AU2002252826B2

AU2002252826B2 - Modification of metal-handling in plants

Info

Publication number: AU2002252826B2
Application number: AU2002252826A
Authority: AU
Inventors: Michael Emmerling; Eng Kok Ong; Timothy Ivor Sawbridge; German Spangenberg
Original assignee: Agriculture Victoria Services Pty Ltd
Current assignee: Agriculture Victoria Services Pty Ltd
Priority date: 2001-05-07
Filing date: 2002-05-07
Publication date: 2008-01-17
Anticipated expiration: 2022-05-07
Also published as: NZ529211A; NZ560526A; WO2002090491A3; AUPR482001A0; WO2002090491A2

Description

WO 02/090491 PCT/AU02/00565 1 MODIFICATION OF METAL-HANDLING IN PLANTS The present invention relates to nucleic acids encoding amino acid sequences for metal-handling enzymes, such as copper chaperones, high affinity potassium transporters, metallothioneins, aluminium-stress induced proteins, cadmium resistance proteins, copper transporting ATPases, blue copper binding proteins, copper transporters, cadmium induced proteins, zinc transporters, zinc binding proteins and phytochelatin synthases, in plants and the use thereof for modifying metal handling in plants, including modification of plant heavy metal detoxification; modification of plant tolerance to metals such as copper, zinc, cadmium and aluminium; modification of plant capacity for accumulation or hyperaccumulation of metals such as cadmium; modification of plant intracellular metal trafficking pathways such as intracellular copper-delivery systems for the delivery of copper to ethylene receptors and transport of copper from senescing leaves; modification of plant uptake of nutrients such as potassium, zinc, manganese and copper; modification of plant capacity of essential heavy metal homeostasis; modification of plant metabolism and/or development associated with heavy metals; and modification of plant responses to toxic or suboptimal levels of metals.

One of the most important mechanisms in the heavy metal tolerance of higher plants is the prevention of toxic heavy metal concentrations in the cytoplasm and organelles. Heavy metal toxicity can elicit a variety of adaptation responses in plants. A mechanism for heavy metal detoxification is the chelation of the metal ion by a ligand. A variety of metal binding ligands exist in plants.

Furthermore, plant cells must accumulate certain levels of metals, such as copper, and distribute them to the cellular components that require them while preventing their toxic effects. A range of metal-handling enzymes such as proteins induced by heavy metals, heavy metal binding proteins and high affinity metal transporters are known in plants. They include copper chaperones (CCH), high affinity potassium transporters (HAK), metallothioneins aluminium-stress induced proteins (WALl), cadmium resistance proteins (YCF), copper transporting ATPases (CTA), blue copper binding proteins (BCB), copper transporters (CTR), cadmium induced proteins zinc transporters zinc binding proteins (ZB) and phytochelatin synthases (PCS).

WO 02/090491 PCT/AU02/00565 2 While nucleic acid sequences encoding some CCH, HAK, MT, WALI, YCF, CTA, BCB, CTR, CI, ZT, ZB and PCS have been isolated for certain species of plants, there remains a need for materials useful in modifying metal handling in plants, for example plant heavy metal detoxification, plant tolerance to metals, plant capacity for accumulation or hyper-accumulation of metals, plant intracellular metal trafficking pathways, plant uptake of nutrients, plant capacity of essential heavy metal homeostasis, plant metabolism and/or development associated with heavy metals, and/or plant responses to toxic or suboptimal levels of metals, in a wide range of plants, particularly in forage and turf grasses and legumes, including ryegrasses, fescues and clovers, and for methods for their use.

It is an object of the present invention to overcome, or at least alleviate, one or more of the difficulties or deficiencies associated with the prior art.

In one aspect, the present invention provides substantially purified or isolated nucleic acids or nucleic acid fragments encoding CCH, HAK, MT, WALl, YCF, CTA, BCB, CTR, CI, ZT, ZB and PCS from a ryegrass (Lolium) or fescue (Festuca) species and functionally active fragments and variants thereof.

The present invention also provides substantially purified or isolated nucleic acids or nucleic acid fragments encoding amino acid sequences for a class of proteins which are related to CCH, HAK, MT, WALI, YCF, CTA, BCB, CTR, CI, ZT, ZB and PCS and functionally active fragments and variants thereof. Such proteins are referred to herein as CCH-like, HAK-like, MT-like, WALI-like, YCFlike, CTA-like, BCB-like, CTR-like, Cl-like, ZT-like, ZB-like and PCS-like, respectively.

The individual or simultaneous enhancement or otherwise manipulation of CCH, HAK, MT, WALl, YCF, CTA, BCB, CTR, CI, ZT, ZB and/or PCS or like gene activities in plants may modify metal handling in plants, for example it may enhance or otherwise alter plant heavy metal detoxification, for example detoxification of copper, zinc and/or aluminium; enhance or otherwise alter plant tolerance to metals, for example tolerance to cadmium, aluminium, and/or zinc; enhance or otherwise alter the plant capacity for accumulation or hyper- WO 02/090491 PCT/AU02/00565 3 accumulation of metals, for example the capacity to hyper-accumulate copper, cadmium and/or zinc; enhance or otherwise alter plant intracellular metal trafficking pathways, for example alter intracellular copper-delivery systems for the delivery of copper to ethylene receptors and transport/mobilisation of copper from senescing leaves; enhance or otherwise alter plant uptake of nutrients, for example the uptake of potassium, copper, manganese and/or zinc; enhance or otherwise alter the plant capacity of essential heavy metal homeostasis, for example homeostasis of copper and/or zinc; enhance or otherwise alter plant metabolism and/or development associated with heavy metals, for example ethylene response and/or sulphur metabolism; enhance or otherwise alter the plant responses to toxic or suboptimal levels of metals in a wide range of plants.

The individual or simultaneous enhancement or otherwise manipulation of CCH, HAK, MT, WALI, YCF, CTA, BCB, CTR, Cl, ZT, ZB and/or PCS or like gene activities in plants has significant consequences for a range of applications in, for example, plant production and phytoremediation of contaminated soils. For example, it has applications in increasing plant tolerance to metals such as cadmium, such as copper, zinc, aluminium; in increasing plant performance to a wide range of environmental stresses due to heavy metals; in reducing plant damage caused by environmental stresses such as exposure to heavy metals; in improving plant biomass productivity on soils contaminated with heavy metals, especially toxic levels of heavy metals; in protecting plant cells against toxic effects of heavy metals; in phytoremediation of soils contaminated with heavy metals such as cadmium, zinc, copper, nickel, mercury, lead, arsenate and selenite; in altering plant response to ethylene; in enhancing plant nutrition in nutrient deficient soils; and in altering metal transport and mobilisation from senescing leaves.

Methods for the manipulation of CCH, HAK, MT, WALl, YCF, CTA, BCB, CTR, CI, ZT, ZB and/or PCS or like gene activities in plants, including grass species such as ryegrasses (Lolium species) and fescues (Festuca species), and legumes such as clovers (Trifolium species) may facilitate the production of, for example, pasture and turf grasses and pasture legumes with modified metal handling such as enhanced tolerance to metals such as cadmium, copper, zinc WO 02/090491 PCT/AU02/00565 4 and/or aluminium, enhanced uptake of nutrients, modified response to ethylene, and/or enhanced tolerance to a range of heavy metals.

The ryegrass (Lolium) or fescue (Festuca) species may be of any suitable type, including Italian or annual ryegrass, perennial ryegrass, tall fescue, meadow fescue and red fescue. Preferably the species is a ryegrass, more preferably perennial ryegrass perenne). Perennial ryegrass (Lolium perenne is a key pasture grass 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 bases, and combinations thereof.

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 nucleic acid fragment present in a living plant is not isolated, but the same nucleic acid or nucleic acid fragment separated from some or all of the coexisting materials in the natural system, is isolated. Such nucleic acids or nucleic acid fragments could be part of a vector and/or such nucleic acids or nucleic acid fragments could be part of a composition, and still be isolated in that such a vector or composition is not part of its natural environment.

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 nucleotide sequence that is assembled from two or more constituent nucleotide sequences that share common or overlapping regions of sequence homology. For example, the nucleotide sequence of two or more 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 corresponding nucleic acids or nucleic acid fragments) can be assembled into a single contiguous nucleotide sequence.

005065110 O In a preferred embodiment the present invention provides a substantially purified or isolated nucleic acid or nucleic acid fragment encoding a zinc binding Sprotein (ZB) from a Lolium species; or a functionally active fragment or variant thereof.

Preferably, the Lolium species is Lolium perenne or Lolium arundinaceum.

(N

0, In a preferred embodiment, the present invention provides a substantially purified or isolated nucleic acid or nucleic acid fragment, encoding ZB and including a nucleotide sequence selected from the group consisting of sequences shown in c- Figures 104, 106, 107, 109 and 186 hereto (Sequence ID Nos: 538, 540 to 547, 548, 550 to 551, and 582, respectively); complements of the sequences recited in sequences antisense to the sequences recited in and and functionally active fragments and variants of the sequences recited in and Preferably, the functionally active fragments and variants have at least approximately 90% identity to the relevant part of the sequences recited in (b) and and have a size of at least 20 nucleotides.

