embodiments of the invention the functionally active fragment is at least 200 nucleotides (nt), at least 500 nt, at least 1000 nt, at least 1500 nt or at least 2000 nt in length. In some embodiments, the fragment comprises at least 200 nt, at least 500 nt, at least 1000 nt or at least 1500 nt 30 contiguous bases from the nucleotide sequence set forth in any of SEQ ID NO: 3, SEQ WO 2010/012034 PCT/AU2009/000968 -21 ID NO: 10, SEQ ID NO: 15, SEQ ID NO: 20, SEQ ID NO: 25, SEQ ID NO: 30 or SEQ ID NO: 35. "Functionally active variants" of the transcriptional control sequence of the invention 5 include orthologs, mutants, synthetic variants, analogs and the like which are capable of effecting transcriptional control of an operably connected nucleotide sequence in a plant flower and/or seed (or a particular cell or tissue type thereof as hereinbefore described) in at least one plant type. The term "variant" should be considered to specifically include, for example, orthologous transcriptional control sequences from 10 other organisms; mutants of the transcriptional control sequence; variants of the transcriptional control sequence wherein one or more of the nucleotides within the sequence has been substituted, added or deleted; and analogs that contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as 15 inosine. In some embodiments, the functionally active fragment or variant comprises at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 85%, at least 87%, at least 90%, at least 92%, at least 95%, at least 96%, 20 at least 97%, at least 98% or at least 99% nucleotide sequence identity to the nucleotide sequence set forth in any of SEQ ID NO: 3, SEQ ID NO: 10, SEQ ID NO: 15, SEQ ID NO: 20, SEQ ID NO: 25, SEQ ID NO: 30 or SEQ ID NO: 35. When comparing nucleic acid sequences to calculate a percentage identity, the compared nucleotide sequences should be compared over a comparison window of at least 200 nucleotide residues, at 25 least 400 nucleotide residues, at least 1000 nucleotide residues, at least 1500 nucleotide residues or over the full length of any of SEQ ID NO: 3, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 20, SEQ ID NO: 25, SEQ ID NO: 30 or SEQ ID NO: 35. The comparison window may comprise additions or deletions (i.e. gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) 30 for optimal alignment of the two sequences. Optimal alignment of sequences for WO 2010/012034 PCT/AU2009/000968 -22 aligning a comparison window may be conducted by computerised implementations of algorithms such the BLAST family of programs as, for example, disclosed by Altschul et al. (1997, supra). A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al. (1998, supra). 5 In some embodiments, the functionally active fragment or variant comprises a nucleic acid molecule which hybridises to a nucleic acid molecule defining a transcriptional control sequence of the present invention under stringent conditions. In some embodiments, the functionally active fragment or variant comprises a nucleic acid 10 molecule which hybridises to a nucleic acid molecule comprising the nucleotide sequence set forth in any of SEQ ID NO: 3, SEQ ID NO: 10, SEQ ID NO: 15, SEQ ID NO: 20, SEQ ID NO: 25, SEQ ID NO: 30 or SEQ ID NO: 35 under stringent conditions. As used herein, "stringent" hybridisation conditions will be those in which the salt 15 concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least 30'C. Stringent conditions may also be achieved with the addition of destabilising agents such as formamide. In some embodiments, stringent hybridisation conditions may be low stringency conditions, medium stringency conditions or high stringency 20 conditions. Exemplary low stringency conditions include hybridisation with a buffer solution of 30 to 35% formamide, I M NaCl, 1% SDS (sodium dodecyl sulphate) at 37 0 C, and a wash in 1x to 2xSSC (2OxSSC=3.0 M NaCI/0.3 M trisodium citrate) at 50 to 55 0 C. Exemplary moderate stringency conditions include hybridisation in 40 to 45% formamide, 1.0 M NaCI, 1% SDS at 37 0 C., and a wash in 0.5x to 1xSSC at 55 to 60'C. 25 Exemplary high stringency conditions include hybridisation in 50% formnamide, 1 M NaCi, 1% SD S at 37'C., and a wash in 0,1xSSC at 60 to 65 0 C. Optionally, wash buffers may comprise about 0.1% to about 1% SDS. Duration of hybridisation is generally less than 24 hours, usually 4 to 12 hours. 30 Specificity of hybridisation is also a function of post-hybridisation washes, with the WO 2010/012034 PCT/AU2009/000968 - 23 critical factors being the ionic strength and temperature of the final wash solution. For DNA--DNA hybrids, the Tm can be approximated from the equation of Meinkoth and Wahl (Anal. Biochen. 138: 267-284, 1984), i.e. Tm, =81.5 0 C +16.6 (log M)+0.41 (% GC)-0.61 (% form)-500/L; where M is the molarity of monovalent cations, % GC is the percentage 5 of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridisation solution, and L is the length of the hybrid in base pairs. The Tm, is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridises to a perfectly matched probe. 'I'm is reduced by about IC for each 1% of mismatching; thus, Tm, hybridisation., and/or wash 10 conditions can be adjusted to hybridise to sequences of different degrees of complementarity, For example, sequences with .90% identity can be hybridised by decreasing the Tm by about 10 0 C. Generally, stringent conditions are selected to be about 5 0 C lower than the thermal melting point (Tm) for the specific sequence and its complement at a defined ionic strength and pH. However, high stringency conditions 15 can utilise a hybridisation and/or wash at, for example, 1, 2, 3, or 4 0 C lower than the thermal melting point (Tm); medium stringency conditions can utilise a hybridisation and/or wash at, for example, 6, 7, 8, 9, or 10'C lower than the thermal melting point (Tm); low stringency conditions can utilise a hybridisation and/or wash at, for example, 11, 12, 13, 14, 15, or 20'C lower than the thermal melting point (Tm). Using the equation, 20 hybridisation and wash compositions, and desired Tm., those of ordinary skill will understand that variations in the stringency of hybridisation and/or wash solutions are inherently described. If the desired degree of mismatching results in a Tm of less than 45 0 C (aqueous solution) or 32 0 C (formamide solution), it is preferred to increase the SSC concentration so that a higher temperature can be used. An extensive guide to the 25 hybridisation of nucleic acids is found in Tijssen (Laboratory Techniques in Biochenistry and Molecular Biology-yf-hbridisation with Nucleic Acid Probes, Pt i, Chapter 2, Elsevier, New York, 1993), Ausubel et al., eds. (Current Protocols in Molecular Biology, Chapter 2, Greene Publishing and Wiley-Interscience, New York, 1995) and Sanbrook et al. (Molecular Cloning: A Laboratory A1hnual, 2,-- ed., Cold Spring Harbor Laboratory Press, 30 Plainview, NY, 1989).

