AU2007201050B2 - Materials and methods for the modification of plant lignin content - Google Patents

Abstract

(57) Abstract Novel isolated DNA sequences associated with the lignin biosynthetic pathway are provided, together with DNA constructs including such sequences. Methods for the modulation of lignin content in plants are also disclosed, the methods comprising incorporating one or more of the inventive DNA sequences or a sequence complementary to an inventive DNA sequence into the genome of a plant.

Description

Regulation 3.2 Revised 2/98 AUSTRALIA Patents Act, 1990 ORIGINAL COMPLETE SPECIFICATION TO BE COMPLETED BY THE APPLICANT NAME OF APPLICANTS: Rubicon Forests Holdings Limited and Arborgen, LLC ACTUAL INVENTORS: Leonard Nathan Bloksberg Alistair Wallace Grierson lIkka Jaako Havukkala ADDRESS FOR SERVICE: Peter Maxwell and Associates Level 6 60 Pitt Street SYDNEY NSW 2000 INVENTION TITLE: MATERIALS AND METHODS FOR THE MODIFICATION OF PLANT LIGNIN CONTENT DETAILS OF ASSOCIATED APPLICATION NO(S): Divisional of Australian Patent Application No. 2003 203 517 filed on 8 April 2003 which is a divisional of Australian Patent Application No. 756,359 (57,975/01) filed on 10 August 2001, which is a divisional of Australian Appin. No. 733,388 (44,036/97) filed on 10 September 1997 The following statement is a full description of this invention including the best method of performing it known to us: m:\docs\971243\1 16493.doc MATERIALS AND METHODS FOR THE MODIFICATION OF PLANT LIGNIN CONTENT 5 Technical Field of the Invention This invention relates to the field of modification of lignin content and composition in plants. More particularly, this invention relates to enzymes involved in the lignin biosynthetic pathway and nucleotide sequences encoding such enzymes. 10 Backrround of the Invention Lignin is an insoluble polymer which is primarily responsible for the rigidity of plant sterns. Specifically, lignin serves as a matrix around the polysaccharide components of some plant cell walls. The higher the lignin content. the more rigid the plant. For example, tree species synthesize large quantities of lignin, with lignin Is constituting between 20% to 30% of the dry weight of wood. In addition to providing rigidity, lignin aids in water transport within plants by rendering cell walls hydrophobic and water impermeable. Lignin also plays a role in disease resistance of plants by impeding the penetration and propagation of pathogenic agents. The high concentration of lignin in trees presents a significant problem. in the 20 paper industry wherein considerable resources must be employed to separate lignin from the cellulose fiber needed for the production of paper. Methods typically employed for the removal of lignin are highly energy- and chemical-intensive, resulting in increased costs and increased levels of undesirable waste products. In the U.S. alone, about 20 million tons of lignin are removed from wood per year. 25 ~ . Lignin is largely responsible for the digestibility, or lack thereof, of forage crops, with small increases in plant lignin content resulting in relatively high decreases in digestibility. For example, crops with reduced lignin content provide more efficient forage for cattle, with the yield of milk and meat being higher relative to the amount of forage crop consumed. During normal plant growth; the increase in dry matter content 30 is accompanied by a corresponding decrease in digestibility. When deciding on the optimum time to harvest forage crops, farmers must therefore chose between a high yield of less digestible material and a lower yield of more digestible material. la 2 For some applications, an increase in lignin content is desirable since increasing the lignin content of a plant would lead to increased mechanical strength of wood, changes in its color and increased resistance to rot. 5 Mycorrhizal species composition and abundance may also be favourably manipulated by modifying lignin content and structural composition. As discussed in detail below, lignin is formed by polymerisation of at least three different monolignols which are synthesized in a multistep pathway, each step in the pathway being catalyzed by a different enzyme. It has been 10 shown that manipulation of the number of copies of genes encoding certain enzymes, such as cinnamyl alcohol dehydrogenase (CAD) and caffeic acid 3 0-methyltransferase (COMT) results in modification of the amount of lignin produced; see, for example, U.S. Patent No. 5,451,514 and PCT publication no. WO 94/23044. Furthermore, it has been shown that antisense expression 15 of sequences encoding CAD in poplar leads to the production of lignin having a modified composition (Grand, C. et al. Plant (Berl.) 163:232-237 (1985)). While DNA sequences encoding some of the enzymes involved in the lignin biosynthetic pathway have been isolated for certain species of plants, genes encoding many of the enzymes in a wide range of plant species have 20 not yet been identified. Thus there remains a need in the art for materials useful in the modification of lignin content and composition in plants and for methods for their use. According to one aspect of the invention there is provided an isolated DNA sequence comprising a nucleotide sequence selected from the group 25 consisting of: (a) coding sequence recited in SEQ ID NO: 3, 13, 16-18, 22-52, 56, 58-70 and 72-88; 13/03/07 3 (b) complements of the coding sequences recited in SEQ ID NO: 3, 13,16-18, 22-52, 56,58-70 and 72-88; (c) reverse complements of the coding sequences recited in SEQ ID NO: 3,13, 16-18, 22-52, 56, 58-70 and 72-88; 5 (d) reverse sequences of the coding sequences recited in SEQ ID NO: 3, 13, 16-18, 22-52, 56, 58-70 and 72-88; and (e) sequences having at least 50% identity, at least 70% identity or at least 90% identity to a coding sequence recited in SEQ ID NO: 3, 13, 16-18, 22-52, 56,58-70 and 72-88; and wherein said 10 sequences of (e) encode an enzyme involved in the lignin biosynthetic pathway as encoded by SEQ ID NO: 3, 13, 16-18, 22-52, 56, 58-70 and 72-88. According to another aspect of the invention there is provided a DNA construct comprising, in the 5'-3' direction: 15 (a) a gene promoter sequence, (b) an open reading frame coding for at least a functional portion of an enzyme encoded by a nucleotide sequence selected from the group consisting of: (1) coding sequences recited in SEQ ID NO: 3, 13, 16-18, 22-52, 56, 58-70 and 72-88; and (2) sequences 20 having at least 50% identity, at least 70% identity or at least 90% identity to a coding sequence of SEQ ID NO: 3, 13, 16-18, 22-52, 56, 58-70 and 72-88; wherein said sequences of (2) encode an enzyme involved in the lignin biosynthetic pathway as encoded by SEQ ID NO: 3, 13, 16-18, 22-52, 56, 58-70 and 72-88; and 25 (c) a gene termination sequence. According to another aspect of the invention there is provided a DNA construct comprising, in the 5'-3' direction: 13/03/07 4 (a) a gene promoter sequence, (b) a non-coding region of a gene coding for an enzyme encoded by a nucleotide sequence selected from the group consisting of: (1) coding sequences recited in SEQ ID NO: 3, 13, 16-18, 22-52, 56, 5 58-70 and 72-88; and (2) sequences having at least 50% identity, at least 70% identity or at least 90% identity to a coding sequence of SEQ ID NO: 3, 13,16-18, 22-52, 56, 58-70 and 72-88; wherein said sequences of (2) encode an enzyme involved in the lignin biosynthetic pathway as encoded by SEQ ID NO: 3, 13,16-18, 10 22-52, 56, 58-70 and 72-88; and (c) a gene termination sequence. According to a further aspect of the invention there is provided a transgenic plant cell comprising a DNA construct, the DNA construct comprising, in the 5'-3' direction: 15 (a) a gene promoter sequence; (b) an open reading frame coding for at least a functional portion of an enzyme encoded by a nucleotide sequence selected from the group consisting of: (1) coding sequences recited in SEQ ID NO: 3, 13, 16-18, 22-52, 56, 58-70 and 72-88; and (2) coding 20 sequences having at least 50% identity, at least 70% identity or at least 90% identity to a sequence of SEQ ID NO: 3, 13, 16-18, 22 52, 56, 58-70 and 72-88; wherein said sequences of (2) encode an enzyme involved in the lignin biosynthetic pathway as encoded by SEQ ID NO: 3, 13, 16-18, 22-52, 56, 58-70 and 72-88; and 25 (c) a gene termination sequence. 13/03/07 4a According to a still further aspect of the invention there is provided a transgenic plant cell comprising a DNA construct, the DNA construct comprising, in the 5'-3' direction: (a) a gene promoter sequence; 5 (b) a non-coding region of a gene coding for an enzyme encoded by a nucleotide sequence selected from the group consisting of: (1) coding sequences recited in SEQ ID NO: 3, 13, 16-18, 22-52, 56, 58-70 and 72-88; and (2) coding sequences having at least 50% identity, at least 70% identity or at least 90% identity to a 10 sequence of SEQ ID NO: 3, 13, 16-18, 22-52, 56, 58-70 and 72 88; wherein said sequences of (2) encode an enzyme involved in the lignin biosynthetic pathway as encoded by SEQ ID NO: 3, 13, 16-18, 22-52, 56, 58-70 and 72-88; and (c) a gene termination sequence. 