AU2008203173A1 - Materials and methods for the modification of plant lignin content - Google Patents

Description

17.Jul. 20DE 14:2C BALDWINS 0064 4 4736712 Nio.3982 P 3 -1-

AUSTRALIA

Patents Act 1990

SPECIFICATION

Name of Applicant: Rubicon Forests Holdings Limited and ArborGen,

LLC

Actual Inventors: Leonard Nathan Bloksberg, Ilkka Jaakko Havukkala Address for Service: Baldwins Intellectual Property 16 Chisholm Street North Ryde Sydney Invention Title: Materials and methods for the modification of plant lignin content The following statement is a full description of this invention, including the best method of performing it known to us:- 100B20172_1.DOCJC:kd COMS ID No: ARCS-198654 Received by IP Australia: Time 13:23 Date 2008-07-17 17.Jul. 2008 14:20 BALDWINS 0064 4 4736712 No.3982 P. 4 -la- 00

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0 MATERIALS AND METHODS FOR c' THE MODIFICATION OF PLANT LUGNN CONTENT The present application is a divisional application of Australian Application No.

10936/00, which is incorporated in its entirety herein by reference.

Cc Technical Field of the Invention 0 ]10 This invention relates to polynucleotides believed to be novel, including partial and 00 etended sequences as well as probes and primers, construts comprising the polynuleotides, biological materials (including plants, microorganisms and multicellular organisms) incorporating the poynucleotides, polypeptides encoded by the polynucleotides, and methods for using the polynucleotides and polypeptide. The invention relates, more particularly, to the modification of lignin content and composition in biological materials including plants, to polypeptides involved in the lignin biosynthetic pathway, and to polynuoleotides encoding such enzymes.

Background of the Ivention Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field Lignin is an insoluble polymer that is primarily responsible for the rigidity of plant stems.

Specifically, lignin serves as a matrix around the polysacharide 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 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 oflignin in trees presents a significant problem in the paper industy were considerable resources must be employed to separate lignin from 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.

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.

(Followed by Page 2) COMS ID No: ARCS-198654 Received by IP Australia: Time 13:23 Date 2008-07-17 17.Ju 2008 14:20 BALDWINS 0064 4 4736712 No.3982 P. -2- O0 0 For example, crops with reduced lignin content provide more efficient forage for cattle, with Sthe 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 is accompanied by a corresponding decrease in digestibility. When deciding on the optimum time to harvest forage crops, farmers must therefore choose between a high yield of less digestible material and a lower yield of more digestible material For some applications, an increase in lignin content is desirable since increasing the n lignin content of a plant wonld lead to increased mechanical strength of wood, changes in its color and increased resistance to rot Mycorrhi2al species composition and abundance may S I 0 also be favorably manipulated by modifying lignin content and structural composition.

00 As discussed in detail below, lignin is formed by polymerization of at least three o different monolignols that are synthesized in a multistep pathway, each step in the pathway being catalyzed by a different enzyme. It has been shown that manipulation of the number of copies of genes encoding certain enzymes, such as cinnamyl alcohol dehydrogenase

(CAD)

and caffeic acid 3 -O-methyltransferase (COMT) result in modification of the amount oflignin 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 of sequences encoding CAD in poplar leads to the production of lignin having a modified composition (Grand C et al., Plana (Berl.) 163:232-237, 1985).

While polynucleotides encoding come 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 not yet been identifies. 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.

Summary of the Invention Unless the context clearly requires otherwise, throughout the description and the claims, the words 'comprise', 'comprising' and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say in the sense of "including but not limited to".

Briefly, the present disclosure includes isolated polynueleotides identified in the attached Sequence Listing as SEQ ID NOS: 1-266 and 350-375, variant of those sequences, constructs comprising such sequences, extended sequences comprising the sequences of SEQ ID NOS: 1-266 and 350-375, and their variants, probes and primers corresponding to the sequences set out in SEQ ID NOS: 1-266, 350-375 and their variants, and polynucleotides comprising at least a specified number of contiguous residues of any of the polynucleotides identified as SEQ ID NOS: 1-266 and 350-375 (x-mers) all of which are r red to herein COMS ID No: ARCS-198654 Received by IP Australia: Time 13:23 Date 2008-07-17 17.Jul. 2008 14:21 BALDWINS 0064 4 4736712 No.3982 P. 6 -3- 00 o collectively, as "poynucleotides of the present invention." Polynucleotides of the present Sinvention are preferably obtainable from eucalyptus and pine species, and preferably encode enzymes involved in the lignin biosynthetic pathway. Constructs incorporating such Ssequences, methods for using such sequences and constructs, and biological materials, including plant cells and plants having an altered genomic and/or lignin content and composition are disclosed. The present disclosure also includes isolated polypeptide sequences identified in the attached Sequence Listing as SEQ ID NOS: 267-349 and 376-401; polypeptide variants of those sequences; and polypeptides comprising the polypeptide en sequences and variants of those sequences.

S ~0 In one aspect, the present disclosure includes isolated polynucleotides encoding the O0 following enzymes, or portions of the following enzymes; cinnamate 4-hydroxylase (C4H), 0 coumarate 3-hydroxylase (C3H), phenolase (PNL), O-methyl transferase (OMT), cinnamyl alcohol dehydrogenase (CAD), cinnamoyl-CoA reductase (CCR), phenylalanine ammonialyase (PAL), 4-coumarate; CoA ligase (4CL), coniferol glucosyl transferase

(CGT),

coniferin beta-glucosidase (CBG), laccase (LAC), peroxidase (POX), hydroxylase (F5H), alpha amylase, caffeic acid methyl tansferase, caffeoyl CoA methyl transforase, coumerate 6A ligase, cytochrome P450 LXXLA, diphenol oxidase, flavonol glucosyl transferase, flavonoid hydroxylase, and isoflavone reductase.

In one embodiment, polynucleotides of the present disclosure encompass polynucleotides comprising a nucleotide sequence selected from the group consisting of polynucleotides recited in SEQ ID NOS: 1-266 and 350-375; complements of the polynucleotides recited in SEQ ID NOS: 1-266 and 350-375; reverse complements of the sequences recited in SEQ ID NOS: 1-266 and 350-375; reverse sequences of the sequences recited in SEQ ID NOS: 1-266 and 350-375; and variants of the polynucleotides recited in SEQ ID NOS: 1-266 and 350-375. In another embodiment of the present disclosure, polynucleotides comprise at least a specified number of contiguous residues (x-mers) of any of the polynucleotides of SEQ ID NOS: 1-266 and 350-375. In yet another aspect, polynucleotides comprise probes and primers corresponding to any of the polynucleotides of SEQ ID NOS: 1-266 and 350-375.

In another aspect, the present disclosure includes constructs comprising a polvnucleotide of the present invention, either alone or in combination with one or more of the inventive sequences, or in combination with one or more known polynucleotides; together with host cells and transgenic cells comprising such constructs.

In a related aspect, the present disclosure includes constructs 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 polynucleotide of the present invention; and a gene termination sequence. An open reading frame may be orientated in either a sense or antisense COMS ID No: ARCS-198654 Received by IP Australia: Time 13:23 Date 2008-07-17 17.Jul. 2008 14:21 BALDWINS 0064 4 4736712 No.3982 P. 7 -4-

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o direction. DNA constructs comprising a non-coding region of a gene coding for an enzyme o encoded by the above polynucleotides or a polynucleotide complementary to a non-coding region, together with a gene promoter sequence and a gene termination sequence, are also 1- provided. Preferably, the gene promoter and termination sequences are functional in a host cell, such as a plant cell. Most preferably, the gene promoter and termination sequences are those of the original enzyme genes but others generally used in the art, such as the CC Cauliflower Mosaic Virus (CMV) promoter, with or without enhancers, such as the Kozak sequence or Omega enhancer, and Agnobacterium tunzefacins nopalin synthase imminator ^n may be usefully employed in the present invention. Tissue-specific promoters may be O 10 employed in order to target expression to one or more desired tissues. In a preferred 00 embodiment, the gene promoter sequence provides for transcription in xylem. The construct 0 may further include a marker for the identification of transformed cells.

In a further aspect, transgenic cells, such as transgenic plant cells, comprising the constructs of the present invention are disclosed, together with plants comprising snob transgenic cells, and fruits and seeds of such plants.

In yet another aspect, methods for modulating the lignin content and composition of a target organism such as a plant are disclosed, such methods including stably incorporating into the genome of the target plant a construct comprising a polynucleotide of the present invention. In a preferred embodiment, the target plant is a woody plant, preferably selected from the group consisting of eucalyptus and pine species, most preferably from the group consisting of Eucalyptus grandis and Pirus radiana. In a related aspect, a method for producing a plant having altered ligin content is disclosed, the method comprising transforming a plant cell with a construct comprising a polynucleotide of the present invention to provide a transgenic cell, and cultivating the transgenic cell under conditions conducive to regeneration and mature plant growth.

In yet a funher aspect, the present disclosure includes methods for modifying the activity of an enzyme in a target organism such as a plant, comprising stably incorporating into the genome of the target organism a construct of the present invention. In a preferred embodiment, the target plant is a woody plant, preferably selected from the group consisting of eucalyptus and pine species, most preferably from the group consisting ofEucalptus grandis and Pinus radiata.

The present disclosure also includes polypeptides comprising the isolated polypeptides identified as SEQ ID NOS: 267-349,376-401 and variants of those polypeptides.

Further disclosed is a purified and isolated polynucleotide consisting essentially of a polynucleotide encoding Pinus radiata O-methyl transferase (OMT).

COMS ID No: ARCS-198654 Received by IP Australia: Time 13:23 Date 2008-07-17 17.Ju 2008 14:21 BALDWINS 0064 4 4736712 No.3982 P. 8 o Still further disclosed is a purified and isolated polynucleotide consisting 0 o essentially of a polynucleotide encoding Eucalyptus grandis 0-methyl transferase cxl

S(OMT).

Yet further disclosed is a wood obtained from a transgenic tree which has been r-.

transformed with the construct o'f the invention.

And yet further disclosed is a wood pulp obtained from a transgenic tree which CC) has been transformed with the construct of any one of the invention.

-Disclosed also is a method of making wood, comprising: transforming a plant en3 Swith the construct of the invention; culturing the transformed plant under conditions Cxl 00 10 that promote growth of a plant; and obtaining wood from the plant.

o Additionally encompassed is a method of making wood pulp, comprising: C N Iransforming a plant with the construct of the invention; culturing the transformed plant under conditions that promote growth of a plant; and obtaining wood pulp from the plant In one particular aspect, the invention comprises an isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of: sequences recited in SEQ ID NOs: 89, 91, 93-101, 104-123, 126-146, 148-152, 164-168, 172-192,201-264, and 350-375 or portions thereof; complements of the sequences recited in reverse complements of the sequences recited in reverse sequences of the sequences recited in nucleotide sequences producing an Expectation value of 0.01 or less when compared to a sequence recited in one of the sequences of(l); nucleotide sequences having at least 50 identity to a sequence recited in one of the sequences of(l); nucleotide sequences having at least 75% identity to a sequence recited in one of the sequences of(l); nucleotide sequences having at least 90 identity to a sequence recited in one of the sequences of nucleotide sequences that hybridize to a sequence recited in one of the sequences of the complements, reverse complements, and reverse sequences thereof under stringent hybridization conditions; (Followed by page )a) (Followed by page COMS ID No: ARCS-198654 Received by IP Australia: Time 13:23 Date 2008-07-17 17.Jul. 200 14:22 BALDWINS 0064 4 4736712 No.3982 P. 9 8 (10) nucleotide sequences that are 200-mers of a sequence recited in one of the sequences of the complements, reverse complements and reverse sequences thereof; (II) nucleotide sequences that are I00-mers of a sequence recited in one of the sequences of the complements, reverse complements and reverse sequences thereof; Ce (12) nucleotide sequences that are 40-mers of a sequence recited in one of the sequences of the complements, reverse complements and reverse sequences o thereof; 00 10 (13) nueleotide sequences that are degeneratively equivalent to a sequence recited in 0 Sone of the sequences of(I); and (14) nucleotide sequences that are allelic variants of a sequence recited in one of the sequences of wherein said sequences encode an enzyme involved in the lignin biosynthetic pathway.

In another aspect, the invention comprises an isolated oligonucleotide probe or primer comprising at least 20 contiguous residues complementary to 20 contiguous residues of a nuceotide sequence recited in one of SEQ. ID NOs: 89, 91, 93-101, 104- 123, 126-146, 148-152, 164-168,172-192,201-264, and 350-375.

Also included is a kit comprising a plurality of oligonuclotide probes or primers of a preceding aspect.

In yet another aspect, the invention comprises a construct comprising a polynucleotide of a preceding aspect In still another aspect, the invention comprises a construct comprising, in the direction: a gene promoter sequence; a polynucleotide sequence comprising at least one of the following: (1) a polynucleotide coding for at least a functional portion of a polypeptide encoded by a nucleotide sequence of a preceding aspect; and a polynucleotide comprising a non-coding region of a gene coding for a polypeptide encoded by a nucleotide sequence selected from the group consisting of sequences according to a preceding aspect; and a gene termination sequence.

The polynucleotide may be in a sense orientation.

(Followed by page COMS ID No: ARCS-198654 Received by IP Australia: Time 13:23 Date 2008-07-17 17-Jul. 2008 14:212 BALDWINS 0064 4 4736712 U -3982 P. 5b 0 The polynucleotide may be in an antisense orientation.

o The gene promoter sequence may be functional in a plant host to provide for transcription in xylem.

c' In an additional aspect, the invention comprises a transgenic plant cell comprising a construct of a preceding aspect.

In yet an additional aspect, the invention comprises a plant comprising a eM transgenic plant cell according to a preceding aspect, or a part or propagule or progeny thereof.

In still an additional aspect, the invention comprises a method for modulating 00 10 one or more of the lignin content, the lignin composition and the lignin structure of a O plant, comprising stably incorporating into the genome of the plant a polynucleotide of Sa preceding aspect.

The plant may be selected from the group consisting of eucalyptus and pine species.

The method may comprise stably incorporating into the genome of the plant a construct of a preceding aspect.

In a further aspect, the invention comprises a method for producing a plant having one or more of altered lignin content, altered lignin composition and altered lignin structure, comprising: transforming a plant cell with a construct of a preceding aspect to provide a transgenic cell; and cultivating the transgenic cell under conditions conducive to regeneration and mature plant growth.