WO 2010/012034 PCT/AU2009/000968 -24 In a second aspect, the present invention also provides a nucleic acid construct comprising an isolated nucleic acid according to the first aspect of the invention. 5 The nucleic acid construct of the second aspect of the present invention may comprise any polyribonucleotide or polydeoxyribonucleotide, which may be urnodified RNA or DNA or modified RNA or DNA. For example, the nucleic acid construct of the invention may comprise single- and/or double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA 10 that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, the nucleic acid construct may comprise triple-stranded regions comprising RNA or DNA or both RNA and DNA. The nucleic acid construct may also comprise one or more modified bases or 15 DNA or RNA backbones modified for stability or for other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus the term "nucleic acid construct" embraces chemically, enzymatically, or metabolically modified forms. 20 In some embodiments, the nucleic acid construct comprises DNA. Accordingly, the nucleic acid construct of the present invention may comprise, for example, a linear DNA molecule, a plasmid, a transposon, a cosmid, an artificial chromosome or the like. Furthermore, the nucleic acid construct of the present invention may be a separate nucleic acid molecule or may be a part of a larger nucleic acid molecule. 25 In some embodiments, the nucleic acid construct further comprises a nucleotide sequence of interest that is heterologous with respect to the transcriptional control sequence or the functionally active fragment or variant thereof; wherein the nucleotide sequence of interest is operably connected to the transcriptional control sequence or 30 functionally active fragment or variant thereof.

WO 2010/012034 PCT/AU2009/000968 -25 The term "heterologous with respect to the transcriptional control sequence" refers to the nucleotide sequence of interest being any nucleotide sequence other than that which the transcriptional control sequence (or functionally active fragment or variant 5 thereof) is operably connected to in its natural state. For example, in its natural state, SEQ ID NO: 3 is operably corrected to the nucleotide sequence set forth in SEQ ID NO: 4. AccordingIY, in this example, any nucleotide sequence other than a nucleotide sequence consisting of the nucleotide sequence set forth in SEQ ID NO: 4 should be considered heterologous with respect to SEQ ID NO: 3. 10 In a further example, SEQ ID NO: 10 is operably connected to SEQ ID NO: 11 in its natural state. As such, any nucleotide sequence other than a nucleotide sequence consisting of the nucleotide sequence set forth in SEQ ID NO: 11 should be considered heterologous with respect to SEQ ID NC): 10. 15 In a further example, SEQ ID NO: 15 is operably connected to SEQ ID NO: 16 in its natural state. As such, any nucleotide sequence other than a nucleotide sequence consisting of the nucleotide sequence set forth in SEQ ID NO: 16 should be considered heterologous with respect to SEQ ID NO: 15. 20 In a further example, SEQ ID NO: 20 is operably connected to SEQ ID NO: 21 in its natural state. As such, any nucleotide sequence other than a nucleotide sequence consisting of the nucleotide sequence set forth in SEQ ID NO: 21 should be considered heterologous with respect to SEQ ID NO: 20. 25 In a further example, SEQ ID NO: 25 is operably connected to SEQ ID NO: 26 in its natural state. As such, any nucleotide sequence other than a nucleotide sequence consisting of the nucleotide sequence set forth in SEQ ID NO: 26 should be considered heterologous with respect to SEQ ID NO: 25. 30 WO 2010/012034 PCT/AU2009/000968 -26 In a further example, SEQ ID NO: 30 is operably connected to SEQ ID NO: 31 in its natural state. As such, any nucleotide sequence other than a nucleotide sequence consisting of the nucleotide sequence set forth in SEQ ID NO: 31 should be considered heterologous with respect to SEQ ID NO: 30. 5 In a further example, SEQ ID NO: 35 is operably connected to SEQ ID NO: 36 in its natural state. As such, any nucleotide sequence other than a nucleotide sequence consisting of the nucleotide sequence set forth in SEQ ID NO: 36 should be considered heterologous with respect to SEQ ID NO: 35. 10 In accordance with the definition above, it would be recognised that a nucleotide sequence of interest which is heterologous to the transcriptional control sequence (or functionally active fragment or variant thereof) may be derived from an organism of a different taxon to the transcriptional control sequence (or functionally active fragment 15 or variant thereof) or the nucleotide sequence of interest may be a heterologous sequence from an organism of the same taxon. In some enbodiments, the nucleic acid construct may further comprise a nucleotide sequence defining a transcription terminator. The term "transcription terminator" or 20 "terminator" refers to a DNA sequence at the end of a transcriptional unit which signals termination of transcription. Terminators are generally 3-non-translated DNA sequences and may contain a polyadenylation signal, which facilitates the addition of polyadenylate sequences to the 3-end of a primary transcript. As with promoter sequences, the terminator may be any terminator sequence which is operable in the 25 cells, tissues or organs in which it is intended to be used. Examples of suitable terminator sequences which may be useful in plant cells include: the nopaline synthase (ios) terminator, the CaMV 35S terminator, the octopine synthase (ocs) terminator, potato proteinase inhibitor gene (pin) terminators, such as the pinIh and pill terminators and the like. 30 WO 2010/012034 PCT/AU2009/000968 - 27 In some embodiments the nucleic acid construct comprises an expression cassette comprising the structure: ([N], - TCS - [N]x - SoI - [N]y - TT - [N]z ) 5 wherein: [Niw comprises one or more nucleotide residues, or is absent; TCS comprises a nucleic acid according to any one of the first aspect of the invention; [N]x comprises one or more nucleotide residues, or is absent; 10 Sol comprises a nucleotide sequence of interest which is operable connected to TCS; [N]Y comprises one or more nucleotide residues, or is absent; TT comprises a nucleotide sequence defining a transcription terminator;

[N]

