WO2008052285A1

WO2008052285A1 - Transcriptional control sequences

Info

Publication number: WO2008052285A1
Application number: PCT/AU2007/001683
Authority: WO
Inventors: Stefanie Sprunck; Marina Gebert; Thomas Dresselhaus
Original assignee: Adelaide Research & Innovation Pty Ltd; Grains Research & Development Corporation
Priority date: 2006-11-03
Filing date: 2007-11-02
Publication date: 2008-05-08

Abstract

The present invention relates generally to transcriptional control sequences and the present invention is predicated, in part, on the identification of transcriptional control sequences which are derived from ARO1 genes. In some embodiments, the present invention relates to transcriptional control sequences that specifically or preferentially direct expression of a nucleotide sequence of interest in a plant gametophyte.

Description

TRANSCRIPTIONAL CONTROL SEQUENCES

FIELD OF THE INVENTION

The present invention relates generally to transcriptional control sequences. More particularly, the present invention relates to transcriptional control sequences that specifically or preferentially direct expression of a nucleotide sequence of interest in a plant gametophyte.

BACKGROUND OF THE INVENTION

This International Patent Application claims priority to Australian Provisional Patent Application 2006906135, the specification of which is hereby incorporated by reference.

The primary emphasis in genetic modification has been directed to prokaryotes and mammalian cells. For a variety of reasons, plants have proven more intransigent than other eukaryotic cells to genetically manipulate. However, in many instances, it is desirable to effect transcription of an introduced nucleotide sequence of interest either specifically or preferentially in a particular plant part or at a particular developmental stage of the plant. Accordingly, there is substantial interest in identifying transcriptional control sequences, such as promoters or enhancers, which specifically or preferentially direct transcription in particular plant organs, tissues or cell types or at particular developmental stages of the plant.

Expression of heterologous DNA sequences in a plant is dependent upon the presence of an operably linked transcriptional control sequence, such as a promoter or enhancer, which is functional within the plant. The choice of transcriptional control sequence will determine when and where within the organism the heterologous DNA sequence is expressed. For example, where continuous expression is desired throughout the cells of a plant, constitutive promoters are utilized. In contrast, where gene expression in response to a stimulus is desired, an inducible promoter may be used. Where expression in specific tissues or organs is desired, a tissue-specific promoter may be used.

Frequently, it is desirable to effect expression of a DNA sequence in particular cells, tissues or organs of a plant. For example, male and/or female sterility in a plant might be accomplished by genetic manipulation of the plant's genome with a male or female gametophyte or gamete specific promoter operably linked to a toxic protein.

Alternatively, it might be desirable to inhibit expression of a native DNA sequence within particular plant tissues to achieve a desired phenotype. In this case, such inhibition might be accomplished by transformation of the plant with a tissue-specific promoter operably linked to an antisense or RNAi nucleotide sequence, such that expression of these sequences produces an RNA transcript that interferes with translation of the mRNA of the native DNA sequence.

However, promoter sequences that can be used to drive, for example, egg cell specific expression of a nucleotide sequence of interest in higher plants are not well known in the literature. This may be at least in part attributed to the difficulty in isolating female gametes from seed plants. As a consequence of this difficulty, the transcripts of plant egg cells are poorly represented in current databases of expressed sequence tags (ESTs), which have been mainly generated through sequencing from cDNA libraries produced from complex tissues, e.g. whole floral organs. Though more than 1.5 million Poaceae ESTs were present in the public EST database (by March 2004) the use of complex tissues resulted in under representation of genes expressed at low levels and in only one or a few cell types.

Accordingly, the isolation and characterization of gametophyte-speάfic or gametophyte-preferential transcriptional control sequences would be desirable for use in the genetic manipulation of plants.

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 OF THE INVENTION

The present invention is predicated, in part, on the identification of transcriptional control sequences which are derived from AROl genes.

Accordingly, in a first aspect, the present invention provides an isolated nucleic add comprising:

(i) a nucleotide sequence defining a transcriptional control sequence, wherein said transcriptional control sequence is derived from a gene which encodes an AROl polypeptide; or

(ii) a nucleotide sequence defining a functionally active fragment or variant of (i).

In some embodiments of the invention, the isolated nucleic adds provided by the present invention comprise a transcriptional control sequence which specifically or preferentially directs expression in a plant gametophyte, or a functionally active fragment or variant thereof. In further embodiments of the invention, the isolated nudeic acids provided by the present invention comprise a transcriptional control sequence which spedfically or preferentially directs expression in a plant egg cell and/or a plant pollen grain, or a functionally active fragment or variant thereof.

In a second aspect, the present invention provides a nucleic add construct comprising the isolated nucleic add of the first aspect of the invention.

In a third aspect, the present invention provides a cell comprising:

(i) the nucleic acid construct of the second aspect of the invention; and/or (ii) a genomically integrated form of the construct mentioned at (i).

The cells contemplated by the third aspect of the invention include any prokaryotic or eukaryotic cell. In one embodiment, the cell is a plant cell.

In a fourth aspect, the present invention provides a multicellular structure comprising one or more cells of the third aspect of the invention. In one embodiment, the multicellular structure comprises a plant or a part, organ or tissue thereof.

In a fifth aspect, the present invention provides a method for specifically or preferentially expressing a nucleotide sequence of interest in a plant gametophyte, the method comprising effecting transcription of the nucleotide sequence of interest in a plant under the transcriptional control of the nucleic add of the first aspect of the invention, wherein said nucleic acid comprises a transcriptional control sequence which spedfically or preferentially directs expression in a plant gametophyte.

In a sixth aspect, the present invention provides a method for promoting sterility in a plant, the method comprising expressing a nucleotide sequence encoding a cytotoxic or cytostatic protein or a cytotoxic or cytostatic non-translated RNA, spedfically or preferentially in a plant gametophyte; wherein said nudeotide sequence is operably connected to a nudeic acid of the first aspect of the invention which comprises a transcriptional control sequence which spedfically or preferentially directs expression in a plant gametophyte.

In a seventh aspect, the present invention also provides a method for inhibiting or eliminating self-incompatibility in a plant, the method comprising expressing a nucleotide sequence which encodes an inhibitor of one or more self-incompatibility determinants in a plant gametophyte, wherein said nucleotide sequence which encodes an inhibitor of one or more self-incompatibility determinants is operably connected to a nucleic acid of the first aspect of the invention which comprises a transcriptional control sequence which specifically or preferentially directs expression in a plant gametophyte.

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.

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.

TABLE 1 - Summary of Sequence Identifiers

Sequence Sequence Identifier in Identifier sequence listing

DESCRIPTION OF EXEMPLARY EMBODIMENTS

It is to be understood that the following description is for the purpose of describing particular embodiments only and is not intended to be limiting with respect to the above description.

As set out above, the present invention is predicated, in part, on the identification of transcriptional control sequences which are derived from AROl genes.

The term "transcriptional control sequence" should be understood as any nucleotide sequence that modulates at least the transcription of an operably connected nucleotide sequence. Furthermore, the transcriptional control sequence of the present invention may comprise any one or more of, for example, a leader, promoter, enhancer or upstream activating sequence.

In one embodiment, the "transcriptional control sequence" at least includes a promoter. A "promoter", as referred to herein, encompasses any nucleic acid that confers, activates or enhances expression of 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 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/nudeotide sequence of interest combinations, it is generally preferred to position the promoter at a distance from the transcription start site that is approximately the same as the distance between that promoter and the gene it controls in its natural setting, ie. 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. Accordingly, in a first aspect, the present invention provides an isolated nucleic add comprising:

(i) a nucleotide sequence defining a transcriptional control sequence, wherein said transcriptional control sequence is derived from a gene which encodes an AROl polypeptide; or (ii) a nucleotide sequence defining a functionally active fragment or variant of

In the present invention, "isolated" refers to material removed from its original environment (eg. the natural environment if it is naturally occurring), and thus is altered "by the hand of man" from its natural state. For example, an isolated polynucleotide 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 polynucleotide. An "isolated" nucleic add molecule should also be understood to include a synthetic nudeic acid molecule, induding those produced by chemical synthesis using known methods in the art or by in-vitro amplification (eg. polymerase chain reaction and the like).

The isolated nudeic acids of the present invention may comprise any polyribonudeotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. For example, the isolated nudeic acids 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 a 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 isolated nudeic acids may comprise triple-stranded regions comprising RNA or DNA or both RNA and DNA. The isolated nudeic add molecules may also 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 inosine. A variety of modifications can be made to DNA and RNA; thus, "polynucleotide" embraces chemically, enzymatically, or metabolically modified forms.

The nucleic adds of the present invention comprise nucleotide sequences defining a transcriptional control sequence which is "derived from a gene which encodes an AROl polypeptide". The term "derived from", as it is used herein, refers to a source or origin for the transcriptional control sequence. As such, a transcriptional control sequence "derived from a gene which encodes an AROl polypeptide" refers to a transcriptional control sequence which, in its native state, exerts at least some transcriptional control over a gene which encodes an AROl polypeptide in an organism.

As set out above, the present invention contemplates, among other things, a nucleotide sequence defining a transcriptional control sequence, wherein said transcriptional control sequence is derived from a gene which encodes an AROl polypeptide.