15 According to a still further aspect of the invention there is provided a method for modulating the lignin content of a plant comprising stably incorporating into the genome of the plant a DNA construct comprising, in the 5'-3' direction: (a) a gene promoter sequence; 20 (b) an open reading frame coding for at least a functional portion of an enzyme encoded by a nucleotide sequence selected from the group consisting of: (1) coding sequences recited in SEQ ID NO: 3, 13, 16-18, 22-52, 56, 58-70 and 72-88; and (2) sequences having at least 50% identity, at least 70% identity or at least 90% 25 identity to a coding sequence of SEQ ID NO: 3, 13, 16-18, 22-52, 56,58-70 and 72-88; wherein said sequences of (2) encode an 13/03/07 4b enzyme involved in the lignin biosynthetic pathway as encoded by SEQ ID NO: 3,13, 16-18, 22-52, 56,58-70 and 72-88; and (c) a gene termination sequence. According to a still further aspect of the invention there is provided a 5 method for modulating the lignin content of a plant comprising stably incorporating into the genome of the plant a DNA construct comprising, in the 5'-3' direction: (a) a gene promoter sequence; (b) a non-coding region of a gene coding for an enzyme encoded by 10 a nucleotide sequence selected from the group consisting of: (1) coding sequences recited in SEQ ID NO: 3, 13, 16-18, 22-52, 56, 58-70 and 72-88; and (2) sequences having at least 50% identity, at least 70% identity or at least 90% identity to a coding sequence of SEQ ID NO: 3, 13, 16-18, 22-52, 56, 58-70 and 72-88; wherein 15 said sequences of (2) encode an enzyme involved in the lignin biosynthetic pathway as encoded by SEQ ID NO: 3, 13, 16-18, 22-52, 56, 58-70 and 72-88; and (c) a gene termination sequence. According to a still further aspect of the invention there is provided a 20 method for producing a plant having altered lignin structure comprising: (a) transforming a plant cell with a DNA construct comprising, in the 5'-3' direction, a gene promoter sequence, an open reading frame coding for at least a functional portion of an enzyme encoded by a nucleotide sequence selected from the group consisting of: (1) 25 coding sequences recited in SEQ ID NO: 3,13,16-18, 22-52, 56, 58-70 and 72-88; and (2) sequences having at least 50% identity, at least 70% identity or at least 90% identity to a coding sequence 13/03/07 4c of SEQ ID NO: 3,13,16-18, 22-52, 56, 58-70 and 72-88; wherein said sequences of (2) encode an enzyme involved in the lignin biosynthetic pathway as encoded by SEQ ID NO: 3, 13, 16-18, 22-52, 56, 58-70 and 72-88, and a gene termination sequence to 5 provide a transgenic cell; and (b) cultivating the transgenic cell under conditions conducive to regeneration and mature plant growth. According to a still further aspect of the invention there is a method for producing a plant having altered lignin structure comprising: 10 (a) transforming a plant cell with a DNA construct comprising, in the 5'-3' direction, a gene promoter sequence, a non-coding region of a gene coding for an enzyme encoded by a nucleotide sequence selected from the group consisting of: (1) coding sequences recited in SEQ ID NO: 3, 13, 16-18, 22-52, 56, 58-70 and 72-88; 15 and (2) sequences having at least 50% identity, at least 70% identity or at least 90% identity to a coding sequence of SEQ ID NO: 3, 13, 16-18, 22-52, 56, 58-70 and 72-88; wherein said sequences of (2) encode an enzyme involved in the lignin biosynthetic pathway as encoded by SEQ ID NO: 3, 13, 16-18, 20 22-52, 56, 58-70 and 72-88, and a gene termination sequence to provide a transgenic cell; and (b) cultivating the transgenic cell under conditions conducive to regeneration and mature plant growth. According to a still further aspect of the invention there is a method of 25 modifying the activity of an enzyme in a plant comprising stably incorporating into the genome of the plant a DNA construct including (a) a gene promoter sequence; 13/03107 4d (b) an open reading frame coding for at least a functional portion of an enzyme encoded by a nucleotide sequence selected from the group consisting of: (1) coding sequences recited in SEQ ID NO: 3, 13, 16-18, 22-52, 56, 58-70 and 72-88; and (2) sequences 5 having at least 50% identity, at least 70% identity or at least 90% identity to a coding sequence of SEQ ID NO: 3, 13, 16-18, 22-52, 56, 58-70 and 72-88; wherein said sequences of (2) encode an enzyme involved in the lignin biosynthetic pathway as encoded by SEQ ID NO: 3, 13, 16-18, 22-52, 56, 58-70 and 72-88; and 10 (c) a gene termination sequence. According to a still further aspect of the invention there is a method of modifying the activity of an enzyme in a plant comprising stably incorporating into the genome of the plant a DNA construct including: (a) a gene promoter sequence; 15 (b) a non-coding region of a gene coding for an enzyme encoded by a nucleotide sequence selected from the group consisting of: (1) coding sequences recited in SEQ ID NO: 3, 13, 16-18, 22-52, 56, 58-70 and 72-88; and (2) sequences having at least 50% identity, at least 70% identity or at least 90% identity to a coding sequence 20 of SEQ ID NO: 3, 13, 16-18, 22-52, 56, 58-70 and 72-88; wherein said sequences of (2) encode an enzyme involved in the lignin biosynthetic pathway as encoded by SEQ ID NO: 3, 13, 16-18, 22-52, 56, 58-70 and 72-88, and a gene termination sequence to provide a transgenic cell; and 25 (c) a gene termination sequence. According to a still further aspect of the invention there is a method for producing a transgenic plant cell having altered lignin structure comprising: 13/03/07 4e (a) transforming a plant cell with a DNA construct comprising, in the 5'-3' direction, a gene promoter sequence, an open reading frame coding for at least a functional portion of an enzyme encoded by a nucleotide sequence selected from the group consisting of: (1) 5 coding sequences recited in SEQ ID NO: 3, 13,16-18,22-52, 56, 58-70 and 72-88; and (2) sequences having at least 50% identity, at least 70% identity or at least 90% identity to a coding sequence of SEQ ID NO: 3, 13, 16-18, 22-52, 56, 58-70 and 72-88; wherein said sequences of (2) encode an enzyme involved in the lignin 10 biosynthetic pathway as encoded by SEQ ID NO: 3,13, 16-18, 22-52, 56, 58-70 and 72-88, and a gene termination sequence to provide a transgenic cell; and (b) cultivating the transgenic cell. According to a still further aspect of the invention there is a method for 15 producing a transgenic plant cell having altered lignin structure comprising: (a) transforming a plant cell with a DNA construct comprising, in the 5'-3' direction, a gene promoter sequence, a non-coding region of a gene coding for an enzyme encoded by a nucleotide sequence selected from the group consisting of: (1) coding sequences 20 recited in SEQ ID NO: 3,13, 16-18, 22-52, 56, 58-70 and 72-88; and (2) sequences having at least 50% identity, at least 70% identity or at least 90% identity to a coding sequence of SEQ ID NO: 3,13, 16-18, 22-52, 56, 58-70 and 72-88; wherein said sequences of (2) encode an enzyme involved in the lignin 25 biosynthetic pathway as encoded by SEQ ID NO: 3,13, 16-18, 22-52, 56, 58-70 and 72-88, and a gene termination sequence to provide a transgenic cell; and 13/03/07 4f (b) cultivating the transgenic cell. Brief Description of the Figures Fig. 1 is a schematic overview of the lignin biosynthetic pathway. 