7 comprises one or more nucleotide residues, or is absent. 15 The nucleic acid constructs of the present invention may further comprise other nucleotide sequences as desired. For example, the nucleic acid construct may include an origin of replication for one or more hosts; a selectable marker gene which is active in one or more hosts or the like. 20 As used herein, the term "selectable marker gene" includes any gene that confers a phenotype on a cell, in which it is expressed, to facilitate the identification and/or selection of cells which are transfected or transformed with a nucleic acid construct of the invention. A range of nucleotide sequences encoding suitable selectable markers are known in the art. Exemplary nucleotide sequences that encode selectable markers 25 include: antibiotic resistance genes such as ampicillin-resistance genes, tetracycline resistance genes, kanamycin-resistance genes, the AURI-C gene which confers resistance to the antibiotic aureobasidin A, neomycin phosphotransferase genes (e.g. npti and nptii) and hygromycin phosphotransferase genes (e.g. kipt); herbicide resistance genes including glufosinate, phosphinothricin or bialaphos resistance genes 30 such as phosphinothricin acetyl transferase-encoding genes (e.g. bar), glyphosate WO 2010/012034 PCT/AU2009/000968 -28 resistance genes including 3-enoyl pyruvyl shikimate 5-phosphate synthase-encoding genes (e.g. aroA), bromyxnil resistance genes including bromyxil nitrilase-encoding genes, sulfonamide resistance genes including dihydropterate synthase-encoding genes (e.g. sul) and sulfonylurea resistance genes including acetolactate synthase 5 encoding genes; enzyme-encoding reporter genes such as GUS-encoding and chloramphenicolacetyltransferase (CAT)-encoding genes; fluorescent reporter genes such as the green fluorescent protein-encoding gene; and luminescence-based reporter genes such as the luciferase gene, amongst others. 10 The constructs described herein may further include nucleotide sequences intended for the maintenance and/or replication of the construct in prokaryotes or eukaryotes and/or the integration of the construct or a part thereof into the genome of a eukaryotic or prokaryotic cell. 15 in some embodiments, the construct of the invention is adapted to be at least partially transferred into a plant cell via Agrobacterium-mediated transformation. Accordingly, in one specific embodiment, the nucleic acid construct of the present invention comprises left and/or right T-DNA border sequences. Suitable T-DNA border sequences would be readily ascertained by one of skill in the art. However, the term "T-DNA border 20 sequences" should be understood to at least include, for example, any substantially homologous and substantially directly repeated nucleotide sequences that delimit a nucleic acid molecule that is transferred from an Agrobacterium sp. cell into a plant cell susceptible to Agrobacteriuni-mediated transformation. By way of example, reference is made to the paper of Peralta and Ream (Proc. Natl. Acad. Sci. LISA, 82(15): 5112-5116, 25 1985) and the review of Gelvin (Microbiology and Molecular Biology Reviews, 67(1): 16-37, 2003). In some embodiments, the present invention also contemplates any suitable modifications to the construct which facilitate bacterial mediated insertion into a plant 30 cell via bacteria other than Agrobacteriurn sp., for example, as described in Broothaerts et WO 2010/012034 PCT/AU2009/000968 -29 al. (Nature 433: 629-633, 2005). In some embodiments, the constructs of the second aspect of the invention may also comprise nucleotide sequences that encode regulatory nicroRNAs which may further 5 modulate the expression pattern determined by the nucleotide sequence of the first aspect of the invention. A discussion of the regulatory activity of microRNAs in plants may be found in the review of Jones-Rhoades et a!. (Annual Review of Plant Biology 57: 19-53, 2006) 10 Those skilled in the art will be aware of how to produce the constructs described herein, and of the requirements for obtaining the expression thereof, when so desired, in a specific cell or cell-type under the conditions desired. In particular, it will be known to those skilled in the art that the genetic manipulations required to perform the present invention may require the propagation of a construct described herein or a 15 derivative thereof in a prokaryotic cell such as an E. coli cell or a plant cell or an animal cell. Exemplary methods for cloning nucleic acid molecules are described in Sambrook et al. (Molceular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 2000). 20 In a third aspect, the present invention provides a cell comprising a nucleic acid construct according to the second aspect of the invention. The nucleic acid construct may be maintained in the cell as a nucleic acid molecule, as an autonomously replicating genetic element (e.g. a plasmid, cosmid, artificial 25 chromosome or the like) or it may be integrated into the genomic DNA of the cell. As used herein, the term "genomic DNA" should be understood in its broadest context to include any and all endogenous DNA that makes up the genetic complement of a cell. As such, the genomic DNA of a cell should be understood to include 30 chromosomes, mitochondrial DNA, plastid DNA, chloroplast DNA, endogenous WO 2010/012034 PCT/AU2009/000968 -30 plasmid DNA and the like. As such, the term "genomically integrated" contemplates chromosomal integration, mitochondrial DNA integration, plastid DNA integration, chloroplast DNA integration, endogenous plasmid integration, and the like. A "genomically integrated form" of the construct may be all or part of the construct. 5 However, in one particular embodiment the genomically integrated form of the construct at least includes the nucleic acid molecule of the first aspect of the invention. The cells contemplated by the third aspect of the invention include any prokarvotic or eukaryotic cell. In some embodiments, the cell is a plant cell. In some embodiments the 10 cell is a monocot plant cell. In some embodiments the cell is a cell from a plant in the family Poaceae. In some embodiments the cell is a cereal crop plant cell and in one particular embodiment the cell is a wheat cell. In some embodiments the cell is a rice cell. 15 in some embodiments, the cell may also comprise a prokaryotic cell. For example, the prokaryotic cell may include an Agrobacteriun sp. cell (or other bacterial cell), which carries the nucleic acid construct and which may, for example, be used to transform a plant. In another exemplary embodiment, the prokaryotic cell may be a cell used in the construction or cloning of the nucleic acid construct (e.g. an E. coli cell). 20 in a fourth aspect, the present invention contemplates a multicellular structure comprising one or more cells according to the third aspect of the invention. In some embodiments, the multicellular structure comprises a plant or a part, organ or 25 tissue thereof. As referred to herein, "a plant or a part, organ or tissue thereof" should be understood to specifically include a whole plant; a plant tissue; a plant organ; a plant part; a plant embryo; and cultured plant tissue such as a callus or suspension culture. 30 in some specific embodiments of the fourth aspect of the invention, the plant or part, WO 2010/012034 PCT/AU2009/000968 -31 organ or tissue thereof comprises reproductive material for a plant including, for exarnpl, flowers or parts thereof (including anthers and ovaries), seeds, vegetative plant material, explants, plant tissue in culture including callus or suspension culture and the like. 5 As would be appreciated, the plant or a part, organ or tissue thereof contemplated in the fourth aspect of the invention may include, for example, any of a monocot, a plant in the family Poaceae, a cereal crop plant, a wheat plant or a rice plant or a part, organ or tissue of any of the foregoing. 10 In some embodiments of the fourth aspect of the invention, the plant or part, organ or tissue thereof comprises a flower and/or seed. In some embodiments of the fourth aspect of the invention, a nucleotide sequence of 15 interest may be operably connected to the transcriptional control sequence or the functionally active fragment or variant thereof, such that the nucleotide sequence of interest is specifically or preferentially expressed in a flower and/or seed, or in a particular cell or tissue type thereof, and optionally at a particular developmental stage, as described above with respect to the first aspect of the invention. 20 In a fifth aspect, the present invention provides a method for specifically or preferentially expressing a nucleotide sequence of interest in one or more parts of a plant flower and/or seed, the method comprising effecting transcription of the nucleotide sequence of interest in a plant under the transcriptional control of a nucleic 25 acid according to the first aspect of the invention. As set out above, in its fifth aspect, the present invention is predicated, in part, on effecting transcription of the nucleotide sequence of interest under the transcriptional control of a transcriptional control sequence of the first aspect of the invention. In some 30 embodiments, this is effected by introducing a nucleic acid molecule comprising the WO 2010/012034 PCT/AU2009/000968 -32 transcriptional control sequence, or a functionally active fragment or variant thereof, into a cell of the plant, such that the nucleotide sequence of interest is operably connected to the transcriptional control sequence. The nucleic acid molecule may be introduced into the plant via any method known in the art. For example, art explant or 5 cultured plant tissue may be transformed with a nucleic acid molecule, wherein the explant or cultured plant tissue is subsequently regenerated into a mature plant including the nucleic acid molecule; a nucleic acid may be directly transformed into a plant, either stably or transiently; a nucleic acid may be introduced into a plant via plant breeding using a parent plant that carries the nucleic acid molecule; and the like. 10 In some embodiments, the nucleic acid molecule is introduced into a plant cell via transformation. Plants may be transformed using any method known in the art that is appropriate for the particular plant species. Common methods include Agrobacterium mediated transformation, microprojectile bombardment based transformation methods 15 and direct DNA uptake based methods. Roa-Rodriguez et al. (Agrobacteriunm-ediated transformation of plants, 3r Ed. CAMBIA Intellectual Property Resource, Canberra, Australia, 2003) review a wide array of suitable Agrobacteriium-mediated plant transformation methods for a wide range of plant species. Other bacterial-mediated plant transformation methods may also be utilised, for example, see Broothaerts et ad. 20 (2005, supra). Microprojectile bombardment may also be used to transform plant tissue and methods for the transformation of plants, particularly cereal plants, reviewed by Casas et al. (Plant Breeding Rev. 13: 235-264, 1995). Direct DNA uptake transformation protocols such as protoplast transformation and electroporation are described in detail in Galbraith et al. (eds.), Methods in Cell Biology Vol. 50, Academic Press, San Diego, 25 1995). In addition to the methods mentioned above, a range of other transformation protocols may also be used. These include infiltration, electroporation of cells and tissues, electroporation of embryos, microinjection, pollen-tube pathway-, silicon carbide- and liposome mediated transformation. Methods such as these are reviewed by Rakoczy-Trojanowska (Cell. Mol. Biol. Lett. 7: 849-858, 2002). A range of other plant 30 transformation methods may also be evident to those of skill in the art and, WO 2010/012034 PCT/AU2009/000968 - 33 accordingly, the present invention should not be considered in any way limited to the particular plant transformation methods exemplified above. As set out above, the transcriptional control sequence of the present invention is 5 introduced into a plant cell such that the nucleotide sequence of interest is operably connected to the transcriptional control sequence and the present invention contemplates any method to effect this. For example, the subject transcriptional control sequence and a nucleotide sequence of interest may be incorporated into a nucleic acid molecule such that they are operably connected, and this construct may be introduced 10 into the target cell. In another example, the nucleic acid sequence of the present invention may be inserted into the genome of a target cell such that it is placed in operable connection with an endogenous nucleic acid sequence. As would be recognised by one of skill in the art, the insertion of the transcriptional control sequence into the genorne of a target cell may be either by non-site specific insertion 15 using standard transformation vectors and protocols or by site-specific insertion, for example, as described in Terada et al (Nat Biotechnol 20: 1030-1034, 2002). The nucleotide sequence of interest, which is placed under the regulatory control of the transcriptional control sequence of the present invention, may be any nucleotide 20 sequence of interest. General categories of nucleotide sequences of interest include nucleotide sequences which encode, for example: reporter proteins, such as, GUS, GFP and the like; proteins involved in cellular metabolism such as Zinc finger proteins, kinases, heat shock proteins and the like; proteins involved in agronomic traits such as disease or pest resistance or herbicide resistance; proteins involved in grain 25 characteristics such as grain biomass, nutritional value, post-harvest characteristics and the like; heterologous proteins, such as proteins encoding heterologous enzymes or structural proteins or proteins involved in biosynthetic pathways for heterologous products; "terminator" associated proteins such as barnase, barstar or diphtheria toxin. Furthermore, the nucleotide sequence of interest may alternatively encode a non 30 translated RNA, for example an siRNA, miRNA, antisense RNA and the like.