In one embodiment, an "AROl polypeptide" includes a polypeptide comprising one or more Armadillo repeat (ARM) domains. Armadillo repeat domains are described in detail in the reviews of Hatzfeld (Int. Rev. Cytol. 186: 179-224, 1999) and Coates (Trends Cell Biol. 13: 463-471, 2003). In another embodiment, the term "AROl polypeptide" refers to a polypeptide that comprises 5 ARM domains. In yet another embodiment, an "AROl polypeptide" does not comprise any functional domain other than one or more ARM domains.

In some embodiments, an "AROl polypeptide" refers to a polypeptide which comprises at least 35%, at least 45%, at least 55%, at least 65%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% amino add sequence identity, to SEQ ID NO: 1.

AROl polypeptides are expressed in a range of organisms including, for example, a range of plants. As used herein, the term "plant" should be understood to at least include monocotyledonous angiosperm plants ('monocots'), dicotyledonous angiosperm plants ('dicots') and gymnosperm plants.

Exemplary dicots include, for example, Arabidopsis spp., Medicago spp., Nicotiana spp., Lotus spp. (including Lotus japonicus), Vitis spp. (including Vitis vinifera). Glycine spp. (including Glycine max), Brassica spp. (including Brassica campestris, Brassica rapa and Brassica napus), sugar beet, sunflower, potato, safflower, cassava, yams, sweet potato, other Brassicaceae such as Thellungiella halophila, among others.

In a further embodiment, the plant is a monocot and in a yet further embodiment, the plant is a cereal crop plant. As used herein, the term "cereal crop plant" includes members of the order Poales, and including the family Poaceae, which produce edible grain for human or animal food. Examples of cereal crop plants that in no way limit the present invention include barley, wheat, rice, maize, millets, sorghum, rye, triticale, oats, teff, rice, spelt and the like. However, the term cereal crop plant should also be understood to include a number of non-Poales species that also produce edible grain, which are known as pseudocereals, and include, for example, amaranth, buckwheat and quinoa.

Exemplary AROl polypeptide sequences from plants include AROl polypeptides comprising the amino acid sequences set forth in:

• SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 (each from

Arabidopsis thaliana)

• SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 (each from Oryza sativa), • SEQ ID NO: 9, SEQ ID NO: 54, SEQ ID NO: 55 (each from Medicago truncatula), • SEQ ID NO: 10 (from Triticum aestivum)

• SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51 (each from Zea mays)

• SEQ ID NO: 52, SEQ ID NO: 53 (each from Vitis vinifera) • SEQ ID NO: 56 (from Lotus japonicus)

Thus, in some embodiments, the transcriptional control sequence is derived from a gene which encodes an AROl polypeptide comprising the amino acid sequence set forth in any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53 SEQ ID NO: 54, SEQ ID NO: 55 or SEQ ID NO: 56

Accordingly, the isolated nucleic adds (or fragments or variants thereof) of the present invention may be derived from any suitable source. For example, the nucleic acids may be derived from an organism, such as a plant. In further embodiments, the present invention also contemplates synthetic nucleic adds.

Exemplary transcriptional control sequences that may be derived a gene which encodes an AROl polypeptide indude:

• SEQ ID NO: 22 (from Arabidopsis thaliana)

• SEQ ID NO: 23, SEQ ID NO: 24 (each from Oryza sativa)

• SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60 (each from Zea mays) • SEQ ID NO: 61, SEQ ID NO: 62 (each from Vitis vinifera)

• SEQ ID NO: 63, SEQ ID NO: 64 (each from Medicago truncatula)

• SEQ ID NO: 65 (from Lotus japonicus)

Thus, in one embodiment, the isolated nucleic add of the first aspect of the invention comprises a nudeotide sequence as set forth in any one of SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64 or SEQ ID NO: 65, or a functionally active fragment or variant thereof.

As set out above, the present invention also provides a nucleic add comprising a nucleotide sequence defining a "functionally active fragment" or "functionally active variant" of a transcriptional control sequence derived from a gene which encodes an AROl polypeptide. As referred to herein, a "functionally active fragment" or "functionally active variant" refers to a fragment or variant which retains the functional activity of a transcriptional control sequence derived from a gene which encodes an AROl polypeptide.

"Functionally active fragments", as contemplated herein, may be of any length wherein the transcriptional control sequence retains the capability to affect expression of an operably connected nucleotide sequence. In various embodiments, the fragment may be at least 50 nucleotides (nt), at least 100 nt, or at least 200 nt in length. For example, in one specific embodiment, a fragment at least 50 nt in length comprises fragments which include 50 or more contiguous bases from, for example, the nucleotide sequence of any of SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64 or SEQ ID NO: 65.

"Functionally active variants" of the transcriptional control sequence of the invention include orthologs, mutants, synthetic variants, analogs and the like which retain the capability to affect expression of an operably connected nucleotide sequence. As such, "variants" specifically include, for example, orthologous transcriptional control sequences from 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 inosine.

In some embodiments, the transcriptional control sequence or functionally active fragment or variant thereof comprises at least 50% sequence identity, at least 65% sequence identity, at least 80% sequence identity or at least 95% sequence identity to the nucleotide sequence set forth in any of SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64 or SEQ ID NO: 65.

When comparing nucleotide sequences to calculate a percentage identity, the nucleotide sequences should be compared over a comparison window of at least 50 nucleotide residues, at least 100 nucleotide residues, at least 200 nucleotide residues, at least 500 nucleotide residues or over the full length of any of SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64 or SEQ ID NO: 65. The comparison window may comprise additions or deletions (ie. 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 alignment of sequences for aligning a comparison window may be conducted by computerized 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 al ("Current Protocols in Molecular Biology" John Wiley & Sons Inc, 1994-1998, Chapter 15, 1998).

In another embodiment, the transcriptional control sequence or functionally active fragment or variant thereof comprises a nucleotide sequence which hybridises to the nucleotide sequence set forth in any of SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64 or SEQ ID NO: 65 under stringent conditions.

As referred to herein, "stringent" hybridisation conditions will be those in which the salt 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 30⁰C. Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Stringent hybridisation conditions may be low stringency conditions, medium stringency conditions or high stringency conditions. Exemplary low stringency conditions include hybridisation with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37°C, and a wash in Ix to 2xSSC (20xSSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55°C. Exemplary moderate stringency conditions include hybridisation in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37°C, and a wash in 0.5x to IxSSC at 55 to 60⁰C. Exemplary high stringency conditions include hybridisation in 50% formamide, 1 M NaCl, 1% SDS at 37°C, and a wash in 0. IxSSC at 60 to 65°C. Optionally, wash buffers may comprise about 0.1% to about 1% SDS. Duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours.

Specificity of hybridisation is also affected by post-hybridization washes, the 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. Biochem. 138: 267-284, 1984), ie. Tm =81.5°C +16.6 (log M)+0.41 (% GQ-0.61 (% form)-500/L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization 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 hybridizes to a perfectly matched probe. Tm is reduced by about 1°C for each 1% of mismatching; thus, Tm, hybridization, and/or wash conditions can be adjusted to hybridize to sequences of different degrees of complementarity. For example, sequences with >90% identity can be hybridised by decreasing the Tm by about 10⁰C. Generally, stringent conditions are selected to be about 5°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 can utilize a hybridization and/or wash at, for example, 1, 2, 3, or 4°C lower than the thermal melting point (Tm); medium stringency conditions can utilize a hybridization and/or wash at, for example, 6, 7, 8, 9, or 10⁰C lower than the thermal melting point (Tm); low stringency conditions can utilize a hybridization and/or wash at, for example, 11, 12, 13, 14, 15, or 20⁰C lower than the thermal melting point (Tm). Using the equation, hybridization and wash compositions, and desired Tm, those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a Tm of less than 45°C (aqueous solution) or 32°C (formamide solution), it is preferred to increase the SSC concentration so that a higher temperature can be used. An extensive guide to the hybridization of nucleic adds is found in Tijssen (Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes, Pt I, Chapter 2, Elsevier, New York, 1993), Ausubel et at, eds. (Current Protocols in Molecular Biology, Chapter 2, Greene Publishing and Wiley-Interscience, New York, 1995) and Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2^nd ed., Cold Spring Harbor Laboratory Press, Plainview, NY, 1989).

In some embodiments, the isolated nucleic acids comprise a transcriptional control sequence which specifically or preferentially directs expression in a plant gametophyte, or a functionally active fragment or variant thereof.

As referred to herein, "a plant gametophyte" refers to a male and/or female gametophyte. As such, the transcriptional control sequences may specifically or preferentially direct expression in a male and/or female gametophyte of a plant. In one embodiment, however, the transcriptional control sequences (or functionally active fragments or variants thereof) specifically or preferentially direct expression in both the male and female gametophytes of a plant.

As used herein, a "transcriptional control sequence which specifically directs expression in a plant gametophyte" refers to a transcriptional control sequence which directs the expression of an operably connected nucleotide sequence of interest substantially only in a plant gametophyte in at least one plant taxon. A "transcriptional control sequence which preferentially directs expression in a plant gametophyte" refers to a transcriptional control sequence which directs the expression of an operably connected nucleotide sequence at a higher level in a plant gametophyte than in one or more other tissues of the plant, eg. sporophytic tissues such as leaf tissue or root tissue, in at least one plant taxon. In one embodiment, preferential expression in a plant gametophyte includes expression of a nucleotide sequence of interest in a plant gametophyte at a level of at least twice, at least 5 times or at least 10 times the level of expression seen in at least one sporophytic plant tissue.

