CA2578311A1 - Altering wood density - Google Patents

Altering wood density Download PDF

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CA2578311A1
CA2578311A1 CA 2578311 CA2578311A CA2578311A1 CA 2578311 A1 CA2578311 A1 CA 2578311A1 CA 2578311 CA2578311 CA 2578311 CA 2578311 A CA2578311 A CA 2578311A CA 2578311 A1 CA2578311 A1 CA 2578311A1
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Paulo Arruda
Fabio Papes
Isabel Rodrigues Gerhardt
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Monsanto do Brasil Ltda
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Alellyx SA
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8255Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving lignin biosynthesis
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88Lyases (4.)
    • DTEXTILES; PAPER
    • D21PAPER-MAKING; PRODUCTION OF CELLULOSE
    • D21HPULP COMPOSITIONS; PREPARATION THEREOF NOT COVERED BY SUBCLASSES D21C OR D21D; IMPREGNATING OR COATING OF PAPER; TREATMENT OF FINISHED PAPER NOT COVERED BY CLASS B31 OR SUBCLASS D21G; PAPER NOT OTHERWISE PROVIDED FOR
    • D21H17/00Non-fibrous material added to the pulp, characterised by its constitution; Paper-impregnating material characterised by its constitution
    • D21H17/005Microorganisms or enzymes
    • DTEXTILES; PAPER
    • D21PAPER-MAKING; PRODUCTION OF CELLULOSE
    • D21CPRODUCTION OF CELLULOSE BY REMOVING NON-CELLULOSE SUBSTANCES FROM CELLULOSE-CONTAINING MATERIALS; REGENERATION OF PULPING LIQUORS; APPARATUS THEREFOR
    • D21C5/00Other processes for obtaining cellulose, e.g. cooking cotton linters ; Processes characterised by the choice of cellulose-containing starting materials
    • D21C5/005Treatment of cellulose-containing material with microorganisms or enzymes
    • DTEXTILES; PAPER
    • D21PAPER-MAKING; PRODUCTION OF CELLULOSE
    • D21HPULP COMPOSITIONS; PREPARATION THEREOF NOT COVERED BY SUBCLASSES D21C OR D21D; IMPREGNATING OR COATING OF PAPER; TREATMENT OF FINISHED PAPER NOT COVERED BY CLASS B31 OR SUBCLASS D21G; PAPER NOT OTHERWISE PROVIDED FOR
    • D21H11/00Pulp or paper, comprising cellulose or lignocellulose fibres of natural origin only
    • D21H11/16Pulp or paper, comprising cellulose or lignocellulose fibres of natural origin only modified by a particular after-treatment
    • D21H11/20Chemically or biochemically modified fibres
    • DTEXTILES; PAPER
    • D21PAPER-MAKING; PRODUCTION OF CELLULOSE
    • D21HPULP COMPOSITIONS; PREPARATION THEREOF NOT COVERED BY SUBCLASSES D21C OR D21D; IMPREGNATING OR COATING OF PAPER; TREATMENT OF FINISHED PAPER NOT COVERED BY CLASS B31 OR SUBCLASS D21G; PAPER NOT OTHERWISE PROVIDED FOR
    • D21H27/00Special paper not otherwise provided for, e.g. made by multi-step processes
    • D21H27/002Tissue paper; Absorbent paper

Abstract

In angiosperm and gymnosperm plants, overexpressing a SAMdc nucleotide sequence can decrease lignin content and, for plants with woody tissue, increase wood density.

Description

Atty. Dkt. No.: 059994-0114 ALTERING WOOD DENSITY

FIELD OF THE INVENTION

[00011 The present invention relates to the field of molecular biology and regulation of wood composition. Thus, the invention relates, inter alia, to methodology and constructs for increasing wood density in plants.

BACKGROUND OF THE INVENTION
[0002] Wood Quality, as used by the pulp and paper industries, refers to a series of wood components that, at the end of the processing, affect cellulose yield.
The most studied wood components affecting wood quality include lignin content, the proportion of monolignols siryngil and guaiacyl (S/G), the module of elasticity, spiral grain, fiber characteristics and wood density. T. Markussen et al., Wood Density Silvae Genetica 53: 45- 50 (2004). Of these components, lignin content and wood density significantly impact cellulose yield.

Lignin Content [0003] Lignin is one of the major products of the general phenylpropanoid pathway, and it is one of the most abundant organic molecules in the biosphere.
Lignin accounts for 20-30% of the dry weight of trees and through a process called lignification, lignin is deposited in the cell walls of supporting and conductive tissues, thereby providing rigidity to the wood and structural integrity to tracheary elements.
Baucher et al., Crit. Rev. Biochem. Mol. Biol. 38: 305-50 (2003).
Lignification also occurs following microbial infection or wounding and protects tissues from pathogen penetration. Baucher et al., (2003); Boerjan et al., Annu. Rev. Plant. Biol.
54: 519-46 (2003); Crawford, LIGNIN BIODEGRADATION AND TRANSFORMATION, New York: John Wiley and Sons (1981).
[0004] Lignin's resistance to degradation significantl.y limits the use of lignocellulosic materials, as lignin must be removed during pulping and papermaking and this requires environmentally hazardous chemicals. Baucher et al., (2003);
WASH1814745.1 WASH_1815926_.1 Atty. Dkt. No.: 059994-0114 Whetten et al., Forest Ecol. Management 43: 301 (1991). Current research efforts aim to develop trees with reduced lignin content, thereby reducing the amount of chemicals needed for kraft pulping. Baucher et al., (2003); Sederoff et al., GENETIC
ENGINEERING OF PLANT SECONDARY METABOLISM, New York, Plenum Press (1994).
[0005] Lignins are the result of dehydrogenative polymerization of monolignols, notably p-coumaryl, coniferyl and synapyl alcohols. Reviewed in Boerjan et al., Annu. Rev. Plant. Biol. 54: 519-46 (2003). Different plant species or cell types harbor lignin polymers composed of varying proportions of these three monolignols.
For example, gymnosperm lignin is primarily composed of guaiacyl (coniferyl-derived) units, whereas angiosperm lignin is primarily composed of guaiacyl and syringyl (synapyl-derived) units. Grass lignin, on the other hand, is a mixture of guaiacyl, syringyl and p-hydroxylphenyl (coumaryl-derived) units. Campbell and Sederoff, Plant. Physiol. 110: 3-13 (1996). It is well known that the monomeric composition of lignin has a significant effect on its chemical degradation during industrial pulping.
Baucher et al., Plant Physiol. 112: 1479-1490 (1996); O'Connell et al., Transgenic Res. 11: 495-503 (2002); Baucher et al., Critical Reviews in Biochemistry and Molecular Biology 38: 305-50 (2003).
[0006] Several steps in the monolignol biosynthesis pathway, leading to lignin synthesis, represent SAM-dependent methylation reactions, evidencing the importance of this methionine-derived substrate in lignin biosynthesis. SAM is synthesized from methionine by the action of one or more SAM synthetase isoforms and is used as a cofactor in many processes in plant cells besides lignification, such as DNA methylation and ethylene, biotin and polyamine biosynthesis. Ravanel et al., Proc. Nati. Acad. Sci. USA 95: 7805-7812 (1998).

Wood density [0007] In the assessment of raw-material quality for pulping, wood density is another important parameter. Wood density significantly influences the yield and quality of fibrous and solid wood products, as well as strength, machinability, conversion, wearability, and paper yield. Bamber and Burley, TrIE WOOD

WASH_1814745.1 WASH 1815926.1 Atty. Dkt. No.: 059994-0114 PROPERTIES OF RADIATA PItvE, Commonwealth Agricultural Bureau. Slough, p. 84 (1983). High wood densities are advantageous because they correspond to higher pulp yields on a raw-material volume basis, and to a better use of digestor capacity.
From the vantage point of forest production, high wood density combined with high volume growth maximizes production on the unit area basis. Miranda et al., Forest Ecology and Management 149: 235-40 (2001).
[0008] While wood density is a critical factor in the profitability of kraft pulp production, increasing wood density is difficult because it is a complex trait that is not easily managed for breeding purposes. For temperate softwood, the average ring density depends on the earlywood and latewood proportion and the relative densities of each of them. Thus, improved wood density is the result of various combinations of components that could be changed by manipulating one or more components.
Louz, Ann. For. Sci. 60: 285-94 (2003). Further adding to the difficulties associated with increasing wood density, hardwood tree improvement is a slow process because of the lengthy time needed for breeding a single generation. This process is made even more difficult by the changes that occur during the transition from juvenility to maturity.
[0009] Although the wood density is a complex trait, it presents great variations between trees as well as high heritability and reduced Genotype x Environment interactions. In analyzing wood densities in different species, it has been shown that wood density has a strong genetic component. Louzada and Fonseca, Ann. For.
Sci.
59: 867-73 (2002).
[0010] There is consensus amongst the forest breeding community that if one could develop methods that allow early selection at the individual level, this would be of considerable value for increasing the genetic gain per unit time. In this regard, current research efforts are focused on identifying molecular markers that cosegregate with complex traits, such as wood basic density. Grattapaglia et al., Genetics 144:
1205-14 (1996). Most of these studies have associated quantitative trait loci (QTLs) with juvenile wood density. For example, four markers showed a consistent WASH1814745.1 WASH_1815926.1 Atty. Dkt. No.: 059994-0114 association with juvenile wood density in segregating populations of Pinus radiata.
Devey et al., Theor Appl Genet 108: 516-24 (2004).

[00111 Accordingly, there is a continuing need to identify genes the expression of which can be affected to increase wood density and decrease lignin in plants, in particular, woody tree species.

SUMMARY OF THE INVENTION

[0012] In one aspect, the invention provides a method for increasing wood density in a plant, comprising (a) introducing into a plant cell a nucleic acid construct comprising, in the 5' to 3' direction, a xylem-preferred promoter operably linked to a SAMdc nucleotide sequence; (b) regenerating transgenic plants from said plant cell;
and (c) selecting a transgenic plant having reduced vessel area and reduced vessel density relative to a control plant. In one embodiment, the plant is an angiosperm.
[0013] In another embodiment, the plant is a gymnosperm. In further embodiments, the plant is Eucalyptus, Populus, and Pinus. In further embodiments, wood pulp and wood fiber are obtained from the transgenic plant.

[00141 In another aspect, the invention provides a method for increasing wood density and decreasing lignin content in a plant, comprising: (a) introducing into a plant cell a nucleic acid construct comprising, in the 5' to 3' direction, a xylem-preferred promoter operably linked to a SAMdc nucleotide sequence; (b) regenerating transgenic plants from said plant cell; and (c) selecting a transgenic plant having increased wood density and decreased lignin content relative to a control plant. In one embodiment, the plant is an angiosperm. In another embodiment, the plant is a gymnosperm. In another embodiment, the plant is Eucalyptus, Populus, and Pinus.
[0015] In another aspect, the invention provides a method of making wood pulp, comprising (a) introducing into a plant cell a nucleic acid construct comprising, in the 5' to 3' direction, a xylem-preferred promoter operably linked to a SAMdc nucleotide sequence; (b) regenerating transgenic plants from said plant cell; (c) selecting a transgenic plant having increased wood density and decreased lignin content relative WASH1814745.1 WASH_18159267.1 Atty. Dkt. No.: 059994-0114 to a control plant; and (d) producing wood pulp from said transgenic plant. In one embodiment, the xylem-preferred promoter is selected from the group consisting of TUB gene promoter, LTP gene promoter, 4CL gene promoter, and C4H gene promoter.

[0016] In another aspect, the invention provides method of making wood fiber, comprising (a) introducing into a plant cell a nucleic acid construct comprising, in the 5' to 3' direction, a xylem-preferred promoter operably linked to a SAMdc nucleotide sequence; (b) regenerating transgenic plants from said plant cell;
(c) selecting a transgenic plant having increased wood density and decreased lignin content relative to a control plant; and (d) producing wood fiber from said transgenic plant.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIGURE 1 illustrates the lignin biosynthesis pathway, showing in details the compounds, reactions and enzymes involved in synthesis of monolignols, the building blocks of the three kinds of lignin, represented at the bottom of the picture as p-hydroxyphenyl, guaiacyl and syringyl lignins.

[0018] FIGURE 2 schematically illustrates the plant expression plasmidial vector pALELLYX-ATG of the invention comprising a cambium/xylem preferred promoter driving the expression of a SAMdc nucleotide sequence of the invention.

[0019] FIGURE 3 schematically illustrates the plant expression plasmidial vector pALELLYX-Nt of the invention comprising a cambium/xylem preferred promoter driving the expression of a SAMdc nucleotide sequence from Nicotiana tabacum (tobacco).

[0020] FIGURE 4 schematically illustrates the plant expression plasmidial vector pALELLYX-Pdl of the invention comprising a cambium/xylem preferred promoter driving the expression of SAMdc nucleotide sequence coding for SAMdc 1 from Populus deltoides.

WASH_1814745.1 WASH_1815926.1 Atty. Dkt. No.: 059994-0114 100211 FIGURE 5 schematically illustrates the plant expression plasmidial vector pALELLYX-Pd2 of the invention comprising a cambium/xylem preferred promoter driving the expression of SAMdc nucleotide sequence coding for SAMdc 2 from Populus deltoides.

[0022] FIGURE 6 schematically illustrates the plant expression plasmidial vector pALELLYX-Pd3 of the invention comprising a cambium/xylem preferred promoter upstream of a SAMdc genomic sequence from Populus deltoides.

[0023] FIGURE 7 schematically illustrates the plant expression plasmidial vector pALELLYX-At of the invention comprising a cambium/xylem preferred promoter driving the expression of a SAMdc nucleotide sequence from Arabidopsis thaliana (thale cress).

[0024] FIGURE 8 schematically illustrates the plant expression plasmidial vector pALELLYX-Xa of the invention comprising a cambium/xylem preferred promoter upstream of an open reading frame coding for SAMdc from the plant pathogen Xanthomonas axonopodis.

[0025] FIGURE 9 schematically illustrates the plant expression plasmidial vector pALELLYX-Ds of the invention comprising a cambium/xylem preferred promoter driving the expression of a SAMdc nucleotide sequence from Datura stramonium.
[0026] FIGURE 10 schematically illustrates the plant expression plasmidial vector pALELLYX-Osl of the invention comprising a cambium/xylem preferred promoter driving the expression of a nucleotide sequence coding for SAMdc 1 from Oryza sativa (rice).

[00271 FIGURE 11 schematically illustrates the plant expression plasmidial vector pALELLYX-Os2 of the invention comprising a cambium/xylem preferred promoter driving the expression of a nucleotide sequence coding for SAMdc 2 from Oryza sativa (rice).

WASH_1814745.1 WASH_1815926.1 Atty. Dkt. No.: 059994-0114 [0028] FIGURE 12 shows hand-sectioned unfixed stems (rosette base level) of Arabidopsis thaliana stained with the lignin-specific dye phloroglucinol. (A) Control non-transgenic plant TUB-SAMdc-Nt22; (B) Transgenic plant TUB-SAMdc-Nt12, which exhibits a high transgene expression level (FIG. 13); (C) Control non-transgenic plant TUB-SAMdc-NtO1; (D) Transgenic plant TUB-SAMdc-Nt09. Insets represent lower magnification images of the respective stem cuts.

[0029] FIGURE 13 exhibits a semi-quantitative RT-PCR experiment showing the transgene expression level (SAMdc) relative to the expression level of a control gene (APTR, adenine phosphoribosyltransferase) in transgenic (PCR +) and control non-transgenic (PCR-) TUB-SAMdc-Nt Arabidopsis thaliana plants.

[0030] FIGURE 14 (A) and (B) show hand-cut unfixed stem sections of 3 month-old Nicotiana benthamiana specimens, stained with the lignin-specific dye phloroglucinol in a control non-transgenic plant (A) and in a transgenic plant SAMdc-Nt30 (B), which exhibits a high transgene expression level. (D) shows a homozygous transgenic plant in the T2 segregating population from the 4CL-SAMdc-Nt30 T1 transformant compared to a control non-transgenic sibling plant (C).
Note the decrease in vessel element number and size in (B) and the general decrease in lignin staining in (D).

[0031] FIGURE 15 shows the relationship between Eucalyptus clones wood density and the number of vessel elements in the xylem.

[0032] FIGURE 16 shows hand-sectioned unfixed stems (base level) of a Tl transgenic Nicotiana benthamiana plants (line 11 B) transformed with the plant expression plasmidial vector pALELLYX-Nt stained with the lignin-specific dye phloroglucinol. (A) sdcsdc genotype; (B) SDCSDC genotype; (C) reduction on the vessel elements number of the TI transgenic plants (line 11B).

[0033] FIGURE 17 shows the area of vessel elements (mean of 100 vessel elements) of three genotypes of a Tl transgenic Nicotiana benthamiana plants (line I1B) transformed with the plant expression plasmidial vector pALELLYX-Nt. (A) WASH1814745.1 WASH 1815926_.1 Atty. Dkt. No.: 059994-0114 sdcsdc genotype; (B) SDCSDC genotype; (C) reduction on the area of vessel elements of the T 1 transgenic plants (line 11 B).

[0034] FIGURE 18 shows the percentage of xylem area occupied by vessel elements of three genotypes of a T1 transgenic Nicotiana benthamiana plants (line 11B) transformed with the plant expression plasmidial vector pALELLYX-Nt.

[0035] FIGURE 19 shows a cross-section of lateral shoot of 6 months old Eucalyptys plants. (A) is a cross-section of the transgenic event A. 12.3 transformed with the plant expression plasmidial vector pALELLYX-Pd; (B) is a cross-section of a wild-type plant.

[0036] FIGURE 20 shows the insoluble (A) and soluble (B) lignin content of three genotypes of a Tl transgenic Nicotiana benthamiana plants (line 11B) transformed with the plant expression plasmidial vector pALELLYX-Nt.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0037] The present inventors realized that overexpressing a polynucleotide encoding SAMdc, an enzyme that converts SAM into decarboxylated SAM, reduces the amount of SAM available for SAM-dependent methylation of intermediates in the monolignol biosynthesis pathway, thereby impairing lignin biosynthesis and deposition, without affecting other plant functions. Moreover, they discovered that overexpressing a polynucleotide encoding SAMdc in a plant causes a reduction in the number of vessels and vessel area in the plant, as well as a reduction in lignin content.
Because vessel number and area negatively correlate with wood density, overexpressing a gene encoding a SAMdc enzyme produces a plant with increased wood density and reduced insoluble lignin content. Thus, the present invention provides constructs and methodology for using a single gene to increase wood density and decrease lignin content in angiosperm and gymnosperm plants.

[0038] Accordingly, the present invention relates to methodology and compositions for reducing lignin and increasing wood density in plant tissues or cells, such as woody angiosperm and gymnosperm xylem, by controlling the activity of WASIi1814745.1 WASH_18159267.1 Atty. Dkt. No.: 059994-0114 SAMdc. Pursuant to this aspect of the invention, a polynucleotide encoding a SAMdc enzyme is introduced into plant cells or whole plants, which sequence, when expressed in vascular cells of angiosperms or xylary tracheids of gymnosperms, reduces the amount of SAM available for the monolignol biosynthesis pathway, leading to reduced lignin and increased wood density.

100391 Because the present inventors have discovered methodology and compositions for increasing wood density and decreasing lignin content, the inventive methods and compositions may be used, for example, to increase pulp and cellulose fiber yields during pulp and paper processing.

[0040] All technical terms in this description are commonly used in biochemistry, molecular biology and agriculture, respectively, and can be understood by those skilled in the field of this invention. Those technical terms can be found in:
MOLECULAR CLONING: A LABORATORY MANUAL, 3rd ed., vol. 1-3, ed. Sambrook and Russel, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001;
CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, ed. Ausubel et al., Greene Publishing Associates and Wiley-Interscience, New York, 1988 (with periodic updates);
SHORT
PROTOCOLS IN MOLECULAR BIOLOGY: A COMPENDIUM OF METHODS FROM CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY, 5h ed., vol. 1-2, ed. Ausubel et al., John Wiley & Sons, Inc., 2002; GENOME ANALYSIS: A LABORATORY MANUAL, vol. 1-2, ed.
Green et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1997.
[0041] Methods involving plant biology techniques are described herein and are described in detail in methodology treatises such as METHODS IN PLANT
MOLECULAR
BIOLOGY: A LABORATORY COURSE MANUAL, ed. Maliga et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1995. Various techniques using PCR
are described, e.g., in Innis et al., PCR PROTOCOLS: A GUIDE TO METHODS AND
APPLICATIONS, Academic Press, San Diego, 1990 and in Dieffenbach and Dveksler, PCR PRIMER: A LABORATORY MANUAL, 2 d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2003. PCR-primer pairs can be derived from known sequences by known techniques such as using computer programs intended for that purpose (e.g., Primer, Version 0.5, 1991, Whitehead Institute for Biomedical WASH1814745.1 WASH_18159267.1 Atty. Dkt. No.: 05 9994-0114 Research, Cambridge, MA). Methods for chemical synthesis of nucleic acids are discussed, for example, in Beaucage and Caruthers, Tetra. Letts. 22: 1859-1862 (1981) and Matteucci and Caruthers, J. Am. Chem. Soc. 103: 3185 (1981).

[0042] Restriction enzyme digestions, phosphorylations, ligations and transformations were done as described in Sambrook et al., MOLECULAR CLONING:
A
LABORATORY MANUAL, 2"d ed. (1989) Cold Spring Harbor Laboratory Press. All reagents and materials used for the growth and maintenance of bacterial cells were obtained from Aldrich Chemicals (Milwaukee, Wis.), DIFCO Laboratories (Detroit, Mich.), Invitrogen (Gaithersburg, Md.), or Sigma Chemical Company (St. Louis, Mo.) unless otherwise specified.

[0043] The term "expression" is used here to denote the production of the protein product encoded by a gene. "Overexpression" refers to the production of a gene product in transgenic organisms that exceeds levels of production in normal or non-transgenic organisms. As in conventional in the art, nucleotide sequences are denoted by italicized font (SAMdc), whereas polypeptide sequences are not italicized (SAMdc).

