NZ529211A

NZ529211A - Modification of metal-handling in plants

Info

Publication number: NZ529211A
Application number: NZ529211A
Authority: NZ
Inventors: German Spangenberg; Timothy Ivor Sawbridge; Eng Kok Ong; Michael Emmerling
Original assignee: Agriculture Victoria Serv Pty; Agres Ltd
Priority date: 2001-05-07
Filing date: 2002-05-07
Publication date: 2007-09-28
Also published as: WO2002090491A2; AU2002252826B2; NZ560526A; AUPR482001A0; WO2002090491A3

Abstract

Disclosed is 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. Also disclosed is constructs and vectors comprising the nucleic acid or nucleic acid fragment.

Description

005013249 , 52 92 1 1 I M AUG 2007 MODIFICATION OF METAL-HANDLING IN PLANTS [ RECPf \/ £ This application is the parent of a divisional application entitled "Modification of metal handling in plants (2).

The present invention relates to nucleic acids encoding amino acid sequences for 5 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 10 plant heavy metal detoxification; modification of plant tolerance to metals such as copper, zinc, cadmium and aluminium; modification of plant capacity for accumulation or hyper-accumulation 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 15 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 ^ 20 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 25 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 (MT), aluminium-stress induced proteins (WALI), cadmium resistance proteins (YCF), copper transporting AT Pases (CTA), 30 blue copper binding proteins (BOB), copper transporters (CTR), cadmium induced proteins (CI), zinc transporters (ZT), zinc binding proteins (ZB) and phytochelatin synthases (PCS). 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, 5 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 0 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, WALI, 5 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, 0 ZT, ZB and PCS and functionally active fragments and variants thereof. Such proteins are referred to herein as CCH-like, HAK-like, MT-iike, WALI-like, YCF-like, 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 5 CCH, HAK, MT, WALI, 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; 0 enhance or otherwise alter the plant capacity for accumulation or hyper- 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 5 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 10 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, CI, ZT, ZB and/or PCS or like gene activities in plants has significant consequences for a range of applications in, for 15 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 20 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 25 nutrient deficient soils; and in altering metal transport and mobilisation from senescing leaves.

Methods for the manipulation of CCH, HAK, MT, WALI, 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 30 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 5 fescue and red fescue. Preferably the species is a ryegrass, more preferably perennial ryegrass (L perenne). Perennial ryegrass (Lolium perenne L.) 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 10 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, 15 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 20 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 25 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 30 contiguous nucleotide sequence. 005013249 Preferably, the present invention provides 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.

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

In a preferred embodiment the present invention provides a substantially purified or isolated nucleic acid or nucleic acid fragment encoding ZB, or complementary or antisense to a sequence encoding ZB, and including a nucleotide sequence selected from the group consisting of (a) 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 (a); (c) sequences antisense to the sequences recited in (a) and (b); and (d) functionally active fragments and variants of the sequences recited in (a), (b) and (c).

Preferably, the functionally active fragments and variants have at least approximately 90% identity to the relevant part of the sequences recited in (a), (b) and 15 (c), respectively, 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 20 13,14,16, 18 to 20, and 560, respectively); (b) complements of the sequences recited in (a); (c) sequences antisense to the sequences recited in (a) and (b); and (d) functionally active fragments and variants of the sequences recited in (a), (b) and (c).

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 25 protein includes a nucleotide sequence selected from the group consisting of (a) sequences shown in Figures 12 and 14 hereto (Sequence ID Nos: 21 and 23 to 24, respectively); (b) complements of the sequences recited in (a); (c) sequences antisense to the sequences recited in (a) and (b); and (d) functionally active fragments and variants of the sequences recited in (a), (b) and (c).

[""SSSttopmrvomcE] ' ♦ AUG 2007 RECEIVED 005013249 5A 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 of (a) sequences shown in Figures 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 32, 33, 35, 36, 38, 39, 41, 42, 5 44, 45, 47, 48, 50, 51, 53, 54, 56, 57, 59, 123, 130, 136 and 143 hereto (Sequence ID Nos: 25, 27 to 100, 101, 103 to 142, 143, 145 to 188, 189, 191 to 214, 215, 217 to 232, 233, 235 to 266, 267, 269 to 308, 309, 311 to 319, 320, 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); (b) complements of the sequences recited in (a) ; (c) 10 sequences antisense to the sequences recited in (a) and (b); and (d) functionally active fragments and variants of the sequences recited in (a), (b) and (c).

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 intellectual property office of n.z. 14 AUG 2007 REf!i=i\/i=n 6 group consisting of (a) 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 5 and 574, respectively); (b) complements of the sequences recited in (a); (c) sequences antisense to the sequences recited in (a) and (b); and (d) functionally active fragments and variants of the sequences recited in (a), (b) and (c).

In a further preferred embodiment of this aspect of the invention, the substantially purified or isolated nucleic acid/or nucleic acid fragment encoding a 10 YCF or YCF-like protein includes a nucleotide sequence selected from the group consisting of (a) sequences shown in Figures 87 and 89 hereto (Sequence ID Nos: 499 and 501 to 502, respectively); (b) complements of the sequences recited in (a); (c) sequences antisense to the sequences recited in (a) and (b); and (d) functionally active fragments and variants of the sequences recited in (a), (b) and 15 (c).

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 (a) sequences shown in Figures 90 and 92 hereto (Sequence ID 20 Nos: 503 and 505 to 509, respectively); (b) complements of the sequences recited in (a) ; (c) sequences antisense to the sequences recited in (a) and (b); and (d) functionally active fragments and variants of the sequences recited in (a), (b) and (c).