In a preferred embodiment of this aspect of the invention, the substantially purified or isolated nucleic acid or nucleic acid fragment encoding a CCH or CCH-like protein includes a nucleotide sequence selected from the group consisting of (a) sequences shown in Figures 1, 3, 4, 6, 7, 9, 11 and 116 hereto (Sequence ID Nos: 1, 3 to 6, 7, 9 to 13, 14, 16, 18 to 20, and 560, respectively); complements of the sequences recited in sequences antisense to the sequences recited in (a) and and functionally active fragments and variants of the sequences recited in and In a further preferred embodiment of this aspect of the invention, the substantially purified or isolated nucleic acid or nucleic acid fragment encoding a HAK or HAK-like protein includes a nucleotide sequence selected from the group consisting of sequences shown in Figures 12 and 14 hereto (Sequence ID Nos: 21 and 23 to 24, respectively); complements of the sequences recited in (c) 005065110 r- 0 sequences antisense to the sequences recited in and and functionally N active fragments and variants of the sequences recited in and In a further preferred embodiment of this aspect of the invention, the substantially purified or isolated nucleic acid or nucleic acid fragment encoding a MT or MT-like protein includes a nucleotide sequence selected from the group consisting c of sequences shown in Figures 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 32, 33, 00 N 35, 36, 38, 39, 41, 42, 44, 45, 47, 48, 50, 51, 53, 54, 56, 57, 59, 123, 130, 136 and cN 143 hereto (Sequence ID Nos: 25, 27 to 100, 101, 103 to 142, 143, 145 to 188, 189, 0 191 to 214, 215, 217 to 232, 233, 235 to 266, 267, 269 to 308, 309, 311 to 319, 320, 0 cN 10 322 to 339, 340, 342 to 357, 358, 360 to 365, 366, 368 to 383, 384, 386 to 388, 389, 391 to 394, 395, 397 to 399, 562, 564, 566 and 568, respectively); complements of the sequences recited in sequences antisense to the sequences recited in and and functionally active fragments and variants of the sequences recited in and In a further preferred embodiment of this aspect of the invention, the substantially purified or isolated nucleic acid or nucleic acid fragment encoding a WALI or WALI-like protein includes a nucleotide sequence selected from the WO 02/090491 PCT/AU02/00565 6 group consisting of sequences shown in Figures 60, 62, 63, 65, 66, 68, 69, 71, 72, 74, 75, 77, 78, 80, 81, 83, 84, 86, 150, 156 and 163 hereto (Sequence ID Nos: 400, 402 to 416, 417, 419 to 439, 440, 442 to 450, 451, 453 to 462, 463, 465 to 480, 481, 483 to 486, 487, 489 to 490, 491, 493 to 494, 495, 497 to 498, 570, 572 and 574, respectively); complements of the sequences recited in (c) sequences antisense to the sequences recited in and and functionally active fragments and variants of the sequences recited in and In a further preferred embodiment of this aspect of the invention, the substantially purified or isolated nucleic acid/or nucleic acid fragment encoding a YCF or YCF-like protein includes a nucleotide sequence selected from the group consisting of sequences shown in Figures 87 and 89 hereto (Sequence ID Nos: 499 and 501 to 502, respectively); complements of the sequences recited in sequences antisense to the sequences recited in and and (d) functionally active fragments and variants of the sequences recited in and In a still further preferred embodiment of this aspect of the invention, the substantially purified or isolated nucleic acid or nucleic acid fragment encoding a CTA or CTA-like protein includes a nucleotide sequence selected from the group consisting of sequences shown in Figures 90 and 92 hereto (Sequence ID Nos: 503 and 505 to 509, respectively); complements of the sequences recited in sequences antisense to the sequences recited in and and (d) functionally active fragments and variants of the sequences recited in and In a still further preferred embodiment of this aspect of the invention, the substantially purified or isolated nucleic acid or nucleic acid fragment encoding a BCB or BCB-like protein includes a nucleotide sequence selected from the group consisting of sequences shown in Figures 93, 95 and 170 hereto (Sequence ID Nos: 510, 512 to 513 and 576, respectively); complements of the sequences recited in sequences antisense to the sequences recited in and and functionally active fragments and variants of the sequences recited in (b) and WO 02/090491 PCT/AU02/00565 7 In a still further preferred embodiment of this aspect of the invention, the substantially purified or isolated nucleic acid or nucleic acid fragment encoding a CTR or CTR-like protein includes a nucleotide sequence selected from the group consisting of sequences shown in Figures 96 and 176 hereto (Sequence ID Nos: 514 and 578, respectively); complements of the sequences recited in sequences antisense to the sequences recited in and and (d) functionally active fragments and variants of the sequences recited in and In a still further preferred embodiment of this aspect of the invention, the substantially purified or isolated nucleic acid or nucleic acid fragment encoding a CI or Cl-like protein includes a nucleotide sequence selected from the group consisting of sequences shown in Figures 98 and 100 hereto (Sequence ID Nos: 516 and 518 to 531, respectively); complements of the sequences recited in sequences antisense to the sequences recited in and and (d) functionally active fragments and variants of the sequences recited in and In a still further preferred embodiment of this aspect of the invention, the substantially purified or isolated nucleic acid or nucleic acid fragment encoding a ZT or ZT-like protein includes a nucleotide sequence selected from the group consisting of sequences shown in Figures 101, 103 and 182 hereto (Sequence ID Nos: 532, 534 to 537, and 580, respectively); complements of the sequences recited in sequences antisense to the sequences recited in (a) and and functionally active fragments and variants of the sequences recited in and In a still further preferred embodiment of this aspect of the invention, the substantially purified or isolated nucleic acid or nucleic acid fragment encoding a ZB or ZB-like protein includes a nucleotide sequence selected from the group consisting of sequences shown in Figures 104, 106, 107, 109 and 186 hereto (Sequence ID Nos: 538, 540 to 547, 548, 550 to 551, and 582, respectively); (b) complements of the sequences recited in sequences antisense to the sequences recited in and and functionally active fragments and variants WO 02/090491 PCT/AU02/00565 8 of the sequences recited in and In a still further preferred embodiment of this aspect of the invention, the substantially purified or isolated nucleic acid or nucleic acid fragment encoding a PCS or PCS-like protein includes a nucleotide sequence selected from the group consisting of sequences shown in Figures 110, 112 and 113 hereto (Sequence ID Nos: 552, 554 to 557, and 558, respectively); complements of the sequences recited in sequences antisense to the sequences recited in (a) and and functionally active fragments and variants of the sequences recited in and By "functionally active" in relation to nucleic acids it is meant that the fragment or variant (such as an analogue, derivative or mutant) is capable of modifying metal handling in plants, such as plant heavy metal detoxification, plant tolerance to metals, plant capacity for accumulation or hyper-accumulation of metals, plant intracellular metal trafficking pathways, plant uptake of nutrients, plant capacity of essential heavy metal homeostasis, plant metabolism and/or development associated with heavy metals, and/or plant responses to toxic or suboptimal levels of metals. Such variants include naturally occurring allelic variants and non-naturally occurring variants. Additions, deletions, substitutions and derivatizations of one or more of the nucleotides 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 at least approximately 80% identity to the relevant 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 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 nucleotides, more preferably at least 15 nucleotides, most preferably at least nucleotides.

Nucleic acids or nucleic acid fragments encoding at least a portion of several CCH, HAK, MT, WALl, YCF, CTA, BCB, CTR, CI, ZT, ZB and PCS have WO 02/090491 PCT/AU02/00565 9 been isolated and identified. The nucleic acid fragments of the present invention may be used to isolate cDNAs and genes encoding homologous proteins from the same or other plant species. Isolation of homologous genes using sequencedependent protocols, such as methods of nucleic acid hybridisation, and methods of DNA and RNA amplification as exemplified by various uses of nucleic acid amplification technologies polymerase chain reaction, ligase chain reaction), is well known in the art.

For example, genes encoding other CCH, HAK, MT, WALl, YCF, CTA, BCB, CTR, CI, ZT, ZB and PCS, or like proteins, either as cDNAs or genomic DNAs, may be isolated directly by using all or a portion of the 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 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 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.

In addition, short segments of the nucleic acids or nucleic acid fragments of the present invention may be used in amplification protocols to amplify longer 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 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.

005065110 O For example, those skilled in the art can follow the RACE protocol [Frohman c et al. (1988) Proc. Natl. Acad Sci. 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; l Loh et al. (1989) Science 243:217; the entire disclosures of which are incorporated 00 C herein by reference]. Products generated by the 3' and 5' RACE procedures may be c combined to generate full-length cDNAs.

c 10 In a second aspect of the present invention there is provided a substantially purified or isolated polypeptide from a ryegrass (Lolium) or fescue (Festuca) species, selected from the group consisting of CCH and CCH-like, HAK and HAK-like, MT and MT-like, WALl and WALl-like, YCF and YCF-like, CTA and CTA-like, BCB and BCB-like, CTR and CTR-like, CI and Cl-like, ZT and ZT-like, ZB and ZB-like and PCS and or PCS-like proteins; and functionally active fragments and variants thereof.

The ryegrass (Lolium) or fescue (Festuca) species may be of any suitable type, including Italian or annual ryegrass, perennial ryegrass, tall fescue, meadow fescue and red fescue. Preferably the species is a ryegrass, more preferably perennial ryegrass perenne).

In a preferred embodiment, the present invention provides a substantially purified or isolated ZB polypeptide from a Lolium species; or a functionally active fragment or variant thereof.

Preferably, the Lolium species is Lolium perenne or Lolium arundinaceum.

In a preferred embodiment the present invention provides a substantially purified or isolated ZB polypeptide including an amino acid sequence selected from the group consisting of sequences shown in Figures 105, 108 and 187 hereto (Sequence ID Nos: 539, 549 and 583, respectively); and functionally active fragments and variants thereof.

005065110 O Preferably, the functionally active fragments and variants have at least C approximately 90% identity with the recited sequences and have a size of at least a amino acids.

C1 In a preferred embodiment of this aspect of the invention, the substantially purified or isolated CCH or CCH-like polypeptide includes an amino acid sequence c selected from the group consisting of sequences shown in Figures 2, 5, 8, 10 and 00 c 117 hereto (Sequence ID Nos: 2, 8, 15, 17 and 561, respectively) and functionally C active fragments and variants thereof.

SIn a further preferred embodiment of this aspect of the invention, the substantially purified or isolated HAK or HAK-like polypeptide includes an amino acid sequence shown in Figure 13 hereto (Sequence ID No: 22) and functionally active fragments and variants thereof.

WO 02/090491 PCT/AU02/00565 11 In a further preferred embodiment of this aspect of the invention, the substantially purified or isolated MT or MT-like polypeptide includes an amino acid sequence selected from the group consisting of sequences shown in Figures 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 124, 131, 137 and 144 hereto (Sequence ID Nos: 26, 102, 144, 190, 216, 234, 268, 310, 321, 341, 359, 367, 385, 390, 396, 563, 565, 567, and 569, respectively) and functionally active fragments and variants thereof.

In a still further preferred embodiment of this aspect of the invention, the substantially purified or isolated WALI or WALI-like polypeptide includes an amino acid sequence selected from the group consisting of sequences shown in Figures 61, 64, 67, 70, 73, 76, 79, 82, 85, 151, 157 and 164 hereto (Sequence ID Nos: 401, 418, 441, 452, 464, 482, 488, 492, 496, 571, 573, and 575, respectively) and functionally active fragments and variants thereof.