1. Increasing wood density by overexpressing SAMdc [0044] The present invention provides methodology and constructs for increasing wood density. Wood is essentially a matrix of cell walls and cellular air spaces from secondary xylem. Megraw, WOOD QUALITY FACTORS IN LOBLOLLY PINES, Tappi Press, Atlanta, p. 88 (1985). In this sense, wood density is determined by the cell wall thickness, the cross-sectional area of the lumen of the vessels, and the number of the vessels involved in water transport through the stem. Roderick and Berry, New Phytol. 149: 473 (2001); Preston et al., New Phytologist. 170: 807-18 (2006).
It has been shown in Eucalyptus and other angiosperm species that wood density negatively correlates with hydraulic conductivity and the cross-sectional area of the vessels.
Thomasa et al., Forest Ecology and Management 193: 157-65 (2004); Preston et al., New Phytologist, 170: 807-18 (2006).

WASH1814745.1 WASH_18159287.1 Atty. Dkt. No.: 059994-0114 100451 The influence of vessels on wood density can be decomposed into two components, vessel area and vessel density. "Vessel area" refers to the transverse lumen area of individual vessels. "Vessel density" refers to the number of vessels per transverse area. Vessel lumen area strongly affects the capacity of wood to conduct water. Zimmermann, XYLEM STRUCTURE AND THE ASCENT OF SAP. Berlin, Germany:
Springer-Verlag. (1983). Wider vessels are generally more vulnerable to cavitation as stems freeze and thaw, particularly when xylem water is under tension. Davis et al., American J. Botany 86: 1367-72 (1999). By contrast, the number of vessels in a given transverse area should have a relatively small effect on sapwood conductance.
These component traits, vessel area and vessel density, contribute to wood density by affecting the amount of lumen space in the wood.

II. Concurrently reducing lignin and increasing wood density by overexpressing SAMdc [0046] The present invention provides methodology and constructs for concurrently reducing lignin and increasing wood density in a plant, particularly a woody tree. Pursuant to this aspect of the invention, a nucleic acid sequence comprising a xylem-preferred promoter operably linked to a SAM decarboxylase (SAMdc) encoding sequence is introduced into a plant and transgenic plants are selected that have reduced lignin content and increased wood density. SAM is converted to decarboxylated SAM (dSAM), a precursor in the polyamine biosynthetic pathway, by the action of SAM decarboxylase (EC. number 4.1.1.50). Malmberg et al., Crit. Rev. Plant Sci. 17: 199-224 (1998).

100471 As described below, therefore, one way to reduce SAM levels in a plant cell and hence its lignin content is by overexpressing a SAMdc encoding gene in lignin-synthesizing tissues. Vascular tissues, which are the major lignin deposition sites in angiosperms, have low SAMdc gene expression levels, leading to the assumption that specific and localized SAMdc gene overexpression in these tissues would affect the local concentrations of SAM and thus impair lignin biosynthesis and deposition rates.
WASH 1814745.1 WASH 1815926.1 Atty. Dkt. No.: 059994-0114 SAMdcNucleotide and Polyaentide Sequences [0048] Illustrative SAMdc sequences include but are not limited to the sequences set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 24, and 26, respectively, as well as nucleic acid molecules comprised of variants of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 24, and 26, with one or more bases deleted, substituted, inserted, or added, which variant codes for a polypeptide with SAMdc enzyme activity.

[0049] A "variant" is a nucleotide or amino acid sequence that deviates from the standard, or given, nucleotide or amino acid sequence of a particular gene or protein.
The terms "isoform," "isotype," and "analog" also refer to "variant" forms of a nucleotide or an amino acid sequence. An amino acid sequence that is altered by the addition, removal, or substitution of one or more amino acids, or a change in nucleotide sequence, may be considered a "variant" sequence. The variant may have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties, e.g., replacement of leucine with isoleucine. A variant may have "nonconservative" changes, e.g., replacement of a glycine with a tryptophan.
Analogous minor variations may also include amino acid deletions or insertions, or both. Guidance in determining which amino acid residues may be substituted, inserted, or deleted may be found using computer programs well known in the art such as Vector NTI Suite (InforMax, MD) software. "Variant" may also refer to a "shuffled gene" such as those described in Maxygen-assigned patents.

[004750] Exemplary SAMdc polypeptide sequences include but are not limited to the sequences set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 25, and 27, as well as polypeptide sequences having one or more amino acids substituted, deleted, inserted, or added yet retain SAMdc enzyme activity. Additionally, multiple forms of SAMdc may exist, which may be due to post-translational modification of a gene product, or to multiple forms of the respective SAMdc genes. Sequences that have such modifications and that code for a SAMdc enzyme are included within the scope of the present invention.
WASH1814745.1 WASH_18159267.1 Atty. Dkt. No.: 059994-0114 [0051] Accordingly, "SAMdc nucleotide sequence" refers to a polynucleotide sequence encoding a polypeptide with SAMdc enzyme activity. In this description, moreover, the phrase "SAMdc enzyme activity" connotes a protein that catalyzes the conversion of SAM into decarboxylated SAM, and that can be assayed by measuring the release of 14COZ from S-adenosyl-L-[14C] as described, for example, in Hanfrey et al., J. Biol. Chem. 277: 44121-129 (2002). SAMdc protein levels in ground plant tissue can be quantified, using conventional protein assays, such as the Bradford method, Anal. Biochem. 72: 248-54 (1976), and enzyme activity typically is expressed as nanomole of C02/h/mg of protein.

Sequence Analysis [004852] Included in the category of "variant" sequences are sequences that hybridize to a reference SAMdc sequence. For the purpose of the invention, two sequences hybridize when they form a double-stranded complex in a hybridization solution of 6X SSC, 0.5% SDS, 5X Denhardt's solution and 100 g of non-specific carrier DNA. See Ausubel et al., supra, at section 2.9, supplement 27 (1994).
Sequences may hybridize at "moderate stringency," which is defined as a temperature of 60 C in a hybridization solution of 6X SSC, 0.5% SDS, 5X Denhardt's solution and 100 g of non-specific carrier DNA. For "high stringency" hybridization, the temperature is increased to 68 C. Following the moderate stringency hybridization reaction, the nucleotides are washed in a solution of 2X SSC plus 0.05% SDS
for five times at room temperature, with subsequent washes with 0.1X SSC plus 0.1% SDS
at 60 C for 1 hour. For high stringency, the wash temperature is increased to 68 C.
One with ordinary skill in the art can readily select such conditions by varying the temperature during the hybridization reaction and washing process, the salt concentration during the hybridization reaction and washing process, and so forth.
For the purpose of the invention, hybridized nucleotides are those that are detected using 1 ng of a radiolabeled probe having a specific radioactivity of 10,000 cpm/ng, where the hybridized nucleotides are clearly visible following exposure to X-ray film at -70 C for no more than 72 hours.
WASH_1814745.1 WASH 1815926.1 Atty. Dkt. No.: 059994-0114 [0053] The present application is directed to such nucleic acid molecules that are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%
identical to a nucleic acid sequence described in any of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 24, and 26. Preferred are nucleic acid molecules which are at least 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequence shown in any of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 24, and 26. Differences between two nucleic acid sequences may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.

[0054] As a practical matter, stating whether any particular nucleic acid molecule is at least 95%, 96%, 97%, 98% or 99% identical to a reference nucleotide sequence implicates a comparison made between two molecules. Such a comparison can be effected conventionally, using publicly available computer programs such as the BLASTN algorithm. See, e.g., Altschul et al., Nucleic Acids Res. 25: 3389-402 (1997).

Nucleic Acid Constructs [0055] In accordance with an aspect of the invention, a sequence that increases wood density and reduces lignin content is incorporated into a nucleic acid construct that is suitable for introduction into a plant or cell. Thus, such a nucleic acid construct can be used to overexpress SAMdc in a plant or plant cell.

[0056] The wood density and lignin content and composition of plant parts may be modified by introducing a nucleic acid construct according to the invention. The invention also provides plant cells containing such constructs; plants derived therefrom having modified SAMdc gene expression; and progeny of such plants.

[0057] Nucleic acid constructs according to the invention may comprise a base sequence of a minimum length to generate a mRNA and consequently a polypeptide retaining SAMdc enzymatic activity. For convenience, it will generally be found suitable to use sequences between about 100 and about 1000 bases in length but there WASH 1814745.1 WASH_181592671 Atty. Dkt. No.: 059994-0114 is no theoretical upper limit to the base sequence length. The preparation of such constructs is described in more detail below.

100581 As a source of the nucleic acid sequence for transcription, a suitable cDNA
or genomic DNA or synthetic polynucleotide may be used. Methods for the isolation of suitable SAMdc sequences are described, supra. Sequences coding for the whole, or substantially the whole, of the enzyme may thus be obtained. Suitable lengths of this DNA sequence may be cut out for use by means of restriction enzymes. When using genomic DNA as the source of a partial base sequence for transcription, it is possible to use either intron or exon regions or a combination of both.

100591 To obtain constructs suitable for modifying expression of SAMdc in plant cells, the cDNA sequence as found in the enzyme cDNA or the gene sequence as found in the chromosome of the plant may be used. Recombinant nucleic acid constructs may be made using standard techniques. For example, the nucleic acid sequence for transcription may be obtained by treating a vector containing said sequence with restriction enzymes to cut out the appropriate segment. The nucleic acid sequence for transcription may also be generated by annealing and ligating synthetic oligonucleotides or by using synthetic oligonucleotides in a polymerase chain reaction (PCR) to give suitable restriction sites at each end. The nucleic acid sequence then is cloned into a vector containing suitable regulatory elements, such as upstream promoter and downstream terminator sequences.

[00601 An important aspect of the present invention is the use of nucleic acid constructs wherein a SAMdc-encoding sequence is operably linked to one or more regulatory sequences, which drive expression of the SAMdc-encoding sequence in certain cell types, organs, or tissues without unduly affecting normal development or plant physiology.

[0061] "Promoter" connotes a region of DNA upstream from the start of transcription that is involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. A "constitutive promoter" is one that is active throughout the life of the plant and under most environmental conditions.
Tissue-WASH 1814745.1 WASH 1815926.1 Atty. Dkt. No.: 059994-0114 specific, tissue-preferred, cell type-specific, and inducible promoters constitute the class of "non-constitutive promoters". "Operably linked" refers to a functional linkage between a promoter and a second sequence, where the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence. In general, "operably linked" means that the nucleic acid sequences being linked are contiguous.

[0062] Promoters useful for expression of a nucleic acid sequence introduced into a cell to increase expression of SAMdc may be constitutive promoters, such as the cauliflower mosaic virus (CaMV) 35S promoter, or tissue-specific, tissue-preferred, cell type-specific, and inducible promoters. By using vascular system-specific, xylem-specific, or xylem-preferred promoters, for example, one can modify SAMdc activity specifically in many tissues such as vascular tissues, especially xylem. The use of a constitutive promoter in general affects enzyme levels and functions in all parts of the plant, while use of a tissue-preferred promoter permits targeting of the modified gene expression to specific plant parts, leading to a more controllable phenotypes.

100631 Thus, in using the invention it may be found convenient to use a promoter that will give expression during xylem development and/or xylem lignification, whereby the SAMdc enzyme would only be overproduced in the organ(s) or tissue(s) or cell type(s) in which its action is required. In this context, a nucleic acid sequence expressed under the control of a "xylem-preferred promoter" is present or more active in the xylem than other plant tissues. The category of xylem-preferred promoters, suitable for the present invention, includes but is not limited to the xylem-preferred tubulin (TUB) gene promoter, the xylem-preferred lipid transfer protein (LTP) gene promoter, and the xylem-preferred coumarate-4-hydroxylase (C4H) gene promoter.
[0064] The vectors of the invention also may contain termination sequences, which are positioned downstream of the nucleic acid molecules of the invention, such that transcription of mRNA is terminated, and polyA sequences added. Exemplary of such terminators are the cauliflower mosaic virus (CaMV) 35S terminator and the WASH 1814745.1 WASH_1815926.1 Atty. Dkt. No.: 05 9994-0114 nopaline synthase gene (Tnos) terminator. The expression vector also may contain enhancers, start codons, splicing signal sequences, and targeting sequences.

[0065] Expression vectors of the invention may contain a selection marker, by which transformed cells can be identified in culture. The marker may be associated with the heterologous nucleic acid molecule, i.e., the gene operably linked to a promoter. In this regard, the term "marker" refers to a gene encoding a trait or a phenotype that permits the selection of, or the screening for, a plant or cell containing the marker. In plants, for example, the marker gene will encode antibiotic or herbicide resistance. This allows for selection of transformed cells from among cells that are not transformed or transfected.

[0066J Examples of suitable selectable markers include adenosine deaminase, dihydrofolate reductase, hygromycin-B-phosphotransferase, thymidne kinase, xanthine-guanine phospho-ribosyltransferase, glyphosate and glufosinate resistance, and amino-glycoside 3'-O-phosphotranserase (kanamycin, neomycin and G418 resistance). These markers may include resistance to G418, hygromycin, bleomycin, kanamycin, and gentamicin. The construct may also contain the selectable marker gene Bar that confers resistance to herbicidal phosphinothricin analogs like ammonium gluphosinate. Thompson et al., EMBO J. 9: 2519-23 (1987). Other suitable selection markers are known as well.

[0067] Visible markers such as green florescent protein (GFP) may be used.
Methods for identifying or selecting transformed plants based on the control of cell division have also been described. See WO 2000/052168 and WO 2001/059086.

[0068) Replication sequences, of bacterial or viral origin, may also be included to allow the vector to be cloned in a bacterial or phage host. Preferably, a broad host range prokaryotic origin of replication is used. A selectable marker for bacteria may be included to allow selection of bacterial cells bearing the desired construct. Suitable prokaryotic selectable markers also include resistance to antibiotics such as kanamycin or tetracycline.

WASH 1814745.1 WASH 1815926_.1 Atty. Dkt. No.: 059994-0114 [0069] Other nucleic acid sequences encoding additional functions may also be present in the vector, as is known in the art. For instance, when Agrobacterium is the host, T-DNA sequences may be included to facilitate the subsequent transfer to and incorporation into plant chromosomes.

Plants for Genetic Engineering [0070) The present invention comprehends the genetic manipulation of angiosperm and gymnosperm plants for increasing wood density and decreasing insoluble lignin content via overexpressing a polynucleotide sequence that encodes SAMdc. In this regard, angiosperm refers to vascular plants having seeds enclosed in an ovary and are divided into dicotyledonous and monocotyledonous plants.
Gymnosperm refers to a seed plant that bears seed without ovaries.
Illustrative gymnosperms include conifers, cycads, ginkgos, and ephedras.

[0071] Genetically engineered (GE) encompasses any methodology for introducing a nucleic acid or specific mutation into a host organism. For example, a Eucalyptus plant is genetically engineered when it is transformed with a polynucleotide sequence that increases expression of a gene, such as SAMdc, and thereby increases wood density. In contrast, a Eucalyptus plant that is not transformed with a polynucleotide sequence is a control plant and is referred to as a "non-transformed" plant.

[0072] In the present context, the "genetically engineered" category includes "transgenic" plants and cells (see defmition, infra), as well as plants and cells produced by means of targeted mutagenesis effected, for example, through the use of chimeric RNA/DNA oligonucleotides, as described by Beetham et al., Proc. Natl Acad. Sci. USA 96: 8774-78 (1999), and Zhu et al., loc. cit. at 8768-73, or so-called "recombinagenic olionucleobases," as described in PCT application WO
03/013226.
Likewise, a genetically engineered plant or cell may be produced by the introduction of a modified virus, which, in turn, causes a genetic modification in the host, with results similar to those produced in a transgenic plant, as described herein.
See, e.g., U.S. patent No. 4,407,956. Additionally, a genetically engineered plant or cell may WASH -81a7a5.t WASH 1815926.1 Atty. Dkt. No.: 059994-0114 be the product of any native approach (i.e., involving no foreign nucleotide sequences), implemented by introducing only nucleic acid sequences derived from the host species or from a sexually compatible species. See, e.g., U.S. published application No. 2004/0107455.

[0073] "Plant" is a term that encompasses whole plants, plant organs (e.g.
leaves, stems, roots, etc.), seeds, differentiated or undifferentiated plant cells, and progeny of the same. Plant material includes, without limitation, seeds suspension cultures, embryos, meristematic regions, callus tissues, leaves, roots, shoots, stems, fruit, gametophytes, sporophytes, pollen, and microspores. The class of plants which can be used in the present invention is generally as broad as the class of higher plants amenable to genetic engineering techniques, including angiosperms, both monocotyledonous and dicotyledonous plants, as well as gymnosperms.

[0074] While any plant may be used, the present invention contemplates plants used in the pulp and paper industry. Preferably, the plants are woody trees, including, but not limited to, Eucalyptus species such as E. alba, E. albens, E.
amygdalina, E.
aromaphloia, E. baileyana, E. balladoniensis, E. bicostata, E. botryoides, E.
brachyandra, E. brassiana, E. brevistylis, E. brockwayi, E. camaldulensis, E.
ceracea, E. cloeziana, E. coccifera, E. cordata, E. cornuta, E. corticosa, E. crebra, E.
croajingolensis, E. curtisii, E. dalrympleana, E. deglupta, E. delegatensis, E. delicata, E. diversicolor, E. diversifolia, E. dives, E. dolichocarpa, E. dundasii, E.
dunnii, E.
elata, E. erythrocorys, E. erythrophloia, E. eudesmoides, E. falcata, E.
gamophylla, E. glaucina, E. globulus, E. globulus subsp. bicostata, E. globulus subsp.
globulus, E.
gongylocarpa, E. grandis, E. grandis x urophylla, E. guilfoylei, E. gunnii, E.
hallii, E.
houseana, E. jacksonii, E. lansdowneana, E. latisinensis, E. leucophloia, E.
leucoxylon, E. lockyeri, E. lucasii, E. maidenii, E. marginata, E. megacarpa, E.
melliodora, E. michaeliana, E. microcorys, E. microtheca, E. muelleriana, E.
nitens, E. nitida, E. obliqua, E. obtusijlora, E. occidentalis, E. optima, E. ovata, E.
pachyphylla, E. paucijlora, E. pellita, E. perriniana, E. petiolaris, E.
pilularis, E.
piperita, E. platyphylla, E. polyanthemos, E. populnea, E. preissiana, E.
pseudoglobulus, E. pulchella, E. radiata, E. radiata subsp. radiata, E.
regnans, E.

WASH 1814745.1 WASH 1815926.1 Atty. Dkt. No.: 059994-0114 risdonii, E. robertsonii, E. rodwayi, E. rubida, E. rubiginosa, E. saligna, E.
salmonophloia, E. scoparia, E. sieberi, E. spathulata, E. staeri, E. stoatei, E. tenuipes, E. tenuiramis, E. tereticornis, E. tetragona, E. tetrodonta, E. tindaliae, E.
torquata, E.
umbra, E. urophylla, E. vernicosa, E. viminalis, E. wandoo, E. wetarensis, E.
willisii, E. willisii subsp. falciformis, E. willisii subsp. willisii, and E.
woodwardii.

[0075] The invention also contemplates Populus species such as P. alba, P.
alba x P. grandidentata, P. alba x P. tremula, P. alba x P. tremula var. glandulosa, P. alba x P. tremuloides, P. balsamifera, P. balsamifera subsp. trichocarpa, P.
balsamifera subsp. trichocarpa x P. deltoides, P. ciliata, P. deltoides, P. euphratica, P.
euramericana, P. kitakamiensis, P. lasiocarpa, P. laurifolia, P. maximowiczii, P.
maximowiczii x P. balsamifera subsp. trichocarpa, P. nigra, P. sieboldii x P.
grandidentata, P. suaveolens, P. szechuanica, P. tomentosa, P. tremula, P.
tremula x P. tremuloides, P. tremuloides, P. wilsonii, P. canadensis, P. yunnanensis and Conifers as, for example, loblolly pine (Pinus taeda), slash pine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta), and Monterey pine (Pinus radiata); Douglas-fir (Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood (Sequoia sempervirens); true firs such as silver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedars such as Western red cedar (Thuja plicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis).

[0076] Other plants that may be modified by the process of the invention include all flowering plants. It is understood that the word "plant" includes any plant or plant material used in the pulp and paper industry.

Methods for Genetic Eneineerine [00771 For the purposes of this description, a SAMdc sequence operably linked to a promoter may be introduced into a plant or cell. For example, an illustrative vector may comprise a SAMdc sequence operably linked to a xylem-preferred promoter.

WASH1814745.1 WASH 1815926.1 Atty. Dkt. No.: 059994-0114 Plant transformation [00781 "Transgenic plant ' refers to a plant that comprises a nucleic acid sequence that also is present per se in another organism or species or that is optimized, relative to host codon usage, from another organism or species. Both monocotyledonous and dicotyledonous angiosperm or gymnosperm plant cells may be transformed in various ways known to the art. For example, see Klein et al., Biotechnology 4: 583-590 (1993); Bechtold et al., C. R. Acad. Sci. Paris 316: 1194-1199 (1993); Bent et al., Mol. Gen. Genet. 204: 383-396 (1986); Paszowski et al., EMBO J. 3: 2717-2722 (1984); Sagi et al., Plant Cell Rep. 13: 262-266 (1994). Agrobacterium species such as A. tumefaciens and A. rhizogenes can be used, for example, in accordance with Nagel et al., Microbiol Lett 67: 325 (1990). Additionally, plants may be transformed by Rhizobium, Sinorhizobium or Mesorhizobium transformation. Broothaerts et al., Nature 433: 629-633 (2005).

100791 For example, Agrobacterium may be transformed with a plant expression vector via, e.g., electroporation, after which the Agrobacterium is introduced to plant cells via, e.g., the well known leaf-disk method. Additional methods for accomplishing this include, but are not limited to, electroporation, particle gun bombardment, calcium phosphate precipitation, and polyethylene glycol fusion, transfer into germinating pollen grains, direct transformation (Lorz et al., Mol. Genet.
199: 179-182 (1985)), and other methods known to the art. If a selection marker, such as kanamycin resistance, is employed, it makes it easier to determine which cells have been successfully transformed. Marker genes may be included within pairs of recombination sites recognized by specific recombinases such as cre or flp to facilitate removal of the marker after selection. See U. S. published application No.
2004/0143874.

