# Patent

## Title
Altering lignin and wood density

## Abstract

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

## Background
CROSS-REFERENCE TO RELATED PATENT APPLICATIONS
This application is a continuation of U.S. non-provisional patent application Ser. No. 11/676,078 filed Feb. 16, 2007 and incorporated herein by reference in its entirety, which is a continuation-in-part filed under 35 U.S.C. §371 of PCT/BR2005/000162, filed Aug. 5, 2005, which was published in English and which claims benefit under 35 USC §119 of U.S. provisional application Serial No. 60/602,440, filed Aug. 18, 2004. The contents of PCT/BR2005/000162 are incorporated herein by reference.


FIELD OF THE INVENTION
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 and reducing lignin content in plants.
BACKGROUND OF THE INVENTION
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
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).
Lignin's resistance to degradation significantly 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); 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).
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).
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 iso forms 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. Natl. Acad. Sci. USA 95: 7805-7812 (1998).
Wood Density
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, The wood properties of Radiata Pine. 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).
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.
Although the wood density is a complex trait, it presents great variations between trees as well as high heritability and reduced Genotype×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).
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 association with juvenile wood density in segregating populations of Pinus radiata. Devey et al., Theor Appl Genet 108: 516-24 (2004).
Accordingly, there is a continuing need to identify genes whose expression can be affected to increase wood density and decrease lignin in plants, in particular, woody tree species.
SUMMARY OF THE INVENTION
In one aspect, the invention provides a nucleic acid construct comprising a SAMdc nucleotide sequence operably linked to a xylem-preferred promoter. In one embodiment, a plant comprises the nucleic acid construct. In further embodiments, the plant is an angiosperm or gymnosperm.
In another aspect, the invention provides a plant or part thereof that overexpresses a nucleotide sequence encoding a SAMdc enzyme, which nucleotide sequence is under the control of a xylem-preferred promoter, such that said plant has reduced lignin levels compared with a control plant. In further embodiments, the plant is an angiosperm or gymnosperm. In even further embodiments, the plant is a woody tree, Eucalyptus, Populus, and Pinus. In another embodiment, the part of the plant is selected from the group consisting of a leaf, a stem, a flower, an ovary, a fruit, a seed, and a callus. In another embodiment, the invention provides progeny of the plant.
In another aspect, the invention provides a method for 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 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 further embodiments, the plant is Eucalyptus, Populus, and Pinus. In another embodiment, the nucleic acid is selected from the group consisting of: (a) a nucleotide sequence set forth in any of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 24, and 26; (b) a nucleotide sequence that encodes a polypeptide having the amino acid sequence set forth in any of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 25, and 27 (c) a nucleotide sequence that differs from the nucleotide sequences of (a) or (b) due to degeneracy of the genetic code and encodes a polypeptide with SAMdc enzyme activity.
In another aspect, the invention provides a method for increasing the ratio of syringyl lignin to guaiacyl lignin in a woody plant, comprising overexpressing a SAMdc nucleotide sequence with a xylem-preferred promoter.
In another 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.
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.
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. 
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 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.
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
FIG. 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.
FIG. 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.
FIG. 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).
FIG. 4 schematically illustrates the plant expression plasmidial vector pALELLYX-Pd1 of the invention comprising a cambium/xylem preferred promoter driving the expression of SAMdc nucleotide sequence coding for SAMdc 1 from Populus deltoides. 
FIG. 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. 
FIG. 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. 
FIG. 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).
FIG. 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. 
FIG. 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. 
FIG. 10 schematically illustrates the plant expression plasmidial vector pALELLYX-Os1 of the invention comprising a cambium/xylem preferred promoter driving the expression of a nucleotide sequence coding for SAMdc 1 from Oryza sativa (rice).
FIG. 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).
FIG. 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-Nt01; (D) Transgenic plant TUB-SAMdc-Nt09. Insets represent lower magnification images of the respective stem cuts.
FIG. 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.
FIGS. 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 4CL-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).
FIG. 15 shows the relationship between Eucalyptus clones wood density and the number of vessel elements in the xylem.
FIG. 16 shows hand-sectioned unfixed stems (base level) of a T1 transgenic Nicotiana benthamiana plants (line 11B) 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 T1 transgenic plants (line 11B).
FIG. 17 shows the area of vessel elements (mean of 100 vessel elements) of three genotypes of a T1 transgenic Nicotiana benthamiana plants (line 11B) transformed with the plant expression plasmidial vector pALELLYX-Nt. (A) sdcsdc genotype; (B) SDCSDC genotype; (C) reduction on the area of vessel elements of the T1 transgenic plants (line 11B).
FIG. 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.
FIG. 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.
FIG. 20 shows the insoluble (A) and soluble (B) lignin content of three genotypes of a T1 transgenic Nicotiana benthamiana plants (line 11B) transformed with the plant expression plasmidial vector pALELLYX-Nt.



DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
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.
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 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.
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.
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, 5th 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.
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, 2nd 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 Research, Cambridge, Mass.). 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).
Restriction enzyme digestions, phosphorylations, ligations and transformations were done as described in Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd 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.
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).
I. Reducing Insoluble Lignin Content by Overexpressing SAMdc
In one aspect, the present invention provides genetic engineering methodology and constructs for reducing lignin content in plants, particularly wood tree species, by overexpressing a polynucleotide sequence encoding SAM decarboxylase (SAMdc; EC. number 4.1.1.50) in a xylem-prefered manner. SAM is converted to decarboxylated SAM (dSAM), a precursor in the polyamine biosynthetic pathway, by the action of SAM decarboxylase (SAMdc; EC. number 4.1.1.50). Malmberg et al., Crit. Rev. Plant Sci. 17: 199-224 (1998). Therefore, and as described below, 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.
II. Increasing Wood Density by Overexpressing SAMdc
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 pine, 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).
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.
III. Concurrently Reducing Lignin and Increasing Wood Density by Overexpressing SAMdc
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 SAMdc encoding sequence is introduced into a plant and transgenic plants are selected that have reduced lignin content and increased wood density.
SAMdc Nucleotide and Polypeptide Sequences
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.
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.
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.
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 14CO2 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 CO2/h/mg of protein.
Sequence Analysis
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 6× SSC, 0.5% SDS, 5× Denhardt's solution and 100 μg 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 6× SSC, 0.5% SDS, 5× Denhardt's solution and 100 μg 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 2× SSC plus 0.05% SDS for five times at room temperature, with subsequent washes with 0.1× 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.
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.
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, using algorithms known in the art and can be determined conventionally using publicly available computer programs such as the BLASTN algorithm. See Altschul et al., Nucleic Acids Res. 25: 3389-402 (1997).
Nucleic Acid Constructs
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.
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.
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 is no theoretical upper limit to the base sequence length. The preparation of such constructs is described in more detail below.
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.
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.
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.
“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-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.
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. For example, by using vascular system-specific, xylem-specific, or xylem-preferred promoters, 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.
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. As used herein, “xylem-preferred promoter” means that the nucleic acid molecules of the current invention are more active in the xylem than in any other plant tissue. Xylem-preferred promoters that could be used include, but are 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.
The vectors of the invention may also 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 nopaline synthase gene (Tnos) terminator. The expression vector also may contain enhancers, start codons, splicing signal sequences, and targeting sequences.
Expression vectors of the invention may also 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. As used herein, 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.
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.
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.
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.
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
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.
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.
In the present context, the “genetically engineered” category includes “transgenic” plants and cells (see definition, 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. Pat. No. 4,407,956. Additionally, a genetically engineered plant or cell may 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.
“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.
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. marginate, E. megacarpa, E. melliodora, E. michaeliana, E. microcorys, E. microtheca, E. muelleriana, E. nitens, E. nitida, E. obliqua, E. obtusiflora, E. occidentalis, E. optima, E. ovata, E. pachyphylla, E. pauciflora, E. pellita, E. perriniana, E. petiolaris, E. pilularis, E. piperita, E. platyphylla, E. polyanthemos, E. populnea, E. preissiana, E. pseudo globulus, E. pulchella, E. radiata, E. radiata subsp. radiata, E. regnans, E. 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. tetragons, 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. 
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).
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 Engineering
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.
Plant Transformation
“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).
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.
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 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.
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.
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.
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
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 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.
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).
“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. Iiyama et al., Wood Sci. Technol. 22: 271-80 (1988).
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 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, N.Y. (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.
* * *
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
RNA was extracted from leaf tissue of Nicotiana tabacum using Trizol reagent (Invitrogen). A cDNA 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
A DNA sequence coding for SAMdc from Nicotiana 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.
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_Nt1 Length: 24



