AU2004236280B2 - Plant CAD3-like genes and their use - Google Patents

Description

WO 2004/099414 PCT/AU2004/000608 PLANT CAD3-LIKE GENES AND THEIR USE The present invention relates to nucleic acid fragments encoding amino acid sequences for lignification-related enzymes in plants, and the use thereof for the modification of plant cell walls and/or defence response in plants. 5 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. Three monolignol precursors, sinapyl, coniferyl and p-coumaryl alcohol combine by dehydrogenative polymerisation to produce respectively the syringyl (S), guaiacyl (G) and hydroxyl 10 (H) 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 15 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 20 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 (F5H). The 5-hydroxyferulate produced by F5H is then 0-methylated by OMT, the same enzyme that carries out the 0-methylation of caffeic acid. The cinnamic acids are converted by action of the 4-coumarate:CoA 25 ligase (4CL) and caffeoyl-CoA 3-0-mehtyltransferase (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, 30 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 SUBSTITUTE SHEET (RULE 26) WO 2004/099414 PCT/AU2004/000608 2 location. Cinnamyl alcohol dehydrogenase (CAD) governs the last committed step of the lignin biosynthesis pathway, converting the hydroxycinnamaldehydes to their corresponding cinnamyl alcohols (monolignols). Different isoforms of CAD have 5 been reported. CADI is monomeric and able to utilise a range of substituted and unsubstituted benzaldehydes. CAD2 is a homo- or heterodimer that has been found in all plants examined and the angiosperm enzyme uses all three cinnamaldehydes whereas the gymnosperm enzyme has a poor affinity for sinapaldehyde. Defence-responsive isoforms (CAD3) have also been reported. 10 Lignification of plant cell walls has effects on their structural, conductive or defensive roles. Dry matter digestibility of forages 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 15 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. Lignification also affects efficiency of cellulose extraction in the pulping process of wood for paper production. Cell wall digestibility, pulping efficiency and 20 feed (grazed, cut hay, silage) quality can thus be increased by the manipulation of enzymes involved in the biosynthesis of lignins. Perennial ryegrass (Lolium perenne L.) 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 lignification in plants. For 25 example it may be desirable to reduce the activity of key lignin biosynthetic related enzymes in order to reduce lignin content and/or alter lignin composition for enhancing dry matter digestibility and improving herbage quality. For other applications it may be desirable to enhance lignin biosynthesis to increase lignin content and/or alter lignin composition, for example to increase mechanical 30 strength of wood, to increase mechanical strength, to reduce plant height, to reduce lodging and to improve disease resistance. SUBSTITUTE SHEET (RULE 26) 3 While nucleic acid sequences encoding some of the enzymes involved in lignification have been isolated for certain species of plants, there remains a need for materials useful in the modification of lignification in a wide range of plants, and for methods for their use. 5 It is an object of the present invention to overcome, or at least alleviate, one or more of these needs in light of the prior art. In a preferred embodiment, the present invention provides a substantially purified or isolated nucleic acid or nucleic acid fragment encoding a cinnamyl alcohol dehydrogenase 3-like polypeptide (CAD3L) from a Lolium species, or complementary or 0 antisense to a sequence encoding a CAD3L polypeptide from a Lolium species, and including a nucleotide sequence selected from the group consisting of (a) sequences shown in Figures 1, 2, 3, 4, 5 and 9 hereto (SEQ ID NOS 1 to 6, respectively); (b) complements of the sequences recited in (a); (c) sequences antisense to the sequences recited in (a) or (b); (d) functionally active fragments and variants of the sequences 15 recited in (a), (b) and (c); and (e) RNA sequences corresponding to the sequences recited in (a), (b), (c) and (d). Preferably, said Lolium species is Lolium perenne or Lolium arundinaceum. In another aspect, the present invention provides substantially isolated nucleic acids or nucleic acid fragments encoding amino acid sequences for novel cinnamyl !0 alcohol dehydrogenase 3 like enzymes (CAD3L). These CAD3L sequences are identified on the basis of the similarity of their expression patten to CAD3, not on the basis of sequence similarity. The genes which encode these polypeptides are expressed in a similar manner to CAD3L. As used herein the term "cinnamyl alcohol dehydrogenase 3 like" polypeptides 25 (CAD3L) relates to polypeptides that are produced in the plant in same organs and at the same developmental stages and processes as CAD3. That is, the expression of the genes encoding CAD3L polypeptides is coordinated with the expression of the CAD3 gene. It is likely that such CAD3L polypeptides are involved in the same developmental processes as CAD enzymes. 30 The present invention also provides substantially isolated nucleic acids or nucleic acid fragments encoding amino acid sequences for a class of polypeptides, which are related to CAD3L. Such polypeptides are referred to herein as CAD3L-like. This CAD3L- 3A like class of polypeptides includes functionally active fragments or variants of CAD3L polypeptides and non-CAD3L polypeptides having similar functional activity to CAD3L polypeptides. The nucleic acid fragments may be obtained from ryegrass (Lolium) or fescue 5 (Festuca) species. These 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). Nucleic acids according to the invention may be full-length genes or part thereof, and are also referred to as "nucleic acid fragments" and "nucleotide WO 2004/099414 PCT/AU2004/000608 4 sequences" on this specification. The nucleic acid fragment may be of any suitable type and includes DNA (such as cDNA or genomic DNA) and RNA (such as mRNA) that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide 5 bases, and combinations thereof. The RNA is readily obtainable, for example, by transcription of a DNA sequence according to the present invention, to produce a RNA corresponding to the DNA sequence. The RNA may be synthesised in vivo or in vitro or by chemical synthesis to produce a sequence corresponding to a DNA sequence by methods well known in the art. In this specification, where the 10 degree of sequence similarity between an RNA and DNA is such that the strand of the DNA could encode the RNA, then the RNA is said to "correspond" to that DNA. The term "isolated" means that the material is removed from its original environment (eg. the natural environment if it is naturally occurring). For example, a naturally occurring nucleic acid fragment or polypeptide present in a living plant 15 is not isolated, but the same nucleic acid fragment or polypeptide separated from some or all of the coexisting materials in the natural system, is isolated. Such an isolated nucleic acid fragment could be part of a vector and/or such nucleic acid fragments could be part of a composition, and still be isolated in that such a vector or composition is not part of its natural environment. 20 By "functionally active" in respect of a nucleotide sequence is meant that the fragment or variant (such as an analogue, derivative or mutant) is 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 25 nucleotides are contemplated so long as the modifications do not result in loss of functional activity of the fragment or variant. Preferably the functionally active fragment or variant has at least approximately 80% identity to the relevant part of the above mentioned sequence, more preferably at least approximately 90% identity, most preferably at least approximately 95% identity. Such functionally 30 active variants and fragments include, for example, those having nucleic acid changes which result in conservative amino acid substitutions of one or more SUBSTITUTE SHEET (RULE 26) WO 2004/099414 PCT/AU2004/000608 5 residues in the corresponding amino acid sequence. Preferably the fragment has a size of at least 30 nucleotides, more preferably at least 45 nucleotides, most preferably at least 60 nucleotides. By "functionally active" in the context of a polypeptide is meant that the 5 fragment or variant has one or more of the biological properties for the enzymes CAD3L and CAD3L-like. 