In a still further preferred embodiment of this aspect of the invention, the 25 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 (a) sequences shown in Figures 93,95 and 170 hereto (Sequence ID Nos: 510, 512 to 513 and 576, respectively); (b) complements of the sequences recited in (a); (c) sequences antisense to the sequences recited in (a) and (b); and 30 (d) functionally active fragments and variants of the sequences recited in (a), (b) and (c). 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 (a) sequences shown in Figures 96 and 176 hereto (Sequence ID 5 Nos: 514 and 578, respectively); (b) complements of the sequences recited in (a); (c) sequences antisense to the sequences recited in (a) and (b); and (d) functionally active fragments and variants of the sequences recited in (a), (b) and (c).

In a still further preferred embodiment of this aspect of the invention, the 10 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 (a) sequences shown in Figures 98 and 100 hereto (Sequence ID Nos: 516 and 518 to 531, respectively); (b) complements of the sequences recited in (a); (c) sequences antisense to the sequences recited in (a) and (b); and (d) 15 functionally active fragments and variants of the sequences recited in (a), (b) and (c).

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 20 consisting of (a) sequences shown in Figures 101,103 and 182 hereto (Sequence ID Nos: 532, 534 to 537, and 580, respectively); (b) complements of the sequences recited in (a); (c) sequences antisense to the sequences recited in (a) and (b); and (d) functionally active fragments and variants of the sequences recited in (a), (b) and (c).

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 (a) 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) 30 complements of the sequences recited in (a); (c) sequences antisense to the sequences recited in (a) and (b); and (d) functionally active fragments and variants 8 of the sequences recited in (a), (b) and (c).

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 5 consisting of (a) sequences shown in Figures 110,112 and 113 hereto (Sequence ID Nos: 552, 554 to 557, and 558, respectively); (b) complements of the sequences recited in (a); (c) sequences antisense to the sequences recited in (a) and (b); and (d) functionally active fragments and variants of the sequences recited in (a), (b) and (c). 0 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, 5 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 0 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 5 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 10 nucleotides, more preferably at least 15 nucleotides, most preferably at least 20 nucleotides.

Nucleic acids or nucleic acid fragments encoding at least a portion of several CCH, HAK, MT, WALI, YCF, CTA, BCB, CTR, CI, ZT, ZB and PCS have 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 sequence-dependent protocols, such as methods of nucleic acid hybridisation, and methods 5 of DNA and RNA amplification as exemplified by various uses of nucleic acid amplification technologies (e.g. polymerase chain reaction, ligase chain reaction), is well known in the art.

For example, genes encoding other CCH, HAK, MT, WALI, 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 25 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 30 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. 005013249 For example, those skilled in the art can follow the RACE protocol [Frohman 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 5 and 5' RACE systems (BRL), specific 3' or 5' cDNA fragments may be isolated [Ohara et al. (1989) Proc. Natl. Acad Sci USA 86:5673; Loh et al. (1989) Science 243:217; the entire disclosures of which are incorporated herein by reference]. Products generated by the 3' and 5' RACE procedures may be combined to generate full-length cDNAs.

In a second aspect of the present invention there is provided a substantially purified 10 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, WALI and WALI-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 (L. perenne).

In a preferred embodiment the present invention provides a substantially purified 20 or isolated ZB polypeptide from a Lolium species, or a functionally active fragments and variants 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 25 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.

Preferably, functionally active fragments and variants have at least approximately 95% identity with the recited sequences and have a size of at least 20 amino acids. intellectual property office of n.z. 14 AUG 2007 RI=riCi\/crt 005013249 10A 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 selected from the group consisting of sequences shown in Figures 2, 5, 8,10 and 117 hereto (Sequence ID Nos: 2, 8, 15, 17 and 561, respectively) and functionally active fragments and variants 5 thereof.

In 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. intellectual property office of n.2. t 4 AUG 2007 received 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 5 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 10 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 15 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 20 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 25 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 CI or Cl-like polypeptide includes an amino acid 5 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 10 102 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, 15 108 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 20 111 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, CCH-like, HAK, HAK-like, MT, MT-like, WALI, WALI-like, YCF, YCF-like, CTA, CTA-like, 25 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 5 corresponding amino acid sequence. Preferably the fragment has a size of at least 10 amino acids, more preferably at least 15 amino acids, most preferably at least 20 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 10 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 15 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 25 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 30 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 5 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, 50, 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 10 188,191 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 25 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, 5 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 10 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 15 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 20 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 25 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 30 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 5 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 10 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 15 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 20 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, non-25 chromosomal and synthetic nucleic acid sequences, eg. derivatives of plant viruses; bacterial plasmids; derivatives of the Ti plasmid from Agrobacterium tumefaciens, derivatives of the Ri plasmid from Agrobacterium rhizogenes; phage DNA; yeast artificial chromosomes; bacterial artificial chromosomes; binary bacterial artificial chromosomes; vectors derived from combinations of plasmids 30 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. 10 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 15 may be from the same gene as the promoter sequence or a different gene. Particularly suitable terminators are polyadenylation signals, such as the CaMV 35S polyA and other terminators from the nopaline synthase (nos), the octopine synthase (ocs) and the rbcS genes.