5 Detailed Description Lignin is formed by polymerisation of at least three different monolignols, primarily para-coumaryl alcohol, coniferyl alcohol and sinapyl alcohol. While these three types of lignin subunits are well known, it is possible that slightly 10 different variants of these subunits may be involved in the lignin biosynthetic pathway in various 13/03/07 plants. The relative concentration of these residues in lignin varies between different plant species and within species. In addition, the composition of lignin may also vary between different tissues within a specific plant. The three monolignols are derived from phenylalanine in a multistep process and are believed to be polymerized into 5 lignin by a free radical mechanism. Fig. I shows the different steps in the biosynthetic pathway for coniferyl alcohol together with the enzymes responsible for catalyzing each step. para-Coumaryl alcohol and sinapyl alcohol are synthesized by similar pathways. Phenylalanine is first deaminated by phenylalanine ammonia-lyase (PAL) to give cinnamate which is then 10 hydroxylated by cinnamate 4-hydroxylase (C4H) to form p-coumarate. p-Coumarate is hydroxylated by coumarate 3-hydroxylase to give caffeate. The newly added hydroxyl group is then methylated by 0-methyl transferase (OMT) to give ferulate which is conjugated to coenzyme A by 4-coumarate:CoA ligase (4CL) to form feruloyl-CoA. Reduction of feruloyl-CoA to coniferaldehyde is catalyzed by cinnamoyl-CoA is reductase (CCR). Coniferaldehyde is further reduced by the action of cinnamyl alcohol dehydrogenase (CAD) to give coniferyl alcohol which is then converted into its glucosylated form for export from the cytoplasm to the cell wall by coniferol glucosy transferase (CGT). Following export, the de-glucosylated form of coniferyl alcohol is obtained by the action of coniferin beta-glucosidase (CBG). Finally, polymerization of 20 the three monolignols to provide lignin is catalyzed by phenolase (PNL), laccase (LAC) and peroxidase (POX). The formation of sinapyl alcohol involves an additional enzyme, ferulate-5 hydroxylase (FSH). For a more detailed review of the lignin biosynthetic pathway, see: Whetton, R. and Sederoff, R., The Plant Cell, 7:1001-1013 (1995). 25 Quantitative and qualitative modifications in plant lignin content are known to be induced by external factors such as light stimulation, low calcium levels and mechanical stress. Synthesis of new types of lignins, sometimes in tissues not normally lignified, can also be induced by infection with pathogens. In addition to lignin, several other classes of plant products are derived from phenylalanine. including flavonoids, 30 coumarins, stilbenes and benzoic acid derivatives, with the initial steps in the synthesis of all these compounds being the same. Thus modification of the action of PAL, C4H and 4CL may affect the synthesis of other plant products in addition to lignin. 5 6 Using the methods and materials of the present invention, the lignin content of a plant can be increased by incorporating additional copies of genes encoding enzymes involved in the lignin biosynthetic pathway into the genome of the target plant. Similarly, a decrease in lignin content can be obtained by 5 transforming the target plant with antisense copies of such genes. In addition, the number of copies of genes encoding for different enzymes in the lignin biosynthetic pathway can be manipulated to modify the relative amount of each monolignol synthesized, thereby leading to the formation of lignin having altered composition. The alteration of lignin composition would be 10 advantageous, for example, in tree processing for paper, and may also be effective in altering the palatability of wood materials to rotting fungi. In one embodiment, the present invention provides isolated complete or partial DNA sequences encoding, or partially encoding, enzymes involved in the lignin biosynthetic pathway, the DNA sequences being obtainable from the 15 eucalyptus and pine. Complements of such isolated DNA sequences, reverse complements of such isolated DNA sequences and reverse sequences of such isolated DNA sequences, together with variants of such sequences, are also provided. DNA sequences encompassed by the present invention include cDNA, genomic 20 DNA, recombinant DNA and wholly or partially chemically synthesized DNA molecules. The definition of the terms "complement", "reverse complement" and "reverse sequence", as used herein, is best illustrated by the following example. For the sequence 5' AGGACC 3', the complement, reverse 25 complement and reverse sequence are as follows: 13/03/07 complement 3' TCCTGG 5' reverse complement 3' GGTCCT 5' reverse sequence 5' CCAGGA 3'. As used herein, the term "variant" covers any sequence which exhibits at least 5 about 50%, more preferably at least about 70% and, more preferably yet. at least about 90% identity to a sequence of the present invention. Most preferably, a variant" is any sequence which has at least about a 99% probability of being the same as the inventive sequence. The probability for DNA sequences is measured by the computer algorithm FASTA (version 2.0u4, February 1996; Pearson W. R. et al.. 10 Proc, Natl. Acad. Sci., 85:2444-2448, 1988), the probability for translated DNA sequences is measured by the computer algorithm TBLASTX and that for protein sequences is measured by the computer algorithm BLASTP (Altschul, S. F. et al. J_ Mol. Biol., 215:403-410, 1990). The term "variants" thus encompasses sequences wherein the probability of finding a match by chance (smallest sum probability) in a 15 database, is less than about 1% as measured by any of the above tests. Variants of the isolated sequences from other eucalyptus and pine species, as well as from other commercially important species utilized by the lumber industry, are contemplated. These include the following gymnosperms, by way of example: loblolly pine Pinus taeda, slash pine Pinus elliotti, sand pine Pinus clausa, longleaf pine 20 Pinuspalustrus, shortleaf pine Pinus echinata, ponderosa pine Pinusponderosa, Jeffrey pine Pinus jeffrey, red pine Pinus resinosa, pitch pine Pinus rigida, jack pine Pinus banksiana, pond pine Pinus serotina, Eastern white pine Pinus strobus, Western white pine Pinus monticola, sugar pine Pinus lambeniana, Virginia pine Pinus virginiana, lodgepole pine Pinus contorla, Caribbean pine Pinus cadbaea, F. pinaster, Calabrian 25 pine P. brutia, Afghan pine P. eldarica, Coulter pine P. coulteri, European pine P nigra and P. sylvestris; Douglas-fir Pseudosuga menziesii; the hemlocks which include Western hemlock Tsuga heterophylla, Eastern hemlock Tsuga canadensis, Mountain hemlock Tsuga mertensiana; the spruces which include the Norway spruce Ficea abies, red spruce Picea rubens, white spruce Picea glauca, black spruce Picea mariana, Sitka 30 spruce Picea sitchensis, Englemann spruce Picea engelmanni, and blue spruce Picea pungens; redwood Sequoia sempervirens; the true firs include the Alpine fir Abies lasiocarpa, silver fir Abies amabilis, grand fir Abies grandis. noble fir Abies procera, white fir Abies concolor, California red fir Abies magnifica, and balsam fir Abies balsamea, the cedars which include the Western red cedar Thuja plicata, incense -7cedar libocednisdecurrens, Northern white cedar Thuja occidentalis, Port Orford cedar Chamaecyparis lawsoniona, Atlantic white cedar Chanaecyparis uhyoides, Alaska yellow-cedar Chamaecyparis noorkatensis. and Eastern red cedar Hunpems virginiana; the larches which include Eastern larch Larix laricina, Western larch Larix 5 occidentalis, European larch Larix decidua, Japanese larch Larix leprolepis, and Siberian larch Larir siberica; bold cypress Taxodium distichum and Giant sequoia Sequoia gigantea; and the following angiosperms, by way of example: Eucalypus alba. E. bancroftii, E. botyroides, E. bridgesiana, E. calophylla, E. 10 camaldulensis. E. citriodora. E cladocalyx, E. coccifera, E currisii, E. dabrympleana. E. deglupra. E. delagatensis, E. diversicolor, E. dunnii, E. ficifolia, E. globulus, E gomphocephala. E gunni, . hemyi, E. laevopinea, E macarthurd, E. macrorhynchta. E. maculata. E. marginata, E. megacarpa . Emelliodora. E nicholl, E. nirens, E. nova anglica. E. obliqua, E. obrusiflora. E. oreades, E. pauciflora. E. polybracrea, E. regnans, 15 E. resinifera E. robusta . rudis, E. saligna. E. sideroxylon, E. stuaniana, E. tereticornis, E. torelliana. E. unigera, E. urophylla. E viminais, E. viridi4, E. wandoo and E. youmanni. The inventive DNA sequences may be isolated by high throughput sequencing of cDNA libraries such as those prepared from Eucalyprus grandis and Pinus radiata 20 as described below in Examples 1 and 2. Alternatively, oligonucleotide probes based on the sequences provided in SEQ ID NO: 1-13 and 16-88 can be synthesized and used to identify positive clones -in either cDNA or genomic DNA libraries from Eucalyptus grandis and Pinus radiata, or from other gymnosperms and angiosperms including those identified above, by means of hybridization or PCR techniques. 