SEQ ID NO: 20



ATCCCATGGATTCGGCCTTGCCTG


 



SAMdc_Nt2 Length: 34



SEQ ID NO: 21



GTCTAGACTACTCCTTCTCTTCTTTCTCTTCATC






(c) PCR Amplification of SAMdc from Nicotiana tabacum 
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 Nicotiana Plants
The gene obtained in Example 1 above was introduced into a plant host to produce transgenic Arabidopsis and Nicotiana plants.
(1) Preparation of Constructs and Transformation of Agrobacterium 
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 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 Mar. 28, 2005, which claims for the priority date of Ser. No. 60/560,227, filed Apr. 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, GV3101 or the like.
(2) Agrobacterium-Mediated Transformation of Arabidopsis thaliana 
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, 383-396 (1986), with the construct containing the tobacco SAMdc gene obtained in Example 1 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. 9:2519-2523 (1987).
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.
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 and the bacterial pellet was ressuspended 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.
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/l 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.
(3) Agrobacterium-Mediated Transformation of Nicotiana benthamiana 
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.
(4) PCR Verification of Foreign Gene Insertion into the Host Plant Genome
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 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 1.8 kb fragment:









Tub_check1 Length: 25



SEQ ID NO: 22



TATCGTTTTACTTCACTGGTCGGTG


 



SAMdc_Nt2 Length: 34



SEQ ID NO: 21



GTCTAGACTACTCCTTCTCTTCTTTCTCTTCATC






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


 



SAMdc_Nt2 Length: 34



SEQ ID NO: 21



GTCTAGACTACTCCTTCTCTTCTTTCTCTTCATC






The PCR reaction mixture contained 100 ng genomic DNA of transformed plant, and 0.2 μM of each primer, 100 μM of each deoxyribonucleotide triphosphate, 1×PCR buffer and 2.5 Units of AmpliTaq DNA polymerase (Applied Biosystems) in a total volume of 50 μL. 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.
(5) Determination of Transgene Expression Level in Transgenic Plants
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 T1 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).
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 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
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 HCl) for 30 minutes. Zhong et al., Plant Physiol., 123, 59-69 (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
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.
Arabidopsis transgenic plants were made following the methods described in Example 2. The first generation of transformants (T1) 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. PCT/BR2005/000041, filed Mar. 28, 2005, which claims for the priority date of Serial No. 60/560,227, filed Apr. 6, 2004, supra.
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.
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.
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).
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 (T1) 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 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.
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 II 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.
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:









SAMDC_NCO Length: 26



SEQ ID NO: 28



CCATGGCGCT GCCAGTCTCT GCAATC


 