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 10 the functional part of the above mentioned sequence, more preferably at least approximately 80% identity, most preferably at least approximately 90% identity. Such functionally active variants and fragments include, for example, those having conservative amino acid substitutions of one or more residues in the corresponding amino acid sequence. Preferably the fragment has a size of at least 15 10 amino acids, more preferably at least 15 amino acids, most preferably at least 20 amino acids. By "operatively linked" is meant that said regulatory element is capable of causing expression of said nucleic acid in a plant cell and said terminator is capable of terminating expression of said nucleic acid in a plant cell. Preferably, 20 said regulatory element is upstream of said nucleic acid and said terminator is downstream of said nucleic acid. 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 25 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. 30 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 SUBSTITUTE SHEET (RULE 26) WO 2004/099414 PCT/AU2004/000608 6 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 acknowledgment that they form part of the common general knowledge in the relevant art. 5 In a preferred embodiment of this aspect of the invention, the substantially purified or isolated nucleic acid fragment encoding a CAD3L or CAD3L-like protein includes a nucleotide sequence selected from the group consisting of (a) sequences shown in Figures 1, 2, 3, 4, 5 and 9 hereto (SEQ ID NOS I to 6); (b) complements of the sequences recited in (a); (c) sequences antisense to the 10 sequences recited in (a) or (b); (d) functionally active fragments and variants of the sequences recited in (a), (b) and (c); and (e) RNA sequences corresponding to the sequences recited in (a), (b), (c), and (d). 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 15 species. Additionally, genes encoding other CAD3L or CAD3L-like enzymes, either as cDNAs or genomic DNAs, may be isolated directly by using all or a portion of the nucleic acid fragments of the present invention as hybridisation probes to screen libraries from the desired plant employing the methodology well known to 20 those skilled in the art. Specific oligonucleotide probes based upon the nucleic acid sequences of the present invention can be designed and synthesized by methods known in the art. Moreover, the entire sequences can 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 25 using available in vitro transcription systems. In addition, specific primers can be designed and used to amplify a part or all of the sequences of the present invention. The resulting amplification products can 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 30 stringency. In addition, two short segments of the nucleic acid fragments of the present SUBSTITUTE SHEET (RULE 26) 7 invention may be used in polymerase chain reaction protocols to amplify longer nucleic acid fragments encoding homologous genes from DNA or RNA. The polymerase chain reaction may also be performed on a library of cloned nucleic acid fragments wherein the sequence of one primer is derived from the nucleic acid fragments of the present 5 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. Nat/. Acad Sci. USA 85:8998, the entire disclosure of 10 which is incorporated herein by reference) to generate cDNAs by using PCR to amplify copies of the region between a single point in the transcript and the 3' or 5' end. Using commercially available 3' RACE and 5' RACE systems (BRL), specific 3' or 5' cDNA fragments can be isolated (Ohara et al. (1989) Proc. Nat/. Acad Sci USA 86:5673; Loh et al. (1989) Science 243:217). Products generated by the 3' and 5' RACE procedures 15 can be combined to generate full-length cDNAs. In a further aspect of the present invention there is provided a substantially purified or isolated polypeptide from a ryegrass (Lolium) or fescue (Festuca) species, selected from the group consisting of CAD3L and CAD3L-like enzymes. The ryegrass (Lolium) or fescue (Festuca) species may be of any suitable type, 20 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 or isolated CAD3L polypeptide from a Lolium species, including an amino acid 25 sequence shown in Figure 10 hereto (SEQ ID NO 7); or a functionally active fragment or variant thereof. Preferably, said Lolium species is Lolium perenne or Lolium arundinaceum. In a preferred embodiment of this aspect of the invention, there is provided a substantially purified or isolated CAD3L and CAD3L-like polypeptide that includes an 30 amino acid sequence selected from the group consisting of (a) the amino acid sequence shown in Figure 10 hereto (SEQ ID NO 7), (b) sequences translated from nucleotide 7A sequences shown in Figures 1, 2, 3, 4, 5 and 9 hereto (SEQ ID NOS 1 to 6); and functionally active fragments and variants of (a) and (b). In a further embodiment of this aspect of the invention, there is provided a WO 2004/099414 PCT/AU2004/000608 8 polypeptide produced (eg recombinantly) from a nucleic acid according to the present invention. Techniques for recombinantly producing polypeptides are known to those skilled in the art. Availability of the nucleotide sequences of the present invention and 5 deduced amino acid sequences facilitates immunological screening of cDNA expression libraries. Synthetic peptides representing portions of the instant amino acid sequences may be synthesized. These peptides can be used to immunise animals to produce polyclonal or monoclonal antibodies with specificity for peptides and/or proteins comprising the amino acid sequences. These antibodies 10 can 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 essential in commercial breeding programs, in determining parentage, in diagnostics and fingerprinting, and the like. Genotypes can be 15 readily described in terms of genetic markers. A genetic marker identifies a specific region or locus in the genome. The more genetic markers, the finer defined is the genotype. A genetic marker becomes particularly useful when it is allelic between organisms because it then may serve to unambiguously identify an individual. Furthermore, a genetic marker becomes particularly useful when it is 20 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, 25 can be used as perfect markers or candidate genes for the given trait. In a further aspect of the present invention there is provided a method of isolating a nucleic acid of the present invention including a single nucleotide polymorphism (SNP), said method including sequencing nucleic acid fragments from a nucleic acid library. The nucleic acid library may be of any suitable type and 30 is preferably a cDNA library. The nucleic acid fragments may be isolated from recombinant plasmids or may be amplified, for example using polymerase chain SUBSTITUTE SHEET (RULE 26) WO 2004/099414 PCT/AU2004/000608 9 reaction. The sequencing may be performed by techniques known to those skilled in the art. In a further aspect of the present invention, there is provided use of nucleic acids of the present invention including SNP's, and/or nucleotide sequence 5 information thereof, as molecular genetic markers. In a still further aspect of the present invention there is provided use of a nucleic acid according to the present invention, and/or nucleotide sequence information thereof, as a molecular genetic marker. More particularly, nucleic acids according to the present invention and/or nucleotide sequence information thereof 10 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 according to the present invention and/or nucleotide sequence information thereof may be used as molecular genetic markers in forage and turf grass improvement, 15 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 of the present invention and/or nucleotide sequence information thereof may be used as molecular genetic markers for QTL 20 tagging and mapping and in marker assisted selection, particularly in ryegrasses and fescues. In a further aspect of the present invention there is provided a construct including a nucleic acid according to the present invention. The construct may be a vector. In a preferred embodiment of this aspect of the invention, the vector may 25 include a regulatory element such as a promoter, a nucleic acid according to the present invention and a terminator; said regulatory element, nucleic acid and terminator being operatively linked. 