WO 2010/012034 PCT/AU2009/000968 -34 In some embodiments, the nucleotide sequence of interest may comprise, for example, a pathogen responsive (PR) gene, a resistance (R) gene or a defensin gene. 5 In some embodiments, the nucleotide sequence of interest may encode a protein such as PDR5 or TRI101. Such proteins may be expressed in a flower or seed-specific manner in crop plants such as wheat in order to lower the incidence of diseases such as head blight (caused by Fusarium graminearum or Gibberella zeac) and/or reduce mycotoxin levels within the seed. 10 The method of the present invention may be applicable to effect specific or preferential expression of a nucleotide sequence of interest in a range of different plant flowers and/or seeds. For example, in some embodiments, the plant may be a monocotyledonous plant. In some embodiments, the plant may be a plant in the family 15 Poaceae. In some embodiments, the plant may be a cereal crop plant. In some embodiments, the method of the present invention may be applicable to effect specific or preferential expression of a nucleotide sequence of interest in a wheat flower and/or seed. 20 In some embodiments, the method of the present invention may be applicable to effect specific or preferential expression of a nucleotide sequence of interest in a rice flower and/or seed. 25 As set out above, in some embodiments, the method of the present invention may be employed to specifically or preferentially effect expression of an operably connected nucleotide sequence in one or more parts of a plant flower and/or seed or at one or more developmental stages of a plant flower and/or seed as hereinbefore described. 30 In some embodiments of the method of the fifth aspect of the invention, the nucleotide WO 2010/012034 PCT/AU2009/000968 -35 sequence of interest is heterologous with respect to the transcriptional control sequence, as defined svra. Finally, reference is made to standard textbooks of molecular biology that contain 5 methods for carrying out basic techniques encompassed by the present invention, including DNA restriction and ligation for the generation of the various constructs described herein. See, for example, Maniatis et al., Aiolecuiar Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, New York, 1982) and Sambrook et al. (2000, supra). 10 The present invention is further described by the following non-limiting examples: BRIEF DESCRIPTION OF THE FIGURES 15 Figure 1 shows (A) Northern blot hybridization of TaPRPI cDNA with total RNA isolated from different wheat tissues and grain at different stages of development. (B) Expression of OsPRPI in different rice tissues demonstrated by Quantitative PCR. (C) Unrooted phylogenetic tree of wheat and rice PRPI protein sequences and known or putative homologues from other plants: (CADEF1 (Acc. AF442388), LePi (Acc. 20 AJ133601.1), pSUN (Acc. ABO36641), HaI (Acc. P82659), GmPI (Acc. AAC97524), CajI (Acc. X95363), CaPI1 (Acc. 065740), AtPDFI.2 (Acc. NP 199255), NaDI (Acc. AAN70999), Tad1 (Acc. BAC10287), TaAILP (Acc. ABX84378). (D) Alignment of wheat and rice PRPI protein sequences deduced from cDNAs and genomic clones identified in this paper. Identical amino acids are in black boxes, similar amino acids are in grey 25 boxes. Figure 2 shows Spatial and temporal GUS expression in wheat directed by the TdPRPI 10 promoter. A1-A8 - TRPI-10 promoter activity in pericarp tissues before fertilization: Al and A5 - two magnifications of wheat flower 1--2 days before 30 pollination (control flower is on the right hand side of each picture); A2-A4 and A6-A8 WO 2010/012034 PCT/AU2009/000968 -36 - longitudinal sections of the female gametophyte 1-2 days before pollination. A9-A16 TdPRPI-10 promoter activity at different stages of grain development in uncut (A9 A12) and hand-cut grain (A13-A16) at 3 DAP (A9 and A10), 8 DAP (All, A13, and A14) and 13 DAP (A12, A15, A16). Control grain is on the right hand side of each 5 picture. A17-A20 - transverse (A17, A19, and A20) and longitudinal (A18) sections of T1 grain at 3 DAP (A17), T2 grain at 2 DAP (A19) and 5 DAP (A20) DAP, and Ti grain at 5 DAP (A18). Figure 3 shows spatial and temporal GUS expression in rice directed by the TdPRPI-10 10 promoter. GUS activity in rice detected in floral and grain tissues shortly before anthesis (1), at anthesis (132),5 DAP (B3), 8 D AP (134), 15 DAP (135) and IS DAP (136). Control flowers and grain at the same stage of development are shown on the right hand side of each panel. B7-B11 -- Histochemical GUS assay counterstained with safranin in: 10 n thick longitudinal sections of anthers (137) and ovule at anthesis (138), 15 grain at 5 DAP (139, B10), and in the vascular bundle of lemma (B11). GUS expression in the vascular bundle of the anther and lemma and in the ovary is indicated with arrows. Figure 4 shows TdPRPI-I promoter activity in rice flower and grain. GUS activity in the lemma (Ci) and the rest of the flower (C2) before anthesis, and in grain at 5 DAP (C3), 20 8 DAP (C4), 10 DAIP (C5 and C6), 16 DAIP (C7-C9), and 26 DAP (C1 0-C12) shown as the whole grain or hand-cut transverse (C4 and C6) and longitudinal (CS, C9, C11 and C12) sections. C12 -GUS expression in the embryo. Figure 5 shows TdIPRPI- 11 promoter activity in rice flower and grain. GUS expression 25 in flowers of two independent transgenic lines before an thesis (D1 and D2), in lemma at I DAP (D3), in flowers with removed lemma at 2 DAP (D4), 5 DAP (D5), and 9 DAP (D6). Figure 6 shows the activity of wheat promoters in germinating rice grain. GUS 30 expression in germinating T2 grain at 3 days after sowing of two independent WO 2010/012034 PCT/AU2009/000968 - 37 transgenic lines for each of the TdPRPI-I (El and E4), idPRPI-10 (E2 and E5), and TdPR PI-- (E3 and E6) promoters. GUS expression in roots (E7-E9), coleoptile and first leaf (EIO-E12), of transgenic plants with TdPRPI-1 (E7 and E10), TdPIRPI-1 (ES and E11)., and TdPR P!-i2 (E9 and E12) promoters. In each panel the control plant is shown 5 on the left hand side. Figure 7 shows the activity of OsPRPI promoter in transgenic rice plants. GUS activity detected 2-3 days before anthesis in vascular bundles of lemma and palea (F1), the vascular bundles of filament, anthers, stamen and both poles of ovary of transgenic 10 (£2) plants, but not in control plants. GUS expression in developing rice caryopsis of transgenic (F4-F9) plants at 2 (F4), 4 (F5), 6 (F7), 10 (F7), 12 (F8), and 20 (F9) DAP. No staining in control grain at 12 (F10) and 20 (F11) DAP. Activity of OsPRPI promoter in germinating rice grain (F12-F14, £17, and F18) was detected in vascular tissue of developing first leaf (F12) coleoptiles (F14) of transgenic rice plant. Developing first 15 leaf of the control plant is shown in the upper part of F12, control coleoptiles cart be seen in F13. The whole seedling at the 3 day after germination (£17 and F18): GUS activity was detected in vascular tissue of coleoptiles and was not detected in root and the first leaf. GUS expression in the first leaf (F15) and root (F16) of transgenic rice transformed with the wheat TdP1RPi--1 promoter is shown for comparison. 