[0080] Transgenic plants without marker genes may be produced using a second plasmid comprising a nucleic acid encoding the marker, distinct from a first plasmid that comprises a SAMdc sequence. The first and second plasmids or portions thereof are introduced into the same plant cell, such that the selectable marker gene that is transiently expressed, transformed plant cells are identified, and transformed plants WASH 1814745.1 WASH 1815926.1 Atty. Dkt. No.: 059994-0114 are obtained in which the SAMdc sequence is stably integrated into the genome and the selectable marker gene is not stably integrated. See U. S. published application No. 2003/0221213.

[0081] The Agrobacterium transformation methods discussed above are known to be useful for transforming dicots. Additionally, de la Pena et al., Nature 325: 274-276 (1987), Rhodes et al., Science 240: 204-207 (1988), and Shimamato et al., Nature 328: 274-276 (1989) have transformed cereal monocots using Agrobacterium. Also see Bechtold et al., C.R. Acad. Sci. Paris 316 (1994), illustrating vacuum infiltration for Agrobacterium-mediated transformation.

[0082] Plant cells may be transformed with nucleic acid constructs of the present invention without the use of a selectable or visible marker and transgenic organisms may be identified by detecting the presence of the introduced construct. The presence of a protein, polypeptide, or nucleic acid molecule in a particular cell can be measured to determine if, for example, a cell has been successfully transformed or transfected.
For example, and as routine in the art, the presence of the introduced construct can be detected by PCR or other suitable methods for detecting a specific nucleic acid or polypeptide sequence. Additionally, transformed cells may be identified by recognizing differences in the growth rate or a morphological feature of a transformed cell compared to the growth rate or a morphological feature of a non-transformed cell that is cultured under similar conditions. See WO 2004/076625.

[0083] Methods of regenerating a transgenic plant from a transformed cell or culture vary according to the plant species but are based on known methodology. For example, methods for regenerating of transgenic Nicotiana and Eucalyptus plants are well-known.

Selection and Analysis of Genetically Engineered Plants [0084] Genetically engineered plants of the invention are selected that have increased expression of SAMdc relative to a control, non-transgenic plant of the same species. Additionally, the inventive genetically engineered plants may have increased wood density and decreased lignin content. For example, an inventive transgenic WASH1814745.1 WASH 1815926_.1 Atty. Dkt. No.: 059994-0114 plant may have a phenotype characterized by (1) an ability of the whole plant to accumulate less compounds derived from monolignols; (2) an altered vessel number and vessel lumen area such that wood density is increased because vessel number and vessel area negatively correlate with wood density; and (3) an altered lignin content relative to an untransformed host plant.

(0085] The phrase "increased wood density" refers to a quantitative increase of wood density relative to a wild-type or control plant of the same species. The wood density of the engineered plant of the invention can be increased from 5% to about 70%, preferably from 10% to about 60%, even more preferably from 15% to about 50% of the wood density of a wild type plant. A most preferred embodiment of the engineered plant of the invention has a wood density of about 20% to about 40%
of a wild type plant. Wood density is determined by methods known in the art, such as those described in Chave et al., Ecol. Appl. 16:2356-2367 (2006). For example, wood density may be determined by collecting wood samples at breast height and calculating oven dry weight (kg)/oven dry volume (m3).

[0086] "Reduced insoluble lignin content" and "decreased insoluble lignin content" refer to a quantitative reduction in the amount of insoluble lignin in the plant when compared to the amount of insoluble lignin in a wild-type or control plant. The insoluble lignin content in the engineered plant of the invention can be reduced to levels of about 5% to about 90%, preferably about 10% to about 75%, even more preferably about 15% to about 65% of the lignin content of a wild-type plant.
A most preferred embodiment of the plant of the invention has a lignin content of about 10%
to about 60% of a wild-type lignin content. A quantitative reduction of lignin content can be assayed by several methods, as for examples the Klason lignin assay, Kirk et al., Method in Enzymol. 161: 87-101 (1988), or acetyl bromide assay of lignin.
liyama et al., Wood Sci. Technol. 22: 271-80 (1988).

(0087] The phrase "altered lignin composition" refers to quantitative alteration in the relative amounts of syringyl and guaiacyl lignin units in the engineered plant compared to a wild-type or control plant. Preferably, a plant having altered syringyl and guaiacyl lignin composition exhibits a reduced guaiacyl lignin content compared WASH_1814745.1 WASH_1815926.1 Atty. Dkt. No.: 059994-0114 to the guaiacyl lignin content of a wild-type plant; more preferably, it exhibits an increased pairwise syringyl lignin/guaiacyl lignin ratio, i.e., "S/G ratio,"
compared to the S/G ratio of a wild-type plant. Plants of the present invention can be assayed to determine their S/G ratios in comparison to the ratio of a wild-type plant using several different assay methods, including those described by Rolando et al., METHODS
IN
LIGNIN CHEMISTRY, Springer, New York (1992). Plants of the present invention exhibit an overall decrease in lignin content when compared to a wild-type or control plant, while exhibiting an increased S/G ratio.

****************
[0088] Specific examples are presented below of methods for obtaining SAMdc encoding genes as well as methods for introducing a SAMdc gene to produce plant transformants. They are meant to illustrate and not to limit the present invention.

EXAMPLE 1: Isolation of the tobacco cDNA encoding SAMdc enzyme (a) Preparation of mRNA from tobacco leaves and cDNA synthesis [0089] RNA was extracted from leaf tissue of Nicotiana tabacum using Trizol reagent (Invitrogen). A eDNA pool was prepared from the isolated total RNA
using a commercially available cDNA Superscript II Amplification Kit (Invitrogen) or the like. The cDNA pool can then be used in RT-PCR experiments in which the isolated total RNA is used as template, and Superscript II reverse transcriptase (Invitrogen) and oligo(dT) primer are used to synthesize the first-strand cDNA and double-stranded cDNA is obtained by the subsequent polymerase reaction, using gene-specific primers.

(b) Design of PCR Primers [0090] A DNA sequence coding for SAMdc from Nicotrana tabacum has already been determined and deposited in the GenBank under accession number AF033100.
Based on this sequence, DNA oligomers were synthesized as primers for PCR, including either the region around the first codon ATG or around the termination codon of the main ORF encoding the SAMdc enzyme.

WASH_1814745.1 WASH 1815926.1 Atty. Dkt. No.: 059994-0114 100911 Primers were designed to amplify the entire coding region of the SAMdc main ORF, i.e., from the ATG through the translation stop codon. The sequences of the primers are given below for the tobacco SAMdc gene:
SAMdc_Ntl Length: 24 SEQ ID N0:20 ATCCCATGGATTCGGCCTTGCCTG
SAMdc_Nt2 Length: 34 SEQ ID NO:21 GTCTAGACTACTCCTTCTCTTCTTTCTCTTCATC

(c) PCR amplification of SAMdc from Nicotiana tabacum [0092] The cDNA pool obtained in (a) was used as template, and the primers designed in (b) were used for PCR. The PCR steps involved 40 cycles of 1 minute at 94 C., 1 minute at 52 C., and 2 minutes at 72 C followed by an extra step of elongation at 72 C for 7 minutes. The PCR products were isolated by gel electrophoresis on 1.0% agarose followed by ethidium bromide staining of the electrophoresed gel and detection of amplified bands on a UV transilluminator.
The detected amplified band was verified and cut out of the agarose gel with a razor. The pieces of gel were transferred to 1.5 mL microtubes, and the DNA fragments were isolated and purified using a GFX PCR clean-up and gel band purification kit (Amersham). The recovered DNA fragments were subcloned to the pGEM-T cloning vector (Promega), transformed into E. coli, and then used to prepare plasmid DNA in the usual manner, which was then sequenced by the dideoxy method, Messing, Methods in Enzymol. 101: 20-78 (1983), using the BigDye chemistry (Applied Biosystems), yielding the DNA sequence disclosed herein under SEQ ID NO: 1 for use according to embodiments described in this patent.

EXAMPLE 2: Preparation of Transgenic Arabidopsis and Nicotiaaa Plants [0093] The gene obtained in Example I above was introduced into a plant host to produce transgenic Arabidopsis and Nicotiana plants.

(a) Preparation of constructs and transformation ofAgrobacterium [0094] Expression constructs can be prepared by cleaving the SAMdc genes obtained in 1 above with suitable restriction enzymes so as to include all of the open reading frame and inserting the gene into the plant transformation vector WASH 1814745.1 WASH 1815926.1 Atty. Dkt. No.: 059994-0114 pALELLYX-ATG (FIG. 2) together with an appropriate promoter. For example, the tobacco SAMdc gene obtained in Example 1 was cloned into the aforementioned expression vector downstream to a xylem-preferred tubulin gene (TUB) promoter from Populus deltoides as set forth in PCT patent application No.
PCT/BR2005/000041, filed March 28, 2005, which claims for the priority date of Serial No. 60/560,227, filed April 6, 2004 (FIG. 3). The resulting expression construct is amplified in E. coli, and then transformed by tripartite conjugation, Bevan, Nucleic Acid Research, 12, 8711 (1984), freeze thawing, electroporation, chemical transformation or the like into Agrobacterium tumefaciens C58, GV3 101 or the like.

(b) Agrobacterium-mediated transformation of Arabidopsis thaliana [00951 Arabidopsis thaliana Columbia plants were transformed using an Agrobacterium tumefaciens mediated transformation protocol, Bechtold et al., C. R.
Acad Sci. Paris 316:1194-1199 (1993); Bent et al., Mol. Gen. Genet. 204:383-(1986), with the construct containing the tobacco SAMdc gene obtained in Example I
operably linked to the promoter of a xylem-preferred gene (TUB). The construct also contains the selectable marker gene Bar that confers resistance to herbicidal phosphinothricin analogs like ammonium gluphosinate. Thompson et al., EMBO J., 9: 2519-23 (1987).

100961 Seeds of Arabidopsis thaliana ecotype Columbia were sown in pots containing vermiculite. Plants were grown at 16/8 hours dark/light regime at 22 C.
After 4-5 weeks plants were transformed with the Agrobacterium tumefaciens strain GV3101 (C58C1 rifampicin) pMP90 (gentamicin), Bent et al., Mol. Gen. Genet.
204:383-396 (1986), harboring the plasmid vector comprising the SAMdc gene of interest operably linked to the TUB promoter.

[0097] For plant transformation, 1 liter of LB medium containing rifampicin, gentamycin and kanamicin was inoculated with an aliquot of overnight starter Agrobacterium culture. The culture was grown overnight at 28 C in a rotatory shaker, until OD600 is ? 0.8. The Agrobacterium was precipitated by centrifugation WASH1814745.1 WASH_1815926_.1 Atty. Dkt. No.: 059994-0114 and the bacterial pellet was resuspended in -300 ml of 5% sucrose and 0.03%
Silwet L-77 (Witco). This Agrobacterium suspension was sprayed onto the plants. The pots were then placed in a tray which is covered with plastic wrap to maintain humidity.
The plants were grown at 16/8 hours dark/light regime at 22 C through maturity to set seeds.

[0098] Seeds were harvested, surface-sterilized in a solution containing 50%
bleach and 0.02% Triton X-100 for 7 minutes. Seeds were then rinsed 3 times in sterile distilled water and plated out in MS medium containing 6 mg/i of Finale (Bayer) as selective agent. After 5 to 7 days, transformants were visible as green plants. Transformed plants were transferred onto new selection plates and after 6-10 days were transferred to pots containing vermiculite and grown under conditions of 16 hours light/8 hours dark at 22 C. After three weeks, the inflorescence stems were cut close to their bases twice a week for a period of one month in order to induce secondary growth at the base of the rosette before the plants were analyzed.

(c) Agrobacterium-mediated transformation of Nicotiana benthamiana [0099] Transformation of Nicotiana benthamiana was accomplished using the leaf disk method of Horsch et al., Science 227: 1229 (1985), using a construct comprising the tobacco SAMdc gene obtained in Example 1, operably linked to the promoter of a xylem-preferred gene (4CL; Hu et al. Proc. Natl. Acad. Sci. USA
95:5407-5412 (1998)). The transformants were selected by growing on Murashige and Skoog medium (Sigma, St. Louis, Mo.) containing 100 milligrams/liter of BASTA herbicide and 500 mg/L carbenicillin (Sigma). The transformed tobacco shoots are allowed to root on the Murashige and Skoog medium, and are subsequently transferred to soil and grown in the greenhouse.

(d) PCR verification offoreign gene insertion into the host plant genome [0100] PCR can be used to verify the integration of the gene construct in the genome of transgenic plants. Two specific primers are synthesized for the construct and used to PCR-amplify the corresponding construct from genomic DNA of Arabidopsis or Nicotiana transformants. For the TUB-SAMdc-Nt construct, which WASH1814745.1 WASH 1815926_.1 Atty. Dkt. No.: 059994-0114 contains the tobacco SAMdc main ORF under the control of the Populus xylem-preferred tubulin gene promoter, two specific primers were synthesized that amplify a 1.8kb fragment:
Tub_checkl Length: 25 SEQ ID NO:22 TATCGTTTTACTTCACTGGTCGGTG
SAMdc_Nt2 Length: 34 SEQ ID NO:21 GTCTAGACTACTCCTTCTCTTCTTTCTCTTCATC

[0101] For the 4CL-SAMdc-Nt construct, which contains the tobacco SAMdc main ORF under the control of the Populus xylem-preferred 4CL gene promoter, two specific primers were synthesized that amplify a 1.6 kb fragment:
4CL seq Length: 20 SEQ ID NO:23 ~ AATCTCACCAACCCAACTCC
SAbIDC NT2 Length: 34 SEQ ID NO:21 rGTCTAGACTACTCCTTCTCTTCTTTCTCTTCATC

101021 The PCR reaction mixture contained 100 ng genomic DNA of transformed plant, and 0.2 M of each primer, 100 M of each deoxyribonucleotide triphosphate, 1 xPCR buffer and 2.5 Units of AmpliTaq DNA polymerase (Applied Biosystems) in a total volume of 50 L. The cycling parameters were as follows: 94 C. for 1 minute, 50 C. for 1 minute and 72 C for 3 minutes, for 40 cycles, with 5 minutes at 72 C.
extension. The PCR products were electrophoresized on an 1% agarose gel.

(e) Determination of transgene expression level in transgenic plants [0103] Semi-quantitative RT-PCR was used to detect the accumulation of tobacco SAMdc transcripts in stem tissue of the transgenic plants. Total RNA was isolated from 5-cm inflorescence stem cuts from transgenic Arabidopsis TI plants (primary transformants) using Trizol reagent (GibcoBRL, USA) according to the manufacturer's instructions or from stem cuts of 3-months old transgenic Nicotiana benthamiana T1 plants using the CTAB method. Aldrich and Cullis, Plant Mol.
Biol.
Report. 11:128-141(1993).

[0104] cDNA was synthesized from 500 ng of total RNA using Superscript II
RNase H- RT (Invitrogen, USA). Primers for the constitutive gene encoding adenine phosphoribosyltransferase (APRT), Moffatt et al., Gene 143, 1211-1216 (1994), were used as an internal control to normalize the quantity of total RNA used in each sample. The PCR was done with a 12.5-fold dilution of the first-strand cDNA
under WASH1814745.1 WASH 1815926.1 Atty. Dkt. No.: 059994-0114 the following conditions: 94 C for 3 minutes and 27 cycles of 94 C for 1 minute, 52 to 60 C for 45 seconds, and 72 C for 1 minute and 30 seconds.

EXAMPLE 3: Histochemical Analysis of Transgenic Plants [01051 Histological staining of lignin can be performed to analyze the amount of lignin in the vascular system of transformed and control plants. Briefly, stems of Arabidopsis TUB-SAMdc-Nt transgenic, Nicotiana benthamiana 4CL-SAMdc-Nt and control non-transgenic plants were free-hand sectioned with a razor blade, and the resulting sections were stained for total lignin with phloroglucinol-HCl (1%
phloroglucinol in 6N HCI) for 30 minutes. Zhong et al., Plant Physiol. 123: 59-(2000). Alternatively, tissues were sectioned in a microtome (Leica RM2255) and subsequently stained with phloroglucinol. The histologically stained sections are observed under a dissection microscope using bright- and dark-field illumination (FIGs. 12 and 14).

EXAMPLE 4: Reduction in Lignin Content in Transgenic Plants Over-expressing SAMdc in the Vascular Tissue [0106] The effect of over-expression of SAMdc in lignin biosynthesis in plant species was investigated in Arabidopsis and Nicotiana. It was found that over-expressing SAMdc in a xylem-preferred manner in transgenic Arabidopsis plants resulted in a reduction in lignin content (FIG. 12). Similar findings were obtained in two generations of transformed Nicotiana plants (FIG. 14). This finding indicates that over-expression of SAMdc is an efficient means for genetically engineering trees with low lignin content.

[0107] Arabidopsis transgenic plants were made following the methods described in Example 2. The first generation of transformants (T 1) was analyzed using the methods described in Example 3 to assay lignin content, as well as vessel structure and vascular tissue anatomy. Several independent transformants were produced that contain a DNA expression construct comprising the Nicotiana tabacum SAMdc main ORF under the control of the Populus xylem-preferred tubulin gene promoter described in PCT patent application No. PCTBR2005/000041, filed March 28, 2005, which claims for the priority date of Serial No. 60/560,227, filed April 6, 2004, supra.

WASH1814745.1 WASH 1815926_.1 Atty. Dkt. No.: 05 9994-0114 [0108] SAMdc gene expression levels were measured in twenty independent T1 plants, using the methods set forth in Example 2. Stems of transgenic plants were used for RNA extraction, followed by a semi-quantitative RT-PCR. SAMdc gene expression levels in transgenic plants were expressed as a percentage of the maximum expression level observed.

[01091 As shown in FIG. 13, the transgenic Arabidopsis plant TUB-SAMdc-Nt12 showed a high transgene expression level (95% of the maximum level observed), which was accompanied by a decrease in phloroglucinol pinkish red staining intensity (FIG. 12B) compared to a comparable control non-transformed plant (FIG. 12A), reflecting a reduction in lignin content, which provides evidence that enhanced SAMdc expression in specific vascular cell types leads to a reduction in the availability of the methyl group donor SAM and therefore impairs lignin monomer biosynthesis during xylem lignification. Notably, although it exhibits a marked reduction in lignin content compared to a wild-type plant, the Arabidopsis transformant TUB-SAMdc-Nt12 has been grown normally under standard conditions, with no visible abnormal growth or morphology being observed.

[0110) Other T1 plants with low or non-existent expression of the SAMdc transgene, like TUB-SAMdc-Nt09, failed to show any difference in phloroglucinol staining compared to comparable control non-transformed plants (FIGs. 12C, D).

[0111) Similar results were obtained when the construct comprising the Nicotiana tabacum SAMdc main ORF under the control of the Populus xylem-preferred 4CL
gene promoter, Hu et al., Proc. Natl. Acad. Sci. USA, 95, 5407-5412 (1998), was transformed into Nicotiana benthamiana plants. Nicotiana benthamiana transgenic plants were made following the methods described in Example 2. Several independent first-generation transformants (Tl) were analyzed using the methods described in Example 3 to assay lignin content, as well as vascular tissue anatomy.
As shown in FIG. 14, the transgenic Nicotiana benthamiana plant 4CL-SAMdc-Nt30 showed a marked decrease in the number of vessel elements in the vascular tissue (FIG. 14B) compared to a control non-transformed plant (FIG. 14A). This might reflect a reduction in the plant's ability to deposit lignin in the cell wall.
Seeds from WASH 1514745.1 WAS H 1815926.1 Atty. Dkt. No.: 059994-0114 this transgenic plant were germinated to obtain a segregating population of plants in the following generation (T2). These segregants could be separated into three groups according to transgene expression level and plant size. Heterozygous and homozygous T2 plants possessed a reduction in size and a strong reduction in lignin content as assessed by phloroglucinol staining according to Example 3 (FIG.
14D), as compared to non-transgenic sibling plants (FIG. 14C), providing strong evidence that enhanced SAMdc expression in specific vascular cell types leads to a reduction in the availability of the methyl group donor SAM and therefore impairs lignin monomer biosynthesis during xylem lignification.

EXAMPLE 5: Isolation of Populus cDNA encoding SAMdc enzyme (a) Preparation of RNA from Populus deltoides cambium/xylem and cDNA
synthesis [0112] Bark was removed from stem cuttings of one-year-old Populus deltoides trees. The inner part of the stem, containing cambium, xylem, and pith, was cut into small pieces, frozen in liquid nitrogen and used for RNA extraction using the cetyltrimethyl-ammonium bromide (CTAB) extraction method. Aldrich and Cullis, Plant Mol. Biol. Report, 11, 128-41 (1993). A cDNA pool was used in RT-PCR
experiments in which the isolated total RNA was used as template, and Superscript Il reverse transcriptase (Invitrogen) and oligo(dT) primer were used to synthesize the first-strand cDNA. Double-stranded cDNA was obtained by the subsequent polymerase reaction, using gene-specific primers, as described below.

(b) Design of PCR Primers and RT-PCR reaction.

[0113] Oligomers based on SEQ ID NO: 26 were synthesized as primers for PCR, including either the region around the first ATG codon or around the termination codon of the main ORF encoding the polypeptide to amplify the entire coding region of the main ORF. The sequences of the primers are:

SAMC NCO Length: 26 SEQ ID NO: 28 CCATGGCGCT GCCAGTCTCT GCAATC

SA14DC 7BA Length: 27 SEQ ID NO: 29 WASH1814745.1 WASH_18159267.1 Atty. Dkt. No.: 059994-0114 TCTAGACTAC TTCTCTTCAG CTTCCTC

(0114] The eDNA pool obtained in (A) was used as the template in a PCR
reaction with the primers of SEQ ID NOs: 28 and 29. The PCR involved 40 cycles of 1 minute at 94 C, 1 minute at 51 C, and 2 minutes at 72 C followed by an extra step of elongation at 72 C for 7 minutes. The PCR products were isolated by gel electrophoresis on 1.0% agarose followed by ethidium bromide staining of the electrophoresed gel and detection of amplified bands on a UV transilluminator.
The detected amplified band was verified and cut out of the agarose gel with a razor. The pieces of gel were transferred to 1.5mL microtubes, and the DNA fragments were isolated and purified using a GFX PCR clean-up and gel band purification kit (Amersham). The recovered DNA fragments were subcloned in a commercially available cloning vector, transformed into E. coli, and then used to prepare plasmid DNA, which was then sequenced by the dideoxy method, Messing, Methods in Enzymol. 101, 20-78 (1983), using standard methods.

