WO2014137312A1 - Method for increasing wood density using transcription factor genes - Google Patents

Abstract

The present invention provides methods for increasing wood density, modulus of elasticity or modulus of rupture, in trees of industrial interest by regulating the expression of transcription factors in the trees, while reducing undesirable growth effects associated with the expression of the transcription factors.

Description

METHOD FOR INCREASING WOOD DENSITY USING

TRANSCRIPTION FACTOR GENES

FIELD OF THE INVENTION

[0001] The present invention relates to methods of increasing wood density, modulus of elasticity or modulus of rupture, in softwood and hardwood trees of industrial interest that involve the use of pine transcription factors.

BACKGROUND OF THE INVENTION

[0002] Wood density is a crucial functional trait in trees, as it is an indicator of mechanical strength and a main factor in demarking a subtle balance between rate of growth and survival in the environment. High density wood trees tend to have a slower growth and a higher survival rate than low density wood trees, and the physical strength of wood is closely correlated with its density. In tropical forests, for example, tree species that depend on light for their survival have a rapid growth rate to reach height, but their wood has low density and, as a consequence, these species have high mortality rates due to stem breakage. The low density of the wood in these species may be due to the development of large cells and/or thin cell walls. In contrast, the growth of shade-tolerant tree species is slow, but their wood is dense and strong, and thus the mortality rate of these species is low (Putz et al, 1983; Muller- Landau, 2004; van Gelder et al., 2006). Wood density also varies vertically along the main axis of the stem and/or radially from the pith to the bark (Grabner and Wimmer, 2006). This is probably due to a shift in allocation from low density wood and rapid height growth early on in tree development to denser wood and structural reinforcement as trees increase in size, age and height (Wiemann and Williamson, 1989b).

[0003] Fast-growing cultivated tree species often have a lower density wood compared to the wood of corresponding wild tree species. This phenomenon is reflected in the quality of wood and wood products obtained from these cultivated species. Thus, the quality of Pinus radiata wood has recently fallen below the standards of construction grade in the timber industry in New Zealand (Watt et al. 2009. For. Ecol. Manag. 258: 1479-1488.). Similar effects have been found in the wood obtained from Southern Pine in the United States (Southern Pine Inspection Bureau, Proposed Design Values for Visually Graded Southern Pine, October 2011).

[0004] There is therefore a need to improve the wood density of cultivated tree species. Wood with increased density would have increased strength and better elasticity and would be less susceptible to damage. In addition, cultivated tree species with increased wood density would produce increased wood yield/ ton or acre, thus producing more biofuel. More mass per volume would provide additional benefits, including lower transportation costs.

[0005] Thus, the present application addresses the need for improving wood yield and lowering cost by presenting methods to increase wood density and thus biomass in cultivated hardwood and softwood tree species.

SUMMARY OF THE INVENTION

[0006] It is therefore an object of the present invention to provide solutions to the aforementioned deficiencies in the art.

[0007] To this end, the invention provides methods of increasing wood density, modulus of elasticity or modulus of rupture, in trees of industrial interest by regulating the expression of transcription factors under the control of different promoters. Specifically, the invention provides enhanced and regulated expression of a DOF (DNA binding with one finger) gene encoding a transcription factor containing the highly conserved DOF-type zinc finger motif, an ERF gene encoding a transcription factor containing the highly conserved DNA-binding ETS domain, or an NF-YC gene encoding a nuclear transcription factor Y subunit gamma in targeted tissues in trees of industrial interest, by transforming tree cells with DNA constructs in which a vascular tissue- preferred promoter or a strong and constitutive promoter mediates the expression of each of these genes.

[0008] Thus, in one embodiment the invention provides a DNA construct comprising the DOF gene operably linked to a vascular tissue-preferred promoter. In a preferred

embodiment, the promoter is the pine 4CL promoter, and the DNA construct comprises a DOF gene-4CL promoter cassette of SEQ ID NO: 3. In an additional preferred embodiment, the DNA construct comprises SEQ ID NO: 4.

[0009] In a different embodiment the invention provides a DNA construct comprising the ERF gene operably linked to a vascular tissue-preferred promoter. In a preferred

embodiment, the promoter is the pine 4CL promoter, and the DNA construct comprises an ERF gene-4CL promoter cassette of SEQ ID NO: 9. In an additional preferred embodiment, the DNA construct comprises SEQ ID NO: 10.

[0010] In yet another embodiment, the invention provides a DNA construct comprising the NF-YC gene operably linked to a vascular tissue-preferred promoter. In a preferred embodiment, the promoter is the pine 4CL promoter, and the DNA construct comprises an NF-YC gene-4CL promoter cassette of SEQ ID NO: 13. In an additional preferred embodiment, the DNA construct comprises SEQ ID NO: 15.