The vector, in addition to the regulatory element, the nucleic acid or nucleic 20 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 25 genes [such as the neomycin phosphotransferase (npt2) gene, the hygromycin phosphotransferase (hph) gene, the phosphinothricin acetyltransferase (bar or paf) 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, 5 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 10 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 15 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) 20 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 25 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 30 transformation) are well known to those skilled in the art. Such techniques include Agrobacterium mediated introduction, electroporation to tissues, cells and 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 10 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 20 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 25 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 0 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 5 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. 0 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 5 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 0 tolerance to metals, plant capacity for accumulation or hyper-accumulation of 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 5 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 10 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 15 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: 20 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 25 ID No: 7).

Figure 5 shows the deduced amino acid sequence of LpCCHb (Sequence ID No: 8). 22 PCT/ATJ02/00565 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 No: 15).

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: 10 17).

Figure 11 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpCCHd (Sequence ID Nos: 18 to 20).

Figure 12 shows the consensus contig nucleotide sequence of LpHAKa 15 (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 20 24).

Figure 15 shows the consensus contig nucleotide sequence of LpMTa (Sequence ID No: 25).

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 5 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 10 142).

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 ID No: 215). 24 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 5 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: 10 234).

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 15 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 20 308).

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 5 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 ID No: 366). 26 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 5 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: 10 385).

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 15 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 20 394).

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 5 (Sequence ID No: 400).

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

Figure 62 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpWALIa (Sequence ID Nos: 402 10 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 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 5 contributing to the consensus contig sequence LpWALId (Sequence ID Nos: 453 to 462).

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

Figure 73 shows the deduced amino acid sequence of LpWALIe (Sequence ID 10 No: 464).

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

Figure 75 shows the consensus contig nucleotide sequence of LpWALIf 15 (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 20 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 5 (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 10 to 494).

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

Figure 85 shows the deduced amino acid sequence of LpWALIi (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 (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 5 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: 10 511).

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 LpCIa (Sequence ID No: 516).

Figure 99 shows the deduced amino acid sequence of LpCIa (Sequence ID No: 20 517).

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

Figure 101 shows the consensus contig nucleotide sequence of LpZTa (Sequence ID No: 532). 31 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 5 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: 10 539).

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 15 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 20 551).

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). 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 15 pKYLX71:35S2 binary transformation vector.

Figure 120 shows plasmid maps of sense and antisense constructs of LpCCHa in pPZP221:35S2 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 5 pKYLX71:35S2 binary transformation vector.

Figure 127 shows plasmid maps of sense and antisense constructs of LpMTb in pPZP221:35S2 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 piasmid maps of sense and antisense constructs of LpMTg in pPZP221:35S2 binary transformation vector.

Figure 134 shows screening by Southern hybridisation for RFLPs using LpMTg as 20 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 pKYLX71:35S2 binary transformation vector.

Figure 140 shows plasmid maps of sense and antisense constructs of LpMTh in pPZP221:35S2 binary transformation vector.

Figure 141 shows screening by Southern hybridisation for RFLPs using LpMTh as 10 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 15 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 pKYLX71:35S2 binary transformation vector.

Figure 147 shows plasmid maps of sense and antisense constructs of LpMTj in pPZP221:35S2 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 10 pPZP221:35S2 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 20 pDH51 transformation vector.

Figure 159 shows plasmid maps of sense and antisense constructs of LpWALIf in pKYLX71:35S2 binary transformation vector.

Figure 160 shows plasmid maps of sense and antisense constructs of LpWALIf in pPZP221:35S2 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 10 pDH51 transformation vector.

Figure 166 shows plasmid maps of sense and antisense constructs of LpWALIh in pKYLX71:35S2 binary transformation vector.

Figure 167 shows plasmid maps of sense and antisense constructs of LpWALIh in pPZP221:35S2 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 pPZP221:35S2 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 pPZP221:35S2 binary transformation vector.

Figure 180 shows screening by Southern hybridisation for RFLPs using LpCTRa 15 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 20 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 pPZP221:35S2 binary transformation vector.

Figure 190 shows screening by Southern hybridisation for RFLPs using LpERa as 10 a probe.

Figure 191 shows A, infiltration of Arabidopsis plants; B, selection of transgenic Arabidopsis plants on medium containing 75 jL/g/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 15 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. 39 EXAMPLE 1 Preparation of cDNA libraries, isolation and sequencing of cDNAs coding for CCH, CCH-like, HAK, HAK-like, MT, MT-like, WALI, WALI-like, YCF, YCF-like, CTA, CTA-like, BCB, BCB-like, CTR, CTR-like, CI, Cl-like, ZT, ZT-like, ZB, ZB-5 like, 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) 05rg Senescing leaves from mature plants 06rg Whole etiolated seedlings (1-5 day old and 17 days old) 07rg Roots from mature plants grown in hydroponic culture 08rg Senescent leaf tissue ! o CO —t CQ Whole tillers and sliced leaves (0,1,3,6,12 and 24 h after harvesting) 10rg Embryogenic suspension-cultured cells 11 rg Non-embryogenic suspension-cultured cells 12rg Whole tillers and sliced leaves (0,1,3,6,12 and 24 h after harvesting) 13rg Shoot apices including vegetative apical meristems 14rg Immature inflorescences including different stages of inflorescence meristem and inflorescence development 15rg Defatted pollen 16rg Leaf blades and leaf sheaths (rbcL, rbcS, cab, wir2A subtracted) 40 17rg Senescing leaves and tillers 18rg Drought-stressed tillers (pseudostems from plants subjected to PEG-simulated drought stress) 19rg Non-embryogenic suspension-cultured cells subjected to osmotic stress (grown in media with half-strength salts) (1,2,3,4,5, 6, 24 and 48 h after transfer) 20rg Non-embryogenic suspension-cultured cells subjected to osmotic stress (grown in media with double-strength salts) (1,2,3,4,5, 6,24 and 48 h after transfer) 21 rg Drought-stressed tillers (pseudostems from plants subjected to PEG-simulated drought stress) 22rg Spikelets with open and maturing florets 23rg Mature roots (specific subtraction with leaf tissue) 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 5 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 10 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 XL10-Gold ultra competent cells (Stratagene, USA) according to the protocol provided by Stratagene.