25 Probes can be shorter than the sequences provided herein but should be at least about 10, preferably at least about 15 and most preferably at least about 20 nucleotides in length. Hybridization and PCR techniques suitable for use with such oligonucleotide probes are well known in the art. Positive clones may be analyzed by restriction enzyme digestion, DNA sequencing or the like. 30 In addition, the DNA sequences of the present invention may be generated by synthetic means using techniques well known in the art. Equipment for automated synthesis of oligonucleotides is commercially available from suppliers such as Perkin Elmer/Applied Biosystems Division (Foster City, CA) and may be operated according to the manufacturer's instructions. -8- In one embodiment, the DNA constructs of the present invention include an open reading frame coding for at least a functional portion of an enzyme encoded by a nucleotide sequence of the present invention or a variant thereof. As used herein, the "functional portion" of an enzyme is that portion which contains the active site 5 essential for affecting the metabolic step, i.e. the portion of the molecule that is capable of binding one or more reactants or is capable of improving or regulating the rate of reaction. The active site may be made up of separate portions present on one or more polypeptide chains and will generally exhibit high substrate specificity. The term "enzyme encoded by a nucleotide sequence" as used herein, includes enzymes encoded to by a nucleotide sequence which includes the partial isolated DNA sequences of the present invention. For applications where amplification of lignin synthesis is desired, the open reading frame is inserted in the DNA construct in a sense orientation, such- that transformation of a target plant with the DNA construct will lead to an increase- in the 15 number of copies of the gene and therefore an increase in the amount of enzyme. When down-regulation of lignin synthesis is desired, the open reading frame is inserted in the DNA construct in an antisense orientation, such that the tNA produced by transcription of the DNA sequence is complementary to the endogenous mRNA sequence. This, in turn, will result in a decrease in the number of copies of the gene and therefore a 20 decrease in the amount of enzyme. Alternatively, regulation can be achieved by inserting appropriate sequences or subsequences (e.g. DNA or RNA) in ribozyme constructs. In a second embodiment, the inventive DNA constructs comprise a nucleotide sequence including a non-coding region of a gene coding for an enzyme encoded by a 25 DNA sequence of the present invention, or a nucleotide sequence complementary to such a non-coding region. As used herein the term "non-coding region" includes both transcribed sequences which are not translated, and non-transcribed sequences -within about 2000 base pairs 5' or 3' of the translated sequences or open reading frames. Examples of non-coding regions which may be usefully employed in the inventive 30 constructs include introns and 5'-non-coding leader sequences. Transformation of a target plant with such a DNA construct may lead to a reduction in the amount of lignin synthesized by the plant by the process of cosuppression, in a manner similar to that 9 discussed, for example, by Napoli et al. (Plant Cell 2:279-290, 1990) and de Carvalho Niebel et al. (Plant Cell 7:347-358. 1995). The DNA constructs of the present invention further comprise a gene promoter sequence and a gene termination sequence, operably linked to the DNA sequence to be 5 transcribed, which control expression of the gene. The gene promoter sequence is generally positioned at the 5' end of the DNA sequence to be transcribed, and is employed to initiate transcription of the DNA sequence. Gene promoter sequences are generally found in the 5' non-coding region of a gene but they may exist in introns (Luehrsen, K. R., Mol. Gen. Genet. 225:81-93, 1991) or in the coding region, as for to example in PAL of tomato (Bloksberg, 1991. Studies on the Biology of Phenylalanine Ammonia Lyase and Plant Pathogen Interaction. Ph.D. Thesis. Univ. of California, Davis, University Microfilms International order number 9217564). When the construct includes an open reading frame in a sense orientation, the gene promoter sequence also initiates translation of the open reading frame. For DNA constructs 15 comprising either an open reading frame in an antisense orientation or a non-coding region, the gene promoter sequence consists only of a transcription initiation site having a RNA polymerase binding site. A variety of gene promoter sequences which may be usefully employed in the DNA constructs of the present invention are well known in the art. The promoter gene 20 sequence, and also the gene termination sequence. may be endogenous to the target plant host or may be exogenous, provided the promoter is functional in the target host. For example, the promoter and termination sequences may be from other plant species, plant viruses, bacterial plasmids and. the like. Preferably, gene promoter and termination sequences are from the inventive sequences themselves. 25 Factors influencing the choice of promoter include the desired tissue specificity of the construct, and the timing of transcription and translation. For example, constitutive promoters, such as the 35S Cauliflower Mosaic Virus (CaMV 35S) promoter, will affect the activity of the enzyme in all parts of the plant. Use of a tissue specific promoter will result in production of the desired sense or antisense RNA only 30 in the tissue of interest. With DNA constructs employing inducible gene promoter sequences, the rate of RNA polymerase binding and initiation can be modulated by external stimuli, such as light, heat, anaerobic stress, alteration in nutrient conditions 10 and the like. Temporally regulated promoters can be employed to effect modulation of the rate of RNA polymerase binding and initiation at a specific time during development of a transformed cell. Preferably. the original promoters from the enzyme gene in question, or promoters from a specific tissue-targeted gene in the organism to 5 be transformed, such as eucalyptus or pine are used. Other examples of gene promoters which may be usefully employed in the present invention include, mannopine synthase (mas), octopine synthase (ocs) and those reviewed by Chua et al. (Science, 244:174 181. 1989). The gene termination sequence, which is located 3' to the DNA sequence to be 10 transcribed. may come from the same gene as the gene promoter sequence or may be from a different gene. Many gene termination sequences known in the art may be usefully employed in the present invention, such as the 3' end of the Agrobacterium tumefaciens nopaline synthase gene. However, preferred gene terminator sequences are those from the original enzyme gene or from the target species to be transformed. 15 The DNA constructs of the present invention may also contain a selection marker that is effective in plant cells, to allow for the detection of transformed cells containing the inventive construct. Such markers, which are well known in the art, typically confer resistance to one or more toxins. One example of such a marker is the NPTII gene whose expression results in resistance to kanamycin or hygromycin, 20 antibiotics which is usually toxic to plant cells at a moderate concentration (Rogers et al. in Methods for Plant Molecular Biology, A. Weissbach and H. Weissbach. eds., Academic Press Inc., San Diego, CA (1988)). Alternatively, the presence of the desired construct in transformed cells can be determined by means of other techniques well known in the art, such as Southern and Western blots. 25 Techniques for operatively linking the components of the inventive DNA constructs are well known in the art and include the use of synthetic linkers containing one or more restriction endonuclease sites as described, for example, by Maniatis et al., (Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratories, Cold Spring Harbor, NY, 1989). The DNA construct of the present invention may be linked 30 to a vector having at least one replication system, for example, E. coli, whereby after each manipulation, the resulting construct can be cloned and sequenced and the correctness of the manipulation determined. 