EXAMPLE 6: Preparation of Transgenic Populus and Eucalyptus Plants (0115] The gene obtained in Example 5 above was introduced into a plant host to produce transgenic Populus and Eucalyptus plants.

(a) Preparation of constructs and transformation of Agrobacterium 101161 Expression constructs can be prepared by cleaving the SAMdc gene obtained in Example 5 above with suitable restriction enzymes so as to include the entire open reading frame and inserting the gene into the plant transformation vector pALELLYX-ATG (FIG. 2) together with an appropriate promoter. For example, the tobacco SAMdc gene obtained in Example 5 was cloned into the aforementioned expression vector downstream to a xylem-preferred 4CL gene promoter, Hu et al., Proc. Natl. Acad. Sci. USA, 95, 5407-5412 (1998), from Populus deltoides. The resulting expression construct is amplified in E. coli, and then transformed by tripartite conjugation, Bevan, Nucleic Acid Research, 12, 8711-8721 (1984), freeze thawing, electroporation, chemical transformation or the like into Agrobacterium tumefaciens C58, GV3101, or the like.

WASH_1814745.1 WASH_1815926.1 Atty. Dkt. No.: 059994-0114 (b) Agrobacterium-mediated transformation of Populus [0117] Wild-type aspen was transformed with Agrobacterium tumefaciens carrying a construct comprising a Populus deltoides SAMdc gene obtained in Example operably linked to the promoter of a xylem-preferred gene (C4H). Petioles and internodal stem segments from in vitro micropropagated plants were used as explants.
Transformed shoots are selected on regeneration medium containing 100mg/L of kanamycin and allowed to root on the Murashige and Skoog medium. Selected plants are subsequently transferred to soil and grown in the greenhouse.

(c) Agrobacterium-mediated transformation of Eucalyptus [0118] Transformation of hybrid Eucalyptus (Eucalyptus grandis x Eucalyptus urophylla) was accomplished by cocultivation of hypocotyl segments with Agrobacterium tumefaciens containing a construct comprising a Populus deltoides SAMdc gene obtained in Example 5 operably linked to the promoter of a xylem-preferred gene (C4H). AHAS-resistant lines were allowed to root on the Murashige and Skoog medium and selected plants were subsequently transferred to soil and grown in the greenhouse.

EXAMPLE 7: Reduction of Vessel Number and Vessel Lumen Area in Transgenic Plants Over-expressing SAMdc in the Vascular Tissue [0119] Stem sections of 10 m thick were cut from the Nicotiana benthamiana segregating population of plants described in Example 2. These sections were subjected to safranin-astra blue coloration and observed under a light microscope (Leica DMIL) fitted with a camera (Sony) linked to a personal computer.

[0120] The number of vessels per mm2 of xylem area and the average lumen area of 100 vessels were measured directly on the screen using the "Image Tool"
software.
101211 The homozygous dominant plants and the hemizygous plants presented a significant decrease in the number of vessels, compared with the homozygous recessive plants. There is a reduction of 48% in the number of vessels of homozygous dominant plants when compared to homozygous recessive plants (FIG. 17). The WASH 1814745.1 WASH 1815926.1 Atty. Dkt. No.: 059994-0114 Tukey test showed that the difference between the two populations is highly significant with P < 0.001.

[0122] Observations of the homozygous dominant population also revealed that the measured average area of 100 vessels in these plants was 57% smaller when compared to the average area of homozygous recessive plants (FIG. 18). The Tukey test showed that the difference between the two populations is highly significant with P < 0.001. No significant difference was observed between homozygous dominant and hemizygous plants.

EXAMPLE 8: Histochemical Analysis of Transgenic Plants 101231 Lateral shoots of 6 months old Eucalyptus plants were cross-sectioned (5 m thick) from wild-type and the transgenic lines with a microtome (LEICA
RM2255) equipped with a steel knife. These sections were subjected to astra-blue staining and were observed under a light microscope just after staining.
Figure 19A
shows a stem section of transgenic event A.12.3 presenting strong reduction in the number and the size of vessel elements when compared to a wild-type plant (FIG.
19B).

EXAMPLE 9: Reduction of Insoluble Lignin and Increase of Soluble Lignin in Transgenic Plants [0124] The main stems of T1 segregating population of the Nicotiana transgenic event 11 B transformed with a construct comprising the Nicotiana tabacum SAMdc gene under the control of the xylem-preferred Populus deltoides 4CL promoter were collected and air-dried for two weeks. Dried stems were cut in pieces and pulverized on a knife mill using a 30-mesh sieve. Stem powder samples were then subjected to chemical analyses to determine lignin content. In brief, to a sample of extracted tobacco was added an aliquot of 72% (w/w) H2S04 and was thoroughly mixed for 1 minute. After 2 hours of hydrolysis, the content was transferred to a serum bottle and the serum bottles (containing HZSO4 at 4% (w/w) plus tobacco) were autoclaved at 121 C for 60 minutes. Samples were allowed to cool, and the hydrolysates were vacuum-filtered, washed with 200 ml warm (approximately 50 C) nanopure H20 to remove residual acid and sugars and dried overnight at 105 C. The dry crucibles WASH 1814745.1 WASH 1815926.1 Atty. Dkt. No.: 059994-0114 were weighed to determine Klason (acid-insoluble lignin) lignin gravimetrically. The filtrate was also analysed for acid-soluble lignin by absorbance at 205 nm.
Patzlaff et al., Plant J. 36: 743-754 (2003).

[01251 As shown in Figure 20A, the homozygous dominant plants presented a decrease of 11 % in their insoluble lignin content, when compared to homozygous recessive plants. The homozygous dominant plants also presented an increase of 13%
in their soluble lignin content, compared to homozygous recessive plants (FIG.
20B) WASH 1814745.1 WASH 1815926.1 Atty. Dkt. No.: 059994-0114 SEQUENCE LISTING

<110> PAPES, Fabio ARRUDA, Paulo GERHARDT, Isabel Rodrigues <120> Altering Wood Density <130> ALEL 203.1 PCT
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WASH1814745.1 WASH 1815926.1 Atty. Dkt. No.: 059994-0114 <400> 2 Met Asp Ser Ala Leu Pro Val Ser Ala Ile Gly Phe Glu Gly Phe Glu Lys Arg Leu Glu Ile Ser Phe Phe Glu Pro Gly Leu Phe Ala Asp Pro Asn Gly Lys Gly Leu Arg Ser Leu Ser Lys Ala Gln Leu Asp Glu Ile Leu Gly Pro Ala Glu Cys Thr Ile Val Asp Ser Leu Ser Asn Asp Asp Val Asp Ser Tyr Val Leu Ser Glu Ser Ser Leu Phe Val Tyr Ser Tyr Lys Ile Ile Ile Lys Thr Cys Gly Thr Thr Lys Leu Leu Leu Ala Ile Pro Pro Ile Leu Lys Leu Ala Glu Thr Leu Ser Leu Lys Val Gln Asp Val Arg Tyr Thr Arg Gly Ser Phe Ile Phe Pro Gly Ala Gln Ser Phe Pro His Arg His Phe Ser Glu Glu Val Ala Val Leu Asp Gly Tyr Phe Gly Lys Leu Ala Ala Gly Ser Lys Ala Val Ile Met Gly Ser Pro Asp Lys Ala Gin Lys Trp His Val Tyr Ser Ala Ser Ala Gly Pro Ile Gln Ser Asn Asp Pro Val Tyr Thr Leu Glu Met Cys Met Thr Gly Leu Asp Arg Glu Lys Ala Ser Val Phe Tyr Lys Thr Glu Gly Ser Ser Ala Ala His Met Thr Val Arg Ser Gly Ile Arg Lys Ile Leu Pro Asn Ser Giu Ile Cys Asp Phe Glu Phe Glu Pro Cys Gly Tyr Ser Met Asn Ser Ile Glu Gly Ala Ala Leu Ser Thr Ile His Ile Thr Pro Glu Asp Gly Phe Ser Tyr Ala Ser Phe Glu Ala Val Gly Tyr Asp Met Lys Thr Met Lys Leu Gly Pro Leu Val Glu Arg Val Leu Ala Cys Phe Gln Pro Asp Glu Phe Ser Ile Ala Leu His Ala Asp Val Ala Thr Lys Leu Leu Glu Arg Val Cys Ser Leu Asp Val Lys Gly Tyr Ser Leu Ala Glu Trp Ser Pro Glu Glu Phe Gly Lys Gly Gly Ser Ile Val Tyr Gln Lys Phe Thr Arg Thr Pro Phe Cys Gly Ser Pro Lys Ser Val Leu Lys Gly Cys Trp Lys Glu Asp Glu Glu Lys Glu Glu Lys Glu WASH 1814745.1 WASH 1815926.1 Atty. Dkt. No.: 059994-0114 <210> 3 <211> 1777 <212> DNA
<213> Populus deltoides <220>
<221> CDs <222> (536)...(1621) <400> 3 tcggacacaa aaaatattgc aagtttaggg tcataaaaat ctctatcatc acaaacaaat 60 atagggtttg ttgatttggt ggctgttttc gccgtctttg ctttcaaaat tcaatcttga 120 cctgtttctt ttatgctttt tgaataggtt ttttttttga acttgagaaa gagggggttt 180 tcaaaggaga ctagcttttc aagatttgat atattttatt ggactgaatg atctaatgga 240 gtctaaaggt ggcaagaaga agtctagtag tagtagtagt agtaaatcct tatgttacga 300 agctcccctc ggttacagca ttgaagacct cagacctgct ggtggaatca agaagttcag 360 atctgctgca tactccaact gcgttcgaaa accatcctga gattttccaa gtgttgacat 420 aaccccattt tagctatttc gcacgctcaa ttgtctttag tctgtttttc tgttctgctt 480 tcctcgttct cttgtactct ttgctgcact tttcatttgt tgactgtgag gtcag atg 538 Met gcg ctg cca gtc tct gca atc gga ttt gaa ggt tac gaa aaa agg ctt 586 Ala Leu Pro Val Ser Ala Ile Gly Phe Glu Gly Tyr Glu Lys Arg Leu gaa ata tct ttc cta gag cct ggc ttc ttt tct gac cct gaa ggg aag 634 Glu Ile Ser Phe Leu Glu Pro Gly Phe Phe Ser Asp Pro Glu Gly Lys ggc ctg agg tct ttg tcc aag gct caa ttg gac gag att ctc aga cca 682 Gly Leu Arg Ser Leu Ser Lys Ala Gln Leu Asp Glu Ile Leu Arg Pro gct gaa tgt act att gtt gat tcg cta tca aat gac cag gtt gat tct 730 Ala Glu Cys Thr Ile Val Asp Ser Leu Ser Asn Asp Gln Val Asp Ser tat gtc ctg tcg gaa tcc agt ctc ttt gtg tac cct tac aaa gtt att 778 Tyr Val Leu Ser Glu Ser Ser Leu Phe Val Tyr Pro Tyr Lys Val Ile atc aaa aca tgt ggg act acc aaa ctg ctt ctc tct ata ccg gtg att 826 Ile Lys Thr Cys Gly Thr Thr Lys Leu Leu Leu Ser Ile Pro Val Ile ctt aag ctc gct gat gcc ctt tca ctc act gta tgt tct gtg agg tat 874 Leu Lys Leu Ala Asp Ala Leu Ser Leu Thr Val Cys Ser Val Arg Tyr act cgt ggg agc ttt cta tgc cct ggg gct cag cca ttt cca cac cgc 922 Thr Arg Gly Ser Phe Leu Cys Pro Gly Ala Gln Pro Phe Pro His Arg aac ttc tgt gag gag gta gct gtc ctt gat ggc cat ttc agt aaa ctt 970 Asn Phe Cys Glu Glu Val Ala Val Leu Asp Gly His Phe Ser Lys Leu ggt ttg aac agt gtg gca tat gtg atg ggt ggt ctt gac aaa act cag 1018 Gly Leu Asn Ser Val Ala Tyr Val Met Gly Gly Leu Asp Lys Thr Gln WASH 1814745.1 WASH 1815926.1 Atty. Dkt. No.: 059994-0114 aaa tgg cat gtt tac tct gcc tct gcc gat ata gag agc cat tct ggc 1066 Lys Trp His Val Tyr Ser Ala Ser Ala Asp Ile Glu Ser His Ser Gly cct gtt tac act ctg gaa atg tgc atg act ggt ttg ggc agg aaa caa 1114 Pro Val Tyr Thr Leu Glu Met Cys Met Thr Gly Leu Gly Arg Lys Gln gca tct gtt ttc tac aaa aca cat tcc agt tca gct gct gcg atg act 1162 Ala Ser Val Phe Tyr Lys Thr His Ser Ser Ser Ala Ala Ala Met Thr gag gat tcc ggc ata agg aaa atc ctt cca cag tct gag atc tgt gat 1210 Glu Asp Ser Gly Ile Arg Lys Ile Leu Pro Gln Ser Glu Ile Cys Asp ttt gat ttt gac cct tgt ggt tac tct atg aat gcc att gaa ggg agt 1258 Phe Asp Phe Asp Pro Cys Gly Tyr Ser Met Asn Ala Ile Glu Gly Ser gca att tcc aca atc cac gtc act cca gaa gat ggt ttc agc tat gca 1306 Ala Ile Ser Thr Ile His Val Thr Pro Glu Asp Gly Phe Ser Tyr Ala agt ttt gag gct gtg ggc tat gat ctt caa gat ttg aat ttg agt cag 1354 Ser Phe Glu Ala Val Gly Tyr Asp Leu Gln Asp Leu Asn Leu Ser Gln ctg ctt gaa agg gtt ttg gct tgc ttt gaa ccg acc gag ttc tcc gtt 1402 Leu Leu Glu Arg Val Leu Ala Cys Phe Glu Pro Thr Glu Phe Ser Val gct ttg cat tct aat atc aag ggt gcc gaa ctt cga gca aag ttt ccc 1450 Ala Leu His Ser Asn Ile Lys Gly Ala Glu Leu Arg Ala Lys Phe Pro ctg gat gtg gaa ggt tac tct ggt gga gga ggg aac tat gaa atg ctt 1498 Leu Asp Val Glu Gly Tyr Ser Gly Gly Gly Gly Asn Tyr Glu Met Leu ggg aaa gga gga tcg atc atc tac cac agc ttt gca agg act gga ggc 1546 Gly Lys Gly Gly Ser Ile Ile Tyr His Ser Phe Ala Arg Thr Gly Gly agt gca tct ccc agg tct atc ctg aaa tgt tgt tgg agt gag gat gag 1594 Ser Ala Ser Pro Arg Ser Ile Leu Lys Cys Cys Trp Ser Glu Asp Glu aag gac gag gaa gct gaa gag aag tag ttcttttcag ctatttgttt 1641 Lys Asp Glu Glu Ala Glu Glu Lys *

tttcctttta ttttttcctt gaataaattt catggggtta tgattctgag ttcttagagg 1701 catttgtcca tgccttgtgt ctttcattat caatttagtt ttgactttgg attaataaag 1761 gggtttgtgt tatcag 1777 <210> 4 <211> 361 <212> PRT
<213> Populus deltoides <220>
<400> 4 Met Ala Leu Pro Val Ser Ala Ile Gly Phe Glu Gly Tyr Glu Lys Arg Leu Glu Ile Ser Phe Leu Glu Pro Gly Phe Phe Ser Asp Pro Glu Gly WASH 1814745.1 WASH 1815926_.1 Atty. Dkt. No.: 059994-0114 Lys Gly Leu Arg Ser Leu Ser Lys Ala Gln Leu Asp Glu Ile Leu Arg Pro Ala Glu Cys Thr Ile Val Asp Ser Leu Ser Asn Asp Gln Val Asp Ser Tyr Val Leu Ser Glu Ser Ser Leu Phe Val Tyr Pro Tyr Lys Val Ile Ile Lys Thr Cys Gly Thr Thr Lys Leu Leu Leu Ser Ile Pro Val Ile Leu Lys Leu Ala Asp Ala Leu Ser Leu Thr Val Cys Ser Val Arg Tyr Thr Arg Gly Ser Phe Leu Cys Pro Gly Ala Gln Pro Phe Pro His Arg Asn Phe Cys Glu Glu Val Ala Val Leu Asp Gly His Phe Ser Lys Leu Gly Leu Asn Ser Val Ala Tyr Val Met Gly Gly Leu Asp Lys Thr Gln Lys Trp His Val Tyr Ser Ala Ser Ala Asp Ile Glu Ser His Ser Gly Pro Val Tyr Thr Leu Glu Met Cys Met Thr Gly Leu Gly Arg Lys Gln Ala Ser Val Phe Tyr Lys Thr His Ser Ser Ser Ala Ala Ala Met Thr Glu Asp Ser Gly Ile Arg Lys Ile Leu Pro Gln Ser Glu Ile Cys Asp Phe Asp Phe Asp Pro Cys Gly Tyr Ser Met Asn Ala Ile Glu Gly Ser Ala Ile Ser Thr Ile His Val Thr Pro Glu Asp Gly Phe Ser Tyr Ala Ser Phe Glu Ala Val Gly Tyr Asp Leu Gln Asp Leu Asn Leu Ser Gln Leu Leu Glu Arg Val Leu Ala Cys Phe Glu Pro Thr Glu Phe Ser Val Ala Leu His Ser Asn Ile Lys Gly Ala Glu Leu Arg Ala Lys Phe Pro Leu Asp Val Glu Gly Tyr Ser Gly Gly Gly Gly Asn Tyr Glu Met Leu Gly Lys Gly Gly Ser Ile Ile Tyr His Ser Phe Ala Arg Thr Gly Gly Ser Ala Ser Pro Arg Ser Ile Leu Lys Cys Cys Trp Ser Glu Asp Glu Lys Asp Glu Glu Ala Glu Glu Lys <210> 5 <211> 1763 <212> DNA
<213> Populus deltoides <220>

WASH 1814745.1 WASH 1815926.1 Atty. Dkt. No.: 059994-0114 <221> CDs <222> (548) ... (1624) <400> 5 acaaacaaga acccttcacg ttctttaaca atcttgcaga tttaagctct taaaaatctc 60 tctcttcaaa aacaaatcta gagtttgttg attttgtcat tggttttgcc cttttttttt 120 ctataaaaaa ccaatcttga tttgtttctt gaacagagtt tagtttggag tttgagaaag 180 agggagtttt tgaaggagat taatttttca agatttcata taatttattg acaagactga 240 atgatccatg gagtctaaag gtggcaagaa gtctagtagt agtagtagta gtagtaaatc 300 cttattgtac gaagcacctc tcggctacag cattgaagac atcagacctg ccggtggaat 360 caagaagttc cgatctgctg cttactccaa ctgcgttcgg aagccatcct gagattttcc 420 aagtgttgag ataacccctt tttagctttt tcatactgtc tgttgtcctt agtctttttt 480 tcttttctgc tttcctcgtt ctctcgcact cttttctgca attgtcatct gtttactgtg 540 aggtgag atg gca ctg ccg gtc tct gca atc gga ttt gaa ggt tac gaa 589 Met Ala Leu Pro Val Ser Ala Ile Gly Phe Glu Gly Tyr Glu aaa agg ctt gaa att tgc ttt tta gag cct ggc ttc ttt tct gac cct 637 Lys Arg Leu Glu Ile Cys Phe Leu Glu Pro Gly Phe Phe Ser Asp Pro gaa gga aag ggc ctc agg tct ttg tcc aag gct caa ttg gac gag att 685 Glu Gly Lys Gly Leu Arg Ser Leu Ser Lys Ala Gin Leu Asp Glu Ile ctc aaa cca gct gaa tgc act ata gtt gat tcg ctt tca aat gac gag 733 Leu Lys Pro Ala Glu Cys Thr Ile Val Asp Ser Leu Ser Asn Asp Glu gtt gat tcg tat gtt ctg tcg gaa tcc agt ctc ttt gta tac cct tac 781 Val Asp Ser Tyr Val Leu Ser Glu Ser Ser Leu Phe Val Tyr Pro Tyr aaa gtt atc atc aaa act tgt ggg act acc aaa ctg ctt ctt tcg atc 829 Lys Val Ile Ile Lys Thr Cys Gly Thr Thr Lys Leu Leu Leu Ser Ile cca gtg atc ctt gag ctc gct gat gcc ctt tca ctc act gta tgt tct 877 Pro Val Ile Leu Glu Leu Ala Asp Ala Leu Ser Leu Thr Val Cys Ser gtg agg tat act cgt ggg agc ttc ata tgt ccc ggg gcg cag cca ttt 925 Val Arg Tyr Thr Arg Gly Ser Phe Ile Cys Pro Gly Ala Gln Pro Phe ccg cat cgt aac ttc tgt gag gag gta act gtc ctc gat ggc cat ttc 973 Pro His Arg Asn Phe Cys Glu Giu Val Thr Val Leu Asp Gly His Phe agt aaa ttt ggt tta gag agt gtg gca tat gtg atg gga agt ccc aac 1021 Ser Lys Phe Gly Leu Glu Ser Val Ala Tyr Val Met Gly Ser Pro Asn tca act cag aaa tgg cat gtt tac tct gct gct gct ggt gtg aag agc 1069 Ser Thr Gln Lys Trp His Val Tyr Ser Ala Ala Ala Gly Val Lys Ser cat tct ggc cct gtt tac act ctg gaa atg tgc atg act ggt ttg gac 1117 His Ser Gly Pro Val Tyr Thr Leu Glu Met Cys Met Thr Gly Leu Asp agg aag cga gca tct gtt ttc tac aaa aca cac gcc agt tca gct act 1165 WASH_1814745.1 WASH_1815926.1 Atty. Dkt. No.: 059994-0114 Arg Lys Arg Ala Ser Val Phe Tyr Lys Thr His Ala Ser Ser Ala Thr gtt atg act gag gat tct ggt ata agg aag atc ctt ccg caa tct gag 1213 Val Met Thr Glu Asp Ser Gly Ile Arg Lys Ile Leu Pro Gln Ser Glu atc tgc gat ttt gat ttt gac cct tgt ggt tac tct atg aat gcc att 1261 Ile Cys Asp Phe Asp Phe Asp Pro Cys Gly Tyr Ser Met Asn Ala Ile gaa ggg agt gca att tcc aca atc cat gtc acc cca gaa gat ggt ttc 1309 Glu Gly Ser Ala Ile Ser Thr Ile His Val Thr Pro Glu Asp Gly Phe agc tat gca agt ttt gag gct gtg ggt tat gat ttt caa gaa tta aat 1357 Ser Tyr Ala Ser Phe Glu Ala Val Gly Tyr Asp Phe Gin Glu Leu Asn ttg aag cag ctc ctt tat agg gtt ttg gtt tgc ttt gaa ccg acc gag 1405 Leu Lys Gln Leu Leu Tyr Arg Val Leu Val Cys Phe Glu Pro Thr Glu ttc tcc att gcg ttg cat tct aat gtt gag tgt gac gaa ctt gga gcg 1453 Phe Ser Ile Ala Leu His Ser Asn Val Glu Cys Asp Glu Leu Gly Ala atg ttt tcc ctg gat gtg aaa ggt tac tct tgt gga ggg ggg aac tat 1501 Met Phe Ser Leu Asp Val Lys Gly Tyr Ser Cys Gly Gly Gly Asn Tyr gaa atg ctc ggg aag ggt gga tcg att gtc tac cac agc ttt gca gcg 1549 Glu Met Leu Gly Lys Gly Gly Ser Ile Val Tyr His Ser Phe Ala Ala act gga ggc tgc tca tct ccc agg tca atc ctg aaa tgt tgt tgg agt 1597 Thr Gly Gly Cys Ser Ser Pro Arg Ser Ile Leu Lys Cys Cys Trp Ser gag gac gag gaa gct gaa gag aag tag ttcttttcag caatgtgttt 1644 Glu Asp Glu Glu Ala Glu Glu Lys *

