AU2013202738A1 - Manipulation of organic acid biosynthesis and secretion (4) - Google Patents

Abstract

The present invention relates to nucleic acid fragments encoding amino acid sequences for organic acid biosynthetic enzymes in plants, and the use thereof for the modification of, for example, organic acid biosynthesis and secretion in plants. 5 In particularly preferred embodiments, the invention relates to the combinatorial expression of citrate synthase (CS) and/or malate dehydrogenase (MDH) and/or phosphoenolpyruvate carboxylase (PEPC) in plants to modify, for example, organic acid synthesis and secretion.

Description

P/00/001 Regulation 3.2 AUSTRALIA Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Invention title: Manipulation of organic acid biosynthesis and secretion (4) The following statement is a full description of this invention, including the best method of performing it known to us: 2 MANIPULATION OF ORGANIC ACID BIOSYNTHESIS AND SECRETION (4) This application is a divisional of Australian patent application No. 2012216840, which in turn is a divisional of Australian patent application 2009200866, which in 5 turn is a divisional of Australian patent application 2004228859, the entire disclosures of which are incorporated herein by reference. The present invention relates to nucleic acid fragments encoding amino acid sequences for organic acid biosynthetic enzyme polypeptides in plants, and the use thereof for the modification of organic acid biosynthesis and secretion in plants. In 10 particularly preferred embodiments, the invention relates to the combinatorial expression of malate dehydrogenase (MDH) and/or phosphoenolpyruvate carboxylase (PEPC) and/or citrate synthase (CS) in plants to modify organic acid biosynthesis and secretion. Organic acids, such as citrate and malate, are key metabolites in plants. They are 15 involved in numerous processes, including C4 and Crassulacean acid metabolism (CAM) photosynthesis, stomatal and pulvinual movement, nutrient uptake, respiration, nitrogen assimilation, fatty acid oxidation, and providing energy to bacteroids in root nodules. For example, malate plays a key role in root nodule metabolism and nitrogen fixation, serving as the primary carbon source for 20 bacteroid maintenance and nitrogenase activity, and is also tightly linked to nodule nitrogen assimilation. Furthermore, the complexing role of organic acids produced and excreted from plant roots has also been associated with tolerance to the aluminium cation A13+ which is toxic to many plants at micromolar concentrations. Aluminium toxicity has been recognized as a major limiting factor of plant 25 productivity on acidic soils, which account for approximately 40% of the earth's arable land. The tricarboxylic acid cycle (TCA), also known as Krebs cycle (after its discoverer Hans Krebs) or citric acid cycle, moves electrons from organic acids to the oxidized redox cofactors NAD* and FAD, forming NADH, FADH 2 , and carbon dioxide (C0 2 ). 30 The reaction sequence of the TCA cycle involves: in a reaction catalysed by citrate synthase (CS), acetyl-CoA formed by the pyruvate dehydrogenase complex 3 combines with oxaloacetate to produce the C 6 tricarboxylic acid, citrate. In the overall cycle, the citrate is oxidized to produce two molecules of CO 2 in a series of reactions that leads to the formation of one oxaloacetate, three NADH, one FADH 2 , and one ATP. The resulting oxaloacetate reacts with another molecule of acetyl 5 CoA to continue the cycle. The oxidative decarboxylation of pyruvate yields an additional CO 2 and NADH. Thus the TCA cycle brings about the complete oxidation of pyruvate to three CO 2 plus 10 electrons, which are stored temporarily as 4 NADH and 1 FADH 2 . Cytosolic reactions generate products that are transported into the mitochondria to 10 feed the TCA cycle. The nature of the end product of the glycolytic reactions in the cytosol of plants is determined by the relative activities of the three enzymes that can utilize phosphoenol-pyruvate (PEP) as substrate. Both pyruvate kinase and PEP-phosphatase form pyruvate; while PEP-carboxylase (PEPC) generates oxaloacetate. Pyruvate is transported directly into the mitochondrion. Oxaloacetate 15 is either transported directly into the mitochondrion or first reduced to malate by cytosolic malate dehydrogenase (MDH). Before entering the TCA cycle proper, pyruvate is oxidised and decarboxylated by the pyruvate dehydrogenase enzyme complex to form CO 2 , acetyl-CoA, and NADH. The pyruvate dehydrogenase enzyme complex, which requires the bound cofactors 20 thiamine pyrophosphate, lipoic acid, and FAD as well as free coenzyme A (CoASH) and NAD*, links the TCA cycle to glycolysis. It is known that the TCA cycle includes the following enzymes: pyruvate dehydrogenase, citrate synthase, citrate hydrolase, isocitrate dehydrogenase, oxoglutarate dehydrogenase, succinyl-CoA synthetase, succinate dehydrogenase, 25 fumarase, malate dehydrogenase, NAD-malic enzyme and phosphoenolpyruvate carboxylase. In particular, citrate synthase (CS) catalyses the condensation of acetyl-CoA and oxaloacetate to form the C6 molecule citrate and free CoASH, as the TCA cycle proper begins.

4 Malate dehydrogenase (MDH) catalyses the final step of the TCA cycle, oxidizing malate to oxaloacetate and producing NADH. This reaction catalysed by MDH is reversible, thus allowing also for the reversible reduction of oxaloacetate to malate. The enzyme MDH is important in several metabolic pathways, and higher plants 5 contain multiple forms that differ in co-enzyme specificity and subcellular localization. Chloroplasts contain an NADP*-dependent MDH that plays a critical role in balancing reducing equivalents between the cytosol and stroma. Plants also contain NAD-dependent MDHs which are found in a) mitochondria as part of the TCA cycle; b) cytosol and peroxisomes involved in malate-aspartate shuttles; and c) 10 glyoxisomes functioning in p-oxidation. In root nodules of nitrogen-fixing legumes, such as white clover (Trifolium repens) and alfalfa (Medicago sativa), malate serves as the primary carbon source to support the respiratory needs of the bacterial microsymbiont and the fixation of N 2 by nitrogenase, and a nodule-enhanced MDH is thus critical for nodule function. 15 Phosphoenolpyruvate carboxylase (PEPC) catalyses the reaction of phosphoenol pyruvate with HC0 3 releasing the phosphate and producing the C4 product, oxaloacetate. Oxaloacetate is commonly reduced to malate by NADH through the action of malate dehydrogenase (MDH). PEPC is a homotetrameric enzyme widely distributed in most plant tissues. In plants, PEPC fulfils various physiological roles 20 such as the photosynthetic CO 2 fixation in C 4 and Crassulacean Acid Metabolism (CAM) plants, and the anaplerotic pathway. While nucleic acid sequences encoding some organic acid biosynthetic enzymes have been isolated for certain species of plants, there remains a need for materials useful in modifying organic acid biosynthesis; in modifying organic acid secretion; in 25 modifying phosphorus acquisition efficiency in plants; in modifying aluminium and acid soil tolerance in plants; in modifying nitrogen fixation and nodule function, particularly in forage legumes and grasses, including alfalfa, medics, clovers, ryegrasses and fescues, and for methods for their use. This invention is directed towards overcoming, or at least alleviating, one or more of 30 the difficulties or deficiencies associated with the prior art.

5 In one aspect, the present invention provides substantially purified or isolated nucleic acids or nucleic acid fragments encoding the organic acid biosynthetic polypeptides CS, MDH and PEPC, from a clover (Trifolium), medic (Medicago), ryegrass (Lolium) or fescue (Festuca) species, or functionally active fragments or 5 variants of these polypeptides. The present invention also provides substantially purified or isolated nucleic acids or nucleic acid fragments encoding amino acid sequences for a class of polypeptides which are related to CS, MDH and PEPC (from a clover (Trifolium), medic (Medicago), ryegrass (Lolium) or fescue (Festuca) species) of CS, MDH and PEPC, 10 or functionally active fragments or variants of CS, MDH and PEPC. Such polypeptides are referred to herein as CS-like, MDH-like and PEPC-like respectively and include polypeptides having similar functional activity. The present invention also relates to individual or simultaneous enhancement or otherwise manipulation of CS, MDH and/or PEPC or like gene activities in plants to 15 enhance or otherwise alter organic acid biosynthesis; to enhance or reduce or otherwise alter organic acid secretion; to enhance or reduce or otherwise alter phosphorous acquisition efficiency in plants; to enhance or reduce or otherwise alter aluminium and acid soil tolerance in plants; and/or to enhance or reduce or otherwise alter nitrogen fixation and nodule function in legumes. 20 The individual or simultaneous enhancement or otherwise manipulation of CS, MDH and/or PEPC or like gene activities in plants has significant consequences for a range of applications in, for example, plant production, plant performance, plant nutrition and plant tolerance. For example, it has applications in increasing plant tolerance to aluminium-toxic acid soils; in improving plant nutrient acquisition 25 efficiency for example in increasing acquisition of phosphorus from soils; in increasing nodule function in nitrogen-fixing legumes for example leading to enhanced nitrogen fixation; in modifying the accumulation of organic acids such as citrate in fruits; in modifying the secretion of organic acids for example citrate and/or malate from plant roots. 30 Manipulation of CS, MDH and/or PEPC or like gene activities in plants, including legumes such as clovers (Trifolium species), lucerne (Medicago sativa) and grass 6 species such as ryegrasses (Lolium species) and fescues (Festuca species) may be used to facilitate the production of, for example, forage legumes and forage grasses and other crops with enhanced tolerance to aluminium toxic soils; enhanced nutrient acquisition efficiency; forage legumes with enhanced nitrogen 5 fixation; fruits with enhanced organic acid content leading to enhanced flavour and health benefits. The clover (Trifolium), medic (Medicago), ryegrass (Lolium) or fescue (Festuca) species may be of any suitable type, including white clover (Trifolium repens), red clover (Trifolium pratense), subterranean clover (Trifolium subterraneum), alfalfa 10 (Medicago sativa), Italian or annual ryegrass (Lolium multiflorum), perennial ryegrass (Lolium perenne), tall fescue (Festuca arundinacea), meadow fescue (Festuca pratensis) and red fescue (Festuca rubra). Preferably the species is a clover or a ryegrass, more preferably white clover (T. repens) or perennial ryegrass (L. perenne). White clover (Trifolium repens L.) and perennial ryegrass (Lolium 15 perenne L.) are key pasture legumes and grasses, respectively, in temperate climates throughout the world. Perennial ryegrass is also an important turf grass. The nucleic acid or nucleic acid fragment may be of any suitable type and includes DNA (such as cDNA or genomic DNA) and RNA (such as mRNA) that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide 20 bases, and combinations thereof. The RNA is readily obtainable, for example, by transcription of a DNA sequence according to the present invention, to produce a RNA corresponding to the DNA sequence. The RNA may be synthesised in vivo or in vitro or by chemical synthesis to produce a sequence corresponding to a DNA sequence by methods well known in the art. In this specification, where the degree 25 of sequence similarity between an RNA and DNA is such that the strand of the DNA could encode the RNA, then the RNA is said to "correspond" to that DNA. The term "isolated" means that the material is removed from its original environment (eg. the natural environment if it is naturally occurring). For example, a naturally occurring nucleic acid or polypeptide present in a living plant is not isolated, but the 30 same nucleic acid or polypeptide separated from some or all of the coexisting materials in the natural system, is isolated. Such an isolated nucleic acid could be 7 part of a vector and/or such a nucleic acid could be part of a composition, and still be isolated in that such a vector or composition is not part of its natural environment. An isolated polypeptide could be part of a composition and still be isolated in that such a composition is not part of its natural environment. 5 By "functionally active" in respect of a nucleic acid it is meant that the fragment or variant is capable of modifying organic acid biosynthesis in a plant. A variant in this context can be an analogue, derivative or mutant and includes naturally occurring allelic variants and non-naturally occurring variants. Additions, deletions, substitutions and derivatizations of one or more of the nucleotides are contemplated 10 so long as the modifications do not result in loss of functional activity of the fragment or variant. Preferably the functionally active fragment or variant has at least approximately 80% identity to the functional part of the above mentioned sequence, more preferably at least approximately 90% identity, most preferably at least approximately 95% identity. Such functionally active variants and fragments 15 include, for example, those having nucleic acid changes which result in conservative amino acid substitutions of one or more residues in the corresponding amino acid sequence. Preferably the fragment has a size of at least 30 nucleotides, more preferably at least 45 nucleotides, most preferably at least 60 nucleotides. By "functionally active" in respect of a polypeptide it is meant that the fragment or 20 variant has one or more of the biological properties of the proteins CS, CS-like, MDH, MDH-like, PEPC and PEPC-like. A variant in this context includes additions, deletions, substitutions and derivatizations of one or more of the amino acids are contemplated so long as the modifications do not result in loss of functional activity of the fragment or variant. Preferably the functionally active fragment or variant has 25 at least approximately 60% identity to the functional part of the above mentioned sequence, more preferably at least approximately 80% identity, most preferably at least approximately 90% identity. Such functionally active variants and fragments include, for example, those having conservative amino acid substitutions of one or more residues in the corresponding amino acid sequence. Preferably the fragment 30 has a size of at least 10 amino acids, more preferably at least 15 amino acids, most preferably at least 20 amino acids.