In yet a further aspect, the invention comprises a method for modifying the activity of a polypeptide involved in a lignin biosynthetic pathway in a plant comprising stably incorporating into the genome of the plant a construct of a preceding aspect.

In still a further aspect, an isolated polypeptide comprising an amino acid sequence selected from the group consisting of: sequences of SEQ ID NOs: 267- 275, 284-347, and 376-401; sequences having at least 50% identity to a sequence recited in sequences having at least 75% identity to a sequence recited in and sequences having at least 90% identity to a sequence recited in (Followed by page COMS ID No: ARCS-198654 Received by IP Australia: Time 13:23 Date 2008-07-17 17.Jul. 2008 14:22 BALDWINS 0064 4 4736712 No.3982 P. 11 0 Also encompassed is an isolated polypeptide encoded by an isolated polynucleotide sequence of a preceding aspect.

S In another aspect, the invention comprises a wood obtained from a transgenic tree which has been transformed with the construct of any one ofthe preceding aspects.

In yet another aspect, the invention comprises a wood pulp obtained from a transgenic tree which has been transformed with the construct of any one of the Cpreceding aspects.

-In still another aspect, the invention comprises a method of making wood or en Swood pulp, comprising: transforming a plant with the construct of any one of the 00 10 preceding aspects; culturing the transformed plant under conditions that promote Sgrowth of a plant; and obtaining wood or wood pulp from the plant.

Additionally encompassed is a method for modifying the activity of a polypeptide involved in a lignin biosynthetic pathway in a plant, comprising introducing into cells of the plant antisense corresponding to a polynucleotide of a preceding aspect or portions thereof encoding a polypeptide having activity in a lignin biosynthetic pathway of a plant thereby inhibiting expression of a polypeptide encoded by the polynucleotide.

Brief Description of the Figures The above-mentioned and additional features of the present invention and the manner of obtaining them will become apparent, and the invention will be best understood by reference to the following more detailed description, read in conjunction with the accompanying drawing.

Figure I is a schematic overview of the lignin biosqlthetie pathway.

Figure 2 illustrates genomic DNA samples from tobacco plants created in a tagging experiment using a unique sequence identifier from Sinus (left panel) and a unique sequence identifier from Eucalyptus (right panel). In both panels, lanes A and B contain DNA samples from empty-vector transformed control plants and lanes C-E contain DNA samples from plants transformed with a unique sequence identifier.

(Followed by page COMS ID No: ARCS-198654 Received by IP Australia: Time 13:23 Date 2008-07-17 17 JAl. 200E 14:22 BALDWINS 0064 4 4736712 N o- 39 2 P 12 Figure 3 demonstrates detection ofa Pinus unique sequence identifier in transformed tobacco plants. Lanes A and B show the hybridization of a probe fiom SEQ ID NO: 402 to the genomic DNA of tobacco plants which lack the Pi7lus unique sequence identifier (emptyvector transformed control plants). Lanes C-E show the hybridization of the probe to the genomic DNA of tobacco plants containing one to three copies of the Pions unique sequence identifier.

Figure 4 demonstrates detection of a Eucalyp&t unique sequence identifier in transformed tobacco plants. Lanes A and B show the hybridization of a probe from SEQ ID NO: 403 to the genomic DNA of tobacco plants which lack the Eucalyptus unique sequence identifier (empty-vector transformed control plants). Lanes C-E show the hybridization of the probe to the genomic DNA of tobacco plants containing one to two copies of the Eucalyptus unique sequence identifier.

Figure 5 shows the amount of extractable lignin, as a percentage of wild type lignin content, present in tobacco plants transformed with sense and anti-sense genetic constructs of the present invention.

Detailed Description (Followed by page 6) COMS ID No: ARCS-198654 Received by IP Australia: Time 13:23 Date 2008-07-17 17.Jul. 2008 14:23 BALDWINS 0064 4 4736712 No.3982 P. 13 00

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-6- Lignin is formed by polymerization of at least three different mionolignols, primarily para-couriaryl alcohol, coniferyl alcohol and sinapyl alcohol. While these three types of lignin subunits are well Thown, it is possible that slightly diffhrent variants of these subunits may be involved in the lignin biosynthetic pathway in various plants. The relative S concentration of these residues in lignin varies among different plant species and within species. In addition, the composition of lignin may also vary among different tissues within a, C) specific plant. The three monolignols are derived from phenylalanine in a multistep process 0 and are believed to be polymerized into lignin by a f-re radical mechanism.

SFig. I shows different steps in the biosynthetic pathway for conifeyl alcohol together O to with the enzymes responsible for catalyzing each step. para-Cournaryl alcohol and sitiapyl alcohol are synthesized by similar pathways. Phenylalatnine is first deaminated by phenylalanine amnmonia-lyase (PAL) to give cinnamate which is then hydroxylated by cimnnamate 4-hydroxylase (C4) to form p-coumarate. p-Counarate is hydroxylated by counarate 3-bydroxylase to give caffeate. The newly added hydroxyl group is then methylated by O-methyl transferase (OMT) to give ferulate which is conjugated to coenzyme A by 4-coumarate:CoA ligase (4CL) to form feniruloyl-CoA. Reduction of feruloyl- CoA to coniferaldehyde is catalyzed by cinnamoyl-CoA 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 glucosyl transfrase (CGT). Following export, the de-glucosylated form of conifbryl alcohol is obtained by the action of coniferin beta-glucosidase (CBG).

Finally, polymerization of the three monolignois to provide ignin is catalyzed by phenolase (PNL), laccase (LAC) and peroxidase (POX).

The formation of sinapyl alcohol involves an additional enzyme, hydroxylase (F5H). For a more detailed review of the lignin biosynthetic pathway, see Whettou R and SederoffR, The Plant Cell, 7:1001-1013, 1995.

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 pathogns. In addition to lignin, several other classes of plant products are derived from phenylalanine. including flavonoids, coumarins, stilbenes and benzoic acid derivatives, wifnth the initial steps in the synthesis of all these compounds being the same. Thus modification ofthe action ofPAL, C4H, 4CL and other eniymes involved in COMS ID No: ARCS-198654 Received by IP Australia: Time 13:23 Date 2008-07-17 17.Ju 2008 14:23 BALDWINS 0064 4 4736712 No.3982 P. 14 -7- 00 o the lignin biosynthetic pathway may affect the synthesis of other plant products in addition to 0 C, lignin.

Z Using the methods and materials of the present invention, the lignin content of a plant may be modulated by modulating expression ofpolynucleotides of the present invention, or by modifying the polypeptides encoded by polynucleotides or the polynucleotides. The lignin content of a target organism, such as a plant, may be modified, for example, by incorporating additional copies of genes encoding enzymes involved in the lignin biosynthetic pathway into e the genome of the target plant. Similarly, a modified lignin content can be obtained by 0 transforming the target plant with antisense copies of such genes. In addition, the number of 00 10 copies of genes encoding for difference enzymes in the lignin biosynthetic pathway can be o manipulated to modify the relative amount of each monolignol synthesized, thereby leading to Cl the formation of lignin having altered composition. The alteration of lignin composition would be advantageous, for example, in applications of wood processing for paper, and may also be effective in altering the palatability of wood materials to rotting fungi.

The polynucleotide disclosed herein were derived from forestry plant sources, namely from Eucalyptus grandis and Pimes radiata. Some the nucleotides of the present invention are "partial" sequences, in that they do not represent a full length gene encoding a fulf length polypeptide. Such partial sequences may be extended by analyzing and sequencing various DNA libraries using primers and/or probes and well known hybridization and/or PCT techniques. Partial sequences may be extended until an open reading frame encoding a COMS ID No: ARCS-198654 Received by IP Australia: Time 13:23 Date 2008-07-17 17-Jul. 2008 14:23 BALDWINS 0064 4 4736712 No.3982 P. O 0 o -8 polypeptide, a full length polynucleotide and/or gene capable of expressing a polypeptide, or Sanother useful portion of the genome is identified. Such extended sequences, including full I> length polynucleotides and genes, are described as "corresponding to" a sequence identified as one of the sequences of SEQ ID NOS: 1-266. 350-375, or a variant thereof, or a portion of 4 one of the sequences of SEQ ID NOS: 1-266, 350-375, or a varant thereof, when the extended polynueleotide comprises an identified sequence or its variant, or an identified c ~contiguous portion (x-mer) of one of the sequences of SEQ ID NOS: 1-266, 350-375, or a O variant thereof Similarly, RNA sequences, reverse sequences, complementary sequences, 00 antisense sequences, and the like, corresponding to the polynocieotides of the present 0 0 10 invention, may be routinely ascertained and obtained using the cDNA sequences identified as SEQ ID NOS: 1-266 and 350-375.

The polynucleotides identified as SEQ ID NOS: 1-266 and 350-375 may contain open icading frames ("ORFs") or partial open reading frames encoding polypeptides.

Additionally, open reading frames encoding polypeptides may be identified in extended or full length sequences corresponding to the sequences set out as SEQ ID NOS: 1-266 and 350- 375. Open reading frames may be identified using techniques that are well known in the art.

These techniques include, for example, analysis for the location of known start and stop codons, most likely reading frame identification based on codon frequencies, etc. Suitable tools and software for ORFP analysis are available, for example, on the Internet at htto://wwwncbi.nlm_nih.gov/gororforhtml. Open reading frames and portions of open reading frames may be identified in the polynucleotides of the present invention. Once a partial open reading frame is identified, the polynucleotide may be extended in the area of the partial. open reading frame using techniques that are well known in the art until the polynucleotide for the full open reading fr-ame is identified. Thus, open reading frames encoding polypeptides may be identified using the polynucieotides of the present invention.

Once open reading frames are identified in the polynucleotides of the present invention, the open reading frames may be isolated and/or synthesized. Expressible genetic constructs comprising the open reading frames and suitable promoters, initiators, terminators, etc., which are well known in the art, may then be constructed. Such genetic constructs may be introduced into a host cell to express the polypeptide encoded by the open reading frame.

Suitable host cells may include various prokaryotic and eukaryotic cells, including plant cells, mammalian cells, bacterial cells, algae and the like.

COMS ID No: ARCS-198654 Received by IP Australia: Time 13:23 Date 2008-07-17 17.Jul. 2008 14:24 BALDWINS 0064 4 4736712 No.3982 P. 16 00

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O -9- Polypeptides encoded by the polynucleotides of the present invention may be exoressed and used in various assays to determine their biological activity, Such polypeptides may be used to raise antibodies, to isolate corresponding interacting proteins or other compounds, and to quantitatively determine leves of interacting proteins or other en, 5 compounds.

SThe present invention also contemplates methods for modulating the polynucleotide r and/or polypeptide content and composition of a forestry species, such methods involving Cl stably incorporating into the genome of the organism a genetic construct comprising one or 00 o more polynucleotides of the present invention. In one embodiment, the target organism is a q I10 forestry species, preferably a woody plant, more preferably a woody plant of the Pinus or Eucalyptus species, and most preferably Eucalyptus grandis or Pinus radiara. In a related aspect, a method for producing a forestry plant having an altered genotype or phenotype is provided, the method comprising transforming a plant cell with a genetic construct of the present invention to provide a transgenic cell, and cultivating the transgenic cell under conditions conducive to regeneration and mature plant growth. Forestry plants having an altered genotype or phenotype as a consequence of modulation of the level or content of a polynucleotide or polypeptide of the present invention compared to a wild-type organism, as well as components (seeds, etc.) of such forestry plants, and the progeny of such forestry plants, are contemplated by and encompassed within the present invention.

The isolated polynucleotides of the present invention also have utility in genome mapping, in physical mapping, and im positional cloning of genes. Additionally, the polynucleotide sequences identified as SEQ ID NOS: 1-266, 350-375, and their variants, may be used to design oligonucleotide probes and primers. OligonucIeotide probes and primers have sequences that are substantially complementary to the polynucleotide of interest over a certain portion of the polynucleotide. Oligonucleotide probes designed using the polynucleotides of the present invention may be used to detect the presence and examine the expression patterns of genes in any organism having sufficiently similar DNA and RNA sequences in their cells using techniques that are well known in the art, such as slot blot DNA hybridization techniques- Oligonucleotide primers designed using the polynucleotides of the present invention may be used for PCR amplifications. Oligonucleotide probes and primers designed using the polynucleotides of the present invention may also be used in connection with various microarray technologies, including the microarray technology used by Synteni (Palo Alto, California).

COMS ID No: ARCS-198654 Received by IP Australia: Time 13:23 Date 2008-07-17 17Juil. 2008 14:24 BALDWINS 0064 4 4736712 No.3982 P. 17 00

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o The polynuclcotides of the present invention rmay also be used to tag or identify an organism or reproductive material therefrom Such tagging may be accomplished, for example, by stably introducing a non-disruptive non-fincional heterologous polynucleotide identifier into an organism, the polynucleoride comprising one of the polvnucleotides of the present invention.

The polypeptides of the present invention and the polynucleotides encoding the M polypeptides have activity in linin biosynthetic pathways in plants- The polynucleotides.

were putatively identified by DNA and polypeptide similarity searches. The polynucleotides 00 and polypeptides of the present invention have demonstrated sinmiiarity to the following o o10 polypeptides that are known to be involved in lignin biosynthetic processes: COMS ID No: ARCS-198654 Received by IP Australia: Time 13:23 Date 2008-07-17 17-Jul. 200 14:24 BALDWINS 0064 4 4736712 ko3982 P. 1: 11 TABLE 1 POLYPEPTIE IENTITY POLYNXJCLEOTIDE POLYPEPTDE.

SEQ ID NO. .SEQ ID No.