[0011] In a different embodiment the NF-YC gene is operably linked to a constitutive promoter. In a preferred embodiment, the promoter is the pine superubiquitin promoter, and the DNA construct comprises an NF-YC gene-SUB promoter cassette of SEQ ID NO: 14. In an additional preferred embodiment, the DNA construct comprises SEQ ID NO: 16.

[0012] In a different aspect, the invention provides isolated tree cells comprising any of the DNA constructs described above.

[0013] In a further aspect, the invention provides a transgenic tree comprising the tree cells comprising any of the DNA constructs described above. The transgenic tree may be a hardwood tree or a softwood tree. Preferably, the transgenic tree is selected from the group consisting of eucalyptus, pine, Cottonwood, poplar, citrus, papaya, avocado, nutmeg, pistachio, acacia, teak, spruce, cedar, fir, larch, douglas-fir, hemlock, cypress, redwood, yew, mahogany, walnut, oak, ash, elm, aspen, birch, maple, palm, cherry, magnolia, hickory, balsa, buckeye, butternut, chestnut, alder, basswood, beech, boxelder, catalpa, corkwood, dogwood, gum, hornbeam, ironwood, laurel, locust, sassafras, sycamore and willow. In one aspect of the invention, the eucalyptus is a Eucalyptus species selected from the group consisting of Eucalyptus amplifolia, Eucalyptus benjensis, Eucalyptus benthamii, Eucalyptus

calmaldulensis, Eucalyptus dorrigoensis, Eucalyptus dunnii, Eucalyptus globulus, Eucalyptus grandis, Eucalyptus gunnii, Eucalyptus macarthurii, Eucalyptus nitens, Eucalyptus urophylla, Eucalyptus viminalis and hybrids thereof.

[0014] In a different embodiment, the invention provides a wood product of the transgenic tree. The wood product may be selected from the group consisting of wood, wood pulp, wood pellets, paper, lumber, veneer, charcoal, extractives, tall oil, biofuel and bioenergy.

[0015] In one embodiment, the invention provides a method for increasing wood density, modulus of elasticity or modulus of rupture, in a tree comprising (i) isolating cells from a tree; (ii) transforming the isolated tree cells with a DNA construct comprising a

polynucleotide comprising a gene selected from the group consisting of the DOF gene of SEQ ID NO: 1, the ERF gene of SEQ ID NO: 5 and the NF-YC gene of SEQ ID NO: 11, operably linked to a promoter; and (iii) culturing the isolated plant cells under conditions that promote growth of a transgenic tree that expresses the DNA construct; wherein the tree has increased wood density, modulus of elasticity or modulus of rupture, compared to a tree of the same age and/or genotype which does not express the DNA construct, and wherein the tree has no reduced growth compared to a tree of the same species which does not express the DNA construct. In a preferred aspect of the invention, the promoter is the pine 4CL promoter. In another preferred aspect of the invention, the promoter is the pine polyubiquitin promoter. Preferably, the transgenic tree is a hardwood tree or a softwood tree. Even more preferably, the transgenic tree is selected from the group consisting of eucalyptus, pine, cottonwood, poplar, citrus, papaya, avocado, nutmeg, pistachio, acacia, teak, spruce, cedar, fir, larch, douglas-fir, hemlock, cypress, redwood, yew, mahogany, walnut, oak, ash, elm, aspen, birch, maple, palm, cherry, magnolia, hickory, balsa, buckeye, butternut, chestnut, alder, basswood, beech, boxelder, catalpa, corkwood, dogwood, gum, hornbeam, ironwood, laurel, locust, sassafras, sycamore and willow. In a preferred embodiment, the eucalyptus is a Eucalyptus species selected from the group consisting of Eucalyptus amplifolia, Eucalyptus benjensis, Eucalyptus benthamii, Eucalyptus calmaldulensis, Eucalyptus dorrigoensis, Eucalyptus dunnii, Eucalyptus globulus, Eucalyptus grandis, Eucalyptus gunnii, Eucalyptus macarthurii, Eucalyptus nitens, Eucalyptus urophylla, Eucalyptus viminalis and hybrids thereof.

[0016] In yet another embodiment, the invention provides a method of making a wood product with increased density, modulus of elasticity or modulus of rupture from the transgenic trees of the invention, comprising obtaining wood from the transgenic trees and making a wood product selected from the group consisting of wood, wood pulp, wood pellets, paper, lumber, veneer, charcoal, extractives, tall oil, biofuel and bioenergy from the wood.

[0017] In a different embodiment, the invention provides a method to identify a gene involved in wood density comprising (i) isolating cells from a tree; (ii) transforming the isolated tree cells with a DNA construct according to the invention; (iii) culturing the isolated plant cells under conditions that promote growth of a transgenic tree that expresses the DNA construct; and (iv) identifying a gene whose expression is upregulated or downregulated compared to its expression in a tree of the same age and/or genotype which does not express the DNA construct.