Alternatively, the cDNAs may be introduced into plasmid vectors for first preparing the cDNA libraries in Uni-ZAP XR vectors according to the manufacturer's protocol (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 20 plasmid vector pBluescript. In addition, the cDNAs may be introduced directly into 41 precut pBIuescript II SK(+) vectors (Stratagene) using T4 DNA (igase (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 5 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 10 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 ABI3700 sequence analyser.

EXAMPLE 2 DNA sequence analyses The cDNA clones encoding CCH, CCH-like, HAK, HAK-like, MT, MT-like, WALI, WALI-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 proteins were identified by conducting BLAST (Basic Local Alignment Search Tool; Altschul et al. (1993) J. 20 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 25 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 30 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 5 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, Ml 48108, USA). The original plasmid was then used to transform chemically competent XL-1 cells (prepared in-20 house, 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 25 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 5 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 CCHa, MTb, MTg, MTh, MTj, WALIc, WALIf, WALIh, BCBa, CTRa, ZTa and ZBa cDNAs encoding metal handling proteins were obtained (Figures 115,116,122,123,129, 10 130, 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 cDNAs gene name clone ID sequencing primer primer sequence (5'>3') LpBCBa 10rg1TsF11 10rg1TsF11.f1 ACTTCATCTGCGACGTCC LpMTh 19rg1 DsF11 19rg1DsF11.f1 AGCAAGTGCAACTGCGGC LpZBa 19rg2QsD02 19rg2QsD02.f1 TAGAGATACTTGGCTTGC LpZTa 20rg1 RsD04 20rg1 RsD04.f1 TCTCTATGGGCAGCTGGC 20rg1 RsD04.f2 TCATGATGCCGAAGAACCG 20rg1 RsD04.f3 ACAGTCAAGATGATTCGG EXAMPLE 4 Development of transformation vectors containing chimeric genes with CCHa, MTb, MTg, MTh, MTj, WALIc, WALIf, WALIh, BCBa, CTRa and ZBa cDNA sequences from perennial ryegrass To alter the expression of the proteins involved in metal handling CCHa, 20 MTb, MTg, MTh, MTj, WALIc, WALIf, WALIh, BCBa, CTRa and ZBa cDNAs, 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 5 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 pUC18-based transformation vector 10 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 full-length open reading frame cDNAs of perennial ryegrass CCHa, MTb, MTg, MTh, MTj, WALIc, WALIf, WALIh, BCBa, CTRa and ZBa cDNAs encoding metal 15 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 (5'->3') BCBa 10rg1TsF11 10rg1TsF11.f GAATTCTAGATAATCAGCGAAGGATTTTGCTCG 10rg1TsF11.r GAATTCTAGATACGGCAAAGACTCTTGTAAGC CCHa 03rg1AsE04 03rg1AsE04.f GCATTCTAGATCGCCGTCCGCCGATCTCGAG 03rg1AsE04.r GCATTCTAGAACATACTAGGGTTCATATATATGG CTRa 08rg1YsD02 08rg1YsD02.f GAATTCTAGAAAACTCAGCTGAAATGGCCATGC 08rg1YsD02.r GAATTCTAGAAGCACCTAGTAATCATGCCACAGC MTb 07rg2FsG10 07rg2FsG10.f GAATTCTAGATTCGCCATAATCTCATCCTTGACC 07rg2FsG10.r GAATTCTAGATCTCACACCATCACACAAGTGCG MTg 17rg1QsA01 17rg1QsA01.f GAATTCTAGATGTGTTGAGAGCTTCATCATGTCG 17rg1QsA01.r GAATTCTAGAACTCATCGATGATCCATCCATCG MTh 19rg1DsF11 19rg1DsF11.f GAATTCTAGATCGTCCAGTCTCAAGCTCGAACCG 45 19rg1DsF11.r GAATTCTAGATAGGCGCACACATACACAGGC MTj 08rg2CsC08 08rg2CsC08.f GAATTCTAGATCACAAGCCAAGTCCACCATGTCG 08rg2CsC08.r GAATTCTAGATTGGGTACTAGTACGTGTAGACGG WALIc 06rg2ZsF08 06rg2ZsF08.f GCATTCTAGAAGCAGCCGGGACAAGGCCAGAGC 06rg2ZsF08.r GCATTCTAGAACGCACATGTACGAAGCGTGTATGC WALIf 18rg1CsG05 18rg1CsG05.f GAATTCTAGAAGTACAGGAGACTAGCAAGCTTTGC 18rg1CsG05.r GAATTCTAGATGCTCATCTCATCTCAGCTCAGC WALIh 17rg1RsG06 17rg1RsG06.f GAATTCTAGATGCTCACTCGAAAAGCTTCGAAGC 17rg1RsG06.r GAATTCTAGATAAGTATCTTCATGTCGTCGTCC ZBa 19rg2QsD02 19rg2QsD02.f GAATTCTAGAACCACCACCACCAGGCGCTCGAGG 19rg2QsD02.r GAATTCTAGAACATGAAGACTGATCAGCAAGCAGG EXAMPLE 5 Development of binary transformation vectors containing chimeric genes with CCHa, MTb, MTg, MTh, MTj, WALIc, WALIf, WALIh, BCBa, CTRa and ZBa 5 cDNA sequences from perennial ryegrass To alter the expression of the metal handling proteins CCHa, MTb, MTg, MTh, MTj, WALIc, WALIf, WALIh, BCBa, CTRa and ZBa, 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 10 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 15 appropriate restriction enzyme (usually Xbal), the cDNA fragments were cloned into the corresponding site in pKYLX71:35S2, 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 20 and an rbcS terminator (An et al., 1985; Schardl et ah, 1987). Alternatively, the 46 PCR fragments were cloned into a modified pPZP binary vector (Hajdukiewicz et al., 1994). The pPZP221 vector was modified to contain the 35S2 cassette from pKYLX71:35S2 as follows. pKYLX71:35S2 was cut with Clal. The 5' overhang was filled in using Klenow and the blunt end was A-tailed with Taq polymerase. After 5 cutting with EcoRI, the 2kb fragment with an EcoRI-compatible and a 3'-A tail was gel-purified. pPZP221 was cut with Hindlll and the resulting 5' overhang filled in and T-tailed with Taq polymerase. The remainder of the original pPZP221 multi-cloning site was removed by digestion with EcoRI, and the expression cassette cloned into the EcoRI site and the 3' T overhang restoring the Hindlll site. This 10 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 pKYLX71:35S2-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 15 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, 20 173,179 and 189).