Cinmate 4-hydroxylase (C4E) 2 3 ,17, 48, 49,92,_ 126, 127, 153-163 Gournarate 3-hydroxylase (CHI) 4,18,50-52,93,101, Phenolase PNL) 1 5, 35,3 81, 116, 183 0-methyl tnsferase (OMT) 6:22-25, 53-55, 94, 104407, 173-175 Cinnamyl alcohol dehydrogenasc 1, 7, 30, 71, (CAD) 112, 164 Cinnamoyl-CoA reductase (COR) 87 26-29, 58-70, 96, 108-111, 128-134,167 Phenylalanine ammonia-lyase (PAL) 1 9-11, 16, 45-47, 97, 98, 190,122, 123,176 242-248 325-331 4-coumaracl:CoA ligase (4CL) 2. 56-57, 90, 147, 158 265-266 348-349 Coniferol glucosyl ansferase (CGT) 31-33,72, 113-115, 135, 168 1 Coniferin beta-glucosidase (CEO) 34, 73-80, 1360141, 165, 166 Laccase (LAG) 37-41,'82-84, 117, 118, 142-144,172 Peroxidase (POX) F 13, 42-44, 85-89, 91, 119-121, 14 146, 332-333 177-182, 249-250: 347, 376-401 264, 350-375 (F53) 19-21, 102, 103, 169-171 Alpha avlase 1 24-186 267-269 Caffeic acid methyl transfaase 187-192 270-275 Caffeoyl CoA methyl transferase 193-195 276-278 Coumerate CoA ligase 196-200 279-283 CytachromeP450 LX2Aj 201-206 284-289 iphnol oxidase 207-217 290-300 251-263 3-34-346 Flavono glucosyl transferae 21 301 Flavnoid h d ]ase 219-3 302-316 i solavon reductase 234-241 I 317-324 COMS ID No: ARCS-198654 Received by IP Australia: Time 13:23 Date 2008-07-17 17.Jul. 2008 14:24 BALDWINS 0064 4 4736712 No.3982 P. 19 00 0O o 12 C,1 In one embodiment, isolated polynucleotides of the present invention comprise a Z sequence selected 5fom the group consisting of: sequences recited in SEQ ID NOS: 1-266 7- and 350-375; complements ofthe sequences recited in SEQ ID NOS; 1-266 and 350-375; reverse complements of the sequences recited in SEQ ID -NOS 1-266 and 350-375; reverse sequences of the sequences recited in SEQ ID NOS: 1-266 and 350-375; and S(e) sequences having at least 50%, 75%, 90%, or 98% identity, as defined herein, to a sequence of(a) or a specified region of a sequence of(a) SIn a further aspect, isolated polypeptides encoded by the polynucleotides of the 00 present invention are provided- In one embodiment, such polypepides comprise an amino o 10 acid sequence recited in SEQ ID NOS: 267-349 and 376-401, and variants thereof, as wel as po]ypeptides expressed by polynucleotides of the present invention, including polynucleotides comprising a sequence of SEQ ID NOS: 1-266 and 350-375.

In another aspect, the invention provides genetic constructs comprising a polynucleotide of the present invention, either alone, in combination with one or more additional polynucleotides of the present invention, or in combination with one or more known polynucleotides, together with cells and target organisms comprising such constructs.

In a related aspect, the present invention provides genetic constructs comprising, in the direction, a gene promoter sequence, an open reading frame coding for at least a functional portion of a polypeptide encoded by a polynucleotide of the present invention, and a gene termination sequence. The open reading frame may be oriented in either a sense or antisense direction. Genetic constructs comprising a gene promoter sequence, a polynucleotide of the present invention, and a gene termination sequence are also contemplated, as are genetic constructs comprising a gene promoter sequence, an untranslated region of a polynucleotide of the present invention, or a nucleotide sequence complementary to an untranslated region, and a gene termination sequence. The genetic construct may further include a marker for the identification of transformed cells The gene promoter and termination sequences are preferably functional in a host plant and, most preferably, are those native to the host plant. Promoter and termination sequences that are generally used in the art. such as the Cauliflower Mosaic Virus (CMV) promoter, with or without enhancers such as the Kozak sequence or Omega enhancer, and Ag-obaczerium tumfacieris nopaline synthase terminator, are useful. Tissue-specific promoters may be employed in order to target expression to one or more desired tissues.

COMS ID No: ARCS-198654 Received by IP Australia: Time 13:23 Date 2008-07-17 17.Ju 2008 14:25 BALDWINS 0064 4 4736712 No.3982 P. 00 0O O- 13 SIn a further aspect, methods for producing forestry plants having a modified content of a polynucleotide or polypeptide of the present invention compared to a native organism are provided The methods involve transforming a target forestry plant with a genetic construct of the present invention to provide a transgenic cell, and cultivating the ransgenic cell under conditions conducive to regeneration and mature plant growth. Cells comprising the genetic constructs of the present invention are provided, ogether with tissues and forestry plants comprising such transgenic cells, and fruits, seeds and other products, derivatives, or progeny Sof such forestry plants. Propagules of the inventive transgenic plants are included in the O present invention. As used herein, the word "propagule" means any part of a plant that may O 10 be used in reproduction or propagation, sexual or asexual, including cuttings.

Plant varieties, particularly registrable plant varieties according to Plant Breeders' Rights, may be excluded from the present invention. A plant need not be considered a "plant variety" simply because it contains stably within its genome a transgene, inroduced into a cell of the plant or an ancestor thereof.

The- word "polyhucleotide(s)," as used herein, meart a polymeric collection of nucleotides and includes DNA and corresponding RNA molecules and both single and double stranded molecules, including HnRNA and mRNA molecules, sense and anti-sense strands of DNA and RNA molecules, and eDNA, genomic DNA, and wholly or partially synthesized polynucleotides. An HnRNA molecule contains introns and "corresponds to" a DNA molecule in a generally one-to-one manner. An mRNA molecule "corresponds to" an EnRNA and DNA molecule from which the intTons have been excised. A polynuc]eotide of the present invention may be an entire gene, or any portion thereof. A gene is a DNA sequence which codes for a functional protein or RNA molecule. Operable anti-sense polynucleotides may comprise a fragmenr of the corresponding polynucleotide, and the definition of "polyncleotide" therefore includes all operable anti-sense fragments. Antisense polynucleotides and techniques involving anti-sense polynucleotides are well known in the art and are described, for example, in Robinson-Benion et al., "Antisense techniques," Methods in Enzymol. 254(23):363-375, 1995; and Kawasaki et aL, Artific. Organs 20(8):836-848, 1996.

Complements of such isolated polynucleotides, reverse complements of such isolated polynucleotides, and reverse sequences of such isolated polynucleotides, togelher with variants of such sequences. are also provided. The definition of the terms "complemen", "reverse complement" and "reverse sequence", as used herein, is best illustrated by the COMS ID No: ARCS-198654 Received by IP Australia: Time 13:23 Date 2008-07-17 17-Jul. 2008 14:25 BALDWINS 0064 4 4736712 No.3982 P. 21 O 00 14 C14 following example. "For the sequence 5' AGGACC the complement, reverse complement Z and reverse sequence are as follows: complement 3 TCCTGG reverse complement 3' GGTCCT Sreverse sequence 5' CCACGA 3'.

As used herein, the term "oligonucleotide" refers to a relatively short segment of a Spolynucleotide sequence, generally comprising between 6 and 60 nucleotides, and o comprehends both probes for use in hybridization- assays and primers for use in the 00 amplification of DNA by polymerase chain reaction.

Identification of genomic DNA and heterologous species DNAs can be accomplished by standard DNA/DNA hybridization techniques, under appropriately stringent conditions, using all or part of a cDNA sequence as a probe to screen an appropriate library.

Alternatively, PCR techniques using oligonucicotide primers that are designed based on known genomic DNA, cDNA and protein sequences can be used to amplify and identify genomic and cDNA sequences. Synthetic DNAs corresponding to the identified sequences and variants may be produced by conventional synthesis methods. Al of the polynucleotides described herein are isolated and purified, as those tecms are commonly used in the art.

In another aspect, the present invention provides isolated polypeptides encoded, or partially encoded, by the above polynucleotides. As used herein, the term "polypeptide" encompasses amino acid chains of any length, including full length proteins, wherein the amino acid residues are linked by covalent peptide bonds. The term "polypeptide encoded by a polynucleotide" as used herein, includes polypeptides encoded by a polynucleotide which comprises an isolated DNA sequence or variant provided herein. In specific embodiments, the inventive polypeptides comprise an amino acid sequence selected from the group consisting of sequences provided in SEQ ID NOS: 267-349 and 376-401, as well as variants of such sequences- Polypeptides of the present invention may be produced recombinantly by inserting a DNA sequence that encodes the polypeptide into an expression vector and expressing the polypeptide in an appropriate host. Any of a variety of expression vectors known to those of ordinary skill in the art may be employed. Expression may be achieved in any appropriate host cell that has been transformed or transfected with an expression vector containing a DNA molecule that encodes a recombinam polypeptide. Suitable host cells include prokaryores, yeast and higher eukaryoic cells. Preferably, the host cells employed are E.

COMS ID No: ARCS-198654 Received by IP Australia: Time 13:23 Date 2008-07-17 17-Ju 200S 14:25 BALDWINS 0064 4 4736712 No.3982 P. 22 00 o -15-

O

0 coli, insect, yeast or a mammalian cell line such as COS or CHO. The DNA sequences expressed ii this manner may encode naturally occurrng polypeptides, portions of naturally occurring polypeptides, or other variants thereof.

In a related aspect, polypeptides are provided that comprise at least a functional portion of a polypeptide having an amino acid sequence selected from the group consisting of sequences provided in SEQ ID NOS:267-349 ard 376-401, and variants thereof. As used ~herein, the "functional portion" of a polypeptide is that portion which contains the active site essential for affecting the function of the polypepide, for example, the portion of the 0 0 molecule that is capable of binding one or more reactants. The active site may be made up of 0 10 separate portions presen oan one or more polypeptide chairs and will generally exhibit high binding affinity.

Functional portions of a polypeptide may be identified by first preparing fragments of the polypeptide by either chemical or enzymatic digestion of the polypeptide, or by mutation analysis of the polynucleptide that encodes the polypeptide and subsequent expression of the resulting mutant polypetides. The polypeptide fragments or mutant polypeptides are then tested to determine which portions retain biological activity, using, for example, the representative assays provided below.

A functional portion comprising an active site may be made up of separate portions present on one or more polypeptide chains and generally exhibits high substrate specificity.

The term "polypeptide encoded by a polynucleotide" as used herein, includes polypeptides encoded by a polynucleotide comprising a partial isolated polynucleotide of the present invention.

Portions and other variants of the inventive polypeptides may also be generated by synthetic or recombinant means. Synthetic polypeptides having fewer than about 100 amino acids, and generally fewer than about 50 amino acids, may be generated using techniques well known to those of ordinary skill in the art. For example, such polypeptides may be synthesized using any of the commercially available solid-phase techniques, such as the Merrifield solid-phase synthesis method, where amino acids are sequentially added to a growing amino acid chain. See Merrifield, J. Am. Chem. Soc. 85: 2149-2146, 1963.

Equipment for automated synthesis ofpolypeptides is commercially available fiom suppliers such as Perkin Elmer Applied Biosystems, Inc. (Foster City, California), and may- be operated according to the manufacturer's instructions. Variants of a native polypeptide may be prepared using standard mutagenesis techniques, such as. oligonucleotide-directed site- COMS ID No: ARCS-198654 Received by IP Australia: Time 13:23 Date 2008-07-17 17-Jul 2008 14:26 BALDWINS 0064 4 4736712 No.3982 P. 23 00 S- 16 specific muragensis (Kunkel, Proc. Naz. Acad. Sd. USA 82: 488-492, 1985). Sections of DNA sequences may also be removed using standard techniques to pernit preparation of truncated polypeptides.

In general, the polypeptides disclosed herein are prepared in an isolated, substantially pure form. Preferably, the polypeptides are at least about g0% pare; more preferably at least about 90% pure; and most preferably, at least about 99% pure. In certain preferred embodiments, described in detail below, the isolated polypeptides are incorporated into o hannaceutical comnositions or vaccines for use in the treatment of skin disorders.

00 As used herein, the term "variant" comprehends nucleotide or amino acid sequences o 10 different from the specifically identified sequences, wherein one or more nucleotides or amino acid residues is deleted, substituted, or addd. Variants may be naturally occurring allelic variants, or non-naturally occurring variants. Variant sequences (polynucleotide or polypeptide) preferably exhibit at least 50%, more preferably at least 75%, and most preferably at least 90% identity to a sequence of the present invention- The percentage identity is determined by aligning the two sequences to be compared as described below, determining the number of identical residues in the aligned portion, dividing that number by the total number of residues in the inventive (queried) sequence, and multiplying the result by 100.

Polynucleotide and polypeptide sequences may be aligned, and percentage of identical nucleotides in a specified region may be determined against another polynucleotide, using computer algorithms that are publicly available. Two exemplary algorithms for aligning and identifying the similarity of polynucleotide sequences are the BLASTN and FASTA algorithms. Polynucleotides may also be analyzed using the BLASTX algorithm, which compares the six-frame conceptual translation products of a nucleotide query sequence (both strands) against a protein sequence database. The similarity of polypeptide sequences may be examined using the BLASTP algorithm. The BLASTN, BLASTX and BLASTP programs are available on the NCBI anonymous FTP server (ft://ncbi.nlm.nih._ov) under /blastlexecutables/. The BLASTN algorithm Version 2.0.4 [Feb-24-1998] and Version 2.0.6 [Sep-16-1998], set to the default parameters described in the documentation and distributed with the algorithm, are preferred for use in the determination of polynucleotide variants according to the present invention. The BLAST? algorithm, set to the default parameters described in the documentation and distributed with the program, is preferred for use in' the derennination of polypeptide variants according to the present invention. The use of the COMS ID No: ARCS-198654 Received by IP Australia: Time 13:23 Date 2008-07-17 17-Ju 2008 14:26 BALDWINS 0064 4 4736712 No.3982 P. 24 O 00 17 3 BLAST family of algorithms, including BLASTN, BLASTP, and BLASTX, is described at C, NCBI's website at URL htto://iww.ncbi.nl.nih.iov!BLASTinewblast.htm] and in the publication of Altschul Stephen F, et al, "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs," Nucleic Acids Res. 25: 3389-3402, 1997.

¢n 5 The computer algorithm FASTA is available on the Internet at the ftp site ftD://ftp.dvrsinia.edut/ub/fasta/. Version 2.0.4, February 1996, set to the default parameters Cc J described in the documentation and distributed with the algorithm, may be used in the l determination of variants according to the present invention. The use of the FASTA 00 o algorithm is described in Pearson WR and Lipman DJ, "Improved Tools for Biological Sequence Analysis," Proc. Natl. Acad. Sci. USA 85: 2444-2448, 1988; and Pearson WR, "Rapid and Sensitive Sequence Comparison with FASTP and FASTA," Methods in Enzymology 183: 63-98, 1990.