[0018] The foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the invention as claimed. Other objects, advantages and novel features will be readily apparent to those skilled in the art from the following detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 depicts the plasmid map of the construct pOX32 comprising the DOF gene- pine 4CL promoter cassette.

[0020] Figure 2 depicts the plasmid map of the construct pOX41 comprising the NF-YC gene-pine 4CL promoter cassette.

[0021] Figure 3 depicts the plasmid map of the construct pOX55 comprising the NF-YC gene-pine polyubiquitin promoter cassette.

[0022] Figure 4 depicts the plasmid map of the construct pOX63 comprising the ERF gene- pine 4CL promoter cassette.

[0023] Figure 5 depicts the plasmid map of the construct pOX73 comprising the ERF gene- pine polyubiquitin promoter cassette. DETAILED DESCRIPTION OF THE INVENTION

[0024] This invention relates to methods for increasing the density, modulus of elasticity or modulus of rupture, of wood in hardwood and softwood trees by separately or simultaneously expressing genes encoding different transcription factors under the control of promoters with different characteristics in commercially important plant species. This invention thus provides strategies to increase wood yield in softwood and hardwood tree species without detrimentally affecting their growth.

[0025] A few research groups in recent years have focused their research on the

identification of genes that could be involved in wood development in plants. However, reported attempts to overexpress these genes in plants have made use of constitutive promoters and have resulted in strong reduction in plant growth. Thus, Goicoechea et al. 2005 describes transgenic tobacco plants in which the over-expression of the Eucalyptus gunnii MYB2 factor was accompanied by an increase in secondary wall thickness and an alteration in lignin composition, as well as by a reduction in plant size, suggesting that higher carbohydrate consumption for more lignin synthesis may have a negative effect on growth rate. Similarly, Zhong et al. 2006 showed that overexpression of the SND1 transcriptional activator in Arabidopsis stimulates secondary cell wall biosynthesis, but the leaves present stunted growth and severely upward-curling blades , which is probably caused by the increased deposition of lignified wall.

[0026] DOF proteins are a family of plant-specific transcription factors characterized by the presence of various zinc-finger DNA-binding domains. Members of this family have been found to play diverse roles in gene regulation of processes restricted to the plants. The DNA binding with one finger (DOF) domain is a conserved region of 50 amino acids with a C2-C2 Zn-fmger structure, associated with a basic region, that binds specifically to DNA sequences with a 5'-T/AAAAG-3' core. DOF proteins have been reported to participate in the regulation of gene expression in a variety of processes, including seed storage protein synthesis in the developing endosperm, light regulation of genes involved in carbohydrate metabolism, plant defense mechanisms, seed germination, gibberellin response in post-germinating aleurone, auxin response and stomata guard cell specific gene regulation. [0027] Genes in the ERF family encode transcriptional regulators with a variety of functions involved in the developmental and physiological processes in plants. The ERF family is a large gene family of transcription factors characterized by the presence of a single AP2/ERF domain, which consists of about 60 to 70 amino acids and is involved in DNA binding. The ERF family is sometimes further divided into two major subfamilies, the ERF subfamily and the CBF/DREB subfamily. Increased expression of CBF/DREB proteins has been shown to confer drought tolerance in a growing number of plant species. However, undesirable developmental phenotypes, such as stunted growth, are often associated with high constitutive expression of the CBF/DREB genes. The ERF domain was first identified as a conserved motif in four DNA-binding proteins from tobacco {Nicotiana tabacum), namely, ethylene-responsive element-binding proteins 1, 2, 3, and 4 (EREBP1, 2, 3, and 4, currently renamed ERF1, 2, 3, and 4), and was shown to specifically bind to a GCC box, which is a DNA sequence involved in the ethylene-responsive transcription of genes (Ohme- Takagi and Shinshi, 1995). Several proteins in the ERF family are believed to be involved in cellular processes, including hormonal signal transduction, response to stress and metabolism in various plant species. However, the specific biological function of each of the ERF genes is not known.

[0028] Nuclear Factor Y (NF-Y) is a conserved heterotrimeric complex consisting of three subunits NF-YA, NF-YB and NF-YC, which binds with high specificity to ubiquitous CCAAT motifs in the promoters of a variety of genes. The three subunits NF-YA, NF-YB and NF-YC are encoded by single genes in yeast, fungi and animals, including mammals. However, in plant species, such as Arabidopsis and rice, multiple genes encode each subunit. NF-Y transcription factors typically act with other regulatory factors to modulate gene expression in a highly controlled manner. Some NF-YC and NF-YB subunits of the trimeric complex have been implicated in the recruitment of CONST ANS-Like transcription factors to their DNA targets in plants, potentially mediating the effect of CONSTANS-like proteins on flowering time. Although overexpression of NFYA5 has been found to improve drought resistance in Arabidopsis (Li et al. 2008 The Plant Cell 20: 2238-2251), the biological roles of most of the NF-Y family members in plants are not understood.