EXAMPLE 6 Production and analysis of transgenic Arabidopsis plants carrying chimeric perennial ryegrass genes CCHa, MTb, MTg, MTh, MTj, WALIc, WALIf, WALIh, 25 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. 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 5 enhancer region (35S2) 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, WALIh, BCBa, CTRa and 10 ZBa cDNAs under the control of the CaMV 35S promoter with duplicated enhancer region (35S2) 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 15 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 9C. After 48 hours the punnets were transferred to a growth room at 22 aC under fluorescent light (constant 20 illumination, 55 //molm'V1) 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 jwg/ml rifampicin and 50 //g/ml kanamycin and grown at 27 SC for 48 hours. A single colony was used to inoculate 5 ml of LB medium containing 50 //g/ml rifampicin and 50 /vg/ml kanamycin and grown over night at 27 eC 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 //g/ml kanamycin only. Incubation was over night at 27 eC and 250 rpm on an orbital shaker in a 21 Erlenmeyer flask.

The overnight cultures were centrifuged for 15 min at 5500 xg and the 5 supernatant discarded. The cells were resuspended in 1 I of infiltration medium [5% (w/v) sucrose, 0.03% (v/v) Silwet-L77 (Vac-ln-Stuff, Lehle Seeds #VIS-01)] and immediately used for infiltration.

Vacuum infiltration The Agrobacterium suspension was poured into a container (Decor 10 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 15 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 fj\ 70% ethanol were added for 2 min and replaced by 500 //I sterilisation solution (H20:4% chlorine:5% SDS, 15:8:1). After vigorous shaking, the tube was left for 10 min after which time the sterilisation solution was replaced 25 with 500 //I 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 //I sterile water.