In a still further preferred embodiment of this aspect of the invention, the substantially purified or isolated YCF or YCF-like polypeptide includes an amino acid sequence shown in Figure 88 hereto (Sequence ID No: 500) and functionally active fragments and variants thereof.

In a still further preferred embodiment of this aspect of the invention, the substantially purified or isolated CTA or CTA-like polypeptide includes an amino acid sequence shown in Figure 91 hereto (Sequence ID No: 504) and functionally active fragments and variants thereof.

In a still further preferred embodiment of this aspect of the invention, the substantially purified or isolated BCB or BCB-like polypeptide includes an amino acid sequence selected from the group consisting of the sequences shown in Figures 94 and 171 hereto (Sequence ID Nos: 511 and 577, respectively) and functionally active fragments and variants thereof.

In a still further preferred embodiment of this aspect of the invention, the substantially purified or isolated CTR or CTR-like polypeptide includes an amino acid sequence selected from the group consisting of the sequences shown in WO 02/090491 PCT/AU02/00565 12 Figures 97 and 177 hereto (Sequence ID Nos: 515 and 579, respectively) and functionally active fragments and variants thereof.

In a still further preferred embodiment of this aspect of the invention, the substantially purified or isolated Cl or Cl-like polypeptide includes an amino acid sequence shown in Figure 99 hereto (Sequence ID No: 517) and functionally active fragments and variants thereof.

In a still further preferred embodiment of this aspect of the invention, the substantially purified or isolated ZT or ZT-like polypeptide includes an amino acid sequence selected from the group consisting of the sequences shown in Figures 102 and 183 (Sequence ID Nos: 533 and 581, respectively) hereto and functionally active fragments and variants thereof.

In a still further preferred embodiment of this aspect of the invention, the substantially purified or isolated ZB or ZB-like polypeptide includes an amino acid sequence selected from the group consisting of sequences shown in Figures 105, 108 and 187 hereto (Sequence ID Nos: 539, 549 and 583, respectively) and functionally active fragments and variants thereof.

In a still further preferred embodiment of this aspect of the invention, the substantially purified or isolated PCS or PCS-like polypeptide includes an amino acid sequence selected from the group consisting of sequences shown in Figures 111 and 114 hereto (Sequence ID Nos: 553 and 559, respectively) and functionally active fragments and variants thereof.

By "functionally active" in relation to polypeptides is meant that the fragment or variant has one or more of the biological properties of the proteins CCH, CCHlike, HAK, HAK-like, MT, MT-like, WALI, WALl-like, YCF, YCF-like, CTA, CTA-like, BCB, BCB-like, CTR, CTR-like, CI, Cl-like, ZT, ZT-like, ZB, ZB-like, PCS and PCS-like, respectively. 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 at least approximately 60% identity to WO 02/090491 PCT/AU02/00565 13 the relevant 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 has a size of at least amino acids, more preferably at least 15 amino acids, most preferably at least amino acids.

In a further embodiment of this aspect of the invention, there is provided a polypeptide recombinantly produced from a nucleic acid or nucleic acid fragments according to the present invention. Techniques for recombinantly producing polypeptides are well known to those skilled in the art.

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 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 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 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), WO 02/090491 PCT/AU02/00565 14 variations in single nucleotides between allelic forms of such nucleotide sequence, can be used as 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.

See for example, Figures 3, 6, 11, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 53, 56, 59, 62, 65, 68, 71, 74, 77, 83, 86, 89, 92, 95, 100, 103, 106, 109 and 112 hereto (Sequence ID Nos: 3 to 6, 9 to 13, 18 to 20, 27 to 100, 103 to 142, 145 to 188, 191 to 214, 217 to 232, 235 to 266, 269 to 308, 311 to 319, 322 to 339, 342 to 357, 360 to 365, 368 to 383, 386 to 388, 391 to 394, 397 to 399, 402 to 416, 419 to 439, 442 to 450, 453 to 462, 465 to 480, 483 to 486, 493 to 494, 497 to 498, 501 to 502, 505 to 509, 512 to 513, 518 to 531, 534 to 537, 540 to 547, 550 to 551, and 554 to 557, respectively).

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, or complements or sequences antisense thereto, and functionally active fragments and variants thereof.

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 nucleic acid library may be of any suitable type and is preferably a cDNA 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.

WO 02/090491 PCT/AU02/00565 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 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.

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 ryegrasses and fescues. Even more particularly, nucleic acids or nucleic acid fragments according to the present invention optionally including SNPs and/or nucleotide sequence information thereof may be used as molecular genetic markers in forage and turf grass improvement in relation to plant metal handling, for example plant heavy metal detoxification, plant tolerance to metals, plant capacity for accumulation or hyper-accumulation of metals, plant intracellular metal trafficking pathways, plant uptake of nutrients, plant capacity of essential heavy metal homeostasis, plant metabolism and/or development associated with heavy metals, and/or plant responses to toxic or suboptimal levels of metals in plants, e.g. tagging QTLs for tolerance to metals such as aluminium; copper, cadmium and/or zinc. 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 ryegrasses and fescues.

In a still further aspect of the present invention there is provided a construct including a nucleic acid or nucleic acid fragment according to the present invention.

WO 02/090491 PCT/AU02/00565 16 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 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.

In a still further aspect of the present invention there is provided a vector including a nucleic acid or nucleic acid fragment according to the present invention.

In a preferred embodiment of this aspect of the invention, the vector may include a regulatory element such as a promoter, a nucleic acid or nucleic acid fragment according to the present invention and a terminator; said regulatory element, nucleic acid or nucleic acid fragment and terminator being operatively linked.

By "operatively linked" is meant that said regulatory element is capable of causing expression of said nucleic acid or nucleic acid fragment in a plant cell and 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.

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, nonchromosomal 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 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 WO 02/090491 PCT/AU02/00565 17 replicable, integrative or viable in the plant cell.

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 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 Mosaic Virus 35S (CaMV 35S) promoter, the maize Ubiquitin promoter, and the rice Actin promoter.

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 the same gene as the promoter sequence or a different gene.

Particularly suitable terminators are polyadenylation signals, such as the CaMV polyA and other terminators from the nopaline synthase (nos), the octopine synthase (ocs) and the rbcS genes.

The vector, in addition to the regulatory element, the nucleic acid or nucleic acid 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 the neomycin phosphotransferase (npt2) gene, the hygromycin phosphotransferase (hph) gene, the phosphinothricin acetyltransferase (bar or pat) 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 amplifying expression.

WO 02/090491 PCT/AU02/00565 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 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 the use of linkers, such as synthetic linkers, for example including one or more restriction enzyme sites.

The constructs and 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, sugarcane, wheat and barley), dicotyledons (such as arabidopsis, tobacco, white clover, red clover, subterranean clover, alfalfa, eucalyptus, potato, sugarbeet, canola, soybean, chickpea) and gymnosperms. In a preferred embodiment, the constructs and vectors may be used to transform monocotyledons, preferably grass species such as ryegrasses (Lolium species) and fescues (Festuca species), even more preferably perennial ryegrass, including forage- and turf-type cultivars. In an alternate preferred embodiment, the constructs and 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 lucerne (Medicago sativa). Clovers, lucerne and medics are key pasture legumes in temperate climates throughout the world.

Techniques for incorporating the constructs and vectors of the present invention into plant cells (for example by transduction, transfection or transformation) are well known to those skilled in the art. Such techniques include Agrobacterium mediated introduction, electroporation to tissues, cells and WO 02/090491 PCT/AU02/00565 19 protoplasts, protoplast fusion, injection into reproductive organs, injection into immature embryos and high velocity projectile 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 constructs and vectors of the present invention may be selected, as described above, and then cultured in an appropriate medium to regenerate 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.

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 construct, 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 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), even 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 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 lucerne (Medicago sativa).

The present invention also provides a plant, plant seed or other plant part derived from a plant cell of the present invention.

The present invention also provides a plant, plant seed or other plant part derived from a plant of the present invention.

WO 02/090491 PCT/AU02/00565 In a further aspect of the present invention there is provided a method of modifying metal handling in a plant, said method including introducing into said plant an effective amount of a nucleic acid or nucleic acid fragment, construct and/or a vector according to the present invention.

Thus, such a method may provide a means to increase plant tolerance to metals such as cadmium, copper, zinc, and/or aluminium; increase plant performance to a wide range of environmental stresses due to heavy metals; reduce plant damage caused by environmental stresses such as exposure to heavy metals; improve plant biomass productivity on soils contaminated with heavy metals, especially toxic levels of heavy metals; protect plant cells against toxic effects of heavy metals; phytoremediate soils contaminated with heavy metals such as cadmium, zinc, copper, nickel, mercury, lead, arsenate and selenite; alter plant response to ethylene; enhance plant nutrition in nutrient deficient soils; and alter metal transport and/or mobilisation from senescing leaves in plants.

The present invention also provides a method for phytoremediation of soils contaminated with heavy metals, said method including growing on said soils plants including an effective amount of a nucleic acid or nucleic acid fragment, construct and/or a vector according to the present invention.

By "an effective amount" it is meant an 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 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.

Using the methods and materials of the present invention, metal handling in plants may be modified, for example plant heavy metal detoxification, plant tolerance to metals, plant capacity for accumulation or hyper-accumulation of WO 02/090491 PCT/AU02/00565 21 metals, plant intracellular metal trafficking pathways, plant uptake of nutrients, plant capacity of essential heavy metal homeostasis, plant metabolism and/or development associated with heavy metals, plant responses to toxic or suboptimal levels of metals, in a wide range of plants, may be increased, decreased or otherwise modified relative to an untransformed control plant. For example, tolerance to metals such as copper, cadmium, and/or zinc, or uptake of nutrients such as potassium, or accumulation of heavy metals, 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 decreased or otherwise modified, for example, by incorporating an antisense nucleic acid or nucleic acid fragment of the present invention.

The present invention will now be more fully described with reference to the 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 Figure 1 shows the consensus contig nucleotide sequence of LpCCHa (Sequence ID No: 1).

Figure 2 shows the deduced amino acid sequence of LpCCHa (Sequence ID No: 2).

Figure 3 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpCCHa (Sequence ID Nos: 3 to 6).

Figure 4 shows the consensus contig nucleotide sequence of LpCCHb (Sequence ID No: 7).

Figure 5 shows the deduced amino acid sequence of LpCCHb (Sequence ID No: 8).