8 The term "construct" as used herein refers to an artificially assembled or isolated nucleic acid molecule which includes the gene of interest. In general a construct may include the gene or genes of interest, a marker gene which in some cases can also be the gene of interest and appropriate regulatory sequences. It should be 5 appreciated that the inclusion of regulatory sequences in a construct is optional, for example, such sequences may not be required in situations where the regulatory sequences of a host cell are to be used. The term construct includes vectors but should not be seen as being limited thereto. The term "vector" as used herein encompasses both cloning and expression 10 vectors. Vectors are often recombinant molecules containing nucleic acid molecules from several sources. By "operatively linked" in respect of a regulatory element, nucleic acid or nucleic acid fragment and terminator, it meant that the regulatory element is capable of causing expression of said nucleic acid or nucleic acid fragment in a plant cell and 15 said terminator is capable of terminating expression of said nucleic acid or nucleic acid fragment in a plant cell. Preferably, said regulatory element is upstream of said nucleic acid or nucleic acid fragment and said terminator is downstream of said nucleic acid or nucleic acid fragment. By "an effective amount" of a nucleic acid or nucleic acid fragment it is meant an 20 amount sufficient to result in an identifiable phenotypic trait in said plant, or a plant, plant seed or other plant part derived therefrom. Such amounts can be readily determined by an appropriately skilled person, taking into account the type of plant, the route of administration and other relevant factors. Such a person will readily be able to determine a suitable amount and method of administration. See, for 25 example, Maniatis et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, the entire disclosure of which is incorporated herein by reference. Such nucleic acids or nucleic acid fragments could be assembled to form a consensus contig. As used herein, the term "consensus contig" refers to a 30 nucleotide sequence that is assembled from two or more constituent nucleotide sequences that share common or overlapping regions of sequence homology. For 9 example, the nucleotide sequence of two or more nucleic acids or nucleic acid fragments can be compared and aligned in order to identify common or overlapping sequences. Where common or overlapping sequences exist between two or more nucleic acids or nucleic acid fragments, the sequences (and thus their 5 corresponding nucleic acids or nucleic acid fragments) can be assembled into a single contiguous nucleotide sequence. In a preferred embodiment of this aspect of the invention, the substantially purified or isolated nucleic acid or nucleic acid fragment encodes a CS or CS-like polypeptide and including a nucleotide sequence selected from the group consisting 10 of (a) sequences shown in Figures 1, 3, 4, 6, 7, 9, 99, 101, 102, 104, 114, 118 and 122 hereto (SEQ ID NOS 1, 3 to 10, 11, 13 to 16, 17, 19, 327, 329 to 335, 336, 338 to 344, 349, 351, 353 respectively); (b) complements of the sequences recited in (a); (c) sequences antisense to the sequences recited in (a) and (b); (d) functionally active fragments and variants of the sequences recited in (a), (b) and (c); and (e) 15 RNA sequences corresponding to the sequences recited in (a), (b), (c) and (d). In a further preferred embodiment of this aspect of the invention, the substantially purified or isolated nucleic acid or nucleic acid fragment encodes a MDH or MDH like polypeptide and including a nucleotide sequence selected from the group consisting of (a) sequence shown in Figures 11, 13, 14, 16, 17, 19, 21, 23, 25, 26, 20 28, 30, 31, 33, 35, 37, 38, 40, 55, 57, 58, 60, 61, 63, 64, 66, 67, 69, 70, 72, 73, 75, 76, 78, 79, 81, 82 and 84 hereto (SEQ ID NOS. 21, 23 to 29; 30, 32 to 33, 34, 36, 38, 40, 42 to 43, 44, 46, 48 to 110, 111, 113, 115, 117 to 182, 183, 185, 205, 207 to 217, 218, 220 to 251, 252, 254 to 270, 271, 273 to 275, 276, 278 to 287, 288, 290 to 292, 293, 295 to 296, 297, 299 to 301, 304 to 305, 306, 308); (b) complements of 25 the sequences recited in (a); (c) sequences antisense to the sequences recited in (a) and (b); (d) functionally active fragments and variants of the sequences recited in (a), (b) and (c); and (e) RNA sequences corresponding to the sequences recited in (a), (b), (c) and (d). In a further preferred embodiment of this aspect of the invention, the substantially 30 purified or isolated nucleic acid or nucleic acid fragment encodes a PEPC or PEPC like polypeptide and including a nucleotide sequence selected from the group 10 consisting of (a) sequences shown in Figures 42, 44, 46, 47, 49, 51, 53, 86, 88, 89, 91, 92, 94, 95, 97 and 110 hereto (SEQ ID NOS 187, 189, 191 to 197, 199, 201, 203, 310, 312 to 314, 315, 317 to 318, 319, 321 to 322, 323, 325 and 347 respectively); (b) complements of the sequences recited in (a); (c) sequences 5 antisense to the sequences recited in (a) and (b); (d) functionally active fragments and variants of the sequences recited in (a), (b) and (c); and (e) RNA sequences corresponding to the sequences recited in (a), (b), (c) and (d). In a particularly preferred embodiment, the present invention provides a substantially purified or isolated nucleic acid or nucleic acid fragment from a 10 Trifolium species, said nucleic acid or nucleic acid fragment encoding phosphoenolpyruvate carboxylase (PEPC), or complementary or antisense to a sequence encoding PEPC, said nucleic acid or nucleic acid fragment including a nucleotide sequence selected from the group consisting of (a) sequences shown in Figures 86, 88, 89, 91, 92, 94, 95, 97 and 110 hereto (SEQ ID NOS 310, 312 15 to 314, 315, 317 to 318, 319, 321 to 322, 323, 325 and 347 respectively); (b) complements of the sequences recited in (a); (c) sequences antisense to the sequences recited in (a) and (b); (d) functionally active fragments and variants having at least approximately 95% identity to the sequences recited in (a), (b) and (c); and (e) RNA sequences corresponding to the sequences recited in (a), 20 (b), (c) and (d). Nucleic acids or nucleic acid fragments encoding at least a portion of several CS, MDH and PEPC polypeptides have been isolated and identified. Genes encoding other CS or CS-like, MDH or MDH-like and PEPC or PEPC-like proteins, either as cDNAs or genomic DNAs, may be isolated directly by using all or a portion of the 25 nucleic acids or nucleic acid fragments of the present invention as hybridisation probes to screen libraries from the desired plant employing the methodology well known to those skilled in the art. Specific oligonucleotide probes based upon the nucleic acid sequences of the present invention may be designed and synthesized by methods known in the art. Moreover, the entire sequences may be used directly 30 to synthesize DNA probes by methods known to the skilled artisan such as random primer DNA labelling, nick translation, or end-labelling techniques, or RNA probes using available in vitro transcription systems. In addition, specific primers may be 11 designed and used to amplify a part or all of the sequences of the present invention. The resulting amplification products may be labelled directly during amplification reactions or labelled after amplification reactions, and used as probes to isolate full length cDNA or genomic fragments under conditions of appropriate stringency. 5 In addition, short segments of the nucleic acids or nucleic acid fragments of the present invention may be used in protocols to amplify longer nucleic acids or nucleic acid fragments encoding homologous genes from DNA or RNA. For example, polymerase chain reaction may be performed on a library of cloned nucleic acid fragments wherein the sequence of one primer is derived from the nucleic acid 10 sequences of the present invention, and the sequence of the other primer takes advantage of the presence of the polyadenylic acid tracts to the 3' end of the mRNA precursor encoding plant genes. Alternatively, the second primer sequence may be based upon sequences derived from the cloning vector. For example, those skilled in the art can follow the RACE protocol (Frohman et al. (1988) Proc. Natl. Acad Sci. 15 USA 85:8998, the entire disclosure of which is incorporated herein by reference) to generate cDNAs by using PCR to amplify copies of the region between a single point in the transcript and the 3' or 5' end. Using commercially available 3' RACE and 5' RACE systems (BRL), specific 3' or 5' cDNA fragments may be isolated (Ohara et al. (1989) Proc. Natl. Acad Sci USA 86:5673; Loh et al. (1989) Science 20 243:217, the entire disclosures of which are incorporated herein by reference). Products generated by the 3' and 5' RACE procedures may be combined to generate full-length cDNAs. In a further aspect of the present invention there is provided a substantially purified or isolated polypeptide from a clover (Trifolium), medic (Medicago), ryegrass 25 (Lolium) or fescue (Festuca) species, selected from the group consisting of CS or CS-like, MDH or MDH-like and PEPC or PEPC-like polypeptides; and functionally active fragments and variants of these polypeptides. The clover (Trifolium), medic (Medicago), ryegrass (Lolium) or fescue (Festuca) species may be of any suitable type, including white clover (Trifolium repens), red 30 clover (Trifolium pratense), subterranean clover (Trifolium subterraneum), alfalfa (Medicago sativa), Italian or annual ryegrass (Lolium multiflorum), perennial 12 ryegrass (Lolium perenne), tall fescue (Festuca arundinacea), meadow fescue (Festuca pratensis) and red fescue (Festuca rubra). Preferably the species is a clover or a ryegrass, more preferably white clover (T. repens) or perennial ryegrass (L. perenne). 5 In a preferred embodiment of this aspect of the invention, the substantially purified or isolated CS or CS-like polypeptide includes an amino acid sequence selected from the group consisting of sequences shown in Figures 2, 5, 8, 10, 100, 103, 115, 119, 123 hereto (SEQ ID NOS 2, 12, 18, 20, 328, 337, 350, 352 and 354 respectively); and functionally active fragments and variants thereof. 10 In a further preferred embodiment of this aspect of the invention, the substantially purified or isolated MDH or MDH-like polypeptide includes an amino acid sequence selected from the group consisting of sequences shown in Figures 12, 15, 18, 20, 22, 24, 27, 29, 32, 34, 36, 39, 41, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83 and 85 hereto (SEQ ID NOS 22, 31, 35, 37, 39, 41, 45, 47, 112, 114, 116, 184, 186, 206, 15 219, 253, 272, 277, 289, 294, 297, 303, 307 and 309, respectively) and functionally active fragments and variants thereof. In a further preferred embodiment of this aspect of the invention, the substantially purified or isolated PEPC or PEPC-like polypeptide includes an amino acid sequence selected from the group consisting of sequences shown in Figures 43, 20 45, 48, 50, 52, 54, 87, 90, 93, 96, 98 and 111 hereto (SEQ ID NOS 188, 190, 198, 200, 202, 204, 311, 316, 320, 324, 326, and 348 ,respectively); and functionally active fragments and variants thereof. In a particularly preferred embodiment, the present invention provides a substantially purified or isolated PEPC polypeptide from a Trifolium species, said 25 polypeptide including an amino acid sequence selected from the group consisting of (a) sequences shown in Figures 87, 90, 93, 96, 98 and 111 hereto (SEQ ID NOS 311, 316, 320, 324, 326, and 348, respectively); and (b) functionally active fragments and variants having at least approximately 90% identity to the sequences recited in (a).

