AU2001293497B9 - Manipulation of plant cell walls - Google Patents

Description

WO 02/26994 PCT/AU01/01221 1 MANIPULATION OF PLANT CELL WALLS The present invention relates to nucleic acid fragments encoding amino acid sequences for lignification and lignification-related enzymes, and for cellulases and cellulase-like enzymes in plants, and the use thereof for the modification of lignin biosynthesis and cellulose degradation in plants to manipulate plant cell walls.

Lignins are complex phenolic polymers that strengthen plant cell walls against mechanical and chemical degradation. The process of lignification typically occurs during secondary thickening of the walls of cells with structural, conductive or defensive roles. Three monolignol precursors, sinapyl, coniferyl and p-coumaryl alcohol combine by dehydrogenative polymerisation to produce respectively the syringyl guaiacyl and hydroxyl subunits of the lignin polymer, which can also become linked to cell-wall polysaccharides through the action of peroxidases and other oxidative enzymes.

Biosynthesis of the monolignol precursors is a multistep process beginning with the aromatic amino-acids phenylalanine (and tyrosine in grasses). Lignin biosynthesis is initiated by the conversion of phenylalanine into cinnamate through the action of phenylalanine ammonia lyase (PAL). The second enzyme of the pathway is cinnamate-4-hydroxylase (C4H), responsible for the conversion of cinnamate to p-coumarate. The second hydroxylation step in the pathway is catalyzed by p-coumarate-3-hydroxylase (C3H) producing caffeic acid. Caffeic acid is then O-methylated by caffeic acid O-methyltransferase (OMT) to form ferulic acid. Ferulic acid is subsequently converted into 5-hydroxyferulate through the last hydroxylation reaction of the general phenylpropanoid pathway catalised by ferulate-5-hydroxylase The 5-hydroxyferulate produced by F5H is then O-methylated by OMT, the same enzyme that carries out the O-methylation of caffeic acid. The cinnamic acids are converted by action of the 4-coumarate:CoA ligase (4CL) and WO 02/26994 PCT/AU01/01221 2 caffeoyl-CoA 3-O-methyltransferase (CCoAMT) into the corresponding CoA derivatives. It is the final two reduction/dehydrogenation steps of the pathway, catalysed by cinnamoyl CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) that are considered to be specific to lignin biosynthesis. The three monolignols, sinapyl, coniferyl and p-coumaryl alcohols, are then polymerised by extracellular peroxidases (PER) and laccases (LAC) to yield lignins. The proportions of monolignols incorporated into the lignin polymers vary depending on plant species, tissue, developmental stage and sub-cellular location.

Cellulose, a main component of plant cell walls, is degraded in maturing plant organs by the action of cellulases (CELL). Cellulases play an important role in softening of plant organs and in fruit ripening.

Dry matter digestibility of forage grasses and legumes has been negatively correlated with lignin content. In addition, natural mutants of lignin biosynthetic enzymes in maize, sorghum and pearl millet that have higher rumen digestibility have been characterised as having lower lignin content and altered S/G subunit ratio. Lignification of plant cell walls is the major factor identified as responsible for lowering digestibility of forage tissues as they mature.

Furthermore, cellulose in forage grasses contributes significantly to the readily available energy in the feed for grazing ruminant animals. The fermentation processes in the rumen require considerable readily available energy. The improvement of the readily available energy in the rumen can increase the efficiency of rumen digestion. An increased efficiency in rumen digestion leads to an improved conversion of the forage protein fed to the ruminant animal into milk or meat, and to a reduction in nitrogenous waste as environmental pollutant.

WO 02/26994 PCT/AU01/01221 3 Perennial ryegrass (Lolium perenne is a key pasture grass in temperate climates throughout the world. Perennial ryegrass is also an important turf grass.

It would be desirable to have methods of altering lignin biosynthesis and/or cellulose degradation in plants, including grass species such as ryegrasses and fescues. For example it may be desirable to reduce the activity of key lignin biosynthetic enzymes in order to reduce lignin content and/or alter lignin composition and to increase cellulose degradation for enhancing dry matter digestibility and/or improving herbage quality. Cell wall digestibility and feed (grazed, cut hay, silage) quality could thus be increased by the manipulation of enzymes involved in the biosynthesis of lignins and in the degradation of cellulose. For other applications it may be desirable to enhance lignin biosynthesis to increase lignin content and/or alter lignin composition and to reduce cellulose degradation, for example to increase mechanical strength of wood, to increase mechanical strength of turf grasses, to reduce plant height, to reduce lodging, to improve disease resistance, to reduce fruit ripening and/or to reduce plant organ softening.

While nucleic acid sequences encoding some of the enzymes involved in the lignin biosynthetic pathway and in cellulose degradation have been isolated for certain species of plants, there remains a need for materials useful in the modification of lignin biosynthesis and cellulose degradation in a wide range of plants, particularly in forage grasses and legumes including ryegrasses and fescues, 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 amino acid sequences for the following enzymes from a ryegrass (Lolium) or fescue WO 02/26994 PCT/AU01/01221 4 (Festuca) species, or functionally active fragments or variants thereof: caffeoyl-CoA 3-O-methyltransferase (CCoAMT), cinnamyl alcohol dehydrogenase (CAD), caffeic acid O-methyltransferase (OMT), cinnamate-4hydroxylase (C4H), cinnamoyl-CoA reductase (CCR), peroxidase (PER), cellulase (CELL), ferulate-5-hydroxylase (F5H), phenylalanine ammonia lyase (PAL) and 4-coumarate:CoA ligase (4CL).

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 CCoAMT, CAD, OMT, C4H, CCR, PER, CELL, F5H, PAL and 4CL, or functionally active fragments or variants thereof.

Such proteins are referred to herein as CCoAMT-like, CAD-like, OMT-like, C4H-like, CCR-like, PER-like, CELL-like, F5H-like, PAL-like and 4CL-like, respectively.

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

The nucleic acid or nucleic acid fragment may be of any suitable type and includes DNA (such as cDNA or genomic DNA) and RNA (such as mRNA) that is single- or double-stranded, optionally containing synthetic, nonnatural or altered nucleotide bases, and combinations thereof.

The term "isolated" means that the material is removed from its original environment (eg. the natural environment if it is naturally occurring). For example, a naturally occurring nucleic acid present in a living plant is not isolated, but the same nucleic acid separated from some or all of the coexisting materials in the natural system, is isolated. Such nucleic acids could be part of a vector and/or such nucleic acids could be part of a WO 02/26994 PCT/AU01/01221 composition, and still be isolated in that such a vector or composition is not part of its natural environment.

Such nucleic acids or nucleic acid fragments could be assembled to form a consensus contig. As used herein, the term "consensus contig" refers to a nucleotide sequence that is assembled from two or more constituent nucleotide sequences that share common or overlapping regions of sequence homology. For example, the nucleotide sequence of two or more nucleic acids or nucleic acid fragments can be compared and aligned in order to identify common or overlapping sequences. Where common or overlapping sequences exist between two or more nucleic acids or nucleic acid fragments, the sequences (and thus their corresponding nucleic acids or nucleic acid fragments) can be assembled into a single contiguous nucleotide sequence.

