WO1996031113A1

WO1996031113A1 - Plants with altered mitochondrial function

Info

Publication number: WO1996031113A1
Application number: PCT/AU1996/000194
Authority: WO
Inventors: Jian Wei Yu; Murray Badger; David Day; G. Dean Price; Jim Whelan
Original assignee: The Australian National University
Priority date: 1995-04-07
Filing date: 1996-04-04
Publication date: 1996-10-10
Also published as: AUPN225695A0

Abstract

The present invention relates generally to a process for the generation of male-sterile plants comprising the selective inhibition of the metabolism, functioning and/or biogenesis of plant mitochondria, by expressing in said plants a genetic construct which targets the expression of nuclear genes encoding polypeptide components of the mitochondrial respiratory electron transport chain. When expression of the genetic construct is controlled by a tissue-specific promoter, only the targeted tissue is affected by the disruption of mitochondrial function and other parts of the plant are normal. In particular, the present invention provides a method for the production of male-sterile plants, said method comprising the selective inhibition of mitochondrial function and/or biogenesis using genetic constructs which target the expression of nuclear genes encoding the mitochondrial Rieske iron-sulfur polypeptide or ATP synthase polypeptide in floral organs.

Description

PLANTS WITH ALTERED MITOCHONDRIAL FUNCTION

The present invention relates generally to a process for the selective inhibition of the metabolism, functioning and/or biogenesis of plant mitochondria, using a genetic construct which targets the expression of nuclear genes encoding polypeptide components of the mitochondrial respiratory electron transport chain. When expression of the genetic construct is controlled by a tissue-specific promoter, only the targeted tissue is affected by the disruption of mitochondrial function and other parts of the plant are normal. In particular, the present invention provides a method for the production of male -sterile plants, said method comprising the selective inhibition of mitochondrial function and/or biogenesis using genetic constructs which target the expression of nuclear genes encoding the mitochondrial Rieske iron-sulfur polypeptide and ATP synthase polypeptides in floral organs.

Throughout this specification and the claims which follow, 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 integer or group of integers but not the exclusion of any other integer or group of integers.

Bibliographic details of the publications referred to by author in this specification are collected at the end of the description.

Mitochondrial metabolism is essential for plant growth and development. Mitochondria are the major site of ATP synthesis in the cell and as such mitochondrial carbon metabolism and electron transport are vital processes. In addition to generating most of the ATP required by aerobic cells, mitochondria also have, to varying degrees, secondary functions related to tissue or organism needs. Most plant mitochondria have "alternative" electron transport chains which are non-phosphorylating and not subject to adenylate control. Mitochondria also have specific roles in other metabolic processes, such as photorespiration in C3 leaves, photosynthesis in some C4 and CAM plants, and fat metabolism of some germinating seeds (Douce and Neuburger, 1989). Mitochondrial biogenesis plays an important, but not yet clarified, role in flower development and also in fruit ripening. Genetic manipulation of mitochondrial function, using recombinant DNA technology, offers considerably scope of the improvement and control of plant growth, with applications in horticulture and agriculture.

Plant mitochondria possess a traditional TCA cycle which is supplemented by NAD-malic enzyme (all tissues) and glycine decarboxylase (photosynthetic tissues only). Malic enzyme (ME) allows plant mitochondria to utilise malate as a glycolytic alternative to pyruvate. and the TCA cycle to function anaplerotically (Hanson and Day, 1980). NADH and succinate produced by TCA cycle oxidations provide reductants to the respiratory electron transport chain (see Figure 1). The latter consists of the four major complexes found in mitochondria from other organisms and transfers electrons from NADH or succinate to oxygen via the cytochromes. The Rieske iron sulphur protein (RISP) is a core protein of complex III - the cytochrome b/c1 complex (Figure 1). This complex is not only an integral part of the electron transport pathway, it also plays an essential role in the import and processing of cytosolically-synthesised mitochondrial proteins (Moore et al., 1994). Concomitant proton translocation generates the required proton motive force to drive oxidative phosphorylation via the ATP synthase. In addition to these components, plants possess an alternative ubiquinol oxidase and non-proton pumping NAD(P)H dehydrogenases on both surfaces of the inner membrane. The function of these bypasses of the energy-conserving respiratory chain are not clear although a variety of roles have been proposed for the alternative oxidase (Mclntosh 1994).

Alteration of the concentration of respiratory components has the potential to alter cellular energy levels and ancillary metabolism, with some tissues likely to be more susceptible than others. The phenomenon of cytoplasmic male sterility highlights the important role of mitochondria in flower development in general and pollen production in particular (Hanson, 1991). Other studies of transcripts of nuclear encoded mitochondrial proteins have also indicated that mitochondrial function is important for floral development (Huang et al., 1994). Furthermore, antisense inhibition of citrate synthase gene expression results in aborted ovary development without apparent effect on vegetative plant growth (Landschutze et al., 1995). Expression of a mutated form of the mammalian β-subunit of the ATP synthase in plants can also lead to male sterility (International Patent Application WO 90/08831). However, although the prior art teaches the importance of mitochondria to the development of fertility in plants, until the present invention there has been no specific disclosure of a method of producing male-sterile plants by altering the expression of a specific mitochondrial electron transport chain component in the floral organs or tissues of said plant.

Accordingly, one aspect of the present invention provides a method of producing a male- sterile plant, said method comprising the steps of:

(i) introducing to a plant cell a genetic construct encoding an inhibitory molecule capable of inhibiting the expression of a nuclear gene encoding a mitochondrial polypeptide which functions normally as a component of the mitochondrial electron transport chain and is selected from the list comprising alternative oxidase polypeptide, Rieske iron-sulfur polypeptide or ATP synthase subunit polypeptide;

(ii) regenerating a whole plant from said plant cell; and

(iii) expressing said inhibitory molecule in the anthers, pollen, tapetum or other tissue required for male-fertility or for normal development of male floral structures.

According to this aspect of the invention, the term "inhibitory molecule" shall be taken to refer to an antisense, ribozyme or co-supression molecule which is capable of reducing, diminishing or otherwise lower ing the level of expression of a gene in a plant or alternatively, capable of reducing, delaying or otherwise diminishing the level of activation of expression of said gene or alternatively, delaying the activation of expression of said gene.

According to one embodiment of the invention, a nucleic acid molecule comprising a sequence of nucleotides complementary to the coding region of said nuclear gene or a part therof, is useful for the construction of an antisense or ribozyme molecule, which is capable of inhibiting the expression of said nuclear gene. By targeting an endogenous plant nuclear gene encoding a mitochondrial electron transport chain polypeptide, the expression of said nuclear gene is reduced, diminished or otherwise lowered, or the activation of expression of said nuclear gene is reduced, diminished or otherwise lowered, or the time taken for activation of expression of said nuclear gene is extended. By reducing expression, or reducing the level of activated expression, or extending the time taken to activate expression, of said nuclear gene, the ribozyme or antisense molecule of the present invention prevents the synthesis of a mitochondrial electron transport chain polypeptide which is required for mitochondrial metabolism, biogenesis or respiration. Preferably, the alteration to mitochondrial metabolism, biogenesis or respiration produces novel phenotypic traits in the plant carrying said ribozyme or antisense molecule, in particular male sterility.

In the context of the present invention, an antisense molecule is an RNA molecule which is transcribed from the complementary strand of a nuclear gene to that which is normally transcribed to produce a "sense" mRNA molecule capable of being translated into a polypeptide component of the mitochondrial electron transport chain. The antisense molecule is therefore complementary to the sense mRNA, or a part thereof. Although not limiting the mode of action of the antisense molecules of the present invention to any specific mechanism, the antisense RNA molecule possesses the capacity to form a double- stranded mRNA by base pairing with the sense mRNA, which may prevent translation of the sense mRNA and subsequent synthesis of a polypeptide gene product. In a particularly preferred embodiment, the antisense or ribozyme molecule of the present invention is able to reduce or inhibit the expression of a plant gene which encodes the Rieske iron-sulfur polypeptide or the ATP synthase subunit polypeptide. F1-ATPβ.

Those skilled in the relevant art will be aware that antisense molecules may comprise a sequence of nucleotides which is complementary to a sequence selected from any region of an mRNA encoding a polypeptide, the only requirement being that said antisense molecule is sufficiently long to hybridise to said mRNA molecule.

Preferred antisense molecules should be capable of hybridising to at least about 10 to 20 nucleotides of the targetsense mRNA molecule, however the present invention extends to molecules capable of hybridising to a target molecule comprising at least about 50-100 nucleotide bases in length, or a molecule capable of hybridising to a full-length or substantially full-length mRNA target molecule encoding a mitochondrial electron transport chain polypeptide selected from the list comprising the Rieske iron-sulfur polypeptide, alternative oxidase polypeptide or ATP synthase subunit polypeptide, amongst others.