20 Figure 8 shows the activity of the OsPRPJ promoter in developing grain demonstrated by histochemical GUS assay counterstained with safranin (GI-G4) in: 10 um thick longitudinal section (Gi) and transverse sections (G2-G3) T2 grain of transgenic rice at 16 DAP. GUS expression can be seen in the main vascular bundle and adjacent to 25 vascular bundle cell layers. Magnification is shown in the corner of each picture. EXAMPLE I Cloning of the TPRI and OsPRI genes 30 The full length cDNA of TaPRPI (SEQ ID NO: 7) was isolated from a cDNA library WO 2010/012034 PCT/AU2009/000968 - 38 prepared from the whole grain of Triticum aestivum at 0-6 DAP. A subsequent comparison of the nucleotide sequence of TaPRPI cDNA with wheat expressed sequence tags (ESTs) identified multiple ESTs with a high level of sequence identity (data not shown). Most of them originated from cDNA libraries prepared from ovary, 5 pre-antliesis spikes, and 5-15 DAP spikes. The flower and grain specific expression of the gene was confirmed by northern blot hybridisation with RNA isolated from different wheat tissues (Figure 1A). A database search using TaPRPI protein sequence (SEQ ID NO: 1) identified this protein as a member of the Yv-thionin (defensin) subfamily. 10 The full length cDNA of TaPRPI (SEQ ID NO: 7) was used as a probe to screen a bacterial artificial chromosome (BAC) library prepared from genomic DNA of Triticun durum cv. Langdon using Southern blot hybridisation. Eleven BAC clones were selected for further analysis on the basis of the strength of the hybridisation signals. 15 BAC DNA was isolated and used as a template for PCR with primers derived from the beginning and the end of the coding region of TaPRP. Five BAC clones gave PCR products. Sequencing of the PCR products revealed that the cloned inserts are genomic clones of close homologues from T. durnm of TaPRPI, which were designated TdPRPI , TdPRPi-5, TdPRPI[-7, TdPRPI-8, TdPRPI-10, and TdPRPI-1L2. One BAC clone 20 contained two very similar genes, TdPRPI-7 and TdPRPI-8, positioned one after another in tandem. The coding regions of all cloned genes are interrupted with- single introns of slightly different length. The nucleotide sequences of each of the TdPRPI genes, together with the deduced protein and cDNA sequences, as well as the upstream promoter regions were identified and designated sequence identifiers as 25 shown in Table 2, below. Southern blot hybridization of nullisomic-tetrasomic lines of hexaploid wheat with full length cDNAs or 3' UTRs (where it was possible) of PRPI genes as probe revealed that TaPRPI and TdPRPli-5 are located on chromosomes 1A and 2B of hexaploid wheat, 30 respectively. The location of other cloned genes could not be identified precisely WO 2010/012034 PCT/AU2009/000968 -39 because several close homologues cross-hybridized with the probe. The amino acid sequence of TaPRPI also allowed the identification of a rice homologue, designated as OsPRPI, in EST databases. ESTs for OsPRPI originated 5 mainly from cDNA libraries prepared from the panicle, flower, endosperm or pistil 1-4 weeks time after anthesis (Accession Numbers of ESTs: C1342038, C1495264, CI719169, C1493678, C1454367, CI454009, C16756628, CI453631, CI481346, C1469382, respectively). Quantitative PCR analysis confirmed preferential expression of OsPRPI in early panicles aid demonstrated temporal similarity of expression to TaPRPI (Figure 1B). 10 The nucleotide sequence of the EST contig was used to identify the translational start site of the respective gene in the rice genomic database. A DNA fragment of 164.0 bp, upstream of the translational start site of OsPRPI, was isolated by PCR using rice (Oryza sativa ssp. japonica cv. Nipponbare) genomic DNA as template. This sequence was designated the OsPRPT promoter (SEQ HID NO: 10) 15 The phylogenetic relationships based on TaPRPI, OsPRPI, TdPRPI amino acid sequences deduced from genomic clones, and the sequences of the closest homologous proteins from other plant species derived from NCBI databases, are shown in Figure IC. An alignment of OsPRPI to the wheat PRPI protein sequences is shown in Figure 20 1D. Table 2 - Sequence Identifiers for PRPI nucleotide and amino acid sequences TaPRPI 6 7 TdPRPI-1 18 19 20 21 22 TdPRPI-5 23 24 25 26 27 TdPRPI-7 28 29 30 31 32 TdPRPI-8 33 34 35 36 37 WO 2010/012034 PCT/AU2009/000968 -40 ''dPRPI-10 1 21 3 4 5 TdPRPI-11 3 14 15 16 17 OsPRPI 8 9 10 1 12 EXAMPLE 2 Spatial and temporal activity of the PRPI promoters in developing grain 5 Expression of TaPRiPi in different wheat (T. acstivui cv. Chinese Spring) tissues was analysed using northern blot hybridisation. In wheat, the expression of the TaPRPI gene was detected in spikes starting from the very early stage of development, progressively increasing until it reached maximum at 2-5 DAP, and quickly diminishing after 11 DAP (Figure IA). Very low level of TaPRPI expression was also 10 observed in shoots of young seedlings (Figure 1A). The expression of the OsPRPI gene in different rice tissues was analysed using quantitative PCR (Figure 113). Very low expression of OsPRPI was detected in all tissues tested except panicles, where expression was very strong before 11 DAP, but 15 undetectable after 11 DAP. Computer analysis of the TdPRPI and OsPRPI promoters using PLACE software (http://www.dn.affrc.go.jlp/PLACE/signalscan.htni) and a database of plant cis-acting regulatory DNA elements revealed no cis-elements which are known to be responsible 20 for specific gene expression in the pericarp and anthers. Two of the analysed PRPI promoters contained elements required for specific expression in the vascular system. The TdPRPT-7 promoter contained the BS1 element, AGCGGG-, which is required for specific expression in vascular tissue (Lacombe et al., 25 Plant J, 23: 663-676, 2000). The TdPR PI-5 promoter has the cis-element identified among the promoters of the "core xylem gene set" in Arabidopsis (Ko et al., Molecular Genetics and Genonics 276: 517-531, 2006).