WO 02/090491 PCT/AU02/00565 22 Figure 6 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpCCHb (Sequence ID Nos: 9 to 13).

Figure 7 shows the nucleotide sequence of LpCCHc (Sequence ID No: 14).

Figure 8 shows the deduced amino acid sequence of LpCCHc (Sequence ID Figure 9 shows the consensus contig nucleotide sequence of LpCCHd (Sequence ID No:16).

Figure 10 shows the deduced amino acid sequence of LpCCHd (Sequence ID No: 17).

Figure 11 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpCCHd (Sequence ID Nos: 18 to Figure 12 shows the consensus contig nucleotide sequence of LpHAKa (Sequence ID No: 21).

Figure 13 shows the deduced amino acid sequence of LpHAKa (Sequence ID No: 22).

Figure 14 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpHAKa (Sequence ID Nos: 23 to 24).

Figure 15 shows the consensus contig nucleotide sequence of LpMTa (Sequence ID No: Figure 16 shows the deduced amino acid sequence of LpMTa (Sequence ID No: 26).

WO 02/090491 PCT/AU02/00565 23 Figure 17 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpMTa (Sequence ID Nos: 27 to 100).

Figure 18 shows the consensus contig nucleotide sequence of LpMTb (Sequence ID No: 101).

Figure 19 shows the deduced amino acid sequence of LpMTb (Sequence ID No: 102).

Figure 20 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpMTb (Sequence ID Nos: 103 to 142).

Figure 21 shows the consensus contig nucleotide sequence of LpMTc (Sequence ID No: 143).

Figure 22 shows the deduced amino acid sequence of LpMTc (Sequence ID No: 144).

Figure 23 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpMTc (Sequence ID Nos: 145 to 188).

Figure 24 shows the consensus contig nucleotide sequence of LpMTd (Sequence ID No: 189).

Figure 25 shows the deduced amino acid sequence of LpMTd (Sequence ID No: 190).

Figure 26 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpMTd (Sequence ID Nos: 191 to 214).

Figure 27 shows the consensus contig nucleotide sequence of LpMTe (Sequence WO 02/090491 PCT/AU02/00565 24 ID No: 215).

Figure 28 shows the deduced amino acid sequence of LpMTe (Sequence ID No: 216).

Figure 29 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpMTe (Sequence ID Nos: 217 to 232).

Figure 30 shows the consensus contig nucleotide sequence of LpMTf (Sequence ID No: 233).

Figure 31 shows the deduced amino acid sequence of LpMTf (Sequence ID No: 234).

Figure 32 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpMTf (Sequence ID Nos: 235 to 266).

Figure 33 shows the consensus contig nucleotide sequence of LpMTg (Sequence ID No: 267).

Figure 34 shows the deduced amino acid sequence of LpMTg (Sequence ID No: 268).

Figure 35 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpMTg (Sequence ID Nos: 269 to 308).

Figure 36 shows the consensus contig nucleotide sequence of LpMTh (Sequence ID No: 309).

Figure 37 shows the deduced amino acid sequence of LpMTh (Sequence ID No: 310).

WO 02/090491 PCT/AU02/00565 Figure 38 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpMTh (Sequence ID Nos: 311 to 319).

Figure 39 shows the consensus contig nucleotide sequence of LpMTi (Sequence ID No: 320).

Figure 40 shows the deduced amino acid sequence of LpMTi (Sequence ID No: 321).

Figure 41 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpMTi (Sequence ID Nos: 322 to 339).

Figure 42 shows the consensus contig nucleotide sequence of LpMTj (Sequence ID No: 340).

Figure 43 shows the deduced amino acid sequence of LpMTj (Sequence ID No: 341).

Figure 44 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpMTj (Sequence ID Nos: 342 to 357).

Figure 45 shows the consensus contig nucleotide sequence of LpMTk (Sequence ID No: 358).

Figure 46 shows the deduced amino acid sequence of LpMTk (Sequence ID No: 359).

Figure 47 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpMTk (Sequence ID Nos: 360 to 365).

Figure 48 shows the consensus contig nucleotide sequence of LpMTI (Sequence WO 02/090491 PCT/AU02/00565 26 ID No: 366).

Figure 49 shows the deduced amino acid sequence of LpMTI (Sequence ID No: 367).

Figure 50 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpMTI (Sequence ID Nos: 368 to 383).

Figure 51 shows the consensus contig nucleotide sequence of LpMTm (Sequence ID No: 384).

Figure 52 shows the deduced amino acid sequence of LpMTm (Sequence ID No: 385).

Figure 53 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpMTm (Sequence ID Nos: 386 to 388).

Figure 54 shows the consensus contig nucleotide sequence of LpMTn (Sequence ID No: 389).

Figure 55 shows the deduced amino acid sequence of LpMTn (Sequence ID No: 390).

Figure 56 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpMTn (Sequence ID Nos: 391 to 394).

Figure 57 shows the consensus contig nucleotide sequence of LpMTo (Sequence ID No: 395).

Figure 58 shows the deduced amino acid sequence of LpMTo (Sequence ID No: 396).

WO 02/090491 PCT/AU02/00565 27 Figure 59 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpMTo (Sequence ID Nos: 397 to 399).

Figure 60 shows the consensus contig nucleotide sequence of LpWALIa (Sequence ID No: 400).

Figure 61 shows the deduced amino acid sequence of LpWALla (Sequence ID No: 401).

Figure 62 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpWALla (Sequence ID Nos: 402 to 416).

Figure 63 shows the consensus contig nucleotide sequence of LpWALIb (Sequence ID No: 417).

Figure 64 shows the deduced amino acid sequence of LpWALIb (Sequence ID No: 418).

Figure 65 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpWALIb (Sequence ID Nos: 419 to 439).

Figure 66 shows the consensus contig nucleotide sequence of LpWALIc (Sequence ID No: 440).

Figure 67 shows the deduced amino acid sequence of LpWALIc (Sequence ID No: 441).

Figure 68 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpWALIc (Sequence ID Nos: 442 to 450).

Figure 69 shows the consensus contig nucleotide sequence of LpWALId WO 02/090491 PCT/AU02/00565 28 (Sequence ID No: 451).

Figure 70 shows the deduced amino acid sequence of LpWALId (Sequence ID No: 452).

Figure 71 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpWALId (Sequence ID Nos: 453 to 462).

Figure 72 shows the consensus contig nucleotide sequence of LpWALle (Sequence ID No: 463).

Figure 73 shows the deduced amino acid sequence of LpWALle (Sequence ID No: 464).

Figure 74 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpWALle (Sequence ID Nos: 465 to 480).

Figure 75 shows the consensus contig nucleotide sequence of LpWALIf (Sequence ID No: 481).

Figure 76 shows the deduced amino acid sequence of LpWALIf (Sequence ID No: 482).

Figure 77 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpWALIf (Sequence ID Nos: 483 to 486).

Figure 78 shows the consensus contig nucleotide sequence of LpWALIg (Sequence ID No: 487).

Figure 79 shows the deduced amino acid sequence of LpWALIg (Sequence ID No; 488).

WO 02/090491 PCT/AU02/00565 29 Figure 80 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpWALIg (Sequence ID Nos: 489 to 490).

Figure 81 shows the consensus contig nucleotide sequence of LpWALIh (Sequence ID No: 491).

Figure 82 shows the deduced amino acid sequence of LpWALIh (Sequence ID No: 492).

Figure 83 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpWALIh (Sequence ID Nos: 493 to 494).

Figure 84 shows the consensus contig nucleotide sequence of LpWALli (Sequence ID No: 495).

Figure 85 shows the deduced amino acid sequence of LpWALli (Sequence ID No: 496).

Figure 86 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpWALIi (Sequence ID Nos: 497 to 498).

Figure 87 shows the consensus contig nucleotide sequence of LpYCFa (Sequence ID No: 499).

Figure 88 shows the deduced amino acid sequence of LpYCFa (Sequence ID No: 500).

Figure 89 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpYCFa (Sequence ID Nos: 501 to 502).

Figure 90 shows the consensus contig nucleotide sequence of LpCTAa WO 02/090491 PCT/AU02/00565 (Sequence ID No: 503).

Figure 91 shows the deduced amino acid sequence of LpCTAa (Sequence ID No: 504).

Figure 92 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpCTAa (Sequence ID Nos: 505 to 509.

Figure 93 shows the consensus contig nucleotide sequence of LpBCBa (Sequence ID No: 510).

Figure 94 shows the deduced amino acid sequence of LpBCBa (Sequence ID No: 511).

Figure 95 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpBCBa (Sequence ID Nos: 512 to 513).

Figure 96 shows the nucleotide sequence of LpCTRa (Sequence ID No: 514).

Figure 97 shows the deduced amino acid sequence of LpCTRa (Sequence ID No: 515).

Figure 98 shows the consensus contig nucleotide sequence of LpCla (Sequence ID No: 516).

Figure 99 shows the deduced amino acid sequence of LpCla (Sequence ID No: 517).

Figure 100 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpCla (Sequence ID Nos: 518 to 531).

Figure 101 shows the consensus contig nucleotide sequence of LpZTa (Sequence WO 02/090491 PCT/AU02/00565 31 ID No: 532).

Figure 102 shows the deduced amino acid sequence of LpZTa (Sequence ID No: 533).

Figure 103 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpZTa (Sequence ID Nos: 534 to 537).

Figure 104 shows the consensus contig nucleotide sequence of LpZBa (Sequence ID No: 538).

Figure 105 shows the deduced amino acid sequence of LpZBa (Sequence ID No: 539).

Figure 106 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpZBa (Sequence ID Nos: 540 to 547).

Figure 107 shows the consensus contig nucleotide sequence of LpZBb (Sequence ID No: 548).

Figure 108 shows the deduced amino acid sequence of LpZBb (Sequence ID No: 549).

Figure 109 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpZBb (Sequence ID Nos: 550 to 551).

Figure 110 shows the consensus contig nucleotide sequence of LpPCSa (Sequence ID No: 552).

Figure 111 shows the deduced amino acid sequence of LpPCSa (Sequence ID No: 553).

WO 02/090491 PCT/AU02/00565 32 Figure 112 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpPCSa (Sequence ID Nos: 554 to 557).

Figure 113 shows the nucleotide sequence of LpPCSb (Sequence ID No: 558).

Figure 114 shows the deduced amino acid sequence of LpPCSb (Sequence ID No: 559).