tttctttttc ttttcttttt tgtgtttagt tgttgatgtc atggggttat gattctgagt 1704 tttagaggca tttgtccatg ccttgagtct ttcattacaa tttatctcat gtcttgttc 1763 <210> 6 <211> 358 <212> PRT
<213> Populus deltoides <220>
<400> 6 Met Ala Leu Pro Val Ser Ala Ile Gly Phe Glu Giy Tyr Glu Lys Arg Leu Glu Ile Cys Phe Leu Glu Pro Gly Phe Phe Ser Asp Pro Glu Gly Lys Gly Leu Arg Ser Leu Ser Lys Ala Gln Leu Asp Glu Ile Leu Lys Pro Ala Glu Cys Thr Ile Val Asp Ser Leu Ser Asn Asp Glu Val Asp Ser Tyr Val Leu Ser Glu Ser Ser Leu Phe Val Tyr Pro Tyr Lys Val WASH 1814745.1 WASH 1815926.1 Atty. Dkt. No.: 05 9994-0114 Ile Ile Lys Thr Cys Gly Thr Thr Lys Leu Leu Leu Ser Ile Pro Val Ile Leu Glu Leu Ala Asp Ala Leu Ser Leu Thr Val Cys Ser Val Arg Tyr Thr Arg Gly Ser Phe Ile Cys Pro Gly Ala Gln Pro Phe Pzo His Arg Asn Phe Cys Glu Glu Val Thr Val Leu Asp Gly His Phe Ser Lys Phe Gly Leu Glu Ser Val Ala Tyr Val Met Gly Ser Pro Asn Ser Thr Gln Lys Trp His Val Tyr Ser Ala Ala Ala Gly Val Lys Ser His Ser Gly Pro Val Tyr Thr Leu Glu Met Cys Met Thr Gly Leu Asp Arg Lys Arg Ala Ser Val Phe Tyr Lys Thr His Ala Ser Ser Ala Thr Val Met Thr Glu Asp Ser Gly Ile Arg Lys Ile Leu Pro Gln Ser Glu Ile Cys Asp Phe Asp Phe Asp Pro Cys Gly Tyr Ser Met Asn Ala Ile Glu Gly Ser Ala Ile Ser Thr Ile His Val Thr Pro Glu Asp Gly Phe Ser Tyr Ala Ser Phe Glu Ala Val Gly Tyr Asp Phe Gln Glu Leu Asn Leu Lys Gln Leu Leu Tyr Arg Val Leu Val Cys Phe Glu Pro Thr Glu Phe Ser Ile Ala Leu His Ser Asn Val Glu Cys Asp Glu Leu Gly Ala Met Phe Ser Leu Asp Val Lys Gly Tyr Ser Cys Gly Gly Gly Asn Tyr Glu Met Leu Gly Lys Gly Gly Ser Ile Val Tyr His Ser Phe Ala Ala Thr Gly Gly Cys Ser Ser Pro Arg Ser Ile Leu Lys Cys Cys Trp Ser Glu Asp Glu Glu Ala Glu Glu Lys <210> 7 <211> 2523 <212> DNA
<213> Populus deltoides <220>
<221> CDs <222> (1283)...(2368) <400> 7 ttaaccctat atttctctca gaactcacat aaaaaatatt gcgagtttag ggtcataaaa 60 atctctatca tcacaaacaa atatagggtt tgttgatttg gtggctgttt tcgccgtctt 120 tgctttcaaa attcaatctt gacctgtttc ttttatgctt tttgaatagg taatatatac 180 WASH_1814745.1 WASH_1815926.1 Atty. Dkt. No.: 059994-0114 attgtttgtt ttgcttgctt tggttttgta atctggtgat cttgtttaat tctttttggc 240 tttggtttgt aggttttttt ttttgaacyt gagaaagagg ggggttttca arggagacta 300 gcttttcaag awttgatata ttttattggt aagtttttnt tttccgtttt ttggtaatag 360 ttgcatggat tcttggtgta gatgttgatt tggacttgtt tttgtattgt tgtcctttat 420 gttttatgtg tatgttaatt gttctgatcc ttgatataat gtaaatctga ggcttttatt 480 tttatggatt cgttaaggtg tatggacttg gtttatggtt ttggtcttga aaaaaaaatt 540 aggcttagca agttttcttc tgagaataac ttattttatt ttattattta aaattaaatg 600 gtgtttggga atttgaattc tttctctttg gtattttttc tatccttgtg aatctgttta 660 gtcttttaaa ttcaatattt tattttattt tacttatgat ttggtgattg attagttatt 720 tttgtagctt gtattgtctg ttgattttta atctccaata tgtcttactt ctcagttttt 780 ttcaatttgc tattggaatt ttgctattat ttttaagatt gtagactagt ttgctttgag 840 ttttgtgcta attgcaatcc ttcttgtgtg attcttacag gactgaatga tctaatggag 900 tctaaaggtg gcaagaagaa gtctagtagt agtagtagta gtaaatcctt atgttacgaa 960 gctcccctcg gttacagcat tgaagacctc agacctgctg gtggaatcaa gaagttcaga 1020 tctgctgcat actccaacgt gagtttgaag ttaatcgatc tgtttttcct ttggcatttc 1080 attgtgcgac tggtttccta atcttgtgtc atgtttattt gtgcaqtgcg ttcgaaaacc 1140 atcctgagat tttccaagtg ttgacataac cccattttag ctatttcgca cgctcagttg 1200 tctttaktct gtttttctgt tctgctttcc tcgttctctt gtactctttg ctgcactttt 1260 catttgttga ctgtgaggtc ag atg gcg ctg cca gtc tct gca atc gga ttt 1312 Met Ala Leu Pro Val Ser Ala Ile Gly Phe gaa ggt tac gaa aaa agg ctt gaa ata tct ttc cta gag cct ggc ttc 1360 Glu Gly Tyr Glu Lys Arg Leu Glu Ile Ser Phe Leu Glu Pro Gly Phe ttt tct gac cct gaa ggg aag ggc ctg agg tct ttg tcc aag gct caa 1408 Phe Ser Asp Pro Glu Gly Lys Gly Leu Arg Ser Leu Ser Lys Ala Gln ttg gac gag att ctc aga cca gct gaa tgt act att gtt gat tcg cta 1456 Leu Asp Glu Ile Leu Arg Pro Ala Glu Cys Thr Ile Val Asp 3er Leu tca aat gac cag gtt gat tct tat gtc ctg tcg gaa tcc agt ctc ttt 1504 Ser Asn Asp Gln Val Asp Ser Tyr Val Leu Ser Glu Ser Ser Leu Phe gtg tac cct tac aaa gtt att atc aaa aca tgt ggg act acc aaa ctg 1552 Val Tyr Pro Tyr Lys Val Ile Ile Lys Thr Cys Gly Thr Thr Lys Leu ctt ctc tcg ata ccg gtg att ctt aag ctc gct gat gcc ctt tca ctc 1600 Leu Leu Ser Ile Pro Val Ile Leu Lys Leu Ala Asp Ala Leu Ser Leu act gta tgt tct gtg agg tat act cgt ggg agc ttt cta tgc cct ggg 1648 Thr Val Cys Ser Val Arg Tyr Thr Arg Gly Ser Phe Leu Cys Pro Gly gct cag cca ttt cca cac cgc aac ttc tgt gag gag gta gct qtc ctt 1696 Ala Gln Pro Phe Pro His Arg Asn Phe Cys Glu Glu Val Ala Val Leu WASH_ I814745.1 WASH_1815926.1 Atty. Dkt. No.: 059994-0114 gac gat cac ttc agt aaa ctt ggt tta aac agt gtg gca tat gtg atg 1744 Asp Asp His Phe Ser Lys Leu Gly Leu Asn Ser Val Ala Tyr Val Met ggt ggt ctt gac aaa act cag aaa tgg cat gtt tac tct gcc tct gcc 1792 Gly Gly Leu Asp Lys Thr Gln Lys Trp His Val Tyr Ser Ala Ser Ala gat ata gag agc cat tct ggc cct gtt tac act ctg gaa atg tgc atg 1840 Asp Ile Glu Ser His Ser Gly Pro Val Tyr Thr Leu Glu Met Cys Met act ggt ttg ggc agg aaa caa gca tct gtt ttc tac aaa aca cat tcc 1888 Thr Gly Leu Gly Arg Lys Gln Ala Ser Val Phe Tyr Lys Thr His Ser agt tca gct gct gcg atg act gag gat tcc ggt ata agg aaa atc ctt 1936 Ser Ser Ala Ala Ala Met Thr Glu Asp Ser Gly Ile Arg Lys Ile Leu cca cag tct gag atc tgt gat ttt gat ttt gac cca tgt ggt tac tct 1984 Pro Gln Ser Glu Ile Cys Asp Phe Asp Phe Asp Pro Cys Gly Tyr Ser atg aat gcc att gaa ggg agt gca att tcc aca atc cac gtc act cca 2032 Met Asn Ala Ile Glu Gly Ser Ala Ile Ser Thr Ile His Val Thr Pro gaa gat ggt ttc agc tat gca agt ttt gag gct gtg ggc tat gat ctt 2080 Glu Asp Gly Phe Ser Tyr Ala Ser Phe Glu Ala Val Gly Tyr Asp Leu caa gat ttg aat ttg agt cgg ctg ctt gaa agg gtc ttg gct tgc ttt 2128 Gln Asp Leu Asn Leu Ser Arg Leu Leu Glu Arg Val Leu Ala Cys Phe gaa ccg acc atg ttc tcc gtt gcc ttg cat tct aat atc aag ggt gcc 2176 Glu Pro Thr Met Phe Ser Val Ala Leu His Ser Asn Ile Lys Gly Ala gaa ctt aga gca aag ttt ccc ctg gac gtg gaa ggt tac tct ggc gga 2224 Glu Leu Arg Ala Lys Phe Pro Leu Asp Val Glu Gly Tyr Ser G1y Gly gga ggg aac tat gaa atg ctt ggg aaa ggt gga tcg atc atc tac cac 2272 Gly Gly Asn Tyr Glu Met Leu Gly Lys Gly Gly Ser Ile Ile Tyr His agc ttt gca agg act gga ggc agt gca tct ccc agg tct atc ctg aaa 2320 Ser Phe Ala Arg Thr Gly Gly Ser Ala Ser Pro Arg Ser Ile Leu Lys tgt tgt tgg agt gag gat gag aag gac gag gaa gct gaa gag aag tag 2368 Cys Cys Trp Ser Glu Asp Glu Lys Asp Glu Glu Ala Glu Glu Lys *

ttcttttcag ctatttgttt tttcctttta ttttttcctt gaataaatgt catggggtta 2428 tgattctgag ttcttgaggc atttgtccat gcctcgtgtc tttcattatc gatttagttt 2488 tgacttcgga ttaataaagg ggtttgtgtt atcag 2523 <210> 8 <211> 361 <212> PRT
<213> Populus deltoides <220>

<400> 8 WASH_1814745.1 WASH_1815926,1 Atty. Dkt. No.: 059994-0114 Met Ala Leu Pro Val Ser Ala Ile Gly Phe Glu Gly Tyr Glu Lys Arg Leu Glu Ile Ser Phe Leu Glu Pro Gly Phe Phe Ser Asp Pro Glu Gly Lys Gly Leu Arg Ser Leu Ser Lys Ala G1n Leu Asp Glu Ile Leu Arg Pro Ala Glu Cys Thr Ile Val Asp Ser Leu Ser Asn Asp Gln Val Asp Ser Tyr Val Leu Ser Giu Ser Ser Leu Phe Val Tyr Pro Tyr Lys Val Ile Ile Lys Thr Cys Gly Thr Thr Lys Leu Leu Leu Ser Ile Pro Val Ile Leu Lys Leu Ala Asp Ala Leu Ser Leu Thr Val Cys Ser Val Arg Tyr Thr Arg Gly Ser Phe Leu Cys Pro Gly Ala Gln Pro Phe Pro His Arg Asn Phe Cys Glu Glu Val Ala Val Leu Asp Asp His Phe Ser Lys Leu Gly Leu Asn Ser Val Ala Tyr Val Met Gly Gly Leu Asp Lys Thr Gln Lys Trp His Val Tyr Ser Ala Ser Ala Asp Ile Glu Ser His Ser Gly Pro Val Tyr Thr Leu Glu Met Cys Met Thr Gly Leu Gly Arg Lys Gln Ala Ser Val Phe Tyr Lys Thr His Ser Ser Ser Ala Ala Ala Met Thr Glu Asp Ser Gly Ile Arg Lys Ile Leu Pro Gln Ser Glu Ile Cys Asp Phe Asp Phe Asp Pro Cys Gly Tyr Ser Met Asn Ala Ile Glu Gly Ser Ala Ile Ser Thr Ile His Val Thr Pro Glu Asp Gly Phe Ser Tyr Ala Ser Phe Glu Ala Val Gly Tyr Asp Leu Gln Asp Leu Asn Leu Ser Arg Leu Leu Glu Arg Val Leu Ala Cys Phe Glu Pro Thr Met Phe Ser Val Ala Leu His Ser Asn Ile Lys Gly Ala Glu Leu Arg Ala Lys Phe Pro Leu Asp Val Glu Gly Tyr Ser Gly Gly Gly Gly Asn Tyr Glu Met Leu Gly Lys Gly Gly Ser Ile Ile Tyr His Ser Phe Ala Arg Thr Gly Gly Ser Ala Ser Pro Arg Ser Ile Leu Lys Cys Cys Trp Ser Glu Asp Glu Lys Asp Glu Glu Ala Glu Glu Lys <210> 9 <211> 1878 WASH_1814745.1 WASH 1815926.1 Atty. Dkt. No.: 059994-0114 <212> DNA
<213> Arabidopsis thaliana <220>
<221> CDs <222> (540) ... (1878) <400> 9 gaacgcatct cattgcttca tcattaccaa atcatcaact taatcgtttc tctcaaattt 60 agggttttct cttttctcga aagtcttgcg gttttctgaa tcatctctat ctggtttgag 120 ggtttcgttt gatatctgga gaaaggggtt tctggaaaca aggagttcat aattcgcgat 180 cttgatctat cgatcttcat ttatatataa aagcgtgaat gagattatga tggagtcgaa 240 aggtggtaaa aagaagtcca gcagtagtag ttccttattt tacgaagctc ccctcggtta 300 cagcattgaa gacgttcgtc caaacggtgg aatcaagaaa ttcaaatctt ctgtctactc 360 aaactgctcc aagaggccat cctgagtacc aqcgtgcacc gatcttcata atatagttat 420 agctttcttt actttccagt ttataatttt cttctttcaa agctcctttt ctgctggttc 480 ccggatccaa tcgttctctc ctcctactac aagtcctgtc gctcacacaa caaggcgag 539 atg gcc tta tct gca atc ggt ttc gaa ggt tac gag aaa cgg ctc gag 587 Met Ala Leu Ser Ala Ile Gly Phe Glu Gly Tyr Glu Lys Arg Leu Glu gtg act ttc ttt gag cca agc atc ttt caa gac tcc aag gga ctg gga 635 Val Thr Phe Phe Glu Pro Ser Ile Phe Gln Asp Ser Lys Gly Leu Gly ctc cgt gct ctg acc aag tcc cag ctt gat gaa att ctt aca cct gct 683 Leu Arg Ala Leu Thr Lys Ser Gln Leu Asp Glu Ile Leu Thr Pro Ala gca tgc acg atc gtt tca tct ctc tcc aac gat caa ttg gac tct tac 731 Ala Cys Thr Ile Val Ser Ser Leu Ser Asn Asp Gln Leu Asp Ser Tyr gta ctc tct gag tcc agc ttc ttt gtc tac ccc tac aaa gtc atc atc 779 Val Leu Ser Glu Ser Ser Phe Phe Val Tyr Pro Tyr Lys Val Ile Ile aag act tgc ggt acc act aag ctc ctc ctc tct atc cca cca ctt cta 827 Lys Thr Cys Gly Thr Thr Lys Leu Leu Leu Ser Ile Pro Pro Leu Leu aag ctg gct ggt gag ctc tct ctg agt gtc aag tct gtg aag tac act 875 Lys Leu Ala Gly Glu Leu Ser Leu Ser Val Lys Ser Val Lys Tyr Thr cgc ggc tCc ttc ctc tgc ccc gga ggc cag cct ttt cct cac cgc agc 923 Arg Gly Ser Phe Leu Cys Pro Gly Gly Gln Pro Phe Pro His Arg Ser ttc tct gaa gaa gtc tct gtt ctt gat ggg cac ttt act cag ctg ggc 971 Phe Ser Glu Glu Val Ser Val Leu Asp Gly His Phe Thr Gln Leu Gly ttg aac agc gta gcc tac ttg atg ggc aat gat gat gag act aag aaa 1019 Leu Asn Ser Val Ala Tyr Leu Met Gly Asn Asp Asp Glu Thr Lys Lys tgg cat gtc tat gct gcc tct gcc cag gac tcc agc aac tgc aac aac 1067 Trp His Val Tyr Ala Ala Ser Ala Gln Asp Ser Ser Asn Cys Asn Asn aat gtc tac act ctc gag atg tgc atg act ggt ctg gac aga gag aaa 1115 WASH1814745.1 WASH_1815926.1 Atty. Dkt. No.: 059994-0114 Asn Val Tyr Thr Leu Glu Met Cys Met Thr Gly Leu Asp Arg Glu Lys gct gct gtc ttc tac aag gat gaa gct gac aag act ggg taa atg act 1163 Ala Ala Val Phe Tyr Lys Asp Glu Ala Asp Lys Thr Gly Ser Met Thr gat aac tct gga atc aga aag atc ctt ccc aag tct gag atc tgc gac 1211 Asp Asn Ser Gly Ile Arg Lys Ile Leu Pro Lys Ser Glu Ile Cys Asp ttt gaa ttc gag ccc tgc ggc tac tct atg aac tca att gaa ggg gat 1259 Phe Glu Phe Glu Pro Cys Gly Tyr Ser Met Asn Ser Ile Glu Gly Asp gca atc tcc acg atc cat gtg acc cct gaa gat ggg ttt agc tac gct 1307 Ala Ile Ser Thr Ile His Val Thr Pro Glu Asp Gly Phe Ser Tyr Ala agc ttc gaa gct gtg ggt tac gac ttc aac acc ctt gac ctt agc cag 1355 Ser Phe Glu Ala Val Gly Tyr Asp Phe Asn Thr Leu Asp Leu Ser Gln ctg gtg aca agg gtt ctc tct tgc ttc gag ccc aag caa ttc tct gta 1403 Leu Val Thr Arg Val Leu Ser Cys Phe Glu Pro Lys Gln Phe Ser Val gct gtg cac tcg agc gtt gga gcg aac tca tac aag cca gag att act 1451 Ala Val His Ser Ser Val Gly Ala Asn Ser Tyr Lys Pro Glu Ile Thr gta gac ttg gaa gac tat ggg tgc aga gag agg aca ttt gag tct cta 1499 Val Asp Leu Glu Asp Tyr Gly Cys Arg Glu Arg Thr Phe Glu Ser Leu gga gaa gag agt gga aca gtg atg tat cag acg ttt gag aag ctt ggt 1547 Gly Glu Glu Ser Gly Thr Val Met Tyr Gln Thr Phe Glu Lys Leu Gly aag tac tgt gga tcg cct aga tct acc ttg aag tgt gaa tgg agc agc 1595 Lys Tyr Cys Gly Ser Pro Arg Ser Thr Leu Lys Cys Glu Trp Ser Ser aac aat agc tgc agc agc gag gac gag aag gac gag gga atc tagcagaatt 1647 Asn Asn Ser Cys Ser Ser Glu Asp Glu Lys Asp Glu Gly Ile tttcttccta ataactattt tcgagctttc tgtttttgtt ctttcttttt aaaaaactta 1707 ttaagttctt atgaataatg acttgtgaag tttgagttcg tctccttcac aagcaagttg 1767 tattggtgtt ttctacttta tgaatatggg ttttatatac ctaaagactt gttatgttat 1827 tattcttaaa tgttgctgct atgatgatta ctattatcga tttttactaa a 1878 <210> 10 <211> 366 <212> PRT
<213> Arabidopsis thaliana <220>
<400> 10 Met Ala Leu Ser Ala Ile Gly Phe Glu Gly Tyr Glu Lys Arg Leu Glu Val Thr Phe Phe Glu Pro Ser Ile Phe Gln Asp Ser Lys Gly Leu Gly Leu Arg Ala Leu Thr Lys Ser Gln Leu Asp Glu Ile Leu Thr Pro Ala WASH 1814745.1 WASH 1815926.1 Atty. Dkt. No.: 059994-0114 Ala Cys Thr Ile Val Ser Ser Leu Ser Asn Asp Gln Leu Asp Ser Tyr Val Leu Ser Glu Ser Ser Phe Phe Val Tyr Pro Tyr Lys Val Ile Ile Lys Thr Cys Gly Thr Thr Lys Leu Leu Leu Ser Ile Pro Pro Leu Leu Lys Leu Ala Gly Glu Leu Ser Leu Ser Val Lys Ser Val Lys Tyr Thr Arg Gly Ser Phe Leu Cys Pro Gly Gly Gln Pro Phe Pro His Arg Ser Phe Ser Glu Glu Val Ser Val Leu Asp Gly His Phe Thr Gln Leu Gly Leu Asn Ser Val Ala Tyr Leu Met Gly Asn Asp Asp Glu Thr Lys Lys Trp His Val Tyr Ala Ala Ser Ala Gln Asp Ser Ser Asn Cys Asn Asn Asn Val Tyr Thr Leu Glu Met Cys Met Thr Gly Leu Asp Arg Glu Lys Ala Ala Val Phe Tyr Lys Asp Glu Ala Asp Lys Thr Gly Ser Met Thr Asp Asn Ser Gly Ile Arg Lys Ile Leu Pro Lys Ser Glu ile Cys Asp Phe Glu Phe Glu Pro Cys Gly Tyr Ser Met Asn Ser Ile Glu Gly Asp Ala Ile Ser Thr Ile His Val Thr Pro Glu Asp Gly Phe Ser Tyr Ala Ser Phe Glu Ala Val Gly Tyr Asp Phe Asn Thr Leu Asp Leu Ser Gln Leu Val Thr Arg Val Leu Ser Cys Phe Glu Pro Lys Gln Phe Ser Val Ala Val His Ser Ser Val Gly Ala Asn Ser Tyr Lys Pro Glu Ile Thr Val Asp Leu Glu Asp Tyr Gly Cys Arg Glu Arg Thr Phe Glu Ser Leu Gly Glu Glu Ser Gly Thr Val Met Tyr Gln Thr Phe Glu Lys Leu Gly Lys Tyr Cys Gly Ser Pro Arg Ser Thr Leu Lys Cys Glu Trp Ser Ser Asn Asn Ser Cys Ser Ser Glu Asp Glu Lys Asp Glu Gly Ile <210> 11 <211> 795 <212> DNA
<213> Xanthomonas axonopodis pv. citri <220>
<221> CDs <222> (1)...(795) <400> 11 WASH_1814745.1 WASH_1815926.1 Atty. Dkt. No.: 059994-0114 atg gtc aag ccg ttg cct cgt ctg agg cta cag ggg ttc aac aac ctc 48 Met Val Lys Pro Leu Pro Arg Leu Arg Leu Gln Gly Phe Asn Asn Leu acc aag gcg ttg agc ttc aac atc tac gac gtc tgt tat gcg cgt acc 96 Thr Lys Ala Leu Ser Phe Asn Ile Tyr Asp Val Cys Tyr Ala Arg Thr gaa gag gag cgt cag cgc tac atc gag tat atc gat gag caa tac gac 144 Glu Glu Glu Arg Gln Arg Tyr Ile Glu Tyr Ile Asp Glu Gln Tyr Asp gcc gat cgt ctg acg cag atc ctg acc gat gtg gcc gag atc att ggc 192 Ala Asp Arg Leu Thr Gln Ile Leu Thr Asp Val Ala Glu Ile Ile Gly gcc aac atc ctc aat atc gca cgt cag gac tac gat ccg cag ggt gca 240 Ala Asn Ile Leu Asn Ile Ala Arg Gln Asp Tyr Asp Pro Gln Gly Ala tcg gtg acg atc ctg atc tcc gaa gag ccg gtg atc gac aag aag cag 288 Ser Val Thr Ile Leu Ile Ser Glu Glu Pro Val Ile Asp Lys Lys Gln gcc ggc aag gaa ttg atc tcc gat gcc gtg gtt gct cat atg gat aag 336 Ala Gly Lys Glu Leu Ile Ser Asp Ala Val Val Ala His Met Asp Lys agt cat atc act gtc cat aca tat ccg gaa acg cat ccg cag gaa ggc 384 Ser His Ile Thr Val His Thr Tyr Pro Glu Thr His Pro Gln Glu Gly atc gcc acg ttc cgc gcc gac atc gac gtc gcg acc tgc ggc gtg att 432 Ile Ala Thr Phe Arg Ala Asp Ile Asp Val Ala Thr Cys Gly Val Ile tcg ccg ttg aaa gcg ctc aac tac ctg atc gag agt ctg gaa tcg gac 480 Ser Pro Leu Lys Ala Leu Asn Tyr Leu Ile Glu Ser Leu Glu Ser Asp att gtg atc atg gat tac cgc gtg cgt ggc ttc acg cgc gat gtg aag 528 Ile Val Ile Met Asp Tyr Arg Vai Arg Gly Phe Thr Arg Asp Val Lys ggc aag aag cat tac atc gat cac aag atc aac tcg att cag caa ttt 576 Gly Lys Lys His Tyr Ile Asp His Lys Ile Asn Ser Ile Gln Gln Phe ctc gcc aag aac gtg aag tcg cgc tac gag atg ttc gac gtc aac gtc 624 Leu Ala Lys Asn Val Lys Ser Arg Tyr Glu Met Phe Asp Val Asn Val tac cag gaa aat atc ttc cac acg aag atg cac ctg aag gac ttc gac 672 Tyr Gln Glu Asn Ile Phe His Thr Lys Met His Leu Lys Asp Phe Asp ctg gat cag tac ctg ttc gaa gag cgc gcc aag aac ctc tcg ttc aag 720 Leu Asp Gln Tyr Leu Phe Glu Glu Arg Ala Lys Asn Leu Ser Phe Lys gag cgc atg aag atc gaa acg ctg ctc aag cgc gaa atc gaa gaa ctg 768 Glu Arg Met Lys Ile Glu Thr Leu Leu Lys Arg Glu Ile Glu Glu Leu ttt cac gga cgc aac ctg agc gaa taa 795 Phe His Gly Arg Asn Leu Ser Glu *