13 In a further embodiment of this aspect of the invention, there is provided a polypeptide produced (e.g. recombinantly) from a nucleic acid or nucleic acid fragment according to the present invention. Techniques for recombinantly producing polypeptides are known to those skilled in the art. 5 Availability of the nucleotide sequences of the present invention and deduced amino acid sequences facilitates immunological screening of cDNA expression libraries. Synthetic peptides representing portions of the instant amino acid sequences may be synthesized. These peptides may be used to immunise animals to produce polyclonal or monoclonal antibodies with specificity for peptides and/or proteins 10 including the amino acid sequences. These antibodies may be then used to screen cDNA expression libraries to isolate full-length cDNA clones of interest. A genotype is the genetic constitution of an individual or group. Variations in genotype are important in commercial breeding programs, in determining parentage, in diagnostics and fingerprinting, and the like. Genotypes can be readily 15 described in terms of genetic markers. A genetic marker identifies a specific region or locus in the genome. The more genetic markers, the finer defined is the genotype. A genetic marker becomes particularly useful when it is allelic between organisms because it then may serve to unambiguously identify an individual. Furthermore, a genetic marker becomes particularly useful when it is based on 20 nucleic acid sequence information that can unambiguously establish a genotype of an individual and when the function encoded by such nucleic acid is known and is associated with a specific trait. Such nucleic acids and/or nucleotide sequence information including single nucleotide polymorphisms (SNPs), variations in single nucleotides between allelic forms of such nucleotide sequence, may be used as 25 perfect markers or candidate genes for the given trait. Applicants have identified a number of SNPs of the nucleic acids or nucleic acid fragments of the present invention. These are indicated (marked with grey on the black background) in the figures that show multiple alignments of nucleotide sequences of nucleic acid fragments contributing to consensus contig sequences. 30 See for example, Figures 3, 6, 9, 13, 16, 30, 37, 57, 60, 63, 79, 89, 92 and 104 hereto.

14 Accordingly, in a further aspect of the present invention, there is provided a substantially purified or isolated nucleic acid or nucleic acid fragment including a single nucleotide polymorphism (SNP) from a nucleic acid or nucleic acid fragment according to the present invention, for example a SNP from a nucleic acid sequence 5 shown in Figures 3, 6, 9, 13, 16, 30, 37, 57, 60, 63, 66, 67, 72, 78, 88, 94, 101 and 104 hereto; or complements or sequences antisense thereto, and functionally active fragments and variants thereof. The invention further provides a substantially purified or isolated nucleic acid or nucleic acid fragment including a single nucleotide polymorphism (SNP) isolated by the method of this invention. 10 In a still further aspect of the present invention there is provided a method of isolating a nucleic acid or nucleic acid fragment of the present invention including a SNP, said method including sequencing nucleic acid fragments from a nucleic acid library. The method includes the step of identifying the SNP. The nucleic acid library may be of any suitable type and is preferably a cDNA 15 library. The nucleic acid or nucleic acid fragment may be isolated from a recombinant plasmid or may be amplified, for example using polymerase chain reaction. The sequencing may be performed by techniques known to those skilled in the art. In a still further aspect of the present invention, there is provided use of the nucleic 20 acids or nucleic acid fragments of the present invention including SNPs, and/or nucleotide sequence information thereof, as molecular genetic markers. In a still further aspect of the present invention there is provided use of a nucleic acid or nucleic acid fragment of the present invention, and/or nucleotide sequence information thereof, as a molecular genetic marker. 25 More particularly, nucleic acids or nucleic acid fragments according to the present invention and/or nucleotide sequence information thereof may be used as a molecular genetic marker for quantitative trait loci (QTL) tagging, QTL mapping, DNA fingerprinting and in marker assisted selection, particularly in clovers, alfalfa, ryegrasses and fescues. Even more particularly, nucleic acids or nucleic acid 15 fragments according to the present invention and/or nucleotide sequence information thereof may be used as molecular genetic markers in plant improvement in relation to plant tolerance to abiotic stresses such aluminium toxic acid soils; in relation to nutrient acquisition efficiency including phosphorus; in 5 relation to nitrogen fixation; in relation to nodulation. Even more particularly, sequence information revealing SNPs in allelic variants of the nucleic acids or nucleic acid fragments of the present invention and/or nucleotide sequence information thereof may be used as molecular genetic markers for QTL tagging and mapping and in marker assisted selection, particularly in clovers, alfalfa, ryegrasses 10 and fescues. In a still further aspect of the present invention there is provided a construct or vector including a nucleic acid or nucleic acid fragment according to the present invention. In a particularly preferred embodiment the construct or vector may include nucleic 15 acids or nucleic acid fragments encoding both CS or CS-like and MDH or MDH-like polypeptides. In yet another preferred embodiment the construct or vector may include nucleic acids or nucleic acid fragments encoding both MDH or MDH-like and PEPC or PEPC-like polypeptides. 20 In yet another preferred embodiment the construct or vector may include both CS or CS-like and PEPC or PEPC-like polypeptides. In another preferred embodiment the construct or vector may include nucleic acids or nucleic acid fragments encoding all three of CS or CS-like, MDH or MDH-like and PEPC or PEPC-like polypeptides. 25 In a preferred embodiment of this aspect of the invention, the vector may include one or more regulatory element such as a promoter, one or more nucleic acids or nucleic acid fragments according to the present invention and one or more terminators; said one or more regulatory elements, one or more nucleic acids or nucleic acid fragments and one or more terminators being operatively linked.

16 In a preferred embodiment of the present invention the vector may contain nucleic acids or nucleic acid fragments encoding both CS or CS-like and MDH or MDH-like polypeptides, operatively linked to a regulatory element or regulatory elements, such that both CS or CS-like and MDH or MDH-like polypeptides are expressed. 5 In another preferred embodiment of the present invention the vector may contain nucleic acids or nucleic acid fragments encoding both CS or CS-like and PEPC or PEPC-like polypeptides, operatively linked to a regulatory element or regulatory elements, such that both CS or CS-like and PEPC or PEPC-like polypeptides are expressed. 10 In yet another particularly preferred embodiment of the present invention the vector or construct may contain nucleic acids or nucleic acid fragments encoding both MDH or MDH-like and PEPC or PEPC-like polypeptides, operatively linked to a regulatory element or regulatory elements, such that both MDH or MDH-like and PEPC or PEPC-like polypeptides are expressed. 15 In another particularly preferred embodiment of the present invention the vector may contain nucleic acids or nucleic acid fragments encoding all three of CS or CS like, MDH or MDH-like and PEPC or PEPC-like, operatively linked to a regulatory element or regulatory elements, such that all three of CS or CS-like, MDH or MDH like and PEPC or PEPC-like polypeptides are expressed. 20 The vector may be of any suitable type and may be viral or non-viral. The vector may be an expression vector. Such vectors include chromosomal, non chromosomal and synthetic nucleic acid sequences, eg. derivatives of plant viruses; bacterial plasmids; derivatives of the Ti plasmid from Agrobacterium tumefaciens, derivatives of the Ri plasmid from Agrobacterium rhizogenes; phage DNA; yeast 25 artificial chromosomes; bacterial artificial chromosomes; binary bacterial artificial chromosomes; vectors derived from combinations of plasmids and phage DNA. However, any other vector may be used as long as it is replicable, integrative or viable in the plant cell.