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 30 chromosomal and synthetic nucleic acid sequences, eg. derivatives of plant viruses; bacterial plasmids; derivatives of the Ti plasmid from Agrobacterium SUBSTITUTE SHEET (RULE 26) WO 2004/099414 PCT/AU2004/000608 10 tumefaciens, derivatives of the Ri plasmid from Agrobacterium rhizogenes; phage DNA; yeast artificial chromosomes; bacterial artificial chromosomes; binary bacterial artificial chromosomes; vectors derived from combinations of plasmids and phage DNA. However, any other vector may be used as long as it is 5 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. In another embodiment, the vector may include more than one nucleic acid. 10 The nucleic acids within the same vector may have identical or differing sequences. In one preferred embodiment, the vector has at least two nucleic acids encoding functionally similar enzymes. For example, as disclosed in this specification, the nucleic acids may be selected from the group consisting of LpCAD3La, LpCAD3Lb, LpCAD3Lc, LpCAD3Ld and LpCAD3Le. Moreover, a 15 second nucleotide sequence may encode another cinnamyl alcohol dehydrogenase or another lignification-related enzyme. 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 20 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 promoters include but are not limited to the constitutive Cauliflower Mosaic Virus 35S (CaMV 35S) promoter and derivatives thereof, the maize Ubiquitin promoter and the rice Actin promoter. 25 A variety of terminators which may be employed in the vectors and constructs of the present invention are also well known to those skilled in the art. The terminator may be from the same gene as the promoter sequence or a different gene. Particularly suitable terminators are polyadenylation signals, such as the CaMV 35S polyA and other terminators from the nopaline synthase (nos), 30 the octopine synthase (ocs) and the rbcS genes. The vector, in addition to the regulatory element, the nucleic acid of the SUBSTITUTE SHEET (RULE 26) WO 2004/099414 PCT/AU2004/000608 11 present invention and the terminator, may include further elements necessary for expression of the nucleic acid, 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 5 genes and other selectable marker genes [such as the neomycin phosphotransferase (npt2) gene, the hygromycin phosphotransferase (hph) gene, the phosphinotricin acetyltransferase (bar or pat) gene and the gentamycin acetyl transferase (aacCl) gene], and reporter genes [such as beta-glucuronidase (GUS) gene (gusA) and green fluorescent protein (gfp)]. The vector may also contain a 10 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, 15 such as PCR (polymerase chain reaction), Southern blot hybridisation analysis, histochemical GUS assays, visual examination including microscopic examination of fluorescence emitted by gfp, 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. 20 Techniques for operatively linking the components of the vector of the present invention are well known to those skilled in the art. Such techniques include the use of linkers, such as synthetic linkers, for example including one or more restriction enzyme sites. The vectors of the present invention may be incorporated into a variety of 25 plants, including monocotyledons (such as grasses from the genera Lolium, Festuca, Paspalum, Pennisetum, Panicum and other forage and turfgrasses, corn, rice, sugarcane, oat, wheat and barley), dicotyledons (such as arabidopsis, tobacco, soybean, canola, cotton, potato, chickpea, medics, white clover, red clover, subterranean clover, alfalfa, eucalyptus poplar, hybrid aspen) and 30 gymnosperms (pine tree). In a preferred embodiment, the vectors are used to transform monocotyledons, preferably grass species such as ryegrasses (Lolium SUBSTITUTE SHEET (RULE 26) WO 2004/099414 PCT/AU2004/000608 12 species) and fescues (Festuca species), even more preferably a ryegrass, most preferably perennial ryegrass, including forage- and turf-type cultivars. Techniques for incorporating the constructs and vectors of the present invention into plant cells (for example by transduction, transfection or 5 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 10 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 15 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 20 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 is from a monocotyledon, preferably a grass species, more preferably a ryegrass (Lolium 25 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 30 invention. In a further aspect of the present invention there is provided a method of SUBSTITUTE SHEET (RULE 26) WO 2004/099414 PCT/AU2004/000608 13 modifying one or more of lignification, defence response and cell walls in a plant, said method including introducing into said plant an effective amount of a nucleic acid and/or a vector according to the present invention. Using the methods and materials of the present invention, plant lignification 5 and defence may be increased or decreased. They may be increased, for example, by incorporating additional copies of a sense nucleic acid of the present invention. They may be decreased, for example, by incorporating an antisense nucleic acid or dsRNA or small interfering RNA (siRNA) derived from the nucleotide sequences of the present invention. In addition, the number of copies of 10 genes encoding for different enzymes involved in lignification and defence may be manipulated to modify the composition of lignin and plant cell walls. 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. 15 Such lignins may be modified from naturally occurring lignins in terms of 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 the degree of polymerisation (number of units), 20 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. In a further aspect of the present invention there is provided a preparation for transforming a plant comprising at least one nucleic acid according to the present invention. The preparation may contain vectors or other constructs to 25 facilitate administration to and/or transformation of the plant with the nucleic acid. 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. 30 In the Figures Figure 1 shows the nucleotide sequence of LpCAD3La. SUBSTITUTE SHEET (RULE 26) WO 2004/099414 PCT/AU2004/000608 14 Figure 2 shows the nucleotide sequence of LpCAD3Lb. Figure 3 shows the nucleotide sequence of LpCAD3Lc. Figure 4 shows the nucleotide sequence of LpCAD3Ld. Figure 5 shows the nucleotide sequence of LpCAD3Le. 5 Figure 6 shows the microarray based expression profile of LpCAD3 in perennial ryegrass as log ratio of its expression values. Figure 7 shows the expression profiling of LpCAD3L genes using LpCAD3 as template gene in perennial ryegrass as log ratios of genes matching LpCAD3 at an Euclidian distance of 4.4393. 10 Figure 8 shows the plasmid map of pGEMLpCAD3Lb. Figure 9 shows the nucleotide sequence of the full length cDNA of LpCAD3Lb. Figure 10 shows the putative amino acid sequence of LpCAD3Lb. Figure 11 shows the binary transformation vector pPZP221 LpCAD3Lb. 15 Figure 12 shows Agrobacterium-mediated transformation and selection of Arabidopsis. SUBSTITUTE SHEET (RULE 26) WO 2004/099414 PCT/AU2004/000608 15 EXAMPLE I Preparation of cDNA libraries, isolation and sequencing of cDNAs for identification of cDNAs coding for CAD3L from perennial ryegrass (Lolium perenne) 5 cDNA libraries representing mRNAs from various organs and tissues of perennial ryegrass (Lo/ium perenne) were prepared. The characteristics of the libraries are described below (Table 1). TABLE I 10 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 09rg Whole tillers and sliced leaves (0, 1, 3, 6, 12 and 24 h after harvesting) 1 Org Embryogenic suspension-cultured cells 11 rg Non-embryogenic suspension-cultured cells 12rg Whole tillers and sliced leaves (0, 1, 3, 6, 12 and 24 h after harvesting) 13rg Shoot apices including vegetative apical meristems 14rg Immature inflorescences including different stages of inflorescence meristem and inflorescence development I 5rg 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 SUBSTITUTE SHEET (RULE 26) WO 2004/099414 PCT/AU2004/000608 16 (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. 