As set out above, in some embodiments, the present invention provides transcriptional control sequences which specifically or preferentially direct expression in a plant gametophyte in at least one plant taxon. This should be understood to mean that the specific or preferential expression pattern effected by the transcriptional control sequence is manifest in at least one plant taxon, but not necessarily all plant taxa. For example, in some taxa, the transcriptional control sequences may effect specific or preferential expression in other, or additional, plant parts. Alternatively, in some taxa the transcriptional control sequences may effect constitutive expression.

In further embodiments of the invention, the isolated nucleic acids provided comprise a transcriptional control sequence which specifically or preferentially directs expression in particular cells within the male and/or female gametophyte of a plant. For example, in one embodiment, the transcriptional control sequence (or functionally active fragment or variant thereof) specifically or preferentially directs expression in a cell selected from a plant egg cell, a pollen grain cell and/or a pollen tube cell. A "plant egg cell" is a component of the female gametophyte in a plant and in one embodiment, the term "plant egg cell" should be understood to specifically refer to the female gamete in the female gametophyte.

"Pollen grains" comprise the microgametophytes (male gametophytes) of a plant and produce the male gametes of seed plants. Pollen grains include, among other things, a tube nucleus which produces the pollen tube, and a generative nucleus that divides to form two sperm cells. Pollen grain cells are generally surrounded by a cellulose cell wall and a thick, tough outer wall made of sporopollenin. In some flowering plants, germination of the pollen grain may begin before it leaves the microsporangium, with the generative cell forming the two gametes. A "pollen grain cell" as referred to herein, refers to any cell which forms part of the pollen grain in a plant. As such, a "pollen grain cell" may include, for example, a generative cell, a pollen tube cell, a gamete or the like.

In further embodiments, the isolated nucleic acid which comprises a transcriptional control sequence which specifically or preferentially directs expression in a plant gametophyte is derived from a gene which encodes a polypeptide comprising the amino add sequence set forth in any of SEQ ID NO: 1, SEQ ID NO: 6 or SEQ ID NO: 10.

In yet further embodiments, the isolated nucleic acid which comprises a transcriptional control sequence which specifically or preferentially directs expression in a plant gametophyte comprises a nucleotide sequence motif comprising the nucleotide sequence set forth in any of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21.

In yet another embodiment, the isolated nucleic acid which comprises a transcriptional control sequence which specifically or preferentially directs expression in a plant gametophyte comprises a nucleotide sequence as set forth in SEQ ID NO: 22 or a functionally active fragment or variant thereof.

In yet another embodiment, the isolated nucleic acid which comprises a transcriptional control sequence which specifically or preferentially directs expression in a plant gametophyte comprises a nucleotide sequence as set forth in SEQ ID NO: 23 or a functionally active fragment or variant thereof.

As referred to herein, a functionally active fragment or variant of a transcriptional control sequence which specifically or preferentially directs expression in a plant gametophyte refers to a functionally active fragment or variant of a transcriptional control sequence (as hereinbefore defined) which retains the ability to specifically or preferentially direct expression in a plant gametophyte in at least one plant taxon.

A "nucleic acid construct" as referred to herein refers to an artificially constructed nudeic add molecule. However, as mentioned below, a nudeic add construct may be incorporated into a naturally occurring nudeic acid molecule (such as the genome of an organism).

The nucleic add construct of the present invention may comprise any polyribonudeotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. For example, the nudeic add 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 that is a 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 nudeic add 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 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, enzymaticaUy, or metabolically modified forms.

In one embodiment, the nucleic add construct comprises DNA. Accordingly, the nucleic add construct of the present invention may comprise, for example, a linear DNA molecule, a plasmid, a transposon, a cosmid, an artifidal chromosome and the like. Furthermore, the nucleic add construct of the present invention may be a separate nudeic acid molecule or may be a part of a larger nudeic acid molecule.

In another embodiment, the isolated nudeic add comprises a nudeotide sequence defining a transcriptional control sequence and further comprises a nudeotide sequence of interest operably connected to the transcriptional control sequence.

In yet another embodiment, the nudeotide sequence of interest is heterologous with respect to the transcriptional control sequence. As used herein, the term "heterologous with respect to the transcriptional control sequence" refers to the nudeotide sequence of interest being a nudeotide sequence other than that which the transcriptional control sequence is operably connected to in its natural state. For example, in its natural state, pAtAROl (SEQ ID NO: 22) is operably connected to an Arabidopsis gene which encodes a protein consisting of the amino add sequence set forth in SEQ ID NO: 1. Accordingly, in this example, any nudeotide sequence other than the Arabidopsis nudeotide sequence which encodes a protein consisting of the amino add sequence set forth in SEQ ID NO: 1 should be considered heterologous with respect to SEQ ID NO: 22. Similarly, any nudeotide sequence other than the rice nudeotide sequence which encodes a protein consisting of the amino add sequence set forth in SEQ ID NO: 6 should be considered heterologous with respect to SEQ ID NO: 23 and any nudeotide sequence other than the rice nucleotide sequence which encodes a protein consisting of the amino acid sequence set forth in SEQ ID NO: 7 should be considered heterologous with respect to SEQ ID NO: 24.

Accordingly, a sequence that is heterologous with respect to a particular transcriptional control sequence may therefore include, for example, orthologous or paralogous AROl sequences, reporter genes, selectable marker genes, heterologous protein-encoding nucleic acid sequences; heterologous non-translated RNA encoding nucleic acid sequences and the like.

The nucleic acid construct may further comprise a nucleotide sequence defining a transcription terminator. The term "transcription terminator" or "terminator" refers to a DNA sequence at the end of a transcriptional unit which signals termination of transcription. Terminators are 3'-non-translated DNA sequences generally containing 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 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 (nos) terminator, the CaMV 35S terminator, the octopine synthase (ocs) terminator, potato proteinase inhibitor gene (pin) terminators, such as the γinll and γinlll terminators and the like.

In one specific embodiment, the nucleic add construct of the third aspect of the invention comprises an expression cassette comprising the structure:

( [N]w - TCS - [N]_x - SoI - [N]_y - TT - [N]z )

wherein:

[N]w comprises one or more nucleotide residues, or is absent; TCS comprises the nucleic acid of the first aspect of the invention; [N]_x comprises one or more nucleotide residues, or is absent;

SoI comprises a nucleotide sequence of interest which encodes an mRNA or non- translated RNA, wherein the nucleotide sequence, SoI, is operably connected to TCS; [N]_y comprises one or more nucleotide residues, or is absent; TT comprises a nucleotide sequence defining a transcription terminator; [N]z comprises one or more nucleotide residues, or is absent.

The nucleic acid constructs of the present invention may further comprise nucleotide sequences such as: an origin of replication for one or more hosts; a selectable marker gene which is active in one or more hosts and the like.

"Selectable marker genes" include any nucleotide sequences which, when expressed by a cell, confer a phenotype on the cell that facilitates the identification and/or selection of transformed cells. A range of nucleotide sequences encoding suitable selectable markers are known in the art. Exemplary nucleotide sequences that encode selectable markers include: antibiotic resistance genes such as ampicillin-resistance genes, tetracydine-resistance genes, kanamycin-resistance genes, the AURI-C gene which confers resistance to the antibiotic aureobasidin A, neomycin phosphotransferase genes (eg. nγtl and nptIT) and hygromycin phosphotransferase genes (eg. kpt); herbicide resistance genes including glufosinate, phosphinothricin or bialaphos resistance genes such as phosphinothricin acetyl transferase encoding genes (eg. bar), glyphosate resistance genes including 3-enoyl pyruvyl shikimate 5-phosphate synthase encoding genes (eg. aroA), bromyxnil resistance genes including bromyxnil nitrilase encoding genes, sulfonamide resistance genes including dihydropterate synthase encoding genes (eg. sul) and sulfonylurea resistance genes including acetolactate synthase encoding genes; enzyme-encoding reporter genes such as GUS and chloramphenicol acetyltransferase (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. The genetic constructs of the present invention may also include further nucleotide sequences intended for the maintenance and/or replication of the genetic construct in prokaryotes or eukaryotes and/or the integration of the genetic construct or a part thereof into the genome of a eukaryotic or prokaryotic cell.

In one embodiment, 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 add 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 sequences" should be understood to include any substantially homologous and substantially directly repeated nucleotide sequences that delimit a nucleic add molecule that is transferred from an Agrobαcterium sp. cell into a plant cell susceptible to Agrobacterium-mediated transformation. By way of example, reference is made to the paper of Peralta and Ream (Proc. Nαtl. Acαd. Sd. USA, 82(15): 5112-5116, 1985) and the review of Gelvin (Microbiology and Molecular Biology Reviews, 67(1): 16-37, 2003).

The present invention also contemplates any suitable modifications to the genetic construct which facilitate bacterial mediated insertion into a plant cell via bacteria other than Agrobacterium sp., for example, as described in Broothaerts et al. (Nature 433: 629- 633, 2005).

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 genetic construct described herein or a derivative thereof in a prokaryotic cell such as an E. coli cell or a plant cell or an animal cell. Exemplary methods for cloning nudeic acid molecules are described in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 2000).

In a third aspect, the present invention provides a cell comprising:

(i) an isolated nucleic add of any one of claims 1 to 10; (ii) a nucleic add construct of any one of daims 11 to 14; and/or

(iii) a genomically integrated form of the nudeic add mentioned at (i) and/or the construct mentioned at (ii).