17 The regulatory element and terminator may be of any suitable type and may be endogenous to the target plant cell or may be exogenous, provided that they are functional in the target plant cell. Preferably the regulatory element is a promoter. A variety of promoters which may 5 be employed in the vectors of the present invention are well known to those skilled in the art. Factors influencing the choice of promoter include the desired tissue specificity of the vector, and whether constitutive or inducible expression is desired and the nature of the plant cell to be transformed (eg. monocotyledon or dicotyledon). Particularly suitable constitutive promoters include the Cauliflower 10 Mosaic Virus 35S (CaMV 35S) promoter, the maize Ubiquitin promoter, and the rice Actin promoter. Particularly suitable tissue-specific promoters include root-prevalent promoters. A variety of terminators which may be employed in the vectors of the present invention are also well known to those skilled in the art. The terminator may be from 15 the same gene as the promoter sequence or a different gene. Particularly suitable terminators are polyadenylation signals, such as the CaMV 35S polyA and other terminators from the nopaline synthase (nos) and the octopine synthase (ocs) genes. The vector, in addition to the regulatory element, the nucleic acid or nucleic acid 20 fragment of the present invention and the terminator, may include further elements necessary for expression of the nucleic acid or nucleic acid fragment, in different combinations, for example vector backbone, origin of replication (ori), multiple cloning sites, spacer sequences, enhancers, introns (such as the maize Ubiquitin Ubi intron), antibiotic resistance genes and other selectable marker genes [such as 25 the neomycin phosphotransferase (npt2) gene, the hygromycin phosphotransferase (hph) gene, the phosphinothricin acetyltransferase (bar or pat) gene, the phospho mannose isomerase (pmi) gene], and reporter genes (such as beta-glucuronidase (GUS) gene (gusA)]. The vector may also contain a ribosome binding site for translation initiation. The vector may also include appropriate sequences for 30 amplifying expression.

18 As an alternative to use of a selectable marker gene to provide a phenotypic trait for selection of transformed host cells, the presence of the vector in transformed cells may be determined by other techniques well known in the art, such as PCR (polymerase chain reaction), Southern blot hybridisation analysis, histochemical 5 GUS assays, northern and Western blot hybridisation analyses. Those skilled in the art will appreciate that the various components of the vector are operatively linked, so as to result in expression of said nucleic acid or nucleic acid fragment. Techniques for operatively linking the components of the vector of the present invention are well known to those skilled in the art. Such techniques include 10 the use of linkers, such as synthetic linkers, for example including one or more restriction enzyme sites. The vectors of the present invention may be incorporated into a variety of plants, including monocotyledons (such as grasses from the genera Lolium, Festuca, Paspalum, Pennisetum, Panicum and other forage and turfgrasses, corn, oat, 15 sugarcane, wheat and barley), dicotyledons (such as Arabidopsis, tobacco, clovers, medics, eucalyptus, potato, sugarbeet, canola, soybean, chickpea) and gymnosperms. In a preferred embodiment, the vectors may be used to transform monocotyledons, preferably grass species such as ryegrasses (Lolium species) and fescues (Festuca species), more preferably perennial ryegrass, including forage 20 and turf-type cultivars. In an alternate preferred embodiment, the vectors may be used to transform dicotyledons, preferably forage legume species such as clovers (Trifolium species) and medics (Medicago species), more preferably white clover (Trifolium repens), red clover (Trifolium pratense), subterranean clover (Trifolium subterraneum) and alfalfa (Medicago sativa). Clovers, alfalfa and medics are key 25 pasture legumes in temperate climates throughout the world. Techniques for incorporating the vectors of the present invention into plant cells (for example by transduction, transfection or transformation) are known to those skilled in the art. Such techniques include Agrobacterium mediated introduction, electroporation to tissues, cells and protoplasts, protoplast fusion, injection into 30 reproductive organs, injection into immature embryos and high velocity projectile 19 introduction to cells, tissues, calli, immature and mature embryos. The choice of technique will depend largely on the type of plant to be transformed. Cells incorporating the vectors of the present invention may be selected, as described above, and then cultured in an appropriate medium to regenerate 5 transformed plants, using techniques well known in the art. The culture conditions, such as temperature, pH and the like, will be apparent to the person skilled in the art. The resulting plants may be reproduced, either sexually or asexually, using methods well known in the art, to produce successive generations of transformed plants. 10 In a further aspect of the present invention there is provided a plant cell, plant, plant seed or other plant part, including, e.g. transformed with, a vector, nucleic acid or nucleic acid fragment of the present invention. The plant cell, plant, plant seed or other plant part may be from any suitable species, including monocotyledons, dicotyledons and gymnosperms. In a preferred 15 embodiment the plant cell, plant, plant seed or other plant part may be from a monocotyledon, preferably a grass species, more preferably a ryegrass (Lolium species) or fescue (Festuca species), more preferably perennial ryegrass, including both forage- and turf-type cultivars. In an alternate preferred embodiment the plant cell, plant, plant seed or other plant part may be from a dicotyledon, preferably 20 forage legume species such as clovers (Trifolium species) and medics (Medicago species), more preferably white clover (Trifolium repens), red clover (Trifolium pratense), subterranean clover (Trifolium subterraneum) and alfalfa (Medicago sativa). The present invention also provides a plant, plant seed or other plant part, or a plant 25 extract derived from a plant cell of the present invention. The present invention also provides a plant, plant seed or other plant part, or a plant extract derived from a plant of the present invention. In a further aspect of the present invention there is provided a method of modifying organic acid biosynthesis; of modifying organic acid secretion; of modifying 30 phosphorous and other nutrients acquisition efficiency in plants; of modifying 20 aluminium and acid soil tolerance in plants; of modifying nitrogen fixation and nodule function, said method including introducing into said plant an effective amount of a nucleic acid or nucleic acid fragment according to the present invention. Preferably the nucleic acid or nucleic acid fragment is part of a vector. 5 Using the methods and products of the present invention, organic acid biosynthesis; organic acid secretion; phosphorous and other plant nutrient acquisition efficiency; aluminium and acid soil tolerance; nitrogen fixation and nodule function, may be increased or otherwise altered, for example by incorporating additional copies of a sense nucleic acid or nucleic acid fragment of the present invention. They may be 10 decreased or otherwise altered, for example by incorporating an antisense nucleic acid or nucleic acid fragment of the present invention. In a particularly preferred embodiment the method may include introducing into said plant nucleic acids or nucleic acid fragments encoding both CS or CS-like and MDH or MDH-like polypeptides. 15 In another preferred embodiment the method may include introducing into said plant nucleic acids or nucleic acid fragments encoding both CS or CS-like and PEPC or PEPC polypeptides. In yet another preferred embodiment the method may include introducing into said plant nucleic acids or nucleic acid fragments encoding both MDH or MDH-like and 20 PEPC or PEPC-like polypeptides. In an even more preferred embodiment the method may include introducing into said plant nucleic acids or nucleic acid fragments encoding all three of CS or CS like, MDH or MDH-like and PEPC or PEPC-like polypeptides. The present invention will now be more fully described with reference to the 25 accompanying Examples and drawings. It should be understood, however, that the description following is illustrative only and should not be taken in any way as a restriction on the generality of the invention described above. In the Figures 21 Figure 1 shows the consensus contig nucleotide sequence of LpCSa. Figure 2 shows the deduced amino acid sequence of LpCSa. Figure 3 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpCSa. 5 Figure 4 shows the consensus contig nucleotide sequence of LpCSb. Figure 5 shows the deduced amino acid sequence of LpCSb. Figure 6 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpCSb. Figure 7 shows the nucleotide sequence of LpCSc. 10 Figure 8 shows the deduced amino acid sequence of LpCSc. Figure 9 shows the nucleotide sequence of LpCSd. Figure 10 shows the deduced amino acid sequence of LpCSd. Figure 11 shows the consensus contig nucleotide sequence of LpMDHa. Figure 12 shows the deduced amino acid sequence of LpMDHa. 15 Figure 13 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpMDHa. Figure 14 shows the consensus contig nucleotide sequence of LpMDHb. Figure 15 shows the deduced amino acid sequence of LpMDHb. Figure 16 shows the nucleotide sequences of the nucleic acid fragments 20 contributing to the consensus contig sequence LpMDHb. Figure 17 shows the nucleotide sequence of LpMDHc. Figure 18 shows the deduced amino acid sequence of LpMDHc. Figure 19 shows the nucleotide sequence of LpMDHd.

22 Figure 20 shows the deduced amino acid sequence of LpMDHd. Figure 21 shows the nucleotide sequence of LpMDHe. Figure 22 shows the deduced amino acid sequence of LpMDHe. Figure 23 shows the consensus contig nucleotide sequence of LpMDHf. 5 Figure 24 shows the deduced amino acid sequence of LpMDHf. Figure 25 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpMDHf. Figure 26 shows the nucleotide sequence of LpMDHg. Figure 27 shows the deduced amino acid sequence of LpMDHg. 10 Figure 28 shows the consensus contig nucleotide sequence of LpMDHh. Figure 29 shows the deduced amino acid sequence of LpMDHh. Figure 30 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpMDHh. Figure 31 shows the nucleotide sequence of LpMDHi. 15 Figure 32 shows the deduced amino acid sequence of LpMDHi. Figure 33 shows the nucleotide sequence of LpMDHj. Figure 34 shows the deduced amino acid sequence of LpMDHj. Figure 35 shows the consensus contig nucleotide sequence of LpMDHk. Figure 36 shows the deduced amino acid sequence of LpMDHk. 20 Figure 37 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpMDHk. Figure 38 shows the nucleotide sequence of LpMDHI.

23 Figure 39 shows the deduced amino acid sequence of LpMDHI. Figure 40 shows the nucleotide sequence of LpMDHm. Figure 41 shows the deduced amino acid sequence of LpMDHm. Figure 42 shows the nucleotide sequence of LpPEPCa. 5 Figure 43 shows the deduced amino acid sequence of LpPEPCa. Figure 44 shows the consensus contig nucleotide sequence of LpPEPCb. Figure 45 shows the deduced amino acid sequence of LpPEPCb. Figure 46 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpPEPCb. 10 Figure 47 shows the nucleotide sequence of LpPEPCc. Figure 48 shows the deduced amino acid sequence of LpPEPCc. Figure 49 shows the nucleotide sequence of LpPEPCd. Figure 50 shows the deduced amino acid sequence of LpPEPCd. Figure 51 shows the nucleotide sequence of LpPEPCe. 15 Figure 52 shows the deduced amino acid sequence of LpPEPCe. Figure 53 shows the nucleotide sequence of LpPEPCf. Figure 54 shows the deduced amino acid sequence of LpPEPCf. Figure 55 shows the consensus contig nucleotide sequence of TrMDHa. Figure 56 shows the deduced amino acid sequence of TrMDHa. 20 Figure 57 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence TrMDHa. Figure 58 shows the consensus contig nucleotide sequence of TrMDHb.