15 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 precut pBluescript II SK(+) vectors (Stratagene) using T4 DNA ligase (New England Biolabs), followed by transfection into E. coli DH10B cells according to the manufacturer's protocol (GiBCO BRL Products). SUBSTITUTE SHEET (RULE 26) WO 2004/099414 PCT/AU2004/000608 17 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 5 DNA preparation may be performed robotically using the Qiagen QiaPrep Turbo kit (Qiagen, Germany) according to the protocol provided by Qiagen. Amplified insert DNAs are sequenced in dye-terminator sequencing reactions to generate partial cDNA sequences (expressed sequence tags or "ESTs"). The resulting ESTs are analyzed using an Applied Biosystems ABI 3700 sequence analyser. 10 EXAMPLE 2 Microarray-based expression profiling and identification of CAD3L genes from perennial ryegrass (Lolium perenne) The function of a gene may be inferred by its co-expression with other genes involved in the same cellular processes. The use of cDNA microarrays 15 allows the expression of thousands of genes to be monitored in one experiment. In this technology a microscope slide is spotted with DNA, each spot containing copies of one gene sequence. The spotted DNA is immobilised on the slide and interrogated with labelled cDNA in solution. The cDNA is prepared from RNA extracted from a tissue of interest and fluorescence-labelled. The amount of 20 fluorescence remaining on the spot after hybridisation with the probe and washing is a measure of the mRNA level of that gene in the tissue of interest. Thousands of these measurements are made from one slide. The DNA spotted on the perennial ryegrass cDNA slide is derived from EST sequences. Each spot contains an EST that is unique or representative of a unique 25 cluster of ESTs. The putative function of the gene spotted has been inferred by homology searching of the EST sequence against public DNA and protein databases. The results of theses searches demonstrated that around 40% of the genes tagged cannot be assigned a function based on a homology search. However, comparisons of the expression profiles of these unknown genes with 30 those of known genes allows an inference of their function to be made. In the first experiment mRNA was isolated from the following tissues: SUBSTITUTE SHEET (RULE 26) WO 2004/099414 PCT/AU2004/000608 18 * 4 day old light grown perennial ryegrass seedlings, 0 5 day old light grown perennial ryegrass seedling leaves, * 7 day old light grown perennial ryegrass seedling leaves, * 10 day old light grown perennial ryegrass seedling leaves, 5 0 5 day old dark adapted perennial ryegrass seedling leaves, * 7 day old dark adapted perennial ryegrass seedling leaves, * 10 day old dark adapted perennial ryegrass seedling leaves, * 5 day old light grown perennial ryegrass seedling roots, 0 7 day old light grown perennial ryegrass seedling roots, 10 0 10 day old light grown perennial ryegrass seedling roots, * 5 day old dark adapted perennial ryegrass seedling roots, * 7 day old dark adapted perennial ryegrass seedling roots, * 10 day old dark adapted perennial ryegrass seedling roots. Fluorescence-labelled cDNA was prepared from each RNA preparation. 15 cDNA prepared from 4 day old seedling was used as a common reference probe and used in all the hybridisations. Each of the other treatments was used as a co hybridisation probe at least once. In the second experiment RNA was prepared from * leaf blades from mature plants, 20 e roots from mature plants, " pseudostems from mature plants. Fluorescence-labelled cDNA was prepared from each RNA preparation. cDNA prepared from pseudostems was used as a common reference probe and used in all the hybridisations. Each of the other treatments was used as a co 25 hybridisation probe at least once. The protocols for these processes are described below. SUBSTITUTE SHEET (RULE 26) WO 2004/099414 PCT/AU2004/000608 19 1. RNA isolation and probe preparation 1.1 Total RNA isolation The quality of RNA influences the efficiency of the labelling processes, hybridisation performance and background level. The CTAB protocol has been 5 modified to extract total plant RNA of high purity and quality. The number of extraction steps with chloroform (step 5) is critical to the purity and yield of the isolated RNA. The quality of the isolated RNA is measured using the ratio of absorbance at 230:280, with high quality RNA having a value of around 0.88-1.0. RNA samples that do not meet the quality requirement can be further purified using 10 an RNeasy mini column (Qiagen, Germany) to give microarray quality RNA. However these factors may not be universally applicable and methods for extraction of microarray quality RNA should be optimised for each organism or tissue. The CTAB protocol [A simple and efficient method for isolating RNA from 15 pine trees. Plant Molecular Biology Reporter (1993) 11(2): 113-116]: 1. Warm 15 ml extraction buffer plus 300 pl p-mercaptoethanol to 650C in a water bath. 2. Homogenise 2-3 g tissues in liquid N 2 with cooled mortar and pestle. 3. Transfer ground tissues into a 50 ml tube and immerse in liquid N 2 bath. 20 4. Quickly add warmed extraction buffer and mix completely by inverting tube. 5. Add equal volume of chloroform:IAA and vortex the mixture. Spin the tube for 20 min at 5000 rpm at 40C in a bench top centrifuge using a swing out rotor (Sigma, USA). 6. Pipette the supernatant (top layer) into a new 50 ml tube. Step 5 is repeated 25 until the interface is clear. 7. Pipette the supernatant into a new 50 ml tube and add 0.25 volume 10 M LiCI. Mix thoroughly and precipitate at 40C overnight. 8. Spin the tube for 30 min at 5000 rpm at 40C in a bench top centrifuge using a swing out rotor (Sigma, USA). SUBSTITUTE SHEET (RULE 26) WO 2004/099414 PCT/AU2004/000608 20 9. Discard supernatant and dissolve pellet with 500 pl SSTE buffer. Transfer to 1.5 ml Eppendorf tube. 10.Add equal volume of chloroform:IAA and vortex the mixture. Spin the tube for 10 min at 13,000 rpm. 5 11.Pipette the supernatant into a new 1.5ml tube and add 2 volumes 100% ethanol. Mix thoroughly and precipitate at -700C for 1 h. 12. Spin the tube for 20 min at 13,000 rpm at 40C. 13.Wash twice with 80% ethanol for 10 min at 13,000 rpm at 4 0 C. 14.Discard supernatant and air dry pellet. Resuspend pellet in RNase free 10 water. 1.2 Precipitation of total RNA Total RNA is concentrated as follows: 1. Add 0.1 volume 3M sodium acetate pH 5.2 to the RNA solution. 2. Add 3 volumes ethanol to the mixture. 15 3. Mix thoroughly by inverting the tube. 4. Precipitate at -700C for at least I h. 5. Centrifuge at 13000 rpm at 40C for 30 minutes. 6. Discard supernatant and add 1 ml 70% ethanol. 7. Centrifuge at 13000 rpm at 40C for 10 minutes. 20 8. Discard supernatant and air-dry pellet. 9. Resuspend pellet in RNase free water. 1.3 Probe labelling by direct incorporation Probes are either generated by directly incorporating fluorescent nucleotides during reverse transcription reaction or by indirect methods. Cy3 and Cy5 (either 25 dUTP or dCTP) are generally used as fluorophores as they are compatible with the excitation and emission wavelength of most slide scanners. These fluorochromes SUBSTITUTE SHEET (RULE 26) WO 2004/099414 PCT/AU2004/000608 21 are light-sensitive and measures should be taken to minimise their exposure to light. 5 1. Prepare a 5OX low-C dNTP mixture dATP (100 mM) 25 pl dGTP (100 mM) 25 pl dTTP (100 mM) 25 pl dCTP (100 mM) 10 l Water 15 pl Total volume 100 l 10 2. Mix total RNA with oligo-d(T) in a 200 pi microfuge tube as follows: Total RNA (20 pg) 10.9 pl Oligo dT12-18 (Gibco, 0.5 pg/pl) 1.5 pl 3. Incubate at 70 0 C for 10 minutes in a thermal cycler to disrupt RNA secondary structure. Snap cool on ice. 15 4. While waiting for denaturation, prepare a master mix of the following: 1 sample 2.5 samples SUBSTITUTE SHEET (RULE 26) WO 2004/099414 PCT/AU2004/000608 22 5X Superscript 6 pl 15 pl 1 st strand buffer DTT (0.1M) 3 pl 7