The isolated nucleic add and/or nudeic add construct may be maintained in the cell as a nucleic acid molecule, as an autonomously replicating genetic element (eg. a plasmid, cosmid, artifidal 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 DNA that makes up the genetic complement of a cell. As such, the genomic DNA of a cell should be understood to indude chromosomes, mitochondrial DNA, plastid DNA, chloroplast DNA, endogenous 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.

The cells contemplated by the third aspect of the invention indude any prokaryotic or eukaryotic cell.

In one embodiment, the cell is a plant cell. As used herein, the term "plant" includes any plant. In one embodiment, the plant is a vascular plant. In another embodiment the plant is a seed plant including, for example, dicotyledonous or monocotyledonous angiosperms and gymnosperms.

In another embodiment, the plant cell is a dicotyledonous plant cell, for example, an Arabidopsis sp. cell. In yet another embodiment the cell is a monocotyledonous plant cell or a cereal crop plant cell.

In further embodiments, the cell is a plant gametophyte cell such as a plant egg cell, a plant pollen grain cell and/or a pollen tube cell. In yet further embodiments, the level, rate and/or pattern of expression of at least one nucleotide sequence is altered in the plant gametophyte cell relative to a wild type form of said plant gametophyte cell.

In another embodiment, the cell may also comprise a prokaryotic cell. For example the prokaryotic cell may include an Agrobacterium sp. cell which carries the nucleic add construct and which may, for example, be used to transform a plant. In another embodiment, the prokaryotic cell may include an E. coli cell, which may, for example, be used in the construction or cloning of a nucleic acid construct.

In a fourth aspect, the present invention provides a multicellular structure comprising one or more cells of the third aspect of the invention.

In one embodiment, the multicellular structure comprises a plant or a part, organ or 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; plant reproductive material (including, for example, cuttings, seed, flowers, pollen grains and the like); and cultured plant tissue such as a callus or suspension culture. In further embodiments, the multicellular structure comprises one or more plant gametophytes or cells thereof. For example, the multicellular structure may comprise a plant, a flower, a carpel or pistil, a plant ovary, an ovule, an embryo sac, an anther or a pollen grain. In one embodiment, the level, rate and/or pattern of expression of at least one nucleotide sequence may be altered in said one or more plant gametophyte cells of the multicellular structure relative to a wild type form of said plant gametophyte cell. In a fifth aspect, the present invention provides a method for specifically or preferentially expressing a nucleotide sequence of interest in a plant gametophyte, the method comprising effecting transcription of the nucleotide sequence of interest in a plant under the transcriptional control of the nucleic add of the first aspect of the invention, wherein said nucleic acid comprises a transcriptional control sequence which specifically or preferentially directs expression in a plant gametophyte.

As set out above, the present invention is predicated, in part, on effecting transcription of the nucleotide sequence of interest under the transcriptional control of the nucleic acid of the first aspect of the invention, wherein said nucleic add comprises a transcriptional control sequence which spedfically or preferentially directs expression in a plant gametophyte. In one embodiment, this is effected by introducing said nudeic acid into a plant cell, such that the nucleotide sequence of interest is operably connected to the transcriptional control sequence.

The present invention contemplates any method to effect operable connection of a nudeotide sequence of interest to the transcriptional control sequence. For example, a nudeotide sequence of interest may be incorporated into the nudeic add molecule that comprises the transcriptional control sequence, and be operably connected thereto. In this way, the nudeotide sequence of interest and transcriptional control sequence are both introduced into the plant. Alternatively, the nudeic acid sequence of the present invention may be inserted into the plant genome such that it is placed in operable connection with an endogenous AROl transcriptional control sequence. As would be recognised by one of skill in the art, the insertion of the transcriptional control sequence into the plant genome may be either by non-site specific insertion or by site- specific insertion (for an example of site-specific insertion see Terada et al._r Nat Biotechnol 20: 1030-1034, 2002).

The nudeic acid may be introduced into a plant via any suitable method. For example, an explant or cultured plant tissue may be transformed with a nudeic add molecule, and the transformed explant or cultured plant tissue subsequently regenerated into a mature plant which produces seed including the nucleic add molecule; a nucleic acid may be directly transformed into a plant, either stably or transiently; a nucleic add may be introduced into a plant via breeding using a parent plant that carries the nucleic acid molecule; and the like.

In one embodiment, the nudeic add molecule is introduced into a plant cell via transformation. Plant cells 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 and direct DNA uptake based methods. Roa-Rodriguez et αl. (Agrobacte^ήum-mediαted transformation of plants, 3^rd Ed. CAMBIA Intellectual Property Resource, Canberra, Australia, 2003) review Agrobacterium-mediated plant transformation methods suitable for a range of plant species. Transformation may also be effected using bacteria other than Agrobαcterium spp., for example, as described by Broothaerts et αl. (2005, supra). Microprojectile bombardment may also be used to transform plant tissue and methods for the transformation of plants, particularly cereal plants, and examples of such methods are reviewed by Casas et al. (Plant Breeding Rev. 13: 235-264, 1995). Exemplary 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, 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. MoI. Biol. Lett. 7: 849-858, 2002). A range of other plant transformation methods may also be evident to those of skill in the art and, accordingly, the present invention should not be considered in any way limited to the particular plant transformation methods exemplified above. The nucleotide sequence of interest, which is placed under the regulatory control of the transcriptional control sequence of the present invention, may include any nucleotide sequence of interest. General categories of nucleotide sequences of interest may include, for example: (i) cytotoxin genes such as barnase, RNase or diphtheria toxin which may be used to induce male and/or female sterility and/or embryo-less fruits in a plant;

(ii) genes encoding transcriptional regulators acting during later stages of embryo development such as BBM or LEC1/LEC2, AP2 transcription factors which may be used to modify embryo development and/or increase embryo size; (iii) genes encoding cell cycle regulators such as RB or E2F, transcriptional regulators acting during later stages of embryo development such as BBM, LEC1/LEC2 or chromatin remodelling factors such as DNA methyltransferases, histone modifying enzymes and the like, which may be used to effect apomictic embryo development (eg. parthenogenesis; autonomous embryogenesis); (iv) reporter genes, such as those encoding GUS, GFP and the like;

(v) genes involved in cellular metabolism such as Zinc finger proteins, kinases, heat shock proteins and the like;

(vi) genes involved in agronomic traits such as disease or pest resistance or herbicide resistance; (vii) genes involved in grain characteristics such as grain biomass, nutritional value, post-harvest characteristics and the like;

(viii) genes encoding heterologous proteins, such as proteins encoding heterologous enzymes or structural proteins or proteins involved in biosynthetic pathways for heterologous products; (ix) nucleotide sequences encoding non-translated RNA, for example an siRNA, miRNA, antisense RNA and the like.

In some embodiments of the invention, the nucleotide sequence of interest is heterologous with respect to the transcriptional control sequence. In a sixth aspect, the present invention provides a method for promoting sterility in a plant, the method comprising expressing a nucleotide sequence encoding a cytotoxic or cytostatic protein or a cytotoxic or cytostatic non-translated RNA, specifically or preferentially in a plant gametophyte; wherein said nucleotide sequence is operably connected to a nucleic acid of the first aspect of the invention which comprises a transcriptional control sequence which specifically or preferentially directs expression in a plant gametophyte.

The term "promoting sterility", as used herein, refers to any reduction in the production of fertile seed by a plant and does not necessarily require a plant to become completely sterile (ie. unable to produce any fertile seed).

A "cytotoxic or cytostatic protein" or "cytotoxic or cytostatic non-translated RNA" refers to any protein or non-translated RNA that inhibits or prevents the growth, division, metabolic function of male or female gametophyte or inhibits or prevents successful fertilisation of the egg cell by the male gamete. In some embodiments of the invention, the nucleotide sequence encodes a cytotoxic protein selected from the list consisting of a barnase, an RNAse or a diphtheria toxin.

As referred to herein, a "self-incompatibility determinant" refers to any gene or gene product (eg. RNA or protein) which is involved in establishing self -incompatibility in a plant. In some embodiments, the "self-incompatibility determinant" comprises a gene in the S-locus of a plant. In one embodiment, the S-locus gene contemplated by the present invention is a gene which is expressed in one or more cells of the pollen grain. Exemplary S-locus genes which may be targeted by the method of the ninth aspect of the invention include for example the S gene described by Ii et al. (Plant MoI Biol 34(2): 223-232, 1997); S-glycoproteins/S-RNases (for examples see the review of Newbigin et al, Plant Cell 5:1315-1324, 1993), SLF (Entani et al. Genes Cells 8: 203-213, 2003) and the like.

As referred to herein, an "inhibitor of one or more self-incompatibility determinants" refers to any nucleotide sequence, RNA or protein which is expressed in a gametophyte and the expression of which inhibits or eliminates self-incompatibility in a plant. For example, the inhibitor can be an inhibitor of the expression or activity of a male or female gametophyte self-incompatibility determinant. Exemplary inhibitors of one or more self-incompatibility determinants may include, for example:

(i) an antisense construct directed against a nucleotide sequence encoding a self-incompatibility protein, such as an S-locus gene (for examples of antisense suppression in plants see van der Krol et al., Nature 333: 866-869; van der Krol et al., BioTechniques 6: 958-967; and van der Krol et al, Gen.