In a more particularly preferred embodiment, the antisense molecule is capable of hybridising to a substantially full-length mRNA molecule encoding the Rieske iron- sulfur polypeptide or the F1-ATPβ polypeptide.

In a most particularly preferred embodiment, the present invention provides an antisense molecule comprising at least 20 nucleotides in length contained within any one or more of the sequences set forth in SEQ ID NO: 1, SEQ ID NO:2 or SEQ ID NO:3 or at least 80% identical thereto.

For the purposes of nomenclature, the nucleotide sequence set forth in SEQ ID NO: 1 is the complement of the nucleotide sequence containing the open reading frame of the tobacco Rieske iron-sulfur polypeptide RISP1. The construction of a genetic construct which expresses an antisense molecule comprising the nucleotide sequence set forth in SEQ ID NO: 1, is described in the non-limiting Example 4 of the specification. The nucleotide sequence set forth in SEQ ID NO: 2 is the complement of a nucleotide sequence encoding a partial tobacco F1-ATPβ polypeptide of the mitochondrial ATP synthase. The construction of a genetic construct which expresses an antisense molecule comprising the nucleotide sequence set forth in SEQ ID NO:2 is described in non- limiting Example 11. The nucleotide sequence set forth in SEQ ID NO: 3 is the complement of a nucleotide sequence encoding the full-length tobacco F1-ATPβ polypeptide of the mitochondrial ATP synthase. Cloning of the cDNA encoding the full- length tobacco F1-ATPβ polypeptide of the mitochondrial ATP synthase is described by Chua and Boutry (1985).

Ribozymes are synthetic RNA molecules which comprise a hybridising region complementary to two regions, each of at least 5 contiguous nucleotide bases in the target sense mRNA. In addition, ribozymes possess highly specific endoribonuclease activity, which autocatalytically cleaves the target sense mRNA. A complete description of the function of ribozymes is presented by Haseloff and Gerlach (1988) and contained in International Patent Application No. WO89/05852. The present invention extends to ribozymes which target a sense mRNA encoding a plant mitochondrial electron transport chain polypeptide, thereby hybridising to said sense mRNA and cleaving it, such that it is no longer capable of being translated to synthesise a functional polypeptide product.

Preferably, the ribozyme molecule of the present invention targets a sense mRNA encoding a polypeptide selected from the list of mitochondrial electron transport chain and energy transduction system components including, but not limited to the alternative oxidase polypeptide, Rieske iron-sulfur polypeptide or ATP synthase subunit polypeptide, amongst others. More preferably, the ribozyme molecule of the invention targets the tobacco Rieske iron-sulfur polypeptide RISP1 or the tobacco ATP synthase polypeptide F1-ATPβ. Those skilled in the relevant art will be aware that, in accordance with the present invention, the hybridising region of a ribozyme molecule must comprise two regions. each of which in turn comprises a sequence of nucleotides complementary to an mRNA encoding a mitochondrial electron transport chain polypeptide selected from the list comprising alternative oxidase polypeptide, Rieske iron-sulfur polypeptide or ATP synthase subunit polypeptide, the only requirement being that each of said two regions is sufficiently long to hybridise to said mRNA molecule.

The present invention contemplates the use of a genetic construct which expresses a ribozyme molecule in which the hybridising region comprises two regions of at least 5 nucleotides in length derived from the sequence of nucleotides set forth in SEQ ID NO: 1 or SEQ ID NO:2 or SEQ ID NO:3 or is at least 80% identical thereto.

Those skilled in the art will be aware that a suitable length for a hybridising region in a ribozyme molecule will vary depending on the mRNA species to which it hybridises and the hybridisation conditions employed and is determined empirically. Accordingly, although a hybridising region comprising two regions of 5 contiguous nucleotides may be sufficient to hybridise to an mRNA molecule, the invention extends to the use of longer hybridising regions which may be more effective in hybridising to particular regions of mRNA. Accordingly, the present invention further contemplates ribozyme molecules in which the hybridising region comprises two regions of at least 10 nucleotides in length derived from the sequence of nucleotides set forth in SEQ ID NO: 1 or SEQ ID NO:2 or SEQ ID NO:3 or at least 80% identical thereto.

According to the foregoing embodiments, the present invention extends to any genetic constructs capable of expressing antisense or ribozyme molecules which may form a hydrogen-bonded complex with a sense mRNA molecule encoding an alternative oxidase polypeptide, Rieske iron-sulfur polypeptide or ATP synthase subunit polypeptide, amongst others. It is understood in the art that certain modifications, including nucleotide substitutions amongst others, may be made to the antisense and/or ribozyme molecules of the present invention without destroying the efficacy of said molecules in inhibiting the expression of a nuclear gene encoding a mitochondrial electron transport chain polypeptide component. It is therefore within the scope of the present invention to include any nucleotide sequence variants, homologues, analogues, or fragments of a nuclear gene encoding a mitochondrial electron transport chain polypeptide, the only requirement being that said nucleotide sequence variant, when transcribed, produces an antisense or ribozyme molecule which is capable of hybridising to the said sense mRNA molecule.

Accordingly, the present invention is directed to an antisense or ribozyme molecule which comprises a sequence of nucleotides complementary to, or preferably, having at least 40% similarity to all or a part thereof, a sense mRNA molecule encoding a mitochondrial electron transport chain polypeptide, for example alternative oxidase polypeptide, Rieske iron-sulfur polypeptide or ATP synthase subunit polypeptide, amongst others.

More preferably, the percentage similarity is at least 60-65 %, still more preferably, the percentage similarity is at least 70-75%. Yet, still more preferably, the percentage similarity is at least 80-90%, including at least 91 % or 93% or 95% similarity to any one or more of said sense mRNA molecules.

In a particularly preferred embodiment, the present invention extends to antisense or ribozyme molecules comprising a sequence of nucleotides which are complementary to, or at least 40% similar to the sense mRNA encoding the Rieske iron sulfur polypeptide, or a variant, homologue, analogue or fragment thereof.

A related embodiment of the present invention contemplates a ribozyme and/or antisense molecule comprising a sequence of nucleotide capable of hybridising under at least low stringency conditions to a nucleic acid molecule encoding, or complementary to a nucleic acid molecule encoding a polypeptide component of the mitochondrial electron transport chain, for example alternative oxidase polypeptide, Rieske iron-sulfur polypeptide or ATP synthase polypeptide, amongst others. For the purposes of defining the level of stringency, a low stringency is defined herein as being a hybridisation and/or a wash carried out in 6xSSC buffer, 0.1 % (w/v) SDS at 28ºC. Generally, the stringency is increased by reducing the concentration of SSC buffer, and/or increasing the concentration of SDS and/or increasing the temperature of the hybridisation and/or wash.

In an alternative embodiment, the present invention provides a method of producing a male-sterile plant, said method comprising inhibition of the expression of a nuclear gene encoding a mitochondrial polypeptide which functions normally as a component of the mitochondrial electron transport chain, wherein said inhibition is achieved using a co- supression molecule.

Co-suppression is the reduction in expression of an endogenous gene that occurs when one or more copies of said gene, or one or more copies of a substantially similar gene are introduced into the cell. The present invention also extends to the use of co- suppression to inhibit the expression of a nuclear gene which encodes a mitochondrial electron transport chain polypeptide.

Preferably, the nuclear gene which is targeted by a co-suppression molecule in the present invention is selected from the list of mitochondrial electron transport chain and energy transduction system genes encoding the alternative oxidase polypeptide, Rieske iron-sulfur polypeptide and ATP synthase subunit , amongst others.

The co-supression molecules of the present invention generally comprise a sequence of nucleotides which encode a functional alternative oxidase polypeptide, Rieske iron-sulfur polypeptide or ATP synthase subunit polypeptide. Although said co-supression molecules generally comprise an entire open-reading frame of the subject genetic sequence, those skilled in the art will appreciate that this may not always be essential in order to obtain a functional polypeptide product, following transcription and translation of said genetic sequence. In a preferred embodiment, the present invention extends to co-supression molecules which comprise a sequence of nucleotides containing the complete open reading frame of a Rieske iron-sulfur polypeptide or an ATP synthase subunit polypeptide. More preferably, said co-supression molecule comprises a sequence of nucleotides substantially as set forth in SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO: 8 or a variant, homologue, analogue or derivative thereof.

Wherein the co-supression molecule of the invention comprises a sequence of nucleotides substantially the same as the sequence set forth in SEQ ID NO:4 or a variant, homologue, analogue or derivative thereof, said co-supression molecule will encode the Rieske iron-sulfur polypeptide set forth in SEQ ID NO:5 or a variant, homologue, analogue or derivative thereof.