24 Figure 59 shows the deduced amino acid sequence of TrMDHb. Figure 60 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence TrMDHb. Figure 61 shows the consensus contig nucleotide sequence of TrMDHc. 5 Figure 62 shows the deduced amino acid sequence of TrMDHc. Figure 63 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence TrMDHc. Figure 64 shows the consensus contig nucleotide sequence of TrMDHd. Figure 65 shows the deduced amino acid sequence of TrMDHd. 10 Figure 66 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence TrMDHd. Figure 67 shows the consensus contig nucleotide sequence of TrMDHe. Figure 68 shows the deduced amino acid sequence of TrMDHe. Figure 69 shows the nucleotide sequences of the nucleic acid fragments 15 contributing to the consensus contig sequence TrMDHe. Figure 70 shows the consensus contig nucleotide sequence of TrMDHf. Figure 71 shows the deduced amino acid sequence of TrMDHf. Figure 72 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence TrMDHf. 20 Figure 73 shows the consensus contig nucleotide sequence of TrMDHg. Figure 74 shows the deduced amino acid sequence of TrMDHg. Figure 75 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence TrMDHg.

25 Figure 76 shows the consensus contig nucleotide sequence of TrMDHh. Figure 77 shows the deduced amino acid sequence of TrMDHh. Figure 78 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence TrMDHh. 5 Figure 79 shows the consensus contig nucleotide sequence of TrMDHi. Figure 80 shows the deduced amino acid sequence of TrMDHi. Figure 81 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence TrMDHi. Figure 82 shows the nucleotide sequence of TrMDHj. 10 Figure 83 shows the deduced amino acid sequence of TrMDHj. Figure 84 shows the nucleotide sequence of TrMDHk. Figure 85 shows the deduced amino acid sequence of TrMDHk. Figure 86 shows the consensus contig nucleotide sequence of TrPEPCa. Figure 87 shows the deduced amino acid sequence of TrPEPCa. 15 Figure 88 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence TrPEPCa. Figure 89 shows the consensus contig nucleotide sequence of TrPEPCb. Figure 90 shows the deduced amino acid sequence of TrPEPCb. Figure 91 shows the nucleotide sequences of the nucleic acid fragments 20 contributing to the consensus contig sequence TrPEPCb. Figure 92 shows the consensus contig nucleotide sequence of TrPEPCc. Figure 93 shows the deduced amino acid sequence of TrPEPCc.

26 Figure 94 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence TrPEPCc. Figure 95 shows the nucleotide sequence of TrPEPCd. Figure 96 shows the deduced amino acid sequence of TrPEPCd. 5 Figure 97 shows the nucleotide sequence of TrPEPCe. Figure 98 shows the deduced amino acid sequence of TrPEPCe. Figure 99 shows the consensus contig nucleotide sequence of TrCSa. Figure 100 shows the deduced amino acid sequence of TrCSa. Figure 101 shows the nucleotide sequences of the nucleic acid fragments 10 contributing to the consensus contig sequence TrCSa. Figure 102 shows the consensus contig nucleotide sequence of TrCSb. Figure 103 shows the deduced amino acid sequence of TrCSb. Figure 104 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence TrCSb. 15 Figure 105 shows the plasmid map in pGEM-T Easy of TrMDH. Figure 106 shows the nucleotide sequence of TrMDH. Figure 107 shows the deduced amino acid sequence of TrMDH. Figure 108 shows the plasmid map of sense construct of TrMDH in the binary vector pPZP221:35S 2 . 20 Figure 109 shows the plasmid map in pGEM-T Easy of TrPEPC. Figure 110 shows the nucleotide sequence of TrPEPC. Figure 111 shows the deduced amino acid sequence of TrPEPC.

27 Figure 112 shows the plasmid map of sense construct of TrPEPC in the binary vector pPZP221:35S 2 . Figure 113 shows the plasmid map in pGEM-T Easy of TrCSa. Figure 114 shows the nucleotide sequence of TrCSa. 5 Figure 115 shows the deduced amino acid sequence of TrCSa. Figure 116 shows the plasmid map of sense construct of TrCSa in the binary vector pPZP221:35S 2 . Figure 117 shows the plasmid map in pGEM-T Easy of TrCSb. Figure 118 shows the nucleotide sequence of TrCSb. 10 Figure 119 shows the deduced amino acid sequence of TrCSb. Figure 120 shows the plasmid map of sense construct of TrCSb in the binary vector pPZP221:35S 2 . Figure 121 shows the plasmid map in pGEM-T Easy of TrCSd. Figure 122 shows the nucleotide sequence of TrCSd. 15 Figure 123 shows the deduced amino acid sequence of TrCSd. Figure 124 shows the plasmid map of sense construct of TrCSd in the binary vector pPZP221:35S 2 . Figure 125 shows the plasmid maps of the modular vector system comprising a binary base vector and 7 auxiliary vectors. 20 Figure 126 shows an example of the modular binary transformation vector system comprising plasmid maps of the binary transformation vector backbone and 4 expression cassettes for combinatorial expression of chimeric CS and MDH and PEPC genes in auxiliary vectors (A) and the plasmid map of the T-DNA region of the final binary transformation vector (B).

28 Figure 127 shows the results of RT-PCR experiments performed as described in Example 6. Samples were isolated from: L, leaf; S, stolon; St, stolon tip; R, root; Rt, root tip. -C: negative (no reverse transcriptase) control; +C, positive (plasmid) control. The numbers indicate cycle numbers. A: phosphate transporter homolog; B: 5 root iron transporter homolog. Figure 128 shows the screening of a white clover BAC library using the phosphate transporter cDNA as a probe (A); Southern hybridisation blot of six BAC clones identified in A using the same probe (B); physical map of the phosphate transporter genomic region including the coding region and the promoter region (C). 10 Figure 129 shows white clover cotyledons, various stages of selection of plantlets transformed with a binary transformation vector constructed as described in Examples 4 and 5, transgenic white clover on root-inducing medium, and white clover plants transformed with genes involved in organic acid biosynthesis. Figure 130 shows the molecular analysis of transgenic white clover plants for the 15 presence of the chimeric MDH gene with real time PCR amplification plot and agarose gel of PCR product. Figure 131 shows the molecular analysis of transgenic white clover plants for the presence of the chimeric PEPC gene with real time PCR amplification plot and agarose gel of PCR product. 20 Figure 132 shows the molecular analysis of transgenic white clover plants for the presence of the chimeric CS gene with real time PCR amplification plot and agarose gel of PCR product. EXAMPLE 1 Preparation of cDNA libraries, isolation and sequencing of cDNAs coding for 25 CS, CS-like, MDH, MDH-like, PEPC and PEPC-like polypeptides from white clover ( Trifolum repens) and perennial ryegrass (Lolum perenne) cDNA libraries representing mRNAs from various organs and tissues of white clover (Trifolium repens) and perennial ryegrass (Lolium perenne) were prepared. The 29 characteristics of the white clover and perennial ryegrass libraries, respectively, are described below (Tables 1 and 2). TABLE 1 cDNA libraries from white clover ( Trifolium repens) Library Organ/Tissue 01wc Whole seedling, light grown 02wc Nodulated root 3, 5, 10, 14, 21 &28 day old seedling 03wc Nodules pinched off roots of 42 day old rhizobium inoculated white clover 04wc Nodulated white clover cut leaf and stem collected after 0, 1, 4, 6 &14 h after cutting 05wc Non-nodulated Inflorescences: <50% open, not fully open and fully open 06wc Dark grown etiolated 07wc Inflorescence - very early stages, stem elongation, < 15 petals, 15-20 petals 08wc seed frozen at -80'C, imbibed in dark overnight at 1 0 0 C 09wc Drought stressed plants 1 Owc AMV infected leaf 1 1wc WCMV infected leaf 12wc Phosphorus starved plants 13wc Vegetative stolon tip 14wc stolon root initials 15wc Senescing stolon 16wc Senescing leaf 5 30 TABLE 2 cDNA libraries from perennial ryegrass (Lolumperenne) Library Organ/Tissue 01 rg Roots from 3-4 day old light-grown seedlings 02rg Leaves from 3-4 day old light-grown seedlings 03rg Etiolated 3-4 day old dark-grown seedlings 04rg Whole etiolated seedlings (1-5 day old and 17 days old) 05rg Senescing leaves from mature plants 06rg Whole etiolated seedlings (1-5 day old and 17 days old) 07rg Roots from mature plants grown in hydroponic culture 08rg Senescent leaf tissue 09rg Whole tillers and sliced leaves (0, 1, 3, 6, 12 and 24 h after harvesting) 1 Org Embryogenic suspension-cultured cells 11 rg Non-embryogenic suspension-cultured cells 12rg Whole tillers and sliced leaves (0, 1, 3, 6, 12 and 24 h after harvesting) 13rg Shoot apices including vegetative apical meristems 14rg Immature inflorescences including different stages of inflorescence meristem and inflorescence development 15rg Defatted pollen 16rg Leaf blades and leaf sheaths (rbcL, rbcS, cab, wir2A subtracted) 17rg Senescing leaves and tillers 18rg Drought-stressed tillers (pseudostems from plants subjected to PEG simulated drought stress) 19rg Non-embryogenic suspension-cultured cells subjected to osmotic stress (grown in media with half-strength salts) (1, 2, 3, 4, 5, 6, 24 and 48 h after transfer) 20rg Non-embryogenic suspension-cultured cells subjected to osmotic stress (grown in media with double-strength salts) (1, 2, 3, 4, 5, 6, 24 and 48 h after transfer) 21rg Drought-stressed tillers (pseudostems from plants subjected to PEG simulated drought stress) 22rg Spikelets with open and maturing florets 23rg Mature roots (specific subtraction with leaf tissue) 31 The cDNA libraries may be prepared by any of many methods available. For example, total RNA may be isolated using the Trizol method (Gibco-BRL, USA) or the RNeasy Plant Mini kit (Qiagen, Germany), following the manufacturers' instructions. cDNAs may be generated using the SMART PCR cDNA synthesis kit 5 (Clontech, USA), cDNAs may be amplified by long distance polymerase chain reaction using the Advantage 2 PCR Enzyme system (Clontech, USA), cDNAs may be cleaned using the GeneClean spin column (Bio 101, USA), tailed and size fractionated, according to the protocol provided by Clontech. The cDNAs may be introduced into the pGEM-T Easy Vector system 1 (Promega, USA) according to 10 the protocol provided by Promega. The cDNAs in the pGEM-T Easy plasmid vector are transfected into Escherichia coli Epicurian coli XL10-Gold ultra competent cells (Stratagene, USA) according to the protocol provided by Stratagene. Alternatively, the cDNAs may be introduced into plasmid vectors for first preparing the cDNA libraries in Uni-ZAP XR vectors according to the manufacturer's protocol 15 (Stratagene Cloning Systems, La Jolla, CA, USA). The Uni-ZAP XR libraries are converted into plasmid libraries according to the protocol provided by Stratagene. Upon conversion, cDNA inserts will be contained in the plasmid vector pBluescript. In addition, the cDNAs may be introduced directly into precut pBluescript II SK(+) vectors (Stratagene) using T4 DNA ligase (New England Biolabs), followed by 20 transfection into E. coli DH10B cells according to the manufacturer's protocol (GIBCO BRL Products). Once the cDNA inserts are in plasmid vectors, plasmid DNAs are prepared from randomly picked bacterial colonies containing recombinant plasmids, or the insert cDNA sequences are amplified via polymerase chain reaction using primers specific 25 for vector sequences flanking the inserted cDNA sequences. Plasmid DNA preparation may be performed robotically using the Qiagen QiaPrep Turbo kit (Qiagen, Germany) according to the protocol provided by Qiagen. Amplified insert DNAs are sequenced in dye-terminator sequencing reactions to generate partial cDNA sequences (expressed sequence tags or "ESTs"). The resulting ESTs are 30 analysed using an Applied Biosystems ABI 3700 sequence analyser.