The seeds were then evenly spread on 150 mm petri dishes containing 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 jt/g/ml timetin and 75 //g/ml gentamycin. After vernalisation for 48 hours at 4 2C the plants were grown under continuous fluorescent light (55 5 jumol m-2s-1) at 22 BC 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 10 DNA was isolated from the supernatant using Wizard Magnetic 96 DNA Plant System kits (Promega) on a Biomek FX (Beckman Coulter). 5 /j\ of the sample (50 jj\) 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 35S2 promoter region and the reverse primer within the transgene to amplify products of approximately 150 bp as recommended. The positioning of the forward primer 20 within the 35S2 promoter region guaranteed that homologous genes in Arabidopsis were not detected. /j\ of each genomic DNA sample was run in a 50 jj\ PCR reaction including SYBR Green on an ABI (Applied Biosystems) together with samples containing DNA isolated from wild type Arabidopsis plants (negative control), 25 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. 50 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 rTGGAGAGGACACGCTGAAATCAC ATCCCAGCCATGCCCATCG LpBCBaanti ITGGAGAGGACACGCTGAAATCAC TGCTAGGTTTCTTCCGTTGGTTTG LpCCHasense CGCACAATCCCACTATCCTTCG CAACACCTTCCATTTTGCCGAG LpCCHaanti rCATTTCATTTGGAGAGGACACGC GACGCCAGATGCTGTTCTTCAGAC LpCTRasense ATTTCATTTGGAGAGGACACGCTG CATCGGAGGCATGTTCCAGACC LpCTRaanti rTGGAGAGGACACGCTGAAATCAC GCCACCCCTAAGGATGACGC LpMTbsense rTGGAGAGGACACGCTGAAATCAC GCAGTTTGAGCCGCAGTTGC LpMTbanti CATTTGGAGAGGACACGCTGAAATC AAGTGCAACCCCTGCAACTGC LpMTgsense CATTTGGAGAGGACACGCTGAAATC rGAGAGTCTGAGAGGTGGTCACCCC LpMTganti ATTTCATTTGGAGAGGACACGCTG GAGAACGGAGGCTGCAAGTGC LpMThsense rTGGAGAGGACACGCTGAAATCAC GGTACATGCCGCAACCGTTG LpMThanti rTGGAGAGGACACGCTGAAATCAC rCAGTTGTTGCCACTGGTCGC LpMTjsense ATTTCATTTGGAGAGGACACGCTG TCTTGGGGCAGTCGTTCTTGTTAG LpMTjanti ATTTCATTTGGAGAGGACACGCTG TTCAGGAGGTCACTGAGAACGACG LpWALIcsense rTGGAGAGGACACGCTGAAATCAC 3ATAGCCTGGAGAACAAGGATCGC LpWALIcanti TCCCACTATCCTTCGCAAGACC CTCGCCATGTCCGCCTTG LpWALIfsense rTGGAGAGGACACGCTGAAATCAC rGGAGAACAAGGATCGCCACG LpWALIfanti ATTTCATTTGGAGAGGACACGCTG CCCGCCCTGCAACCAGTAGTG LpWALIhsense rTGGAGAGGACACGCTGAAATCAC rGCAGTTGTCGCAGCACGG LpWALIhanti AATCCCACTATCCTTCGCAAGACC CGTGACGACTAACCCCCAGACG LpZBasense CACAATCCCACTATCCTTCGCAAG CGCCTCTTTCTCCGACGCC LpZBaanti rTGGAGAGGACACGCTGAAATCAC TTGGAGGCAGGCAGATGAACTG 51 EXAMPLE 7 Genetic mapping of perennial ryegrass genes involved En metal stress protection The cDNAs representing genes involved in metal stress protection were 5 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 Fi (first generation) population, NA6 x AU6- This population was made by crossing an individual (NA6) from a North African ecotype with an individual (AU6) from the cultivar Aurora, which is derived from a Swiss 10 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 ng) of both parents and 5 Fi progeny restricted with the enzymes Dral, EcoRI, EcoRV or Hindlll. Hybridisations were carried out using the method of 15 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 20 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 2.0.