WO 2010/012034 PCT/AU2009/000968 -41 The promoter sequences of TdPRP1-i, TdP RPi-0, TdPRP-1-1, and OsPRPI were cloned into the plant transformation vector pMDC164 to provide four transcriptional GUS fusion promoter constructs (pTdPRP-,-10,-I1, and pOsPRFI), which were used to 5 transform rice. One of the promoters, pTdPRP ,-70 was also linearised by restriction of the unique Piel site and transformed into wheat using microprojectile bombardment. The integration of promoter-GUS fusions in transgenic plants was confirmed by PCR using primers derived from promoter and GUS sequences. Fourteen transgenic To 10 wheat lines as confirmed by PCR were analysed. Six To wheat lines were selected using the GUS staining assay, from which three demonstrated strong GUS expression and three showed weak expression of the reporter gene. Three lines, two with strong transgene expression and one with weak expression, were selected for further analysis. All positive lines demonstrated the same pattern of GUS expression. 15 14 To lines of transgenic rice were also analysed for GUS activity. Three lines demonstrated strong promoter activity and the same pattern of gene expression. One of them had strong GUS expression, two demonstrated moderate levels of expression. No expression was found in eight lines and three more lines were sterile. The T1 20 generation was analysed for two lines (six plants for each). All positive plants had the same patterns of transgene expression as To plants. Wild type plants and/or plants transformed with a vector containing only the selectable marker cassette were used as negative controls. No differences were found between wild type plants and plants transformed with the control vector. 25 TdPRPI 70 is one of the closest homologues of TaPRP[ isolated so far from T. drim (Figure 1 C and D). Protein sequence alignment shows differences in ten amino acid residues in unprocessed proteins and only three amino acid residues are different in mature proteins (Figure ID). Analysis of the activity of the TdPRPI-10 promoter in 30 transgenic wheat plants using a promoter-GUS fusion construct demonstrated good WO 2010/012034 PCT/AU2009/000968 -42 spatial and temporal correlation between the activity of the TdPRPI-10 promoter and expression of TiPRPL The activity of the TdPRPi-10 promoter was observed in ovules prior to fertilisation 5 (Figure 2 Al- AS) and later in grain with maximum activity between 5 and 8 DAP (Figure 2 A9-A16). In ovules, GUS activity was detected in pericarp tissue with the strongest activity in epidermis (Figure 2 A2-A4, A6-AS) and in the main vascular bundle (Figure 2 A2 and A6). GUS activity became weak after 11 DAP, at 13 DAP remained mainly around the crease (Figure 2 A16). 3-5 days later., No GUS activity was 10 observed. In addition, no GUS activity was detected in the endosperm and embryo. In grain, GUS was detected mainly in the upper epidermis layer; GUS expression was weaker in the underlying cell layers of the pericarp (Figure 2 A17 --- A20). The activity of the promoter in T1 grain was stronger than in T2 grain (Figure 2 Al 9-A20). 15 Beyond 20 DAP, no GUS activity was detected in floral tissues other than the ovule or in the grain. Neither was GUS activity observed in mature leaves or stems of transgenic wheat plants. 20 A very similar pattern of TdPRP--l0 promoter activity was found in transgenic rice plants, except that the activity of the promoter was less specific than in wheat. Before and at anthesis strong GUS expression was detected in ovaries and the vascular tissue of anthers, glume, lemma and palea, and in lodicules (Fig 3 B1 and B2). After anthesis the GUS activity was found mainly in the pericarp (Figure 3 B3-B6). During grain 25 elongation (2-7 DAI) strong activity was detected close to the grain poles and was not found in the middle part of the grain (Figure 3 B3). In contrast to rice, in elongating wheat grain GUS expression was detected mainly in the lower part of grain (Figure 2 A9 and A10). At the end of rice grain elongation (at 7-8 DAP) and later the promoter was active everywhere in the pericarp, and the GUS activity strongly decreased at 15 30 DAP and was detectible mainly along the crease at 18 DAI (Figure 3 B3). This is WO 2010/012034 PCT/AU2009/000968 - 43 consistent with the lifetime of the pericarp tissue, which dries as the seed matures. No GUS expression was observed in the embryo, endosperm, aleurone, testa, and mature vegetative tissue. No GUS activity has been detected anywhere in grain beyond 20-23 DAP. 5 Minor differences were observed in the spatial patterns of activity of the TdPRPI-10., Td1PRPi-i and TdPRPI-11 promoters in rice. Comparison of the lines with strongest expression revealed that the TdPRPI-1 promoter is stronger than the TdPIRPI-10 promoter. A short time before fertilization very strong GUS activity was detected in the 10 lemma, palea, anthers and ovules of plants transformed with the TdPRP-10 promoter (Figure 4 C1 and C2). No differences in the spatial pattern of GUS expression driven by TdPRP1-i and TdPRPI-10 promoters was found during the first 16 DAP, except the activity of the TdPRPi-1 promoter was much stronger than the TdPRP1-10 promoter (Figure 3 C3-C8). However, at 16 DAP and later the activity of the T'PRP1-1 promoter 15 diminished in the pericarp, while very low GUS expression appeared in the starchy endosperm (Figure 3 C9). At 26 DAP strong activity of the TdPRPI-1 promoter was detected in vascular tissue of the embryo (Figure 3 C10-C12). After 30-35 DAP no activity of TdPRPT-1 promoter was detected in any grain tissues (data not shown). Additionally, no activity of the promoter was detected in vegetative tissues of mature 20 plants, except very weak activity in the vascular bundles of the youngest part of stems. In contrast, the TdPRI-11 promoter was weaker than TdPRPi-10 promoter. Before fertilization strong GUS activity was detected in the ovule and very weak activity was observed in anthers (Figure 4 D1 and D2). GUS expression in anthers increased a short 25 time before pollination and remained on the same level until anthers became dry (Figure 4 D4). For the first two days after fertilization GUS was strongly expressed in the vascular tissue of the lemma and palea, everywhere in the pericarp and in anthers (Figure 4 D3). A polarised pattern of expression similar to that observed for other PRPI promoters was detected between 3 and 7 DAP (Figure 4 D5). However, in contrast to 30 other promoters, the polarised pattern of expression of the T'PRPI-11 promoter WO 2010/012034 PCT/AU2009/000968 -44 disappeared from 7 DAP first from the tip (Figure 4 D6) and a few days later from the rest of the grain. EXAMPLE 3 5 Activity of the PRI promoters in germinating grain and seedlings All three wheat promoters were active during grain germination and one-two weeks after germination (Figure 5 Ei-E12). A short time after the transfer of grain to wet filter paper strong activity of all three wheat promoters was detected in the germinating 10 embryo axis (Figure 5 E1-E6). However, in contrast to the TaPRPI-2 and TaPRPI-10 promoters, the TaPRPI-11 promoter also showed strong activation in the scutellum (Figure 5 E3 and E6). Relatively strong activity has been detected in the coleoptile and the tip of the first leaf (Figure 5 E10-E12) and weaker activity was observed in roots (Figure 5 E7-E9). The strength of GUS expression in roots correlated with the strength 15 of the respective promoters in grain. The TdPRPI-20 and TdPRPi-21 promoters were active in root vascular tissue and the TPRPI-2 promoter active in all root tissues. In the first 3-4 days after sowing no GUS expression was detected in the elongation zone of the root, but was observed in the root tip where it gradually diminished and vanished after 3-4 days. No GUS activity was detected in root tips of seedlings where GUS was 20 driven by the TaPRPi-I promoter (Figure 5 E7). At the end of the second week after seed sowing, GUS activity eventually became restricted to the vascular bundle of the mature part of the root and finally disappeared in both roots and coleoptiles. No activity of wheat promoters has been detected in the leaves of three week old seedlings of transgenic rice. 25 EXAMPLE 4 Discussion The full length cDNA of wheat defensin TaPRPI was identified as a gene whose 30 product frequently generated false positives with a number of different unrelated bait WO 2010/012034 PCT/AU2009/000968 - 45 proteins in the yeast 2-hybrid screen (data not shown). It was isolated from a cDNA library prepared from whole developing grain. Northern blot hybridization revealed specific expression of this gene in flowers and grain until 12 DAPR 5 The full length cDNA of TaPRPI was used as a probe to screen a BAC library prepared from tetraploid wheat and the screen resulted in the isolation of six dose homologues to TaPRP. Because of the flower and early grain specific expression of TdPRPs (except TdPRPi-5, 10 ESTs for which originated mainly from cDNA libraries prepared from root tissue) the promoters of TaPRPT homologues were characterised. The promoter of a TaPRPI homologue, designated OsPRPI, with a similar pattern of expression (Figure 1 A and B) was also cloned and characterised. Taking into consideration the high similarity of wheat genes and their products, the promoters of three representative wheat genes, 1 5 TdPRPT-1, TdPRPI-10, and TdPRPI-11, were selected for characterisation in transgenic rice. The promoter of TdPRPI-10 was also characterised in transgenic wheat. In addition, rice plants were transformed with an OsPREI promoter-GUS construct, providing the opportunity to compare in one species, rice, the activity of the promoters of similar wheat and rice genes. 20 All the promoters analysed demonstrated similar spatial patterns of activity in wheat and rice grain. In flowers, they were activated in ovary tissues and after fertilisation were expressed in pericarp. During 3-4 days of intensive grain elongation the promoters were active only around both grain poles in rice and closer to the 25 micropylar pole in wheat. No GUS staining was detected in extending cells in the middle part of the pericarp during grain elongation. Several small differences in GUS expression driven by different wheat promoters were found after grain elongation was completed. TaPRPI-1 and TaPRi-P-10 promoter 30 activities were detected everywhere in pericarp about 2-2.5 weeks longer and were riot WO 2010/012034 PCT/AU2009/000968 -46 observed in other grain tissues. However, in contrast to the other tested promoters, the TaPRPL-1 promoter was slightly activated in starchy endosperm and strongly activated in the vascular tissue of the embryo after 16 DAP (Figure 4). The TaPRPI-11 promoter was, at the end of grain elongation, the weakest promoter. Despite being strongly 5 expressed everywhere in the pericarp before pollination and up to 2-3 DAP, TaPRPI-JI promoter activity during grain elongation was not sustained in the middle part of the pericarp and quickly vanished at the end of the second week after pollination, first at the top end of grain and then everywhere (Figure 4). 10 The pattern of GUS expression driven by the TaPRPI-10 promoter in transgenic wheat flowers was more specific than the pattern driven by the same promoter in rice flowers. In wheat, the activity of the promoter was localised only to ovaries and later mainly to the tipper epidermis layer of the pericarp (Figure 2). No GUS activity was detected in other flower tissues. In rice, activity of the TaPRPI-10 promoter was 15 observed in the vascular tissue of anthers, lemma and palea (Figure 3 and 4). At least at some stages of rice grai development the activity of the promoter was also detected in several layers of seed coat and even in aleurone cells. Expression of PRPI genes was not restricted to flower and pericarp tissues, but was 20 also observed in the embryo axis and, in the case of TaPRP-1-1, in the scutellum of germinating grain. In one week-old seedlings germinated on filter paper it was detected in coleoptiles and the vascular tissue of roots. An interesting observation was the absence of promoter activity not only in the elongating region of the pericarp, but also in the elongation zone of root, coleoptile, and developing first leaf (Figure 6 E7 25 E12). In summary, the specific spatial and temporal patterns of expression of PRPI gene promoters in developing and germinating grain makes them valuable tools for the targeted expression of, for example, defensin, PR and R genes for engineered plant 30 protection.