Figure 115 shows a plasmid map of the cDNA encoding perennial ryegrass CCHa.

Figure 116 shows the nucleotide sequence of perennial ryegrass CCHa cDNA (Sequence ID No: 560).

Figure 117 shows the deduced amino acid sequence of perennial ryegrass CCHa cDNA (Sequence ID No: 561).

Figure 118 shows plasmid maps of sense and antisense constructs of LpCCHa in pDH51 transformation vector.

Figure 119 shows plasmid maps of sense and antisense constructs of LpCCHa in pKYLX71:35S 2 binary transformation vector.

Figure 120 shows plasmid maps of sense and antisense constructs of LpCCHa in pPZP221:35S 2 binary transformation vector.

Figure 121 shows screening by Southern hybridisation for RFLPs using LpCCHa as a probe.

Figure 122 shows a plasmid map of the cDNA encoding perennial ryegrass MTb.

Figure 123 shows the nucleotide sequence of perennial ryegrass MTb cDNA (Sequence ID No: 562).

Figure 124 shows the deduced amino acid sequence of perennial ryegrass MTb WO 02/090491 PCT/AU02/00565 33 cDNA (Sequence ID No: 563).

Figure 125 shows plasmid maps of sense and antisense constructs of LpMTb in pDH51 transformation vector.

Figure 126 shows a plasmid map of the antisense construct of LpMTb in pKYLX71:35S 2 binary transformation vector.

Figure 127 shows plasmid maps of sense and antisense constructs of LpMTb in pPZP221:35S 2 binary transformation vector.

Figure 128 shows screening by Southern hybridisation for RFLPs using LpMTb as a probe.

Figure 129 shows a plasmid map of the cDNA encoding perennial ryegrass MTg.

Figure 130 shows the nucleotide sequence of perennial ryegrass MTg cDNA (Sequence ID No: 564).

Figure 131 shows the deduced amino acid sequence of perennial ryegrass MTg cDNA (Sequence ID No: 565).

Figure 132 shows plasmid maps of sense and antisense constructs of LpMTg in pDH51 transformation vector.

Figure 133 shows plasmid maps of sense and antisense constructs of LpMTg in pPZP221:35S 2 binary transformation vector.

Figure 134 shows screening by Southern hybridisation for RFLPs using LpMTg as a probe.

Figure 135 shows a plasmid map of the cDNA encoding perennial ryegrass MTh.

Figure 136 shows the nucleotide sequence of perennial ryegrass MTh cDNA (Sequence ID No: 566).

WO 02/090491 PCT/AU02/00565 34 Figure 137 shows the deduced amino acid sequence of perennial ryegrass MTh cDNA (Sequence ID No: 567).

Figure 138 shows plasmid maps of sense and antisense constructs of LpMTh in pDH51 transformation vector.

Figure 139 shows plasmid maps of sense and antisense constructs of LpMTh in pKYLX71:35S 2 binary transformation vector.

Figure 140 shows plasmid maps of sense and antisense constructs of LpMTh in pPZP221:35S 2 binary transformation vector.

Figure 141 shows screening by Southern hybridisation for RFLPs using LpMTh as a probe.

Figure 142 shows a plasmid map of the cDNA encoding perennial ryegrass MTj.

Figure 143 shows the nucleotide sequence of perennial ryegrass MTj cDNA (Sequence ID No: 568).

Figure 144 shows the deduced amino acid sequence of perennial ryegrass MTj cDNA (Sequence ID No: 569).

Figure 145 shows plasmid maps of sense and antisense constructs of LpMTj in pDH51 transformation vector.

Figure 146 shows plasmid maps of sense and antisense constructs of LpMTj in pKYLX71:35S 2 binary transformation vector.

Figure 147 shows plasmid maps of sense and antisense constructs of LpMTj in pPZP221:35S 2 binary transformation vector.

Figure 148 shows screening by Southern hybridisation for RFLPs using LpMTj as a probe.

WO 02/090491 PCT/AU02/00565 Figure 149 shows a plasmid map of the cDNA encoding perennial ryegrass WALIc.

Figure 150 shows the nucleotide sequence of perennial ryegrass WALIc cDNA (Sequence ID No: 570).

Figure 151 shows the deduced amino acid sequence of perennial ryegrass WALIc cDNA (Sequence ID No: 571).

Figure 152 shows plasmid maps of sense and antisense constructs of LpWALIc in pDH51 transformation vector.

Figure 153 shows plasmid maps of sense and antisense constructs of LpWALIc in pPZP221:35S 2 binary transformation vector.

Figure 154 shows screening by Southern hybridisation for RFLPs using LpWALIc as a probe.

Figure 155 shows a plasmid map of the cDNA encoding perennial ryegrass WALIf.

Figure 156 shows the nucleotide sequence of perennial ryegrass WALIf cDNA (Sequence ID No: 572).

Figure 157 shows the deduced amino acid sequence of perennial ryegrass WALIf cDNA (Sequence ID No: 573).

Figure 158 shows plasmid maps of sense and antisense constructs of LpWALIf in pDH51 transformation vector.

Figure 159 shows plasmid maps of sense and antisense constructs of LpWALIf in pKYLX71:35S 2 binary transformation vector.

Figure 160 shows plasmid maps of sense and antisense constructs of LpWALIf in pPZP221:35S 2 binary transformation vector.

WO 02/090491 PCT/AU02/00565 36 Figure 161 shows screening by Southern hybridisation for RFLPs using LpWALIf as a probe.

Figure 162 shows a plasmid map of the cDNA encoding perennial ryegrass WALIh.

Figure 163 shows the nucleotide sequence of perennial ryegrass WALIh cDNA (Sequence ID No: 574).

Figure 164 shows the deduced amino acid sequence of perennial ryegrass WALIh cDNA (Sequence ID No: 575).

Figure 165 shows plasmid maps of sense and antisense constructs of LpWALIh in pDH51 transformation vector.

Figure 166 shows plasmid maps of sense and antisense constructs of LpWALIh in pKYLX71:35S 2 binary transformation vector.

Figure 167 shows plasmid maps of sense and antisense constructs of LpWALIh in pPZP221:35S 2 binary transformation vector.

Figure 168 shows screening by Southern hybridisation for RFLPs using LpWALIh as a probe.

Figure 169 shows a plasmid map of the cDNA encoding perennial ryegrass BCBa.

Figure 170 shows the nucleotide sequence of perennial ryegrass BCBa cDNA (Sequence ID No: 576).

Figure 171 shows the deduced amino acid sequence of perennial ryegrass BCBa cDNA (Sequence ID No: 577).

Figure 172 shows plasmid maps of sense and antisense constructs of LpBCBa in pDH51 transformation vector.

WO 02/090491 PCT/AU02/00565 37 Figure 173 shows plasmid maps of sense and antisense constructs of LpBCBa in pPZP221:35S 2 binary transformation vector.

Figure 174 shows screening by Southern hybridisation for RFLPs using LpBCBa as a probe.

Figure 175 shows a plasmid map of the cDNA encoding perennial ryegrass CTRa.

Figure 176 shows the nucleotide sequence of perennial ryegrass CTRa cDNA (Sequence ID No: 578).

Figure 177 shows the deduced amino acid sequence of perennial ryegrass CTRa cDNA (Sequence ID No: 579).

Figure 178 shows plasmid maps of sense and antisense constructs of LpCTRa in pDH51 transformation vector.

Figure 179 shows plasmid maps of sense and antisense constructs of LpCTRa in pPZP221:35S 2 binary transformation vector.

Figure 180 shows screening by Southern hybridisation for RFLPs using LpCTRa as a probe.

Figure 181 shows a plasmid map of the cDNA encoding perennial ryegrass ZTa.

Figure 182 shows the nucleotide sequence of perennial ryegrass ZTa cDNA (Sequence ID No: 580).

Figure 183 shows the deduced amino acid sequence of perennial ryegrass ZTa cDNA (Sequence ID No: 581).

Figure 184 shows screening by Southern hybridisation for RFLPs using LpZTa as a probe.

Figure 185 shows a plasmid map of the cDNA encoding perennial ryegrass ZBa.

WO 02/090491 PCT/AU02/00565 38 Figure 186 shows the nucleotide sequence of perennial ryegrass ZBa cDNA (Sequence ID No: 582).

Figure 187 shows the deduced amino acid sequence of perennial ryegrass ZBa cDNA (Sequence ID No: 583).

Figure 188 shows plasmid maps of sense and antisense constructs of LpZBa in pDH51 transformation vector.

Figure 189 shows plasmid maps of sense and antisense constructs of LpZBa in pPZP221:35S 2 binary transformation vector.

Figure 190 shows screening by Southern hybridisation for RFLPs using LpERa as a probe.

Figure 191 shows A, infiltration of Arabidopsis plants; B, selection of transgenic Arabidopsis plants on medium containing 75 pg/ml gentamycin; C, young transgenic Arabidopsis plants; D, E, two representative results of real-time PCR analysis of Arabidopsis transformed with chimeric genes involved in metal stress protection.

Figure 192 shows the genetic map detailing the relation of perennial ryegrass genes involved in metal stress protection.

Figure 193 shows a subgrid of a microarray for the expression profiling of perennial ryegrass genes involved in metal stress protection.

WO 02/090491 PCT/AU02/00565 39 EXAMPLE 1 Preparation of cDNA libraries, isolation and sequencing of cDNAs coding for CCH, CCH-like, HAK, HAK-like, MT, MT-like, WALl, WALl-like, YCF, YCF-like, CTA, CTA-like, BCB, BCB-like, CTR, CTR-like, CI, Cl-like, ZT, ZT-like, ZB, ZBlike, PCS and PCS-like proteins from perennial ryegrass (Lolium perenne) cDNA libraries representing mRNAs from various organs and tissues of perennial ryegrass (Lolium perenne) were prepared. The characteristics of the libraries are described below (Table 1).