Loci representing genes involved in metal handling mapped to the linkage 25 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. 52 TABLE 5 MAP locations of ryegrass genes encoding metal handling enzymes across two genetic linkage maps of perennial ryegrass Probe Polymorphic Mapped with Locus Linkage group NA6 AU6 LpCCHa Y Mnaflll LpCCHa 7 7 LpHAKa Y Oral LpHAKa 4 LpHAKI Y Oral LpHAKI 3 3 Lpn/a Y Dra 1 Lpn/a 2 2 LpMTc Y Oral LpMTc 3 3 LpMTd Y Hind III LpMTd 1 1 LpMTg Y Dra 1 LpMTg 3 3 LpMTj Y Dra 1 LpMTj.1 3 LpMTj.2 8 LpMTi Y EcoRV LpMTI.1 3 LpMTI.2 3 3 LpMTn Y Eco RV LpMTI.1 3 LpMTI.2 3 3 LpMTo Y Dra 1 LpMTo 3 3 LpWALIb Y Dra 1 LpWALIb 2 LpWALIc Y Hind III LpWALIc 1 LpWALIe Y HindlW LpWALIe 1 LpWALIg Y Dra 1 LpWALIg 1 LpWALIh Y EcoRI LpWALIh 2 LpCTAa Y Hind III LpCTAa 6 LpCTRa Y EcoRI LpCTRa 6 LpCIa Y EcoRI LpCIa 7 7 LpZTa Y Dral LpZTa 3 LpZBa Y EcoRV LpZBa 4 4 LpZBb Y Hind 111 LpZBb. 1 7- 7 LpZBb.2 7 LpPCSa Y EcoRV LpPCSa 1 EXAMPLE 8 Expression profiling of cDNAs encoding proteins involved in metal stress protection using microarray technology cDNAs encoding proteins involved in metal stress protection were PCR 53 amplified and purified. The amplified products were spotted on each amino-silane coated glass slide (CMT-GAPS, Corning, 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 5 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 10 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 grown hvdroponicallv) Leaf blade Sheat Root Seed Seedling grown under light condition -dav old shoot (5LS) 7-dav old shoot (7LS) -dav old shoot (10LS) -dav old root (5LR) 7-dav old root (7LR) -dav old root (10LR) Seedling grown under dark condition -dav old shoot (5DS) 7-dav old shoot (7DS) -dav old shoot (10DS) -dav old root (5DR) 7-dav old root (7DR) -dav old root (10DR) TABLE 7 o Results of expression profiling of ryegrass genes encoding metal handling enzymes O b> vo o Clone name in patent Clone id Gene id length sheath Leaf Root seed 5LS 7LS 10LS 5LR 7LR 10LR 5DS 7DS 10DS 5DR 7DR 10DR CCHb 10rg1UsH07 UxxLYS7_YEAST10803 + + ++++ + + + ++ +++ +++ 4-4- 4- - 4-4- 4-4- +4-4- CCHc 17rg1YsE04 UxxLYS7_YEAST10803 + + ++++ + + + ++ ++ +++ +++ 4-4- 4- - 4-4- ++ 4-4*4- CCHd 07rg1AsB05 UxxLYS7_YEAST10803 + + ++++ + + + ++ ++ +++ +++ 4-4- + - 4-4- ++ 4*4-4* HAKa 09rg1GsE01 UioHAK1_DEBOC14701 Full? ++ + - + - + - - - - - - - - - - MTc 11rg1ZsA10 DdtMT3_MUSAC-16793 Full + +++ + ++ + ++ - ++ ++ +++ 4-4- + - ++ 4-4- ++ MTd 07rg1HsB07 DdtMT1_HORVU-13517 Full + ++ ++-H- + +++ ++ ++ ++++ ++++ ++++ 4-4- 4-4- 4- ++-H- +-H-4- -H-4-4- Mte 08rg1GsH12 MlsMT22_ORYSA11075 Full +++ ++++ ++ ++ ++++ ++4-4- +4"t*+ ++ ++++ ++++ 4-4-4-4- +-H-+ 4-4-4-4- 4-4-4*4- ++++ 4-+4H- MTf 08rg2EsA04 DdtMT1_FESRU-10703 Full ++++ ++++ ++++ ++ ++++ ++++ ++++ ++++ ++++ ++++ 4-4-4- 4-4-4*4- ++++ -H-H- 4-4-4-4* 4*4-4*4- MTg 17rg1QsA01 EpsMAOX_MYCTU1095 5 Full ++++ 4*4-4-4- + + +++ ++++ ++++ ++++ + ++++ 4- 4-4-4-4- 4-4-+4- 4-4-4- 4-4-4*4* 4-4- MTh 19rg1DsF11 XnsDCI 3_DROME10947 Full + ++ + -H- + ++ ++ + ++ ++ 4-4- -H- 4- +4- 4- 4-4- MTj 08rg2CsC08 DdtMT3_MUSAC-16793 Full + +++ + ++ + ++ - ++ ++ +4-+ 4-4- 4- - 4-4- ++ 4-4- MTk 07rg2GsH09 MIsNRAMJAKIEI 3885 Full ++ +++ ++ +++ + +++ ++ ++ ++ 4-4- 4-4- 4-4- 4-4- -H- 4-4-4- MTI 19rg2JsD05 DdtMT3_MUSAC-16793 Full + +++ + ++ + ++ ++ ++ +4-4- -H- 4- - 4-4- 4-4- 4-4- WALIa 10rg2GsC05 XnslWP1_MAIZE10914 Full ++ ++++ ++++ ++++ ++++ ++ ++++ i i t i TTTT +++ 4-4-4-4- 4-4- 4-H- WALIb 17rg1QsG03 XnsVGLLPRVR111962 Full + +++ +++ ++++ +++ ++++ ++++ ++++ ■H-H- 4-4-4-4- ++++ + 4-4-4- ++++ ++++ WALId 07rg2DsE09 DsrNR43_RAT—16545 Partial ++ ++++ + ++ ++ ++ ++ ++ +++ +4-4- 4-4- + + ++ ++ 4-H- WALIe 17rg1MsG02 XnslWP1_MAIZE10914 Full ++ ++++ ++++ ++++ ++++ ++++ ++ ++++ ++++ 4-4-4- ++++ -H-++ 4-4- 4-4-4- ++++ 4-4-H* WAUg 10rg1VsH06 QpdlWP1_MAIZE16026 Full + ++ + + + + - - - + + 4- - 4* 4- WALIi 17rg1LsD12 XnslWP1_MAIZE10914 Full 4-4* 4-+++ ++++ ++++ ++++ ++++ ++ ++++ ++++ +4-4- 4-+-H- ++++ 4-4- 4-4-4- ++++ ++++ Oi 4^ TABLE 7 (cont.) Results of expression profiling of ryegrass genes encoding metal handling enzymes Clone name in patent Clone id Gene id length sheath Leaf root seed 5LS 7LS 10LS 5LR 7LR 10LR 5DS 70S 10DS 5DR 7DR 10DR YCFa 10rg1FsA03 DdtYCFLYEAST15645 Partial - + - ++ - + - ++ + ++ - - - + + + CTAa 07rg1KsG07 UatAT7A_MOUSE16157 Partial - + - - - - - - + + - - - - - - BCBa 10rg1TsF11 EetBCP_PEA—15745 Full + ++ + + - + - - + + - - - + + CTRa 08rg1YsD02 UioCOPT_ARATH14471 Full + ++ + + + + + + + ++ + + - ++ + + Cla 11 rg1 LsA06 DdtCDI3_ARATH11697 Partial ++ +++ - ++ +++ ++ + ++ H—I—h ++ + - + + ++ ZTa 06rg1VsF01 ZhyYGLBJ3ACST11983 Full +++ ++++ ++++ ++4-f ++ + ++++ ++++ ++++ ++++ +++ + H-++ ++ +++ ZBb 08rg1UsE09 SmeZB 14_MAIZE 14313 Full + - + + + + + - + + + + - - + + 56 1 REFERENCES An, G., Watson, B.D., Stachel, S., Gordon, M.P., Nester, E.W. (1985) New cloning vehicles for transformation of higher plants. The EMBO Journal 4,227-284 Feinberg, A.P., Vogelstein, B. (1984). A technique for radiolabelling DNA 5 restriction endonuclease fragments to high specific activity. Anal. Biochem. 132:6-13.

Frohman etal. (1988) Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc. Natl. Acad Sci. USA 85:8998 Gish and States (1993) Identification of protein coding regions by database similarity search. Nature Genetics 3:266-272 Lander, E.S., Green P., Abrahamson, J., Barlow, A., Daly, M.J., Lincoln, S.E., Newburg, L. (1987). MAPMAKER: an interactive computer package for constructing primary linkage maps of experimental and natural populations.