11 The DNA constructs of the present invention may be used to transform a variety of plants, both monocotyledonous (e.g. grasses. com, grains, oat, wheat and barley), dicotyledonous (e.g. Arabidopsis, tobacco, legumes, alfalfa, oaks, eucalyptus, maple), and Gyrnnosperms (e.g. Scots pine (Aronen, Finnish Forest Res. Papers, vol. 595, 5 1996), white spruce (Ellis et al., Biotechnology 11:94-92, 1993), larch (Huang et al., In Vitro Cell 27:201-207, 1991). In a preferred embodiment, the inventive DNA constructs are employed to transform woody plants, herein defined as a tree or shrub whose stem lives for a number of years and increases in diameter each year by the addition of woody tissue. Preferably the target plant is selected from the group to consisting of eucalyptus and pine species, most preferably from the group consisting of Eucalyptus grandis and Pinus radiara. As discussed above, transformation of a plant with a DNA construct including an open reading frame coding for an enzyme encoded by an inventive DNA sequence wherein the open reading frame is orientated in a sense direction will lead to an increase in lignin content of the plant or, in some cases, toa Is decrease by cosuppression. Transformation of a plant with a DNA construct comprising an open reading frame in an antisense orientation or a non-coding (untranslated) region of a gene will lead to a decrease in the lignin content of the transformed plant. Techniques for stably incorporating DNA constructs into the genome of target 20 plants are well known in the art and include Agrobacterium tumefaciens mediated introduction, electroporation, protoplast fusion, injection into reproductive organs, injection into immature embryos, high velocity projectile introduction and the like. The choice of technique will depend upon the target plant to be transformed. For example, dicotyledonous plants and certain monocots and gymnosperms may be transformed by 25 Agrobacterium Ti plasmid technology, as described, for example by Bevan (NucI. Acid Res. 12:8711-8721, 1984). Targets for the introduction of the DNA constructs of the present invention include tissues, such as leaf tissue, disseminated cells, protoplasts, seeds, embryos, meristematic regions; cotyledons, hypocotyls, and the like. One preferred method for transforming eucalyptus and pine is a -biolistic method using 30 pollen (see, for example, Aronen 1996, Finnish Forest Res. Papers vol. 595, 53pp) or easily regenerable embryonic tissues. Other transformation techniques which may be usefully employed in the inventive methods include those taught by Ellis et al. (Plant 12 Cell Reports. 8:16-20, 1989), Wilson et al-(Plant Cell Reports 7:704-707, 1989) and Tautorus et al. (Theor. Apl. Genet. 8:531-536, 1.9-89). Once the cells are transformed, cells having the inventive DNA construct incorporated in their genome may be selected by means of a marker, such as the 5 kanamycin resistance marker discussed above. Transgenic cells may then be cultured in an appropriate medium to regenerate whole plants, using techniques well known in the art. In the case of protoplasts, the cell wall is allowed to reform under appropriate osmotic conditions. In the case of seeds or embryos, an appropriate germination or callus initiation medium is employed. For explants. an appropriate regeneration i0 medium is used. Regeneration of plants is well established for. many species. For a review of regeneration of forest trees see Dunstan et al., Somatic embryogenesis in woody plants. In: Thorpe, T.A. ed., 1995: in vitro embryogenesis of plants. Vol. 20 in Current Plant Science and Biotechnology in Agriculture, Chapter 12, pp. 471-540. Specific protocols for the regeneration of spruce are discussed by Roberts et al., 1S (Somatic Embryogenesis of Spruce. In: Synseed. Applications ofsynrhetic seed to crop improvement. Redenbaugh, K, ed. CRC Press, Chapter 23, pp. 427-449, 1993). The resulting transformed plants may be reproduced sexually or asexually, using methods well known in the art, to give successive generations of transgenic plants. As discussed above, the production of RNA in target plant cells can be 20 controlled by choice of the promoter sequence, or by selecting the number of functional copies or the site of integration of the DNA sequences incorporated into the genome of the target plant host. A target plant may be transformed with more than one DNA construct of the present invention, thereby modulating the lignin biosynthetic pathway for the activity of more than one enzyme, affecting enzyme activity in more than one 25 tissue or affecting enzyme activity at more than one expression time. Similarly, a DNA construct may be assembled containing more than one open reading frame coding for an enzyme encoded by a DNA sequence of the present invention or more than one non coding region of a gene coding for such an enzyme. The DNA sequences of the present inventive may also be employed in combination with other known sequences encoding 30 enzymes involved in the lignin biosynthetic pathway. In this manner, it may be possible to add a lignin biosynthetic pathway to a non-woody plant to produce a new woody plant. 13 The isolated DNA sequences of the present invention may also be employed as probes to isolate DNA sequences encoding enzymes involved in the lignin synthetic pathway from other plant species, using techniques well known to those of skill in the art. 5 The following examples are offered by way of illustration and not by way of limitation. Example I Isolation and Characterization of cDNA Clones from Eucalvorus grandis 10 Two Eucalyprus grandis cDNA expression libraries (one from a mixture of various tissues from a single tree and one from leaves of a single tree) were constructed and screened as follows. mRNA was extracted from the plant tissue using the protocol of Chang et-a!. (Plant Molecular Biology Reporter fl:1 13-116 (1993)) with mixior modifications. is Specifically, samples were dissolved in CPC-RNAXB (100 rmM Tris-Cl, pH 8,0; 25 mM EDTA; 2.0 M NaCl; 2%CTAB; 2% PVP and 0.05% Spermidine*3 HCI)and extracted with Chloroform:isoamyl alcohol, 24:1. mRNA was precipitated with ethanol and the total RNA preparate was purified using a Poly(A) Quik mRNA Isolation Kit (Stratagene, La Jolla, CA). A cDNA expression library was constructed from the 20 purified mRNA by reverse transcriptase synthesis followed by insertion of the resulting cDNA clones in Lambda ZAP using a ZAP Express cDNA Synthesis Kit (Stratagene), according to the manufacturer's protocol. The resulting cDNAs were packaged using a Gigapack II Packaging Extract (Stratagene) employing 1 pl of sample DNA from the 5 p1 ligation mix. Mass excision of the library was done using XLI-Bluc MRF' cells and 25 XLOLR cells (Stratagene) with ExAssist helper phage (Stratagene). The excised phagernids were diluted with NZY broth (Gibco BRL, Gaithersburg, MD) and plated out onto LB-kanamycin agar plates containing X-gal and isopropylthio-beta-galactoside (IPTG). Of the colonies plated and picked for DNA miniprep, 99% contained an insert 30 suitable for sequencing. Positive colonies were cultured in NZY broth with kanamycin and cDNA was purified by means of alkaline lysis and polyethylene glycol (PEG) precipitation. Agarose gel at 1% was used to screen sequencing templates for 14 chromosomal contamination. Dye primer -sequences were prepared using a Turbo Catalyst 800 machine (Perkin Elmer/Applied Biosystems Foster City, CA) according to the manufacturer's protocol. DNA sequence for positive clones was obtained using an Applied Biosystems s Prism 377 sequencer. cDNA clones were sequenced first from both the 5' end and, in some cases, also from the 3' end. For some clones, internal sequence was obtained using subcloned fragments. Subcloning was performed using standard procedures of restriction mapping and subcloning to pBluescript II SK+ vector. The determined cDNA sequence was compared to known sequences in the 10 EMBL database (release 46, March 1996) using the FASTA algorithm of February 1996 (version 2.0u4) (available on the Internet at the ftp site ftp://ftp.virginia.edu/pub/fasta/). Multiple alignments of redundant sequences were used to build up reliable consensus sequences. Based on similarity to known sequences from other plant species, the isolated DNA sequence (SEQ ID NO: 1) was identified as 15 encoding a CAD enzyme. In further studies, using the procedure described above, cDNA sequences encoding the following Eucalyptus grandis enzymes were isolated: PAL (SEQ ID NO: 16); C4H (SEQ ID NO: 17); C3H (SEQ ID NO: 18); F5H (SEQ ID NO: 19-21); OMT (SEQ ID NO: 22-25); CCR (SEQ ID NO: 26-29); CAD (SEQ ID NO: 30); CGT (SEQ 20 ID NO: 31-33); CBG (SEQ ID NO: 34); PNL (SEQ ID NO: 35, 36); LAC (SEQ ID NO: 37-41); and POX (SEQ ID NO: 42-44). Exa-mple 2 Isolatiot and Characterization of cDNA Clones from Pinus radiata 25 a) Isolation ofeDNA clones by hih thuhut screening A Pinus radiata cDNA expression library was constructed from xylem and screened as described above in Example 1. DNA sequence for positive clones was obtained using forward and reverse primers on an Applied Biosystems Prism 377 30 sequencer and the determined sequences were compared to known sequences in the database as described above. 15 Based on similarity to known sequences from other plant species, the isolated DNA sequences were identified as encoding the enzymes C4H (SEQ ID NO: 2 and 3), CMH (SEQ ID NO: 4), PNL (SEQ ID NO: 5), OMT (SEQ ID NO: 6), CAD (SEQ ID NO: 7), CCR (SEQ ID NO: 8), PAL (SEQ ID NO: 9-11) and 4CL (SEQ ID NO: 12). 