[0029] The present invention is based on the unexpected and surprising discovery that wood density, modulus of elasticity or modulus of rupture, may be increased in mature softwood and hardwood trees by transforming tree cells with DNA constructs comprising the ERF, NF-YC or DOF transcription factors or homologous genes thereof operably linked to xylem-specific promoters, without affecting normal growth. Thus, homologous NF-YC genes include, but are not limited to, the Genbank BT 108746 from the Picea glauca mRNA and the Genbank EF084647 from the Pinus sitchensis mRNA. Accordingly, the present application provides softwood and hardwood trees of commercial utility with increased wood density, modulus of elasticity and modulus of rupture.

[0030] The previous work on transcription factor expression in transgenic plants could not presage the present inventors' discovery that ERF, NF-YC or DOF transcription factors and homologous genes thereof may be expressed in transgenic plants while reducing the undesirable effects associated with the expression of these transcription factors.

[0031] Accordingly, methods are provided for improving wood yield and/or increasing wood density, modulus of elasticity or modulus of rupture, in plants, plant cells and plant tissues. Pursuant to this aspect of the invention, hardwood and softwood tree cells are transformed with the ERF, NF-YC or DOF genes or homologous genes thereof, which, when expressed in plant cells or in whole plants, increase wood yield without causing undesirable effects on growth associated with transcription factor expression. In a preferred embodiment, hardwood trees include, but are not limited to, ash, eucalyptus, aspen, birch, cherry, elm, hazel, palm, poplar, mahogany, maple, oak and teak. Softwood trees include, but are not limited to, pine, spruce and cedar.

Definitions

[0032] All technical terms used herein are terms commonly used in biochemistry, molecular biology and agriculture, and can be understood by one of ordinary skill in the art to which this invention belongs. Technical terms can be found in: Molecular Cloning: A Laboratory Manual, 3rd ed., vol. 1-3, ed. Sambrook and Russell, 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. Methodology involving plant biology techniques is described herein and is described in detail in 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 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, Primer, Version 0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge, MA. Methods for chemical synthesis of nucleic acids are discussed, for example, in Beaucage and Caruthers, 1981, Tetra. Letts. 22: 1859-1862, and Matteucci and Caruthers, 1981 J. Am. Chem. Soc. 103: 3185.

[0033] 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, WI), DIFCO Laboratories (Detroit, MI), Invitrogen (Gaithersburg, MD), or Sigma Chemical Company (St. Louis, MO), unless otherwise specified.

[0034] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein, and the laboratory procedures in cell culture, molecular genetics, and nucleic acid chemistry and hybridization described herein, are those well known and commonly employed in the art. Standard techniques are used for recombinant nucleic acid methods, polynucleotide synthesis, microbial culture, cell culture, tissue culture, transformation, transfection, transduction, analytical chemistry, organic synthetic chemistry, chemical syntheses, chemical analysis, and pharmaceutical formulation and delivery. Generally, enzymatic reactions and purification and/or isolation steps are performed according to the manufacturers' specifications. The techniques and procedures are generally performed according to conventional methodology disclosed, for example, in Molecular cloning a laboratory manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989), and Current protocols in molecular biology, John Wiley & Sons, Baltimore, MD (1989).

[0035] Amino acid sequence: as used herein, includes an oligopeptide, peptide, polypeptide, or protein and fragments thereof, that are isolated from, native to, or naturally occurring in a plant, or are synthetically made but comprise the nucleic acid sequence of the endogenous counterpart.

[0036] Artificially manipulated: as used herein, "artificially manipulated" means to move, arrange, operate or control by the hands or by mechanical means or recombinant means, such as by genetic engineering techniques, a plant or plant cell, so as to produce a plant or plant cell that has a different biological, biochemical, morphological, or

physiological phenotype and/or genotype in comparison to unmanipulated, naturally- occurring counterpart.

[0037] Asexual propagation: producing progeny by generating an entire plant from leaf cuttings, stem cuttings, root cuttings, tuber eyes, stolons, single plant cells protoplasts, callus and the like, that does not involve fusion of gametes.

[0038] Consisting essentially of: a composition "consisting essentially of certain elements is limited to the inclusion of those elements, as well as to those elements that do not materially affect the basic and novel characteristics of the inventive composition. Thus, so long as the composition does not affect the basic and novel characteristics of the instant invention, that is, does not contain foreign DNA that is not from the selected plant species or a plant that is sexually compatible with the selected plant species, then that composition may be considered a component of an inventive composition that is characterized by "consisting essentially of language.

[0039] Degenerate primer: a "degenerate primer" is an oligonucleotide that contains sufficient nucleotide variations that it can accommodate base mismatches when hybridized to sequences of similar, but not exact, homology.