Wherein the co-supression molecule of the invention comprises a sequence of nucleotides substantially the same as the sequence set forth in SEQ ID NO:6 or a variant, homologue, analogue or derivative thereof, said co-supression molecule will encode the partial F1-ATPβ polypeptide set forth in SEQ ID NO:7 or a variant, homologue, analogue or derivative thereof. Wherein the co-supression molecule of the invention comprises a sequence of nucleotides substantially the same as the sequence set forth in SEQ ID NO: 8 or a variant, homologue, analogue or derivative thereof, said co-supression molecule will encode the full-length F1-ATPβ polypeptide set forth in SEQ ID NO:9 or a variant, homologue, analogue or derivative thereof.

The term "variant" is used to describe a variant genetic sequence which contains nucleotide substitutions, insertions or deletions which may alter the amino acid sequence of me polypeptide product of said gene, or the expression of said gene in terms of liming, quantity, or quality. Thus, the term "variant" may also be used in the art to describe a variant polypeptide product of a gene which differs by one or more amino acid residues but which is substantially similar to another polypeptide, or has a similar catalytic activity as another polypeptide. Allelic variants may be derived by mutagenesis, gene duplication, unequal crossing-over, and so on. Generally, a derived allelic variant gene contains single or multiple nucleotide substitutions, deletions and or /additions. Nucleotide insertional derivatives of the allelic variant of the present invention include 5' and 3' terminal fusions as well as intra-sequence insertions of single or multiple nucleotides. Insertional nucleotide sequence variants are those in which one or more nucleotides are introduced into a predetermined site in the nucleotide sequence although random insertion is also possible with suitable screening of the resulting product. Deletional variants are characterised by the removal of one or more nucleotides from the sequence. Substitutional nucleotide variants are those in which at least one nucleotide in the sequence has been removed and a different nucleotide inserted in its place. Derivative variant genes may or may not encode derivative variant polypeptides. For example, a substitution may be "silent" in that the substitution does not change the amino acid defined by the codon. Alternatively, nucleotide substituents in a gene may alter one or more amino acid residues encoded therein, changing the amino acid sequence of a polypeptide.

The term "analogue" as used herein shall be taken to refer to a variant genetic sequence or a variant polypeptide which is functionally equivalent to the genetic sequence or polypeptide respectively, of the present invention, but which contains certain non- naturally occurring or modified residues.

The term "homologue" as used herein, in relation to a variant genetic sequence, refers to a gene which encodes a polypeptide which retains its function, although it may contain amino acid substitutions, deletions and/or additions. The term "homologues" in relation to a variant polypeptide refers to a polypeptide containing amino acid substitutions, amino acid deletions and/or amino acid additions without altering its function as a polypeptide influencing mitochondrial function. Furthermore, amino acids may be replaced by other amino acids having similar properties, for example hydrophobicity, hydrophilicity, hydrophobic moment or antigenicity, and so on. Amino acid substitutions are typically of single residues, but may be clustered depending upon functional constraints placed upon the polypeptide; insertions will usually be of the order of about 1-10 amino acid residues; and deletions will range from about 1-20 residues. Deletions or insertions preferably are made in adjacent pairs, i.e: a deletion of 2 residues or insertion of 2 residues.

Amino acid alterations to the peptides contemplated herein include insertions such as amino acid and/or carboxyl terminal fusions as well as intra-sequence insertions of single or multiple amino acids. Generally, insertions within the amino acid sequence will be smaller than amino or carboxyl terminal fusions, of the order of about 1 to 4 residues. Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in the protein. Deletional variants are characterised by the removal of one or more amino acids from the sequence. Substitutional variants are those in which at least one residue in the sequence has been removed and a different residue inserted in its place. Such substitutions may be made in accordance with Table 1.

The amino acid variants referred to in Table 1 may readily be made using peptide synthetic techniques well known in the art, such as solid phase peptide synthesis and the like, or by recombinant DNA manipulations. Techniques for making substitution mutations at predetermined sites in DNA having known sequence are well known, for example through M13 mutagenesis. The manipulation of DNA sequences to produce variant polypeptides which manifest as substitutional, insertional or deletional variants are well known in the art.

Other examples of variant polypeptides contemplated by the present invention include single or multiple substitutions, deletions and/or additions to any molecule associated with a ligand such as a carbohydrate, lipid and/or peptide, polypeptide or protein moiety.

A further aspect of the present invention provides a genetic construct which, when expressed, reduces the expression of a nuclear gene encoding a mitochondrial electron transport chain component. The genetic constructs of the present invention are useful in the generation of transgenic plants with novel useful phenotypic traits selected from the list including male sterility, loss of apical dominance, dwarfing and reduced senescence and aging, amongst others.

The genetic construct of the present invention comprises the foregoing antisense, or ribozyme, or co-suppression nucleic acid molecule encoding or complementary to a nucleic acid molecule encoding a mitochondrial electron transport chain component polypeptide, placed operably under the control of a promoter sequence capable of regulating the expression of the said antisense nucleic acid molecule in a eukaryotic cell, preferably a plant cell. The said genetic construct optionally comprises, in addition to a promoter and antisense, or ribozyme, or co-suppression nucleic acid molecule, a terminator sequence.

In one embodiment, the invention provides a genetic construct comprising a sequence of nucleotides encoding an antisense molecule capable of hybridising under at least low stringency conditions to mRNA encoding a mitochondrial electron transport chain polypeptide selected from the list comprising alternative oxidase polypeptide, Rieske iron-sulfur polypeptide or ATP synthase polypeptide wherein the expression of said antisense molecule under the control of a promoter capable of regulating or directing gene expression in a plant cell and wherein said sequence of nucleotides encoding said antisense molecule is further placed upstream of a transcription termination sequence.

In an alternative embodiment, the present invention provides a genetic construct comprising a first sequence of nucleotides encoding a ribozyme molecule which is capable of hybridising to and cleaving mRNA encoding a mitochondrial electron transport chain polypeptide selected from the list comprising alternative oxidase polypeptide, Rieske iron-sulfur polypeptide or ATP synthase polypeptide, wherein said first sequence of nucleotides is placed operably under the control of a promoter capable of regulating or directing gene expression in a plant cell and wherein said first sequence of nucleotides is further placed upstream of a transcription termination sequence.

In a further alternative embodiment, the present invention provides a genetic construct comprising a first sequence of nucleotides encoding a co-supression molecule which when expressed is capable of inhibiting the expression of a nuclear gene encoding a mitochondrial electron transport chain polypeptide selected from the list comprising alternative oxidase polypeptide, Rieske iron-sulfur polypeptide or ATP synthase polypeptide, wherein said first sequence of nucleotides is placed operably under the control of a promoter capable of regulating or directing gene expression in a plant cell and wherein said first sequence of nucleotides is further placed upstream of a transcription termination sequence.

The term "terminator" or "transcription termination sequence" or similar term refers to a DNA sequence at the end of a transcriptional unit which signals termination of transcription. Terminators are DNA 3 '-non-translated sequences that contain a polyadenylation signal, that causes the addition of polyadenylate sequences to the 3 '-end of a primary transcript. Terminators active in plant cells are known and described in the literature. They may be isolated from bacteria, fungi, viruses, animals and/or plants. Examples of terminators particularly suitable for use in the genetic constructs of the invention include the nopaline synthase terminator of A. tumefaciens, the 35 S terminator of CaMV and the zein terminator from Zea mays.

Reference herein to a "promoter" is to be taken in its broadest context and includes the transcriptional regulatory sequences of a classical genomic gene, including the TATA box which is required for accurate transcription initiation, with or without a CCAAT box sequence and additional regulatory elements (i.e. upstream activating sequences, enhancers and silencers) which alter gene expression in response to developmental and/or environmental stimuli, or in a tissue-specific manner. A promoter is usually, but not necessarily, positioned upstream or 5', of a structural gene, the expression of which it regulates. Furthermore, the regulatory elements comprising a promoter are usually positioned within 2 kb of the start site of transcription of the gene.