In a preferred embodiment of this aspect of the invention, the substantially purified or isolated nucleic acid or nucleic acid fragment encoding a CCoAMT or CCoAMT-like protein includes a nucleotide sequence selected from the group consisting of sequences shown in Figures 1, 3, 4, 6, 7, 82 and 87 hereto (Sequence ID Nos: 1, 3 to 11, 12, 14 to 20, 21, 168 and 170, respectively); complements of the sequences shown in Figures 1, 3, 4, 6, 7, 82 and 87 hereto; sequences antisense to the sequences recited in and and functionally active fragments and variants of the sequences recited in and In a further preferred embodiment of this aspect of the invention, the substantially purified or isolated nucleic acid or nucleic acid fragment encoding a CAD or CAD-like protein includes a nucleotide sequence selected from the group consisting of sequences shown in Figures 9, 11, 13, 15, 16, 18, 19, 21 and 22 hereto (Sequence ID Nos: 23, 25, 27, 29 to 33, 34, 36 to 41, 43 to 44 and 45, respectively); complements of the sequences shown in Figures 9, 11, 13, 15, 16, 18, 19, 21 and 22 hereto; sequences WO 02/26994 PCT/AU01/01221 6 antisense to the sequences recited in and and functionally active fragments and variants of the sequences recited in and In another preferred embodiment of this aspect of the invention, the substantially purified or isolated nucleic acid or nucleic acid fragment encoding an OMT or OMT-like protein includes a nucleotide sequence selected from the group consisting of sequences shown in Figures 24, 26, 27, 29, 30, 93 and 99 hereto (Sequence ID Nos: 47, 49 to 58, 59, 61 to 93, 94, 172 and 174, respectively); complements of the sequences shown in Figures 24, 26, 27, 29, 30, 93 and 99 hereto; sequences antisense to the sequences recited in and and functionally active fragments and variants of the sequences recited in and In a further preferred embodiment of this aspect of the invention, the substantially purified or isolated nucleic acid or nucleic acid fragment encoding a C4H or C4H-like protein includes a nucleotide sequence selected from the group consisting of sequences shown in Figures 32, 34, 36 and 76 hereto (Sequence ID Nos: 96, 98, 100 and 166, respectively); (b) complements of the sequences shown in Figures 32, 34, 36 and 76 hereto; (c) sequences antisense to the sequences recited in and and (d) functionally active fragments and variants of the sequences recited in (b) and In another preferred embodiment of this aspect of the invention, the substantially purified or isolated nucleic acid or nucleic acid fragment encoding a CCR or CCR-like protein includes a nucleotide sequence selected from the group consisting of sequences shown in Figures 38, 40, 41, 43 and 44 hereto (Sequence ID Nos: 102, 104 to 106, 107, 109 to 110 and 111, respectively); complements of the sequences shown in Figures 38, 40, 41, 43 and 44 hereto; sequences antisense to the sequences recited in and and functionally active fragments and variants of the sequences recited in and WO 02/26994 PCT/AU01/01221 7 In a further preferred embodiment of this aspect of the invention, the substantially purified or isolated nucleic acid or nucleic acid fragment encoding a PER or PER-like protein includes a nucleotide sequence selected from the group consisting of sequences shown in Figures 46, 48, 49, 51, 52, 54 and 105 hereto (Sequence ID Nos: 113, 115 to 116, 117, 119 to 120, 121, 123 and 176, respectively); complements of the sequences shown in Figures 46, 48, 49, 51, 52, 54 and 105 hereto; sequences antisense to the sequences recited in and and functionally active fragments and variants of the sequences recited in and In another preferred embodiment of this aspect of the invention, the substantially purified or isolated nucleic acid or nucleic acid fragment encoding a CELL or CELL-like protein includes a nucleotide sequence selected from the group consisting of sequences shown in Figures 56 and 58 hereto (Sequence ID Nos: 125 and 127 to 134, respectively); (b) complements of the sequences shown in Figures 56 and 58 hereto; (c) sequences antisense to the sequences recited in and and (d) functionally active fragments and variants of the sequences recited in (b) and In a still further preferred embodiment of this aspect of the invention, the substantially purified or isolated nucleic acid or nucleic acid fragment encoding a F5H or F5H-like protein includes a nucleotide sequence selected from the group consisting of sequences shown in Figures 59 and 61 hereto (Sequence ID Nos: 135 and 137 to 139, respectively); complements of the sequences shown in Figures 59 and 61 hereto; sequences antisense to the sequences recited in and and functionally active fragments and variants of the sequences recited in and In another preferred embodiment of this aspect of the invention, the substantially purified or isolated nucleic acid or nucleic acid fragment encoding a PAL or PAL-like protein includes a nucleotide sequence selected WO 02/26994 PCT/AU01/01221 8 from a group consisting of sequences shown in Figures 62, 64, 65 and 67 hereto (Sequence ID Nos: 140, 142 to 148, 149 and 151 to 153, respectively); complements of the sequences shown in Figures 62, 64, 65 and 67 hereto; sequences antisense to the sequences recited in and and (d) functionally active fragments and variants of the sequences recited in (b) and In a further preferred embodiment of this aspect of the invention, the substantially purified or isolated nucleic acid or nucleic acid fragment encoding a 4CL or 4CL-like protein includes a nucleotide sequence selected from the group consisting of sequences shown in Figures 68, 70, 71 and 73 hereto (Sequence ID Nos: 154, 156 to 161, 162 and 164, respectively); (b) complements of the sequences shown in Figures 68, 70, 71 and 73 hereto; (c) sequences antisense to the sequences recited in and and (d) functionally active fragments and variants of the sequences recited in (b) and By "functionally active" in relation to nucleic acids it is meant that the fragment or variant (such as an analogue, derivative or mutant) encodes a polypeptide capable of modifying lignin biosynthesis and/or cellulose degradation in a plant. Such variants include naturally occurring allelic variants and non-naturally occurring variants. Additions, deletions, substitutions and derivatizations of one or more of the nucleotides are contemplated so long as the modifications do not result in loss of functional activity of the fragment or variant. Preferably the functionally active fragment or variant has at least approximately 80% identity to the relevant part of the above mentioned nucleotide sequence, more preferably at least approximately 90% identity, most preferably at least approximately identity. Such functionally active variants and fragments include, for example, those having nucleic acid changes which result in conservative amino acid substitutions of one or more residues in the corresponding amino acid 005042824 9 O sequence. Preferably the fragment has a size of at least 10 nucleotides, more CN preferably at least 15 nucleotides, most preferably at least 20 nucleotides.

O

Z In the preferred embodiment, the present invention provides a substantially purified or isolated nucleic acid or nucleic acid fragment encoding caffeoyl-CoA 3-O-methyltransferase (CCoAMT) from a Lolium species.

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 CCoAMT, or complementary or antisense to a CCoAMT-encoding sequence, and including a nucleotide sequence selected from the group consisting of sequences shown in Figures 1, 3, 4, 6, 7, 82 and 87 hereto (Sequence ID Nos: 1, 3 to 11, 12, 14 to 20, 21, 168 and 170, respectively); (b) complements of the sequences in sequences antisense to the sequences recited in and and functionally active fragments and variants of the sequences recited in and Preferably, the functionally active fragments and variants have at least approximately 90% identity to the relevant part of the sequences recited in and and have a size of at least 20 nucleotides.

The nucleic acids or nucleic acid fragments encoding at least a portion of several lignification and lignification-like enzymes and cellulase and cellulase-like enzymes have been isolated and identified. The nucleic acids and 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 is well known in the art. Examples of sequence-dependent protocols include, but are not limited to, methods of nucleic acid hybridisation, and methods of DNA and RNA amplification as exemplified by various uses of 005042824 9A nucleic acid amplification technologies polymerase chain reaction, ligase Cl chain reaction).

0 Z For example, genes encoding other lignification or lignification-like enzymes and other cellulase or cellulase-like enzymes, 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 Swell known to those skilled in the art. Specific oligonucleotide probes based upon the nucleic acid sequences of the present invention may be designed 10 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.

WO 02/26994 PCT/AU01/01221 In addition, short segments of the nucleic acids or nucleic acid fragments of the present invention may be used in amplification protocols to amplify longer nucleic acids or nucleic acid fragments encoding homologous genes from DNA or RNA. For example, the polymerase chain reaction may be performed on a library of cloned nucleic acid fragments wherein the sequence of one primer is derived from a nucleic acid or nucleic acid fragment of the present invention, and the sequence of the other primer takes advantage of the presence of the polyadenylic acid tracts to the 3' end of the mRNA precursor encoding plant genes. Alternatively, the second primer sequence may be based upon sequences derived from the cloning vector. For example, those skilled in the art can follow the RACE protocol (Frohman et al.

(1988) Proc. Natl. Acad Sci. 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 end. Using commercially available 3' RACE and 5' RACE systems (BRL), specific 3' or 5' cDNA fragments may be isolated (Ohara et al. (1989) Proc.

Natl. Acad Sci USA 86:5673; Loh et al. (1989) Science 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 or isolated polypeptide from a ryegrass (Lolium) or fescue (Festuca) species, selected from the group consisting of CCoAMT and CCoAMT-like, CAD and CAD-like, OMT and OMT-like, C4H and C4H-like, CCR and CCR-like, PER and PER-like, CELL and CELL-like, F5H and like, PAL and PAL-like, 4CL and 4CL-like, enzymes; 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 WO 02/26994 PCT/AU01/01221 11 fescue, meadow fescue and red fescue. Preferably the species is a ryegrass, more preferably perennial ryegrass perenne).

In a preferred embodiment of this aspect of the invention, the substantially purified or isolated CCoAMT or CCoAMT-like polypeptide includes an amino acid sequence selected from the group of sequences shown in Figures 2, 5, 8, 83 and 88 hereto (Sequence ID Nos: 2, 13, 22, 169 and 171, respectively); and functionally active fragments and variants thereof.

In a further preferred embodiment of this aspect of the invention, the substantially purified or isolated CAD or CAD-like polypeptide includes an amino acid sequence selected from the group of sequences shown in Figures 12, 14, 17, 20 and 23 hereto (Sequence ID Nos: 24, 26, 28, 35, 42 and 46, respectively); and functionally active fragments and variants thereof.

In another preferred embodiment of this aspect of the invention, the substantially purified or isolated OMT or OMT-like polypeptide includes an amino acid sequence selected from the group of sequences shown in Figures 28, 31, 94 and 100 hereto (Sequence ID Nos: 48, 60, 95, 173 and 175, 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 C4H or C4H-like polypeptide includes an amino acid sequence selected from the group of sequences shown in Figures 33, 35, 37 and 77 hereto (Sequence ID Nos: 97, 99, 101 and 167, respectively); and functionally active fragments and variants thereof.

In another preferred embodiment of this aspect of the invention, the substantially purified or isolated CCR or CCR-like polypeptide includes an amino acid sequence selected from the group of sequences shown in Figures 39, 42 and 45 hereto (Sequence ID Nos: 103, 108 and 112, respectively); and functionally active fragments and variants thereof.

WO 02/26994 PCT/AU01/01221 12 In a further preferred embodiment of this aspect of the invention, the substantially purified or isolated PER or PER-like polypeptide includes an amino acid sequence selected from the group of sequences shown in Figures 47, 50, 53, 55 and 106 hereto (Sequence ID Nos: 114, 118, 122, 124 and 177, respectively); and functionally active fragments and variants thereof.

In another preferred embodiment of this aspect of the invention, the substantially purified or isolated CELL or CELL-like polypeptide includes an amino acid sequence shown in Figure 57 hereto (Sequence ID No: 126); and functionally active fragments and variants thereof.

In another preferred embodiment of this aspect of the invention, the substantially purified or isolated F5H or F5H-like polypeptide includes an amino acid sequence shown in Figure 60 hereto (Sequence ID No: 136); and functionally active fragments and variants thereof.

In a further preferred embodiment of this aspect of the invention, the substantially purified or isolated PAL or PAL-like polypeptide includes an amino acid sequence selected from the group of sequences shown in Figures 63 and 66 hereto (Sequence ID Nos: 141 and 150, respectively); and functionally active fragments and variants thereof.

In another preferred embodiment of this aspect of the invention, the substantially purified or isolated 4CL or 4CL-like polypeptide includes an amino acid sequence selected from the group of sequences shown in Figures 69, 72 and 74 hereto (Sequence ID Nos: 155, 163 and 165, respectively); and functionally active fragments and variants thereof.