The following running parameters are preferred for determination of alignments and similarities using BLASTN that contribute to the E values and percentage identity for polynucleotide sequences: Unix nnming command: blastall -p blasta -d embldb -e 10 -GO EO -r 1 -v 30 -b 30 -i queryseq -o results; the parameters are: -p Program Name [String]; d Database [String]; -e Expectation value [Real]; -G Cost to open a gap (zero invokes default behavior) [Integer]; -E Cost to extend a gap (zero invokes default behavior) [Integer]; -r Reward for a nucleotide match (blastn only) [Integer]; -v Number of one-line descriptions [Integer); -b Number of alignments to show [Integer]; -i Query File [File In]; and -o BLAST report Output File [File Out] Optional. The following running parameters are preferred for detennination of alignments and similarities using BL.ASTP that contribute to the E values and percentage identity of polypeptide sequences: blastall -p blastp -d swissprotdb -e !0 -G 0 -E 0 -v 30 -b 30 -i queryseq -o results; the parameters are: -p Program Name [String]; -d Database [String]; -e Expectation value [Real]; -G Cost to open a gap (zero invokes default behavior) [Integer]; -E Cost to extend a gap (zero invokes default behavior) [Integer]; -v Number of one-line descriptions [Integer]; -b Number of alignments to show [Integer]; -I Query File [File In]; -o BLAST report Output File [File Out] OptionaL The "hits" to one or more database sequences by a queried sequence produced by BLASTN, FASTA, BLASTP or a similar algorithm, align and identify similar portions of sequences. The hits are arranged in order of the degree of similarity and the length of sequence overlap. Hits to a database sequence generally represent an overlap over only a fraction of the sequence length of the queried sequence.

COMS ID No: ARCS-198654 Received by IP Australia: Time 13:23 Date 2008-07-17 17.Jul. 2008 14:26 BALDWINS 0064 4 4736712 No.3982 P. O 00 o 18 The BLASTN, FASTA, and BLASTP algorithms also produce "Expect" values c. for alignments. The Expect value indicates the number of hits one can "expect" to see over a certain number of contiguous sequences by chance when searching a database of a certain size. The Expect value is used as a significance threshold for determining whether the hit to a database, such as the preferred EMBL database, indicates true similarity. For I" example, an E value of 0.1 assigned to a polynuclecide hit is interpreted as meaning that in a Mn database of the size of the EMBL database, one might expect to see 0.1 matches over the Saligned porton of the sequence with a similar score simply by chance. By this criterion, the 0 aligned and matched portions of the polynucleotide sequences then have a probability of O 0o of being the same. For sequences having an E value of 0.01 or less over aligned and matched c-l portions, the probability of finding a match by chance in the EMBL database is 1% or less using the BLASTN or FASTA algorithm.

According to one embodiment, "variant" polynucleotides and polypeptides, with reference to each of the polynucleotides and polypeptides of the present invention, preferably comprise sequences having the same number or fewer nucleic or amino acids than each of the polynucleotides or polypeptides of the present invention and producing an E value of 0.01 or less when compared to the polynucleotide or polypeptide of the present invention. That is, a variant polynucleotide or polypeptide is any sequence that has at least a 99% probability of being the same as the polynucleotide or polypeptide of the present invention, measured as having an E value of 0.01 or less using the BLASTN, FASTA, or BLASTP algorithms set at parameters described above. According to a preferred embodiment, a variant polynucleotide is a sequence having the same number or fewer nucleic acids than a polynucleotide of the present invention that has at least a 99% probability of being the same as the polynucleotide of the present invention, measured as having an E value of 0.01 or less using the BLASTN or FASTA algorithms set at parameters described above. Similarly, according to a preferred embodiment, a variant polypeptide is a sequence having the same number or fewer amino acids than a polypeptide of the present invention that has at least a 99% probability of being the same as a polypeptide of the present invention, measured as having an E value of 0.01 or less using the BLASTP algorithm set at the parameters described above.

Alternatively, variant polynucleotides of the present invention hybridize to the polynucleotide seuences recited in SEQ ID NOS: 1-266 and 350-375, or complements, reverse sequences, or reverse complements of those sequences under stringent conditions. As used herein, "stringent conditions" refers to prewashing in a solution of 6X SSC. 0.2% SDS; COMS ID No: ARCS-198654 Received by IP Australia: Time 13:23 Date 2008-07-17 17.J l. 2008 14:27 BALDWINS 0064 4 4 736712 No.3982 P. 26 00

O

19 hybridizing at 65°C, 6X SSC, 0.2% SDS overnight; followed by two washes of 30 minutes c-s each in IX SSC, 0.1% SDS at 65'C and two washes of 30 minutes each in 0.2X SSC, 0.1% SDS at 650C.

The present invention also encompasses polynucleotides that differ from the disclosed sequences but that, as a consequence of the degeneracy of the genetic code, encode the same amino acid sequence are contemplated by the present invention. Such polynucleotides are C'n said to be "degeneratively equivalent" to a poiynacleotide disclosed herein. Similarly, C0 polynucleotides that differ from the disclosed sequences but that encode a polypeptide having o0 similar enzymatic activity as a polypeptide encoded by a polynuclcotide of the present O 0 invention are included within the present invention. Thus, polynucleotides comprising sequences that differ from the polynucleotide sequences recited in SEQ ID NOS: 1-266 and 350-375, or complements, reverse sequences, or reverse complements of those sequences as a result of conservative substitutions are contemplated by and encompassed within the present invention. Additionally, polynucleotides comprising sequences that differ from the polynucleotide sequences recited in SEQ ID NOS: 1-266 and 350-375, or complements, reverse complements, or reverse sequences as a result of deletions and/or insertions totaling less than 10% of the total sequence length are also contemplated by and encompassed within the present invention. Similarly, polypeptides comprising sequences that differ from the polypeptide sequences recited in SEQ ID NOS: 267-349 and 376-401 as a result of amino acid substitutions, insertions, and/or deletions totaling less than 10% of the total sequence length are contemplated by and encompassed within the present invention, provided the variant polypeptide has activity in a lignin biosynthetic pathway.

The polynucleotides of the present invention, including variants, may be isolated from various libraries assembled from plant or non-plant organisms, or may be synthesized using techniques that are well known in the art. Polynucleotides of the present invention may be isolated by high throughput sequencing of cDNA libraries prepared from Eucalyptus grandis and Pinus radiata as described below in Examples 1 and 2. Alternatively, oligonucleotide probes based on the sequences provided in SEQ ID NO: 1-266 and 350-375 may be synthesized and used to identify positive clones in either cDNA or genomic DNA libraries from Eucalyptus grandis and Pinus radiata by means of hybridization or PCR techniques.

Probes may be shorter than the sequences provided herein but should be at least about 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 COMS ID No: ARCS-198654 Received by IP Australia: Time 13:23 Date 2008-07-17 17A .2666 14:27 BALDWJNS 6664 4 4736712 ko-3982 P. 27 00 200 ___known in the art- Positi-ve cloncs ray be analyzed by restIction enzyme digestion, DNA sequencing or the like.

Variants of the polynaucluotides of the present invention derived from other eucalyptus and pine species, as well1 as frm other commercially important species utilized by the lumbr are contemplated, These include the followinng gymnoospenns, by way of example: loblolly pine Pinus taeds, slash pine Pinscs elliotti, sand pine Pinus cLausa, longleaf pine M'f Pus palus7t=u, shorticaf pinie Pimas echinala, ponderosa pine -Pinus ponderosa, .leffirey pine SPMuns Jeffrey, red pine Pinus resin osa, pitch pine Pinus rig-ida, Jack pine Pinus banksiana.

0o pond pi-ne Pnus serotina, Eastern white,& pine Pinus sn-chus, Western white pine Pinus m7onticvia, sugrar pine Pinus lambertiana, Virginia pine Finus virginiana, lodgepole pine ,Pinzis contorts, Caribbean: pine Pinus caribae, P. pinasrer, Calabria pine P. brufia, Afgban pine P. eldanica, C oulter pine P. cvzteri, European pine P. nigra and P. syi-vesiris; Doug-lasfir Pseudotsuga inenziesii; the hemlocks which include Western hemalock Tsuga hez-opkylta, Eastern hemnlock- zmga. canadensis, Mountain hemlock Thtga rnerzensiana; the spruIces i3 which include the Norway spruce Pica chinsT, red spruce Picea rubans, white spruce Ricea glcauca, black spruce Pica nvaia, Sitka spruce Piece sitchensis, Englemnan spruce.Pica engelnlanni, and blue spruce Pica pungens; redwood Sequoia sempervirens; the true firs include. the Alpine fir A bias issiocarpa, silver fir Abies smabilis, p-xand ai Abies -,vandis, nobel fir Abies proceas, white fir Abtas cancelor, California red fir Abies magniftca, and balsam fir Abies balsama, the cedars which include the W kestern red cedar Thzuja plicaza, incense dcdar liboce-drus decurrens, Northern white cedar Thzpc occidentalis, Port Orford ccdar Chamaacyparis IaWYOn ions, Atlantic white cedar Chwaacyparis thycides; Alaska yellow-cedar Caracacyparis nocikazensis, and Eastern red cedar HuniperIZs virginians; the larches which include Eastern l1=h Larix laricina, Western larch Larix occidental s, Euoenlac -aix decidua, Japanese larch Larix 1uptolepis, and Siberian larch Larix siberiai; bold cypress Thxodiumn disticliwn and Giant sequoia Sequoia gigazuea; and the following angiosperms, by way of eample: Eucalyprus slb, if. ban cro ff, E. botyroides, if.

bi-idgesiana, E. calopkvlla, E- caasdmZensis, ciz-iodora. iE. cladocalyx coccifera curtisit, E. dah-rnpleana. Ef deglupta, if. data gatansis. E. diversicolor, dunnii, ficfflia.

E. globulus, Ef. goniphocepkals. E. pmnnil, E- henrvi, Ef. isevopinesF. a carzhurii, Ef.

fflacy-QrkimclC, if. 7aculae, E. matginarati E. meg2capa. nialiodor-a, nick cuff regnans. -E rasi4fera. E5. ,-obusxa, Ef. rudis. saligns. Ef. siderc'xvyion. Ef. stuazz.isna, Ef.

ARCS-198654 Received by IP Australia: Time 13:23 Date 2008-07-17 17-Ju 2008 14:27 BALDWINS 0064 4 4736712 No.3982 P. 28 O 0 S- 21 ,eretiornis, E. torelliana, E. urnigera, E. urophvlla, E. viminalis. E. viridis. E. wandoo and SE. vournanni.

The polynucleotides of the present invention may alternatively be snathesized, for example, using automated oligonucleotide synthesizers Beckman Oligo 1000M DNA Synthesizer) to obtain polynucleotide segments of up to 50 or more nucleic acids. A plurality of such polynucleotide segments may then be ligated using standard DNA manipulation C techniques that are well known in the art of molecular biology. One conventional and 0 exemplary polynucleotide synthesis technique invoives synthesis of a single stranded 0 polynucleotide segment having, for example, 80 nucleic acids, and hybridizing that segment to a synthesized complementary 85 nucleic acid segment to produce a 5 nucleotide overhang.

The next segment may then be synthesized in a similar fashion, with a 5 nucleotide overhang on the opposite strand. The "sticky" ends ensure proper ligation when the two portions are hybridized. In this way, a complete polynucleotide of the present invention may be synthesized entirely in vitro.

The polynucleotides identified as SEQ ID NOS: 1-266 and 350-375 may be "partial" or full length sequences. Partial sequences do not represent the full coding portion of a gene encoding a naturally occurring polypeptide. The partial polynucleotide sequences disclosed herein may be employed to obtain the corresponding full length genes for various species and organisms by, for example, screening DNA expression libraries using hybridization probes based on the polynucleotides of the present invention, or using PCR amplification with primers based upon the polynucleotides of the present invention. In this way one can, using methods well known in the art, extend a polynucleotide of the present invention upstream and downstream of the corresponding .mRNA, as well as identify the corresponding genomic DNA, including the promoter ind enhancer regions, of the complete gene.

The present invention thus comprehends isolated polynucleotides. comprising a sequence identified in SEQ ID NOS: 1-266 and 350-375, or a variant of one of the specified sequences, that encode a functional polypeptide, including full length genes. Such extended polynucleotides may have a length of from about 50 to about 4,000 nucleic acids or base pairs, and preferably have a length of less than about 4,000 nucleic acids or base pairs, more preferably a length of less than about 3,000 nucleic acids or base pairs, more preferably yet a length of less than about 2,000 nucleic acids or base pairs. Under some circumstances.

extended polynucleotides of the present invention may have a length of less than about 1,800 nucleic acids or base pairs, preferably less than about 1.600 nucleic acids or base pairs, more COMS ID No: ARCS-198654 Received by IP Australia: Time 13:23 Date 2008-07-17 17.Jul. 2008 14:28 BALDWINS 0064 4 4736712 No.3982 P. 29 O 0 S- 22preferably less than about 1,400 nucleic acids or base pairs, more preferably yet less than Z about 1,200 nucleic acids or base pairs, and most preferably less than about 1,000 nucleic acids or base pairs.

Polynucleotides of the present invention also comprehend polynucleotides comprising at least a specified number of contiguous residues (x-mers) of any of the polynucleotides identified as SEQ ID NOS: 1-266 and 350-375 or their variants. According to preferred t embodiments, the value of x is preferably at least 20, more preferably at least 40, more 0 preferably yet at least 60. and most preferably at least 80. Thus. polynucleotides of the OO present invention include polynucleotides comprising a 20-mer, a 40-mer, a 60-ier. an mer, a 100-mer, a 120-mer, a 150-mer, a 180-mcr, a 220-mer a 250-mer, or a 300-mer, 400-

C<]

met, 500-mer or 600-mer of a polynucleotide identifed as SEQ ID NOS: 1-266 and 350-375., or a variant of any x-mer- That is, the definitions for variants described above in terms of E values, similarity and hybridization, apply also to any x-mer of any polynucleotide of the present invention.

is Polynucleotide probes and primers complementary to and/or corresponding to SEQ ID NOS: 1-266 and 350-375, and variants of those sequences, are also comprehended by the present invention. Such oligonucleotide probes and primers are substantially complementary to the polynucleotide of interest. An oligonucleotide probe or primer is described as "corresponding to" a polynucleotide of the present invention, including one of the sequences set out as SEQ ID NOS: 1-266 and- 350-375 or a variant, if the oligonucleotide probe or primer, or its complement, is contained within one of the sequences set out as SEQ ID NOS: 1-266 and 350-375 or a variant of one of the specified sequences.