[0040] Dicotyledonous (dicot): a flowering plant whose embryos have two seed leaves or cotyledons. Examples of dicots include, but are not limited to, tobacco, tomato, potato, sweet potato, cassava, legumes including alfalfa and soybean, carrot, strawberry, lettuce, oak, maple, walnut, rose, mint, squash, daisy, and cactus. [0041] "Encoding" and "Coding": refer to the process by which a gene, through the mechanisms of transcription and translation, provides information to a cell from which a series of amino acids can be assembled into a specific amino acid sequence to produce an active enzyme. Because of the degeneracy of the genetic code, certain base changes in DNA sequence do not change the amino acid sequence of a protein. It is therefore understood that modifications in the DNA sequence encoding transcription factors which do not substantially affect the functional properties of the protein are contemplated.

[0042] Expression: denotes the yield of the protein product encoded by a gene. The term "over-expression" refers to the yield of a gene product in transgenic organisms that exceeds levels of yield in normal or non-transformed organisms.

In this description, the phrases "ERF polynucleotide sequence," "ERF homologous polynucleotide sequence," "NF-YC polynucleotide sequence," "NF-YC homologous polynucleotide sequence," "DOF polynucleotide sequence" and "DOF homologous polynucleotide sequence" also refer to any nucleic acid molecule with a nucleotide sequence capable of hybridizing under stringent conditions with any of the sequences disclosed herein, and coding for a polypeptide with ERF, NF-YC or DOF transcription factor activity equivalent to the polypeptides comprising amino acid sequences disclosed herein under SEQ ID NOS: 2, 6, 8, or 12. The phrases also include sequences which cross-hybridize with SEQ ID NO: 1, SEQ ID NO: 5, or SEQ ID NO: 11, which are at least 70% identical to the nucleotide sequence represented by SEQ ID NOs: 1, 5 or 11. The nucleotide sequences of the invention may encode a polypeptide which is homologous to the polypeptides disclosed herein that comprise an amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 12. Further, the nucleotide sequences of the invention include those sequences that encode a polypeptide having ERF, NF-YC or DOF transcription factor activity having an amino acid sequence which has at least 55%, preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%) and most preferably at least 95% sequence identity to an amino acid sequence disclosed herein as SEQ ID Nos: 2, 6, 8 or 12.

[0043] Foreign: with respect to a nucleic acid, means that that nucleic acid is derived from non-plant organisms, or derived from a plant that is not the same species as the plant to be transformed or is not derived from a plant that is not interfertile with the plant to be transformed, does not belong to the species of the target plant. According to the present invention, foreign DNA or RNA represents nucleic acids that are naturally occurring in the genetic makeup of fungi, bacteria, viruses, mammals, fish or birds, but are not naturally occurring in the plant that is to be transformed. Thus, a foreign nucleic acid is one that encodes, for instance, a polypeptide that is not naturally produced by the transformed plant. A foreign nucleic acid does not have to encode a protein product. According to the present invention, a desired intragenic plant is one that does not contain any foreign nucleic acids integrated into its genome.

[0044] Gene: refers to the coding region and does not include nucleotide sequences that are 5'- or 3'- to that region. A functional gene is the coding region operably linked to a promoter or terminator.

[0045] Genetic rearrangement: refers to the re-association of genetic elements that can occur spontaneously in vivo as well as in vitro which introduce a new organization of genetic material. For instance, the splicing together of polynucleotides at different chromosomal loci, can occur spontaneously in vivo during both plant development and sexual

recombination. Accordingly, recombination of genetic elements by non-natural genetic modification techniques in vitro is akin to recombination events that also can occur through sexual recombination in vivo.

[0046] In frame: nucleotide triplets (codons) are translated into a nascent amino acid sequence of the desired recombinant protein in a plant cell. Specifically, the present invention contemplates a first nucleic acid linked in reading frame to a second nucleic acid, wherein the first nucleotide sequence is a gene and the second nucleotide is a promoter or similar regulatory element.

[0047] Integrate: refers to the insertion of a nucleic acid sequence from a selected plant species, or from a plant that is from the same species as the selected plant, or from a plant that is sexually compatible with the selected plant species, into the genome of a cell of a selected plant species. "Integration" refers to the incorporation of genetic elements into a plant cell genome.

[0048] Introduction: as used herein, refers to the insertion of a nucleic acid sequence into a cell, by methods including infection, transfection, transformation or transduction. [0049] Isolated: "isolated" refers to any nucleic acid or compound that is physically separated from its normal, native environment. The isolated material may be maintained in a suitable solution containing, for instance, a solvent, a buffer, an ion, or other component, and may be in purified, or unpurified, form.

[0050] Leader: Transcribed but not translated sequence preceding (or 5 ' to) a gene.

[0051] Modulus of Elasticity: relates to deformations produced by low stress that are completely recoverable after loads are removed. The three moduli of elasticity defined in trees, which are denoted by EL, ER, and ET, respectively, are the elastic moduli along the longitudinal, radial, and tangential axes of wood. These moduli are usually obtained from compression tests, and vary within and between species and with moisture content and specific gravity.