In the present context, the term "promoter" is also used to describe a synthetic or fusion molecule, or derivative which confers, activates or enhances expression of said antisense, or ribozyme, or co-suppression nucleic acid molecule, in a plant cell. Preferred promoters may contain additional copies of one or more specific regulatory elements, to further enhance expression of the antisense or ribozyme or co-suppression molecule and/or to alter the spatial expression and/or temporal expression of said antisense or ribozyme or co-suppression molecule. For example, regulatory elements which confer copper inducibility may be placed adjacent to a heterologous promoter sequence driving expression of an antisense, or ribozyme, or co-suppression molecule, thereby conferring copper inducibility on the expression of said antisense, or ribozyme, or co-suppression molecule. Placing a ribozyme or antisense or co-suppression molecule under the regulatory control of a promoter sequence means positioning the said ribozyme or antisense or co- suppression molecule such that expression is controlled by the promoter sequence. Promoters are generally positioned 5' (upstream) to the genes that they control. In the construction of heterologous promoter/structural gene combinations it is generally preferred to position the promoter at a distance from the gene transcription start site that is approximately the same as the distance between that promoter and the gene it controls in its natural setting, i.e., the gene from which the promoter is derived. As is known in the art, some variation in this distance can be accommodated without loss of promoter function. Similarly, the preferred positioning of a regulatory sequence element with respect to a heterologous gene to be placed under its control is defined by the positioning of the element in its natural setting, i.e., the genes from which it is derived. Again, as is known in the art, some variation in this distance can also occur.

Examples of promoters suitable for use in genetic constructs of the present invention include viral, fungal, bacterial, animal and plant derived promoters capable of functioning in plant cells. The promoter may regulate the expression of the ribozyme or antisense or co-suppression molecule constitutively, or differentially with respect to the tissue in which expression occurs or, with respect to the developmental stage at which expression occurs, or in response to environmental stimuli such as stress or disease. Preferably, the promoter is capable of regulating expression of a ribozyme or antisense molecule, or a co-suppression molecule, in a plant cell. Particularly preferred promoters include the cauliflower mosaic virus 35S (CaMV 35S) promoter, derivatives thereof, and as promoter inducible after wounding by a disease carrier such as thrips, e.g. a wound inducible promoter. Examples of further suitable promoters include nopaline synthase, octopine synthase and the like. Those skilled in the relevant art will be aware that promoter sequences such as the CaMV 35S promoter, nopaline synthase promoter or octopine synthase promoter are capable of directing the expression of an antisense, ribozyme or co-supression molecule in several different plant cell tissues or organs and not restricted to expression in floral organs. Accordingly, wherein the present invention utilises such promoter sequences, the transgenic plants thus generated exhibit a wide range of novel phentypic traits in the vegetative organs, icluding but not limited to, a loss of apicqal dominance, dwarfing, or reduced senescence, amongst others.

In a particularly preferred embodiment, the invention provides a genetic construct comprising:

(i) a promoter sequence operable in the tapetum, anther , pollen or other plant tissue required for male-fertility;

(ii) a first sequence of nucleotides encoding an antisense molecule which when expressed is capable of hybridising under at least low stringency conditions to mRNA encoding a mitochondrial electron transport chain polypeptide selected from the list comprising alternative oxidase polypeptide, Rieske iron-sulfur polypeptide or ATP synthase polypeptide; and

(iii) a transcription termination sequence,

wherein said promoter sequence, first sequence of nucleotides encoding me antisense molecule and transcription termination sequence are placed operably in relation to each other such that said antisense molecule is capable of being expressed under the control of said promoter.

In a related preferred embodiment, the first sequence of nucleotides encodes a ribozyme molecule which is capable of hybridising to and cleaving mRNA encoding a mitochondrial electron transport chain polypeptide selected from the list comprising alternative oxidase polypeptide. Rieske iron-sulfur polypeptide or ATP synthase polypeptide.

In a further related embodiment, the first sequence of nucleotides encodes a co-supression molecule which is capable of inhibiting the expression of a nuclear gene encoding a mitochondrial electron transport chain polypeptide selected from the list comprising alternative oxidase polypeptide, Rieske iron-sulfur polypeptide or ATP synthase polypeptide.

Wherein said promoter sequence is operable in the tapetum, anther, pollen or other male- floral structure required for the development of male fertility, the genetic construct of the present invention targets the expression of a nuclear gene encoding a mitochondrial electron transport chain polypeptide in floral structures, producing a male sterile plant. To generate male sterile plants for hybrid seed production, expression of the antisense or ribozyme or co-suppression molecule is best controlled by male sporophyte-specific promoter, eg a tapetal specific promoter. Particularly preferred promoters suitable for this purpose include, but are not limited to the tapetum-specific A9 promoter sequence (Wyatt Paul et al. , 1992).

In a more particularly preferred embodiment, the genetic construct comprises either the antisense mitochondrial RISP molecule set forth in SEQ ID NO:1 or the antisense F1- ATPβ molecules set forth in SEQ ID NO: 2 or SEQ ID NO: 3 or a part, homologue, analogue or fragment thereof, placed operably under the control of the A9 promoter sequence and introduced into tobacco plants such that said plant develop floral structures which are male sterile. Utilisation of antisense molecules comprising SEQ ID NO: 1 or SEQ ID NO:2 is exemplified herein.

In accordance with this embodiment of the present invention, a mechanism of restoring fertility to said male sterile plants is also contemplated, wherein male fertility is restored by introducing a heterologous gene from other plant species that is not inhibited by the antisense or ribozyme or co-suppression genetic construct, yet is capable of expressing a functional gene product which complements the function inhibited by said antisense or ribozyme or co-suppression molecule. A still further aspect of the present invention extends to a transgenic plant such as a crop plant, carrying the foregoing antisense or ribozyme or co-suppression molecule and/or genetic constructs comprising the same. Preferably, the transgenic plant is one or more of the following: wheat, barley, rice, canola, cotton, tomato, or potato, amongst others. More preferably, the transgenic plant is tobacco. Additional species are not excluded.

The recombinant DNA molecule carrying the antisense or ribozyme or co-suppression molecule and/or genetic construct comprising the same, may be introduced into plant tissue, thereby producing a "transgenic plant", by various techniques known to those skilled in the art. The technique used for a given plant species or specific type of plant tissue depends on the known successful techniques. Means for introducing recombinant DNA into plant tissue include, but are not limited to, transformation (Paszkowski et al., 1984), electroporation (Fromm et al. , 1985), or microinjection of the DNA (Crossway et al. , 1986), or T-DNA-mediated transfer from Agrobacterium to the plant tissue. Representative T-DNA vector systems are described in the following references: An et al. (1985); Herrera-Estrella et al. (1983a,b); Herrera-Estrella et al. (1985). Once introduced into the plant tissue, the expression of the introduced gene may be assayed in a transient expression system, or it may be determined after selection for stable integration within the plant genome. Techniques are known for the in vitro culture of plant tissue, and in a number of cases, for regeneration into whole plants. Procedures for transferring the introduced gene from the originally transformed plant into commercially useful cultivars are known to those skilled in the art. The present invention further extends to the progeny of said transgenic plant.

The present invention is further described by reference to the following non-limiting Figures and Examples.

In the Figures:

Figure 1 is a schematic representation of the mitochondrial electron transport chain, showing polypeptide components contained therein.

Figure 2 is a photographic representation showing abnormal phenotypes of flowers in antisense RISP mutants. A, B, wild type: C, D, E, antisense mutants. Figure 3 is a photographic representation of a Northern blot showing RISP gene expression in tobacco flowers. Total RNA (20 μg) was used in the blotting. A, double stranded RISP1 cDNA was used for probe preparation. W, whole flowers; V, vegetative parts (sepals and petals); S, stamen (anthers and filaments); P, pistil (stigma, style and ovary). B, single stranded sense RISP1 RNA was used as the probe. Lane 1 to 5 represents total RNA from whole flowers of control, mutant 2FC30C, Tl of 2FC30C, 1FC25.2A, 1FC25.2B, and from the pistil of 1FC25.2A, respectively.

Figure 4 is a photographic representation showing flower development in wild-type and antisense RISP mutant of tobacco plants. The same flower at each stage was dissected to show the development of stamen and pistil.

Figure 5 is a photographic representation showing pollen viability of tobacco plants. Pollens were taken from stage 12 flowers. A. control; B, 1FC25.2A; C, 2FC30C; D, Tl of 2FC30C. Same magnification was used.

Figure 6 is a photographic representation showing plant growth and leaf cell morphology of wild-type and antisense RISP tobacco plants. A, 3 months old plant with wild type on the left and T1 of 2FC30C on the right; B, 7 months old T1 of 2FC30C; C and E, scanning micrographs of A; D and F, scanning micrographs of B. bar = 200 μm Figure 7 is a graphical representation showing specific fresh and dry weight and water content of antisense RISP mutants (T1). Plants were grown under optimal conditions in the glasshouse. Closed symbol, wild type; open symbols, mutants.

Figure 8 is a graphical representation showing the rate of respiratory CO₂ evolution from leaves. The rate was determined by a IRGA in a closed gas exchange system at 25°. Closed symbol, wild type; open symbols, mutants.