.

5 pl 50X dNTP mix 0.6 pi 1.5 pl Rnasin (Promega) 0.5 ptl 1.25 pl Superscript I 1.5 Ll 3.75 p RT (200U/tl) Total volume 11.6 pl 29.0 pl 5. After cooling, add 11.6 pl of master mix from step 4 into each tube. Then add 6 pl of Cy3-dCTP and Cy5-dCTP into the appropriate tubes. Mix well and spin briefly. 5 6. Incubate at 42'C for 2 hours in a thermal cycler. 7. Add 1.5 pIt 1 M NaOH and incubate at 700C for 10 min in a thermal cycler to hydrolyse the RNA. 8. Neutralise the reactions by adding 1.5 pl 1 M HCI. 9. Dry down the reaction volume to 20 p.l by heating in a thermal cycler set at 10 800C for 15 minutes. 10.The labelled probes are purified using DyeEx column (Qiagen, Germany) or S-400 column (Amersham BioSciences, UK) according to the manufacturer's instructions. 11. Pool the Cy3 and Cy5 labelled probes and dry down to 10 pl for half slide or 15 20 pl for full slide microarray by heating in a thermal cycler set at 800C for 30 minutes. (Note: Do not dry probe completely) 12. The pooled probes are now ready for hybridisation. 2. Prehybridisation treatment, hybridisation and washing SUBSTITUTE SHEET (RULE 26) WO 2004/099414 PCT/AU2004/000608 23 Hybridisations can be carried out under a cover slip placed in a humid hybridisation chamber or a fluidic station. Hybridisation chambers can be obtained from Corning (# 2551) or TeleChem International (# AHC-1). Fluidic stations can be purchased from Amersham Pharmacia Biotech, Affymetrix or Genomic Solutions. 5 Hybridisations carried out under a cover slip use 15-45 pl of pooled probe depending on the array size. The potential drawback to this method is that the movement of probes is by diffusion. The fluidic station allows a larger volume of pooled probe (100-200 pl) and agitation during hybridisation. Hybridisations can occur either at 650C, or 420C if 50% formamide is 10 included. General and species specific blocking elements such as CoT-1 DNA, yeast tRNA or poly-d(A) should be included in the hybridisation. 2.1 Immobilising DNA on glass slide I. Bake CMT-GAPS slides (Corning, USA) for 30 minutes at 800C in an oven 15 (This protocol can also be used to immobilise DNA onto polylysine coated slides (Sigma, USA)). 2. UV cross-link DNA to slides with 650x100 Joules in Stratalinker (Stratagene, USA). 3. Immerse slides in 950C water for 5 min. 20 4. Immerse in 95% ethanol for I min. 5. Dry slides by centrifugation at 800 rpm for 3 min using a 96-well plate rotor (Qiagen, Germany) in a bench-top centrifuge (Sigma, USA). 2.2 Prehybridisation In principle, prehybridisation is used to block DNA void spaces on the slide 25 to prevent non-specific binding of probes. Generally, prehybridisation on poly-L lysine or aminosilane coated slide involves two steps. Firstly, the free amine groups on the slide are blocked using succinic anhydride. A condensation reaction takes place and for every succinic anhydride molecule two peptide bonds are formed with the poly-L-lysine and aminosilane. Secondly, DNA void spaces are blocked using SUBSTITUTE SHEET (RULE 26) WO 2004/099414 PCT/AU2004/000608 24 salmon sperm DNA. Non specific hybridisation blocker such as Cot-1 DNA or poly d(A) are also included. Prehybridisation however is not necessary when using CMT-GAPS slide. 2.3 Hybridisation 5 1. The volume required for hybridisation is dependent on the size of array used: 15-20 ptl for half a glass slide (25x75 mm) and 30 pl for a full glass slide. 10 2. Mix the pooled labelled probe with hybridisation solution: Half slide Full slide Labelled probe 11.4 L 22.8 pl 20X SSC 2.63 pl 5.26 ptl 10% SDS 0.45 plt 0.9 pl Poly-adenine (80pg/pl) 0.5 pLl I pl Total volume 15 pl 30 pl 15 3. Denature at 98 0 C for 2 min and then incubate at 370C for 20 min. 4. Pipette the pooled probes on array. 5. Apply cover slip (22x22 mm for half the slide and 22x40 for full slide) with care to avoid introducing air bubbles. 6. Pipette 80 pl of 3X SSC into both reservoirs of the hybridisation cassette 20 (Arrayit, USA). 7. Gently place the microarray onto the hybridisation cassette and seal it by screwing tightly. SUBSTITUTE SHEET (RULE 26) WO 2004/099414 PCT/AU2004/000608 25 8. Hybridise at 650C for 16-24h in a water bath excluding light. 2.4 Slide washing 1. After at least 16h of hybridisation, the slides are washed in 50 ml tubes in a rotating oven with: * 2X SSC, 0.1% SDS for 15 min at RT with shaking * 1X SSC for 15 min at 420C with shaking * O.X SSC for 15 min at 68CC with shaking 5 2. Dry slides by centrifugation at 800 rpm for 3 min using a 96-well plate rotor (Qiagen, Germany) in a bench-top centrifuge (Sigma, USA). 3. The slides are now ready for scanning. 10 3. Image analysis The slides from each experiment were scanned using a ScanArray 3000 confocal laser scanner. Each scan produced 2 tiff image files, one for the reference probe sample and one for the experimental sample. The intensity information from 15 each pair of images was extracted using Biodiscovery Imagene v5 software, using the default settings. This produces text files describing the intensity of each spot, and various background and quality control measurements. These files were imported into the Biodiscovery Genesight v 3.2.21 software. In this software duplicate experiments with respect to treatments, were combined and the data 20 normalised. The normalisation procedure used was the default settings for replicated log ratio experiments. This comprises: * Background subtraction using local background, " Substitution of negative values with a value of 20 " Take the ratio of the corrected experimental signal to that of the reference 25 sample SUBSTITUTE SHEET (RULE 26) WO 2004/099414 PCT/AU2004/000608 26 * Take the log of the ratio to the base 2 * Normalise values by the subtraction of the mean log ratio for the treatment or reference being analysed o Combine the replicated values into one value using the data derived from 5 the median signal values, keeping all replicated values. The log ratios from each experiment are then plotted using the time series tool in the Genesight software. This allows the changes in the expression in the experimental sample relative to the reference sample to be visualised over the time of the experiment. The median signal values are also plotted over time, for both the 10 reference sample and the experimental sample, which allows visual confirmation of the data seen in the log ratio plot. Once these plots have been produced the expression profile of individual genes can be examined by selecting the gene in the software. This produces a line in each graph. The genes showing the most similar pattern of expression ratios to 15 the selected gene can be chosen by specifying a Euclidian distance within which the software will look for matching genes. In these experiments the following known lignification genes were used as template genes: * LpCAD3 (gi2388661) represented by spot NcwCADHLOLPR13222 20 In each experiment the ratios from each of the time points in the series are plotted from left to right as follows 0 5 day old dark adapted perennial ryegrass seedling roots, e 7 day old dark adapted perennial ryegrass seedling roots, * 10 day old dark adapted perennial ryegrass seedling roots 25 a 5 day old light grown perennial ryegrass seedling roots, e 7 day old light grown perennial ryegrass seedling roots, * 10 day old light grown perennial ryegrass seedling roots, * 5 day old dark adapted perennial ryegrass seedling leaves, SUBSTITUTE SHEET (RULE 26) WO 2004/099414 PCT/AU2004/000608 27 0 7 day old dark adapted perennial ryegrass seedling leaves, 0 10 day old dark adapted perennial ryegrass seedling leaves * 5 day old light grown perennial ryegrass seedling leaves, * 7 day old light grown perennial ryegrass seedling leaves, 5 e 10 day old light grown perennial ryegrass seedling leaves, (all versus 4 day old light grown whole seedling) * mature