32 EXAMPLE 2 DNA sequence analyses The cDNA clones encoding CS, CS-like, MDH, MDH-like, PEPC and PEPC-like polypeptides were identified by conducting BLAST (Basic Local Alignment Search 5 Tool; Altschul et al. (1993) J. Mol. Biol. 215:403-410) searches. The cDNA sequences obtained were analysed for similarity to all publicly available DNA sequences contained in the eBioinformatics nucleotide database using the BLASTN algorithm provided by the National Center for Biotechnology Information (NCBI). The DNA sequences were translated in all reading frames and compared for 10 similarity to all publicly available protein sequences contained in the SWISS-PROT protein sequence database using BLASTx algorithm (v 2.0.1) (Gish and States (1993) Nature Genetics 3:266-272) provided by the NCBI. The cDNA sequences obtained and identified were then used to identify additional identical and/or overlapping cDNA sequences generated using the BLASTN 15 algorithm. The identical and/or overlapping sequences were subjected to a multiple alignment using the CLUSTALw algorithm, and to generate a consensus contig sequence derived from this multiple sequence alignment. The consensus contig sequence was then used as a query for a search against the SWISS-PROT protein sequence database using the BLASTx algorithm to confirm the initial identification. 20 EXAMPLE3 Identification and full-length sequencing of cDNAs encoding CS, MDH and PEPC polypeptides To fully characterise for the purposes of the generation of probes for hybridisation experiments and the generation of transformation vectors, a set of cDNAs encoding 25 white clover CS, MDH and PEPC polypeptides was identified and fully sequenced. Full-length cDNAs were identified from our EST sequence database using relevant published sequences (NCBI databank) as queries for BLAST searches. Full-length cDNAs were identified by alignment of the query and hit sequences using Sequencher (Gene Codes Corp., Ann Arbor, MI 48108, USA). The original plasmid 33 was then used to transform chemically competent XL-1 cells (prepared in-house, CaCl 2 protocol). After colony PCR (using HotStarTaq, Qiagen) a minimum of three PCR-positive colonies per transformation were picked for initial sequencing with M13F and M13R primers. The resulting sequences were aligned with the original 5 EST sequence using Sequencher to confirm identity and one of the three clones was picked for full-length sequencing, usually the one with the best initial sequencing result. Sequencing of all cDNAs was completed by primer walking, i.e. oligonucleotide primers were designed to the initial sequence obtained using M13F and M13R 10 oligonucleotide primers and used for further sequencing. The sequences of the oligonucleotide primers are shown in Table 2. Contigs were then assembled in Sequencher. The contigs include the sequences of the SMART primers used to generate the initial cDNA library as well as pGEM-T Easy vector sequence up to the EcoRi cut site both at the 5' and 3' end. 15 Plasmid maps and the full cDNA sequences of TrCSa, TrCSb, TrCSd, TrMDH and TrPEPC polypeptides were obtained (Figures 1, 2, 5, 6, 9, 10, 13, 14, 17, 18, 21, 22, 25, 26, 29 and 30).

34 TABLE 3 List of primers used for sequencing of the full-length cDNAs encoding CS, MDH and PEPC gene name clone ID sequencing primer primer sequence (5'>3') TrCSa 05wclHsBO8 05wclHsBO8.fl TTGCCCGAGGCTATACTGTGGC 05wcl HsB08.f2 CAGCTCACCTAGTTGCTAG 05wcl HsB08.f3 CCATGGCCTAATGTTGATGC 05wclHsBO8.rl TTGGCCTTTCAAGTGGCATTCC 05wcl HsB08.r2 CAGAATGGGAGGCACGACTTC 05wcl HsB08.r3 ATGTGAGCATAGTTTGCACC TrCSb 05wc2HsDO9 05wc2HsD9.fl GACTGCCAGAAAACACTTCCAGG 05wc2HsDO9.f2 ATGACTGCTTTAGTGTGG 05wc2HsD9.rl CTCAAGTTTCTCCAGTGTGACAC 05wc2HsDO9.r2 TGACTTATGTATCCCACC 05wc2HsDO9.r3 GCTCTGAATGGTTTAGCTGG TrCSd 1Owc1BsF1O 1Owc1BsF1O.f1 GCACTGCCTGTTTCTGCTCATCC 1 Owc BsF1 O.f2 AGCCAACTTATGAGGATAGC 1Owc1BsF1O.rl CTCCAATACTCCTCGCGACGCC 1 Owc BsF1 O.r2 AGGCACAACCTGGCCACTG 1Owc1BsF1O.r3 ACGTTGCCACCTTCATGATC TrMDH 13wclNsDO1 13wclNsDOl.fl GTTGTTATACCTGCTGGTGTT 13wclNsDOl.rl CTCACTCAACCCTTGGAGAT TrPEPC 15wc1DsH12 15wclDsH12.f1 TCCTAAGAAACTTGAAGAGCTCGG 15wclDsH12.f2 AGATGTTTGCTTACTAGC 15wclDsH12.rl GCCAGCAGCAATACCCTTCATGG 15wclDsHl2.r2 TTGCTTCTCAACTGTTCC 35 EXAMPLE4 Development of binary transformation vectors containing chimeric genes with cDNA sequences encoding CS, MDH and PEPC 5 To alter the expression of the polypeptides involved in organic acid biosynthesis to improve phosphorus acquisition efficiency as well as aluminium and acid soil tolerance in forage plants, a set of sense binary transformation vectors was produced. The pPZP221 binary transformation vector (Hajdukiewicz et al., 1994) was modified 10 to contain the 35S 2 cassette from pKYLX71:35S 2 (Schardl et al., 1987) as follows: pKYLX71:35S 2 was cut with Clal. The 5' overhang was filled in using Klenow and the blunt end was A-tailed with Taq polymerase. After cutting with EcoRI, the 2kb fragment with an EcoRI-compatible and a 3'-A tail was gel-purified. pPZP221 was cut with Hindlil and the resulting 5' overhang filled in and T-tailed with Taq 15 polymerase. The remainder of the original pPZP221 multi-cloning site was removed by digestion with EcoRI, and the expression cassette cloned into the EcoRi site and the 3' T overhang restoring the HindIII site. This binary vector contains between the left and right border the plant selectable marker gene aacC1 under the control of the 35S promoter and 35S terminator and the pKYLX71:35S 2 -derived expression 20 cassette with a CaMV 35S promoter with a duplicated enhancer region and an rbcS terminator. A GATEWAY* cloning cassette (Invitrogen) was introduced into the multicloning site of the pPZP221:35S2 vector obtained as described following the manufacturer's protocol. 25 cDNA fragments were generated by high fidelity PCR with a proofreading DNA polymerase using the original pGEM-T Easy plasmid cDNA as a template. The primers used (Table 3) contained attB sequences for use with recombinases utilising the GATEWAY* system (Invitrogen). The resulting PCR fragments were used in a recombination reaction with pDONR* vector (Invitrogen) to generate entry 30 vectors. In a further recombination reaction, the cDNAs encoding the open reading 36 frame sequences were transferred from the entry vector to the GATEWAY*-enabled pPZP221:35S 2 vector. The orientation of the constructs (sense or antisense) was checked by restriction enzyme digest and sequencing which also confirmed the correctness of the 5 sequence. Transformation vectors containing chimeric genes using full-length open reading frame cDNAs encoding white clover TrCSa, TrCSb, TrCSd, TrMDH and TrPEPC proteins in sense orientation under the control of the CaMV 35S 2 promoter were generated (Figures 4, 8, 12, 16, 20, 24, 28 and 32).