Figure 9 is a photographic representation of a Western blot showing RISP level in isolated mitochondria from leaves. One hundred μg mitochondrial protein was loaded per lane and separated by SDS-PAGE and RISP was subsequently detected by ECL technique (Amersham).

Figure 10 is a schematic representation of the binary plasmid pA9-RISP comprising the tapetum-specific A9 promoter operably linked to the antisense RISP1 genetic sequence set forth in SEQ ID NO: 1 and nopaline synthase terminator sequence. The genetic construct further comprises the nptll kanamycin-resistance selection marker gene sequence and left and right T-DNA border sequences for integration into the DNA of a host plant.

EXAMPLE 1

Plant Materials Tobacco (Nicotiana tabacum L. cv Wisconsin38) plants were grown under glasshouse conditions (peak irradiance around 800 mmols quanta m^-2 s^{- 1}; temperature: 25ºC day/20° night) in pots containing vermiculite and fertilised with a complete nutrient solution twice a week.

EXAMPLE 2

Isolation of Mitochondria and Assay of Mitochondrial Activity

Mitochondria were purified from tobacco leaves, and assayed using an oxygen electrode, as described by Day et al. (1985).

EXAMPLE 3

Gas Exchange

Gas exchange measurements of the rate of CO₂ evolution were made on attached leaves in a closed gas exchange system. The system was fitted with an aluminium leaf chamber with a glass window of 84 cm². Temperature control was provided by a waterjacket and aided by a circulating fan inside the chamber. An IR gas analyser (UNOR, Maihak) was used to measure the absolute partial pressure of CO₂ in the airstream circulating by a metal bellows pump (Metal Bellows Corp. Mass.). Respiratory CO₂ evolution rate was calculated from the rate of CO₂ increase at air levels of CO₂. All plant materials were left in the dark for 30 min before measurement to avoid photorespiratory CO₂ evolution and effects of previous photosynthesis on the respiratory rates. For measurements of CO₂ evolution from flowers, the leaf chamber was replaced with a 10 ml syringe and the air stream was circulated by the pump from one end to the other in the same enclosed system. All measurements were made at a temperature of 25°C in the dark. Leaf CO₂ compensation points were determined in the same system with the leaf chamber at an irradiance of 500 mmoles quanta m^-2s^-1 and the post-illumination burst was measured from the rate of CO₂ increase immediately after the light was turned off. EXAMPLE 4

Engineering of an Antisense Construct against the Mitochondrial

Rieske Iron-Sulfur Protein (RISP) Gene

A PCR DNA product for the tobacco mitochondrial Rieske iron-sulfur protein (RISP) was obtained by PCR after reverse transcription of total RNA isolated from tobacco leaves. The primers were designed from a published RISP1 sequence (Huang et al., 1994) and have the sequences as follows: 5'-GGCCCGTGAGCTCCGCCGC-3' (5' primer) incorporating a Sac I site and 5'-TTACCTACTTAATCTAGAAGTAACTTG-3' (3' primer) incorporating an Xba I site just after the stop codon. The 0.77 kb PCR fragment, containing essentially the whole coding region sequence of RISP1, was digested with Sac I and Xba I and ligated into pTZ19R. It was subsequently sequenced and verified as a RISP1 sequence. The 0.75 kb Sac l-Xba I fragment was then cloned direcdy into the Sac l-Xba I sites of the binary vector pBI121 (Jefferson et al., 1987) so as to replace the β-glucuronidase gene. The resulting binary plasmid, pBI-RISP, was 11.87 kb in size and contained the antisense RISP1 gene driven by the cauliflower mosaic virus (CaMV) 35S promoter and the NPT II gene conferring kanamycin resistance. The pBI-RISP binary plasmid was then introduced into Agrobacterium tumefaciens (strain LBA4404) by electroporation and used to transform tobacco leaf disks as previously described (Price et al., 1994). Unless otherwise stated, all procedures for manipulating recombinant DNA were carried out by the standardised procedures described in Sambrook et al., (1989). EXAMPLE 5

Antisense RISP Mutants were Identified by

Abnormalities in F1owering After transformation with the antisense RISP1 construct, the resulting kanamycin resistant callus tissues grew much more slowly that those with antisense against proteins involved in photosynthesis, which were transformed at the same time. After a lengthy tissue culture period, a total of 57 separated callus tissues were obtained. Each of these generated 3 to 4 plantlets for transplantation to soil. Leaf morphology of some transgenic plants was altered, but it was not certain at that stage if it was an effect of tissue culture. However, when plants were grown dirough to flowering, differences became obvious among plants. One plant produced curved flowers and 11 others had abnormal flowers which did not set seeds in the normal fashion. Analyses were then focused on these primary transformants and their T1 progeny (obtained by hand-self-fertilisation or cross-pollination with wt pollen). Examples of the difference in flower morphologies detected in the transgenic plants are shown in Figure 2.

EXAMPLE 6

Abnormal Flowers have Reduced Levels of RISP Message and

Contain the Antisense Transcript

Total RNA was isolated from tobacco leaves, whole flowers, and dissected parts of flowers frozen in liquid nitrogen, essentially as previously described (Yu et al., 1992). All DNA probes were ³²P-labelled according to the instruction of the manufacturer (Megaprime DNA labelling systems, Amersham) and blotted as previously described (Yu et al. , 1992). Single-stranded RNA probe, however, was generated using the DIG RNA labelling kit (Boehringer Mannheim) and the subsequent blotting and detection followed the protocol of the DIG nucleic acid detection kit (Boehringer Mannheim).

To establish whether the abnormalities in the flowers of some mutants were associated with the expression of the antisense RISP1 gene, we examined the steady-state mRNA transcript levels in the whole flowers and different parts of the flowers. F1owers at developmental stage 2 (Koltunow et al., 1990). either as whole flowers or dissected into vegetative, stamen and pistil parts, were used for isolation of total RNA. Similar to wt tobacco plants (Huang et al., 1994). flowers of the transgenic control have only a single but diffuse band of RISP transcript at about 1.25 kb (Figure 3A). Among the different parts of the flower, the stamen (anthers and filaments) contained the highest steady state level of RISP transcript, representing about 60% of the total RISP transcripts in the flower. The pistil (stigma, style and ovary) and the remaining vegetative parts (sepals and petals) represented only about 25 and 15%. respectively of transcript levels. The high steady state transcript level in the stamen may indicate that more mitochondria are present in this part of the flower (see Huang et al., 1994) and may be associated with the that energy demand during anther development.

The steady state level of the endogenous 1.25 kb mitochondrial RISP transcript was dramatically reduced in flowers of the antisense mutants, ranging from 61 to 93% when compared with the counterparts of the control (Figure 3A). Associated with the reduced steady state level of the endogenous 1.2 kb transcript, however, was an additional transcript of about 0.9 kb in the mutants. This occurred when double-stranded DNA was used as the template for probe preparation thus detecting both the sense and antisense transcripts. To determine whether the 0.9 kb transcript was a result of the expression of an induced RISP gene in response to the reduction in the 1.25 kb wt message, rather than simply the expression of the antisense gene itself, we prepared a single-stranded sense RNA as the probe by in vitro transcription of RISP1. Only a single band of about 0.9 kb hybridised to the sense probe in the mutant samples but not in the control, thus confirming that this 0.9 kb transcript was the antisense transcript expressed from the engineered construct (Figure 3B). The majority of this 0.9 kb antisense mRNA was found in the female part of the flower and only low levels were detected in the vegetative and male parts. The reason for the failure of excess antisense RISP1 transcript to totally suppress the endogenous sense transcript is not clear yet but may be related to the fact that there are other respiratory RISP genes expressed in the flower (Huang et al., 1994). The result, however, clearly demonstrates that the mutants with abnormal flowers did indeed express the antisense RISPl gene, reducing the steady state transcript level of the endogenous genes. EXAMPLE 7

Antisense Suppression of Mitochondrial RISP Gene Expression

Causes Male Sterility

The stain developed by Alexander (1969) was used to test the viability of pollen. At least 500 pollen grains per plant were scored for viability.

The transgenic tobacco plants carrying the antisense RISP genetic construct were male- sterile. All mutant flowers tested set seeds when hand pollinated, with viable pollens. Without manual pollination, some mutants could not set seed and the flowers eventually senesced.

Two major defects in the male part of the mutant flowers may have contributed to male sterility. These are shortened filaments and reduced numbers and viability of pollen grains. In the majority of mutant flowers, the filaments were substantially shorter than those of the control and consequendy their anthers were below the stigma. The situation developed predominandy in the early stages of the flower development (Figure 4). Thus any viable pollen grains from these anthers would have difficulty in reaching the stigma for fertilisation. When pollen grains were strained (Alexander 1969), more than 90% of the pollen in the control were found to be viable but only 40 to 50% of mutant pollen was viable (Figure 5). Some primary transformants may show normal levels of pollen viability (Figure 5C) but some of their T1 segregants had a significant increase in the presence of aborted pollen (Figure 5D) which was often accompanied by short filaments. In these short filament flowers, the total number of pollen grains produced was also less.