Two single stranded sequences are said to be substantially complementary when the nucleotides of one strand, optimally aligned and compared, with the appropriate nucleotide insertions and/or deletions, pair with at least 80%, preferably at least 90% to 95%, and more preferably at least 98% to 100%, of the nucleotides of the other strand. Alternatively, substantial complementarity exists when a first DNA strand will selectively hybridize to a second DNA strand under stringent hybridization conditions. Stringent hybridization conditions for determining complementarity include salt conditions of less than about 1 M, more usually less than about 500 mM and preferably less than about 200 mM. Hybridization temperatures can be as low as 5°C, but are generally greater than about 22"C, more preferably ereater than about 30"C and most preferably greater than about 37'C. Longer DNA fragments may require higher hybridization temperatures for specific hybridization. Since the COMS ID No: ARCS-198654 Received by IP Australia: Time 13:23 Date 2008-07-17 17JAl. 200E 14:2E BALDWINS 0064 4 4736712 No-3982 P. 23 sringency of hybridization may be affected by other factors such as probe composition, presence of organic solvents and extent of base mismatching, the combination of parameters is more important than the absolute measure of any one alone The DNAs from plants or samples or products containing plant material can be either genomic DNA or DNAs derived M 5 by preparing cDNA from the RNAs present in the sample.

In addition to DNA-DNA hybridization, DNA-RNA or RNA-RNA hybridization CM assays are also possible. In the first case, the mRNAs from expressed genes would then be

O

Sdeteted instead of genomic DNA or cDNA derived 5fom mRNA of the sample. In the o second case, RNA probes could be used- In addition, artificial analogs of DNA hybridizing 0 10 specifically to target sequences could also be used.

In specific embodiments, the oligonucleotide probes and/or primers comprise at least about 6 contiguous residues, more preferably at least about 10 contiguous residues, and most preferably at least about 20 contiguous residues complementary to a polynucleotide sequence of the present invention. Probes and primers of the present invention may be from about 8 to 100 base pairs in length or, preferably, from about 10 to 50 base pairs in length or, more preferably, from about 15 to 40 base pairs in length. The probes can be easily selected using procedures well known in the art, taking into account DNA-DNA hybridization stringencies, annealing and melting temperatures, potential for formation of loops and other factors, which are well known in the art Tools and software suitable for designing probes, and especially suitable for designing PCR primers, are available on the Intemet, for example, at hT l://www.horitonTress.com/pcr/. Preferred techniques for designing PCR primers are also disclosed in Dieffenbach CW and Dvksler GS, PCR primer: a laboratory manual, CSHL Press: Cold Spring Harbor, NY, 1995.

A plurality of oligonucleotide probes or primers corresponding to polynucleotides of the present invention may be provided in a kit form. Such kits generally comprise multiple DNA or oligonucleotide probes, each probe being specific for a polynucleotide sequence.

Kits of the present invention may comprise one or more probes or primers corresponding to a polynucleotide of the present invention, including a polynucleotide sequence identified in SEQ ID NOS: 1-266 and 350-375.

In one embodiment useful for high-throughput assays, the oligonucleotide probe kits of the present invention comprise multiple probes in an array format, wherein each probe is immobilized in a predefined, spatially addressable location on the surface of a solid substrate.

Array formats which may be usefully employed in the present invention are disclosed, for COMS ID No: ARCS-198654 Received by IP Australia: Time 13:23 Date 2008-07-17 17.Jul. 2008 14:28 BALDWINS 0064 4 4736712 No.3982 P. 31 00 O 0 0 24 example, in U.S. Patents No. 5,412,087, 5,545,531, and PCT Publication No. WO 95/00530, o the disclosures of which are hereby incorporated by erfeence.

The significance of high-throughput screening systems is apparent for applications such as plant breeding and quality control operations in which there is a need to identify large numbers of seed lots and plant seedlings, to examine samples or products for unwanted plant Ce¢ materials, to identify plants or samples or products containing plant material for quarantine ~purposes etc. or to ascertain the true origin of plants or samples or products containing plant O material. Screening for the presence or absence of polynucleotides of the present invention 00 used as identifiers for tagging plants is valuable for later detecting the amount of gene flow in 0 o 10 plant breeding, introgression of genes via dispersed pollen, etc.

In this manner, oligonucleotide probe kits of the present invenion may be employed to examine the presence/absence (or relative amounts in case of mixtures) of polynucleotides of the present invention in different samples or products containing different materials rapidly and in a cost-effective manner. Examples of plant species that may be examined using the present invention, include forestry species, such as pine and eucalyptus species, other tree species,.agricultural plants including crop and forage plants, and horticultural plants.

Another aspect of the present invention involves collections of polynucleotides of the present invention. A collection of polynucleotides of the present invention, particularly the polynucleotides identified as SEQ ID NOS: 1-266, 350-375 and variants and x-mers thereof may be recorded and/or stored on a storage medium and subsequently accessed for purposes of analysis, comparison, etc. Suitable storage media indlude magnetic media such as magnetic diskettes, magnetic tapes, CD-ROM storage media, optical storage media, and the like. Suitable storage media and methods for recording and storing information, as well as accessing information such as polynucleotide sequences recorded on such media, are well known in the art. The polynucleotide information stored on the storage medium is preferably computer-readable and may be used for analysis and comparison of the polynucleotide information.

Another aspect of the present invention thus involves storage medium on which are recorded a collection of the polynucleotides of the present invention, particularly a collection of the polynucleotides identified as SEQ ID NOS: 1-266, 350-375 and variants thereof, as well as x-mers of the polynucleotides of SEQ ID NOS: 1-266 and 350-375, and extended sequences, probes and primers comprising or correspond to a polynucleotide of SEQ ID NOS: 1-266 and 350-375. According to one embodiment. the storage medium includes a COMS ID No: ARCS-198654 Received by IP Australia: Time 13:23 Date 2008-07-17 17-Ju 2008 14:29 BALDWINS 0064 4 4736712 No.3982 P. 32 00 0O S- 25 collection of at least 20, preferably at least 50, more preferably at least 100, and most Spreferably at least 200 of the polynucleotides of the present invention, preferably the polynucleotides identified as SEQ ID NOS: 1-266 and 350-375, or variants of those polynuclotides.

5 In another aspect, the preseni invention provides genetic consiructs comprising, in the S5'-3' direction, a gene promoter sequence; an open reading frame coding for at least a Cf functional portion of a polypeptide encoded by a polynucleotide of the present invention; and C' i a gene termination sequence. As used herein, the "functional portion" of an enzyme is a 00 o portion that contains an active site essential for affecting a metabolic step, i.e. a portion of the o molecule that is capable of binding one or more reactants or is capable of improving or regulating the rate of reaction. An 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 by a nucleotide sequence which includes the partial isolated polynucleotides of the present invention.

The open reading frame may be orientated in either a sense or antisense direction. For applications where amplification of lignin synthesis is desired, the open reading frame may be inserted in the construct in a sense orientation, such that transformation of a target organism with the construct will lead to an increase in the number of copies of the gene and therefore an increase in the amount of enzyme. When down-regulation qf lignin synthesis is desired, the open reading frame may be inserted in the construct in an antisense orientation, such that the RNA produced by transcription of the polynucleodide 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 decrease in the amount of enzyme. Alternatively, regulation may be achieved by inserting appropriate sequences or subsequences DNA or RNA) in ribozyme constructs.

Genetic constructs comprising a non-coding region of a gene coding for an enzyme encoded by the above DNA sequences or a nucleotide sequence complementary to a noncoding region, together with a gene promoter sequence and a gene termination sequence, arc also provided. As used herein the term "non-coding region" includes both transcribed sequences which are not -tanslaed, 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 constructs include inmons and.

COMS ID No: ARCS-198654 Received by IP Australia: Time 13:23 Date 2008-07-17 17.Ju 2008 14:29 BALDWINS 0064 4 4736712 No.3982 P. 33 00 0 S- 26 non-coding leader sequences. Transformation of a target plant with such a DNA construct Smay lead to a reduction in the amount of lignin synthesized by the plant by the process of cosuppression, in a manner similar to that discussed, for example, by Napoli et Plant Cell 2:279-290, 1990; and de Carvalho Niebel et al., Plant Cell 7:347-358, 1995.

The genetic constructs of the present invention further comprise a gene promoter sequence and a gene termination sequence, operably linked to the polynucleotide to be Cc transcribed, which control expression of the gene. The gene promoter sequence is generally Spositioned at the 5' end of the polynucleotide to be transcribed, and is employed to initiate 00 transcription of the polynucleotide. Gene promoter sequences are generally found in the 5' non-coding region of a gene but they may exist in introns (Luehrsen KR, Mol. Gen. Gener.

225:81-93, 1991, or in the coding region, as for example in PAL of tomato (Bloksberg, Studies on the Biology of Phenylalanine Ammonia Lyase and Plant Pathogen Irneraction, Ph.D. Thesis, University of California, Davis, 1991, University Microfilms International Order No. 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 comprising either an open reading frame in an andsense 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 lkown in the art. The promoter gene 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.

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 JRNA only 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 and the like. Temporally regulated promoters can be COMS ID No: ARCS-198654 Received by IP Australia: Time 13:23 Date 2008-07-17 17.Jul. 2008 14:29 BALDWINS 0064 4 4736712 No.3982 P. 34 00 27employed to effect modulation of the rate of RNA polymerise 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 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 1> synthase (mas), octopine synthase (ocs) and those reviewed by Chua et al., Science C 244:174181, 1989.

0, The gene termination sequence, which is located 3' to the poiynucleotide to be

CO

00 Stranscribed, may come from the same gene as the gene promoter sequence or may be from a S1o 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.

The genetic 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 NPTI- gene whose expression results in resistance to kanamycin or hygromycin, antibiotics which are usually toxic to plant cells at a moderate concentration (Rogers et al., in Weissbach A and H, eds., Methods for Plant Molecular Biology, Academic Press Inc.: San Diego, CA, 1988).

Altematively, 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.

Techniques for operatively linking the components of the inventive genetic 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, CSHL Press: Cold Spring Harbor, NY, 1989). The DNA construct of the present invention may be linked 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.

The genetic constructs of the present invention may be used to transform a variety of plants, both monocotyledonous grasses, corn, grains. oat, wheat and barley), dicotyledonous Arabidopsis, tobacco, leaumes, alfalfa, oaks, eucalyptus, maple), and Gvmnosperns Scots pine; see Aronen, Finnish Forest Res. Papers, Vol. 595, 1996), COMS ID No: ARCS-198654 Received by IP Australia: Time 13:23 Date 2008-07-17 17 Jul 2008 14:30 BALDWINS 0064 4 4736712 No.3982 P. 00 O 0 28cl white spruce (Ellis et al., Biotechnology. 11:94-92, 1993), and larch (Huang et al., In Vitro SCell 27:201-207, 1991). In a preferred embodiment, the inventive genetic 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 consisting of eucalyptus and pine species, most preferably from the group consisting of Eucalyptus grandis and Pinus radiaa.

n As discussed above, transfonration of a plant with a genetic construct including an open Sreading frame coding for an enzyme encoded by an inventive poiynucleotide wherein the 00 open reading frame is orientated in a sense direction will produce a modified lignin content in o i0 the plant. Transformation of a plant with a genetic construct comprising an open reading C frame in an antisense orientation or a non-coding (untranslated) region of a gene will also produced a modification in the lignin content of the transformed plant.

The production of RNA in target cells may be controlled by choice of the promoter sequence, or by selecting the number of fubctional copies or the site of integration of the polynucleotides incorporated into the genome of the target organism. A target plant may be transformed with more than one 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 tissue or affecting enzyme activity at more than one expression time. Similarly, a construct may be assembled containing more than one open reading frame coding for an enzyme encoded by a polynucleotide of the present invention or more than one non-coding region of a gene coding for such an enzyme. The polynucleotides of the present invention may also be employed in combination with other known sequences encoding 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.

Techniques for stably incorporating DNA constructs into the genome of target plants are well known in the art and include Agrobaclerium turmefaciens 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 transfomned. For example, dicotyledonous plants and certain monocots and gymnosprms may be transformed by Agrobacrerium Ti plasmid technology, as described, for example by Bevan (Nuc. 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, meristeratic regions; COMS ID No: ARCS-198654 Received by IP Australia: Time 13:23 Date 2008-07-17 17.Jul. 2008 14:30 BALDWINS 0064 4 4736712 No.3982 P. 36 00 S- 29cotyledons, bypocotyls, and the like- One preferred method for transforming eucalyptus and pine is a biolistic method using pollen (see. for example, Aronen, Finnish Forest Res. Papers.

Vol. 595:53, 1996) or easily regenerable embryonic tissues. Other transformation techniques which may be usefully employed in the inventive methods include those taught by Ellis et al.

5 (Plant Cell Reports, 8:16-20, 1989), Wilson et at. (Plant Cell Reports 7:704-707, 1989) and Tautous et al. (Theor. Appl. Genet. 78:531-536, 1989).

Once the cells are transformed, cells having the inventive DNA construct incorporated C, in their genome may be selected by means of a marker, such as the kananvcin resistance 00 o marker discussed above. Transgenic cells may then be cultured in an appropriate medium to 1 10 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 Sexplants, an appropriate regeneration 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 TA, ed., In virro embryogenesis of plants, Current Plant Science and Biotechnology in Agriculture 20(12):471-540, 1995.

Specific protocols for the regeneration of spruce are discussed.by Roberts et al., ("Somatic embryogenesis of spruce," in Redenbaugh K, ed., Svnseed: applications ofsynithetic seed to crop improvement, 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.

In yet a further aspect, the present invention provides methods for modifying the level (concentration) or activity of a polypeptide in a host organism, comprising stably incorporating into the genome of the plant a construct comprising a poiynucleotide of the present invention. The genetic NA constructs. of the present invention may be used to transform a variety of organisms. Such organisms include plants, such as monocotyledonous angiosperms grasses, corn, grains, oat, wheat and barley), and dicotyledonous angiosperms Arabidopsis, tobacco, legumes, alfalfa, oaks, eucalyptus, maple), and gymnosperns Scots pine; see Aronen Finnish Forest Res. Papers, Vol. 595, 1996), white spruce (Ellis et al., Biotechnology 11:94-92, 1993), and larch (Huang et al., In Vitro Cell 27:201-207, 1991).