By "functionally active" in relation to polypeptides it is meant that the fragment or variant has one or more of the biological properties of the enzymes CCoAMT, CCoAMT-like, CAD, CAD-like, OMT, OMT-like, C4H, C4H-like, CCR, CCR-like, PER, PER-like, CELL, CELL-like, F5H, 005042824 13 PAL, PAL-like, 4CL and 4CL-like, respectively. Additions, deletions, C 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 Z activity of the fragment or variant. Preferably the functionally active fragment or variant has at least approximately 60% identity to the relevant part of the above mentioned amino acid sequence, more preferably at least t approximately 80% identity, most preferably at least approximately identity. Such functionally active variants and fragments include, for example, Sthose having conservative amino acid substitutions of one or more residues in the corresponding amino acid sequence. Preferably the fragment has a size c of at least 10 amino acids, more preferably at least 15 amino acids, most preferably at least 20 amino acids.

In a preferred embodiment, the present invention provides a substantially purified or isolated CCoAMT polypeptide from a Lolium species.

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

In a preferred embodiment, the present invention provides a substantially purified or isolated CCoAMT polypeptide including an amino acid sequence selected from the group of sequences shown in Figures 2, 5, 8, 83 and 88 hereto (Sequence ID Nos: 2, 13, 22, 169 and 171, respectively); and functionally active fragments and variants thereof having at least approximately 98% identity with the sequences shown in Figures 2, 5, 8, 83 and 88 hereto and have a size of 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 fragment according to the present invention. Techniques for recombinantly producing polypeptides are well known to those skilled in the art.

005042824 13A Availability of the nucleotide sequences of the present invention and c- deduced amino acid sequences facilitates immunological screening of cDNA O expression libraries. Synthetic peptides representing portions of the instant Z amino acid sequences may be synthesized. These peptides can 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 WO 02/26994 PCT/AU01/01221 14 marker identifies a specific region or locus in the genome. The more genetic markers, the finer defined is the genotype. A genetic marker becomes particularly useful when it is allelic between organisms because it then may serve to unambiguously identify an individual. Furthermore, a genetic marker becomes particularly useful when it is based on nucleic acid sequence information that can unambiguously establish a genotype of an individual and when the function encoded by such nucleic acid is known and is associated with a specific trait. Such nucleic acids and/or nucleotide sequence information including single nucleotide polymorphisms (SNPs), 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 SNP's of the nucleic acids and nucleic acid fragments of the present invention. These are indicated (marked with grey on the black background) in the figures that show multiple alignments of nucleotide sequences of nucleic acid fragments contributing to consensus contig sequences. See for example, Figures 3, 6, 15, 18, 26, 29, 58, 61, 64, 67 and 70 (Sequence ID Nos: 3 to 11, 14 to 20, 29 to 33, 36 to 49 to 58, 61 to 93, 104 to 106, 127 to 134, 137 to 139, 142 to 148, 151 to 153 and 156 to 161, 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 single nucleotide polymorphism (SNP), said method including sequencing nucleic acid fragments from a nucleic acid library.

WO 02/26994 PCT/AU01/01221 The nucleic acid library may be of any suitable type and is preferably a cDNA library.

The nucleic acid or nucleic acid fragment may be isolated from a recombinant plasmid or may be amplified, for example using polymerase chain reaction.

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 nucleic acids or nucleic acid fragments of the present invention including SNPs, and/or nucleotide sequence information thereof, as molecular genetic markers.

In a still further aspect of the present invention there is provided use of a nucleic acid or nucleic acid fragment according to the present invention, and/or nucleotide sequence information thereof, as a molecular genetic marker.

More particularly, nucleic acids or nucleic acid fragments according to the present invention and/or nucleotide sequence information thereof may be used as a molecular genetic marker for quantitative trait loci (QTL) tagging, QTL mapping, DNA fingerprinting and in marker assisted selection, particularly in ryegrasses and fescues. Even more particularly, nucleic acids or nucleic acid fragments according to the present invention and/or nucleotide sequence information thereof may be used as molecular genetic markers in forage and turf grass improvement, e.g. tagging QTLs for herbage quality traits, dry matter digestibility, mechanical stress tolerance, disease resistance, insect pest resistance, plant stature, leaf and stem colour. Even more particularly, sequence information revealing SNPs in allelic variants of the nucleic acids or nucleic acid fragments of the present invention and/or WO 02/26994 PCT/AU01/01221 16 nucleotide sequence information thereof may be used as molecular genetic markers for QTL tagging and mapping and in marker assisted selection, particularly in ryegrasses and fescues.

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

The term "construct' as used herein refers to an artificially assembled or isolated nucleic acid molecule which includes the gene of interest. In general a construct may include the gene or genes of interest, a marker gene which in some cases can also be the gene of interest and appropriate regulatory sequences. It should be appreciated that the inclusion of regulatory sequences in a construct is optional, for example, such sequences may not be required in situations where the regulatory sequences of a host cell are to be used. The term construct includes vectors but should not be seen as being limited thereto.

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

The term "vector" as used herein includes both cloning and expression vectors. Vectors are often recombinant molecules including nucleic acid molecules from several sources.

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

WO 02/26994 PCT/AU01/01221 17 By "operatively linked" is meant that said regulatory element is capable of causing expression of said nucleic acid or nucleic acid fragment in a plant cell and said terminator is capable of terminating expression of said nucleic acid or nucleic acid fragment in a plant cell. Preferably, said regulatory element is upstream of said nucleic acid or nucleic acid fragment and said terminator is downstream of said nucleic acid or nucleic acid fragment.

The vector may be of any suitable type and may be viral or non-viral.

The vector may be an expression vector. Such vectors include chromosomal, non-chromosomal and synthetic nucleic acid sequences, eg. derivatives of plant viruses; bacterial plasmids; derivatives of the Ti plasmid from Agrobacterium tumrnefaciens, derivatives of the Ri plasmid from Agrobacterium rhizogenes; phage DNA; yeast artificial chromosomes; bacterial artificial chromosomes; binary bacterial artificial chromosomes; vectors derived from combinations of plasmids and phage DNA. However, any other vector may be used as long as it is replicable or integrative or viable in the plant cell.

The regulatory element and terminator may be of any suitable type and may be endogenous to the target plant cell or may be exogenous, provided that they are functional in the target plant cell.

Preferably the regulatory element is a promoter. A variety of promoters which may be employed in the vectors of the present invention are well known to those skilled in the art. Factors influencing the choice of promoter include the desired tissue specificity of the vector, and whether constitutive or inducible expression is desired and the nature of the plant cell to be transformed (eg. monocotyledon or dicotyledon). Particularly suitable constitutive promoters include the Cauliflower Mosaic Virus 35S (CaMV 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 WO 02/26994 PCT/AU01/01221 18 terminator may be from the same gene as the promoter sequence or a different gene. Particularly suitable terminators are polyadenylation signals, such as the CaMV 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 acid fragment of the present invention and the terminator, may include further elements necessary for expression of the nucleic acid or nucleic acid fragment, in different combinations, for example vector backbone, origin of replication (ori), multiple cloning sites, spacer sequences, enhancers, introns (such as the maize Ubiquitin Ubi intron), antibiotic resistance genes and other selectable marker genes [such as the neomycin phosphotransferase (npt2) gene, the hygromycin phosphotransferase (hph) gene, the phosphinothricin acetyltransferase (bar or pat) gene], and reporter genes (such as betaglucuronidase (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.

As an alternative to use of a selectable marker gene to provide a phenotypic trait for selection of transformed host cells, the presence of the vector in transformed cells may be determined by other techniques well known in the art, such as PCR (polymerase chain reaction), Southern blot hybridisation analysis, histochemical GUS assays, northern and Western blot hybridisation analyses.

Those skilled in the art will appreciate that the various components of the vector are operatively linked, so as to result in expression of said nucleic acid or nucleic acid fragment. Techniques for operatively linking the components of the vector of the present invention are well known to those skilled in the art. Such techniques include the use of linkers, such as synthetic linkers, for example including one or more restriction enzyme sites.

WO 02/26994 PCT/AU01/01221 19 The vectors of the present invention may be incorporated into a variety of plants, including monocotyledons (such as grasses from the genera Lolium, Festuca, Paspalum, Pennisetum, Panicum and other forage and turfgrasses, corn, oat, wheat and barley), dicotyledons (such as arabidopsis, tobacco, white clover, red clover, subterranean clover, alfalfa, eucalyptus) and gymnosperms. In a preferred embodiment, the vectors may be used to transform monocotyledons, preferably grass species such as ryegrasses (Lolium species) and fescues (Festuca species), more preferably perennial ryegrass, including forage- and turf-type cultivars.

Techniques for incorporating the vectors of the present invention into plant cells (for example by transduction, transfection or transformation) are well known to those skilled in the art. Such techniques include Agrobacterium mediated introduction, electroporation to tissues, cells and 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 vectors of the present invention may be selected, as described above, and then cultured in an appropriate medium to regenerate transformed plants, using techniques well known in the art. The culture conditions, such as temperature, pH and the like, will be apparent to the person skilled in the art. The resulting plants may be reproduced, either sexually or asexually, using methods well known in the art, to produce successive generations of transformed plants.

In a further aspect of the present invention there is provided a plant cell, plant, plant seed or other plant part, including, e.g. transformed with, a vector of the present invention.

WO 02/26994 PCT/AU01/01221 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 a ryegrass, most preferably perennial ryegrass, including both forage- and turf-type cultivars.

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.

In a further aspect of the present invention there is provided a method of modifying lignin biosynthesis and/or cellulose degradation 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.

By "an effective amount" is meant an amount sufficient to result in an identifiable phenotypic trait in said plant, or a plant, plant seed or other plant part derived therefrom. Such amounts can be readily determined by an appropriately skilled person, taking into account the type of plant, the route of administration and other relevant factors. Such a person will readily be able to determine a suitable amount and method of administration. See, for example, Maniatis et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, the entire disclosure of which is incorporated herein by reference.