[0052] Modulus of Rupture: also known as flexural strength, relates to the force a material is able to withstand without failing. When a piece of wood is bent, the side of the wood on the outside of the curve is exposed to the greatest tensile stress. Modulus of rupture is a measure of the ability of the material to resist these forces without breaking. As with the modulus of elasticity, the modulus of rigidity varies within and between species and with moisture content and specific gravity.

[0053] Monocotyledon (monocot): a flowering plant whose embryos have one cotyledon or seed leaf. Examples of monocots include, but are not limited to turf grass, maize, rice, oat, wheat, barley, sorghum, orchid, iris, lily, onion, and palm.

[0054] Native: a "native" genetic element refers to a nucleic acid that naturally exists in, originates from, or belongs to the genome of a plant that is to be transformed. Thus, any nucleic acid, gene, polynucleotide, DNA, RNA, mRNA, or cDNA molecule that is isolated either from the genome of a plant or plant species that is to be transformed or is isolated from a plant or species that is sexually compatible or interfertile with the plant species that is to be transformed, is "native" to, i.e., indigenous to, the plant species.

[0055] Naturally occurring nucleic acid: naturally occurring nucleic acid are found within the genome of a selected plant species and may be a DNA molecule or an RNA molecule. The sequence of a restriction site that is normally present in the genome of a plant species can be engineered into an exogenous DNA molecule, such as a vector or oligonucleotide, even though that restriction site was not physically isolated from that genome. Thus, the present invention permits the synthetic creation of a nucleotide sequence, such as a restriction enzyme recognition sequence, so long as that sequence is naturally occurring in the genome of the selected plant species or in a plant that is sexually compatible with the selected plant species that is to be transformed.

[0056] Operably linked: combining two or more molecules in such a fashion that in combination they function properly in a plant cell. For instance, a promoter is operably linked to a structural gene when the promoter controls transcription of the structural gene.

[0057] Plant: includes angiosperms and gymnosperms such as potato, tomato, tobacco, alfalfa, lettuce, carrot, strawberry, sugarbeet, cassava, sweet potato, soybean, maize, turf grass, wheat, rice, barley, sorghum, oat, oak, eucalyptus, walnut, and palm. Thus, a plant may be a monocot or a dicot. The word "plant," as used herein, also encompasses plant cells, seed, plant progeny, propagule whether generated sexually or asexually, and descendents of any of these, such as cuttings or seed. Plant cells include suspension cultures, callus, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes,

sporophytes, pollen, seeds and microspores. Plants may be at various stages of maturity and may be grown in liquid or solid culture, or in soil or suitable media in pots, greenhouses or fields. Expression of an introduced leader, trailer or gene sequences in plants may be transient or permanent. A "selected plant species" may be, but is not limited to, a species of any one of these "plants."

[0058] Plant species: the group of plants belonging to a plant species that display at least some sexual compatibility.

[0059] Plant transformation and cell culture: broadly refers to the process by which plant cells are genetically modified and transferred to an appropriate plant culture medium for maintenance, further growth, and/or further development.

[0060] Recombinant: as used herein, broadly describes various technologies whereby genes can be cloned, DNA can be sequenced, and protein products can be produced. As used herein, the term also describes proteins that have been produced following the transfer of genes into the cells of plant host systems. [0061] Regulatory sequences: refer to those sequences which are standard and known to those in the art, that may be included in the expression vectors to increase and/or maximize transcription of a gene of interest or translation of the resulting RNA in a plant system. These include, but are not limited to, promoters, peptide export signal sequences, introns, polyadenylation, and transcription termination sites. Methods of modifying nucleic acid constructs to increase expression levels in plants are also generally known in the art (see, e.g. Rogers et al, 260 J. Biol. Chem. 3731-38, 1985; Cornejo et al, 23 Plant Mol. Biol. 567: 81,1993). In engineering a plant system to affect the rate of transcription of a protein, various factors known in the art, including regulatory sequences, such as positively or negatively acting sequences, enhancers and silencers, as well as chromatin structure, may have an impact. The present invention provides that at least one of these factors may be utilized in engineering plants to express a protein of interest.

[0062] Selectable marker: a "selectable marker" is typically a gene that codes for a protein that confers some kind of resistance to an antibiotic, herbicide or toxic compound, and is used to identify transformation events. Examples of selectable markers include the streptomycin phosphotransferase (spt) gene encoding streptomycin resistance, the

phosphomannose isomerase (pmi) gene that converts mannose-6-phosphate into fructose-6 phosphate; the neomycin phosphotransferase (nptlT) gene encoding kanamycin and geneticin resistance, the hygromycin phosphotransferase (hpt or aphiv) gene encoding resistance to hygromycin, acetolactate synthase (als) genes encoding resistance to sulfonylurea-type herbicides, genes coding for resistance to herbicides which act to inhibit the action of glutamine synthase such as phosphinothricin or basta (e.g., the bar gene), or other similar genes known in the art.