roots e mature leaves e imbibed seed, 10 (all versus mature pseudostem) The identities of the spots with similar ratios to LpCAD3 are given in Table 2. Table 2: Identities of the spots with similar ratios to LpCAD3 Spot ID Spot annotation XnsCA12_MOUSE15263 hypothetical protein DdrGTH1 WHEAT12860 Glutathione transferase IIl like protein DdrPRI ASPOF-1 5682 asparagus officinalis pathogenesis-related protein 1 (aoprl) DdtGTX1TOBAC13521 glutathione transferase, putative EgyG6PISYNY313053 glucose-6-phosphate isomerase EreATP6_OENBE17301 similar to ATPase subunit 6 MaaMMSACAEEL22499 betaine Aldehyde dehydrogenase, putative UxxACPM ARATH12844 arab-Arabidopsis thaliana Acyl carrier protein precursor XnsAROE BACSU12703 putative dehydroquinase shikimate dehydrogenase MnsPPS2 HUMAN14726 putative ATP dependent RNA helicase NcwCADH LOLPR13222 cinnamyl Alcohol dehydrogenase - like protein XnsIDHP ASPNG12559 arab-Arabidopsis thaliana mRNA for AKIN gamma SkiSRK6_BRAOL21162 protein kinase like protein SkiSRK6 BRAOL12332 putative protein kinase SkiMPKIARATH2IO6 rice-Oryza sativa MAP kinase 3 mRNA, complete cds SUBSTITUTE SHEET (RULE 26) WO 2004/099414 PCT/AU2004/000608 28 SmeP2CBMOUSEI 1914 protein phosphatase 2C, putative UatATC1 DICD116604 arab-Arabldopsis thaliana envelope Ca2+-ATPase XnsRGSE RAT--12689 No known database match XnsYIQ3_CAEELi3123 No known database match XnsPA2EPSEAU12485 No known database match XnsOXYB RABIT21613 No known database match XnsFLOIYEAST12047 No known database match U - 14rg1GsB119414 No known database match XnsADOHUMAN-8611 No known database match XnsBCPPEA---8575 hypothetical protein DsrNR43_RAT--1 6545 nicotiana tabacum (common tobacco). stem-specific protein tsjtl U - 07rg1YsE 113105 hypothetical protein XnsCAPP MEDSA17787 hypothetical protein supported by cDNA: gi_18958014 XnsSR52_LYCES14181 bHLH transcription factor, putative ZhyY810_METJAI9999 unknown protein ZhyZEP1_MOUSE8616 arab-Arabidopsis thaliana putative protein ZhyYDB1_SCHPO21884 putative protein ZunPZ12 LUPPOI 5495 putative protein / hypersensitive-induced response protein HIR3 XnsFATPMOUSEI4I86 unknown protein SUBSTITUTE SHEET (RULE 26) WO 2004/099414 PCT/AU2004/000608 29 The sequence identifiers associated with the spot IDs and the number of ESTs represented by the cluster are shown in Table 3. Table 3: Identification of representative ESTs and size of EST cluster Spot ID Representative EST ESTs in cluster XnsCA12_MOUSE15263 1Org2BsGO4 I DdrGTH1 WHEAT12860 07rgITsBO4 2 DdrPR1_ASPOF-15682 1Org1MsCO4 2 DdtGTXITOBAC13521 07rg2PsF11 1 EgyG6PISYNY313053 07rg1XsCO4 I EreATP6_OENBE17301 11rg2EsAO8 1 MaaMMSACAEEL22499 19rg2PsGO8 1 UxxACPMARATH12844 07rglSsGO6 2 XnsAROEBACSU12703 07rg1NsGO8 I MnsPPS2_HUMAN14726 09rg1EsE11 4 NcwCADH_LOLPR13222 07rg2CsDO1 I XnslDHPASPNG12559 07rgllsFO3 2 SkiSRK6_BRAOL21162 17rg1KsDO4 I SkiSRK6_BRAOL12332 07rg1OsC11 4 SkiMPKIARATH21061 17rgIGsDO1 3 SmeP2CB_MOUSE11914 06rglZsFO8 1 UatATCI_DICD116604 11rg isCO6 2 XnsRGSERAT--12689 07rglNsCO7 2 XnsYlQ3_CAEEL13123 07rglYsH1O 2 XnsPA2EPSEAU12485 07rg1EsGQ5 1 XnsOXYBRABIT21613 17rg1VsC12 11 XnsFLOIYEAST12047 06rg2JsHO3 I U - 14rg1GsB119414 14rg1GsB11 2 XnsADOHUMAN-8611 01rgl1sEl2 1 XnsBCPPEA---8575 OrgIGsFIO I DsrNR43_RAT--16545 11rglGsF04 10 U - 07rgIYsE113105 07rglYsE1O 2 XnsCAPPMEDSA17787 11rg2WsBO6 I XnsSR52_LYCES14181 08rg1SsA02 2 ZhyY810_METJAI9999 14rg2AsHO7 I ZhyZEPIMOUSE8616 O1rgilsGO8 I ZhyYDB1_SCHPO21884 19rglEsDOI I ZunPZ12_LUPP015495 1Org2RsGO7 1 SUBSTITUTE SHEET (RULE 26) WO 2004/099414 PCT/AU2004/000608 30 XnsFATPMOUSE14186 08rg1SsBO2 3 The clusters with no known database match represented by ESTs 07rgl NsC07, 07rglYsH1O, 07rglEsGO5, 17rg1VsC12, 06rg2JsHO3, 14rg1GsB11, 01rgllsE12 allowed the identification of LpCAD3L and LpCAD3L-like genes. 5 4. Identification of CAD3L genes Candidate ESTs identified by microarray expression profiling were further analysed by visual inspection of their sequencing trace files and CAD3L genes were selected based on sequence quality assessment. Table 4 summarises the selected cDNA clones (indicating EST code on microarray) coding for CAD3L 10 enzymes identified by microarray expression profiling and provides the sequence name for the perennial ryegrass CAD3L cDNA sequences. Table 4 Identification of CAD3L genes from perennial ryegrass using LpCAD3 as template gene. EST code on LpCAD3L microarray sequence name 06rg2JsHO3 LpCAD3La 07rglYsHlO LpCAD3Lb 07rg1EsGO5 LpCAD3Lc 14rg1GsB 11 LpCAD3Ld 17rg1VsC12 LpCAD3Le 15 EXAMPLE3 Full-length sequencing of CAD3L genes from perennial ryegrass (Lolium perenne) To fully characterise for the purposes of confirmation of the results of the microarray experiments, the generation of probes for hybridisation experiments 20 and the generation of transformation vectors, perennial ryegrass CAD3L genes were fully sequenced. The original plasmids identified by microarray as described in Example 2 were used to transform chemically competent XL-1 cells (prepared in-house, SUBSTITUTE SHEET (RULE 26) WO 2004/099414 PCT/AU2004/000608 31 CaCl 2 protocol). After colony PCR (using HotStarTaq, Qiagen) a minimum of three PCR-positive colonies per transformation were picked for initial sequencing with M13F and M13R primers. The resulting sequences were aligned with the original EST sequence using Sequencher to confirm identity and one of the three clones 5 was picked for full-length sequencing, usually the one with the best initial sequencing result. A putative open reading frame was determined for LpCAD3Lb. Sequencing of LpCAD3Lb was completed by primer walking, i.e. oligonucleotide primers were designed to the initial sequence and used for further sequencing. In the case of 10 LpCAD3Lb, the sequencing could be completed with one oligonucleotide primer from the 5' end. The sequence of the oligonucleotide primer is shown in Table 5. Contigs were then assembled in Sequencher. The contigs include the sequences of the SMART primers used to generate the initial cDNA library as well as pGEM-T Easy vector sequence up to the EcoRI cut site both at the 5' and 3' 15 end. Out of the 7 EST sequences selected after microarray, the clone carrying the EST ID 17rg1VsC12 was identified as a putative full length open reading frame. A plasmid map and the full length cDNA sequence as well as the putative amino acid sequence for perennial ryegrass CAD3Lb were obtained (Figures 8, 9, 20 10). TABLE 5 List of primers used for sequencing of the full-length cDNA of LpCAD3Lb gene name clone ID sequencing primer primer sequence (5'>3') LpCAD3Lb 17rg1VsC12 17rg1VsC12.f1 TCTTCTGCGGTGTTTGTGC EXAMPLE 4 25 Development of a binary transformation vector containing chimeric genes with the cDNA sequence from perennial ryegrass CAD3Lb To alter the expression of the perennial ryegrass CAD3Lb, a sense binary transformation vector was produced. SUBSTITUTE SHEET (RULE 26) WO 2004/099414 PCT/AU2004/000608 32 The pPZP221 binary transformation vector (Hajdukiewicz et al., 1994) was modified to contain the 35S 2 cassette from pKYLX71:35S 2 (Schardi et al., 1987) as follows: pKYLX71:35S 2 was cut with Clal. The 5' overhang was filled in using Klenow and the blunt end was A-tailed with Taq polymerase. After cutting with 5 EcoRI, the 2kb fragment with an EcoRI-compatible and a 3'-A tail was gel-purified. pPZP221 was cut with Hindill 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 Hindill site. This binary vector 10 contains between the left and right border the plant selectable marker gene aacC1 under the control of the 35S promoter and 35S terminator and the pKYLX71:35S2_ derived expression cassette with a CaMV 35S promoter with a duplicated enhancer region and an rbcS terminator. A GATEWAY* cloning cassette (Invitrogen) was introduced into the 15 multicloning site of the pPZP221:35S2 vector obtained as described following the manufacturer's protocol. The LpCAD3Lb cDNA fragment was generated by high fidelity PCR with a proofreading DNA polymerase using the original pGEM-T Easy plasmid cDNA as a template. The primers used (Table 6) contained attB sequences for use with 20 recombinases utilising the GATEWAY* system (Invitrogen). The resulting PCR fragment was used in a recombination reaction with pDONR* vector (Invitrogen) to generate an entry vector. In a further recombination reaction, the cDNA encoding the open reading frame sequence was transferred from the entry vector to the GATEWAY-enabled pPZP221:35S 2 vector. 