<210> 12 <211> 264 <212> PRT

WASH 1814745.1 WASH_1815926.1 Atty. Dkt. No.: 059994-0114 <213> Xanthomonas axonopodis pv. Citri <220>
<400> 12 Met Val Lys Pro Leu Pro Arg Leu Arg Leu Gln Gly Phe Asn Asn Leu Thr Lys Ala Leu Ser Phe Asn Ile Tyr Asp Val Cys Tyr Ala Arg Thr Glu Glu Glu Arg Gln Arg Tyr Ile Glu Tyr Ile Asp Glu Gln Tyr Asp Ala Asp Arg Leu Thr Gln Ile Leu Thr Asp Val Ala Glu Ile Ile Gly Ala Asn Ile Leu Asn Ile Ala Arg Gln Asp Tyr Asp Pro Gln Gly Ala Ser Val Thr Ile Leu Ile Ser Glu Glu Pro Val Ile Asp Lys Lys Gln Ala Gly Lys Glu Leu Ile Ser Asp Ala Val Val Ala His Met Asp Lys Ser His Ile Thr Val His Thr Tyr Pro Glu Thr His Pro Gln Glu Gly Ile Ala Thr Phe Arg Ala Asp Ile Asp Val Ala Thr Cys Gly Val Ile Ser Pro Leu Lys Ala Leu Asn Tyr Leu Ile Glu Ser Leu Glu Ser Asp Ile Val Ile Met Asp Tyr Arg Val Arg Gly Phe Thr Arg Asp Val Lys Gly Lys Lys His Tyr Ile Asp His Lys Ile Asn Ser Ile Gln Gln Phe Leu Ala Lys Asn Val Lys Ser Arg Tyr Glu Met Phe Asp Val Asn Val Tyr Gln Glu Asn Ile Phe His Thr Lys Met His Leu Lys Asp Phe Asp Leu Asp Gln Tyr Leu Phe Glu Glu Arg Ala Lys Asn Leu Ser Phe Lys Glu Arg Met Lys Ile Glu Thr Leu Leu Lys Arg Glu Ile Glu Glu Leu Phe His Gly Arg Asn Leu Ser Glu <210> 13 <211> 1820 <212> DNA
<213> Datura stramonium <220>
<221> CDs <222> (491) ... (1579) <400> 13 cttgtatact cttccgaatc gccttctcca aatttctagg gtttctctct tcttcggaat 60 tttctggttt ggaaagatcg ttgtttattg gagataaatc ggatttggaa caaatttcaa 120 WASH1814745.1 WASH 1815926_.1 Atty. Dkt. No.: 059994-0114 tttttaaata ttgtgaatga tctaatggag tcaaaaggtg ggaaaaagaa gtctagtagt 180 agttccttat tttacgaagc tcccctcggc tacagcattg aagacgttcg accaaacggt 240 ggagtgaaga agttcagatc tgctgcttac tccaactgcg cgcgcaaacc atcctgatat 300 tccctaaact tctgtcctta aagcgtcaat agacgcaacc aaaaaaaaca aaaaaaattt 360 ctgctttcaa tttctttcgt tgtcaagccc tcactccttt tcttccttct tttactactt 420 cctgctttgc actcgttgct ctgaacattt tctgctttaa cttcttttgc tgctgtgaac 480 ttttttcata atg gaa atg gac ttg cca gtc tct gcc atc ggt ttt gaa 529 Met Glu Met Asp Leu Pro Val Ser Ala Ile Gly Phe Glu ggt ttt gaa aag agg ctc gaa att tct ttt gtc gag cct ggt ctg ttt 577 Gly Phe Glu Lys Arg Leu Glu Ile Ser Phe Val Glu Pro Gly Leu Phe tct gat cct aat gga aaa gga ctt cga tct ctc tca aag gca cag ttg 625 Ser Asp Pro Asn Gly Lys Gly Leu Arg Ser Leu Ser Lys Ala Gln Leu gat gaa att ctc gga cct gct gag tgc acc att gtc gat aac cta tca 673 Asp Glu Ile Leu Gly Pro Ala Glu Cys Thr Ile Val Asp Asn Leu Ser aat gac tat gtt gat tcc tat gtc ctc tct gag tcg agc ctc ttc gtt 721 Asn Asp Tyr Val Asp Ser Tyr Val Leu Ser Glu Ser Ser Leu Phe Val tat tct tac aag ata atc att aaa aga tgt ggc acc aca aag ttg ctt 769 Tyr Ser Tyr Lys Ile Ile Ile Lys Thr Cys Gly Thr Thr Lys Leu Leu ctc gca att ccg ccc att cta agg ttg gct gag acc ttg tct ctc aaa 817 Leu Ala Ile Pro Pro Ile Leu Arg Leu Ala Glu Thr Leu Ser Leu Lys gta caa gat gtg agg tat acc cgt ggg agc ttc att ttc cct ggt gct 865 Val Gln Asp Val Arg Tyr Thr Arg Gly Ser Phe Ile Phe Pro Gly Ala caa tcg ttt cct cac cgt cac ttt tct gaa gaa gtt gct gtc ctc gat 913 Gln Ser Phe Pro His Arg His Phe Ser Glu Glu Val Ala Val Leu Asp ggc tat ttt ggg aag ctt gct gcc ggt agc aag gct gtg att atg ggc 961 Gly Tyr Phe Gly Lys Leu Ala Ala Gly Ser Lys Ala Val Ile Met Gly aac cct gac aaa aca cag aaa tgg cat gtt tac tct gcc tca gct ggg 1009 Asn Pro Asp Lys Thr Gin Lys Trp His Val Tyr Ser Ala Ser Ala Gly cct gtt cag tct aat gac cct gtt tac act cta gag atg tgt atg act 1057 Pro Va1 Gln Ser Asn Asp Pro Val Tyr Thr Leu Glu Met Cys Met Thr ggt ttg gac agg gag aag gca tct gtc ttt tac aag act gaa gga agc 1105 Gly Leu Asp Arg Glu Lys Ala Ser Val Phe Tyr Lys Thr Glu Gly Ser tcg gct gct cac atg act gtt aga tct ggc ata agg aag atc ctc ccc 1153 Ser Ala Ala His Met Thr Val Arg Ser Gly Ile Arg Lys Ile Leu Pro aac tct gag ata tgc gat ttt gag ttt gaa ccc tgt ggt tat tcc atg 1201 Asn Ser Glu Ile Cys Asp Phe Glu Phe Glu Pro Cys Gly Tyr Ser Met WASH 18 14745.1 WASH_1815926_.1 Atty. Dkt. No.: 059994-0114 aat tct att gaa gga gct gct gtt tca acc att cac att aca cca gaa 1249 Asn Ser Ile Glu Gly Ala Ala Val Ser Thr Ile His Ile Thr Pro Glu gac qgc ttt agc tat gct agc ttt gaa tca gtt gga tat gat ctg aaa 1297 Asp Gly Phe Ser Tyr Ala Ser Phe Glu Ser Val Gly Tyr Asp Leu Lys acc atg gag ttg ggt ccc ctg gtt gag agg gtg ctg gca tgt ttt gag 1345 Thr Met Glu Leu Gly Pro Leu Val Glu Arg Val Leu Ala Cys Phe Glu cct gca gag ttc tct att gct ttg cat gct gat gtt gct acc aag tta 1393 Pro Ala Glu Phe Ser Ile Ala Leu His Ala Asp Val Ala Thr Lys Leu ctg gag cgt gtt tgc tgt gtt gat gtt aag ggc tac tct ctt gct gag 1441 Leu Glu Arg Val Cys Cys Val Asp Val Lys Gly Tyr Ser Leu Ala Glu tgg agt cca gaa qaa ttt ggc aag ggt ggt tcc att gtc tac cag aag 1489 Trp Ser Pro Glu Glu Phe Gly Lys Gly Gly Ser Ile Val Tyr Gln Lys ttc act aag act cct tac tgt gca tct ccc aag tcc gtt ctg aag ggc 1537 Phe Thr Lys Thr Pro Tyr Cys Ala Ser Pro Lys Ser Val Leu Lys Gly tgc tgg aaa gag gaa gag gaa gag aaa gaa gaa aag gag tag tgtttgtctt 1589 Cys Trp Lys Glu Glu Glu Glu Glu Lys Glu Glu Lys Glu *

gagggtcgtt ttgttattgt tgttttagtg tctgtcgttt gctcatgttt tacctgtttg 1649 tcagaataaa ggacttaaga ttgtcccatt tgtgtcagtt tggatttgta acgattgtgt 1709 gcaaattctg aactagtccg gcttcttggt gttccaccag aagccctatg tgtctgcatt 1769 ttgttccctg ttccgttgtg gcttctaata aaagtttgtt tgttgtgtgt a 1820 <210> 14 <211> 362 <212> PRT
<213> Datura stramonium <220>

<400> 14 Met Glu Met Asp Leu Pro Val Ser Ala Ile Gly Phe Glu Gly Phe Glu Lys Arg Leu Glu Ile Ser Phe Val Glu Pro Gly Leu Phe Ser Asp Pro Asn Gly Lys Gly Leu Arg Ser Leu Ser Lys Ala Gln Leu Asp Glu Ile Leu Gly Pro Ala Glu Cys Thr Ile Val Asp Asn Leu Ser Asn Asp Tyr Val Asp Ser Tyr Val Leu Ser Glu Ser Ser Leu Phe Val Tyr Ser Tyr Lys Ile Ile Ile Lys Thr Cys Gly Thr Thr Lys Leu Leu Leu Ala Ile Pro Pro Ile Leu Arg Leu Ala Glu Thr Leu Ser Leu Lys Va1 Gln Asp WASH1814745.1 WASH_18159267.1 Atty. Dkt. No.: 059994-0114 Val Arg Tyr Thr Arg Gly Ser Phe Ile Phe Pro Gly Ala Gln Ser Phe Pro His Arg His Phe Ser Glu Glu Val Ala Val Leu Asp Gly Tyr Phe Gly Lys Leu Ala Ala Gly Ser Lys Ala Val Ile Met Gly Asn Pro Asp Lys Thr Gln Lys Trp His Val Tyr Ser Ala Ser Ala Gly Pro Val Gln Ser Asn Asp Pro Val Tyr Thr Leu Glu Met Cys Met Thr Gly Leu Asp Arg Glu Lys Ala Ser Val Phe Tyr Lys Thr Glu Gly Ser Ser Ala Ala His Met Thr Val Arg Ser Gly Ile Arg Lys Ile Leu Pro Asn Ser Glu Ile Cys Asp Phe Glu Phe Glu Pro Cys Gly Tyr Ser Met Asn Ser Ile Glu Gly Ala Ala Val Ser Thr Ile His Ile Thr Pro Glu Asp Gly Phe Ser Tyr Ala Ser Phe Glu Ser Val Gly Tyr Asp Leu Lys Thr Met Glu Leu Gly Pro Leu Val Glu Arg Val Leu Ala Cys Phe Glu Pro Ala Glu Phe Ser Ile Ala Leu His Ala Asp Val Ala Thr Lys Leu Leu Glu Arg Val Cys Cys Val Asp Val Lys Gly Tyr Ser Leu Ala Glu Trp Ser Pro Glu Glu Phe Gly Lys Gly Gly Ser Ile Val Tyr Gln Lys Phe Thr Lys Thr Pro Tyr Cys Ala Ser Pro Lys Ser Val Leu Lys Gly Cys Trp Lys Glu Glu Glu Glu Glu Lys Glu Glu Lys Glu <210> 15 <211> 1971 <212> DNA
<213> Oryza sativa <220>
<221> CDs <222> (357) ... (1724) <400> 15 acgcgtccgt ttcgttttgg tattcccata cactttgttt ctcttccccg tctcgtggtc 60 gagagaaacc gagaggagag agagggagag gagcqctgcc gattttccgg agggggagat 120 ctttttcttc tccctgaagg ttcgagatcg atctgcgacg aggaggagcg gggaatcgta 180 actttatttc cgaggaaatt tcaacaaatt gatgcactaa tggagtccaa gggtggcaaa 240 aagaagtcta gcagtagtcg ttcctccctg atgtacgaag ctcccctcgg ctacagcatt 300 gaggacctcc gacctgccgg cggcatcaag aagttccgct ctgctgctta ctccaactgc 360 gcgaggaagc cctcttgata gcccccttgg ctaccttgac ctagtagttt agttactcct 420 WASH1814745.1 WASH 1815926_.1 Atty. Dkt. No.: 059994-0114 ctgaattctc gttttgggct ttcttacatc tctctggcta gctgcttccc agtgaccggg 480 aaggtcatca gtcctgcttc ctttcttcct catctctggc atcgtccttc gaaaca 536 atg gca gtc ctg tca gtt gct gac tct cca ccg gtc tcg gcg atc ggg 584 Met Ala Val Leu Ser Val Ala Asp Ser Pro Pro Val Ser Ala Ile Gly ttt gag gga tat gag aaa cgc ctc gag atc act ttc tcc gag gcg cct 632 Phe Glu Gly Tyr Glu Lys Arg Leu Glu Ile Thr Phe Ser Glu Ala Pro gtc ttt gct gac ccc aat ggc agg ggg ttg cgt gca ctg tca cgt gct 680 Val Phe Ala Asp Pro Asn Gly Arg Gly Leu Arg Ala Leu Ser Arg Ala cag att gac tct gtt ctt gat ctg gcg agg tgc acc att gtg tct gag 728 Gln Ile Asp Ser Val Leu Asp Leu Ala Arg Cys Thr Ile Val Ser Glu ctc tcc aat gag gtc ttc gac tcg tat gtc ctt tct gaa tcg agc ctc 776 Leu Ser Asn Glu Val Phe Asp Ser Tyr Val Leu Ser Glu Ser Ser Leu ttt gtg tac cca tac aag att gtg atc aag acc tgt ggg act acc aag 824 Phe Val Tyr Pro Tyr Lys Ile Val Ile Lys Thr Cys Gly Thr Thr Lys ctc ctg ctc gcc att ccg agg att ctt gag ctt gct gaa gag cta tct 872 Leu Leu Leu Ala Ile Pro Arg Ile Leu Glu Leu Ala Glu Glu Leu Ser ctg cca ctt gaa gct gtg aag tac tct cgt ggq aca ttc att ttt cct 920 Leu Pro Leu Glu Ala Val Lys Tyr Ser Arg Gly Thr Phe Ile Phe Pro gaa gca cag ccc tct cca cac aag aac ttc tct gag gag gtt gca gtc 968 Glu Ala Gin Pro Ser Pro His Lys Asn Phe Ser Glu Glu Val Ala Val ctg aac cgc tac ttc ggc ggt ctc aaa tct ggt ggc aac gca tac gtg Leu Asn Arg Tyr Phe Gly Gly Leu Lys Ser Gly Gly Asn Ala Tyr Val 1016 att gga gat cct gca aag cca ggg cag aag tgg cat gtc tac tat gcc 1064 Ile Gly Asp Pro Ala Lys Pro Gly Gln Lys Trp His Val Tyr Tyr Ala acc cag cac ccg gag caa cct gtg gtc act ctt gag atg tgc atg act 1112 Thr Gln His Pro Glu Gln Pro Val Val Thr Leu Glu Met Cys Met Thr ggg ctg gac aag aag aaa gct tct gtc ttc ttc aag act tct gct gat 1160 Gly Leu Asp Lys Lys Lys Ala Ser Val Phe Phe Lys Thr Ser Ala Asp gga cac aca aca tat gct aag gaa atg acc aag ctc tca ggt atc tct 1208 Gly His Thr Thr Tyr Ala Lys Glu Met Thr Lys Leu Ser Gly Ile Ser gac atc atc cca gag atg gaa gtc tgc gac ttc gat ttc gag ccc tgc 1256 Asp Ile Ile Pro Glu Met Glu Val Cys Asp Phe Asp Phe Glu Pro Cys ggc tac tcc atg aat gcc atc cac ggc cct gct ttc tca acc att cat 1304 Gly Tyr Ser Met Asn Ala Ile His Gly Pro Ala Phe Ser Thr Ile His gtg act cct gag gat ggc ttc agc tat gcc agc tac gag gtc atg ggc 1352 Val Thr Pro Glu Asp Gly Phe Ser Tyr Ala Ser Tyr Glu Val Met Gly WASH1814745.1 WASH 1815926.1 Atty. Dkt. No.: 059994=0114 ttc aac cct gct tcc ctt gcc tat ggt gac ctt gtc aag agg gtg ttg 1400 Phe Asn Pro Ala Ser Leu Ala Tyr Gly Asp Leu Val Lys Arg Val Leu aga tgc ttt ggc ccg ttg gag ttc tct gtt gcg gtt acc atc ttt ggt 1448 Arg Cys Phe Gly Pro Leu Glu Phe Ser Val Ala Val Thr Ile Phe Gly ggc cgc aac cat gca ggg acc tgg gcc aag ggg ctg gat gtc ggg gcc 1496 Gly Arg Asn His Ala Gly Thr Trp Ala Lys Gly Leu Asp Val Gly Ala tat tct tgc agc aac atg gtt gag cag gag ctg cct tct ggg ggt ttg 1544 Tyr Ser Cys Ser Asn Met Val Glu Gln Glu Leu Pro Ser Gly Gly Leu ctc att tac cag agc ttc act gct act gct gaa atc gcc acc ggg tcg 1592 Leu Ile Tyr Gln Ser Phe Thr Ala Thr Ala Glu Ile Ala Thr Gly Ser cca cga tct gtt ctg cat tgc ttt gct gat gaa aac acg gag aaa gct 1640 Pro Arg Ser Val Leu His Cys Phe Ala Asp Glu Asn Thr Glu Lys Ala ggt aaa atg gag gct ctc tac tgg gaa gac gat gct gtc gag gag ata 1688 Gly Lys Met Glu Ala Leu Tyr Trp Glu Asp Asp Ala Val Glu Glu Ile gat ggc aca gag ggt aag aag atg agg agc tgc tga tgagggaagc 1734 Asp Gly Thr Glu Gly Lys Lys Met Arg Ser Cys *