In preferred embodiments, the genetic constructs of the present invention are employed to transform woody plants, herein defined as a tree or shrub having a stem that COMS ID No: ARCS-198654 Received by IP Australia: Time 13:23 Date 2008-07-17 17-Jul. 2008 14:31 Y7.~I 906 4:31 BALDWINS 0064 4 4736712ko38 P. 7 No.3982 P. 37 00 8 13-lves for a number of years and increases in diameter each year as a consequence of the Z ~addition of woody tissue. The target plant is preferably selected froma the group conasisting of 1>eucalyptus and pine species, most preferably from the group consisting- ofEucalyptus randis and Flints radiata, but also inciuding mny of the species in the following list: Pines: Pinns banlzian-, Pinus bruria, Rinus caribaea, Rinus Clausa, Plnus Cantorta, 17--Pimas caulleri, Finus echinata, Pinus eldarica, Pinus eliiot4 Finus jeffi-cyt Finys Inuberrifaa Finns nxon7ticola, Pias nigra, Pli-s paliastrus, Finius pie-aster, Pim,!s 0 tjqondez-osa. Plius resinasa, .Pinus rigida, Finus serotin,RPim's strobus, Plans svlveszrls,' O o~~1 Other -ymnosperns: Ables ante-ills, Ables balsame-, Abies conco/or, Ahi as gra- 71d Abler losiocarpa, Ables za gnifica, A hi s procera, Chame-ecyparis le-wsoniono, Chaemaceqypari s noorkazenss, Chant aecypezris skvoides, H-uparmus vi-gin Lani2, Larix decidua.

Larix larice-a, Larix lepto/epis, Lat-ix occidentalls, Larix siberica, Libocedrus decza7-ens.

Ricea ables, Rice-a enge/mnanni Pica glae, Ricea mare-na, Ricea pun ge-as, Ricea rubeas, Pie-ew sixcitenis, Fsendomsuge- menie-sit Sequoia gigeantee, Sequoia se-mpen'irens, Tasodium distichuni, Tsuge- ce-natansis, Tinga hate-rap ila, Isg- mertensiana. Thzqa occidealis, ThLuja pie-ta.

B ucalypts-: Eucalyptus a/ba, Eucalypzus ban croftul. Eucalyvtus boon-aides. Eucalyptus bridgeszane-, Eucalyptu ca/op byfla, Eucalyptus camaldulens is, Eucalyptus citriodora, EucaLvptus cladoc-lyx, Eucalyptu coccifer-. Eucalyptus curtisil, Eucalyptus daynnle-n-, .Eucalts dag/upta, EPucalyptus de-legate-nsls. Eucalyptus dh'ersico/or, Eucalip ins dunni4, Eucalyptus ficifoli-, Eucalyptus globulus-, Ezicalypwu gomphocaphaba, Eucalyptu gunnit, EucalyPtus he-n7 yi. Eucalyptus le-eVOolne, Eucalyptus macarilturi, Eucalyptu macrorrynchaz. Eucealyptus- rnacu=era, -Eucalyptus rnargie-ta. Eucabypeus nege-carpa, Eucalyptus ma//bodora, Eucalyptus ale-hold, Eucalyptus Wiens, -Eucalyptus no0va-an glia, Euicahyptvr Oblique-, Eucalyptus chfis pora, Eucalyptus ore-odes, -Eucalyptus poucylora.

Eucalyptms po/ybraea, Eucab~pzus regnans, Eucalyptus resut/2rfe-. Eucalyptus r-ot'ste-, Evcalyptus j-udis, Eucdtvptus se-hgna. Eue-all ptUS sde-roxylon, EuIcalyPtus sinartlo no, Eucalyptus rere-ricorn is-, Eucaijpzus torelliana, Eucalypu urnig-rp, -Eucalyptu urophylla, so Eucalyptus viminaczis. Eucalyptus i'ridis, Eucalypu wadoEucalytsytnOuad hybrids of any of the above species.

Further, the polynucleorides of the present invention have particular applic-ation) for use as non-disruptive tags for marking organisms, particularly plants. Other orgaMnis may, COMS ID No: ARCS-198654 Received by IP Australia: Time 13:23 Date 2008-07-17 17-4l. 2008 14:31 BALDWINS 0064 4 4736712 No.3982 P. 38 00 oO 31 bowever be tagged with the polvnucleotides of the present invention, including commercially valuable animals, fish, bacteria and yeasts.Constracts comprising polynucleotides of the present invention may be stably introduced into an organism as heterologous, non-functional, non-isupTive tags It is then possible to identify the origin or source of the organism at a MS later date by detenrmining the presence or absence of the tag(s) in a sample of material.

Detection of the tag(s) may be accomplished using a variety of conventional teclmiques, and will generally involve the use of nucleic acid pmrobes Sensitivity in assaying ci the presence of probe can be usefully increased by using branched oligonucleoctides, as 004 0aescnribed in Horn T, Chang CA and Urdea MS, "Chemical synthesis and characterization of branched oligodeoxyribonucleotides (bDNA) for use as signal amplifiers in nucleic acid quantification assays," Nucleic Acids Research 25(23):4842-4849, 1997), enabling detection of as few as 50 DNA molecules in the sample.

The following examples are offered by way of illustration and not by way of limitation.

Examle 1 Isolation and Characterization of cDNA Cloies from Eucalvtus randis Two Eucalyptus 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 (Plant Molecular Biology Reporter 11:113-116, 1993) with minor modifications. Specifically, samples were dissolved in CPC-RNAXB (100 mM Tris-Cl, pH 8,0; 25 mM EDTA; 2.0 M NaCl; 2% CTAB; 2% PVP and 0.05% Spennidine*3 HCI) and extracted with 2$ chloroform;isoamyl alcohol, 24:1. mRNA was precipitated with ethanol and the total RNA preparate was purified using a Poly(A) Quil mRNA Isolation Kit (Stratage, La Joll3011a, CA).

A cDNA expression library was constructed from the purified mRNA by reverse transcriptase synthesis followed by insertion of the resulting ODNA clones in Lambda ZAP using a ZAP Express cDNA Synthesis Kit (Straragene), according to the manufacturer's protocol. The resulting cDNAs were packaged using a Gigapack II Packaging Extract (Stratagene) employing 1 il of sample DNA aorn the 5 gl ligation mix. Mass excision of the library was done using XL1-Blue MRF' cells and XLOLR cells (Stratagene) with EXAssisi helper phage (Stratagene). The excised phagemids were diluted with NZY broth (Gibco BRL, COMS ID No: ARCS-198654 Received by IP Australia: Time 13:23 Date 2008-07-17 17Jul 2008 14:31 BALDWINS 0064 4 4736712 No.3982 P. 39 00 oO 32- 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 suitable for sequencing. Positive colonies were cultured in NZY broth with kanamycin and cDNA was purified by means of alkaline lysis and polyethyltne glycol (PEG) precipitation. Agarose gel at 1% was used to screen sequencing templates for clvomosomfal contamination. Dye.primer sequences were prepared using a Turbo Catalyst 800 machine (Perkin Elmer/Applied CA Biosyserfls, Foster City, CA) according to the manufacturer's protocol.

00 DNA sequences for positive clones were obtained using a Perkin Elmer/Applied o 10 Biosystems Prism 377 sequencer. oDNA clones were sequenced first from the 5' end atd in some cases, also from the 3' end. For some clones, internal sequence was obtained using subcloned fragments. Subcloning was pcrforamed using standard procedures of restriction mapping and subcloning to pBluescript II SK+ vector.

The dotermined cDNA sequences were comnipared to known sequences in the EMEBL database (release 46, March 1996) using the FASTA algorithm of February 1996 (Version 2.0.4) (available on the Inteet at the ftp site ft://ftn.viTinia edu/ub/fastta/ or the BLASTN algorithm Version 2.0.4 [Feb-24-1998], or Version 2.0.6 [Sept-16-1998], set to the preferred parameters described above. Multiple alignmnts of redundant sequences were used to build up reliable consensus sequences. Based on similarity to known sequences from other plant species, the isolated polynucleotides of the present invention were identified as encoding a specified enzyme.

Using the procedures described above, cDNA sequences derived from the Eucaivprs grandis library encoding the following polypeptides were isolated: PAL (SEQ ID NOS: 16, 100 242-246); C4H (SEQ ID NOS: 17, 153, 154, and 161); C33H (SEQ ID NOS: 18, 101, 149 and 150); F5H (SEQ ID NOS: 19-21, 102; 103 and 169-171); OMT (SEQ ID NOS: 22-25, 104-107, 173 and 174); CCR (SEQ ID NOS: 26-29 and 108-111); CAD (SEQ ID NOS: 1, and 112); CGT (SEQ ID NOS: 31-33 and 113-115); CBG (SEQ ID NOS: 34, 165 and 166); PNL (SEQ ID NOS: 35, 36 and 116); LAC (SEQ ID NOS: 37-41, 117 and 118); POX (SEQ ID NOS: 42-44, 119-121, 179, 249-250 and 350-358); caffeic acid methyl transferase (SEQ ID NOS: 187-192); caffeoyl CoA methyl transferase (SEQ ID NOS: 193-195); coumarate Co-A ligase (SEQ ID NOS: 196-198); cytochrome P450 LXXIA (SEQ ID NOS: 201-206); diphenol oxidase (SEQ ID NOS: 207-217); flavonol glocosyl transferase (SEQ ID NO: 218); COMS ID No: ARCS-198654 Received by IP Australia: Time 13:23 Date 2008-07-17 lit I. 299H 14:32 BALDWJNS 9964 4 4736712 No.3982 P. 49 00 0 ~33 flavwood bvdrc-xYlaSe (SEQ ID INOS- 219-223); and isoflavone redactase (SEQ ID NOS, ~~Isolation ir-nd Char-acterizaionof cDNA C1loes.from Pim7Ur radiataf a) Isolation of cDNA clones 1- ihthog-ntsrenn 0 A Pbzuy -adiana eDNA expression library wa constructed fromn xyl em- and screrned 00 as described above in Example i. DNA sequences for positive clones were obtained Using forward and reverse primers on a Perkin Elm er/Applied Biosystems Prism 377 sequencetr and the deftermined sequences were c-ompared to kcnown sequences in the EML database as described above.

Based on similarity to known sequecs from orther plant species, the isolated DNA sequences were identified as encodi-ng the enzymes C4Ei (SEQ IID NOS. ,3 8 4,9,14 125. 155-160, 162 and 163); C3H (SEQ ID NOS: 4, 50-52, 93, 126, 127t 151 and 152); PN (SEQ IDNOS: 5, 81 and 183 OMT (SEQ ID NOS- 6, 53-55, 94 and 175); CAD (SEQ ID NOS: 7, 71, 95 and 164); CCR (SE-Q ID NOS: 8, 58-70, 96, 128-134 and 167); PAL (SEQ ID NQS: 9 -11, 45-47, 97, 98, 122,. 123 and 176, 947 and 248); 4CL (SEQ ID NOS: 12, 56, 57, 99, 147, 148, 265 and 266); COT (SEQ ID NOS; 72, 135 and 168); CBG (SEQ ID NOS:- 73-SO and 136-142); LAC (SEQ DD NOS. 82-84, 142-144 and 172); POX (SEQ ED NOS: 89, 91, 145, 146, 177, 178, 180-182, 264, '59-375)- alpha amnylase (SEQ DD NOS: 184-ls6); couxnarate 6A ligase (SEQ ED NOS: 199 and 200); fiavonoid hydroxylase (SIEQ ID NOS: 224-233); isofiavone reductase (SEQ ED NO: 241); and diphenol oxidase (SEQ ID NOS. 253-263)., b)Iisolation of goDNA clones bvPCR Two PCR probes, hereinafter referred to as LNBO1O and LNBO1 I (SEQ ID NO: 14 and 15, respectively) ware designed base d on conserved domnains in the following pe-oxidase sequences previouisly identified in other species vanpox, bvupox6, taepox, hvupoxl, osapox, ntopox2, ntopoxl, lespox, pok-pox, luspox, athpox, brpox, spopox, and Ivepox (Qenbank Accession Nus. D11337, M8367L X56011, X(58396, X66125, 102979, D113396, X71593, D 11102, L07554, M5838 1, X(57564, Z22920, and 73301 1Rrespectively).

COMS ID No: ARCS-198654 Received by IP Australia: Time 13:23 Date 2008-07-17 17.Jul. 2008 14:32 BALDWINS 0064 4 4736712 No.3982 P. 41 00 0 o 34 CA RNA was isolated from pine xylem and first strand cDNA was synthesized as described above. This cDNA was subjected to PCR using 4 jiM LNB010, 4 gM LNBO11, 1 x Kogen's buffer, 0.1 mg/ml BSA, 200 mM dNTP, 2 mIM Mgt, and 0.1 U/p1 of Taq polymerase (Gibco BRL). Conditions were 2 cycles of 2 min at 940C, 1 min at 550C and 1 min at 720C; 25 cycles of I min at 94*C, 1 min at 55'C, and I min at 72*C; and 18 cycles of 1 min at 940C, 1 min at 55CC, and 3 min at 720C in a Stratagene Robocycler. The gene was re-ampnlified in the same manner. A band of about 200 bp was purified from a TAE agarose gel using a Schieicher Schuell E1u-Quik DNA pmurification kit and clones into a T-tailed plluescript vector (Marchuk D et al., Nucleic Acids Res. 19:1154, 1991). Based on similarity O 10 to known sequences, the isolated gene (SEQ ID NO: 13) was identified as encoding pine peroxidase (POX).

Examle 3 Is Use of an O-mnethvyltransferase (OMT) Gene to Modift Linin Biosynthesis a) Transformation of tobacco planis with a Pinhus radiat OMT ene Sense and anti-sense constructs containing a polynucleotide including the coding region of OMT (SEQ ID NO: 53) from Pinus radiaia were inserted into Agrobacterium tumefaciens 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, Mitra A, Ha SB, "Binary Vectors," in Gelvin SB, Schilperoort RA, eds., Plant Molecular Biology Manual, Khuwer Acaderic Publishers: Dordreclht, 1988). The presence and integrity of the transgenic constructs were verified by restriction digestion and DNA sequencing.