It should be noted that, because of the constitutive expression of the antisense gene under control of the 35S promoter sequence in these plants, more severely inhibited ones have taken longer than 7 months after germination to flower. Some of the seeds T1 progeny were so inhibited that they could not germinate properly. So the potential impact on flower and/or male sterility of antisense suppression of mitochondrial RISP cannot be seen in the mutants we have generated here. Without being bound by any theoretical consideration, it is reasonable to surmise that complete male sterility would result if the same antisense gene is expressed specifically in the anther or tapetal tissue using an active tissue specific promoter.

EXAMPLE 8

Antisense Suppression of Mitochondrial RISP Affects

Plant Growth and Cell Development

The antisense plants not only experienced difficulty in flowering, but their growth was also dramatically retarded (Figure 6A). The severely inhibited plants lost their apical dominance and produced many secondary shoots (Figure 6B). Their leaves were thicker, elongated but were slower to senesce (judged as chlorophyll loss).

Cell morphology was further examined under the electron microscope. The epidermal cells of mutant leaves were as much as 5 times larger than those of the control (compare Figure 6C and 6D). In addition, there was a reduced density of stomata in the mutants. From leaf cross sections it was apparent that the mutant mesophyll cells had not clearly differentiated into palisade and spongy mesophyll cells as in the control (Figure 6E and Figure 6F). The mutant leaf was also much thicker (a 2.5 -fold lower magnification had been used for the mutant so that the hairs could be seen). This increased thickness primarily resulted from the enlargement of mesophyll cells, and the number of cell layers across the leaf was not significantly changed (Figure 6F). This result suggests that the total number of cells in the mutant leaves had been dramatically reduced.

This reduction in cell numbers per leaf, was presumably brought about by the inhibition of cell division in the leaf meristem where high mitochondrial activity is required. The plant appears to be trying to maximise the size of the leaf by increasing the size of the limited number of cells initiated from the meristem These larger cells in the mutants were not filled with organic biomass but water (the vacuoles are much larger). On a surface area basis, the leaves of mutants were significantly heavier in fresh weight (Figure 7A) but lighter on a dry weight basis than the control (Figure 7B). This reduction in specific dry weight was apparently correlated with the increase in overall water content of the leaves. Wild-type tobacco plants had about 88 % water in their fresh leaves while the antisense mutants had as much as 95 % water.

EXAMPLE 9

Antisense Suppression of Mitochondrial RISP Affects

Mitochondrion Biogenesis

The above results clearly demonstrate that even a moderate expression of the antisense RISP1 gene causes dramatic changes in many aspects of plant growth and development.

To understand how this occurred, we have examined different aspects of mitochondrial function. First of all, we measured the respiratory CO₂ evolution rate in attached, youngest full-expanded leaves in a large number of T1 segregants from different transgenic lines. Because of the significant change in the cell morphology and its water content (see Figure 7), different impressions can be obtained from the same data set depending on the basis for comparison (Figure 8). On a fresh weight basis, the T1 progeny showed different levels of reduction in respiratory activity, ranging from 100 to 25% of the control activity (Figure 8A). Again the extent of reduction seemed to correlate with the increase in water content in the leaves and also was similar to the decrease in specific dry weight. Thus when comparisons were made on a dry weight basis, no significant changes were observed, except in severely inhibited mutants (Figure

8B).

Mitochondria were then isolated from the severely inhibited plants and state 3 respiratory activity was compared on a mitochondriai protein basis. Surprisingly, it appears that the mutants had normal mitochondrial activity provided that the comparison was made on a mitochondrial protein basis (Table 1). That is, individual mitochondria seem to behave similarly to those from wt plants. No difference in either maximum cytochrome or alternative pathway electron transport was seen. Likewise, no significant differences in immunologically detectable RISP was observed in mitochondria from the leaves of mutant plants except in the severely inhibited plant in which respiration rate was also lower (Figure 9). The implication of this is that the lower respiratory rates seen with whole leaves were due to a decrease in mitochondrial numbers in those leaves. In other words, mitochondrion biogenesis is affected by the antisense RISP gene. This is consistent with the fact that the mitochondrial cytochrome bc₁ complex, of which RISP is a core component, is a bifunctional protein complex, involved in both electron transport and protein processing during protein import. Therefore, reduction in RISP could affect the assembly of the bc₁ complex and thus mitochondrial biogenesis. This is supported by the co-suppression of the ATPase a subunit in the mutants. This subunit is encoded by the mitochondrial genome. Although the suppression was not exactly proportional to the reduction in the level of Rieske FeS message, the general trend is consistent with the lower level of mitochondrial biogenesis in the mutants. Electron microscopy was used to physically count and estimate the number of mitochondria on a per volume basis which demonstrated that the mutant pollen grains did have less number of mitochondria (Table 2). This reduction in mitochondrial number appeared not as much as the reduction in activity in leaves on a fresh weight basis (Figure 8) but it was enough to greatly increase the percentage of aborted pollens (Figure 5).

Stage 1 flowers were used and mitochondria were counted on electron micrographs. Pollen grains were about the same size of 182.5 μm³ in both samples with mitochondrion of 0.6 μm in diameter.

EXAMPLE 10

Expression of a Tapetum-specific antisense RISP1 gene causes male-sterility

The tapetum-specific A9 promoter (Wyatt Paul et al, 1992) was used to specifically drive the expression of the antisense RISP1 genetic sequence set forth in SEQ ID NO: 1 in the tapetum cells of developing anthers of tobacco plants. The A9 promoter was obtained as a 964bp HindlII/ Xbal fragment isolated from the plasmid pWP91 described by Wyatt Paul et al (1992). The promoter fragment was cloned upstream of the antisense RISP1 genetic sequence in the plasmid pBI-RISP described in Example 4, as a HindlI/ Xbal fragment, thereby replacing the CaMV 35S promoter sequence which was previously used to drive general expression of the RISP1 antisense gene. The resulting plasmid, designated pA9-RISP, is shown in Figure 10.

The plasmid pA9-RISP was transformed into tobacco using the Agrobacterium-mediaxed transformation procedure described in Example 4. Transgenic plant material was selected as described in Example 4. Transgenic plants are analysed to determine their phenotypes with respect to anther development and pollen viability as described in Examples 5 and 7. Transgenic plants expressing the pA9-RISP genetic construct are shown to grow normally during the vegetative phase of development, however the RISP antisense phenotype is apparent upon flowering. A range of phenotypes is produced, depending upon the reduction in gene expression which the antisense genetic construct produces. Some plants exhibit no effect or only a slight effect upon the development of anthers and pollen and will be capable of producing viable pollen and self-fertilisation. The most severe phenotype produced is one in which flowers show abnormal development of the anthers and pollen and are male- sterile. In such plants, the female organs of the flower develop normally and can be fertilised successfully using viable pollen derived from non-transgenic or wild-type plants.

Tissue-specific microscopic in situ hybridisation protocols are employed to determine the level of RISP gene expression at the RNA level in transgenic plants. A highly-sensitive detection assay is necessary to detect gene expression in male-sterile plants, because of the small amount of male-gametophytic tissue available. In particular, quantification of mRNA (and protein) on a tissue basis is extremely difficult in such plants. In the most severe phenotypes, the tapetum tissue also expresses RISP mRNA and protein at reduced levels compared to non-transformed control plants.

The male-sterile mutant plants are fertilised with wild-type pollen to produce T1 seed, which is subsequently germinated. Approximately half of the progeny plants thus obtained are heterozygous for the antisense pA9-RISP genetic construct and male-sterile. All fertile plants are shown to lack detectable expression of the pA9-RISP gene. Thus, the male-sterile phenotype is capable of being transmitted through the maternal nuclear DNA of male-sterile plants. EXAMPLE 11

Tapetum-specific expression of an antisense molecule directed against the F1- ATPβ subunit of the mitochondrial ATP synthase causes male sterility in tobacco plants.

A cDNA fragment containing the tobacco F1-ATPβ gene sequence is cloned in the antisense orientation into the pA9-RISP binary vector which is described in Example 10. In this construct, designated pA9-ATPβ, the antisense F1-ATPβ cDNA sequence replaces the antisense RISP1 DNA sequence. Plant material is transformed using Agrobacterium- mediated transformation and transgenic plants are regenerated as described in Example 4.