5 In further studies, using the procedure described above, additional cDNA clones encoding the following Pinus radiata enzymes were isolated: PAL (SEQ ID NO: 45 47); C4H (SEQ ID NO: 48, 49); C3H1 (SEQ ID NO: 50-52); OMT (SEQ ID NO: 53 55); 4CL (SEQ ID NO: 56, 57); CCR (SEQ ID NO: 5 8-70); CAD (SEQ ID NO: 71); CGT (SEQ ID NO: 72); CBG (SEQ ID NO: 73-80); PNL (SEQ ID NO: 81); LAC i (SEQ ID NO: 82-84); and POX (SEQ ID NO: 85-88). b) Isolation of cDNA clones by PCR Two PCR probes, hereinafter referred to as LNBO10 and LNBO I1 (SEQ ID NO: 14 and 15, respectively) were designed based on conserved domains in the-following i5 peroxidase sequences previously identified in 'other species: vanpox, hvupox6, taepox, hvupox1, osapox, ntopox2, ntopoxl, lespox, pokpox, luspox, athpox, hrpox, spopox, and tvepox (Genbank accession nos. D11337, M93671, X56011, X58396, X66125, J02979, D11396, X71593, Dl l102, L07554, M58381, X57564, Z22920, and Z31011, respectively). 20 RNA was isolated from pine xylem and first strand cDNA was synthesized as described above. This cDNA was subjected to PCR using 4 iM LNB010, 4 pM LNB011, 1 x Kogen's buffer, 0.1 mg/ml BSA, 200 mM dNTP, 2 mM Mg, and 0.1 U/pl of Taq polymerase (Gibco BRL). Conditions were 2 cycles of 2 min at 94 *C, 1 min at 55 *C and I min at 72 "C; 25 cycles of I ain at 94 *C, 1 min at 55 "C, and 1 ruin 25 at 72 *C; and 18 cycles of l min at 94 "C, I min at 55 *C, and 3 min at 72 "C in a Stratagene Robocycler. The gene was re-amplified in the same manner. A band of about 200 bp was purified from a TAE agarose gel using a Schleicher & Schuell Elu Quik DNA purification kit and clones into a T-tailed pBluescript vector (Marchuk D. et al., Nucleic Acids Res 19:1154, 1991). Based on similarity to known sequences, the 30 isolated gene (SEQ ID NO: 13) was identified as encoding pine peroxidase (POX). 16 Example 3 Use of2an O-meth vJtranfera e (OMT) Gene to Modify Lienr B o ythe s 5 a) Transformation of tobacco plants with a Pinus radita OMT gene Sense and anti-sense constructs containing a sequence including the codin region of OMT (SEQ ID NO: 53) from Pinus radiate were inserted into Agrobacterium tfZmefaciens LBA4301 (provided as a gift by Dr. C. Kado, University of California, Davis, CA) by direct transformation using published methods (see, An G, Ebert PR 10 Mitra A, Ha SB: Binary Vectors. In: Gelvin SB, Schilperoort RA (eds) Plant Molecular Biology Manual, Kluwer Academic Publishers, Dordrecht (1988)). The presence and integrity of the transgenic constructs-were verified by restriction digestion and DNA sequencing. Tobacco (Nicotlana eabacum cv. Samsun) leaf sections were transformed using 15 the method of Horsch et al. (Science, 227:1229-1231, 1985). ~ Five independent transformed plant lines were established for the sense construct and eight independent transforkned plant lines were established for the anti-sense c o nstruct for OMT. Transformed plants containing the appropriate lignin gene construct were verified using Southern blot experiments. A "+" in the column labeled "SoutheM" 'in Table. Lbfow 20 indicates that the transformed plant lines were confiumed as independent transformed lines. b) Expression of Pinus OMT in transformed plants Total RNA was isolated from each independent transformed plant line created 25 with the OMT sense and anti-sense constructs. The RNA samples were analysed in Northem blot experiments to determine the level of expression of the transgene in each transformed line. The data shown in the column labeled "Northern" in Table I shows that the transformed plant lines containing the sense and anti-sense constructs for OMT all exhibited high levels of expression, relative to the background on the Northern blots. 30 OMT expression in sense plant line number 2 was not measured because the RNA sample showed signs of degradation. There was no detectable hybridisation to RNA samples from empty vector-transformed control plants. 17 c) Modulation of OMTjenzyme activity in transformed plants The total activity of OMT enzyme, encoded by the Pinur OMT gene and by the endogenous tobacco OMT gene, in transformed tobacco plants was analysed for each transformed plant line created with the OMT sense and anti-sense constructs. Crude 5 protein extracts were prepared from each transformed plant and assayed using the method of Zhang et al. (Plant Physiol., _13:65-74, 1997). The data contained in the column labeled "Enzyme" in Table I shows that the transformed plant lines conaining the OMTr sense construct generally had elevated OMT enzyme activity, with a maximum of 199%, whereas the transformed plant lines containing the OMT anti-sense 10 construct generally had reduced OMT .enzyme activity, with a minimum of 35%, relative to empty vector-transformed control .plants. OMT enzyme activity was not estimated in sense plant line number 3. d) Effects ofPinr OMT ondlinin concentration in transformed plants 15 The concentration of lignin in the transformed tobacco plants was determined using the well-established procedure of thioglycolic acid extraction (see, Freudenberg et al. in "Constitution and- Biosynthesis of Lignin", Springer-Verlag, Berlin, 1968). Briefly, whole tobacco plants, of an average age of 38 days, were frozen in liquid nitrogen and ground to a fine powder in a mortar and pestle. 100 mg of frozen.powder 20 from one empty vector-transformed control plant line, the five independent transformed plant lines containing the sense construct for OMT and the eight independent transformed plant lines containing the anti-sense construct for OMT were extracted individually with methanol, followed by 10% thioglycolic.acid and finally dissolved in 1 M NaOH. The final extracts were assayed for absorhance at 280 nm. The data shown 25 in the column labelled "TGA" in Table I shows that the transformed plant lines containing the sense and the anti-sense OMT gene constructs all exhibited significantly decreased levels of lignin, relative to the empty vector-transformed control plant lines. 18 Table I 5 olant line traspene orientation Southern Northern Enzyme I control na blank 100 104 1 OMT sense + 2.9E+6 86 2 OMT sense + Sn 162 58 3 OMT sense. + 4.1E+6 na 63 -o 4 OMT sense + 2.3E+6 142 66 5 OMT sense + 3.6E+5 199 I OMT anti-sense + 1.6E+4 189 66 2 OMT anti-sense + 5.7E+3 35 70 3 OMT anti-sense + 8.OE+3 105 73 IS5 4 OMT anti-sense + 1.4E+4 109 74 5 OMT anti-sense + 2.5E+4 87 78 6 OlMT anti-sense + 2.5E+4 58 84 7 OMT anti-sense + 2.5E+4 97 92 20 OMT anti-sense + 1.LE+4 151 94 These data clearly indicate that lignin concentration, as measured by the TGA assay, can be directly manipulated by either sense or anti-sense expression of a lignin biosynthetic gene such as OMT. 25 Example 4 :CoA ligase (4CL) Gene to Modify Linin Biosynthesis a)--Tra ornatio.n of tobacco plants with a Pinus radiata 4CL ene 30 Sense and anti-sense constructs containing a sequence including the coding region of 4CL (SEQ ID NO: 56) from Pins radiata were inserted into Agrobacterium umefaciens LBA4301 by direct transformation as described above. The presence and integrity of the transgenic constructs were verified by restriction digestion and DNA sequencing. 35 Tobacco (Nicotiana tabacum cv. Samsun) leaf sections were transformed as described above. Five independent transformed plant lines were established for the sense construct and eight independent transformed plant lines were established for the anti-sense construct for 4CL Transformed plants containing the appropriate lignin gene construct were verified using Southern blot experiments. A "-" in the column 19 labeled "Southern" in Table 2 indicates that the transformed plant lines listed were confirmed as independent transformed lines. b) Expression of Pinus 4CL in transformed plants 5 Total RNA was isolated from each independent transformed plant line created with the 4CL sense and anti-sense constructs. The RNA samples were analysed in Northern blot experiments to determine the level of expression of the transgene in each transformed line. The data shown in the column labelled "Northern" in Table 2 below shows that the transformed plant lines containing the sense and anti-sense constructs for 10 4CL all exhibit high levels of expression, relative to the background on the Northern blots. 4CL expression in anti-sense plant line number 1- was not measured because the RNA was not available at the time of the experiment. There was no detectable hybridisation to RNA samples from empty vector-transformed control plants. 