TABLE 1 cDNA libraries from perennial ryegrass (Lolium perenne) 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) 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 1, 3, 6, 12 and 24 h after harvesting) Embryogenic suspension-cultured cells 11rg Non-embryogenic suspension-cultured cells 12rg Whole tillers and sliced leaves 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 Defatted pollen 16rg Leaf blades and leaf sheaths (rbcL, rbcS, cab, wir2A subtracted) WO 02/090491 PCT/AU02/00565 17rg Senescing leaves and tillers 18rg Drought-stressed tillers (pseudostems from plants subjected to PEGsimulated drought stress) 19rg Non-embryogenic suspension-cultured cells subjected to osmotic stress (grown in media with half-strength salts) 2, 3, 4, 5, 6, 24 and 48 h after transfer) Non-embryogenic suspension-cultured cells subjected to osmotic stress (grown in media with double-strength salts) 3, 4, 5, 6, 24 and 48 h after transfer) 21rg Drought-stressed tillers (pseudostems from plants subjected to PEGsimulated drought stress) 22rg Spikelets with open and maturing florets 23rg Mature roots (specific subtraction with leaf tissue) 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 (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 the protocol provided by Promega. The cDNAs in the pGEM-T Easy plasmid vector are transfected into Escherichia coli Epicurian coli 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 (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 WO 02/090491 PCT/AU02/00565 41 precut pBluescript II vectors (Stratagene) using T4 DNA ligase (New England Biolabs), followed by 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 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 analyzed using an Applied Biosystems ABI 3700 sequence analyser.

EXAMPLE 2 DNA sequence analyses The cDNA clones encoding CCH, CCH-like, HAK, HAK-like, MT, MT-like, WALl, WALl-like, YCF, YCF-like, CTA, CTA-like, BCB, BCB-like, CTR, CTR-like, Cl, Cl-like, ZT, ZT-like, ZB, ZB-like, PCS and PCS-like proteins were identified by conducting BLAST (Basic Local Alignment Search 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 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 algorithm. The identical and/or overlapping sequences were subjected to WO 02/090491 PCT/AU02/00565 42 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.

EXAMPLE 3 Identification and full-length sequencing of perennial ryegrass CCHa, MTb, MTg, MTh, MTj, WALIc, WALIf, WALIh, BCBa, CTRa, ZTa and ZBa cDNAs encoding metal handling proteins To fully characterise for the purposes of the generation of probes for hybridisation experiments and the generation of transformation vectors, perennial ryegrass CCHa, MTb, MTg, MTh, MTj, WALIc, WALIf, WALIh, BCBa, CTRa, ZTa and ZBa cDNAs encoding metal handling proteins were 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 was then used to transform chemically competent XL-1 cells (prepared inhouse, CaCl2 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 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 was completed by primer walking, i.e. oligonucleotide primers were designed to the initial sequence and used for further sequencing. In most cases the sequencing could be done from both 5' and 3' end. The sequences of the oligonucleotide primers are shown in Table 2. In some instances, however, an WO 02/090491 PCT/AU02/00565 43 extended poly-A tail necessitated the sequencing of the cDNA to be completed from the 5' end.

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.

Plasmid maps and the full cDNA sequences of perennial ryegrass OCHa, MTb, MTg, MTh, MTj, WALIc, WALlf, WAL~h, BCBa, OTRa, ZTa and ZBa cDNAs encoding metal handling proteins were obtained (Figures 115, 116, 122, 123, 129, 130, 135, 136, 142, 143, 149, 150, 155, 156, 162, 163, 169, 170, 175, 176, 181, 182, 185 and 186).

TABLE 2 List of primers used for sequencing of the full-length oDNAs gene name clone ID sequencing primer primer sequence LpBCBa 1 OrglTsF1 1 1 OrglTsFl 1 .f1 ACTTCATCTGCGACGTCC LpMTh l9rgl DsFll 1l1rgl DsF1 lIfl AGCAAGTGCAACTGCGGC LpZBa 1 Brg2QsDO2 1 9rg2QsDO2.f1 TAGAGATACTTGGCTTGC LpZTa 2Orgl RsDO4 2Orgl RsDO4.fl TCTCTATGGGCAGCTGGC 2Orgl RsDO4.f2 TCATGATGCCGAAGA&CCG 2Orgl RsDO4.f3 ACAGTCAAGATGATTCGG EXAMPLE 4 Development of transformation vectors containing chimeric genes with CCI-a, MTb, MTg, MTh, MTJ, WALIC, WALlf, WAL11h, BCBa, CTRa and ZBa cDNA sequences from perennial ryegrass To alter the expression of the proteins involved in metal handling CCHa, MTb, MTg, MTh, MTj, WAL11c, WALlf, WALlh, BOlBa, CTRa and ZBa cDNAs, WO 02/090491 PCT/AU02/00565 44 through antisense and/or sense suppression technology and for over-expression of these key enzymes in transgenic plants, a set of sense and antisense transformation vectors was produced.

cDNA fragments were generated by high fidelity PCR using the original pGEM-T Easy plasmid cDNA as a template. The primers used (Table 3) contained restriction sites for EcoRI and Xbal for directional and non-directional cloning into the target vector. After PCR amplification and restriction digest with the appropriate restriction enzyme (usually Xbal), the cDNA fragments were cloned into the corresponding site in pDH51, a pUC1 8-based transformation vector containing a CaMV 35S expression cassette. The orientation of the constructs (sense or antisense) was checked by DNA sequencing through the multi-cloning site of the vector. Transformation vectors containing chimeric genes using fulllength open reading frame cDNAs of perennial ryegrass CCHa, MTb, MTg, MTh, MTj, WALlc, WALif, WALh, BCBa, CTRa and ZBa cDNAs encoding metal handling proteins in sense and antisense orientations under the control of the CaMV 35S promoter were generated (Figures 118, 125, 132, 138, 145, 152, 158, 165,172, 178 and 188).

TABLE 3 List of primers used to PCR-amplify the open reading frames gene name clone ID primer primer sequence BCBa 10rg TsF11 1 Orgl TsF11.f GAATTCTAGATAATCAGCGAAGGATTTTGCTCG 10rgTsF1.r GAATTCTAGATACGGCAAAGACTCTTGTAAGC CCHa 03rgl AsEO4 03rg l AsEO4.f GCATTCTAGATCGCCGTCCGCCGATCTCGAG 03rglAsEO4.r GCATTCTAGAACATACTAGGGTTCATATATATGG CTRa 08rglYsDO2 08rglYsDO2.f GAATTCTAGAAAACTCAGCTGAAATGGCCATGC 08rgl1YsDO2.r GAATTCTAGAAGCACCTAGTAATCATGCCACAGC MTb 0g7rg2FsG00 O7rg2FsGl0.f GAATTCTAGATTCGCCATAATCTCATCCTTGACC 07rg2FsGl0.r GAATTCTAGATCTCACACCATCACACAAGTGCG MTg 17rgl QsAO1 17rgl QsAO1 .f GAATTCTAGATGTGTTGAGAGCTTCATCATGTCG 17rglQsAOl.r GAATTCTAGAACTCATCGATGATCCATCCATCG MTh 19rgl DsF11 19rglDsF 1.f GAATTCTAGATCGTCCAGTCTCAAGCTCGAACCG WO 02/090491 WO 02/90491PCT/AU02/00565 1 9rgl DsF1 1 .r GAATTCTAGATAGGCGCACACATACACAGGC MTj O8rg2CsCO8 O8rg2CSCO8.f GAATTCTAGATCACAAGCCAAGTCCACCATGTCG O8rg2CsCO8. r GAATTCTAGATTGGGTACTAGTACGTGTAGACG WAILIc 06rg2ZsFO8 O6rg2ZsFO8.f GCATTCTAGAAGCAGCCGGGACAAGGCCAGAGC O6rg2ZsFO8. r GCATTCTAGAACGCACATGTACGAAGCGTGTATGC WALlf 1 8rgl CsGO5 1 8rgl CSGO5.f GAATTCTAGAAGTACAGGAGACTAGCAAGCTTTGC 1 8rgl CsGO5.r GAATTCTAGATCCTCATCTCATCTCAGCTCAGC WALlh 1 7rgl RsGO6 1 7rgi RsGO6.f GAATTCTAGATGCTCACTCGAAAAGCTTCGAAGC 1 7rg 1 RsGO6.r GAATTCTAGATAAGTATCTTCATGTCGTCGTCC M~a 1 Srg2QsDO2 1 9rg2QsDO2.f GAATTCTAGAACCACCACCACCAGG3CGCTCGAGG 1 9rg2QsDO2.r GAATTCTAGAALCATGAAGACTGATCAGCAAGCAGG EXAMPLE Development of binary transformation vectors containing chimeric genes with CCHa, MTb, MTg, MTh, MTj, WALIc, WALlf, WALIh, BCBa, CTRa and ZBa cDNA sequences from perennial ryegrass To alter the expression of the metal handling proteins COHa, MTb, MTg, MTh, MTj, WALIc, WALlf, WALlh, BOBa, CTRa and ZMa, through antisense and/or sense suppression technology and for over-expression of these key proteins in transgenic plants, a set of sense and antisense transformation vectors was produced.

cDNA fragments were generated by high fidelity POR using the original pGEM-T Easy plasmid cDNA as a template. The primers used (Table 3) contained restriction sites for EcoRl and Xbal for directional and non-directional cloning into the target vector. After PCR amplification and restriction digest with the appropriate restriction enzyme (usually Xbal), the cDNA fragments were cloned into the corresponding site in pKYLX71 :35S 2 a binary transformation vector. The vector contains between the left and the right border the plant selectable marker gene nptll under the control of the nos promoter and nos terminator and an expression cassette with a CaMV 35S promoter with a duplicated enhancer region and an rbcS terminator (An et 1985; Schardl et 1987). Alternatively, the WO 02/090491 PCT/AU02/00565 46 PCR fragments were cloned into a modified pPZP binary vector (Hajdukiewicz et al., 1994). The pPZP221 vector was modified to contain the 35S 2 cassette from pKYLX71:35S 2 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 Hindll and the resulting 5' overhang filled in and T-tailed with Taq polymerase. The remainder of the original pPZP221 multicloning site was removed by digestion with EcoRI, and the expression cassette cloned into the EcoRI site and the 3' T overhang restoring the Hindlll site. This binary vector contains between the left and right border the plant selectable marker gene aaaC1 under the control of the 35S promoter and 35S terminator and the pKYLX71:35S 2 -derived expression cassette with a CaMV 35S promoter with a duplicated enhancer region and an rbcS terminator.