37 TABLE 4 List of primers used to PCR-amplify the open reading frames of cDNAs encoding CS, MDH and PEPC gene name clone ID primer primer sequence (5'>3') TrCSa 05wcl HsB08 05wcl HsB08f GGGGACAAGTTTGTACAAAAAAGC AGGCTTGATCTTAATGGCGTTCTT TCG 05wclHsBO8r GGGGACCACTTTGTACAAGAAAGC TGGGTTTTCAATTTTAGGACGATG CG TrCSb 05wc2HsDO9 05wc2HsDO9f GGGGACAAGTTTGTACAAAAAAGC AGGCTTTGTTGATTGATCTTAATG GC 05wc2HsDO9r GGGGACCACTTTGTACAAGAAAGC TGGGTTAGTAATCCACAGATAACC G TrCSd 1Owc1BsF1O 1Owc1BsF1Of GGGGACAAGTTTGTACAAAAAAGC AGGCTCTAGATTGTTGATTGATCT AAATGGC 1OwclBsF1Or GGGGACCACTTTGTACAAGAAAGC TGGGTCTAGATTCAATTTTAGGAT GATGCACC TrMDH 13wclNsDO1 13wclNsDOlf GGGGACAAGTTTGTACAAAAAAGC AGGCTCTAGAAATTCCCATTACCA TTCATTCC 13wclNsDO1r GGGGACCACTTTGTACAAGAAAGC TGGGTCTAGATTGACATTCTCTCG CATGGACGC TrPEPC 15wc1DsH12 15wclDsHl2f GGGGACAAGTTTGTACAAAAAAGC AGGCTTGAGAAGGAGTGAATTGCT CC 15wclDsHl2r GGGGACCACTTTGTACAAGAAAGC TGGGTATGATATCTTAGCACACAC TTAAC 5 38 EXAMPLE 5 Development of binary transformation vectors containing chimeric genes with a combination of 2 or more cDNA sequences encoding CS, MDH and PEPC To alter the expression of the polypeptides involved in organic acid biosynthesis to 5 improve phosphorus acquisition efficiency as well as aluminium and acid soil tolerance in forage plants, a modular binary transformation vector system was used (Figure 125). The modular binary vector system enables simultaneous integration of up to seven transgenes the expression of which is controlled by individual promoter and terminator sequences into the plant genome (Goderis et al., 2002). 10 The modular binary vector system consists of a pPZP200-derived vector (Hajdukiewicz et al., 1994) backbone containing within the T-DNA a number of unique restriction sites recognised by homing endonucleases. The same restriction sites are present in pUC18-based auxiliary vectors flanking standard multicloning sites. Expression cassettes comprising a selectable marker gene sequence or a 15 cDNA sequence to be introduced into the plant under the control of regulatory sequences like promoter and terminator can be constructed in the auxiliary vectors and then transferred to the binary vector backbone utilising the homing endonuclease restriction sites. Up to seven expression cassettes can thus be integrated into a single binary transformation vector. The system is highly flexible 20 and allows for any combination of cDNA sequence to be introduced into the plant with any regulatory sequence. For example, a selectable marker cassette comprising the nos promoter and nos terminator regulatory sequences controlling the expression of the nptl| gene was PCR-amplified using a proofreading DNA polymerase from the binary vector 25 pKYLX71:35S 2 and directionally cloned into the Agel and Notl sites of the auxiliary vector pAUX3166. Equally, other selectable marker cassettes can be introduced into any of the auxiliary vectors. In another example, the expression cassette from the binary vector pWM5 consisting of the ASSU promoter and the tob terminator was PCR-amplified with a 30 proofreading DNA polymerase and directionally cloned into the Agel and Notl sites 39 of the auxiliary vector pAUX3169. Equally, other expression cassettes can be introduced into any of the auxiliary vectors. In yet another example, the expression cassette from the direct gene transfer vector pDH51 was cut using EcoRi and cloned directly into the EcoRi site of the auxiliary 5 vector pAUX3132. TABLE 5 List of primers used to PCR-amplify plant selectable marker cassettes and the regulatory elements used to control the expression of CS, MDH and PEPC genes expression primer primer sequence (5'>3') cassette nos::nptll-nos forward ATAATAACCGGTTGATCATGAGCGGAGAATTAAG GG reverse ATAATAGCGGCCGCTAGTAACATAGATGACACCG CG 35S::aacCl-35S forward AATAGCGGCCGCGATTTAGTACTGGATTTTGG reverse AATAACCGGTACCCACGAAGGAGCATCGTGG 35S 2 ::rbcS forward ATAATAACCGGTGCCCGGGGATCTCCTTTGCC reverse ATAATAGCGGCCGCATGCATGTTGTCAATCAATT GG assu::tob forward TAATACCGGTAAATTTATTATGRGTTTTTTTCCG reverse TAATGCGGCCGCTAAGGGCAGCCCATACAAATGA AGC 10 The expression cassettes were further modified by introducing a GATEWAY* cloning cassette (Invitrogen) into the multicloning site of the respective pAUX vector following the manufacturer's protocol. In a recombination reaction, the cDNAs encoding the open reading frame sequences were transferred from the entry vector 15 obtained as described in Example 4 to the GATEWAY*-enabled pAUX vector. Any combination of the regulatory elements with cDNA sequences of TrCSa, TrCSb, TrCSd, TrMDH and TrPEPC can be obtained. One typical example is given in Figure 126 with expression cassettes comprising the nptll plant selectable marker, TrPEPC, TrCSa and TrMDH.

40 Complete expression cassettes comprising any combination of regulatory elements and cDNA sequences to be introduced into the plant were then cut from the auxiliary vectors using the respective homing endonuclease and cloned into the respective restriction site on the binary vector backbone. After verification of the 5 construct by nucleotide sequencing, the binary transformation vector comprising a number of expression cassettes was used to generate transgenic white clover plants. EXAMPLE 6 Isolation of regulatory elements to direct expression of chimeric genes 10 encoding CS, MDH and PEPC involved in organic acid biosynthesis To direct the expression of chimeric white clover genes TrCSa, TrCSb, TrCSd, TrMDH and TrPEPC involved in organic acid biosynthesis to specific tissues, regulatory elements showing specificity for expression in root or root tip tissue were identified and isolated. 15 Using the BLASTn algorithm, white clover EST sequence collections prepared as detailed in Examples 1 and 2 were searched with nucleotide sequences representing genes with known root-specific expression identified in GenBank as queries. Suitable candidate ESTs were identified and oligonucleotide primers for reverse transcription-PCR (RT-PCR) were designed (see Table 4). 20 41 TABLE 6 Oligonucleotide primers used in reverse transcription-PCR to confirm tissue specificity of candidate white clover ESTs gene forward primer (5'->3') reverse primer (5'->3') histone (internal CCGATTCCGTTTCAATGGCTCGTA GCCATCCTTAACCCTAAGCACGT control) white clover phosphate TTGCATTTGCTTGGAACAACTAG GCAAGAGCAAACATGAAACCA transporter homolog white clover root iron ATGGGTCTTGGTGGTTGCA GCAGCAAGAAGATCAACCAAAGCCA transporter homolog 5 Total RNA for RT-PCR experiments was isolated from a leaf, stolon, stolon tip, root and root tip of white clover plants grown in the glasshouse using the TRIZOL method. Reverse transcription was performed using SuperScriptil (Invitrogen) following the supplier's instructions. Preliminary PCR reactions using Dynazyme as the DNA polymerase were set up to determine the correct amount of template using 10 the PCR primers for the internal control (histone). The results of this preliminary PCR were used to set up another round of PCR to determine the optimum number of cycles for linear amplification. The final PCR amplifications were performed using the following cycling conditions: 94 OC, 4 min., 1 time; 94 OC, 15 sec., 60 OC, 30 sec., 72 OC, 2 min., x times; 72 OC, 10 min., 1 time. The number of cycles in the 15 amplification (x) was found to be dependent on the relative abundance of transcript and had to be optimised for each template. RT-PCR results using a white clover histone gene as an internal constitutively expressed control confirmed the tissue-specificity of two candidate ESTs to be root prevalent (Figure 127 A and B). These were a phosphate transporter homolog 20 (clone name 02wc1 DsG07) and a root iron transporter homolog (clone name 05wc1 IsB1 1). A spotted white clover BAC library consisting of 50,304 clones with an estimated 99% genome coverage (6.3 genome equivalents) was screened using the phosphate transporter homolog EST nucleotide sequence as a probe. A number of 25 positive BAC clones could be identified (Figure 128 A). After Southern hybridisation 42 blotting (Figure 128 B) a 7.5 kb EcoRV genomic DNA fragment was selected and fully sequenced. The fragment contained the phosphate transporter homolog open reading frame and 4 kb of upstream sequence including the promoter region. A physical map of the genomic DNA fragment including the promoter region is shown 5 in Figure 128 C. EXAMPLE 7 Production by Agrobacterium-mediated transformation and analysis of transgenic white clover plants carrying chimeric genes encoding CS, MDH and PEPC involved in organic acid biosynthesis 10 A set of binary transformation vectors carrying chimeric white clover genes to alter the expression of the polypeptides involved in organic acid biosynthesis to improve phosphorus acquisition efficiency as well as aluminium and acid soil tolerance in forage plants were produced as detailed in Examples 4 and 5. Agrobacterium-mediated gene transfer experiments were performed using these 15 transformation vectors. The production of transgenic white clover plants carrying the white clover TrCSa, TrCSb, TrCSd, TrMDH and TrPEPC cDNAs, either singly or in combination, is described here in detail (Table 7). Preparation of Agrobacterium 20 Agrobacterium tumefaciens strain AGL-1 transformed with one of the binary vector constructs detailed in Example 6 were streaked on LB medium containing 50 pg/ml rifampicin and 50 pg/ml kanamycin and grown at 27 IC for 48 hours. A single colony was used to inoculate 5 ml of LB medium containing 50 pg/ml rifampicin and 50 pg/ml kanamycin and grown over night at 27 IC and 250 rpm on an orbital shaker. 25 The overnight culture was used as an inoculum for 500 ml of LB medium containing 50 pg/ml kanamycin only. Incubation was over night at 27 IC and 250 rpm on an orbital shaker in a 2 I Erlenmeyer flask.

43 Preparation of white clover seeds 1 spoon of seeds (ca. 500) was placed into a 280 pm mesh size sieve and washed for 5 min under running tap water, taking care not to wash seeds out of sieve. In a laminar flow hood, seeds were transferred with the spoon into an autoclaved 100 ml 5 plastic culture vessel. A magnetic stirrer (wiped with 70% EtOH) and ca. 30 ml 70% EtOH were added, and the seeds were stirred for 5 min. The EtOH was discarded and replaced by 50 ml 1.5% sodium hypochlorite. The seeds were stirred for an additional 45 - 60 min on a magnetic stirrer. The sodium hypochlorite was then discarded and the seeds rinsed 3 to 4 times with autoclaved ddH 2 0. Finally 30 ml of 10 ddH 2 0 were added, and seeds incubated over night at 10 - 15'C in an incubator. Agrobacterium-mediated transformation of white clover The seed coat and endosperm layer of the white clover seeds prepared as above were removed with a pair of 18 G or 21 G needles. The cotyledons were cut from the hypocotyl leaving a ca. 1.5 mm piece of the cotyledon stalk. The cotyledons 15 were transferred to a petridish containing ddH 2 0. After finishing the isolation of clover cotyledons, ddH 2 0 in the petridish was replaced with Agrobacterium suspension (diluted to an OD 600 = 0.2 - 0.4). The petridish was sealed with its lid and incubated for 40 min at room temperature. After the incubation period, each cotyledon was transferred to paper towel using the 20 small dissection needle, dried and plated onto regeneration medium RM73. The plates were incubated at 25'C with a 16h light/8h dark photoperiod. On day 4, the explants were transferred to fresh regeneration medium. Cotyledons transformed with Agrobacterium were transferred to RM73 containing cefotaxime (antibacterial agent) and gentamycin. The dishes were sealed with Parafilm and incubated at 25 25'C under a 16/8 h photoperiod. Explants were subcultured every three weeks for a total of nine weeks onto fresh RM 73 containing cefotaxime and gentamycin. Shoots with a green base were then transferred to root-inducing medium RIM. Roots developed after 1 - 3 weeks, and plantlets were transferred to soil when the roots were well established.