Using the methods and materials of the present invention, plant lignin biosynthesis and/or cellulose degradation may be increased, decreased or WO 02/26994 PCT/AU01/01221 21 otherwise modified relative to an untransformed control plant. They may be increased or otherwise modified, for example, by incorporating additional copies of a sense nucleic acid or nucleic acid fragment of the present invention. They may be decreased or otherwise modified, for example, by incorporating an antisense nucleic acid or nucleic acid fragment of the present invention. In addition, the number of copies of genes encoding different enzymes in the lignin biosynthetic pathway may be manipulated to modify the relative amount of each monolignol synthesized, thereby leading to the formation of lignin having altered composition.

In a still further aspect of the present invention there is provided a lignin or modified lignin or a cellulose or modified cellulose substantially or partially purified or isolated from a plant, plant seed or other plant part of the present invention.

Such lignins may be modified from naturally occurring lignins in terms of, for example, their monomeric composition or ratios of individual monolignols, the presence of novel monolignols, the degree of linkage and/or nature of linkages between lignins and other plant cell wall components.

Such cellulose may be modified from naturally occurring cellulose in terms of, for example, the degree of polymerisation (number of units), and/or degree of branching and/or nature of linkages between units and/or nature of linkages between cellulose and other plant cell wall components.

The present invention will now be more fully described with reference to the accompanying Examples and drawings. It should be understood, however, that the description following is illustrative only and should not be taken in any way as a restriction on the generality of the invention described above.

WO 02/26994 PCT/AU01/01221 22 In the Figures Figure 1 shows the consensus contig nucleotide sequence of LpCCoAMTa (Sequence ID No: 1).

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

Figure 3 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpCCoAMTa (Sequence ID Nos: 3 to 11).

Figure 4 shows the consensus contig nucleotide sequence of LpCCoAMTb (Sequence ID No: 12).

Figure 5 shows the deduced amino acid sequence of LpCCoAMTb (Sequence ID No: 13).

Figure 6 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpCCoAMTb (Sequence ID Nos: 14 to Figure 7 shows the nucleotide sequence of LpCCoAMTc (Sequence ID No: 21).

Figure 8 shows the deduced amino acid sequence of LpCCoAMTc (Sequence ID No: 22).

Figure 9 shows the nucleotide sequence of LpCADa (Sequence ID No: 23).

Figure 10 shows the deduced amino acid sequence of LpCADa (Sequence ID No: 24).

WO 02/26994 PCT/AU01/01221 23 Figure 11 shows the nucleotide sequence of LpCADb (Sequence ID No: Figure 12 shows the deduced amino acid sequence of LpCADb (Sequence ID No: 26).

Figure 13 shows the consensus contig nucleotide sequence of LpCADc (Sequence ID No: 27).

Figure 14 shows the deduced amino acid sequence of LpCADc (Sequence ID No: 28).

Figure 15 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpCADc (Sequence ID Nos: 29 to 33).

Figure 16 shows the consensus contig nucleotide sequence of LpCADd (Sequence ID No: 34).

Figure 17 shows the deduced amino acid sequence of LpCADd (Sequence ID No: Figure 18 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpCADd (Sequence ID Nos: 36 to Figure 19 shows the consensus contig nucleotide sequence of LpCADe (Sequence ID No: 41).

Figure 20 shows the deduced amino acid sequence of LpCADe (Sequence ID No: 42).

WO 02/26994 PCT/AU01/01221 24 Figure 21 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpCADe (Sequence ID Nos: 43 and 44).

Figure 22 shows the nucleotide sequence of LpCADf (Sequence ID No: Figure 23 shows the deduced amino acid sequence of LpCADf (Sequence ID No: 46).

Figure 24 shows the consensus contig nucleotide sequence of LpOMTa (Sequence ID No: 47).

Figure 25 shows the deduced amino acid sequence of LpOMTa (Sequence ID No: 48).

Figure 26 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpOMTa (Sequence ID Nos: 49 to 58).

Figure 27 shows the consensus contig nucleotide sequence of LpOMTb (Sequence ID No: 59).

Figure 28 shows the deduced amino acid sequence of LpOMTb (Sequence ID No: Figure 29 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpOMTb (Sequence ID Nos: 61 to 93).

Figure 30 shows the nucleotide sequence of LpOMTc (Sequence ID No: 94).

Figure 31 shows the deduced amino acid sequence of LpOMTc (Sequence ID No: WO 02/26994 PCT/AU01/01221 Figure 32 shows the nucleotide sequence of LpC4Ha (Sequence ID No: 96).

Figure 33 shows the deduced amino acid sequence of LpC4Ha (Sequence ID No: 97).

Figure 34 shows the nucleotide sequence of LpC4Hb (Sequence ID No: 98).

Figure 35 shows the deduced amino acid sequence of LpC4Hb (Sequence ID No: 99).

Figure 36 shows the nucleotide sequence of LpC4Hc (Sequence ID No: 100).

Figure 37 shows the deduced amino acid sequence of LpC4Hc (Sequence ID No: 101).

Figure 38 shows the consensus contig nucleotide sequence of LpCCRa (Sequence ID No: 102).

Figure 39 shows the deduced amino acid sequence of LpCCRa (Sequence ID No: 103).

Figure 40 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpCCRa (Sequence ID Nos: 104 to 106).

Figure 41 shows the consensus contig nucleotide sequence of LpCCRb (Sequence ID No: 107).

Figure 42 shows the deduced amino acid sequence of LpCCRb (Sequence ID No: 108).

WO 02/26994 PCT/AU01/01221 26 Figure 43 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpCCRb (Sequence ID Nos: 109 and 110).

Figure 44 shows the nucleotide sequence of LpCCRc (Sequence ID No: 111).

Figure 45 shows the deduced amino acid sequence of LpCCRc (Sequence ID No: 112).

Figure 46 shows the consensus contig nucleotide sequence of LpPERa (Sequence ID No: 113).

Figure 47 shows the deduced amino acid sequence of LpPERa (Sequence ID No: 114).

Figure 48 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpPERa (Sequence ID No: 115 and 116).

Figure 49 shows the consensus contig nucleotide sequence of LpPERb (Sequence ID No: 117).

Figure 50 shows the deduced amino acid sequence of LpPERb (Sequence ID No: 118).

Figure 51 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpPERb (Sequence ID Nos: 119 and 120).

Figure 52 shows the nucleotide sequence of LpPERc (Sequence ID No: 121).

Figure 53 shows the deduced amino acid sequence of LpPERc (Sequence ID No: 122).

WO 02/26994 PCT/AU01/01221 27 Figure 54 shows the nucleotide sequence of LpPERd (Sequence ID No: 123).

Figure 55 shows the deduced amino acid sequence of LpPERd (Sequence ID No: 124).

Figure 56 shows the consensus contig nucleotide sequence of LpCELL (Sequence ID No: 125).

Figure 57 shows the deduced amino acid sequence of LpCELL (Sequence ID No: 126).

Figure 58 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpCELL (Sequence ID Nos: 127 to 134).

Figure 59 shows the consensus contig nucleotide sequence of (Sequence ID No: 135).

Figure 60 shows the deduced amino acid sequence of LpF5Ha (Sequence ID No: 136).

Figure 61 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpF5Ha (Sequence ID Nos: 137 to 139).

Figure 62 shows the consensus contig nucleotide sequence of LpPALa (Sequence ID No: 140).

Figure 63 shows the deduced amino acid sequence of LpPALa (Sequence ID No: 141).

WO 02/26994 PCT/AU01/01221 28 Figure 64 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpPALa (Sequence ID Nos: 142 to 148).

Figure 65 shows the consensus contig nucleotide sequence of LpPALb (Sequence ID No: 149).

Figure 66 shows the deduced amino acid sequence of LpPALb (Sequence ID No: 150).

Figure 67 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpPALb (Sequence ID Nos: 151 to 153).

Figure 68 shows the consensus contig nucleotide sequence of Lp4CLa (Sequence ID No: 154).

Figure 69 shows the deduced amino acid sequence of Lp4CLa (Sequence ID No: 155).

Figure 70 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence Lp4CLa (Sequence ID Nos: 156 to 161).

Figure 71 shows the nucleotide sequence of Lp4CLb (Sequence ID No: 162).

Figure 72 shows the deduced amino acid sequence of Lp4CLb (Sequence ID No: 163).

Figure 73 shows the nucleotide sequence of Lp4CLc (Sequence ID No: 164).

Figure 74 shows the deduced amino acid sequence of Lp4CLc (Sequence ID No: 165).

WO 02/26994 PCT/AU01/01221 29 Figure 75 shows a plasmid map of the cDNA encoding perennial ryegrass C4H.

Figure 76 shows the nucleotide sequence of perennial ryegrass C4H cDNA (Sequence ID No: 166).

Figure 77 shows the deduced amino acid sequence of perennial ryegrass C4H cDNA (Sequence ID No: 167).

Figure 78 shows plasmid maps of sense and antisense constructs of LpC4H in pDH51 transformation vector.

Figure 79 shows plasmid maps of sense and antisense constructs of LpC4H in pKYLX71:35S2 binary transformation vector.

Figure 80 shows screening by Southern hybridisation for RFLPs using LpC4H as a probe.

Figure 81 shows a plasmid map of the cDNA encoding perennial ryegrass CCoAOMT1.

Figure 82 shows the nucleotide sequence of perennial ryegrass CCoAOMT1 cDNA (Sequence ID No: 168).

Figure 83 shows the deduced amino acid sequence of perennial ryegrass CCoAOMT1 cDNA (Sequence ID No: 169).

Figure 84 shows plasmid maps of sense and antisense constructs of LpCCoAOMT1 in pDH51 transformation vector.

Figure 85 shows screening by Southern hybridisation for RFLPs using LpCCoAOMT1 as a probe.

WO 02/26994 PCT/AU01/01221 Figure 86 shows a plasmid map of the cDNA encoding perennial ryegrass CCoAOMT2.

Figure 87 shows the nucleotide sequence of perennial ryegrass CCoAOMT2 cDNA (Sequence ID No: 170).