Mutant plants expressing the pA9-ATPβ gene are identified and analysed as described in Example 10. Transgenic plants expressing the pA9-ATPβ genetic construct are shown to grow normally during the vegetative phase of development, however the F1-ATPβ antisense phenotype is apparent upon flowering. The observable phenotype of plants varies, depending upon the reduction in gene expression which the antisense genetic construct produces. Some plants exhibit no effect or only a slight effect upon the development of anthers and pollen and will be capable of producing viable pollen and self-fertilisation. The most severe phenotype produced is one in which flowers show abnormal development of the anthers and pollen and are male-sterile. In such plants, the female organs of the flower develop normally and can be fertilised successfully using viable pollen derived from non-transgenic or wild-type plants.

Tissue-specific microscopic in situ hybridisation protocols are employed to determine the level of F1-ATPβ gene expression at the RNA level in transgenic plants. In the most severe phenotypes, the tapetum tissue also expresses F1-ATPβ mRNA and protein at reduced levels compared to non-transformed control plants. The male-sterile mutant plants are fertilised with wild-type pollen to produce T1 seed, which is subsequently germinated. Approximately half of the progeny plants thus obtained are heterozygous for the antisense pA9-ATPβ genetic construct and male-sterile. All fertile plants are shown to lack detectable expression of the pA9-ATPβ gene. Thus, the male-sterile phenotype is capable of being transmitted through the maternal nuclear DNA of male-sterile plants.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically descried. 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 in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

REFERENCES

1. Alexander, M.P. (1969). Differential staining of aborted and non-aborted pollen. Stain Technol. 44. 117-122.

2. An et al. (1985) EMBO J. 4:277-284

3. Crossway et al. (1986) Mol. Gen. Genet. 202: 179-185 4. Day, D.A. , Neuburger, M. and Douce, R. (1985) Biochemical

characterization of cylorophyll-free mitochondria from pea leaves. Australian Journal of Plant Physiology 12:219-228

5. Douce, R. and Neuburger, M. (1989) The uniqueness of plant mitochondria.

Annu. Rev. Plant Physiol. Plant Mol. Biol. 40:371-414

6. Fromm et al. (1985) Proc. Natl. Acad. Sci. USA 82:5824-5828

7. Hanson, J.B. and Day, D.A. (1980). Plant Mitochondria. In The Plant Cell.

Tolbert, N.E. (ed.). 1. New York, Academic Press. 315-357

8. Hanson, M.R. (1991). Plant mitochondrial mutations and male sterility.

Annu. Rev. Genet. 25, 461-486. 9. Haseloff, J.. and Gerlach, W.L. (1988) Nature 334: 586-594

10. Herrera-Estrella et al. (1983a) Nature 303:209-213

11. Herrera-Estrella et al. (1983b) EMBO J. 2:987-995

12. Herrera-Estrella et al. (1985) Plant Genetic Engineering, Cambridge University Press. New York, pp 63-93

13. Huang, J., Struck, F., Matzinger. D.F. , and Levings, C.S., III. (1994).

F1ower-enhanced expression of a nuclear-encoded mitochondrial respiratory protein is associated with changes in mitochondrion number. Plant Cell 6,

439-448.

14. Jefferson, R.A., Kavanagh, T.A., and Bevan, M.W. (1987). GUS fusions: b-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6, 3901-3907.

15. Koltunow. A.M., Truettner, J., Cox, K.H. , Wallroth, M., and Goldberg, R.B. (1990). Different temporal and spatial gene expression patterns occur during anther development. Plant Cell 2, 1201-1224.

16. Kosugi, S., Suzuka, I., Ohashi, Y., Murakami, T. and Arai, Y. (1991)

Upstream sequences of rice proliferating cell nuclear antigen (PCNA) gene mediate expression of PCNA-GUS chimeric gene in meristems of transgenic tobacco plants. Nucleic Acids Research 19: 1571-1576

17. Lambers, H. (1985). Respiration in intact plant tissues: its regulation and dependence on environmental factors, metabolism and invading organisms. In: 'Higher plant cell respiration, encyclopedia of plant physiology (new series)'. (Eds. R. Douce and D.A. Day.) 18, 418-474. Springer-Verlag;

Berlin.

18. Landschutze, V., Willmitzer, L. and Muller-Rober, B. (1995). Inhibition of glower formation by antisense repression of mitochondrial citrate synthase in transgenic potato plants leads to a specific disintegration of the ovary tissues of flowers. EMBO J. 14, 660-666. 19. Mclntosh, L. (1994) Molecular Biology of the alternative oxidase. Plant Physiology 105, 781-786.

20. Moore, A.L. , Wood, C.K. and Watt, F.Z. (1994) Protein import into plant mitochondria. Annu. Rev. Plant Physiol. Plant Mol. Biol. 45:545-576

21. Paszkowski et al. (1984) EMBO J. 3:2717-2722

22. Price. G.D., von Caemmerer, S., Evans, J.R. Yu, J.-W., Lloyd, J., Oja, V., Kell, P. , Harrison, K., Gallagher. A. and Badger, M.R. (1994). Specific reduction of chloroplast carbonic anhydrase activity by antisense RNA in transgenic tobacco plants has a minor effect on photosynthetic CO2 assimilation. Planta 193, 331-340. 23. Sambrook, J., Fritsch, E.F., and Maniatis, I. (1989). Molecule cloning: A laboratory manual, 2nd ed. (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press).

24. Vanlerberghe, G.C., Vanlerberghe A.E. and Mclntosh, L. (1994). Molecular genetaic alteration of plant respiration: transgenic tobacco expressing sense and antisense alternative oxidase genes. Plant Physiology in press.

25. Wyatt Paul et al. (1992) The isolation and characterisation of the tapetum- specific Arabidopsis thaliana A9 gene. Plant Molecular Biology 19, 611-622.

26. Yu, J.-W. , Price, G.D., Song, L., and Badger, M.R. (1992) Isolation of a putative carboxysomal carbonic anhydrase gene from the cyanobacterium Synechococcus PCC7942. Plant Physiol. 100, 794-800.

Claims

CLAIMS:-

1. A method of producing a male-sterile plant, said method comprising the steps of:

(ii) regenerating a whole plant from said plant cell; and

2. The method of claim 1 wherein said step of introducing said genetic construct into a plant cell is by Agrobacterium-mediated transformation, microparticle bombardment, microinjection or electroporation of plant cells, tissues or organs.

3. The method of claim 1 or 2, wherein said plant cell is derived from a

dicotyledonous plant selected from the list comprising cotton, tobacco, tomato or potato, amongst others.

4. The method of claim 1 or 2, wherein said plant cell is derived from a

monocotyledonous plant selected from the list comprising wheat, barley, rice or canola, amongst others.

5. The method of any one of claims 1 to 3, wherein said nuclear gene encodes the tobacco RISP1 polypeptide.

6. The method of any one of claims 1 to 3, wherein said nuclear gene encodes the tobacco mitochondrial F1-ATPβ polypeptide.

7. The method of any one of claims 1 to 6 wherein said inhibitory molecule is an antisense molecule.

8. The method of claim 7, wherein said antisense molecule comprises a sequence of nucleotides which is complementary to at least 20 contiguous nucleotides of an mRNA molecule which encodes a mitochondrial polypeptide selected from the list comprising alternative oxidase polypeptide, Rieske iron-sulfur polypeptide or ATP synthase polypeptide .

9. The method of claim 8, wherein said antisense molecule further comprises the sequence of nucleotides set forth in SEQ ID NO: 1 or its complementary nucleotide sequence or is at least 80% identical to all or a part thereof.

10. The method of claim 8. wherein said antisense molecule further comprises the sequence of nucleotides set forth in SEQ ID NO:2 or its complementary nucleotide sequence or is at least 80% identical to all or a part thereof.

11. The method of claim 8. wherein said antisense molecule further comprises the sequence of nucleotides set forth in SEQ ID NO:3 or its complementary nucleotide sequence or is at least 80% identical to all or a part thereof.

12. The method of any one of claims 1 to 6, wherein said inhibitory molecule is a ribozyme molecule comprising :

(i) a hybridising region comprising two nucleotide sequences, each of at least 5 nucleotides in length complementary to a sequence contained within mRNA encoding said mitochondrial polypeptide; and

(ii) an endoribonuclease activity capable of cleaving said mRNA.

13. The method of claim 12, wherein said hybridising region is further capable of hybridising to said mRNA.

14. The method of claim 13, wherein said hybridising region further comprises a sequence of nucleotides contained within the nucleotide sequence set forth in SEQ ID NO: 1 or its complementary nucleotide sequence or is at least 80% identical to all or a part thereof.