gcactgaaga ttaaagaata ctcaaaactc cagtagcgat cactctgtta ctttgtgaag 1794 cagccagcca gactattcat atagtactag tatatgctat gaccaatgtt ctttattatc 1854 ttgcatatat tctgaataaa agggctctag gtggtctgct ttgtctgcca cagtgagcaa 1914 aatgtatccg aatatcaaac tttaatgttg ggaatataat acaagtcatt ttataat 1971 <210> 16 <211> 395 <212> PRT
<213> Oryza sativa <220>

<400> 16 Met Ala Val Leu Ser Val Ala Asp Ser Pro Pro Val Ser Ala Ile Gly Phe Glu Gly Tyr Glu Lys Arg Leu Glu Ile Thr Phe Ser Glu Ala Pro Val Phe Ala Asp Pro Asn Gly Arg Gly Leu Arg Ala Leu Ser Arg Ala Gln Ile Asp Ser Val Leu Asp Leu Ala Arg Cys Thr Ile Val Ser Glu Leu Ser Asn Glu Val Phe Asp Ser Tyr Val Leu Ser Glu Ser Ser Leu Phe Val Tyr Pro Tyr Lys Ile Val Ile Lys Thr Cys Gly Thr Thr Lys Leu Leu Leu Ala Ile Pro Arg Ile Leu Glu Leu Ala Glu Glu Leu Ser WASH1814745. l WASH 1815926_.1 Atty. Dkt. No.: 059994-0114 Leu Pro Leu Glu Ala Val Lys Tyr Ser Arg Gly Thr Phe Ile Phe Pro Glu Ala Gln Pro Ser Pro His Lys Asn Phe Ser Glu Glu Val Ala Val Leu Asn Arg Tyr Phe Gly Gly Leu Lys Ser Gly Gly Asn Ala Tyr Val Ile Gly Asp Pro Ala Lys Pro Gly Gln Lys Trp His Val Tyr Tyr Ala Thr Gln His Pro Glu Gin Pro Val Val Thr Leu Glu Met Cys Met Thr Gly Leu Asp Lys Lys Lys Ala Ser Val Phe Phe Lys Thr Ser Ala Asp Gly His Thr Thr Tyr Ala Lys Glu Met Thr Lys Leu Ser Gly Ile Ser Asp Ile Ile Pro Glu Met Giu Val Cys Asp Phe Asp Phe Glu Pro Cys Gly Tyr Ser Met Asn Ala Ile His Gly Pro Ala Phe Ser Thr Ile His Val Thr Pro Glu Asp Gly Phe Ser Tyr Ala Ser Tyr Glu Val Met Gly Phe Asn Pro Ala Ser Leu Ala Tyr Gly Asp Leu Val Lys Arg Val Leu Arg Cys Phe Gly Pro Leu Glu Phe Ser Val Ala Val Thr Ile Phe Gly Gly Arg Asn His Ala Gly Thr Trp Ala Lys Giy Leu Asp Val Gly Ala Tyr Ser Cys Ser Asn Met Val Glu Gin Glu Leu Pro Ser Gly Gly Leu Leu Ile Tyr Gln Ser Phe Thr Ala Thr Ala Glu Ile Ala Thr Gly Ser Pro Arg Ser Val Leu His Cys Phe Ala Asp Glu Asn Thr Glu Lys Ala Gly Lys Met Glu Ala Leu Tyr Trp Glu Asp Asp Ala Val Glu Glu Ile Asp Gly Thr Glu Gly Lys Lys Met Arg Ser Cys <210> 17 <211> 2952 <212> DNA
<213> Oryza sativa <220>
<221> CDs <222> (1444) ... (2622) <400> 17 gcctgggcga ttctagaaag atgtctcttt tgctaatggt tattattagc tgaatcgctg 60 atatggtgaa tccaggaaag tttcatgctt tttgtcctat gtcctgtctt tctttgtcat 120 attcgtacct ttcccagttt gcgatgccca agaagcaacc tttctgttgg aaacatctag 180 WASH_1814745.1 WASH 1815926.1 Atty. Dkt. No.: 059994-0114 tgctcaattc attatttcaa attgttgaaa tcttcaagga qcagtttatt gtgcgccctt 240 gttgattgct cttgtttatt tacaagtggg gatttaggcg ctgggttcat atatgttctt 300 tgtttctgcc aggtttcctt gttaggctct agttttgctc gttgtttact gtaaattccc 360 atgtatatta agtttgtgca gtgcgctgtc agcttggcaa atgggctttt cttttcctga 420 tcgaaaaaaa aacaaacctg tgattaacaa ggggaaatta ccttcactgt gcctattggc 480 atcattccaa gttattcatg ctttagttcc tagccattaa cttaagatag aaataagatc 540 ctgccagttc actagttttc ttgtcagttt gatatatact ttgatcttgt cttttctggt 600 tgttttcctt aattttgtct gtgttgttct agtgcttaat tagttgcctc tcagttcttc 660 acattctatg tttttgagta aaactctgtc catatggccg ttctgttatg tatctcagca 720 atctgcttat attttttttt tgcatcccca cacaattcta tttgacgttg taaaaaagaa 780 gagagataac tagataagcg caactgatgc tctatactta gtttcacggt tcttggttta 840 atgcctaaaa attgtgcact atcagtactt caattatgtt atctagtagt gttttgtctc 900 ctctgaataa tgcctgccat tcttatgctg ctctactgct gagaaaacta cactatttga 960 attactgatt gctaattatg tttcgattaa cagcataaag gaacattttg ttgatgttct 1020 aatggagtca aaaggtggca aaaagaagtc tagcagtagt agttccatgt acgaagctcc 1080 cctaggctac aagattgagg acgttcgccc agccggagga atcaagaagt tccagtctgc 1140 tgcttactcc aacgtaagca ccaaaagatt taagcagcat actttaaata acaaaaatat 1200 ttacatttat gaactctggt ttctaatgct tatttggttt atcttttcct ttgcagtgcg 1260 ctcgcaagcc atcctgatat tccttcacat gcctttcccc gtagagtagg aatttcctgc 1320 aatttttcct ttgttttgtt gcaataaatc tcccttggct ggctgctttc tcaatctctt 1380 cccttgtctt ctccgatctc ttctttcctg tcttgtccac tgctccttgc aacacttgat 1440 caa atg tcg atg tcc ttg gct gat tct tgg ggc tct gcc cct gcc tcg 1488 Met Ser Met Ser Leu Ala Asp Ser Trp Gly Ser Ala Pro Ala Ser ccc att ggg ttt gaa ggc tat gag aag cgc ctt gag ata aca ttg tct 1536 Pro Ile Gly Phe Glu Gly Tyr Glu Lys Arg Leu Glu Ile Thr Leu Ser gat gcg ccc gtc ttt gtg gac ccc tgt ggt cgc ggc ctt cgt gcc ctc 1584 Asp Ala Pro Val Phe Val Asp Pro Cys Gly Arg Gly Leu Arg Ala Leu tca cgt gag cag atc gac tcg ttc ttg gat ctt gca aag tgt acc att 1632 Ser Arg Glu Gln Ile Asp Ser Phe Leu Asp Leu Ala Lys Cys Thr Ile gta tcc cat ctt tcg aac aag cac ttc gac tca tat gtg ctt tca gag 1680 Val Ser His Leu Ser Asn Lys His Phe Asp Ser Tyr Val Leu Ser Glu tcg agc ctt ttt gtt tat ccc cac aag gtt gtt ctg aag acc tgt ggt 1728 Ser Ser Leu Phe Val Tyr Pro His Lys Val Val Leu Lys Thr Cys Gly aca aca aag ctt ctg ctc tcc att cct cgc atc ctt gag ctt gct gca 1776 Thr Thr Lys Leu Leu Leu Ser Ile Pro Arg Ile Leu Glu Leu Ala Ala gag ttg tca ctg cct gtt cta tca gtg aag tac tct cgt ggg atg ttc 1824 Glu Leu Ser Leu Pro Val Leu Ser Val Lys Tyr Ser Arg Gly Met Phe WASH1814745.1 WASH_18159267.1 Atty. Dkt. No.: 059994-0114 atc ttc cct gga gcg cag cca tca ccg cat cgc agc ttc ttg gag gag 1872 Ile Phe Pro Gly Ala Gln Pro Ser Pro His Arg Ser Phe Leu Glu Glu gtt tct gtg ctc aac agc ttc ttt ggt ggc Ctc aag tca ggt ggc aat 1920 Val Ser Val Leu Asn Ser Phe Phe Gly Gly Leu Lys Ser Gly Gly Asn gct tat gtc att ggt gat gca ttc aag ccc aag aag aag tgg cat gtc 1968 Ala Tyr Val Ile Gly Asp Ala Phe Lys Pro Lys Lys Lys Trp His Val tac tat gcc aca gaa gag cct gag cag ccc atg gtt aca ctc gag atg 2016 Tyr Tyr Ala Thr Glu Glu Pro Glu Gln Pro Met Val Thr Leu Glu Met tgc atg act ggg ctg gat gct aag aaa gct gag gtg ttc ttc aag gat 2064 Cys Met Thr Gly Leu Asp Ala Lys Lys Ala Glu Val Phe Phe Lys Asp tcc act gat ggc tcc tgc tca tca gct aag gag atg act atg ttc tct 2112 Ser Thr Asp Gly Ser Cys Ser Ser Ala Lys Glu Met Thr Met Phe Ser ggg att tct gaa atc atc cct gag atg gag att tgt gac ttt gag ttt 2160 Gly Ile Ser Glu Ile Ile Pro Glu Met Glu Ile Cys Asp Phe Glu Phe gac ccg tgt ggg tac tca atg aat ggc att tat ggt ccc gcc gtc tcc 2208 Asp Pro Cys Gly Tyr Ser Met Asn Gly Ile Tyr Gly Pro Ala Val Ser acg atc cat gtc act cct gag gaa ggt ttc agc tat gca agc tat gaa 2256 Thr Ile His Val Thr Pro Glu Glu Gly Phe Ser Tyr Ala Ser Tyr Glu gca atg aac ttc aat cct agc tcc ttg gtc tac gat gat ttg atc aag 2304 Ala Met Asn Phe Asn Pro Ser Ser Leu Val Tyr Asp Asp Leu Ile Lys aag gtc ctg gct tgt ttc tgc cct tca gac ttt tcg gtc gct gtt acc 2352 Lys Val Leu Ala Cys Phe Cys Pro Ser Asp Phe Ser Val Ala Val Thr atc ttc ggt ggg cat ggt ttt gcc aaa tca tgg gca aaa ggt gca gag 2400 Ile Phe Gly Gly His Gly Phe Ala Lys Ser Trp Ala Lys Gly Ala Glu gtt gat tcc tac atg tgc gat gat ctt gtt gag caa gag ctt cct ggt 2448 Val Asp Ser Tyr Met Cys Asp Asp Leu Val Glu Gln Glu Leu Pro Gly ggc ggt gtg ctg atg tat cag agt ttt act gct gtt act cct ggt gct 2496 Giy Gly Val Leu Met Tyr Gln Ser Phe Thr Ala Val Thr Pro Gly Ala gtg tca ccg agg tcg acc ttg gat ggc tgg aac agc gat gga gca gag 2544 Val Ser Pro Arg Ser Thr Leu Asp Gly Trp Asn Ser Asp Gly Ala Glu atg gtt gcg aag agc aaa gag atg agt gtc tgc tgg gaa gga gag aag 2592 Met Val Ala Lys Ser Lys Glu Met Ser Val Cys Trp Glu Gly Glu Lys gcg gcg aag aag aaa gat gca gat gcc tga gaaatcggca gttcttcaac 2642 Ala Ala Lys Lys Lys Asp Ala Asp Ala *