Figure 88 shows the deduced amino acid sequence of perennial ryegrass CCoAOMT2 cDNA (Sequence ID No: 171).

Figure 89 shows plasmid maps of sense and antisense LpCCoAOMT2 in pDH51 transformation vector.

Figure 90 shows plasmid maps of sense and antisense LpCCoAOMT2 in pKYLX71:35S2 binary transformation vector.

Figure 91 shows screening by Southern hybridisation for LpCCoAOMT2 as a probe.

constructs of constructs of RFLPs using Figure 92 shows plasmid map of the cDNA encoding perennial ryegrass OMT2 cDNA.

Figure 93 shows the nucleotide sequence of perennial ryegrass OMT2 cDNA (Sequence ID No: 172).

Figure 94 shows the deduced amino acid sequence of perennial ryegrass OMT2 cDNA (Sequence ID No: 173).

Figure 95 shows plasmid maps of sense and antisense constructs of LpOMT2 in pDH51 transformation vector.

Figure 96 shows plasmid maps of sense and antisense constructs of LpOMT2 in pKYLX71:35S2 binary transformation vector.

WO 02/26994 PCT/AU01/01221 31 Figure 97 shows screening by Southern hybridisation for RFLPs using LpOMT2 as a probe.

Figure 98 shows the plasmid map of the cDNA encoding perennial ryegrass OMT3.

Figure 99 shows the nucleotide sequence of perennial ryegrass OMT3 cDNA (Sequence ID No: 174).

Figure 100 shows the deduced amino acid sequence of perennial ryegrass OMT3 cDNA (Sequence ID No: 175).

Figure 101 shows Plasmid maps of sense and antisense LpOMT3 in pDH51 transformation vector.

Figure 102 shows plasmid maps of sense and antisense LpOMT3 in pKYLX71:35S2 binary transformation vector.

Figure 103 shows screening by Southern hybridisation for LpOMT3 as a probe.

constructs of constructs of RFLPs using Figure 104 shows the plasmid map of the cDNA encoding perennial ryegrass Peroxidasel.

Figure 105 shows the nucleotide sequence of perennial ryegrass Peroxidasel cDNA (Sequence ID No: 176).

Figure 106 shows the deduced amino acid sequence of perennial ryegrass Peroxidasel cDNA (Sequence ID No: 177).

Figure 107 shows plasmid maps of sense and antisense constructs of LpPeroxidasel in pDH51 transformation vector.

WO 02/26994 PCT/AU01/01221 32 Figure 108 shows plasmid maps of sense and antisense constructs of LpPeroxidasel in pKYLX71:35S2 binary transformation vector.

Figure 109 shows screening by Southern hybridisation for RFLPs using LpPeroxidasel as a probe.

Figure 110 shows the regeneration of transgenic tobacco plants from direct gene transfer to protoplasts of chimeric ryegrass lignin biosynthesis genes.

Figure 111 shows a subgrid of a microarray for the expression profiling of perennial ryegrass lignin biosynthesis and cellulose degradation genes. Red represents up-regulated expression, green represents down-regulated expression and yellow represents no change in expression. For example, an overlay of microarray images probed with 5LS tissues (red) and 5LR tissues (green). Expression level is relatively expressed as up-regulated in 5LS (red), down-regulated in 5LS (green) and no change in expression (yellow).

Figure 112 shows a genetic linkage map of perennial ryegrass NA6 showing map location of ryegrass genes encoding enzymes involved in lignin biosynthesis and cellulose degradation.

EXAMPLE 1 Preparation of cDNA libraries, isolation and sequencing of cDNAs coding for CCoAMT, CAD, OMT, C4H, CCR, PER, CELL, F5H, PAL and 4CL 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 in Table 1.

WO 02/26994 PCT/AU01/01221 33 TABLE 1 cDNA libraries from perennial ryegrass (Lolium perenne) Library Organ/Tissue 01 rg Roots from 3-4 day old light-grown seedlings 02rg Leaves from 3-4 day old light-grown seedlings 03rg Etiolated 3-4 day old dark-grown seedlings 04rg Whole etiolated seedlings (1-5 day old and 17 days old) Senescing leaves from mature plants 06rg Whole etiolated seedlings (1-5 day old and 17 days old) 07rg Roots from mature plants grown in hydroponic culture 08rg Senescent leaf tissue 09rg Whole tillers and sliced leaves 1, 3, 6, 12 and 24 h after harvesting) Embryogenic suspension-cultured cells 11 rg Non-embryogenic suspension-cultured cells 12rg Whole tillers and sliced leaves 1, 3, 6, 12 and 24 h after harvesting) 13rg Shoot apices including vegetative apical meristems 14rg Immature inflorescences including different stages of inflorescence meristem and inflorescence development Defatted pollen 16rg Leaf blades and leaf sheaths (rbcL, rbcS, cab, wir2A subtracted) 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) 2, 3, 4, 5, 6, 24 and 48 h after transfer) Non-embryogenic suspension-cultured cells subjected to osmotic WO 02/26994 PCT/AU01/01221 Library Organ/Tissue stress (grown in media with double-strength salts) 2, 3, 4, 5, 6, 24 and 48 h after transfer) 21rg Drought-stressed tillers (pseudostems from plants subjected to PEG-simulated drought stress) 22rg Spikelets with open and maturing florets 23rg Mature roots (specific subtraction with leaf tissue) The cDNA libraries may be prepared by any of many methods available. For example, total RNA may be isolated using the Trizol method (Gibco-BRL, USA) or the RNeasy Plant Mini kit (Qiagen, Germany), following the manufacturers' instructions. cDNAs may be generated using the SMART PCR cDNA synthesis kit (Clontech, USA), cDNAs may be amplified by long distance polymerase chain reaction using the Advantage 2 PCR Enzyme system (Clontech, USA), cDNAs may be cleaned using the GeneClean spin column (Bio 101, USA), tailed and size fractionated, according to the protocol provided by Clontech. The cDNAs may be introduced into the pGEM-T Easy Vector system 1 (Promega, USA) according to the protocol provided by Promega. The cDNAs in the pGEM-T Easy plasmid vector are transfected into Escherichia coli Epicurian coli 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 plasmid vector pBluescript. In addition, the cDNAs may be introduced directly into precut pBluescript II vectors (Stratagene) using T4 DNA ligase (New England Biolabs), followed by transfection into E. coli cells according to the manufacturer's protocol (GIBCO BRL Products).

WO 02/26994 PCT/AU01/01221 Once the cDNA inserts are in plasmid vectors, plasmid DNAs are prepared from randomly picked bacterial colonies containing recombinant plasmids, or the insert cDNA sequences are amplified via polymerase chain reaction using primers specific for vector sequences flanking the inserted cDNA sequences. Plasmid DNA preparation may be performed robotically using the Qiagen QiaPrep Turbo kit (Qiagen, Germany) according to the protocol provided by Qiagen. Amplified insert DNAs are sequenced in dyeterminator sequencing reactions to generate partial cDNA sequences (expressed sequence tags or "ESTs"). The resulting ESTs are analyzed using an Applied Biosystems ABI 3700 sequence analyser.

EXAMPLE 2 DNA sequence analyses The cDNA clones encoding CCoAMT, CAD, OMT, C4H, CCR, PER, CELL, F5H, PAL and 4CL were identified by conducting BLAST (Basic Local Alignment Search Tool; Altschul et al. (1993) J. Mol. Biol. 215:403-410) searches. The cDNA sequences obtained were analysed for similarity to all publicly available DNA sequences contained in the eBioinformatics nucleotide database using the BLASTN algorithm provided by the National Center for Biotechnology Information (NCBI). The DNA sequences were translated in all reading frames and compared for similarity to all publicly available protein sequences contained in the SWISS-PROT protein sequence database using BLASTx algorithm (v 2.0.1) (Gish and States (1993) Nature Genetics 3:266- 272) provided by the NCBI.

The cDNA sequences obtained and identified were then used to identify additional identical and/or overlapping cDNA sequences generated using the BLASTN algorithm. The identical and/or overlapping sequences were subjected to a multiple alignment using the CLUSTALw algorithm, and to generate a consensus contig sequence derived from this multiple sequence WO 02/26994 PCT/AU01/01221 36 alignment. The consensus contig sequence was then used as a query for a search against the SWISS-PROT protein sequence database using the BLASTx algorithm to confirm the initial identification.

EXAMPLE 3 Identification and full-length sequencing of perennial ryegrass C4H, CCoAOMT1, CCoAOMT2, OMT2, OMT3 and Peroxidasel cDNAs encoding enzymes involved in cell wall modification To fully characterise for the purposes of the generation of probes for hybridisation experiments and the generation of transformation vectors, a set of perennial ryegrass cDNAs encoding enzymes involved in cell wall modification was identified and fully sequenced.

Full-length cDNAs were identified from our EST sequence database using relevant published sequences (NCBI databank) as queries for BLAST searches. Full-length cDNAs were identified by alignment of the query and hit sequences using Sequencher (Gene Codes Corp., AnnArbor, MI 48108, USA). The original plasmid was then used to transform chemically competent XL-1 cells (prepared in-house, CaCI 2 protocol). After colony PCR (using HotStarTaq, Qiagen) a minimum of three PCR-positive colonies per transformation were picked for initial sequencing with M13F and M13R primers. The resulting sequences were aligned with the original EST sequence using Sequencher to confirm identity and one of the three clones was picked for full-length sequencing, usually the one with the best initial sequencing result.

Sequencing was completed by primer walking, i.e. oligonucleotide primers were designed to the initial sequence and used for further sequencing. In most cases the sequencing could be done from both 5' and 3' end. The sequences of the oligonucleotide primers are shown in Table 2. In WO 02/26994 WO 02/6994PC 1/AU 110 1221 37 some instances, however, an extended poly-A tail necessitated the sequencing of the cDNA to be completed from the 5' end.