SAMDC_XBA Length: 27



SEQ ID NO: 29



TCTAGACTAC TTCTCTTCAG CTTCCTC






The cDNA 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 1.5 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 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
The gene obtained in Examples 5 above was introduced into a plant host to produce transgenic Populus and Eucalyptus plants.
(A) Preparation of Constructs and Transformation of Agrobacterium 
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 Populus SAMdc gene obtained in Example 5 was cloned into the aforementioned expression vector downstream to a xylem-preferred 4CL gene promoter, Hu et al., PNAS 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.
(B) Agrobacterium-Mediated Transformation of Populus 
Wild-type aspen was transformed with Agrobacterium tumefaciens carrying a construct comprising a Populus deltoides SAMdc gene obtained in Example 5 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 100 mg/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 
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
Stem sections of 10 μm 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.
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.
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. 16). The Tukey test showed that the difference between the two populations is highly significant with P<0.001.
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. 17). 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
Lateral shoots of 6 months old Eucalyptus plants described in Example 6 were cross-sectioned (5 μm 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 FIG. 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
The main stems of T1 segregating population of the Nicotiana transgenic event 11B 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) H2SO4 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 H2SO4 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 H2O to remove residual acid and sugars and dried overnight at 105° C. The dry crucibles 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).
As shown in FIG. 20A, the homozygous dominant plants presented a decrease of 13% in their insoluble lignin content, when compared to homozygous recessive plants. The homozygous dominant plants also presented an increase of 11% in their soluble lignin content, compared to homozygous recessive plants (FIG. 20B).

## Claims

1. Wood pulp comprising a recombinant nucleic acid encoding a SAMdc enzyme and, operably linked thereto, a xylem-preferred promoter.

2. The wood pulp of claim 1, wherein said wood pulp is wood pulp of a woody tree.

3. The wood pulp of claim 2, wherein said woody tree is a Eucalyptus species, a Populus species, or a Conifer.

4. The wood pulp of claim 3, wherein said Conifer is a Pinus species.

5. The wood pulp of claim 1, wherein said wood pulp has reduced lignin levels compared with wood pulp of a control plant.

6. The wood pulp of claim 1, wherein said wood pulp has an increased ratio of syringyl lignin to guaiacyl lignin compared with wood pulp of a control plant.

7. The wood pulp of claim 1, wherein said nucleic acid is selected from the group consisting of:
(a) a nucleotide sequence set forth in any of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 24, and 26;
(b) a nucleotide sequence that encodes a polypeptide having the amino acid sequence set forth in any of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, and 18;
(c) a nucleotide sequence that differs from the nucleotide sequences of (a) or (b) due to degeneracy of the genetic code and encodes a polypeptide with SAMdc enzyme activity.

8. The wood pulp of claim 1, 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.

9. A method of making wood pulp, comprising producing wood pulp from a transgenic plant comprising a recombinant nucleic acid encoding a SAMdc enzyme and, operably linked thereto, a xylem-preferred promoter.

10. The method of claim 9, wherein said wood pulp is wood pulp of a woody tree.

11. The method of claim 10, wherein said woody tree is a Eucalyptus species, a Populus species, or a Conifer.

12. The method of claim 11, wherein said Conifer is a Pinus species.

13. The method of claim 9, wherein said wood pulp has reduced lignin levels compared with wood pulp of a control plant.

14. The method of claim 9, wherein said wood pulp has an increased ratio of syringyl lignin to guaiacyl lignin compared with wood pulp of a control plant.

15. The method of claim 9, wherein said nucleic acid is selected from the group consisting of:
(a) a nucleotide sequence set forth in any of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 24, and 26;
(b) a nucleotide sequence that encodes a polypeptide having the amino acid sequence set forth in any of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, and 18;
(c) a nucleotide sequence that differs from the nucleotide sequences of (a) or (b) due to degeneracy of the genetic code and encodes a polypeptide with SAMdc enzyme activity.

16. The method of claim 9, 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.

17. The method of claim 9, wherein said transgenic plant has increased wood density and decreased lignin content relative to a control plant.

18. A method of increasing pulp and cellulose fiber yields during pulp and paper processing, comprising processing wood pulp comprising a recombinant nucleic acid encoding a SAMdc enzyme and, operably linked thereto, a xylem-preferred promoter.

19. The method of claim 18, wherein said wood pulp has reduced lignin levels compared with wood pulp of a control plant.

20. The method of claim 18, wherein said wood pulp has an increased ratio of syringyl lignin to guaiacyl lignin compared with wood pulp of a control plant.