ctcaagttgc aagtttgatt catctgaagt ttctgagacg ccttatttgg ttctgctctt 2702 WASH 1814745.1 WASH 1815926_1 Atty. Dkt. No.: 059994-0114 gcagtttcgg ttaagcagct ggctgggcta ccggcaaagc atatgttact tgttcattgt 2762 tcttttatat ctgtctgccc aaataagtgc tctagatggt ttgctgcgtc tgccatagtg 2822 agcaattcag attgtagtag aattcttatc tgttgccgat gctctttaag ggagaatgat 2882 atgtaactcg attattttca ataactattg caacttccat aattttctgt ccattggtta 2942 tgtttgtttg 2952 <210> 18 <211> 392 <212> PRT
<213> Oryza sativa <220>
<400> 18 Met Ser Met Ser Leu Ala Asp Ser Trp Gly Ser Ala Pro Ala Ser Pro Ile Gly Phe Glu Gly Tyr Glu Lys Arg Leu Glu Ile Thr Leu Ser Asp Ala Pro Val Phe Val Asp Pro Cys Gly Arg Gly Leu Arg Ala Leu Ser Arg Glu Gln Ile Asp Ser Phe Leu Asp Leu Ala Lys Cys Thr Ile Val Ser His Leu Ser Asn Lys His Phe Asp Ser Tyr Val Leu Ser Glu Ser Ser Leu Phe Val Tyr Pro His Lys Val Val Leu Lys Thr Cys Gly Thr Thr Lys Leu Leu Leu Ser Ile Pro Arg Ile Leu Glu Leu Ala Ala Glu Leu Ser Leu Pro Val Leu Ser Val Lys Tyr Ser Arg Gly Met Phe Ile Phe Pro Gly Ala Gln Pro Ser Pro His Arg Ser Phe Leu Glu Glu Val Ser Val Leu Asn Ser Phe Phe Gly Gly Leu Lys Ser Gly Gly Asn Ala Tyr Val Ile Gly Asp Ala Phe Lys Pro Lys Lys Lys Trp His Val Tyr Tyr Ala Thr Glu Glu Pro Glu Gln Pro Met Val Thr Leu Glu Met Cys Met Thr Gly Leu Asp Ala Lys Lys Ala Glu Val Phe Phe Lys Asp Ser Thr Asp Gly Ser Cys Ser Ser Ala Lys Glu Met Thr Met Phe Ser Gly ile Ser Glu Ile Ile Pro Glu Met Glu Ile Cys Asp Phe Glu Phe Asp Pro Cys Gly Tyr Ser Met Asn Gly Ile Tyr Gly Pro Ala Val Ser Thr Ile His Val Thr Pro Glu Glu Gly Phe Ser Tyr Ala Ser Tyr Glu Ala Met Asn Phe Asn Pro Ser Ser Leu Val Tyr Asp Asp Leu Ile Lys Lys WASH 1814745.1 WASH 1815926.1 Atty. Dkt. No.: 059994-0114 Val Leu Ala Cys Phe Cys Pro Ser Asp Phe Ser Val Ala Val Thr Ile Phe Gly Gly His Gly Phe Ala Lys Ser Trp Ala Lys Gly Ala Glu Val Asp Ser Tyr Met Cys Asp Asp Leu Val Glu Gln Glu Leu Pro Gly Gly Gly Val Leu Met Tyr Gln Ser Phe Thr Ala Val T'hr Pro Gly Ala Val Ser Pro Arg Ser Thr Leu Asp Gly Trp Asn Ser Asp Gly Ala Glu Met Val Ala Lys Ser Lys Glu Met Ser Val Cys Trp Glu Gly Glu Lys Ala Ala Lys Lys Lys Asp Ala Asp Ala <210> 19 <211> 2523 <212> DNA
<213> Populus deltoides <220>
<222> (1)...(2523) <400> 19 ttaaccctat atttctctca gaactcacat aaaaaatatt gcgagtttag ggtcataaaa 60 atctctatca tcacaaacaa atatagggtt tgttgatttg gtggctgttt tcgccgtctt 120 tgctttcaaa attcaatctt gacctgtttc ttttatgctt tttgaatagg taatatatac 180 attgtttgtt ttgcttgctt tggttttgta atctqgtgat cttgtttaat tctttttggc 240 tttggtttgt aggttttttt ttttgaacyt gagaaagagg ggggttttca arggagacta 300 gcttttcaag awttgatata ttttattggt aagtttttnt tttccgtttt ttggtaatag 360 ttgcatggat tcttggtgta gatgttgatt tggacttgtt tttgtattgt tgtcctttat 420 gttttatgtg tatgttaatt gttctgatcc ttgatataat gtaaatctga ggcttttatt 480 tttatggatt cgttaaggtg tatggacttg gtttatggtt tt:ggtcttga aaaaaaaatt 540 aggcttagca agttttcttc tgagaataac ttattttatt ttattattta aaattaaatg 600 gtgtttggga atttgaattc tttctctttg gtattttttc tatccttgtg aatctgttta 660 gtcttttaaa ttcaatattt tattttattt tacttatgat ttggtgattg attagttatt 720 tttgtagctt gtattgtctg ttgattttta atctccaata tgtcttactt ctcaqttttt 780 ttcaatttgc tattggaatt ttgctattat ttttaagatt gtagactagt ttgctttgag 840 ttttgtgcta attgcaatcc ttcttgtgtg attcttacag gactgaatga tctaatggag 900 tctaaaggtg gcaagaagaa gtctagtagt agtagtagta gtaaatcctt atgttaagaa 960 gctcccctcg gttacagcat tgaagacctc agacctgctg gtggaatcaa gaagttcaga 1020 tctgctgcat actccaacgt gagtttgaag ttaatcgatc tgtttttcct ttggcatttc 1080 attgtgcgac tggtttccta atcttgtgtc atgtttattt gtgcagtgcg ttcgaaaacc 1140 atcctgagat tttccaagtg ttgacataac cccattttag ctatttcgca cgctcagttg 1200 WASH1814745.1 WASH_1815926_.1 Atty. Dkt. No.: 059994-0114 tctttaktct gtttttctgt tctgctttcc tcgttctctt gtactctttg ctgcactttt 1260 catttgttga ctgtgaggtc agatggcgct gccagtctct qcaatcggat ttgaaggtta 1320 cgaaaaaagg cttgaaatat ctttcctaga gcctggcttc ttttctgacc ctgaagggaa 1380 gggcctgagg tctttgtcca aggctcaatt ggacgagatt ctcagaccag ctgaatgtac 1440 tattgttgat tcgctatcaa atgaccaggt tgattcttat gtcctgtcgg aatccagtct 1500 ctttgtgtac ccttacaaag ttattatcaa aacatgtggg eictaccaaac tgcttctctc 1560 gataccggtg attcttaagc tcgctgatgc cctttcactc actgtatgtt ctgtgaggta 1620 tactcgtggg agctttctat gccctggggc tcagccattt ccacaccgca acttctgtga 1680 ggaggtagct gtccttgacg atcacttcag taaacttggt t.taaacagtg tggcatatgt 1740 gatgggtggt cttgacaaaa ctcagaaatg gcatgtttac tctgcctctg ccgatataga 1800 gagccattct ggccctgttt acactctgga aatgtgcatg actggtttgg gcaggaaaca 1860 agcatctgtt ttctacaaaa cacattccag ttcagctgct gcgatgactg aggattccgg 1920 tataaggaaa atccttccac agtctgagat ctgtgatttt gattttgacc catgtggtta 1980 ctctatgaat gccattgaag ggagtgcaat ttccacaatc cacgtcactc cagaagatgg 2040 tttcagctat gcaagttttg aggctgtggg ctatgatctt caagatttga atttgagtcg 2100 gctgcttgaa agggtcttgg cttgctttga accgaccatg ttctccgttg ccttgcattc 2160 taatatcaag ggtgccgaac ttagagcaaa gtttcccctg gacgtggaag qttactctgg 2220 cggaggaggg aactatgaaa tgcttgggaa aggtggatcg atcatctacc acagctttgc 2280 aaggactgga ggcagtgcat ctcccaggtc tatcctgaaa tgttgttgga gtgaggatga 2340 gaaggacgag gaagctgaag agaagtagtt cttttcagct atttgttttt tccttttatt 2400 ttttccttga ataaatgtca tggggttatg attctgagtt cttgaggcat ttgtccatgc 2460 ctcgtgtctt tcattatcga tttagttttg acttcggatt aataaagggg tttgtgttat 2520 cag 2523 <210> 20 <211> 24 <212> DNA
<213> Artificial sequence <220>
<223> Synthetic oligonucleotide <400> 20 atcccatgga ttcggccttg cctg 24 <210> 21 <211> 34 <212> DNA
<213> Artificial sequence <220>
<223> Synthetic oligonucleotide <400> 21 gtctagacta ctccttctct tctttctctt catc 34 WASH 1814745.1 WAS H 1815926.1 Atty. Dkt. No.: 059994-0114 <210> 22 <211> 25 <212> DNA
<213> Artificial sequence <220>
<223> Synthetic oligonucleotide <400> 22 tatcgtttta cttcactggt cggtg 25 <210> 23 <211> 20 <212> DNA
<213> Artificial sequence <220>
<223> Synthetic oligonucleotide <400> 23 aatctcacca acccaactcc 20 <210> 24 <211> 1658 <212> DNA
<213> Nicotiana tabacum <220>
<222> (364) ... (1449) <223> CDS
<400> 24 atggagtcga aaggtggtaa aaactctagt agtaaatcct taccctacga agcacccctc 60 ggctacagta ttgaagacgt tcggccaaac ggtggaatca agaagttcag atcagctqct 120 tactccaact gcgctcgcaa accatcctga cattccttaa gcttctctcc tgcacgtgtc 1B0 tcctgacaca aaaaagaaaa aatccccaaa aaaagttcct tctgtcaatt gtttttgttg 240 ttaaaccctc actccttttc ctcaatttct tccttctgct gctttctgct cttgctctcc 300 ttggctgtga acaattttct ttaaaagatc atttgttgct gtgaacatat ttttttttat 360 cta atg gat tcg gcc ttg cct gtc tct gcc att ggt ttt gaa ggt ttc 408 Met Asp Ser Ala Leu Pro Val Ser Ala Ile Gly Phe Glu Gly Phe gag aag agg ctt gaa att tct ttt ttc gag cct ggt ctg ttt gct gat 456 Glu Lys Arg Leu Glu Ile Ser Phe Phe Glu Pro Gly Leu Phe Ala Asp ccc aac gga aaa gga ctt cga tct ctc tca aag gca caa ttg gat gag 504 Pro Asn Gly Lys Gly Leu Arg Ser Leu Ser Lys Ala Gln Leu Asp Glu att ctc gga cct gct gag tgc acc ata gtt gat tcc cta tca aat gac 552 Ile Leu Gly Pro Ala Glu Cys Thr Ile Val Asp Ser Leu Ser Asn Asp gat gtt gat tct tat gtc ctc tcc gag tcg agc ctc ttt gtt tat tct 600 Asp Val Asp Ser Tyr Val Leu Ser Glu Ser Ser Leu Phe Val Tyr Ser tac aag ata atc atc aaa acc tgt ggc acc aca aag ttg ctt ctc gca 648 Tyr Lys Ile Ile Ile Lys Thr Cys Gly Thr Thr Lys Leu Leu Leu Ala WASH 18 14745.1 WASH 1815926_.1 Atty. Dkt. No.: 059994-0114 att ccg ccc atc cta aag ttg gct gag acc ctg tct ctc aaa gta caa 696 Ile Pro Pro Ile Leu Lys Leu Ala Glu Thr Leu Ser Leu Lys Val Gin gac gtg agg tat acc cgt ggg agc ttc att ttc cct ggc gct cag tcg 744 Asp Val Arg Tyr Thr Arg Gly Ser Phe Ile Phe Pro Gly Ala Gin Ser ttt cct cat cgt cac ttc tct gaa gaa gtt gct gtc ctc gat ggc tat 792 Phe Pro His Arg His Phe Ser Glu Glu Val Ala Val Leu Asp Gly Tyr ttt gga aag ctt gct gcc ggt agc aag gct gtg att atg ggc agt cct 840 Phe Gly Lys Leu Ala Ala Gly Ser Lys Ala Val Ile Met Gly Ser Pro gac aaa gca cag aaa tgg cat gtt tac tct gcc tct gca gga cct att 888 Asp Lys Ala Gln Lys Trp His Val Tyr Ser Ala Ser Ala Gly Pro Ile cag tct aat gac cct gtt tac act ctt gag atg tgt atg act ggt ttg 936 Gln Ser Asn Asp Pro Val Tyr Thr Leu Glu Met Cys Met Thr Gly Leu gac agg gag aag gca tct gtc ttt tac aag act gaa gga agc tcg gct 984 Asp Arg Glu Lys Ala Ser Val Phe Tyr Lys Thr Glu Gly Ser Ser Ala gct cat atg act gtt cga tct gga ata agg aag atc ctc ccc aat tct 1032 Ala His Met Thr Val Arg Ser Gly Ile Arg Lys Ile Leu Pro Asn Ser gag ata tgc gat ttt gag ttt gaa ccc tgt ggt tat tcc atg aat tca 1080 Glu Ile Cys Asp Phe Glu Phe Glu Pro Cys Gly Tyr Ser Met Asn Ser att gaa gga gct gca ctc tca acc att cac att acc ccg gaa gat ggc 1128 Ile Glu Gly Ala Ala Leu Ser Thr Ile His Ile Thr Pro Glu Asp Gly ttt agc tat gct agc ttt gaa gca gtt ggg tat gac atg aaa acc atg 1176 Phe Ser Tyr Ala Ser Phe Glu Ala Val Gly Tyr Asp Met Lys Thr Met aag ctg ggt ccc ctg gtt gag agg gtg ctg gca tgt ttc cag cca gat 1224 Lys Leu Gly Pro Leu Val Glu Arg Val Leu Ala Cys Phe Gln Pro Asp gag ttc tct att gct ttg cat gct gat gtt gct acc aag tta ctg gag 1272 Glu Phe Ser Ile Ala Leu His Ala Asp Val Ala Thr Lys Leu Leu Glu cgt gtt tgc tct ctt gat gtg aaa ggc tac tct ctt gct gag tgg agt 1320 Arg Val Cys Ser Leu Asp Val Lys Gly Tyr Ser Leu Ala Glu Trp Ser cca gaa gaa ttt ggc aag ggt ggt tcc att gtc tac cag aag ttc acc 1368 Pro Glu Glu Phe Gly Lys Gly Gly Ser Ile Val Tyr Gln Lys Phe Thr aga act cct ttc tgt gga tct ccc aag tcc gtt ctg aag ggc tgc tgg 1416 Arg Thr Pro Phe Cys Gly Ser Pro Lys Ser Val Leu Lys Gly Cys Trp aaa gaa gat gaa gag aaa gaa gag aag gag tag tgtcttgagg 1459 Lys Glu Asp Glu Glu Lys Glu Glu Lys Glu *

gctgtgttgt tttttgtttc agtgtccgtg tctgtctctg tctgtgtcgg tgtcgtttgt 1519 tttttcagtg tttctccgaa taaagtactt gatgtccaag ctgtgtcgtt tggatttgta 1579 WASH_1814745.1 WASH_1815926.1 Atty. Dkt. No.: 059994-0114 atgccgatgt gcaaattctg aactattctt ggctttttgt gttccacccg aagccctatg 1639 aacctgcatt ttgaataaa 1658 <210> 25 <211> 361 <212> PRT
<213> Nicotiana tabacum <220>
<400> 25 Met Asp Ser Ala Leu Pro Val Ser Ala Ile Gly Phe Glu Gly Phe Glu Lys Arg Leu Glu Ile Ser Phe Phe Glu Pro Gly Leu Phe Ala Asp Pro Asn Gly Lys Gly Leu Arg Ser Leu Ser Lys Ala Gln Leu Asp Glu Ile Leu Gly Pro Ala Glu Cys Thr Ile Val Asp Ser Leu Ser Asn Asp Asp Val Asp Ser Tyr Val Leu Ser Glu Ser Ser Leu Phe Val Tyr Ser Tyr Lys Ile Ile Ile Lys Thr Cys Gly Thr Thr Lys Leu Leu Leu Ala Ile Pro Pro Ile Leu Lys Leu Ala Glu Thr Leu Ser Leu Lys Val Gln Asp Val Arg Tyr Thr Arg Gly Ser Phe Ile Phe Pro Gly Ala Gln Ser Phe Pro His Arg His Phe Ser Glu Glu Val Ala Val Leu Asp Gly Tyr Phe Gly Lys Leu Ala Ala Gly Ser Lys Ala Val Ile Met Gly Ser Pro Asp Lys Ala Gln Lys Trp His Val Tyr Ser Ala Ser Ala Gly Pro Ile Gln Ser Asn Asp Pro Val Tyr Thr Leu Glu Met Cys Met Thr Gly Leu Asp Arg Glu Lys Ala Ser Val Phe Tyr Lys Thr Glu Gly Ser Ser Ala Ala His Met Thr Val Arg Ser Gly Ile Arg Lys Ile Leu Pro Asn Ser Giu Ile Cys Asp Phe Glu Phe Glu Pro Cys Gly Tyr Ser Met Asn Ser Ile Glu Gly Ala Ala Leu Ser Thr Ile His Ile Thr Pro Glu Asp Gly Phe Ser Tyr Ala Ser Phe Glu Ala Val Gly Tyr Asp Met Lys Thr Met Lys Leu Gly Pro Leu Val Glu Arg Val Leu Ala Cys Phe Gln Pro Asp Glu Phe Ser Ile Ala Leu His Ala Asp Val Ala Thr Lys Leu Leu Glu Arg Val Cys Ser Leu Asp Val Lys Gly Tyr Ser Leu Ala Glu Trp Ser Pro WASH1814745.1 WASH_1815926.1 Atty. Dkt. No.: 059994-0114 Glu Glu Phe Gly Lys Gly Gly Ser Ile Val Tyr Gln Lys Phe Thr Arg Thr Pro Phe Cys Gly Ser Pro Lys Ser Val Leu Lys Gly Cys Trp Lys Glu Asp Glu Glu Lys Glu Glu Lys Glu <210> 26 <211> 1086 <212> DNA
<213> Populus deltoides <220>
<222> (1)...(1086) <223> CDS
<400> 26 atg gcg ctg cca gtc tct gca atc gga ttt gaa ggt tac gaa aaa agg 48 Met Ala Leu Pro Val Ser Ala Ile Gly Phe Glu Gly Tyr Glu Lys Arg ctt gaa ata tct ttc cta gag cct ggc ttc ttt tct gac cct gaa ggg 96 Leu Glu Ile Ser Phe Leu Glu Pro Gly Phe Phe Ser Asp Pro Glu Gly aag ggc ctg agg tct ttg tcc aag gct caa ttg gac gag att ctc aga 144 Lys Gly Leu Arg Ser Leu Ser Lys Ala Gln Leu Asp Glu Ile Leu Arg cca gct gaa tgt act att gtt gat tcg cta tca aat gac cag gtt gat 192 Pro Ala Glu Cys Thr Ile Val Asp Ser Leu Ser Asn Asp Gln Val Asp tct tat gtc ctg tcg gaa tcc agt ctc ttt gtg tac cct tac aaa gtt 240 Ser Tyr Val Leu Ser Glu Ser Ser Leu Phe Val Tyr Pro Tyr Lys Val att atc aaa aca tgt ggg act acc aaa ctg ctt ctt tcg ata ccg gtg 288 Ile Ile Lys Thr Cys Gly Thr Thr Lys Leu Leu Leu Ser Ile Pro Val att ctt aag ctc gct gat gcc ctt tca ctc act gta tgt tct gtg agg 336 Ile Leu Lys Leu Ala Asp Ala Leu Ser Leu Thr Val Cys Ser Val Arg tat act cgt ggg agc ttt cta tgc cct ggg gct cag cca ttt cca cac 384 Tyr Thr Arg Gly Ser Phe Leu Cys Pro Gly Ala Gln Pro Phe Pro His cga aac ttc tgt gag gag gta gct gtc ctt gac gat cac ttc agt aaa 432 Arg Asn Phe Cys Glu Glu Val Ala Val Leu Asp Asp His Phe Ser Lys ctt ggt tta aac agt gtg gca tat gtg atg ggt ggt ctt gac aaa act 480 Leu Gly Leu Asn Ser Val Ala Tyr Val Met Gly Gly Leu Asp Lys Thr cag aaa tgg cat gtt tac tct gcc tct gcc gat ata ggg agc cat tct 528 Gln Lys Trp His Val Tyr Ser Ala Ser Ala Asp Ile Gly Ser His Ser ggc cct gtt tac acc ctg gaa atg tgc atg act ggt ttg ggc agg aaa 576 Gly Pro Val Tyr Thr Leu Glu Met Cys Met Thr Gly Leu Gly Arg Lys caa gca tct gtt ttc tac aaa aca cat tcc agt tca gct gct gcg atg 624 Gln Ala Ser Val Phe Tyr Lys Thr His Ser Ser Ser Ala Ala Ala Met WASH1814745.1 WASH_18159267.1 Atty. Dkt. No.: 059994-0114 act gag gat tcc ggt ata agg aaa atc ctt cca cag tct gag atc tgt 672 Thr Glu Asp Ser Gly Ile Arg Lys Ile Leu Pro Gln Ser Glu Ile Cys gat ttt gat ttt gac cct tgt ggt tac tcc atg aat gcc att gaa ggg 720 Asp Phe Asp Phe Asp Pro Cys Gly Tyr Ser Met Asn Ala Ile Glu Gly agt gca att tcc aca atc cac gtc act cca gaa gat ggt ttc agc tat 768 Ser Ala Ile Ser Thr Ile His Val Thr Pro Giu Asp Gly Phe Ser Tyr gca agt ttt gag gct gtg ggc tat gat ctt caa gat ttg aat ttg agt 816 Ala Ser Phe Glu Ala Val Gly Tyr Asp Leu Gln Asp Leu Asn Leu Ser cgg ctg ctt gaa agg gtc ttg gct tgc ttt gat ccg acc aag ttc tcc 864 Arg Leu Leu Glu Arg Vai Leu Ala Cys Phe Asp Pro Thr Lys Phe Ser gtt gcc ttg cat tct aat atc aag ggt gcc gaa ctt aga gca aag ttt 912 Val Ala Leu His Ser Asn Ile Lys Gly Ala Glu Leu Arg Ala Lys Phe ccc ctg gac gtg gaa ggt tac tct ggc gga gga ggg aac tat gaa atg 960 Pro Leu Asp Val Glu Gly Tyr Ser Gly Gly Gly Gly Asn Tyr Glu Met ctt ggg aaa ggt gga tcg atc atc tac cac agc ttt gca agg act gga 1008 Leu Gly Lys Gly Gly Ser Ile Ile Tyr His Ser Phe Ala Arg Thr Gly ggc agt gca tct ccc agg tct atc ctg aaa tgt tgt tgg agt gag gat 1056 Gly Ser Ala Ser Pro Arg Ser Ile Leu Lys Cys Cys Trp Ser Glu Asp gag aag gac gag gaa gct gaa gag aag tag 1086 Glu Lys Asp Glu Glu Ala Glu Glu Lys ~

<210> 27 <211> 361 <212> PRT
<213> Populus deltoides <220>
<400> 27 Met Ala Leu Pro Val Ser Ala Ile Gly Phe Glu Gly Tyr Glu Lys Arg Leu Glu Ile Ser Phe Leu Glu Pro Gly Phe Phe Ser Asp Pro Glu Gly Lys Gly Leu Arg Ser Leu Ser Lys Ala Gln Leu Asp Glu Ile Leu Arg Pro Ala Glu Cys Thr Ile Val Asp Ser Leu Ser Asn Asp Gln Val Asp Ser Tyr Val Leu Ser Glu Ser Ser Leu Phe Val Tyr Pro Tyr Lys Val Ile Ile Lys Thr Cys Gly Thr Thr Lys Leu Leu Leu Ser Ile Pro Val Ile Leu Lys Leu Ala Asp Ala Leu Ser Leu Thr Val Cys Ser Val Arg Tyr Thr Arg Gly Ser Phe Leu Cys Pro Gly Ala Gln Pro Phe Pro His WASH1814745.1 WASH 1815926_.1 Atty. Dkt. No.: 059994-0114 Arg Asn Phe Cys Glu Glu Val Ala Val Leu Asp Asp His Phe Ser Lys Leu Gly Leu Asn Ser Val Ala Tyr Val Met Gly Gly Leu Asp Lys Thr Gln Lys Trp His Val Tyr Ser Ala Ser Ala Asp Ile Gly Ser His Ser Gly Pro Val Tyr Thr Leu Glu Met Cys Met Thr Gly Leu Gly Arg Lys Gln Ala Sex Val Phe Tyr Lys Thr His Ser Ser Ser Ala Ala Ala Met Thr Glu Asp Ser Gly Ile Arg Lys Ile Leu Pro Gln Ser Glu Ile Cys Asp Phe Asp Phe Asp Pro Cys Gly Tyr Ser Met Asn Ala Ile Glu Gly Ser Ala Ile Ser Thr Ile His Val Thr Pro Glu Asp Gly Phe Ser Tyr Ala Ser Phe Glu Ala Val Gly Tyr Asp Leu Gln Asp Leu Asn Leu Ser Arg Leu Leu Glu Arg Val Leu Ala Cys Phe Asp Pro Thr Lys Phe Ser Val Ala Leu His Ser Asn Ile Lys Gly Ala Glu Leu Arg Ala Lys Phe Pro Leu Asp Val Glu Gly Tyr Ser Gly Gly Gly Gly Asn Tyr Glu Met Leu Gly Lys Gly Gly Ser Ile Ile Tyr His Ser Phe Ala Arg Thr Gly Gly Ser Ala Ser Pro Arg Ser Ile Leu Lys Cys Cys Trp Ser Glu Asp Glu Lys Asp Glu Glu Ala Glu Glu Lys <210> 28 <211> 26 <212> DNA
<213> Artificial sequence <220>
<223> Synthetic oligonucleotide <400> 28 ccatggcgct gccagtctct gcaatc 26 <210> 29 <211> 27 <212> DNA
<213> Artificial sequence <220>
<223> Synthetic oligonucleotide <400> 29 tctagactac ttctcttcag cttcctc 27 WASH 1814745.1 WASH_18159267.1

Claims (15)

1. A method for increasing wood density in a plant, comprising:
(a) introducing into a plant cell a nucleic acid construct comprising, in the 5' to 3' direction, a xylem-preferred promoter operably linked to a SAMdc nucleotide sequence;
(b) regenerating transgenic plants from said plant cell; and (c) selecting a transgenic plant having reduced vessel area and reduced vessel density relative to a control plant.
2. The method of claim 1, wherein said plant is an angiosperm.
3. The method of claim 1, wherein said plant is a gymnosperm.
4. The method of claim 2, wherein said plant is Eucalyptus or Populus.
5. The method of claim 3, wherein said plant is Pinus.
6. Wood pulp obtained from a transgenic plant produced by the method of claim 1.
7. Wood fiber obtained from a transgenic plant produced by the method of claim 1.
8. A method for increasing wood density and decreasing lignin content in a plant, comprising:
(a) introducing into a plant cell a nucleic acid construct comprising, in the 5' to 3' direction, a xylem-preferred promoter operably linked to a SAMdc nucleotide sequence;
(b) regenerating transgenic plants from said plant cell; and (c) selecting a transgenic plant having increased wood density and decreased lignin content relative to a control plant.
9. The method of claim 8, wherein said plant is an angiosperm.
10. The method of claim 8, wherein said plant is a gymnosperm.
11. The method of claim 9, wherein said plant is Eucalyptus or Populus.
12. The method of claim 10, wherein said plant is Pinus.
13. A method of making wood pulp, comprising:
(a) introducing into a plant cell a nucleic acid construct comprising, in the 5' to 3' direction, a xylem-preferred promoter operably linked to a SAMdc nucleotide sequence;
(b) regenerating transgenic plants from said plant cell;
(c) selecting a transgenic plant having increased wood density and decreased lignin content relative to a control plant; and (d) producing wood pulp from said transgenic plant.
14. The method of claim 13, wherein said xylem-preferred promoter is selected from the group consisting of TUB gene promoter, LTP gene promoter, 4CL gene promoter, and C4H gene promoter.
15. A method of making wood fiber, comprising:
(a) introducing into a plant cell a nucleic acid construct comprising, in the 5' to 3' direction, a xylem-preferred promoter operably linked to a SAMdc nucleotide sequence;
(b) regenerating transgenic plants from said plant cell;
(c) selecting a transgenic plant having increased wood density and decreased lignin content relative to a control plant; and (d) producing wood fiber from said transgenic plant.
CA 2578311 2007-02-22 2007-02-22 Altering wood density Abandoned CA2578311A1 (en)

Priority Applications (1)

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CA 2578311 CA2578311A1 (en) 2007-02-22 2007-02-22 Altering wood density

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Application Number Priority Date Filing Date Title
CA 2578311 CA2578311A1 (en) 2007-02-22 2007-02-22 Altering wood density

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CA2578311A1 true CA2578311A1 (en) 2008-08-22

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