15 c) Modulation of 4CL enzyme activity in transformed plants The total activity of 4CL enzyme, encoded by the Pinus 4CL gene and by the endogenous tobacco 4CL gene, in transformed tobacco plants was analysed for each transfonned plant line created with the 4CL sense and anti-sense constructs. Crude protein extracts were prepared from each transformed plant and assayed using the 20 method of Zhang et al. (ant Physiol., .1:65-74, 1997). The data contained in the column labeled "Enzyme" in Table 2 shows that the transformed plant lines containing the 4CL sense construct had elevated 4CL enzyme activity, with a maximum of 258%, and the transformed plant lines containing the 4CL anti-sense construct had reduced 4CL enzyme activity, with a minimum of 59%, relative to empty vector-transformed 25 control plants. Loinin concentration in transformed plants The concentration of lignin in samples of transformed plant material was determined as described in Example 3. The data shown in the column labelled "TGA" 30 in Table 2 shows that the transformed plant lines containing the sense and the anti sense 4CL gene constructs all exhibited significantly decreased levels of lignin, relative to the empty vector-transformed control plant lines. These data clearly indicate that 20 lignin concentration, as measured by the TGA assay, can be directly manipulated by either sense or anti-sense expression of a lignin biosynthetic gene such as 4CL. 5 Table 2 Plant line trans Northern zyme TGA I control na + blank 100 92 1o 2 control na + blank 100 104 2 4CL sense + 2.3E+4 169 64 2 4CL sense + 4.5E+4 258 73 3 4CL sense + 11E+4 174 77 4 4CL sense + L.7E+4 164 15 5 4CL sense + 1.6E+4 184 92 4C.L anti-sense + na 19 75 2 4L.a 59 75 4CL anti-sense + l.0E+4 70 75 3 4CL anti-sense + 9.6E+3 81 80 4 4CL anti-sense + 1.2E+4 90 83 2 5 4CL anti-sense + 4.7E+3 101 8g 6 4CL anti-sense + 3.9E+3 116 89 7 4CL anti-sense + 1.8E+3 125 94 8 4CL anti-sense + 1.7E+4 106 97 25 Example 5 TransformatinLinin Biosynthetic Genes 30 Sense and anti-sense Constructs :containing sequences including the coding regions of C3H (SEQ ID NO: 18), F5H (SEQ ID NO: 19), CCR (SEQ ID NO: 25) and CGT (SEQ ID NO: 31) from Eucalyptus grandis, and PAL (SEQ ID NO: 45 and 47), C4H (SEQ ID NO: 48 and 49), PNL (SEQ ID NO: 81) and LAC (SEQ ID NO: 83) 35 from Pinus radiata were inserted into Agrobacterium tumefaciens LBA43oI by direct transformation as described above. The presence and integrity of the transgenic constructs were verified by restriction digestion and DNA sequencing. Tobacco (Nicotiana tabacum cv. Samsun) leaf sections were transformed as described in Example 3. Up to twelve independent transformed plant lines were 40 established for each sense construct and each anti-sense construct listed in the preceding paragraph. Transformed plants containing the appropriate lignin gene 21 construct were verified using Southern blot experiments. All of the transformed plant lines analysed were confirmed as independent transformed lines. xample_6 5 Manipulation of Linin.Content in Transformed a. Determination of transgene expression bv Northern blot ex Total RNA was isolated from each independent transformed plant line described in 10 Example 5. The RNA samples were analysed in Northern blot experiments to determine the level of expression of the transgene in each transformed line. The column labelled "Northern" in Table 3 shows the level of transgene expression for all plant lines assayed, relative to the background on the Northern blots. There was no detectable hybridisation to RNA samples from empty vector-transformed control 15 plants. b) Determination of lienin concentration in transformed plants The concentration of lignin in empty vector-transformed control plant lines and in up to twelve independent transformed lines for each sense construct and each anti-sense 20 construct described in Example 5 was determined as described in Example 3. The column labelled "TGA" in Table 3 shows the thioglycolic acid retractable lignins for all plant lines assayed, expressed as the average percentage of TGA retractable lignins in transformed plants versus control plants. The range of variation is shown in parentheses. 22 Table 3 5 trans|ige.... orientation no. of lines Northern TGA control na 3 blank 100 (92-104) C3H sense 5 3.7E+4 74 (67-85) F5H sense 9 5.8E+4 70 (63-79) PiN anti-sense 9 5.8E+4 73 (35-93) 10 CCR sense 2 na 74 Santi-sense 2 na 74 (62-86) PAL sense 5 l.9E+5 77 (71-86) PAL anti-sense 4 i.5E+4 62 (37-77) C4H anti-sense 10 .8E+4 86 (52-113) Is PNL anti-sense 6 1.2E+4 88 (70-114) LAC Sense 2 17E+5 na LAG anti-sense 12 1.7Ei5 88 (73-114) Transformed plant lines containing the sense and the anti-sense lignin 20 biosynthetic gene constructs all exhibited significantly decreased levels of Iignin, relative to the empty vector-transformed control plant lines. The most dramatic effects on lignin concentration were seen in the F5H anti-sense plants with as little as 35% of the amount of lignin in control plants, and in the PAL anti-sense plants with as little as 37% of the amount of lignin in control plants. These data clearly indicate that lignin 25 concentration, as measured by the TGA assay, can be directly manipulated by conventional anti-sense methodology and also by sense over-expression using the inventive lignin biosynthetic genes. Example 7 30Di7 Modulation o LigninEnzyme Activity in Transformed Plants The activities and substrate specificities of selected lignin biosynthetic enzymes were assayed in crude extracts from transformed tobacco plants containing sense and 35 anti-sense constructs for PAL (SEQ ID NO: 45), PNL (SEQ ID NO: 81) and LAC (SEQ ID NO: 83) from Pinus radiata, and CGT (SEQ ID NO: 31) from Eucalyptus grandis. Enzyme assays were performed using published methods for PAL (Southerton, S.G. and Deverall, BJ., Plant Path. 3_:223-230, 1990), CGT (Vellekoop, P. et al., 40 FEBS, 3_0:36-40, 1993), PNL (Espin, C.J. et al., Phytochemistry, 44:17-22, 1997) and 23 LAC (Bao, W. et a, ~j~ncIL 260:672-674, 1993). The data shown in the labelled "Enzyme" in Table 4 shows the average enzyme activity from replicate measures for all plant lines assayed, expressed as a percent of enzyme activity in empty vecxrn-5sfoed control plants. The range of variation is shown in parentheses. 5 Table 4 transeene ono. of eines Enzyme 10 control na 3 100 PAL sense 5 87 (60-124) PAL anti-sense 3 53 (38-80) CGT anti-sense 89 PNL ant-sense 6 144 (41-279) 15 LAC sense 78 (16-240) LAO anti-sense 11 64 (14-106) All of the transformed plant lines, except the PNL anti-sense transformed plant 20 lines, showed average lignin enzyme activities which were ignifcatly lower than the activities observed in empty vector-tnJIsformed control plants. The most dramaic effects on lignin enzyme activities ere seen in the PAL anti-sense transformed plan: lines in which all of the lines showed reduced PAL activity and in the LAO anti-sense transfonned plant lines which showed as little as 14% of the LAC activity in empty 25 vector-transformed control plant lines. Example 8 30 Fo ninBiosynthetic Genes Sense constnucts containing sequences including the coding regions for PAL (SEQ ID NO: 47), OMT (SEQ ID NO: 53), 4CL (SEQ ID NO: 56 and 57) and POX (SEQ ID NO: 86) from Pima radia a, and OMT (SEQ ID NO: 23 and 24) 4 CR (SEQ 35 ID NO: 26-28), COT (SEQ ID NO: 31 and 33) and POX (SEQ ID No: 42 and 4) from Eucalyptus grandis were inserted into the commercially available protein expression vector, pProEX a (Oibco BRL). The resultant constructs were transfonned into E. coli XL I -Blue (Strtagene), which were then induced to produce recombinant protein by the addition of IPT0 Purified proteins were produced for the Pinus OMT and 4CL 40 constructs and the Eucalyprus OMT and POX constructs using Ni column 24 chromatography (Janknecht, R. et al., Proc. Nat. Acad Sci., 88:8972-8976 1991) Enzyme assays for each of the purified proteins conclusively demonstrated the expected substrate specificity and enzymatic activity for the genes tested. The .data for two representative enzyme assay experiments, demonstrating the s verification of the enzymatic activity of a Pinus radiara 4CL gene.(SEQ ID NO: 56) and a Pinus radiata OMT gene (SEQ ID NO: 53), are shown in Table 5. For the 4CL enzyme, one unit equals the quantity of protein required to convert the substrate into product at the rate of 0.1 absorbance units per minute. For the OMT enzyme, one unit equals the quantity of protein required to convert I pole of substrate to product per 10 minute. Table 5 purification total mi total mug total units % yield fold transgene ste extract tein activity activity purification 4CL crude 10 il 51 mg 4200 100 Ni column 4 ml 0-84 mg 3680 88 53 OMT crude 10 mI 7 4 mg 4600 100 1 Ni column 4 m 1.2 mg 4487 98 60 25 The data shown in Table 5 indicate that both the purified 4CL enzyme and the purified OMT enzyme show high activity in enzyme assays, confirming the identification of the 4CL and OMT genes described in this'application. Crude protein preparations from E. coli transformed with empty vector show no activity in either the 4CL or the OMT enzyme assay. 30 Although the present invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, changes and modifications can be carried out without departing from the scope of the invention which is intended to be limited only by the scope of the appended claims. 25