[0063] Stringent conditions: as referred herein, means conditions under which only base sequences coding for a polypeptide with ERF, NF-YC or DOF transcription factor activity equivalent to the transcription factor encoded by a ERF, NF-YC or DOF gene sequence or a ERF, NF-YC or DOF homologous gene sequence form hybrids with the specific ERF, NF- YC or DOF gene sequences or ERF, NF-YC or DOF homologous sequences (referred to as specific hybrids), and base sequences coding for polypeptides with no such equivalent activity do not form hybrids with the specific sequence (referred to as non-specific hybrids). One with ordinary skill in the art can readily select such conditions by varying the temperature during the hybridization reaction and washing process, or the salt concentration during the hybridization reaction and washing process. Specific examples include, but are not limited to, conditions under which hybridization is brought about in 3.5xSSC, lxDenhardt's solution, 25mM sodium phosphate buffer (pH 7.0), 0.5% SDS, and 2mM EDTA for 18 hours at 65°C, followed by 4 washes of the filter at 65°C for 20 minutes, in 2xSSC, 0.1% SDS, and a final wash for up to 20 minutes in 0.5xSSC, 0.1% SDS, or 0.3xSSC and 0.1%) SDS for greater stringency, and O. lxSSC, 0.1%> SDS for even greater stringency. Other conditions may be substituted, as long as the degree of stringency in equal to that provided herein, using a 0.5xSSC final wash.

[0064] Transcribed DNA: DNA comprising both a gene and the untranslated leader and trailer sequence that are associated with that gene, which is transcribed as a single mRNA by the action of a promoter that drives its expression.

[0065] Transformation of plant cells: a process by which DNA is stably integrated into the genome of a plant cell. "Stably" refers to the permanent, or non-transient retention and/or expression of a polynucleotide in and by a cell genome. Thus, a stably integrated

polynucleotide is one that is a fixture within a transformed cell genome and can be replicated and propagated through successive progeny of the cell or resultant transformed plant.

Transformation may occur under natural or artificial conditions using various methods well known in the art. Transformation may rely on any known method for the insertion of nucleic acid sequences into a prokaryotic or eukaryotic host cell, including Agrobacterium-mediatGd transformation protocols, viral infection, whiskers, electroporation, heat shock, lipofection, polyethylene glycol treatment, micro-injection, and particle bombardment.

[0066] Transgene: a gene that will be inserted into a host genome, comprising a protein coding region. In the context of the instant invention, the elements comprising the transgene are isolated from the host genome.

[0067] Transgenic plant: a genetically modified plant which contains at least one transgene. The phrase "transgenic plant" refers to a plant that has incorporated a DNA sequence, including, but not limited, to genes that are not normally present in a host plant genome, DNA sequences not normally transcribed into R A or translated into a protein ("expressed"), or any other genes or DNA sequences normally present in the non-transformed plant, that are genetically engineered or have altered expression. The phrase "transgenic plant" encompasses primary transformants regenerated from calluses obtained from transformed plant cells (Ro plants), as well as their seed-derived Ri and R₂ progenies, and vegetatively-propagated derivatives of the Ro plants and Ri and R₂ progenies. The invention also contemplates yield of hybrids using an Ro, Ri or R₂ plant as a parent.

[0068] It is contemplated that, in some instances, the genome of an inventive transgenic plant will have been augmented through the stable introduction of a transgene. In other instances, however, the introduced gene will replace an endogenous sequence. A preferred gene in the regard, pursuant to the present invention, is a ERF, NF-YC or DOF gene or a ERF, NF-YC or DOF homologous gene.

[0069] Variant: as used herein, is understood to mean 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.

[0070] The ERF, NF-YC or DOF homologous gene sequences contemplated in this invention include fragments and variants of the polynucleotides represented by SEQ ID Nos: 1, 5 or 11, with one or more bases deleted, substituted, inserted, or added, that code for a polypeptide with transcription factor activity. The "base sequences with one or more bases deleted, substituted, inserted, or added" referred to here are widely known by those having ordinary skill in the art to retain physiological activity even when the amino acid sequence of a protein generally having that physiological activity has one or more amino acids substituted, deleted, inserted, or added. For example, the poly A tail or 5 ' or 3' end nontranslation regions may be deleted, and bases may be deleted to the extent that amino acids are deleted. Bases may also be substituted, as long as no frame shift results. Bases also may be "added," as long as such modifications do not result in the loss of transcription factor activity. A modified DNA in this context can be obtained by modifying the DNA base sequences of the invention so that amino acids at specific sites are substituted, deleted, inserted, or added by site-specific mutagenesis, as described in Zoller & Smith, 1982, Nucleic Acid Res. 10: 6487-6500.