Genet. 220: 204-212);

(ii) a co-suppression construct directed against a nucleotide sequence encoding a self -incompatibility protein, such as an S-locus gene (for an example of co- suppression in plants see van der Krol et al, Plant Cell 2(4): 291-299); (iii) a construct encoding a double stranded RNA directed against a nucleotide sequence encoding a self-incompatibility protein, such as an S-locus gene (for an example of dsRNA mediated gene silencing see Waterhouse et al, Proc. Natl Acad. Sd. USA 95: 13959-13964, 1998); and

(iv) a construct encoding an siRNA or hairpin RNA directed against a nucleotide sequence encoding a self-incompatibility protein, such as an S- locus gene (for an example of siRNA or hairpin RNA mediated gene silencing in plants see Lu et at, Nucl. Acids Res. 32(21): el 71; doi:10.1093/nar/gnhl70, 2004).

(v) a nucleic add aptamer that binds to and inhibits a protein self- incompatibility determinant, such as an S-locus gene product.

In addition to the examples above, an inhibitor of one or more self-incompatibility determinants may indirectly modulate the expression or activity of a self- incompatibility determinant. Examples of such inhibitors may include nucleic acid molecules that encode transcription factors or other proteins which promote or suppress the expression or activity of a self-incompatibility determinant; and other non-translated RNAs which directly or indirectly promote or suppress the expression or activity of a self -incompatibility determinant, and the like.

The methods of the fifth, sixth and seventh aspects of the present invention may be performed using any suitable plant. However, in one embodiment, the plant is a dicotyledonous plant and, in another embodiment an Arabidopsis sp. plant. In further embodiments of the invention, the plant is a monocotyledonous plant or a cereal crop plant.

Finally, reference is made to standard textbooks of molecular biology that contain methods for carrying out basic techniques encompassed by the present invention, including DNA restriction and ligation for the generation of the various genetic constructs described herein. See, for example, Maniatis et at, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, New York, 1982) and Sambrook et al. (2000, supra).

The present invention is further described by the following non-limiting examples: BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the expression profile of A06 17 transcripts (TaAROl) originally derived from the wheat egg cell cDNA library. Expression of TaAROl was examined by RT-PCR using DNAse treated total RNA from different tissues of wheat and gene specific primers for A06_17. As controls, cDNA from egg cells, central cells and 2-celled pro-embryos have been used. Quality and quantity of generated cDNA was verified using primers for the ubiquitously expressed GAP-DH. Lane 1: egg cell, 2: pro-embryo,

3: central cell, 4: coleoptile, 5: primary leaf, 6: mature leaf, 7: stem, 8: root without tip, 9: root tip, 10: anther, 11: pistil, 12: kernel 12dap (days after pollination), 13: negative control.

Figure 2 shows the expression profile of AtAROl-4 genes in Arabidopsis. Expression was examined by RT-PCR using DNAse treated mRNA from different tissues of Arabidopsis and gene specific primers for AtAROl, AtAROl₁ AtARO3 and AtARO4. AtAROl is specifically or preferentially expressed in tissues which contain the female and male gametophytes. AtARO2-4 are expressed in all examined tissues. Quality and quantity of generated cDNA was verified using primers for the ubiquitously expressed Actin 3 gene. Lane 1: root, 2: stem, 3: leaf, 4: flower bud, 5: mature flower, 6: silique, 7: immature anther, 8: mature anther, 9: ovary, 10: negative control, 11: genomic DNA

Figure 3 shows the expression profile of AtAROl during anther development and pollen germination in Arabidopsis. mRNA levels of AtAROl at different developmental stages of anthers (anther stages after Smyth et ah, Plant cell 2: 755-767, 1990) and pollen were examined by qRT-PCR with gene specific primers for AtAROl and primers for the ubiquitously expressed gene Actin 3 as a normalization standard. The increase of mRNA levels of AtAROl correlates with maturation of pollen grains in anthers (expression before anther stage 10 was not detectable). Pollen germinated on pollen germination medium for 5 hours (5hai) showed higher AtAROl mRNA levels than dehiscent pollen rehydrated on pollen germination medium for 10 minutes. Abbreviations: (Anth.) anther, (RH) rehydrated, (5hai) 5 hours after incubation.

Figure 4 shows the promoter activity of AtAROl in Arabidopsis. The promoter of AtAROl (pAtAROl; SEQ ID NO: 22) was cloned as a translational fusion 5' upstream of the β-Glucuronidase gene. Cloned constructs were used for stable transformation of Arabidopsis. In the female gametophyte, GUS staining is not visible during the development of the embryo sac (A and B). GUS activity was detected exclusively in egg cells of mature, unfertilized ovules (C). After fertilization, GUS activity was still weakly visible in the zygote (D). However, after the first zygotic division (E), the GUS signal is no longer detectable in AROlp::GUS x WT ovules. (F) Schematic of the Arabidopsis ovule displaying a GUS signal in the egg cell. During anther development, in contrast to microspores (G, floral stage 10), only mature pollen grains were stained (H, floral stage 11 and I, floral stage 12). Pollen grains germinated in vitro showed also strong GUS activity in the pollen tube (K). Floral stages after Smyth et al. (1990, supra). Abbreviations: (ec) egg cell; (em) embryo; (es) mononuclear embryo sac; (ms) megaspore mother cell; (zy) zygote;

Figure 5 shows the expression and subcellular localization of an EGFP-AtAROl fusion protein in Arabidopsis. E-GFP (enhanced green fluorescence protein) was C-terminal fused to the open reading frame of AtAROl, under control of the promoter of AtAROl

(p AtAROl; SEQ ID NO: 22). The construct was used for transformation of Arabidopsis.

Ovules of transgenic plants were analyzed for green fluorescence using bright field (A,

D) and UV microscopy (B, E). (C, F) Merged images of (A+B) and (D+E) respectively. Green fluorescence was visible exclusively in the egg cell of the female gametophyte

(arrows in B and C). In heterozygous plants, the fusion protein could be detected in half of the pollen grains (D-F). CLSM images of female and male gametophytes from

Arabidopsis lines transformed with an AROlp::AROl-GFP construct are shown in panels G to O. (G) Single plane dose up of an egg cell with green fluorescence. The AROl-GFP fusion protein is uniformly distributed in the cytoplasm of the cell but it can not be detected in the egg cell nucleus. (HJ) An Arabidopsis ovule 24 hap. After the first zygotic division AROl-GFP is no longer detectable in the female gametophyte. Only very weak fluorescence of the pollen tube is still visible (arrowhead) in a projection of 32 consecutive confocal sections. (K-N) Single plane image of a AROlp::AROl-GFP pollen grain stained with propidium iodide. The two sperm cell nuclei are visible in the red channel (K). In the green channel (L) and the overlay (N) it is shown that AROl-GFP is not localized in the sperm cells (sc). The vegetative nucleus is not in focus. (O) Germinated AROlp::AROl-GFP pollen. The fusion protein is distributed throughout the cytoplasm of the pollen tube but clearly accumulates in the tube tip (arrowhead). Also, green fluorescence in the vegetative nucleus can be seen (arrow). The sperm cells are not in focus, ac, apical cell; be, basal cell. Scale bars: (HJ) 20μm; (G,K-O) lOμm.

Figure 6 shows a restriction map of the pMG2002 vector.

Figure 7 shows a restriction map of the pLNU-GFP vector.

Figure 8 shows a restriction map of the p95P-Nos vector.

EXAMPLE 1 Identification of egg-cell specific genes and promoters

To identify genes that are specifically expressed in the egg cell, female gametophytes of wheat were microdissected to isolate egg cells. Using the messenger RNA from 12 egg cells, a cDNA library was constructed and single-run partial sequencing of 960 randomly selected cDNA clones was performed. After DNA sequencer trace data passed an automated cleanup pipeline, a total of 735 ESTs were used for bioinformatical analysis. The 735 ESTs formed 404 independent clusters including 310 singletons. The consensus sequences of the dusters were used for BLASTN and BLASTX searches at the NCBI nonredundant database (nr), dbEST and SwissProt data base. Some cDNAs resulted in limited sequence information from non-coding regions. Therefore, BLASTN searches were performed against the TIGR Wheat Gene Index Release 8.0, using the BLASTN algorithm. If a match with >95% sequence identity over the total length of the query sequence was found, the matching sequence was retrieved and used in subsequent BLASTX searches in place of the original EST. BLASTN searches against the NCBI database category of non-mouse and non-human ESTs resulted in 629 egg cell ESTs (333 dusters) matching significantly to annotated ESTs mainly generated from different vegetative tissues of wheat, barley or rice (NCBI dbEST Poaceae). 106 egg cell ESTs (71 clusters) did not match annotated ESTs and were thus considered as uncharacterised transcripts.

Transcripts which did not match to any EST generated from vegetative plant tissues and matched to so-called "hypothetical" proteins (computer-predicted open reading frames from the Arabidopsis and/or rice genome sequences) were selected, as it was assumed that the corresponding genes of some of these transcripts might be specifically or preferentially expressed in egg cells of seed plants. Significant similarities to "hypothetical" proteins of Arabidopsis and/or rice were identified for 98 egg cell dusters. Of these, 11 clusters were not similar to any published EST but only to annotated "hypothetical" genes detected in genomes of Arabidopsis and/or rice and it was concluded that these might be candidate genes that are specifically or preferentially expressed in the female gametes or gametophyte of Arabidopsis and/or rice.