Claims (21)

1. An isolated DNA sequence comprising a nucleotide sequence selected from the group consisting of: (a) coding sequence recited in SEQ ID NO: 25; (b) complements of the coding sequences recited in SEQ ID NO: 25; (c) reverse complements of the coding sequence recited in SEQ ID NO: 25; (d) reverse sequences of the coding sequence recited in SEQ ID NO: 25; and (e) sequences having at least about 80% identity, at least about 90% identity or at least about 99% identity to a coding sequence recited in SEQ ID NO: 25; and wherein said sequences of (e) encode an enzyme involved in the lignin biosynthetic pathway as encoded by SEQ ID NO: 25.

2. A DNA construct comprising a DNA sequence of claim 1.

3. A transgenic cell comprising a DNA construct of claim 2.

4. A DNA construct comprising, in the 5'-3' direction: (a) a gene promoter sequence, (b) an open reading frame coding for at least a functional portion of an enzyme, or a non-coding region of a gene coding for the enzyme, wherein the enzyme is encoded by a nucleotide sequence selected from the group consisting of: (1) coding sequence recited in SEQ ID NO: 25; and (2) sequences having at least about 80% identity, at least about 90% identity or at least about 99% identity to a coding sequence of SEQ ID NO: 25; wherein said 29/04/11 73 sequences of (2) encode an enzyme involved in the lignin biosynthetic pathway as encoded by SEQ ID NO: 25; and (c) a gene termination sequence.

5. The DNA construct of claim 4 wherein the open reading frame is in a sense orientation.

6. The DNA construct of claim 4 wherein the open reading frame is in an antisense orientation.

7. The DNA construct of claim 4 wherein the gene promoter sequence and gene termination sequence are functional in a plant host.

8. The DNA construct of claim 4 wherein the gene promoter sequence provides for transcription in xylem.

9. The DNA construct of claim 4 further comprising a marker for identification of transformed cells.

10. A transgenic plant cell comprising the DNA construct of any one of claims 2 and 4 to 9.

11. A plant comprising a transgenic plant cell of claim 10, or fruit or seeds thereof.

12. The plant of claim 11 wherein the plant is a woody plant. 29/04/11 74

13. The plant of claim 12 wherein the plant is selected from the group consisting of eucalyptus and pine species.

14. A method for modulating the lignin content of a plant comprising stably incorporating into the genome of the plant a DNA construct of any one of claims 2 and 4 to 9.

15. A method for producing a plant having altered lignin structure comprising: (a) transforming a plant cell with a DNA construct of any one of claims 2 and 4 to 9; and (b) cultivating the transgenic cell under conditions conducive to regeneration and mature plant growth.

16. A method of modifying the activity of an enzyme in a plant comprising stably incorporating into the genome of the plant a DNA construct of any one of claims 2 and 4 to 9.

17. A method for producing a transgenic plant cell having altered lignin structure comprising: (a) transforming a plant cell with a DNA construct of any one of claims 2 and 4 to 9; and (b) cultivating the transgenic cell.

18. A wood obtained from a transgenic tree which has been transformed with the DNA construct of any one of claims 2 and 4 to 9. 29/04111 75

19. A wood pulp obtained from a transgenic tree which has been transformed with the DNA construct of any one of claims 2 and 4 to 9.

20. A method of making wood, comprising: transforming a plant with the DNA construct of any one of claims 2 and 4 to 9; culturing the transformed plant under conditions that promote growth of a plant; and obtaining wood from the plant.

21. A method of making wood pulp, comprising: transforming a plant with the DNA construct of any one of claims 2 and 4 to 9; culturing the transformed plant under conditions that promote growth of a plant; and obtaining wood pulp from the plant. Dated this 27th June 2011 Rubicon Forests Holdings Limited and Arborgen, LLC Patent Attorneys for the Applicants PETER MAXWELL AND ASSOCIATES 29/04/11