44 Preparation of genomic DNA for real-time PCR and analysis for the presence of transgenes 3 - 4 leaves of white clover plants regenerated on selective medium were harvested and freeze-dried. The tissue was homogenised on a Retsch MM300 mixer mill, then 5 centrifuged for 10 min at 1700xg to collect cell debris. Genomic DNA was isolated from the supernatant using Wizard Magnetic 96 DNA Plant System kits (Promega) on a Biomek FX (Beckman Coulter). 5 pl of the sample (50 pl) were then analysed on an agarose gel to check the yield and the quality of the genomic DNA. Genomic DNA was analysed for the presence of the transgene by real-time PCR 10 using SYBR Green chemistry. PCR primer pairs were designed using MacVector (Accelrys) or PrimerExpress (ABI). The forward primer was located within the 35S 2 promoter region and the reverse primer within the transgene to amplify products of approximately 150 - 250 bp as recommended. The positioning of the forward primer within the 35S 2 promoter region guaranteed that endogenous genes in white clover 15 were not detected. 5 pl of each genomic DNA sample was run in a 50 pl PCR reaction including SYBR Green on an ABI 7700 (Applied Biosystems) together with samples containing DNA isolated from wild type white clover plants (negative control), samples containing buffer instead of DNA (buffer control) and samples containing the plasmid used for 20 transformation (positive plasmid control). Cycling conditions used were 2 min. at 50 1C, 10 min. at 95 IC and then 40 cycles of 15 sec. at 95 1C, 1 min. at 60 1C. Preparation of genomic DNA and analysis of DNA for presence and copy number of transgene by Southern hybridisation blotting Genomic DNA for Southern hybridisation blotting was obtained from leaf material of 25 white clover plants following the CTAB method. Southern hybridisation blotting experiments were performed following standard protocols as described in Sambrook et al. (1989). In brief, genomic DNA samples were digested with appropriate restriction enzymes and the resulting fragments separated on an agarose gel. After transfer to a membrane, a cDNA fragment representing a 30 transgene or selectable marker gene was used to probe the size-fractionated DNA 45 fragments. Hybridisation was performed with either radioactively labelled probes or using the non-radioactive DIG labelling and hybridisation protocol (Boehringer) following the manufacturer's instructions. Plants were obtained after transformation with all chimeric constructs and selection 5 on medium containing gentamycin. Details of plant analysis are given in Table 5 and Figures 130, 131 and 132.

CL. CLL C6 o - 0 *

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00( I- L (. >~ CCo

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0. 00 (n 0 q m N (D a C- CY w) C: C ~ui' 0 0 i- 0(D ~ - () >~.. .0 >~ ao3 C rCf f) f UY CC Y m~ 0 4 0E 0 a4 - a-) a_ o0 C0 10- 1\ \ 47 REFERENCES Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J. (1990) "Basic local alignment search tool." J. Mol. Biol. 215, 403-410. Frohman, M.A., Dush, M.K., Martin, G.R. (1988) Rapid production of full-length 5 cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc. Natl. Acad. Sci. USA 85, 8998. Gish, W., States, D.J. (1993) Identification of protein coding regions by database similarity search. Nature Genetics 3, 266-272. Goderis, I., De Bolle, M.F.C., Francois, I., Wouters, P.F.J., Broekaert, W.F., 10 and Cammue, B.P.A. (2002) A set of modular plant transformation vectors allowing flexible insertion of up to six expression units. Plant Molecular Biology 50, 17-27. Hajdukiewicz P, Svab Z, Maliga P. (1994) The small, versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol Biol. 25, 989 15 94. Loh, E.Y., Elliott, J.F., Cwirla, S., Lanier, L.L., Davis, M.M. (1989). Polymerase chain reaction with single-sided specificity: Analysis of T-cell receptor delta chain. Science 243, 217-220. Ohara, 0., Dorit, R.L., Gilbert, W. (1989). One-sided polymerase chain 20 reaction: The amplification of cDNA. Proc. Natl. Acad Sci USA 86, 5673-5677 Sambrook, J., Fritsch, E.F., Maniatis, T. (1989). Molecular Cloning. A Laboratory Manual. Cold Spring Harbour Laboratory Press Schardl, C.L., Byrd, A.D., Benzion, G., Altschuler, M.A., Hildebrand, D.F., Hunt, A.G. (1987) Design and construction of a versatile system for the 25 expression of foreign genes in plants. Gene 61, 1-11 Finally, it is to be understood that various alterations, modifications and/or additions may be made without departing from the spirit of the present invention as outlined herein. In this specification, the term 'comprises' and its variants are not intended to 30 exclude the presence of other integers, components or steps.

48 In this specification, references to prior art are not intended to acknowledge or suggest that such prior art is part of the common general knowledge in Australia or that a person skilled in the relevant art could be reasonably expected to have ascertained, understood and regarded it as relevant.

Claims (22)

1. A substantially purified or isolated nucleic acid or nucleic acid fragment from a Trifolium species, said nucleic acid or nucleic acid fragment encoding 5 phosphoenolpyruvate carboxylase (PEPC), or complementary or antisense to a sequence encoding PEPC, said nucleic acid or nucleic acid fragment including a nucleotide sequence selected from the group consisting of (a) sequences shown in Figures 86, 88, 89, 91, 92, 94, 95, 97 and 110 hereto (SEQ ID NOS 310, 312 to 314, 315, 317 to 318, 319, 321 to 322, 323, 325 and 347 respectively); (b) 10 complements of the sequences recited in (a); (c) sequences antisense to the sequences recited in (a) and (b); (d) functionally active fragments and variants having at least approximately 95% identity to the sequences recited in (a), (b) and (c); and (e) RNA sequences corresponding to the sequences recited in (a), (b), (c) and (d). 15

2. A nucleic acid or nucleic acid fragment according to claim 1, wherein said nucleic acid or nucleic acid fragment includes a nucleotide sequence selected from the group consisting of sequences shown in Figures 86, 88, 89, 91, 92, 94, 95, 97 and 110 hereto (SEQ ID NOS 310, 312 to 314, 315, 317 to 20 318, 319, 321 to 322, 323, 325 and 347 respectively).

3. A nucleic acid or nucleic acid fragment according to claim 1 or 2, wherein said Trifolium species is Trifolium repens. 25

4. A construct including one or more nucleic acids or nucleic acid fragments according to any one of claims 1 to 3, excluding a construct including nucleic acids encoding both malate dehydrogenase (MDH) and PEPC.

5. A construct according to claim 4, wherein the one or more nucleic acids 30 or nucleic acid fragments are operably linked to one or more regulatory elements, such that the one or more nucleic acids or nucleic acid fragments are each expressed. 50

6. A construct according to claim 5, wherein the one or more regulatory elements include a promoter and a terminator, said promoter, nucleic acid or nucleic acid fragment and terminator being operably linked. 5

7. A polypeptide encoded by a nucleic acid or nucleic acid fragment according to any one of claims 1 to 3.

8. A plant cell, plant, plant seed or other plant part, transformed with a construct according any one of claims 4 to 6. 10

9. A plant, plant seed or other plant part derived from a plant cell or plant according to claim 8 and transformed with a construct according to any one of claims 4 to 6. 15

10. A method of modifying one or more of organic acid synthesis; organic acid secretion; nutrient acquisition; aluminium and acid soil tolerance; or nitrogen fixation and nodule function; in a plant, said method including introducing into said plant an effective amount of a nucleic acid or nucleic acid fragment according to any one of claims 1 to 3, or a construct according to any one of claims 4 to 6. 20

11. A method according to claim 10, wherein the method is modifying nutrient acquisition and the nutrient is phosphorous.

12. Use of a nucleic acid or nucleic acid fragment according to any one of 25 claims 1 to 3, and/or single nucleotide polymorphisms thereof, as a molecular genetic marker.

13. The use according to claim 12, wherein the molecular genetic marker is for one or more quantitative trait loci (QTL) tagging, QTL mapping, DNA fingerprinting 30 or marker assisted selections.

14. The use according to claim 12 or 13, wherein the molecular marker is used in a Trifolium species. 51

15. The use according to any one of claims 12 to 14, wherein the molecular marker is used to select for plant tolerance to abiotic stresses, nutrient acquisition efficiency, nitrogen fixation or modulation. 5

16. A substantially purified or isolated nucleic acid or nucleic acid fragment including a single nucleotide polymorphism (SNP) from a nucleic acid fragment according to any one of claims 1 to 3.

17. A nucleic acid or nucleic acid fragment including an SNP according to 10 claim 16, wherein said nucleic acid or nucleic acid fragment is from Trifolium repens.

18. A substantially purified or isolated PEPC polypeptide from a Trifolium species, said polypeptide including an amino acid sequence selected from the 15 group consisting of (a) sequences shown in Figures 87, 90, 93, 96, 98 and 111 hereto (SEQ ID NOS 311, 316, 320, 324, 326, and 348, respectively); and (b) functionally active fragments and variants having at least approximately 90% identity to the sequences recited in (a). 20

19. A polypeptide according to claim 18, wherein said polypeptide includes an amino acid sequence selected from the group consisting of sequences shown in Figures 87, 90, 93, 96, 98 and 111 hereto (SEQ ID NOS 311, 316, 320, 324, 326, and 348, respectively). 25

20. A polypeptide according to claim 18 or 19, wherein said Trifolium species is Trifolium repens.

21. A nucleic acid or nucleic acid fragment according to claim 1 substantially as hereinbefore described with reference to any one of the figures or examples. 30

22. A polypeptide according to claim 18 substantially as hereinbefore described with reference to any one of the figures or examples.