25 The orientation of the construct (sense or antisense) was checked by restriction enzyme digest and sequencing which also confirmed the correctness of the sequence. A transformation vector containing chimeric genes using full-length open reading frame cDNA representing perennial ryegrass CAD3Lb in sense orientation under the control of the CaMV 35S 2 promoter was generated (Figure 30 11). SUBSTITUTE SHEET (RULE 26) WO 2004/099414 PCT/AU2004/000608 33 5 TABLE6 List of primers used to PCR-amplify the open reading frame of LpCAD3Lb gene name clone ID primer primer sequence (5'->3') LpCAD3Lb 17rg1VsCl2 17rg1VsC12.f GGGGACAAGTTTGTACAAAAAAGCAGGCTTCTAGA TGAGGCGAGGCGCTGGTGC 17rgIVsC12.r GGGGACCACTTTGTACAAGAAAGCTGGGTTCTAGA AAGGCCCAGAAGGACCTGG EXAMPLE 5 10 Production and analysis of transgenic Arabidopsis plants carrying the chimeric perennial ryegrass gene CAD3Lb A set of transgenic Arabidopsis plants carrying the chimeric perennial ryegrass gene CAD3Lb were produced. A pPZP221-based transformation vector with LpCAD3Lb cDNA comprising 15 the full open reading frame sequence in sense orientation under the control of the CaMV 35S promoter with duplicated enhancer region (35S 2 ) was generated as detailed in Example 6. Agrobacterium-mediated gene transfer experiments were performed using these transformation vectors. 20 The production of transgenic Arabidopsis plants carrying the perennial ryegrass CAD3Lb cDNA under the control of the CaMV 35S promoter with duplicated enhancer region (35S 2 ) is described here in detail. Preparation of Arabidopsis plants Seedling punnets were filled with Debco seed raising mixture (Debco Pty. 25 Ltd.) to form a mound. The mound was covered with two layers of anti-bird netting SUBSTITUTE SHEET (RULE 26) WO 2004/099414 PCT/AU2004/000608 34 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 0C. After 48 hours the punnets were 5 transferred to a growth room at 22 0C under fluorescent light (constant illumination, 55 pmoim 2 s 1 ) and fed with Miracle-Gro (Scotts Australia Pty. Ltd.) once a week. Primary bolts were removed as soon as they appeared. After 4 - 6 days the secondary bolts were approximately 6 cm tall, and the plants were ready for vacuum infiltration. 10 Preparation of Agrobacterium Agrobacterium tumefaciens strain AGL-1 were streaked on LB medium containing 50 pg/mI rifampicin and 50 pg/ml kanamycin and grown at 27 0C for 48 hours. A single colony was used to inoculate 5 ml of LB medium containing 50 pg/ml rifampicin and 50 pg/ml kanamycin and grown over night at 27 0C and 250 15 rpm on an orbital shaker. The overnight culture was used as an inoculum for 500 ml of LB medium containing 50 pg/ml kanamycin only. Incubation was over night at 27 0C and 250 rpm on an orbital shaker in a 2 1 Erlenmeyer flask. The overnight cultures were centrifuged for 15 min at 5500 xg and the supernatant discarded. The cells were resuspended in I I of infiltration medium 20 [5% (w/v) sucrose, 0.03% (v/v) Silwet-L77 (Vac-In-Stuff, Lehle Seeds #VIS-01)] and immediately used for infiltration. Vacuum infiltration The Agrobacterium suspension was poured into a container (D6cor Tellfresh storer, #024) and the container placed inside the vacuum desiccator (Bel 25 Art, #42020-0000). A punnet with Arabidopsis plants was inverted and dipped into the Agrobacterium suspension and a gentle vacuum (250 mm Hg) was applied for 2 min. After infiltration, the plants were returned to the growth room where they were kept away from direct light overnight. The next day the plants were returned to full direct light and allowed to grow until the siliques were fully developed. The 30 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 SUBSTITUTE SHEET (RULE 26) 35 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 5 microfuge tube. 500 pi 70% ethanol were added for 2 min and replaced by 500 pl sterilisation solution (H 2 0:4% chlorine:5% SDS, 15:8:1). After vigorous shaking, the tube was left for 10 min after which time the sterilisation solution was replaced with 500 pl 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 10 with approximately 200 pl sterile water. The seeds were then evenly spread on 150 mm petri dishes containing germination medium (4.61 g Murashige & Skoog salts, 10 g sucrose, 1 ml I M KOH, 2 g Phytagel, 0.5 g MES and I ml 1000x Gamborg's B-5 vitamins per litre) supplemented with 250 pg/ml timetin and 75 pg/ml gentamycin. After vernalisation 15 for 48 hours at 4 *C the plants were grown under continuous fluorescent light (55 pmol m-2s-1) at 22 *C to the 6 - 8 leaf stage, transferred to soil and grown to seeding stage using the Arasystem (Betatech, Belgium). Preparation of genomic DNA 3 - 4 leaves of Arabidopsis plants regenerated on selective medium were 20 harvested and freeze-dried. The tissue was homogenised on a Retsch MM300 mixer mill, then centrifuged for 10 min at 1700xg to collect cell debris. Genomic DNA was isolated from the supernatant using Wizard Magnetic 96 DNA Plant System kits (Promega) on a Biomek FX (Beckman Coulter). 5 pl of the sample (50 pl) were then analysed on an agarose gel to check the yield and the quality of the 25 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 7) were designed using Primer Express 1.5. The forward primer was located within the 35S 2 30 promoter region and the reverse primer within the transgene to amplify products of SUBSTITUTE SHEET (RUI F 291 36 approximately 150 bp as recommended. The positioning of the forward primer within the 35S2 promoter region guaranteed that homologous genes in Arabidopsis were not detected. 5 pl of each genomic DNA sample was run in a 50 pl PCR reaction 5 including SYBR Green on an AB17700 (Applied Biosystems) together with samples containing DNA isolated from wild type Arabidopsis plants (negative control), samples containing buffer instead of DNA (buffer control) and samples containing the plasmid used for transformation (positive plasmid control). Plants were obtained after transformation with all chimeric constructs and 10 selection on medium. containing gentamycin. The transformation and selection process is shown in Figure 12. Table 7 List of primers used for real time PCR analysis of Arabidopsis plants transformed with LpCAD3Lb gene name clone ID primer primer sequence (5'->3') LpCAD3Lb 17rg1VsC12 17rg1VsC12q.f CCACTATCCTTCGCAAGACCC 17rg1VsC12q.r CACGGCCAAGAACTACACCTAAT 15 REFERENCES Frohman, M.A., Dush, M.K., Martin, G.R. (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. 20 Hajdukiewicz P, Svab Z, Maliga P. (1994) The small, versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol Biol. 25, 989-94. Loh, E.Y., Elliott, J.F., Cwirla, S., Lanier, L.L., Davis, M.M. (1989). Polymerase chain reaction with single-sided specificity: Analysis of T-cell receptor delta 25 chain. Science 243, 217-220. Ohara, 0., Dorit, R.L., Gilbert, W. (1989). One-sided polymerase chain reaction: The amplification of cDNA. Proc. Nat/. Acad Sci USA 86, 5673-5677 SUBSTITUTE SHEET (RULE 261 37 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 5 of foreign genes in plants. Gene 61, 1-11 Finally, it is to be understood that various alterations, modifications and/or additions may be made without departing from the spirit of the present invention as outlined herein. SUBSTITUTE SHEET IRIJI F 261