WO 2010/012034 PCT/AU2009/000968 - 47 EXAMPLE 5 Materials and methods 5 Genc cloning and plasnid construction The full length cDNA of TaPRPI was isolated from a yeast 2-hybrid cDNA library prepared from wheat grain at 0-6 DAP (Lopato et al., Plant MAot. Biol. 62: 637-653, 2006) as an occasional false positive gene. The full length cDNA sequence of TaPRPI was used to probe a BAC library prepared from the genomic DNA of Triticum durum cv. 10 Langdon (Cenci et al, Thcorctical n Applied Genctics 107: 931-939, 2003) using Southern blot hybridisation as described elsewhere (Sambrook ct at., Moicular Cloning: a Laboratory Manual, 2' Ed., Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York, USA, 1989). Plasmid DNA from eleven BAC clones, which strongly hybridised with the probe, was isolated using a Large Construct Kit (QIAGEN). Five 15 BAC clones containing T. durun homologues of TaPRPI were identified by PCR using BAC DNAs as templates and primers derived from the ends of the coding region of TaPRPI cDNA. Five BAC DNAs were combined and sequenced using the 454 sequencing method. The obtained gene sequences were subsequently used to design forward and reverse primers for the isolation of the promoter segments. Fragments of 20 promoters of the three genes TdPRPI-1, TdPRPI-10, TdPRPI-11 with full-length 5' untranslated regions were amplified by PCR using AccuPrimeTM Pfx DNA polymerase (Invitrogen) from DNA of BAC clones as a template. They were cloned into the pENTR-D-TOPO vector (Invitrogen); the cloned inserts were verified by sequencing and subcloned into the pMDC164 vector (Curtis and Grossniklaus, Plant Phnysiol. 133: 25 462-469, 2003) using recombination cloning. Selectable marker genes conferred hygromycin resistance in plants and kanamycin resistance in bacteria. The resulting binary vector was introduced into Agrobacteriuni tuniefacicns AGLI strain by electroporation. For wheat transformation, the construct containing the TdPRPI-10 promoter was linearised using the unique Pmel site in the vector sequence. The 30 deduced amino acid sequence of TaPRPI allowed the identification of a rice WO 2010/012034 PCT/AU2009/000968 -48 homologue, designated as OsPRPI, in expressed sequence tag (EST) databases (Acc. CX102611). The nucleotide sequence of the EST contig was used to identify the translational start site of the respective gene (Acc. AP008209, region: 1694297.1694622) in rice genornic databases. A DNA fragment of 1640 bp upstream of the translational 5 start site of OsPRPI was isolated by PCR using rice (Oryza sativa ssp. japonica cv. Nipponbare) genomic DNA as template. The promoter sequence of OsPRPI was cloned into the plant transformation vector pMDC164 to provide a transcriptional GUS fusion promoter construct, designated pOsPRPI, which was transformed into rice. 10 Plant transformation and analysis The constructs pTdPRPI-1, pTdPRPI-10, pTdPRPI-1l and pOsPRPI were transformed into rice (Oryza sativa L. ssp. Japonica cv. Nipponbare) using Agrobacteriun-mediated transformation and the method developed by Tingay et al. (Plant J. 14: 285-295, 1997) and modified by Matthews et al. (Mol. Breed. 7: 195-202, 2001). Wheat (Triticumn aestivum 15 L. cv. Bobwhite) was transformed using biolistic bombardment as described in Kovalchuk et al. (Plant Mol Biol. 2009 Jun 10. [Epub ahead of print]). Transgene integration was confirmed by PCR using GUS specific primers. Histochemical and histological GUS assays were performed as described in Li at al. (Plant Biotechnology Journal 6: 465476, 2008) using To - T2 transgenic plants and Ti -- T3 seeds, respectively. 20 For the analysis of promoter activity in germinating grain, Ti seeds were sterilised by 10 minutes exposure to UV light with repeated shaking of plates containing seeds, and germinated in sterile conditions on wet paper. The whole seedlings were stained at 3, 5, 7 and 14 days after germination. 25 Northern blot hybridisation and Q-PCR RNA isolation and northern blot hybridisation was performed as described elsewhere (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2n Ed., Cold spring Harbor Press, Cold Spring Harbor, New York, USA, 1989). The filter membrane was hybridised with a full length cDNA of TaPRPL Q-PCR analysis of expression of the 30 OsPRPI gene in different tissues of wild type rice was performed as described in Li et WO 2010/012034 PCT/AU2009/000968 -49 al. (Plant Biotechnology journal 6: 465-476, 2008). Those skilled in [he art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is 5 to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to, or indicated in this specification, individually or collectively, and any and all combinations of any two or more of the steps or features. 10 Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers. 15 Also, it must be noted that, as used herein, the singular forms "a", "an" and "the" include plural aspects unless the context already dictates otherwise.