Using this strategy a large duster of transcripts from the wheat egg cell (TaAROl) was identified which did not match to ESTs from any vegetative tissues, but which displays significant similarity to hypothetical proteins from Arabidopsis and rice. In Arabidopsis, there is one TaAROl-like hypothetical gene located on chromosome 4 (AtAROl, SEQ ID NO: 1), which belongs to a small group of four genes. This group is part of a large family of "armadillo repeat only" proteins comprising at least 37 members. The other genes of the sub-group are located on chromosomes 3, 4 and 5 and were termed AtAROl, AtARO3, and AtAROi (SEQ ID Nos: 2, 3 and 4, respectively). Likewise, there is a group of four members in rice which were termed OsARO3G, OsAROδG, OsAROθG and OsAROlOG (SEQ ID NOs: 5, 6, 7 and 8, respectively) and are located on chromosomes 3, 8, 9 and 10 respectively. While OsARO9G has a slightly higher homology to AtAROl on the protein level, the predicted protein of OsAROSG has a higher homology to TaAROl and shows a similar expression pattern in the female and male gametophytes.

The specific expression of TaAROl by RT-PCR was proofed using DNAse treated total RNA from different tissues of wheat and gene specific primers for the egg cell cDNA (ARMfw, SEQ ID NO: 25; ARMrev, SEQ ID NO: 26). Expression of TaAROl was not detected in any vegetative tissues or 12 day old developing caryopsis of wheat. Transcripts were only found in tissues containing the unfertilized egg cell (pistil), in isolated egg cells and in anthers. After fertilization, TaAROl is down-regulated in the ovule (see Figure 1). A similar expression profile was observed for the AtAROl gene in Arabidopsis. Expression was examined by RT-PCR using DNAse treated mRNA from different tissues of Arabidopsis and gene specific primers for AtAROl, AtAROl, AtARO3 and AtAROi. While AtAROl is only expressed in reproductive organs containing male and/or female gametophytes, the other three genes (AtAROl-4) of the gene family are ubiquitously expressed (see Figure 2). qRT-PCR studies using AtAROl specific primers (ARMIa gen for, SEQ ID NO: 27; ARMl gen rev, SEQ ID NO: 28) confirmed expression in the latest stages of anther development with highest transcript amounts in the growing pollen tube (see Figure 3).

Promoter specificity of AtAROl was analysed by cloning a defined 5' upstream region of AtAROl (SEQ ID NO: 22) in front of the β-Glucuronidase (GUS) gene. The cloned construct was used for stable transformation of Arabidopsis. GUS activity was detected exclusively in egg cells of unfertilized ovules and in mature pollen grains as well as in germinating pollen tubes (see Figure 4). After fertilization, GUS activity was still weakly visible in the zygote but no longer detectable in the 2-celled proembryo. Expression in maternal tissues was never observed.

In addition, E-GFP (enhanced green fluorescence protein) was C-terminal fused to the open reading frame of AtAROl (SEQ ID NO: 1), under control of the AtAROl promoter (SEQ ID NO: 22). This construct was used for transformation of Arabidopsis. Ovules and pollen of transgenic plants were analysed for green fluorescence using bright field and UV-microscopy. Green fluorescence was visible in egg cells of unfertilized ovules, in mature pollen and pollen tubes. GFP could neither be detected in other cells of the embryo sac and ovule nor in the maternal tissues of the anther (see Figure 5).

EXAMPLE 2

Isolation of wheat embryo sac cells before and after fertilization

Spikes of Triticum aestivum cv 'Florida' were emasculated 2-4 days before anthesis and covered with bags to prevent fertilization. Egg cells were isolated mechanically from microdissected ovules in 0.55 M sterile mannitol using fine-tipped glass needles and an inverted microscope, as described by Kumlehn et at. (Protoplasma 208: 156-162, 1999). Single cells were transferred into 0.5 ml reaction tubes by using a glass capillary interfaced with a hydraulic system to a micropump. Collected cells were immediately frozen in liquid nitrogen.

EXAMPLE 3 mRNA isolation and cDNA synthesis

mRNA was isolated from 12 egg cells using the Dynabeads^® mRNA DIRECT™ Micro kit (Dynal) following the manufacturer's guidelines, but scaled down to 50 μl. Annealed mRNA was isolated using a magnetic particle transfer device (PickPen™, Bio-Nobile). Subsequently, the SMART™ PCR cDNA synthesis kit (BD Biosciences) was used for cDNA synthesis. First-strand cDNA, long distance-PCR, and determination of optimal cycle numbers for generating a population of representative cDNAs was performed according to the manufacturer's guidelines, but using a digoxigenin-11-dUTP (Roche Applied Science) labeled fragment of wheat GAPDH as a probe.

EXAMPLE 4 Library construction and sequencing

150 μl of cDNA was used for polishing, according to instructions of the SMART™ PCR cDNA synthesis kit (BD Biosciences). Subsequently, 3 μg of EcoRI (Notl) adapters (Invitrogen) were ligated to blunt-end cDNA, using T4 ligase (New England Biolabs). Remaining adapters and fragments below 0.3 kb were removed by electrophoresis in 0.8% low-melting point agarose (Seaplaque GTG). Afterwards, cDNA was extracted using β-agarase I (New England Biolabs). After phosphorylation of EcoRI cohesive ends (10 U/μl T4 polynucleotide kinase, New England Biolabs), a second purification step using Chromaspin™ columns (BD Biosciences) was performed. The cDNA was then ligated into predigested lambda ZAP^® II/EcoRI/CIAP vector (Stratagene). The titre of the unamplified library was 1.43 x 10⁶ pfu/ml. After amplification and in vivo excision, clones were randomly picked and used to generate ESTs. Insert sizes ranged from 300 to 3000 bp, with an average of 900 bp. The average readable sequence length of ESTs was about 500 bp. DNA sequencer trace data subsequently passed an automated cleanup pipeline including PHRED to call bases and assign quality values, followed by CROSS_MATCH to align sequences and to eliminate vector sequences. EXAMPLE 5 Bioinformatics

The sequences were clustered using blastclust (NCBI) and assembled into contigs using Vector NTI 8 (Invitrogen software package). The contig's consensus sequence or the longest representative was used for BLASTN searches against NCBI's nonredundant (nr) database and the EST-database, and for BLASTX searches against NCBI's nr database and SWISSPROT (March 2004). A number of cDNAs resulted in limited sequence information (100 - 250 bp) from non-coding regions. Therefore, BLASTN searches against the TIGR Wheat Gene Index Release 8.0 (Quackenbush et ah, Nucleic Acids Res 29: 159-164, 2001) were performed, using the BLASTN algorithm. If a match with >95% sequence identity over the total length of the query sequence was found, the matching sequence was retrieved and used in subsequent BLASTX searches in place of the original EST. A sequence was considered of interest if it did not show a significant match with a sequence of the NCBI databases (nr, EST) or to the TIGR assembled wheat consensus sequences using the BLASTN algorithm (Altschul et ah, 1997, supra). The significance threshold used for BLASTN searches were: Score > 115, Expect-value < e-25.

For BLASTX searches, the cutoff for a significant match for all but the short sequences was an e-value of < e-15 , Score >= 80. Matches to short query sequences (below 260 bp) were inspected and categorized manually. Clusters encoding proteins of known function were manually categorized into broad functional groups using the MIPS (Munich Information Centre for Protein Sequences) classification as guidance.

EXAMPLE 6 Expression analysis by RT-PCR

Wheat RNA was isolated from vegetative and generative tissues using TRIzol^® reagent (Invitrogen), following the manufacturer's protocol. Starch containing tissues such as caryopsis were extracted twice, using 3 ml of TRIzol^® reagent per 100 mg of tissue. The quality of the total RNA preparation was analyzed by denaturating agarose gel electrophoresis. Before RT-PCR, 1 μg of total RNA was digested with DNAse (RNAse free; Invitrogen) and subsequently used for first-strand cDNA synthesis using Oligo(dT)23 (Sigma) and Superscript II reverse transcriptase (Invitrogen), following the manufacturer's protocol but adding RNAseOUT™ (Invitrogen). Quality and amount of generated cDNAs was analyzed by PCR with intron-spanning primers directed against wheat GAPDH, ie, primers TaGAPl (S'-AGGGTGGTGCCAAGAAGGTCA-S', SEQ ID NO: 29) and TaGAP2 (S'-TATCCCCACTCGTTGTCGTA-S', SEQ ID NO: 30). Expression of TaAROl was analysed using the primer pair ARMfw (5'- GACGAGCACGCGAGGGAGGGATTA-3', SEQ ID NO: 25) and ARMrev (5'- CGGCGGGTGACGTCGGCTTGAA-3', SEQ ID NO: 26). PCR reactions were carried out for 30 cycles (GAPDH) and 38 cycles (TaAROl) respectively, using 2.5 μl of cDNA as template.