The orientation of the constructs (sense or antisense) was checked by restriction enzyme digest. Transformation vectors containing chimeric genes using full-length open reading frame cDNAs of perennial ryegrass CCHa, MTb, MTg, MTh, MTj, WALIc, WALIf, WALIh, BCBa, CTRa and ZBa in sense and antisense orientations under the control of the CaMV 35S2 promoter were generated (Figures 119, 120, 126, 127, 133, 139, 140, 146, 147, 153, 159, 160, 166, 167, 173, 179 and 189).

EXAMPLE 6 Production and analysis of transgenic Arabidopsis plants carrying chimeric perennial ryegrass genes CCHa, MTb, MTg, MTh, MTj, WALIc, WALIf, WALIh, BCBa, CTRa and ZBa in sense and antisense orientations involved in metal stress protection A set of transgenic Arabidopsis plants carrying chimeric perennial ryegrass genes involved in metal stress protection were produced.

WO 02/090491 PCT/AU02/00565 47 pPZP221-based transformation vectors with LpCCHa, LpMTb, LpMTg, LpMTh, LpMTj, LpWALIc, LpWALIh, LpBCBa, LpCTRa and LpZBa cDNAs comprising the full open reading frame sequences in sense and antisense orientations under the control of the CaMV 35S promoter with duplicated enhancer region (35S 2 were generated as detailed in Example 6.

Agrobacterium-mediated gene transfer experiments were performed using these transformation vectors.

The production of transgenic Arabidopsis plants carrying the perennial ryegrass CCHa, MTb, MTg, MTh, MTj, WALIc, WALIf, WALlh, BCBa, CTRa and ZBa cDNAs under the control of the CaMV 35S promoter with duplicated enhancer region (35S 2 is described here in detail.

Preparation of Arabidopsis plants Seedling punnets were filled with Debco seed raising mixture (Debco Pty.

Ltd.) to form a mound. The mound was covered with two layers of anti-bird netting secured with rubber bands on each side. The soil was saturated with water and enough seeds (Arabidopsis thaliana ecotype Columbia, Lehle Seeds #WT-02) sown to obtain approximately 15 plants per punnet. The seeds were then vernalised by placing the punnets at 4 After 48 hours the punnets were transferred to a growth room at 22 "C under fluorescent light (constant illumination, 55 pmolm2s 1 and fed with Miracle-Gro (Scotts Australia Pty. Ltd.) once a week. Primary bolts were removed as soon as they appeared. After 4 6 days the secondary bolts were approximately 6 cm tall, and the plants were ready for vacuum infiltration.

Preparation of Agrobacterium Agrobacterium tumefaciens strain AGL-1 were streaked on LB medium containing 50 yg/ml rifampicin and 50 pg/ml kanamycin and grown at 27 °C for 48 hours. A single colony was used to inoculate 5 ml of LB medium containing pg/ml rifampicin and 50 pug/ml kanamycin and grown over night at 27 2 C and 250 WO 02/090491 PCT/AU02/00565 48 rpm on an orbital shaker. The overnight culture was used as an inoculum for 500 ml of LB medium containing 50 jug/ml kanamycin only. Incubation was over night at 27 °C and 250 rpm on an orbital shaker in a 2 I Erlenmeyer flask.

The overnight cultures were centrifuged for 15 min at 5500 xg and the supernatant discarded. The cells were resuspended in 1 I of infiltration medium sucrose, 0.03% Silwet-L77 (Vac-ln-Stuff, Lehle Seeds #VIS-01)] and immediately used for infiltration.

Vacuum infiltration The Agrobacterium suspension was poured into a container (D6cor Tellfresh storer, #024) and the container placed inside the vacuum desiccator (Bel Art, #42020-0000). A punnet with Arabidopsis plants was inverted and dipped into the Agrobacterium suspension and a gentle vacuum (250 mm Hg) was applied for 2 min. After infiltration, the plants were returned to the growth room where they were kept away from direct light overnight. The next day the plants were returned to full direct light and allowed to grow until the siliques were fully developed. The plants were then allowed to dry out, the seed collected from the siliques and either stored at room temperature in a dry container or used for selection of transformants.

Selection of transformants Prior to plating the seeds were sterilised as follows. Sufficient seeds for one 150 mm petri dish (approximately 40 mg or 2000 seeds) were placed in a 1.5 ml microfuge tube. 500 p/ 70% ethanol were added for 2 min and replaced by 500 /p sterilisation solution (H 2 0:4% chlorine:5% SDS, 15:8:1). After vigorous shaking, the tube was left for 10 min after which time the sterilisation solution was replaced with 500 ul sterile water. The tube was shaken and spun for 5 sec to sediment the seeds. The washing step was repeated 3 times and the seeds were left covered with approximately 200 pl sterile water.

The seeds were then evenly spread on 150 mm petri dishes containing WO 02/090491 PCT/AU02/00565 49 germination medium (4.61 g Murashige Skoog salts, 10 g sucrose, 1 ml 1 M KOH, 2 g Phytagel, 0.5 g MES and 1 ml 1000x Gamborg's B-5 vitamins per litre) supplemented with 250 pg/ml timetin and 75 p/g/ml gentamycin. After vernalisation for 48 hours at 4 °C the plants were grown under continuous fluorescent light /pmol m-2s-1) at 22 "C to the 6 8 leaf stage and transferred to soil.

Preparation of genomic DNA 3 4 leaves of Arabidopsis plants regenerated on selective medium were harvested and freeze-dried. The tissue was homogenised on a Retsch MM300 mixer mill, then 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 pl) were then analysed on an agarose gel to check the yield and the quality of the genomic DNA.

Analysis of DNA using real-time PCR Genomic DNA was analysed for the presence of the transgene by real-time PCR using SYBR Green chemistry. PCR primer pairs (Table 4) were designed using MacVector (Accelrys). The forward primer was located within the 35S 2 promoter region and the reverse primer within the transgene to amplify products of approximately 150 bp as recommended. The positioning of the forward primer within the 35S 2 promoter region guaranteed that homologous genes in Arabidopsis were not detected.

pl of each genomic DNA sample was run in a 50 /ul PCR reaction including SYBR Green on an ABI (Applied Biosystems) together with samples containing DNA isolated from wild type Arabidopsis plants (negative control), samples containing buffer instead of DNA (buffer control) and samples containing the plasmid used for transformation (positive plasmid control).

Plants were obtained after transformation with all chimeric constructs and selection on medium containing gentamycin. The selection process and two representative real-time PCR analyses are shown in Figure 191.

WO 02/090491 PCT/AU02/00565 TABLE 4 List of primers used for Real-time PCR analysis of Arabidopsis plants transformed with chimeric perennial ryegrass genes involved in metal stress protection construct primer 1 (forward) primer 2 (reverse) LpBCBasenSe TGGAGAGGACACGCTGAAATCAC ATCCCAGCCATGCCCATCG LpBCBaanti TGGAGAGGACACGCTGAAATCAC TGCTAGGTTTCTTCCGTTGGTTTG LpCCHasense CGCACAATCCCACTATCCTTCG CAACACCTTCCATTTTGCCGAG LpCCHaanti TCATTTCATTTGGAGAGGACACGC GACGCCAGATGCTGTTCTTCAGAC LpCTRasense ATTTCATTTGGCAGGACACGCTG CATCGGAGcGCATGTTCCAGACC LpCTRaanti TGGAGAGGACACGCTGAAATCAC GCCACCCCTAAGGATGACGC LpMTbsense TGCGAGAGGACACGCTGAMJTCAC GCAGTTTGAGCCGCAGTTGC LpMTbanti CATTTGGAGAGGACACGCTGAAATC AGTGCAACCCCTGCAACTGC LpMTgsense CATTTGGAGAGGACACGCTGAATC TGAGAGTCTGAGAGGTGGTCACCCC LpMrganti ATTTCATTTGGAGAGGACACGCTG GAGAACGGAGGCTGCAAGTGC LpMrhsense TGGAGAGGACACGCTGAAATCAC GGTACATGOCGCAACCGTTG LpMThanti TGGAGAGGACACGCTGAAATCAC TCAGTTGTTGCCACTGGTCGC LpMTjsense ATTTCATTTGGAGAGGACACGCTG TCTTGGGGCACTCGTTCTTGTTAG LpMTjanti ATTTCATTTCGAGAGGACACGCTG TCAGGAGGTCACTGAGAACGACG LpWALlcsense TGGA GAGGACACGCTGAAATCAC CATAGCCTGGAGAACAAGGATCGC LpWALlcanti TCCCACTATCCTTCGCAAGACC CTCGCCATGTCCGCCTTG LpWALfsense TGGAGAGGACACGCTGAAATCAC TGGAGALACALAGGATCGCCACG LpWALlfanti ATTTCATTTGGAGAGGACACGCTG CCCGCCCTGCAACCAGTAGTG LpWALlhsense TGGAGAGGACACGCPGAAATCAC TGCAGTTGTCGCAGCACG LpWALlhanti ATCCCACTATCCTTCGCAAGACC CGTGACGACTAACCCCCAGACG LpZ~asense CACAATCCCACTATCCTTCGCAAG CGCCTCTTTCTCCGACGCC LpZ~aanti TGGAGAGGACACGCTGAAATCAC TGGAGGCAGGCAGATGAACTG WO 02/090491 PCT/AU02/00565 51 EXAMPLE 7 Genetic mapping of perennial ryegrass genes involved in metal stress protection The cDNAs representing genes involved in metal stress protection were amplified by PCR from their respective plasmids, gel-purified and radio-labelled for use as probes to detect restriction fragment length polymorphisms (RFLPs).

RFLPs were mapped in the F 1 (first generation) population, NAe x AU 6 This population was made by crossing an individual (NA 6 from a North African ecotype with an individual (AUe) from the cultivar Aurora, which is derived from a Swiss ecotype. Genomic DNA of the 2 parents and 114 progeny was extracted using the 1 x CTAB method of Fulton et al. (1995).

Probes were screened for their ability to detect polymorphism using the DNA (10 gg) of both parents and 5 F 1 progeny restricted with the enzymes Dral, EcoRI, EcoRV or Hindlll. Hybridisations were carried out using the method of Sharp et al. (1988). Polymorphic probes were screened on a progeny set of 114 individuals restricted with the appropriate enzyme (Figures 121, 128, 134, 141, 148, 154, 161,168, 174, 180, 184 and 190).