Tobacco (Nicotiana tabacun cv. Samsun) leaf sections were transformed using the method ofHorsch et aL (Science, 227:1229-1231, 1985). 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 OMT. Transformed plants containing the appropriate lignin gene construct were verified using Southem blot experiments. A in the column labeled "Southern" in Table 2 below indicates that the transforned plant lines were contined as independent transformed lines.

b) Exnression ofrPinus OMT in transfomed niants COMS ID No: ARCS-198654 Received by IP Australia: Time 13:23 Date 2008-07-17 17-Jul. 2008 14:32 BALDWINS 0064 4 4736712 No.3982 P. 42 00 Total RNA was isolated from each independent transformed plant line created with the OMT sense and antj-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 labeled "Northem" in Table 2 shows that the transformed plant en 5 lines containing the sense and anti-sense constructs for OMT all exhibited high levels of expression, relative to the background on the Northern blots- OMT expression in sense plant line number 2 was not measured because the RNA sample showed signs of degradation.

Ci There was no derectable hybridisation to RNA samples from empty vector-transfonjed 00 0 control plants.

0 1° c) Modulation of OMT enzymne activity in transformed plants The total activity of OMT enzyme, encoded by the Pinus 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 protein ectracts were prepared f-om each transformed plant and assayed -using the method of Zhang et aL (Plant Physio., 113:65-74, 1997). The data contained in the column labeled "Enzyme" in Table 1 shows that the transformed plant lines containing the OMT sense construct generally had elevated OMT enzyme activity, with a maximum of 199%, whereas the transformed plant lines containing the OMT anti-sense 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 ofPinus OMT on lienin concentration in transformed piants The concentration of lignin in the transformed tobacco plants was determined using the well-established procedure of thioglycolic acid extraction (see, Freudenberg et at., Constitution and Biosynthesis of Lignn, 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 from one empty vectortransformed control plant line, the five independent transformed plant lines containing the sense construct for OMT and the eight independent transformed plant lines containing the anii-sense construct for OMT were extracted individually with methanol, followed by thioglycolic acid and finally dissolved in 1 M NaOH. The final extracts were assayed for absorbance at 280 nm. The data shown in the column labelled "TGA" in Table 2 shows that COMS ID No: ARCS-198654 Received by IP Australia: Time 13:23 Date 2008-07-17 17-Ad., 200E 14:32 BALDWINS 0064 4 4736712 No-3982 P. 43 36 the transformed plant lines containin the sense and the anti-sense OMT gene constructs all exhibited significantly decreased levels of liguin, relative to the empty vector-transformed control plant lines.

Table 2 orientation Southern Northern olant line transwene En-m -P Tr-A ulaitlin tanien orenatin outer orhe contol

OMT

OMT

OMT

OMT

OMT

OMT

OMT

OMT

OMT

OMT

OMT

OMT

OMT

na sense sense sense sense sense anti-sense anti-sense anti-sense anti-sense anti-sense anti-sense anti-sense anti-sense blank 2.9E+6 na 4.1E+6 2.3E+6 3.6ES+5 1.6E+4 5.7E+3 8.OE+3 I4E+4 .SE44 25E+4 2.5E+4 IIE+4 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 liain biosynthetic 2s gene such as OMT.

ExamIle 4 JUSe of a 4-CounarateCoA litase (4CL) Gene to Modify Lignin Biosynthesis a) Transformation of tobacco olants with a Pinus radiata 4CL ane Sense and anti-sense constructs containing a polynucleotide including the coding region of 4CL (SEQ ID NO: 56) from Pinus radiata were inserted into Agrobacterjunl nsmefaciens LBA4301 by direct transformation as described above. The presence and integrity of the transgenic constructs were verified by restriction digestion and DNA sequencing.

Tobacco (Nicotiona tabacuin ci'. Samsun) leaf sections were transformed as described above. Five independent transformed plant lines were establish'ned for the sense cosinruct and eight independent transfonrmed plant lines were established for the anti-sense construct for 4CL Transfonned plants containing the appropriate lignin gene constnruct were verified COMS ID No: ARCS-198654 Received by IP Australia: Time 13:23 Date 2008-07-17 17 Jul. 2008 14:33 BALDWINS 0064 4 4736712 No.3982 P. 44 00 S- 37 Susing Southen blot experiments. A in the column labeled "Southern" in Table 3 Z indicates that the transformed plant lines listed were confirmed as independent transformcd lines.

b Expression ofPinus 4CL in ransformed plants Total RNA was isolated from each indendent transformed plant line created with rn the 4CL sense and anti-sense constructs. The RNA samples were analysed in Northern blot experiments to determine the level of expression pf the transgene in each transformed line 0 The data shown in the column labelled "Northem" in Table 3 below shows Ltat the O 10 transformed plant lines containing the sense and and-sense constructs for 4CL all exhibit high levels ofexpression, relative to the background on the Northern blots. 4CL expression in antisense 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 vectortransformed control plants, cl 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 transformed plant line created with the 4CL sense and anti-sense constructs. Crude protein extracts were prepared from each transformed plant and assayed using the method of Zhang et al. (PlantPhysioL, 113:65-74, 1997). The data contained in the column labeled "Enzyme" in Table 3 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 control plants, d) ffects of Pinus 4CL on lenin 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" in Table 3 shows that the transformed plant lines containing the sense and the anti-sense 4CL gene constructs all exhibited signficantly decreased levels ofligmin. relative to the empty vector-transformed control plant lines. These data clearly indicate that linin concentration, as measured by the COMS ID No: ARCS-198654 Received by IP Australia: Time 13:23 Date 2008-07-17 17Jul, 20H8 14:3 BALDWINS 0064 4 4736712 No.3982 P. 38 TGA assay, can be directly manipulated by either sense or anti-sense expression of a lignin biosynthetic gene such as 4CL.

Tale 3 plant line traoseene orientation Southern Northern Euryvme TGr Enzymen TflA control control 4CL 4CL 4CL 4CL 4CL 4CL 4CL 4CL 4CL 4CL 4CL 4CL 4CL na na sense sense sense sense sense anti-sense anti-sense anti-sense anti-sense anti-sense anti-sense anti-sense anti-sense blank blank 2.3E+4 4.5E+4 I I E+4 1.7±E4 1.6E-'4 na 1.0E+4 9,6E-+3 1.2E+4 4.7E+3 3.9E+3 1.8E-3 1.7E+4 100 100 169 258 174 164 184 59 70 81 90 101 116 125 106 92 104 64 73 77 92 83 88 89 94 97 Examnile Transfonrmation of Tobacto usinme the Inventive Lignin Biosynthetic Genes Sense and anti-sense constructs containing polynacleotides including the coding regions of C3H (SEQ ID NO: 18), F5H (SEQ ID NO: 19), CCR (SEQ ID NO: 26) and CGT (SEQ ID NO: 31) from Eucalypnts grandis, and OMT (SEQ ID NO: PAL (SEQ ID NO: 45 and 47), C4H (SEQ ID NO: 48 and 49), PNL (SEQ ID NO: 81) and LA.C (SEQ ID NO: 83) from Pinzs radiata were inserted into Agrobaderiun tumnfaciens LA4301 by direct transformation as described above. The presence and integrity of the trangenic constucts were verified by restriction digestion and DNA seqtencinz.

Tobacco (Nicoiana tabacum cv. Samsun) leaf sections were transformed as described in Example 3. Up to twelve independent transformed plant lines were established for each sense construct and each anti-sense construct listed in the preceding paragraph. Transformed plants containing the appropriate lignin gene constrct were verified using Southern blot experiments. All of the transformed plant lines analysed were confirmed as independent transformed lines.

COMS ID No: ARCS-198654 Received by IP Australia: Time 13:23 Date 2008-07-17 17-Jul. 2008 14:33 BALDWINS 0064 4 4736712 No.3982 P. 46 39 Exarnule 6 Manioulation of inin Content in Traniformed Plants a) Determination of transetoe exnression by Northern blot exceriments Total RNlA was isolated fron each independent transformed plant line describod in 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 4 shows the level of transgene expression for all plant lines assayed, relative to the background on the Northem blots. There was no detectable hybridisation to RNA samples from empty vector-transfortned control plants.

b) Determination of liain concentration in transfrmed plants The concentration of lignin in empty vector-transformed control plant lines and in un to twelve independent tfransfomnned lines for eachb sese construct and each anti-sense construct described in Example 5 was determined as described in Example 3. The column labelled "TGA" in Table 4 shows the thioglycolic acid extractable ligins for plant lines transformed with C31, FSH, CCR, PAL, C4H, PNL and LAC, expressed as the average percentage of TGA extractable lignins in transformed plants versus control plants. The range of Variation is shown in parentheses.

Table 4 transene oe tation no, of lines hnortm TGA North- 'M A Nnrtl, em rrt A control C3H P511

CCR

CCR

PAL

PAL

C41i

PNL

LAC

LAC

na sense sense anti-sense sense anti-sense sense anti-sense anti-sense anti-sense sense anti-sense blank 3.7E 4 5.8E+4 5-8E+4 na na 1.9E+5 1.5E4-4 5.SE+4 1.2E+4 l.7E+5 1.7E+5 100 (92-104) 74 (67-85) 70 (63-79) 73 (35-93) 74 74 (62-86) 77 (71-86) 62 (37-77) 86 (52-113) 88 (70-114) na as (73-114) Fig. 5 illustrates the quantity of extractable lignin, as a percentage of wild type lignin content, in tobacco plants transformed with PAL (sense and anti-sense), C4H (antisense), COMS ID No: ARCS-198654 Received by IP Australia: Time 13:23 Date 2008-07-17 17.Ju 2008 14:34 BALDWINS 0064 4 4736712 No.3982 P. 47 00

O

S- 40 C3H (sense), F5H (sense and antisense), C5H (sense and antisense) C3H (sense; referred to Z as COMT in Fig. OMT (sense and antisense; referred to as CCOMT in Fig. 4CL (souse I> and antisense), CCR (sense and antisnse) and CGT (antisense) constructs as described in Example 5. Thioglycolc acid-extractable lignin quantities were measured in transgenic plants, normalized to empty-vector control plants, Three extracts were independently derived Ce from each of approximately 10 independently derived transgenic plants. The average of the en three extracts is shown by a black dot, as the lignin value for that plant. The average of en Sindependent transgenic plants transformed with a given cDNA construct is shown as a bar.

OO The average of empty vector transformed control plants is shown as an X. The value for the O 10 controls is extrapolated across the field to facilitate comparison. Black bars indicate means cl which are significantly reduced (p 0.05) in lignin content with respect to control plants, Grey bars indicate means which are not significantly changed from control plants.

Transformed plant lines containing the sense and the anti-sense lignin biosynthetic gene constucts exhibited a mean level of ignin content that was significantly lower than that of empty vector-transformed control plant lines. The most dramatic effects on lignin concentration were seen in the OMT sense plants, and in the PAL sense plants. These data clearly indicate that lignin 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.

Examle 7 Modulation ofLicnin Enzvme 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 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 (Southeron SG and 3o Deverall BJ. Plant Path. 39:223-230, 1990), CGT (Vellekoop P et FEBS, 330:36-40, 1993), PNL (Espin CJ et al-, Phytochemistry 44:17-22, 1997) and LAC (Bao W et al., Science, 260:672-674, 1993). The data shown in the column labelled "Enzyme" in Table shows the average enzyme activity from replicate measures for all plant lines assayed, expressed as a percent of enzyme activity in empty vector-transformed control plants, The range of variation is shown in parentheses.

COMS ID No: ARCS-198654 Received by IP Australia: Time 13:23 Date 2008-07-17 17.Jul. 2008 14:34 BALDWINS 0064 4 4736712 No.3982 P. 48 O 00 S- 41 STable Transgene orientation no. of lines enzyme control na 3 100 PAL sense 5 87 (60-124) R PAL anti-sense 3 53 (38-80) SCGT anti-sense 1 89 C 0to PNL ant-sense 6 144 (41-279) o LAC sense 5 7 (16-240) tC LAC anri-sense 11 64 (14-106) 00 All of the transformed plant lines, except the PNL anti-sense transformed plant lines, showed average lignin enzyme activities which were significantly lower than the activities observed in empty vector-transformed control plants. The most diamatic effects on lignin enzyme activities were seen in the PAL anti-sense transformed plant lines in which all of the lines showed reduced PAL activity and in the LAC anti-sense transformed plant lines which showed as little as 14% of the LAC activity in empty vector-transformed control plant lines.

Examnle 8 Functional Identification ofLijnin Biosynthetic Genes Sense constructs containing polynucleotides 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 Pinus radiata, and OMT (SEQ ID NO: 23 and 24), CCR (SEQ 1D NO: 26-28), CGT (SEQ ID NO: 31 and 33) and POX (SEQ ID NO: 42 and 44) from Eucalyptus grandis were inserted into the commercially available protein expression vector, pProEX-1 (Gibco BRL). The resultant constructs were transformed into E. coli XLl-Blue (Stratagene) which were then induced to produce recombinant protein by the addition of IPTG. Purified proteins were produced for the Pinus OVMT and 4CL constructs and the Eucalyptus OMT and POX constructs using Ni column chromatography (Janknecht R et ai., Proc. NatL 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 verification of the enzymatic activity of a Pinus radiata 4CL gene (SEQ ID NO: 56) and a Pinus radicta OMT gene (SEQ ID NO: 53), are shown in Table 6. For the 4CL enzyme, one unit equals the quantity of protein required to convert the substrate into product at the rate of COMS ID No: ARCS-198654 Received by IP Australia: Time 13:23 Date 2008-07-17 17.Ju 2008 14:34 BALDWINS 0064 4 4736712 No.3982 P. 49 O 00 o 42 S0,1 absorbance units per minute. For the OMT enzyme, one unit equals the quantity of Z protein required to convert 1 pmoie of substrate to product per minute.

STable 6 purification total nil total mg total units yield fold T ransgene steo extract protein activity activity purification 4CL crude 10ml 51 mg 4200 100 1 Cf o Ni column 4 ml 0.84 mg 3680 88 53 OMT crude 10 ml 74 mg 4600 100 1 0 Ni column 4 ml 1.2 mg 4487 98 CN The data shown in Table 6 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 protei prearations fiom coli transformed with enpty vector show no activity in either the 4CL or the OMT enzyme assay.