Claims

1. An isolated nucleic acid comprising a nucleotide sequence defining a transcriptional control sequence which specifically or preferential directs expression 5 of an operably connected nucleotide sequence in one or more parts of a plant flower and/or seed, wherein said transcriptional control sequence is derived from aythionin gene which comprises the amino acid sequence set forth in SEQ ID NO: 1L or a homolog thereof, wherein the homolog comprises an amino acid sequence which comprises at least 35% identity to SEQ ID NO:4 10

2. The nucleic acid of claim 1, wherein the homolog of the ythionin gene comprises the amino acid sequence set forth in any of SEQ ID NO: 6 SEQ ID NO: 8, SEQ ID NO: 13,, SEQ ID NO: 18, SEQ ID NO: 23, SEQ ID NO: 28 and SEQ ID N: 33. 15 3 The nucleic acid of clairn I or claim 2, wherein the plant is: (i) a monocotyledonous plant; (ii) a plant in the family Poaceae; (iii) a cereal crop plant; (iv) a wheat plant; or (v) a rice plant.

4. The nucleic acid of any one of claims 1 to 3, wherein the transcriptional control 20 sequence directs expression of an operably connected nucleotide sequence in at least an ovary or ovule of a flower, and/or in at least the pericarp of an immature seed.

5. The nucleic acid of any one of claims I to 4, wherein the transcriptional control sequence is derived from: (i) a monocotyledonous plant; (ii; a plant in the family 25 Poaceae; (ii) a Triticum sp. plant; (iv) a Triticum durum plant; or (v) a rice plant

6. The nucleic acid of any one of claims 1 to 5, wherein the transcriptional control sequence comprises the nucleotide sequence set forth in any of SEQ ID NO: 3, SEQ ID NO: 10, SEQ ID NO: 15 SEQID NO: 20, SEQ ID NO:25, SEQ ID NO: 30 and SEQ ID 30 NO: 35, or a functionally active variant of any of the foregoing, wherein the -51 functionally active variant comprises at least 50% nucleotide sequence identity to any of SEQ ID NO: 3, SEQ ID NO: 10, SEQ ID NO: 15, SEQ ID NO: 20, SEQ ID NO: 25, SEQ ID NO 30. 5 7. A nucleic acid construct comprising the isolated nuclei acid of any one of claims I to 6.

8. The nucleic acid construct of claim 7, wherein the nudeic acid construct further comprises a nucleotide sequence of interest operably connected to the nuclei acid of 10 any one of claims i to 6,

9. The nucleic acid construct of claim 8, wherein the nucleotide sequence of interest is heterologous with respect to the nucleic acid onf any one daims I to 6. 15 10- A cell comprising a nucleic acid construct according to any one of claims 7 to 9. 1L The cell of claim 10, wherein the cell is a plant cell.

12. The cell of claim 1L, wherein the plant is: (i) a monocotydedonous plant; (ii) a 20 plant in the family Poaceae; (iii) a cereal crop plant; (iv) a wheat plant; or (v) a rice plant.

13. A multicellular structure comprising one or more cells according to any one of claims 10 to 12. 25

14. The multicellular structure of clain 13, wherein the multicellular structure comprises a plant or a part, organ or tissue thereof,

15. A method for specifically or preferentially expressing a nucdeotide sequence of 30 interest in one or more parts of a plant flower and/or seed, the method comprising <52 effecting transcription of the nudeotide sequence of interest in a plant inder the transcriptional control of a nuceic acid according to any one of claims I to 6.

16. The method of claim 15, wherein the plant is: (i) a monocotyledonous plant; (ii) 5 a plant in the family Poaceae; (iii) a cereal crop plant; (iv) a wheat plant; or (v) a rice plant 17 The method of claim 15 or daim 16, wherein the nucleotide sequence of interest is expressed in at least an ovary or ovule of a flower. 10 18 The method of any one of aims 15 to 17, wherein the nucleotide sequence of interest is expressed in at least the pericarp of an immature seed.

19. The method of any one of claims 15 to 18, wherein the nucleotide sequence of 15 interest is beterologous with respect to the transcriptional control sequence.

20. An isolated nucleic acid of daim 1, substantially as herein described with reference to any one or more of the Examples and/or Figures.