RFLP bands segregating within the population were scored and the data was entered into an Excel spreadsheet. Alleles showing the expected 1:1 ratio were mapped using MAPMAKER 3.0 (Lander et al. 1987). Alleles segregating from, and unique to, each parent, were mapped separately to give two different linkage maps. Markers were grouped into linkage groups at a LOD of 5.0 and ordered within each linkage group using a LOD threshold of Loci representing genes involved in metal handling mapped to the linkage groups as indicated in Table 5 and in Figure 192. These gene locations can now be used as candidate genes for quantitative trait loci for metal handling-associated traits such as heavy metal detoxification, metal tolerance, metal uptake, and response to toxic or suboptimal levels of metals.

WO 02/090491 PCT/AU02/00565 52 TABLE MAP locations of ryegrass genes encoding metal handling enzymes across two genetic linkage maps of perennial ryegrass Probe Polymorphic Mapped with Locus Linkage group LpCCHa LpHAKa LpHAK1 Lpn/a LpMTc LpMTd LpMTg LpMTj LpMVTI LpMTn LpMTo LpWALIb LpWALIc LpWALIe LpWALIg LpWALI h LpCTAa LpCTRa LpCla LpZTa LpZBa LpZBb LpPCSa Hind Ill Dra I Dra I Dra I Dra I Hind III Dra I Dra I Eco RV Eco RV Dra I Dra I Hind Ill Hind III Dra I Eco RI Hind Ill Eco RI Eco RI Dra I Eco RV Hind II I Eco RV LpCCHa LpHAKa LpHAK1I Lpn/a LpMTc LpMTd LpMTg LpMTj. 1 LpMTj .2 LpMVTI.

LpMVTI.2 LpMTI.1 LpMVTI.2 LpMTo LpWALI b LpWALIc LpWAIe LpWALIg LpWALI h LpCTAa LpCTRa LpCIa LpZTa LpZBa LpZBb. 1 LpZBb.2 LpPCSa NA6 7 4 3 2 3 1 3 3 8 AU6 7 3 3 3 3 3 3 3 3 EXAMPLE 8 Expression profiling of cDNAs encoding proteins involved in metal stress protection using microarray technology oDNAs encoding proteins involved in metal stress protection were PCR WO 02/090491 PCT/AU02/00565 53 amplified and purified. The amplified products were spotted on each amino-silane coated glass slide (CMT-GAPS, Coming, USA) using a microarrayer MicroGrid (BioRobotics, UK). Spotting solution was also spotted in every subgrid of the microarray as negative and background controls. Table 6 gives details on the tissues used to extract total RNA. Fluorescence labelled probes were synthesis by reversed transcribing RNA and incorporating Cyanine 3 or 5 labelled dCTP. The probes were hybridised onto microarrays. In each case the experiment was repeated on two microarrays. After hybridisation for 16 hours (overnight), the microarrays were washed and scanned using a confocal laser scanner (ScanArray 3000, Packard, USA). The images obtained were quantified using Imagene 4.1 (BioDiscovery, USA). Data were scaled to a factor of 2000 across all experiments and judged as not present low expression medium expression high expression and highly expression (Table 7).

TABLE 6 List of hybridization probes used in expression profiling of perennial ryegrass genes encoding metal handling enzymes Hybridisation probe for microarrays Organ specificity (3-months old plants Leaf blade grown hydroponically) Sheat Root Seed Seedling grown under light condition 5-day old shoot 7-day old shoot (7LS) old shoot old root 7-day old root (7LR) old root Seedling grown under dark condition 5-day old shoot 7-day old shoot (7DS) old shoot (1ODS) old root 7-day old root (7DR) old root TABLE 7 Results of expression profiling of ryegrass genes encoding metal handling enzymes Clone name Clone id Gene Id length sheath Leaf Root seed 5LS 7LS 1OLS 513 7LR 1ULR 51S 7DS lUDS 5DR 7DR in patent CCHb lOrglUsHO7 UxxLYS7_YEAST10803 -i -i OCHo 17rglYsEO4 UxxLYS7YEAST10803 CCHd 07rglAsBO5 UxxLYS7_YEAST10803 -ii- i HA~a 09rglGsED1 UioHAKIDEBOC147O1 Full? MTc IlrglZsAlO DdtMT3-MUSAC-16793 Full -f MTd 7rglHsBO7 DdtMT1_HORVU-13517 Full Mte 08rg1GsH12 MIsMT22_ORYSA1 1075 Full MTf O8rg2EsAO4 DdtMT1_FESRU-10703 Full 1. MTO 17rglQsAOl EpsMAOXQMYCTU1095 Full ++44 MTh I9rgDsFt I XnsDC13DR0ME10947 Full MTj C8rq2CsCO8 DdtMT3_MUSAC-16793 Full I44 MTk 07rg2GsHO9 MIsNRAM_IAKIE13885 Full 4-1-4+ +4 .4 MTI 19rg2JsDO5 DdtMT3_MUSAC-16793 Full WALla 1Ory2GsCO5 XnslWPIMAIZE1O914 Full 44- WALlb 17rglQsGO3 XnsVGLLPRVR 11962 Full WALld 07rg2DsEO9 DsrNR43-RAT-1 6545 Partial +I WALle 17rglMsGO2 XnslWPltMAIZE10914 Full WALIg 10rgVsHO6 QpdIWP1-MAIZE16O26 Full +i WALIH 17rgILsD12 XnslWPlMAIZE10914 Full TABLE 7 (cont.) Results of expression profiling of ryegrass genes encoding metal handling enzymes Clone name Clone id Gene id length sheath Leaf root seed 5L-S XLS lOLS 51-R 7LR 1OLR 5DS 7DS lODS 5DR 70IR in patent YCFa 1 Orgi FsAO3 DdtYCFL-YEAST1 5645 Partial CTAa O7rgl KsGO7 UaIAT7A.MOUSE1 6157 Partial BCBa 1 Orgl TsFll1 EetBOPPEA 16745 Full CTRa 08rglYsDO2 UioCOPTLARATH14471 Full +i Cla 11lrgl LsAO6 DdICD13_ARATH1 1697 Partial hi ±4 4+ ZTa O6rgl VsFO1 ZhyYGLB BACST1 1983 Full i i Zlb IO0rgl UsEOQ SmeZBi4 MAIZE14313 Full I- WO 02/090491 PCT/AU02/00565 56

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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.

It will also be understood that the term "comprises" (or its grammatical variants) as used in this specification is equivalent to the term "includes" and should not be taken as excluding the presence of other elements or features.

Documents cited in this specification are for reference purposes only and their inclusion is not an acknowledgment that they form part of the common general knowledge in the relevant art.

Claims

1. A substantially purified or isolated nucleic acid or nucleic acid fragment encoding a zinc binding protein (ZB) from a Lolium species; or a functionally active fragment or variant thereof.

2. A nucleic acid or nucleic acid fragment according to Claim 1, wherein oO Ssaid Lolium species is Lolium perenne or Lolium arundinaceum.

3. A substantially purified or isolated nucleic acid or nucleic acid Sfragment encoding ZB and including a nucleotide sequence selected from the group consisting of sequences shown in Figures 104, 106, 107, 109 and 186 hereto (Sequence ID Nos: 538, 540 to 547, 548, 550 to 551, and 582, respectively); complements of the sequences recited in sequences antisense to the sequences recited in and and functionally active fragments and variants of the sequences recited in and

4. A nucleic acid or nucleic acid fragment according to Claim 3, wherein said functionally active fragments and variants have at least approximately identity to the relevant part of the sequences recited in and and have a size of at least 20 nucleotides. A nucleic acid or nucleic acid fragment according to Claim 3, including a nucleotide sequence selected from the group consisting of sequences shown in Figures 104, 106, 107, 109 and 186 hereto (Sequence ID Nos: 538, 540 to 547, 548, 550 to 551, and 582, respectively).

6. A nucleic acid or nucleic acid fragment according to any one of Claims 3 to 5 wherein said nucleic acid or nucleic acid fragment is from a Lolium species.

7. A construct including a nucleic acid or nucleic acid fragment according to any one of Claims 1 to 6.

8. A vector including a nucleic acid or nucleic acid fragment according 005065110 to any one of Claims 1 to 6. U S9. A vector according to Claim 8, further including a promoter and a Sterminator, said promoter, nucleic acid or nucleic acid fragment and terminator being operatively linked.

10. A plant cell, plant, plant seed or other plant part, including a (N 0 construct according to Claim 7 or a vector according to Claim 8 or 9.

11. A plant, plant seed or other plant part derived from a plant cell or 0 plant according to Claim and including a construct according to Claim 7 or a vector according to Claim 8 or 9.

12. A method of modifying metal handling 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 6, a construct according to claim 7 and/or a vector according to Claim 8 or 9.

13. A method according to Claim 12 wherein said method includes modifying heavy metal detoxification in said plant.

14. A method according to Claim 12 or 13 wherein said metal is selected from the group consisting of cadmium, copper, zinc, aluminium, manganese and copper. A method for phytoremediation of soils contaminated with heavy metals, said method including growing on said soils plants including an effective amount of a nucleic acid or nucleic acid fragment according to any one of Claims 1 to 6, a construct according to claim 7 and/or a vector according to Claim 8 or 9.

16. A method according to Claim 15 wherein said heavy metals are selected from the group consisting of cadmium, zinc, copper, nickel, mercury, lead, arsenate and selenite.

17. Use of a nucleic acid or nucleic acid fragment according to any one 005065110 0 of Claims 1 to 6, and/or nucleotide sequence information thereof, and/or single C nucleotide polymorphisms thereof as a molecular genetic marker.

18. A substantially purified or isolated ZB polypeptide from a Lolium species; or a functionally active fragment or variant thereof. 5 19. A polypeptide according to Claim 18, wherein said Lolium species is (N0 00 Lolium perenne or Lolium arundinaceum. (N A substantially purified or isolated ZB polypeptide including an amino 0 acid sequence selected from the group consisting of sequences shown in Figures 105, 108 and 187 hereto (Sequence ID Nos: 539, 549 and 583, respectively); and functionally active fragments and variants thereof.

21. A polypeptide according to claim 20, wherein said functionally active fragments and variants have at least approximately 90% identity with the recited sequences and have a size of at least 20 amino acids.

22. A polypeptide according to claim 20 including an amino acid sequence selected from the group of sequences shown in Figures 195, 108 and 187 hereto (Sequence ID Nos: 539, 549 and 583, respectively).

23. A polypeptide according to any one of claims 20 to 22 wherein said polypeptide is from a Lolium species.