TABLE 2 List of primers used for sequencing of the full-length cDNAs gene name clone ID sequencing primer sequence primer LpC4H O6rgl BsFO3.3 Q6rgl BsFO3.f 1 TCAACCACCCCAACCTGATG O6rgl BsFO3.rl GAGTAATCGTCTCACTTCGC LpCCoAOMT1 Q6rgl JsFO4. 1 06rgl JsFO4.fl CATCCCAGAAGACGGAAC LpCCoAOMT2 1 2rgl DsEO1 .1 1 2rgl DsEQ1 .f 1 TACGAGACCATCGGCAAG LpQMVT2 O6rgl SsGO2. 1 Qergi SsGQ2.fl1 CGTTCGCTCTCCTCACCC 06rgl SsGO2.rl AAGTCGGCGAAGATGTAGG LpOMT3 O6rg2QsGOl .1 O6rg2QsGOl ii AAGTTCCTCACCCCCAAC 06rg2QsGOl .f2 CGCTGCTCAAGAACTGCTAC O6rg2QsGOl .rl CTCATCTACTTGGTGAACTCG LpPeroxidasel 1 6rg2OsGO7.2 06rg2OsGO7.f 1 CGCCAAGAAGAACCTCAAC 06rg2OsGO7. rl ATTTTGAGCTGCACCACG Contigs were then assembled in Sequencher. The contigs include the sequences of the SMART primers used to generate the initial cDNA library as well as pGEM-T Easy vector sequence up to the EcoRl cut site both at the and 3' end.

Plasmid maps and the full cDNA sequences of perennial ryegrass C4H, CCoAOMT1, CCoAOMT2, OMT2, OMT3 and Peroxidasel were obtained (Figures 75, 76, 81, 82, 86, 87, 92, 93, 98, 99,104 and 105).

WO 02/26994 WO 02/6994PC 1/AU 110 1221 38 EXAMPLE 4 Development of transformation vectors containing chimeric genes with C41-, CCoAOMT1, CCoAOMT2, OMT2, OMT3 and Peroxidasel cDNA sequences from perennial ryegrass To alter the expression of the enzymes involved in lignin biosynthesis C4H, CCoAOMT1, C~oAOMT2, OMT2, OMT3 and Peroxidasel, 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 PCIR using the original pGEM-T Easy plasmid cDNA as a template. The primers used (Table 3) contained restriction sites for Ecofl and Xbal for directional and nondirectional cloning into the target vector.

TABLE 3 List of primers used to PCIR-amplify the open reading frames gene name clone ID primer primer sequence LpO4H O6rgl BsFO3.3 O6rgl BsFO3.f GAATTCTAGACACAGTCACAGAAGCCACCTACC Q6rgl BsFO3.r GAATTCTAGACTCTCGGCACCAGCGTTCACG LpCCoAOMT1 06rgl JsF04. 1 LpCCoAOMT1 f GAATTCTAGAAAGACAGCCCAATCATCC LpC~oAOMT1 r GAATTCTAGAGAAGAAACCAACAACGGC LpCCoAOMT2 1 2rgl DsEO1 .1 1 2rgl DsEQ1 GAATTCTAGAGI'TCCTTCCTCCACCTAAACACC 1 2rgl DsEO1 .r GAATTCTAGAGCGAGCGTCATCAATCAGAC LpOMT2 06rgl SsGO2.1 O6rgl SsGO2.f GAATTCTAGACTAGCAAGCAGCAACAGGTAC O6rgl SsGO2.r GAATTCTAcAAGGTc4AGTTTAGCCTACCAAAAPG LpOMT3 O6rg2QsGOl .1 O6rg2QsGOl .1 GAATTCTAGAGTCTCTCGCTCACACCAAAATCG 06rg2QsGOl .r GAATTCTAGATGTTGCCTGGATGCTTGGAGG LpPeroxidasel O 6rg2OsGO7.2 O6rg2OsGO7f GAAIUTCTAGAGACGCCGCAAkTATAGCTTGG O6rg2OsGO7r CAATTCTAGAGCTGAACTGCTA-ATAGTAGAGTG WO 02/26994 PCT/AU01/01221 39 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 containing a CaMV 35S expression cassette. The orientation of the constructs (sense or antisense) was checked by DNA sequencing through the multicloning site of the vector. Transformation vectors containing chimeric genes using full-length open reading frame cDNAs of perennial ryegrass C4H, CCoAOMT1, CCoAOMT2, OMT2, OMT3 and Peroxidasel in sense and antisense orientations under the control of the CaMV 35S promoter were generated (Figures 78, 84, 89, 95, 101 and 107).

EXAMPLE Development of binary transformation vectors containing chimeric genes with C4H, CCoAOMT2, OMT2, OMT3 and Peroxidasel cDNA sequences from perennial ryegrass To alter the expression of the enzymes involved in lignin biosynthesis C4H, CCoAOMT2, OMT2, OMT3 and Peroxidasel, through antisense and/or sense suppression technology and for over-expression of these key enzymes in transgenic plants, a set of sense and antisense transformation vectors was produced.

cDNA fragments were generated by high fidelity PCR using the original pGEM-T Easy plasmid cDNA as a template. The primers used (Table 3) contained restriction sites for EcoRI and Xbal for directional and nondirectional cloning into the target vector. After PCR amplification and restriction digest with the appropriate restriction enzyme (usually Xbal), the cDNA fragments were cloned into the corresponding site in pKYLX71:35S2, 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 WO 02/26994 PCT/AU01/01221 promoter with a duplicated enhancer region and an rbcS terminator (An et al., 1985; Schardl et al., 1987). The orientation of the constructs (sense or antisense) was checked by restriction enzyme digest. Transformation vectors containing chimeric genes using full-length open reading frame cDNAs of perennial ryegrass C4H, CCoAOMT2, OMT2, OMT3 and Peroxidasel in sense and antisense orientations under the control of the CaMV 35S2 promoter were generated (Figures 79, 90, 96, 102 and 108).

EXAMPLE 6 Production of transgenic tobacco plants carrying chimeric C4H, CCoAOMT1, CCoAOMT2, OMT2, OMT3 and Peroxidasel genes from perennial ryegrass A set of transgenic tobacco plants carrying chimeric C4H, CCoAOMT1, CCoAOMT2, OMT2, OMT3 and Peroxidasel cDNA genes from perennial ryegrass were produced.

pDH51-based transformation vectors with LpC4H, LpCCoAOMT1, LpCCoAOMT2, LpOMT2, LpOMT3 and LpPeroxidasel cDNAs comprising the full open reading frame sequences in sense and antisense orientations under the control of the CaMV 35S promoter were generated.

Direct gene transfer experiments to tobacco protoplasts were performed using these transformation vectors.

The production of transgenic tobacco plants carrying the perennial ryegrass C4H, CCoAOMT1, CCoAOMT2, OMT2, OMT3 and Peroxidasel cDNAs under the control of the constitutive CaMV 35S promoter is described here in detail.

WO 02/26994 PCT/AU01/01221 41 Isolation of mesophyll protoplasts from tobacco shoot cultures 2 to 4 fully expanded leaves of a 6 week-old shoot culture were placed under sterile conditions (work in laminar flow hood, use sterilized forceps, scalpel and blades) in a 9 cm plastic culture dish containing 12 ml enzyme solution cellulase "Onozuka" R10 and 1.0% Macerozyme® The leaves were wetted thoroughly with enzyme solution and the midribs removed. The leaf halves were cut into small pieces and incubated overnight (14 to 18 h) at 25 0 C in the dark without shaking The protoplasts were released by gently pipetting up and down, and the suspension poured through a 100 pm stainless steel mesh sieve on a 100 ml glass beaker. The protoplast suspension was mixed gently, distributed into two 14 ml sterile plastic centrifuge tubes and carefully overlayed with 1 ml solution. After centrifugation for 5 min. at 70g (Clements Orbital 500 bench centrifuge, swing-out rotor, 400 rpm), the protoplasts were collected from the interphase and transferred to one new 14 ml centrifuge tube. 10 ml solution were added, the protoplasts resuspended by gentle tilting the capped tube and pelleted as before. The protoplasts were resuspended in 5 to 10 ml solution and the yield determined by counting a 1:10 dilution in a haemocytometer.

Direct gene transfer to protoplasts using polyethylene glycol The protoplasts were pelleted [70g (Clements Orbital 500 bench centrifuge, 400 rpm) for 5 min.] and resuspended in transformation buffer to a density of 1.6 x 106 protoplasts/ml. Care should be taken to carry over as little as possible W5 solution into the transformation mix. 300 pl samples of the protoplast suspension (ca. 5 x 10 5 protoplasts) were aliquotted in 14 ml sterile plastic centrifuge tubes, 30 pl of transforming DNA were added. After carefully mixing, 300 pl of PEG solution were added and mixed again by careful shaking. The transformation mix was incubated for 15 min. at room temperature with occasional shaking. 10 ml W5 solution were gradually WO 02/26994 PCT/AU01/01221 42 added, the protoplasts pelleted [70g (Clements Orbital 500 bench centrifuge, 400 rpm) for 5 min.] and the supernatant removed. The protoplasts were resuspended in 0.5 ml K3 medium and ready for cultivation Culture of protoplasts, selection of transformed lines and regeneration of transgenic tobacco plants Approximately 5 x 105 protoplasts were placed in a 6 cm petri dish. ml of a pre-warmed (melted and kept in a water bath at 40 to 45 0 C) 1:1 mix of K3:H medium containing 0.6% SeaPlaque T M agarose were added and, after gentle mixing, allowed to set.

After 20 to 30 min the dishes were sealed with Parafilm® and the protoplasts were cultured for 24 h in darkness at 24 0 C, followed by 6 to 8 days in continuous dim light (5 pmol m' 2 s 1 Osram L36 W/21 Lumilux white tubes), where first and multiple cell divisions occur. The agarose containing the dividing protoplasts was cut into quadrants and placed in 20 ml of A medium in a 250 ml plastic culture vessel. The corresponding selection agent was added to the final concentration of 50 mg/l kanamycin sulphate (for npt2 expression) or 25 mg/I hygromycin B (for hph expression) or 20 mg/l phosphinotricin (for bar expression). Samples were incubated on a rotary shaker with 80 rpm and 1.25 cm throw at 24°C in continuous dim light.