Genomics 1:174-181.

Loh, E.Y., Elliott, J.F., Cwirla, S., Lanier, L.L., Davis, M.M. (1989). Polymerase chain reaction with single-sided specificity: Analysis of T-cell receptor delta chain. Science 243:217-220 Ohara, O., Dorit, R.L., Gilbert, W. (1989). One-sided polymerase chain reaction: The amplification of cDNA. Proc. Natl. Acad Sci USA 86:5673-5677 Sambrook, J., Fritsch, E.F., Maniatis, T. (1989). Molecular Cloning. A Laboratory Manual. Cold Spring Harbour Laboratory Press Schardl, C.L., Byrd, A.D., Benzion, G., Altschuler, M.A., Hildebrand, D.F., Hunt, A.G. (1987) Design and construction of a versatile system for the expression of foreign genes in plants. Gene 61,1-11 WO 02/090491 PCT/AU02/00565 57 Sharp, P.J., Kreis, M., Shewry, P.R., Gale, M.D. (1988). Location of a-amylase sequences in wheat and its relatives. Theor. Appl. Genet. 75:286-290.

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 5 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 10 their inclusion is not an acknowledgment that they form part of the common general knowledge in the relevant art. 005013249 58

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. 5 2. A nucleic acid or nucleic acid fragment according to Claim 1, wherein said

Lolium species is Lolium perenne or Lolium arundinaceum.

3. A substantially purified or isolated nucleic acid or nucleic acid fragment encoding ZB, or complementary or antisense to a sequence encoding ZB, and including a nucleotide sequence selected from the group consisting of (a) sequences shown in 10 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 (a); (c) sequences antisense to the sequences recited in (a) and (b); and (d) functionally active fragments and variants of the sequences recited in (a), (b) and (c).

4. A nucleic acid or nucleic acid fragment according to Claim 3 wherein said 15 functionally active fragments and variants have at least approximately 90% identity to the relevant part of the sequences recited in (a), (b) and (c), respectively, and have a size of at least 20 nucleotides.

5. A construct including a nucleic acid or nucleic acid fragment according to any one of Claims 1 to 4. 20

6. A vector including a nucleic acid or nucleic acid fragment according to any one of Claims 1 to 4.

7. A vector according to Claim 6, further including a promoter and a terminator, said promoter, nucleic acid or nucleic acid fragment and terminator being operatively linked. 25

8. A plant cell, plant, plant seed or other plant part, including a construct according to Claim 5 or a vector according to Claim 6 or 7. intellectual property office of n.z. 1 <i AUG 2007 received 005013249 59

9. A plant, plant seed or other plant part derived from a plant cell or plant according to Claim 8 and including a construct according to Claim 5 or a vector according to Claim 6 or 7.

10. A method of modifying metal handling in a plant, said method including 5 introducing into said plant an effective amount of a nucleic acid or nucleic acid fragment according to any one of Claims 1 to 4, a construct according to Claim 5 and/or a vector according to Claim 6 or 7.

11. A method according to Claim 10 wherein said method includes modifying heavy metal detoxification in said plant. 10

12. A method according to Claim 10 wherein said metals are selected from the group consisting of cadmium, copper, zinc, aluminium, manganese and copper.

13. 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 4, a construct 15 according to Claim 5 and/or a vector according to Claim 6 or 7.

14. A method according to Claim 13 wherein said heavy metals are selected from the group consisting of cadmium, zinc, copper, nickel, mercury, lead, arsenate and

15. Use of a nucleic acid or nucleic acid fragment according to any one of 20 Claims 1 to 4, and/or nucleotide sequence information thereof, and/or single nucleotide polymorphisms thereof as a molecular genetic marker.

16. The use according to Claim 15 wherein the molecular genetic marker is for one or more of quantitative trait loci (QTL) tagging, QTL mapping, DNA fingerprinting and marker assisted selections. 25

17. The use according to Claim 15 or 16 wherein the molecular genetic marker is used in a Lolium species. intellectual property office of n.z. 14 AUG 2007 received 005013249 60

18. The use according to any one of Claims 15 to 16 wherein the molecular genetic marker is used in forage and turf grass improvement of plant metal handling, plant tolerance to metals, plant capacity for accumulation or hyper-accumulation of metals, plant intracellular metal trafficking pathways, plant uptake of nutrients, plant 5 capacity of essential heavy metal homeostasis, plant metabolism and/or development associated with heavy metals, and/or responses to toxic or suboptimal levels of metals, in plants.

19. A substantially purified or isolated ZB polypeptide from a Lolium species, or a functionally active fragments and variants thereof. 10 20. A polypeptide according to Claim 19, wherein said Lolium species is

Lolium perenne or Lolium arundinaceum.

21. 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 15 active fragments and variants thereof.

22. A polypeptide according to Claim 21 wherein said functionally active fragments and variants have at least approximately 95% identity with the recited sequences and have a size of at least 20 amino acids.

23. A substantially purified or isolated nucleic acid or nucleic acid fragment 20 encoding a polypeptide according to any one of Claims 19 to 21.

24. A nucleic acid or nucleic acid fragment according to any one of Claims 1 to 4 substantially as hereinbefore described with reference to any one of the figures or examples.

25. A polypeptide according to any one of Claims 19 to 22 substantially as 25 hereinbefore described with reference to any one of the figures or examples. intellectual property office of n.z. 14 AUG 2007 received