Arabidopsis mRNA was isolated from up to 5mg tissue using the Dynabeads^® mRNA DIRECT™ Micro kit (Dynal) following the manufacturer's guidelines. For small amounts of tissues (pistils, anthers and pollen grains), the reaction was scaled down to 50 μl. Annealed mRNA was isolated using a magnetic particle transfer device (PickPen™, Bio-Nobile). Before RT-PCR, the annealed mRNA was treated with DNAse I (RNAse free; MBI Fermentas) in a volume of 12.5 μl. First-strand cDNA synthesis was carried out using Oligo(dT)is (MBI Fermentas) and Reverted Aid H Minus M- MuLV reverse transcriptase (MBI Fermentas), following the manufacturer's protocol but adding RNAse inhibitor (MBI Fermentas). Quality and amount of generated cDNAs was analyzed by PCR with intron-spanning primers directed against Arabidopsis Actin 3 (At2g37620), Act3fw (S'-GATTTGGCATCACACTTTCTACAATG-S', SEQ ID NO: 31) and Act3rev (S'-GTTCCACCACTGAGCACAATG-S', SEQ ID NO: 32). AtAROl- 4 cDNAs were amplified using gene specific primer pairs for each, AtAROl, AtAROl, AtARO3 and AtARO4, as shown in Table 2. 2μl of cDNA was used as template for each PCR reaction.

qRT-PCR analysis was carried out using an iCyder (Biometra) and the corresponding iQ5 software. The amounts of mRNA of AtAROl in different tissues were normalized using primers Actδfw and Actδrev against the housekeeping gene Actin 3. lμl of cDNA was used as template in each PCR reaction. A serial dilution of genomic DNA, isolated from Arabidopsis leaves with the Invisorb Spin Plant Mini Kit (Invitek) according to the manufacturer's guidelines, was used to generate a standard curve for each gene. With the standard curve, the efficiency of each PCR reaction can be determined.

Table 2 - PCR primers for amplification of AtAROl- 4 cDNAs cDNA Primer Sequence (5' - 3') Sequence Identifier

EXAMPLE 7 Cloning of promoter-GUS fusion constructs

Genomic DNA of A. thaliana (ecotype Columbia-0) was isolated according to Li and Chory (In: Methods in Molecular Biology, Volume 82 Eds. Martinez-Zapater and Salinas, pp55-60, 1997). Gene specific primers containing restriction sites were designed to clone the promoter of AtAROl in front of the β-glucuronidase gene (GUS, uidA; Jefferson et at, EMBO J 6: 3901-3907, 1987). A 711 bp genomic fragment 5' upstream of the start codon of AtAROl was amplified from genomic DNA by PCR, using "proofreading" ACCUZYME™ DNA Polymerase (Bioline) with promoter primer ARMl Prom for (5'-TAAAGagatctAAGCTGGTGTC-3', SEQ ID NO: 39, included Bgϊll shown in lower case) and promoter primer ARMl Prom rev (5'- CGCCATGagatctAACAATCAA-3', SEQ ID NO: 40, included BgUl shown in lower case). Genomic amplification products were cloned into Zero Blunt^® TOPO^® PCR Vector (Invitrogen). Ligation and transformation of competent DH5α cells was performed according to the manufacturer's guidelines.

Plasmids of TOPO-pAtAROl and the GUS containing vector pMG2002 (Figure 6) were isolated with the peqGOLD Plasmid Midi Kit (Peqlab) and restricted with BgIII (TOPO- pAtAROl) and Bglll and BamHl (pMG2002), respectively. Thereby, the maize ubiquitin promoter in front of the GUS gene of pMG2002 was removed. The restricted promoter fragment and the vector were purified using the "Easy Pure" DNA purification kit (Biozym). Before ligation the Bglll and BamHl digested plasmid pMG2002 was dephosphorylated using CIAP (Calf Intestine Alkaline Phosphatase; MBI Fermentas), following the manufacturer's guidelines. The promoter was ligated into the digested and dephosphorylated vector using T4 ligase (1 U/μl; MBI Fermentas), following the manufacturer's protocol. The ligation reaction was used for transforming competent DH5α cells. 10 μl of the ligation reaction was diluted 1:5 and mixed with lOOμl of competent cells. The reaction was left on ice for 30 minutes, heat shocked at 42°C for 1 minute and immediately placed on ice again. 250 μl of preheated (37°C) LB-Medium were added to the reaction and placed into a thermoshaker for one hour at 37°C. Afterwards the bacteria were streaked onto a selective LB-plate containing 50μg/ml Spectinomycin. A positive done was selected by colony PCR, using gene specific primers GUS Start rev (S'-ATCCAGACTGAATGCCCACA-S', SEQ ID NO: 41) and AtAROl Prom for (SEQ ID NO: 39). The cloned fragment and construct was verified by sequencing, using the gene specific primers GUS Start rev (SEQ ID NO: 41) and AtAROl Prom for (SEQ ID NO: 39).

EXAMPLE 8

Cloning of a GFP fusion protein construct

E-GFP (enhanced green fluorescence protein; Pang et al., Plant Physiol 112: 893-900, 1996) was C-terminal fused to the open reading frame of AtAROl, under control of the AtAROl promoter. The open reading frame of AtAROl was amplified from genomic DNA using Advantage Genomic Polymerase Mix (Clonetech) and primers ARMl GFP for (5'-TTGTcctaggTCATGGCGGATATTGTGAAACAG-3', SEQ ID NO: 42; Avr II site included) and ARMl GFP rev (5'-CGTCcctaggCAATGAAATCCTCTTGACCCTC-3', SEQ ID NO: 43; Avr II site included). Genomic amplification products were cloned into Zero Blunt^® TOPO^® PCR Vector (Invitrogen). Ligation and transformation of competent DH5α cells was performed according to the manufacturer's guidelines. The plasmid of TOPO-AiAKOI and the vector pLNU-GFP (Figure 7) were restricted with Avr Il The pLNU-GFP vector was dephosphorylated with CIAP (Calf Intestine Alkaline Phosphatase; MBI Fermentas), following the manufacturer's guidelines. The open reading frame of AtAROl was ligated into the vector using T4 ligase (1 U/μl; MBI Fermentas), following the manufacturers protocol. The ligation was transformed into competent XLl- blue cells as described above (see: Cloning of promoter-GUS fusion constructs). The AtAROl promoter was amplified from the pMG2002-AiAKOI construct with primers PArolF (5'-CTCTcggccgcgATCTAAGCTGGTGT-3', SEQ ID NO: 44, Not I site included) and PArolR (5'-CCCCactagtAACAATCAAGAAACTC-3', SEQ ID NO: 45, Spe I site included). The PCR product was digested with the respective enzymes. The pLNU-GFP vector was also digested using Not I and Spe I thereby removing the Ubiquitin promoter and this was replaced by the AtAROl promoter. After restriction of the complete cassette of AtAROl promoter, AtAROl open reading frame and GFP open reading frame with Sfi, the fragment was cloned into the Sfi sites of the vector p95P-Nos (Figure 8). The C-terminal fusion of E-GFP and the sequence of the cloned fragment were verified by sequencing using the primers Ml 3 for (5'- TGTAAAACGACGGCCAGT-3', SEQ ID NO: 46) and AlpromS' (5'- CTTGTTCGTTTT AATAACTCATGA-3', SEQ ID NO: 47).

EXAMPLE 9 Transformation of Arabidopsis

For plant transformation, vectors were transferred into Agrobacterium tumefaciens strain GV3101 pMP90RK (Koncz and Schell, MoI Gen Genet 204: 383-396, 1987). Transformations were performed on ecotype Columbia-0 by a "floral dip" procedure according to Clough and Bent (Plant J 16:735-743, 1998). The seeds obtained from the TO transformants were germinated on soil after a cold-treatment of 2 days at 2°C. Three days after germination, transgenics were selected by spraying 200 mg/1 BAST A^® (Bayer Crop Science) supplemented with 0.1 % Tween. BAST A^® treatment was repeated two times after three days each. Surviving seedlings were transferred to single pots.

Kanamycin selection of respective transformants was performed according to Xiang et al. (Plant MoI Biol Rep 17: 59-65, 1999).

EXAMPLE 10 GUS staining

Activity of β-glucuronidase (GUS; Jefferson et al., 1987, supra) was tested according to a protocol of Vielle-Calzada et al. (Genes Dev 13: 2971-2982, 2000). Inflorescences, siliques, leaves, stems and roots from soil-grown plants were transferred to microtiter wells containing 300 μl of GUS staining buffer. Pistils and siliques were cut open lengthwise with a hypodermic needle (0.4 x 20mm, Braun) before transferring into GUS staining buffer. Microtiter dishes were placed under vacuum for 2 min. After release of vacuum, plates were covered with a lid and incubated at 37°C in the dark over night. Afterwards, ovules were isolated on a glass slide by dissecting the pistils with a syringe in a drop of clearing solution. Ovules were cleared using either Hoyers solution (Liu and Meinke, Plant J 16: 21-31. 1998) or lactatic add clearing buffer (Vielle- Calzada et at. 2000, supra) and analysed under a Zeiss Axioskop microscope under differential interference contrast (DIC) optics. Images were captured on an Axiocam camera (Zeiss) using the Axiovision program AC Release 4.1 (Zeiss).

EXAMPLE 11 Identification of further AROl polypeptides

Bioinformatics was used to identify putative AROl orthologs (AROl polypeptides) in published genomic sequences of Zea mays, Medicago truncatula, Vitis vinifera and Lotus japonicus. In this method, the AtAROl protein sequence (At4g34940) was used as query to run a BLAST search in the Plant Genome Database (www.plantgdb.org), utilizing TBLASTN 2.2.15 with default parameters. Zea mays BAC (Zmbac) was selected as a database.