Example 9 Demonstration of the Presence Absence of Uniue Seauence Identifiers in Plants Transgenic tobacco plants were created using unique identifier sequences which are not found in tobacco. The unique identifier sequences inserted were isolated from Pinus radiata, SEQ ID NO: 402, and Eucalyptus grandis, SEQ ID NO: 403. The unique identifier sequences were inserted into Agrobacterium tumefaciens LBA4301 (provided as a gift by Dr. C. Kado, University of California, Davis, CA) by direct transformation using published methods (see, An 0, Ebert PR, 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 unique identifier sequences in the Agrobacerumn transgenic constructs were verified by restriction digestion and DNA sequencing.

Tobacco (Nicoiana tabacum cv. Samsun) leaf sections were transformed using the method of Horsch et al. (Science, 227:1229-1231, 1985). Three independent transformed plant lines were established for each unique sequence identifier used. Two empty-vector control plant lines were established using an empty gene transfer- vector which lacked a unique sequence identifier.

COMS ID No: ARCS-198654 Received by IP Australia: Time 13:23 Date 2008-07-17 17-.J 2008 14:34 BALDWINS 0064 4 4736712 No.3982 P. O 00 o 43- The uniqueness of the sequence identifiers was assayed using Southern blot analyses Sto test for the presence of the sequence identifier in the genome'of the plants. If the sequence identifier is unique and therefore useful as a tag, then the sequence identifier should be clearly absent in plants which have not been tagged and it should be clearly present in plants which c 5 have been tagged. In the present example, the unique identifiers would be expected to be absent in the empty-vector transformed control plants. The unique identifier would be expected to be present m the transgenic plants transformed with the unique sequence C, identifiers.

00 o Genomic DNA was prepared from empty-vector transformed control plants and plants S0 transformed with unique sequence identifiers using the cetyltrimethyl-ammonium bromide (CTAB) extraction method of Murray and Thompson (Nucleic Acids Research 8:4321-4325, 1980). The DNA samples were digested with the restriction enzyme EcoRJ in the case of the plants transformed with the Pinus unique sequence identifier (SEQ ID NO: 402) and the restriction enzyme XbaI in the case of the plants transformed with the Eucalyptus unique sequence identifier (SEQ ID NO: 403). The DNA fragments produced in the restriction digests were resolved on a 1% agarose gel; the left panel of Figure 2 and the right panel of Figure 2 show the DNA fragment patterns of the DNA samples from the Pinus and Eucalyptus experiments, respectively.

After the agarose gel electrophoresis step, the DNA samples were transferred to Hybond-N+ brand nylon membranes (Amersham Life Science, Little Chalfont, Bucldngamshire, England) using methods established by Southern (J MoL Bio. 98:503- 517). The nylon membranes were probed with radioactively-Iabeled probes for the unique sequence identifiers identified above and washed at high stringency (final wash: 0.5 X salt sodium citrate buffer (SSC) plus 0.1% sodium dodecy] sulfate (SDS), 15 minutes at 65 0

C).

The hybridisation of the probes to complementary sequences in the genomic DNA samples was detected using auto-radiography. The results are shown in Figures 3 and 4.

Figure 3 (corresponding to the left panel of Figure 2) shows the hybridisation pattern detected in the Southern blot analysis using a probe derived from the Pinus sequence identifier (SEQ ID NO: 402). Lanes A-B contain DNA samples from empty-vector transformed control plants and lanes C-E contain DNA from plants transformed with SEQ ID NO: 402. There is no hybridization in lanes A-B indicating that SEQ ID NO: 402 is not present in empty-vector transformed tobacco plants; that is, SEQ ID NO: 402 is a unique tag suitable for unambiguous marking of tobacco plants. There is strong hybridisation in lanes COMS ID No: ARCS-198654 Received by IP Australia: Time 13:23 Date 2008-07-17 17.Jul. 2008 14:35 BALDWINS 0064 4 4736712 No.3982 P. 51 00 O 0- 44 CA C-E indicating that the plants which received SEQ ID NO: 402 via transformation have been Z clearly and unambiguously tagged with the unique sequence contained in SEQ ID NO: 402.

Figure 4 (corresponding to the right panel of Figure 2) shows the hybridization pattern detected in the Southern bloi analysis using a probe derived from the Eucalyptus sequence identifier (SEQ ID NO: 403). Lanes A-B contain DNA samples from empty-vector transformed control plants and Janes C-E contain DNA from plants transformed with SEQ ID NO; 403. There is no hybridisation in lanes A-B indicating that SEQ ID NO: 403 is not 0 present in empty-vector transformed tobacco plants; that is, SEQ ID NO: 403 is a unique tag 00 suitable for unambiguous marking of tobacco plants. There is strong hybridisation in lanes C-E indicating that the plants which received SEQ ID NO: 403 via transformation have been C clearly and unambiguously tagged with the unique sequence contained in SEQ ID NO; 403.

The present example clearly demonstrates the utility of the sequences disclosed in this specificafiorinfor the-Ppurpses ofunambiguously -tagging transgenic materials. A.unique sequence was selected from a large number of potential tags and shown to be absent in the genome of the organism to be tagged. The tag was inserted into the genome of the organism to be tagged and a well-established DNA detection method was used to clearly detect the unique sequence identifier used as the tag.

.Because of the sequence-specific detection methods used in the example, a user of the invention disclosed in this specification has both a high likelihood of finding a sequence identifier, amodg the list which has been disclosed, which will be useful for tagging any given organism and an unequivocal method for demonstrating that a tagged organism could only have acquired a given tag through the deliberate addition of the unique sequence to the genome of the organism to.be tagged. If the user of this invention maintains the precise sequence of the tag used in a given organism as a secret, then any disputes as to the origin and history of the organism can be unambiguously resolved using the tag detection techniques demonstrated in the present example.

SEQ ID NOS: 1-403 are set out in the attached Sequence Listing. The codes for nicieotide sequences used in the attached Sequence Listing, including the symbol conform to WIPO Standard ST.25 (1998), Appendix 2, Table I.

All references cited herein, including patent references and non-patent publications, are hereby incorporated by reference in their entireties.

While in the foregoing specification this invention has been described in relation to certain preferred embodiments, and many details have been set forth for purposes of COMS ID No: ARCS-198654 Received by IP Australia: Time 13:23 Date 2008-07-17 17-M, 20H 14:35 20% 4:3~ BALDWJNS 0064 4 4736712 No.3932 P. 52 ilhstration. it will be apparent to those skilled in7 the art that the inrventIon IS SuscenDtible tCo additional einibodinenzts and thjai certain of the details duscribed herein mnay be varied considerably witho~ut departing fromn the basic principles of the invention.

COMS ID No: ARCS-198654 Received by IP Australia: Time 13:23 Date 2008-07-17

Claims (5)

17.Jul. 2008 15:00 BALD INS 0064 4 4736712 No.3983 P. 1/70 00 -46- 0 3 The Claims Defining the Invention Arc As Follows: 1. An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of: sequences recited in SEQ ID NOs: 89, 91, 93-101, 104-123, 126-146,

148- 152, 164-168,

172-192,

201-264, and 350-3 75 or portions thereof; oc complements of the sequences recited in 0( reverse complements of the sequences recited in reverse sequences of the sequences recited in nucleotide sequences producing an Expectation value of 0.01 or less when compared to a sequence recited in one of the sequences of(1); nucleotide sequences having at least 50% identity to a sequence recited in one of the sequences of(1); nucleotide sequences having at least 75% identity to a sequence recited in one of the sequences of(1); 1s nucleotide sequences having at least 90 identity to a sequence recited in one of the sequences of(1); nucleotide sequences that hybridize to a sequence recited in one of the sequences of the complements, reverse complements, and reverse sequences thereof under stringent hybridization conditions; (10) nucleotide sequences that arc 2 00-mers of a sequence recited in one of the sequences of the complements, reverse complements and reverse sequences thereof; 67702 v I/C COMS ID No: ARCS-198654 Received by IP Australia: Time 13:23 Date 2008-07-17 17 Jul. 2008 15:01 BALDWINS 0064 4 4736712 No.3983 P. 2/70 00 -47- 0 0 ci S(11) nucleotide sequences that are 100-mers of a sequence recited in one of the sequences of the complements, reverse complements and reverse sequences thereof; n (12) nucleotide sequences that are 4 0-menrs of a sequence recited in one of the sequences of the complements, reverse complements and reverse sequences Sthereof; 00 o (13) nucleotide sequences that are degeneratively equivalent to a sequence recited in one of the sequences of(I); and (14) nueleotide sequences that are alllic variants of a sequence recited in onc of the sequences of wherein said sequences encode an enzyme involved in the lignin biosynthetic pathway. 2. An isolated oligonucleotide probe or primer comprising at least contiguous residues complementary to 20 contiguous residues of a nucleotide sequence recited in one ofSEQ. ID NOs: 89, 91, 93-101, 104-123, 126-146, 148- 152,164-168, 172-192,201-264, and 350-375. 3. A kit comprising a plurality of oligonucleotide probes or primers of claim 2. 4. A construct comprising a polynucleotide of claim 1. A construct comprising, in the direction: a gene promoter sequence; a polynucleotide sequence comprising at least one of the following: a polynucleotide coding for at least a functional portion of a polypeptide encoded by a nucleotide sequence of claim 1; and a polynucleotide comprising a non-coding region of a gene coding 67702 vt/IC COMS ID No: ARCS-198654 Received by IP Australia: Time 13:23 Date 2008-07-17 17.Jul 2008 15:01 BALDWINS 0064 4 4736712 No.3983 P. 3/70 00 43 o0 4 8 0 ci Sfor a polypeptide encoded by a nucleotide sequence selected from rthe group consisting of sequences recited in claim 1; and a gene termination sequence. 6. The construct of claim 4 or claim 5, wherein the polynucleotide is in a Ssense orientation. 0 ci 0 7. The construct of claim 4 or claim 5, wherein the polynucleotide is in an 0 antisense orientation. 8. The construct of claim 5, wherein the gene promoter sequence is functional in a plant host to provide for transcription in xylem. 9. A transgenic plant cell comprising a construct of any one of claims 4 to 8. A plant comprising a transgenic plant cell according to claim 9, or a part or propagule or progeny thereof. 11. A method for modulating one or more of the lignin content, the lignin composition and the lignin structure of a plant, comprising stably incoiporating into the genome of the plant a polynucleotide of claim 1. 12. The method of claim II wherein the plant is selected from the group consisting of eucalyptus and pine species. 13. The method of claim 11 or claim 12 comprising stably incorporating into the genome of the plant a construct of any one of claims 5 to 8. 14. A method for producing a plant having one or more of altered lignin content, altered lignin composition and altered lignin structure, comprising: 67702v I/DC COMS ID No: ARCS-198654 Received by IP Australia: Time 13:23 Date 2008-07-17 17.Jul. 2008 15:01 BALDWINS 0064 4 4736712 No.3983 P. 4/70 00 .49.- 0 0 S transforming a plant cell with a construct of any one of claims 5 to 8 to provide a transgcnic cell; and cultivating the transgenic cell under conditions conducive to regeneration and mature plant growth. A method for modifying the activity of a polypeptide involved in a lignin o biosynthetic pathway in a plant comprising stably incorporating into the genome c-,l 0 0S of the plant a construct of any one of claims 5 to 8. 16. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of: sequences of SEQ ID NOs: 267-275, 284-347, and

376-401; sequences having at least 50% identity to a sequence recited in sequences having at least 75% identity to a sequence recited in and sequences having at least 90% identity to a sequence recited in 17. An isolated polypeptide encoded by an isolated polynucleotide sequence of claim 1. 18. A wood obtained from a transgenic tree which has been transformed with the construct of any one of claims 5 to 8. 19. A wood pulp obtained from a transgenic tree which has been transformed with the construct of any one of claims 5 to 8. 20. A method of making wood or wood pulp, comprising: transforming a plant with the construct of any one of claims 5 to 8; culturing the transformed plant under conditions that promote growth of a plant; and obtaining wood or wood pulp from the plant. 67702v I/DC COMS ID No: ARCS-198654 Received by IP Australia: Time 13:23 Date 2008-07-17 17.Jul. 2008 15:01 BALDWINS 0064 4 4736712 No.3983 P. 5/70 00 0 0 ci 3 21. A method for modifying the activity of a polypeptide involved in a lignin biosynthetic pathway in a plant, comprising introducing into cells of the plant antisense corresponding to a polynucleotide of claim 1, or portions thereof encoding a polypeptide having activity in a lignin biosynthetic pathway of a plant thereby inhibiting expression of a polypeptide Sencoded by the polynucleotide. 0 ci 0 22. The isolated polynucleotide according to claim 1, substantially as o described herein with reference to Figures 1 to 5, Examples 1 to 9 and/or the ci Sequence Listing thereof. 23. The isolated oligonucleolide probe or primer according to claim 2 or the kit according to claim 3, substantially as described herein with reference to Figures I to 5, Examples 1 to 9 and/or the Sequence Listing thereof. 24. The construct according to any one of claims 4 to 8, substantially as described herein with reference to Figures 1 to 5, Examples I to 9 and/or the Sequence Listing thereof. 25. The transgcnic plant cell according to claim 9, substantially as described herein with reference to Figures 1 to 5, Examples 1 to 9 and/or the Sequence Listing thereof. 26. The plant according to claim 10, substantially as described herein with reference to Figures 1 to 5, Examples 1 to 9 and/or the Sequence Listing thereof. 27. The method according to any one of claims 11 to 14, substantially as described herein with reference to Figures 1 to 5, Examples 1 to 9 and/or the Sequence Listing thereof. 67702 vl/DC COMS ID No: ARCS-198654 Received by IP Australia: Time 13:23 Date 2008-07-17 17.Jul 2008 15:01 BALDWINS 0064 4 4736712 No.3983 P. 6/70 00 -51 0 28. The method according to claim 15, substantially as described herein with reference to Figures 1 to 5, Examples I to 9 and/or the Sequence Listing thereof. 29. The isolated polypeptide according to claim 16 or claim 17, substantially as described herein with reference to Figures 1 to 5, Examples I to 9 and/or the eSequence Listing thereof. 00 30. The wood according to claim 18 or the wood pulp according to claim 19, osubstantially as described herein with reference to Figures 1 to 5, Examples I to 9 and/or the Sequence Listing thereof. 31. The method according to claim 20 or claim 21, substantially as described herein with reference to Figures 1 to 5, Examples I to 9 and/or the Sequence Listing thereof. 67"702 1/1.C COMS ID No: ARCS-198654 Received by IP Australia: Time 13:23 Date 2008-07-17