Resistant colonies were first seen 3 to 4 weeks after protoplast plating, and after a total time of 6 to 8 weeks protoplast-derived resistant colonies (when 2 to 3 mm in diameter) were transferred onto MS morpho medium solidified with 0.6% agarose in 12-well plates and kept for the following 1 to 2 weeks at 24°C in continuous dim light (5 pmol m" 2

S

1 Osram L36 W/21 Lumilux white tubes), where calli proliferated, reached a size of 8 to 10 mm, differentiated shoots that were rooted on MS hormone free medium leading to the recovery of transgenic tobacco plants (Table 4 and Figure 110).

WO 02/26994 WO 02/6994PC 1/AU 110 1221 43 TABLE 4 Production of transgenic tobacco calli carrying chimeric ryegrass genes (in sense and antisense orientation) for enzymes involved in lignin biosynthesis and cellulose degradation from direct gene transfer to protoplasts construct transfected transformed transformation protoplasts calli eff iciency pDH51 LpC4H 2.3xl 0 6 67 2.91x10pDH51 LpC4H anti 2.3x 0 6 12 0.52X10pDH51 LpCCoAOMT1 1.2xl 0 6 113 9.42x 0pDH51 LpCCoAOMT1 anti 0.6xl Y_ 76 12.67A 0pDH51 LpCCoAQMT2 2.3x1 0 6 41 1 .78x1 pDH51 LpCCoAOMT2 anti 2.3x1 06 61 2.65x1 pDH51 LpOMT2 2.3xl 0 6 115 5.00xl10 pDH51 LpOMT2 anti 2.3x 0'3 136 5.91X10pDH51 LpOMT3 2.3x1 06 64 2.78x1 LpOMT3 anti 2.3xl 0 6 100 4.35xl 0pDH51 LpPeroxidasel 2.3x1 l 260 1 1.30x1 pDH51 LpPeroxidasel anti 2.3x1 l 250 1 0.87x1 EXAMPLE 7 Genetic mapping of perennial ryegrass genes involved in lignin biosynthesis and cellulose degradation The cDNAs representing genes involved in lignin biosynthesis and cellulose degradation were amplified by POR from their respective plasmids, gel-purified and radio-labelled for use as probes to detect restriction fragment length polymorphisms (RFLPs). RFLPs were mapped in the F, (first WO 02/26994 PCT/AU01/01221 44 generation) population, NA 6 x AU 6 This population was made by crossing an individual (NA 6 from a North African ecotype with an individual (AUs) from the cultivar Aurora, which is derived from a Swiss ecotype. Genomic DNA of the 2 parents and 114 progeny was extracted using the 1 x CTAB method of Fulton etal. (1995).

Probes were screened for their ability to detect polymorphism using the DNA (10 [tg) of both parents and 5 F 1 progeny restricted with the enzymes Dral, EcoRI, EcoRV or Hindll. Hybridisations were carried out using the method of Sharp et al. (1988). Polymorphic probes were screened on a progeny set of 114 individuals restricted with the appropriate enzyme (Figures 85, 91, 97, 103 and 109).

RFLP bands segregating within the population were scored and the data was entered into an Excel spreadsheet. Alleles showing the expected 1:1 ratio were mapped using MAPMAKER 3.0 (Lander et al. 1987). Alleles segregating from, and unique to, each parent, were mapped separately to give two different linkage maps. Markers were grouped into linkage groups at a LOD of 5.0 and ordered within each linkage group using a LOD threshold of Loci representing genes involved in lignin biosynthesis and cellulose degradation mapped to the linkage groups as indicated in Table 5 and in Figure 112. These gene locations can now be used as candidate genes for quantitative trait loci for lignin biosynthesis-associated traits such as herbage quality, dry matter digestibility, mechanical stress tolerance, disease resistance, insect pest resistance, plant stature and leaf and stem colour.

WO 02/26994 WO 02/6994PC 1/AU 110 1221 TABLE Map locations of ryegrass genes encoding enzymes for lignin biosynthesis and cellulose degradation across two genetic linkage maps of perennial ryegrass (NA6 and AU6) Probe Polymorphic Mapped Locus Linkage with group NA6 AU6 Lp4CLJa Y Hind III Lp4CLJa 4 4 Lp4CLJb Y Hind III Lp4CLJb 1 1 LpC4H Y EcoR I LpO4H 3 3 LpCADd Y Dra I LpCADd 3 LpCADc Y Dra I LpCADc 1 LpCCoAOMT1-1 Y EcoR V LpCCoAQMT1 -1 7 LpCCRa Y Hind III LpCCRa 6 6 LpCCRb Y Dra I LpCCRb 7 7 LpCELL Y EcoR I LpCELL 4 Y EcoR V LpF5H-.1 3 LpF5H.2 1 LpOMT2 Y Dra I LpOMT2 6 6 LpOMT3 Y Dra I LpOMVT3 4 7 LpPALb Y EcoR I LpPALb 6 LpPeroxidasel Y Hind IlI LpPeroxidasel 2 2 WO 02/26994 PCT/AU01/01221 46 EXAMPLE 8 Expression profiling of cDNAs encoding enzymes for lignin biosynthesis and cellulose degradation using microarray technology cDNAs encoding enzymes for lignin biosynthesis and cellulose degradation were PCR amplified and purified. The amplified products were spotted three times 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. The duplicates were placed about 800 micron apart to prevent competitive hybridisation.

Table 6 gives details on the tissues used to extract total RNA.

TABLE 6 List of hybridization probes used in expression profiling of perennial ryegrass genes encoding enzymes for lignin biosynthesis and cellulose degradation using microarrays Hybridization probe for microarrays Organ specificity (3-month old plants Leaf blade grown hydroponically) Sheath Root Seedling grown under light condition 5-day old shoot 7-day old shoot (7LS) old shoot old root 7-day old root (7LR) WO 02/26994 PCT/AU01/01221 old root Seedling grown under dark condition 5-day old shoot 7-day old shoot (7DS) old shoot old root 7-day old root (7DR) old root Fluorescence labelled probes were synthesis by reversed transcribing RNA and incorporating Cyanine 3 or 5 labelled dCTP. The probes were hybridised onto microarrays. In each case the experiment was repeated on two microarrays. After hybridisation for 16 hours (overnight), the microarrays were washed and scanned using a confocal laser scanner (ScanArray 3000, Packard, USA). The images obtained were quantified and analysed using Imagene 4.1 and GeneSight 2.1 (BioDiscovery, USA). Data were judged as not present low expression medium expression high expression and highly expression (Table 7).

TABLE 7 Results of expression profiling of ryegrass genes for lignin biosynthesis and cellulose degradation plate Row col Gene id clone id gene seq sheath leaf root 51.8 7LS lOLS 5LR 7LR IOLR 5DS 7DS lOBS 5DR 71DR IODR notes bade Array2Urg A I NcwCAMT POPTM7556 l2rglDsEOl LpCCoAOMTa full 4-1 T T1 T i+ (LpGCoAOMT2) Array2Urg A 2 NcwCAMT POPTM7557 06rglJsFO4 LpCCcAOMTa full -Ii4 i- H!1 i4± (LpCCoAQMT1 -1) Array2Urg A 3 NowCAMTPOPTM7558 O3rglAsGl I LpCCoAOMTh partial f- +1- Array2Urg A 4 NcwCAML PETCR7559 06rgI GsAO9 LpO~oAOMTb full i i+ I I -14 44 +i I I ~~~(likely)I II IIII I Array2Urg a 5 NcwCAMTPOPTM7S66 O6rgl CsCO3 LpCCoAQMTb partial I Array2Urg a 0 NcwOAMTPETOR7501 0O3rg2OsDlO LpG~oAOMTb partial i-i-i- tn-n 44 4 Array2Urg a 17 jNcwCAMT PETCR7562 O~rg2RsHOB LpCCoAOMTb partial Array2Urg a 8 NcwCAML -POPTM7563 O7rglXsGO7 LpCCoAOMTb partial ++4-i-ill -i-4i- iii; 4..4 +44 -44 +-4 Array2Urg a 9 NcwCAD2-ARATH7564 11 rgl DsCO2 LpCADc partial Array2Urg a 10 NowCAD3-STYHU75G5 19rg21-sGOl LpCADo partial Array2Urg a 11 NcwCADH-PICA67566 1 Org2T3DO5 LpGAI~c partial Array2Urg a 12 NcwCADH-PICAB7567 2OrglDsGlO LpCADo full Array2Urg b 1 XnsPSS-YEAST-7568 OlrglDsEOll LpCADd partial Array2Urg b 2 NppDFRA-DIACA7559 llrgVsEO4 LpCADd partial +4 4 +4 +4+4 +444++ .4 .4 Array2Urg b 3 NDFRA_CALCH--7570 l8rglHsEOl LpCADd partial +4 44 44 444 Array2Urg b 4 NDFHA CALCH--7571 1 Brg-lKsA12 LpCADd partial -4 +4 +4 +4 +41 4444..

plate Row co[ Gene id clone id geng seq sheath leaf root 5LS 7LS lOLS 5LR 7LR IOLR 5DS 70S 1ODS 5DR 7DR IODR notes bade Array2Urg lb 5 NDFRACGALOH--7572 14rglYsFO3 LpCADe partial Array2Urg b 6 XnsDFRA SYNY37573 14rg2H-sEO9 LpCADe partial H Array2Urg b 7 NcwCOMTLMAIZE7574 06rgI MsAO4 LpOMTI full i± -i Ii 4 4 44 4 H.I 4---4+H Array2Urg b 8 NcwCOMT MAIZE7575 01 rgi lsGC6 LpOMT1 partial 4- 14 444 Array2Urg b 9 NcwCOMTMAIZE7576 01 rgRsG06 LpOMT1 partial I 4-4- 4-4-4-4- H4 4-4- H-.