Significant alignments were produced with sequences of several maize BAC clones derived from chromosome 1, 2, 3, 5 and 9. Highest scores were produced with sequences of one chromosome UNKNOWN done CH201-388C5 (AC197544), chromosome 2 clone CH201 216H15 (AC197262), chromosome 2 done ZMMBBb- 102J21 (AC190494), chromosome 1 done CH201-87C11 (AC202953), chromosome 1 clone CH201-452C8 (AC205545), chromosome 1 done CH201-135E7 (AC186238), chromosome 1 done CH201-1M3 (AC195786).

Open reading frames of the corresponding genomic sequences were identified and translated into proteins using Clone Manager 6 (Scientific & Educational Software) and ORF-Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). Translated protein sequences of Zea mays AROl -like proteins and 5' upstream sequences of Zea mays AROl-like genes were compared using the programs ClustalW (http://www.ebi.ac.uk/Tools/dustalw') and Lasergene MegAlign 4.05 (^®DNASTAR, Inc.).

The Arabidopsis AtAROl protein sequence, the protein sequence of OsARO8g, as well as the predicted ORF of maize chromosome 2 clone CH201 216H15 (AC197262) were used as queries to run BLAST searches with TBLASTN 2.2.17 in the nucleotide collection (nr/nt) database at NCBI (http://www.ncbi.nlm.nih.gov). Open reading frames of corresponding genomic sequences of Medicago truncatula, Vitis vinifera and Lotus japonicus were identified and translated into proteins using Clone Manager 6 (Scientific & Educational Software). Translated protein sequences and 5' upstream sequences of genomic regions were analysed using the programs ClustalW (http://www.ebi.ac.uk/Tools/dustalw) and Lasergene MegAlign 4.05 (DNASTAR, Inc.).

Identified genomic sequences which gave a significant hit to protein sequences of AtAROl were named as follows:

Zea mays

ZmARO-like 1: chromosome 1 dones CH201-87C11 (Accession AC202953) and CH201- 452C8 (Ace. AC205545)

ZmARO-like 2: chromosome 2 dones CH201-216H15 (Ace. AC197262) and ZMMBBb- 102J21 (Ace. AC190494) ZmARO-like 3: chromosome UNKNOWN clone CH201-388C5 (Ace. AC197544)

ZmARO-like 4: chromosome 1 dones CH201-135E7 (Ace. AC186238) and CH201-1M3 (Ace. AC195786).

Vitis vinifera VvARO-like 1: chromosomal location unknown (whole genome shotgun sequencing contig VV78X018362.6; Accession AM432239)

VvARO-like 2: chromosomal location unknown (whole genome shotgun sequencing contig VV78X185794.4; Accession AM477759)

Medicago truncatula

MtARO-like 1: clone mth2-159f24 (Ace. AC147013.4) and clone mth2-211i21 (Ac.

AC138010.13)

MtARO-like 2: chromosome 8 done mth2-75al5 (Ace. AC160096.19)

Lotus japonicus

LjARO-like: chromosome 4, clone LJT45A24 (Ace. AP006113.1)

A transcriptional control was also derived from the genomic nucleotide sequence present upstream of the coding region for each of the above polypeptide sequences. Each polypeptide sequence and the derived transcriptional control sequences are presented in the sequence listing as set out below:

AROl polypeptide Polypeptide sequence Derived transcriptional control sequence

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is 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.

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. Thus, for example, reference to "a nucleotide sequence of interest" includes a single nucleotide sequence as well as two or more nucleotide sequences; "an egg cell" includes a single egg cell as well as two or more egg cells; and so forth.

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS

1. An isolated nucleic acid comprising:

(ii) a nucleotide sequence defining a functionally active fragment or variant of

2. The isolated nucleic acid of claim 1 wherein said transcriptional control sequence is derived from a gene which encodes an AROl polypeptide comprising the amino add sequence set forth in any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53 SEQ ID NO: 54, SEQ ID NO: 55 or SEQ ID NO: 56.

3. The isolated nucleic acid of claim 1 wherein said transcriptional control sequence comprises a nucleotide sequence as set forth in any one of SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64 or SEQ ID NO: 65, or a functionally active fragment or variant thereof.

4. The isolated nucleic acid of any one of claims 1 to 3 wherein said transcriptional control sequence specifically or preferentially directs expression in a plant gametophyte.

5. The isolated nucleic add of any one of claims 1 to 4 wherein said transcriptional control sequence specifically or preferentially directs expression in any of a plant egg cell, a pollen grain cell and a pollen tube cell.

6. The isolated nudeic acid of any one of claims 1 to 5 wherein said transcriptional control sequence specifically or preferentially directs expression in both the male and female gametophytes of a plant.

7. The isolated nudeic add of any one of claims 1 to 6 wherein said transcriptional control sequence is derived from a gene which encodes a polypeptide comprising the amino add sequence set forth in any of SEQ ID NO: 1, SEQ ID NO: 6 or SEQ ID NO: 10.

8. The isolated nudeic acid of any one of daims 1 to 7 wherein said isolated nudeic acid comprises a nucleotide sequence motif comprising the nudeotide sequence set forth in any of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21.

9. The isolated nudeic acid of any one of daims 1 to 8 wherein said isolated nudeic add comprises a nucleotide sequence as set forth in SEQ ID NO: 22 or a functionally active fragment or variant thereof.

10. The isolated nudeic acid of any one of daims 1 to 8 wherein said isolated nudeic add comprises a nucleotide sequence as set forth in SEQ ID NO: 23 or a functionally active fragment or variant thereof.

11. A nudeic add construct comprising the isolated nucleic acid of any one of claims 1 to 10.

12. The nudeic add construct of daim 11, wherein said isolated nucleic acid comprises a nudeotide sequence defining a transcriptional control sequence and said nudeic acid construct further comprises a nudeotide sequence of interest operably connected to said transcriptional control sequence.

13. The nucleic acid construct of claim 12, wherein the nucleotide sequence of interest is heterologous with respect to said transcriptional control sequence.

14. The nucleic acid construct of any one of claims 11 to 13 wherein the nucleic acid construct comprises an expression cassette comprising the structure:

( [N]w - TCS - [N]_* - SoI - [N]_y - TT - [N]z )

wherein: [N]w comprises one or more nucleotide residues, or is absent; TCS comprises a nucleic add of any one of claims 1 to 7; [N]_* comprises one or more nucleotide residues, or is absent;

SoI comprises a nucleotide sequence of interest which encodes an mRNA or non- translated RNA, wherein the nucleotide sequence, SoI, is operably connected to TCS; [N]_y comprises one or more nucleotide residues, or is absent;

TT comprises a nucleotide sequence defining a transcription terminator; [N]z comprises one or more nucleotide residues, or is absent.

15. A cell comprising: (i) an isolated nucleic add according to any one of claims 1 to 10;

(ii) the nudeic acid construct of any one of claims 11 to 14; and/or (iii) a genomically integrated form of the nudeic acid mentioned at (i) and/or the construct mentioned at (ii).

16. The cell of daim 15 wherein the cell is a plant cell.

17. The cell of daim 16 wherein the cell is a dicotyledonous plant cell.

18. The cell of daim 17 wherein the cell is an Arabiάopsis sp. cell.

19. The cell of claim 16 wherein the cell is a monocotyledonous plant cell.

20. The cell of claim 19 wherein the cell is a cereal crop plant cell.

21. The cell of any one of claims 15 to 20 wherein the cell is a plant gametophyte cell.

22. The cell of claim 21 wherein said plant gametophyte cell is a plant egg cell.

23. The cell of claim 21 wherein said plant gametophyte cell is a pollen grain cell or a pollen tube cell.

24. The cell of any one of claims 21 to 23 wherein the level, rate and/or pattern of expression of at least one nucleotide sequence is altered in said plant gametophyte cell relative to a wild type form of said plant gametophyte cell.

25. A multicellular structure comprising one or more cells of any one of claims 15 to 24.

26. The multicellular structure of claim 25 wherein the multicellular structure comprises a plant or a part, organ or tissue thereof.

27. The multicellular structure of claim 25 or 26 wherein said multicellular structure comprises one or more plant gametophyte cells of any one of claims 21 to 24.

28. A method for specifically or preferentially expressing a nucleotide sequence of interest in a plant gametophyte, the method comprising effecting transcription of the nucleotide sequence of interest in a plant under the transcriptional control of the nucleic acid of any one of claims 4 to 10.

29. The method of claim 28 wherein the nucleotide sequence of interest is heterologous with respect to said transcriptional control sequence.

30. A method for promoting sterility in a plant, the method comprising expressing a nucleotide sequence encoding a cytotoxic or cytostatic protein or a cytotoxic of cytostatic non-translated RNA, specifically or preferentially in a plant gametophyte; wherein said nucleotide sequence is operably connected to a nucleic add of any one of claims 4 to 10.

31. The method of claim 30 wherein the nucleotide sequence encodes a cytotoxic protein selected from the list consisting of a barnase, an RNAse or a diphtheria toxin.

32. A method for inhibiting or eliminating self-incompatibility in a plant, the method comprising expressing a nucleotide sequence which encodes an inhibitor of one or more self-incompatibility determinants in a plant gametophyte, wherein said nucleotide sequence which encodes an inhibitor of one or more self-incompatibility determinants is operably connected to nucleic acid of any one of claims 4 to 10.

33. The method of any one of claims 27 to 32 wherein the plant is a dicotyledonous plant.

34. The method of claim 33 wherein the plant is an Arabiάopsis sp. plant.

35. The method of any one of claims 27 to 32 wherein the cell is a monocotyledonous plant.

36. The method of claim 35 wherein the cell is a cereal crop plant.