Claims (24)

1. A substantially purified or isolated nucleic acid or nucleic acid fragment encoding a cinnamyl alcohol dehydrogenase 3-like polypeptide (CAD3L) from a Lolium species, or complementary or antisense to a sequence encoding a CAD3L polypeptide 5 from a Lolium species, and including a nucleotide sequence selected from the group consisting of (a) sequences shown in Figures 1, 2, 3, 4, 5 and 9 hereto (SEQ ID NOS 1 to 6, respectively); (b) complements of the sequences recited in (a); (c) sequences antisense to the sequences recited in (a) or (b); (d) functionally active fragments and variants of the sequences recited in (a), (b) and (c); and (e) RNA sequences 10 corresponding to the sequences recited in (a), (b), (c) and (d).

2. A nucleic acid or nucleic acid fragment according to claim 1, wherein said functionally active fragments and variants have at least approximately 90% identity to the relevant part of the sequences recited in (a), (b) or (c) and have a size of at least 30 nucleotides. 5

3. A nucleic acid or nucleic acid fragment according to claim 1, wherein said functionally active fragments and variants have at least approximately 95% identity to the relevant part of the sequences recited in (a), (b) or (c) and have a size of at least 60 nucleotides.

4. A nucleic acid or nucleic acid fragment according to claim 1, including a !0 nucleotide sequence selected from the group consisting of sequences shown in Figures 1, 2, 3, 4, 5 and 9 (SEQ ID NOS 1 to 6, respectively).

5. A nucleic acid or nucleic acid fragment according to any one of claims 1 to 4 wherein said Lolium species is Lolium perenne or Lolium arundinaceum.

6. A construct including one or more nucleic acids or nucleic acid fragments 25 according to any one of claims 1 to 5.

7. A construct according to claim 6 wherein the one or more nucleic acids or nucleic acid fragments are operably linked to one or more regulatory elements, such that the one or more nucleic acids or nucleic acid fragments are each expressed.

8. A construct according to claim 7, wherein the one or more regulatory 30 elements include an operably linked promoter and an operably linked terminator. 39

9. A plant cell, plant, plant seed or other plant part, including a construct according to any one of claims 6 to 8.

10. A plant, plant seed or other plant part derived from a plant cell or plant according to claim 9 and including a construct according to any one of claims 6 to 8. 5

11. A method of modifying one or more of lignification, defence response, or cell walls in a plant, said method including introducing into said plant an effective amount of a nucleic acid or nucleic acid fragment according to any one of claims 1 to 5, or a construct according to any one of claims 6 to 8.

12. A lignin or cellulose substantially or partially purified or isolated from a 10 plant cell, plant, plant seed or other plant part according to claim 9 or 10.

13. Use of a nucleic acid or nucleic acid fragment according to any one of claims 1 to 5, and/or nucleotide sequence information thereof, and/or single nucleotide polymorphisms thereof as a molecular genetic marker.

14. The use according to claim 13 wherein the molecular genetic marker is for 5 one or more of quantitative trait loci (QTL) tagging, QTL mapping, DNA fingerprinting and marker assisted selections.

15. The use according to claim 13 or 14 wherein the molecular marker is used to select for a herbage quality trait selected from the group consisting of dry matter digestibility, mechanical stress tolerance, disease resistance, insect pest resistance, 20 plant stature and leaf and stem colour.

16. A substantially purified or isolated nucleic acid or nucleic acid fragment including a single nucleotide polymorphism (SNP) from a nucleic acid fragment according to any one of claims 1 to 5.

17. A substantially purified or isolated CAD3L polypeptide from a Lolium 25 species, including an amino acid sequence shown in Figure 10 hereto (SEQ ID NO 7); or a functionally active fragment or variant thereof.

18. A polypeptide according to claim 17, wherein said functionally active fragment or variant has at least approximately 90% identity to the functional part of SEQ ID NO 7 and has a size of at least 20 amino acids. 30

19. A polypeptide according to claim 18 including the amino acid sequence shown in Figure 10 hereto (SEQ ID NO 7). 40

20 A polypeptide according to any one of claims 17 to 19, wherein said Lolium species is Lolium perenne or Lolium arundinaceum.

21. A polypeptide encoded by a nucleic acid or nucleic acid fragment according to any one of claims 1 to 5. 5

22. A preparation for transforming a plant comprising a nucleic acid or nucleic acid fragment according to any one of claims 1 to 5, or a construct according to any one of claims 6 to 8.

23. A nucleic acid or nucleic acid fragment according to claim 1, substantially as hereinbefore described with reference to any one of the examples. 0

24. A polypeptide according to claim 17, substantially as hereinbefore described with reference to any one of the examples.