Array2Urg b 10 NcwCOMT MAIZE7577 O7rgl SsH02 LpQMITI parial +4 44 44+ 4-+4 -I 4 4.

Array2Urg b 11 NcwCOMTF_MAIZE7578 O7rglYsFOO LpOMT1 paria +i f -H-I -h R4 4-444 -444 4 +4 Array2Llrg b 12 NcwCOMTLMAIZE7579 l7rglWsJOB LpOMT1 patial L -H Array2Urg c I NowCOMT_MAIZE7560 O6rqISsQO2 LpOMTZ full I -H I 4 .4-1 44I-4-H -4 Array2Urg c 2 NcwCOMtTMAIZE7581 O6rg2PsEO2 LpOMT2 partial +4 4 -1444-+ 44+ -4 ArrayZUrg c 3 NowTOMO-GLYEC75B2 06rgiBsFO3 LpC4H lull -i-i i- -II-I 4-4- -44--4 4-Hi-4 44-H 44 4 Array2Urg c 4 XnslOrgIDsGO87583 lOrgIDsGO8 LpCORa partial Array2Urg c 5 XnsYGVV1-YEAST7584 lOrg2RsDO6 LpCCRa partial Array2Urg c 6 NppDFRAJ-IORVU7585 11rg1KsG07 LpCCRS partial Array2Urg o 7 NppDFRA.MAIZE7586 l7rglAsCl2 LpCCRb partial Array2Urg c 8 DdtPER1-HQRVU7587 06rgl FsFO9 LpPeroxidasel full 4-1 4-4- 4--4 444 4- -4444 4--4 Array2Urg c F NcwPERX(NICSY7588 06rgITsGO3 LpPeroxidasel partial .H I .44 4 444- 4-4-44 44+4 ++4 Array2Urg c 10 NcwPERX-NICSY7589 05rg2PsAO6 LpPeroxidasql partial 444-44 4- Array2Urq c 11 UioNTPE-ENTHR7590 0Srg1!~sG08 LpPeroxidase2 full -1-4 4-4 4I-+4-4 H -4 -4 -4 Array2Urg c 12 DdrPERI-ORYSA7591 O8rgISsFO9 LpParoxidaso2 partial? -1-4 4 Array2Urg d 1 DdrPER1_ORYSA7502 O8rglTsEO5 LpPeroxidase2 partial? f44 i .4 H +4 Array2Urg d 2 BcwGUN1-PERAE7593 l4rg1PsEOl LpCELL partial 4 44 4 plate Row col Gone id clone id gone seq sheath leaf root 5LS ALS 1OLS 5LR 7LR 1 1OLR 5D8 711S 1 DDS 5DR 71IR I IODU notes bade Array2Urg d 3 N070'LMAlZE--7594- OlrglQs-19 LpF5H- Partial Array2Urg d 4 NG7C4 MAIZE--7595 Orgi P5E04 LpF5H partial f -f Array2Urg d 5 NC7D8&SOYBN--7696 Otirgi DsEO9 LpF5H partial 4+ Array2Urg d 6 NcwPALY-WHEAT7597 OlrgITsBO3 LpPALa partial i+-i 4 4 44- 44-- Array2Urg d 7 NcwPALY-WHEAT7598 07rg2AsFCI4 LpPALa partial 444 H- H- Array2Urg d 8 NcwPALYLWHEAT7599 lOrgi BsDO2 LpPALa partial +±44 4-±i 44 4 Array2Urg d 9 NcwPALY'LWHEAT7600 l6rgl CsDO1 LpPALa partial 4±4 +4-4-4 4+4+ 4± 4. 4 ++4 Array2Urg d 10 NcwPALY-WHEAT7601 16rgi OaFO6 LpPALa partial 4-i± i-i +4+4 444. +4+4 -444 44 Array2Urgq d 11 NcwPALY'LWHEAT7602 17rgi CsEOI LpPALa partial +4 4+1- 4+4 +4 Array2Urg d 12 NcwPDAILY -HEAT7603 11rgl BsAO LpPALa partial Array2Urg e I NcwPALYjNHEAT7604 06rgl KsC02 LpPALb partial +44+ Hi--i- 4+4 4 4+1 +4 4+44+ Array2Urg a 2 NowPAL-ORYSA7605 07rg2OsGO2 LpPALb partial +4 H 4 Array2Urg e 3 NcwPALYWHEAT7606 14rg2lBsGO3 LpPALb partial +4 4 444 4± Array2Urg e 4 ZhyYDID-ECOL]7607 O~rgl BsBO4 Lp4CLJa partial Array2Urg e 5 ZhyYDID-ECOL7608 O6rg2DsBO6 Lp4CLJa partial +4 Array2Urg a 6 ZhyYDIDECOL7S09 06rg2EsBC6 Lp4CLJa partial Array2Urg e 7 DdtAGSAALCEU7610 11lrg2HsFC3 Lp4CL~a partialt +4 4 +4+ shorter Array2Urg e 8 ZhyYDIDECOL7611 19rglOsAO2 Lp4CLJa partial Array2Urg e 9 MisALKlQPSEOL76I2 l9rglSC08 Lp4CL~a partial? Array2Urg e 10 Now4CLPINTA-7613 17rglC5C0S Lp4CLJb partial Array2lUrg e 11 Ncw4CLLSOILTU7614 08rg2GsEO2 Lp4CLlc partial WO 02/26994 PCT/AU01/01221 51

REFERENCES

An, Watson, Stachel, Gordon, Nester, E.W. (1985) New cloning vehicles for transformation of higher plants. The EMBO Journal 4, 227-284 Feinberg, Vogelstein, B. (1984). A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132: 6- 13.

Frohman et al. (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 serach. Nature Genetics 3:266-272 Lander, Green Abrahamson, Barlow, Daly, Lincoln, Newburg, L. (1987). MAPMAKER: an interactive computer package for constructing primary linkage maps of experimental and natural populations.

Genomics 1: 174-181.

Loh, Elliott, Cwirla, Lanier, Davis, M.M. (1989).

Polymerase chain reaction with single-sided specificity: Analysis of T-cell receptor delta chain. Science 243:217-220 Ohara, Dorit, Gilbert, W. (1989). One-sided polymerase chain reaction: The amplification of cDNA. Proc. Natl. Acad Sci USA 86:5673-5677 Sambrook, Fritsch, Maniatis, T. (1989). Molecular Cloning. A Laboratory Manual. Cold Spring Harbour Laboratory Press Schardl, Byrd, Benzion, Altschuler, 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 02/26994 PCT/AU01/01221 52 Sharp, Kreis, Shewry, Gale, M.D. (1988). Location of aamylase 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 as outlined herein.

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

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

Claims (19)

1. A substantially purified or isolated nucleic acid or nucleic acid O z fragment encoding caffeoyl-CoA 3-O-methyltransferase (CCoAMT) from a Lolium species.

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

3. A substantially purified or isolated nucleic acid or nucleic acid Sfragment encoding CCoAMT, or complementary or antisense to a CCoAMT- l encoding sequence, and including a nucleotide sequence selected from the group consisting of sequences shown in Figures 1, 3, 4, 6, 7, 82 and 87 hereto (Sequence ID Nos: 1, 3 to 11, 12, 14 to 20, 21, 168 and 170, respectively); complements of the sequences in sequences antisense to the sequences recited in and and functionally active fragments and variants of the sequences recited in and

4. A nucleic acid or nucleic acid fragment according to claim 3 wherein said functionally active fragments and variants have at least approximately 90% identity to the relevant part of the sequences recited in and and have a size of at least 20 nucleotides.

A nucleic acid or nucleic acid fragment according to claim 3 including a nucleotide sequence selected from the group consisting of sequences shown in Figures 1, 3, 4, 6, 7, 82 and 87 hereto (Sequence ID Nos: 1,3 to 11, 12, 14 to 20, 21, 168 and 170, respectively).

6. A nucleic acid or nucleic acid fragment according to any one of claims 3 to 5 wherein said nucleic acid or nucleic acid fragment is from a Lolium species. 005042824 54

7. A construct including a nucleic acid or nucleic acid fragment cl according to any one of Claims 1-6. 0 Z

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

9. A vector according to Claim 8, further including a promoter and a terminator, said promoter, nucleic acid or nucleic acid fragment and terminator being operatively linked.

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

11. A plant, plant seed or other plant part derived from a plant cell or plant according to Claim 10 and including a construct according to Claim 7 or a vector according to Claim 8 or 9.

12. A method of modifying lignin biosynthesis in a plant, said method including introducing into said plant an effective amount of a nucleic acid or nucleic acid fragment according to any one of Claims 1-6, a construct according to Claim 7, and/or a vector according to Claim 8 or 9.

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

14. A substantially purified or isolated CCoAMT polypeptide from a Lolium species.

A polypeptide according to Claim 14, wherein said Lolium species is Lolium perenne or Lolium arundinuceum.

16. A substantially purified or isolated CCoAMT polypeptide including an amino acid sequence selected from the group of sequences shown in Figures 2, 5, 8, 83 and 88 hereto (Sequence ID Nos: 2, 13, 22, 169 005042824 0 and 171, respectively); and functionally active fragments and variants thereof C having at least approximately 98% identity with the sequences shown in Figures 2, 5, 8, 83 and 88 hereto and have a size of at least 20 amino acids.

17. A polypeptide according to Claim 16 including an amino acid sequence selected from the group of sequences shown in Figures 2, 5, 8, 83 and 88 hereto (Sequence ID Nos: 2, 13, 22, 169 and 171, respectively).

18. A polypeptide according to Claim 16 or 17 wherein said Spolypeptide is from a Lolium species.

19. A lignin or modified lignin substantially or partially purified or isolated from a plant, plant seed or other plant part according to Claim 10 or 11.