15. The method of claim 13, wherein said hybridising region further comprises a sequence of nucleotides contained within the nucleotide sequence set forth in SEQ ID NO: 2 or its complementary nucleotide sequence or is at least 80% identical to all or a part thereof.

16. The method of claim 13, wherein said hybridising region further comprises a sequence of nucleotides contained within the nucleotide sequence set forth in SEQ ID NO: 3 or its complementary nucleotide sequence or is at least 80% identical to all or a part thereof.

17. The method of any one of claims 1 to 6 wherein said inhibitory molecule is a co-supression molecule which encodes said mitochondrial polypeptide.

18. The method of claim 17, wherein said co-supression molecule comprises the sequence of nucleotides set forth in SEQ ID NO:4 or a complement, homologue, analogue or derivative thereof.

19. The method of claim 17, wherein said co-supression molecule comprises the sequence of nucleotides set forth in SEQ ID NO: 6 or a complement, homologue, analogue or derivative thereof.

20. The method of claim 17. wherein said co-supression molecule comprises the sequence of nucleotides set forth in SEQ ID NO: 8 or a complement, homologue, analogue or derivative thereof.

21. A genetic construct comprising a sequence of nucleotides encoding an

antisense molecule capable of hybridising under at least low stringency conditions to mRNA encoding a mitochondrial electron transport chain polypeptide selected from the list comprising alternative oxidase polypeptide, Rieske iron-sulfur polypeptide or ATP synthase polypeptide wherein the expression of said antisense molecule under the control of a promoter capable of regulating or directing gene expression in a plant cell and wherein said sequence of nucleotides encoding said antisense molecule is further placed upstream of a transcription termination sequence.

22. The genetic construct of claim 21, wherein the promoter is selected from the list comprising CaMV 35S, nopaline synthase, octopine synthase or

Arabidopsis thaliana A9 promoters.

23. A genetic construct comprising:

(ii) a first sequence of nucleotides encoding an antisense molecule which when expressed is capable of hybridising under at least low stringency conditions to mRNA encoding a mitochondrial electron transport chain polypeptide selected from the list comprising alternative oxidase polypeptide. Rieske iron-sulfur polypeptide or ATP synthase polypeptide; and

(iii) a transcription termination sequence.

wherein said promoter sequence, first sequence of nucleotides encoding the antisense molecule and transcription termination sequence are placed operably in relation to each other such that said antisense molecule is capable of being expressed under the control of said promoter.

24. The genetic construct of claim 23. wherein the promoter sequence is the tapetum-specific A. thaliana A9 promoter.

25. The genetic construct of claim 21 or 22 or 23 or 24, wherein the antisense molecule comprises a sequence of at least 20 contiguous nucleotides contained within the nucleotide sequence set forth in SEQ ID NO: 1 or its complementary nucleotide sequence or is at least 80% identical thereto.

26. The genetic construct of claim 21 or 22 or 23 or 24, wherein the antisense molecule comprises a sequence of at least 20 contiguous nucleotides contained within the nucleotide sequence set forth in SEQ ID NO: 2 or its complementary nucleotide sequence or is at least 80% identical thereto.

27. The genetic construct of claim 21 or 22 or 23 or 24, wherein the antisense molecule comprises a sequence of at least 20 contiguous nucleotides contained within the nucleotide sequence set forth in SEQ ID NO: 3 or its complementary nucleotide sequence or is at least 80% identical thereto.

28 A genetic construct comprising a first sequence of nucleotides encoding a ribozyme molecule which is capable of hybridising to and cleaving mRNA encoding a mitochondrial electron transport chain polypeptide selected from the list comprising alternative oxidase polypeptide, Rieske iron-sulfur polypeptide or ATP synthase polypeptide, wherein said first sequence of nucleotides is placed operably under the control of a promoter capable of regulating or directing gene expression in a plant cell and wherein said first sequence of nucleotides is further placed upstream of a transcription termination sequence.

29. The genetic construct of claim 28, wherein the promoter is selected from the list comprising CaMV 35S. nopaline synthase. octopine synthase or

Arabidopsis thaliana A9 promoters.

30. A genetic construct comprising:

(ii) a first sequence of nucleotides encoding a ribozyme molecule which is capable of hybridising to and cleaving mRNA encoding a mitochondrial electron transport chain polypeptide selected from the list comprising alternative oxidase polypeptide, Rieske iron-sulfur polypeptide or ATP synthase polypeptide; and

(iii) a transcription termination sequence,

wherein said promoter sequence, first sequence of nucleotides and

transcription termination sequence are placed operably in relation to each other such that said ribozyme molecule is expressed under the control of said promoter.

31. The genetic construct of claim 30, wherein the promoter sequence is the

tapetum-specific A. thaliana A9 promoter.

32. The genetic construct of claim 28 or 29 or 30 or 31, wherein the ribozyme molecule comprises in a first part a hybridising region comprising two nucleotide sequences, each of at least 5 nucleotides in length complementary to a sequence contained within mRNA encoding said mitochondrial polypeptide and in a second part an endoribonuclease activity capable of cleaving said mRNA.

33. The genetic construct of claim 32, wherein said hybridising region further comprises a sequence of nucleotides contained within the nucleotide sequence set forth in SEQ ID NO: 1 or its complementary nucleotide sequence or is at least 80% identical to all or a part thereof.

34. The genetic construct of claim 32. wherein said hybridising region further comprises a sequence of nucleotides contained within the nucleotide sequence set forth in SEQ ID NO: 2 or its complementary nucleotide sequence or is at least 80% identical to all or a part thereof.

35. The genetic construct of claim 32, wherein said hybridising region further comprises a sequence of nucleotides contained within the nucleotide sequence set forth in SEQ ID NO: 3 or its complementary nucleotide sequence or is at least 80% identical to all or a part thereof.

36. A genetic construct comprising a first sequence of nucleotides encoding a co- supression molecule which when expressed is capable of inhibiting the expression of a nuclear gene encoding a mitochondrial electron transport chain polypeptide selected from the list comprising alternative oxidase polypeptide, Rieske iron-sulfur polypeptide or ATP synthase polypeptide, wherein said first sequence of nucleotides is placed operably under the control of a promoter capable of regulating or directing gene expression in a plant cell and wherein said first sequence of nucleotides is further placed upstream of a transcription termination sequence.

37. The genetic construct of claim 36, wherein the promoter is selected from the list comprising CaMV 35S, nopaline synthase, octopine synthase or

Arabidopsis thaliana A9 promoters.

38. A genetic construct comprising:

(ii) a first sequence of nucleotides encoding a co-supression molecule which is capable of inhibiting the expression of a nuclear gene encoding a

mitochondrial electron transport chain polypeptide selected from the list comprising alternative oxidase polypeptide, Rieske iron-sulfur polypeptide or ATP synthase polypeptide: and

(iii) a transcription termination sequence. wherein said promoter sequence, first sequence of nucleotides and

transcription termination sequence are placed operably in relation to each other such that said co-supression molecule is expressed under the control of said promoter sequence.

39. The genetic construct of claim 38. wherein the promoter sequence is the

tapetum-specific A. thaliana A9 promoter.

40. The genetic construct of claim 36 or 37 or 38 or 39, wherein said co- supression molecule comprises a sequence of nucleotides set forth in SEQ ID

NO:4 or a complement, homologue, analogue or derivative thereof.

41. The genetic construct of claim 36 or 37 or 38 or 39, wherein said co- supression molecule comprises the sequence of nucleotides set forth in SEQ ID NO: 6 or a complement, homologue, analogue or derivative thereof.

42. The genetic construct of claim 36 or 37 or 38 or 39, wherein said co- supression molecule comprises the sequence of nucleotides set forth in SEQ ID NO: 8 or a complement, homologue, analogue or derivative thereof.

43. The genetic construct of any one of claims 21 to 42 when used to produce a male-sterile plant.

44. A plant transformed with a genetic construct according to any one of claims 21 to 42.

45. A plant according to claim 44 , wherein said genetic construct is the genetic construct of claim 25.

46. A plant according to claim 44 wherein said genetic construct is the genetic construct of claim 26.

47. The plant of claim 44 or 45 or 46. wherein said plant is a dicotyledonous plant selected from the list comprising cotton, tomato, tobacco or potato, amongst others.

48. The plant of claim 47, wherein said plant is a tobacco plant.

49. The plant of claim 44, wherein said plant is a monocotyledonous plant selected from the list comprising wheat, barley, rice or canola, amongst others.

50. The plant of any one of claims 44 to 49, wherein said plant is male-sterile or at least exhibits abnormal development of anthers, pollen, tapetum or other floral structure required for male-fertility.

51. A male-sterile plant constructed according to the method of any one or more of claims 1 to 20.

52. The progeny of a plant according any one of claims 44 to 51.