[0071] It is understood that the present invention is not limited to the particular

methodology, protocols, vectors, and reagents, etc., described herein, as these may vary. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a gene" is a reference to one or more genes and includes equivalents thereof known to those skilled in the art and so forth. Indeed, one skilled in the art can use the methods described herein to express any native gene (known presently or subsequently) in plant host systems.

Promoters

[0072] An important aspect of the present invention is the use of DNA constructs wherein a ERF, NF-YC or DOF gene or a homologous ERF, NF-YC or DOF sequence is operably linked to one or more promoters that drive the expression of the ERF, NF-YC or DOF gene sequence or ERF, NF-YC or DOF homologous sequence in a xylem-preferred manner or in certain cell types, organs, or tissues, such as the xylem cells, in a transformed plant without unduly affecting its normal development or growth.

[0073] The selected promoter should cause the expression of the ERF, NF-YC or DOF gene or the homologous gene thereof, pursuant to the invention, to improve wood yield and/or increase wood density, modulus of elasticity or modulus of rupture, in the transgenic plant cell, or in the transgenic plant.

[0074] Suitable promoters include, but are not limited to, xylem-preferred promoters, such as Pinus taeda ACL, Eucalyptus grandis arabinogalactan protein (AGP), Eucalyptus grandis CesA, Pinus radiata CesA, populus 4CL, populus CesA, Pinus trichocarpa F5H and Eucalyptus grandis F5H promoters.

Plants for Genetic Engineering

[0075] The present invention comprehends the genetic manipulation of plants, to enhance their wood yield and/or increase their wood density, modulus of elasticity or modulus of rupture, by driving the expression of a ERF, NF-YC or DOF gene or a ERF, NF-YC or DOF homologous gene, preferably under the control of a promoter as described above. The result is enhanced wood yield and/or increased wood density, modulus of elasticity or modulus of rupture.

[0076] The term "plant" denotes any fiber-containing plant material that can be genetically manipulated, including, but not limited to, differentiated or undifferentiated plant cells, protoplasts, whole plants, plant tissues, or plant organs, or any component of a plant such as a leaf, stem, root, bud, tuber, fruit, rhizome, or the like.

[0077] Plants that can be engineered in accordance with the invention include, but are not limited to, hardwood and softwood trees.

[0078] The term "softwood trees" designates gymnosperm trees, including conifers. Soft wood is generally strong in tension but weak in shear, and splits easily. Examples of softwood trees include, but are not limited to, pine, spruce, cedar, fir, larch, douglas-fir, hemlock, cypress, redwood, and yew.

[0079] The term "hardwood trees" designates dicotyledonous angiosperm trees. Hard wood is generally strong in compression, tension and shear. Examples of hardwood trees include, but are not limited to, mahogany, teak, walnut, oak, ash, elm, aspen, poplar, cottonwood, birch, maple, cherry, magnolia, hickory, balsa, eucalyptus, apple, citrus, fig, jujube, mulberry, olive, pawpaw, pear, plum, quince, buckeye, butternut, chestnut, alder, basswood, beech, boxelder, catalpa, corkwood, dogwood, gum, hornbeam, ironwood, laurel, locust, sassafras, sycamore and willow.

[0080] Eucalyptus trees include Eucalyptus species and hybrids thereof, including, but not limited to, E. alba, E. albens, E. amplifolia, E. amygdalina, E. aromaphloia, E. baileyana, E. balladoniensis, E. benjensis, E. benthamii, E. bicostata, E. botryoides, E. brachyandra, E. brassiana, E. brevistylis, E. brockwayi, E. calmaldulensis, 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. dorrigoensis, 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.

macarthurii, E. maidenii, E. marginata, 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. pseudoglobulus, 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. 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, E. woodwardii).

[0081] Poplar refers to Populus species and hybrids thereof which include, but are not limited to, 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 and P. yunnanensis.

[0082] Conifer refers to trees that produce their seeds in cone, such as 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); cedars, such as Western red cedar (Thuja plicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis).

[0083] Further examples of hardwood species include, but are not limited to, Citrus species, including C. medica, C. aurantifolia, C. latipes, C. Union; C. reticulata, C. sinensis, C.

paradisi, C. aurantium, C.jambhiri, C. grandis, C. indica, C. ichangensis, C. tachibana, C. micrantha; Citrus hybrids, including, Palestine sweet lime, bergamot and Volkamer lemon, Rangpur lime and Rough lemon; avocado (Persea americana Mill), sweetgum (Liquidambar styraciflua), tulip tree (Liriodendron tulipifera), papaya (Carica papaya), nutmeg (Myristica insipida) and pistachio (Pistacio vera).

[0084] Fiber-producing plants also are included in this context. Illustrative crops are cotton (Gossipium spp.), flax (Linum usitatissimum), stinging nettle (Urtica dioica), hop (Humulus lupulus), linden trees (Tilia cordata, T. x. europaea and T. platyphyllus), Spanish broom (Spartium junceum), ramie (Boehmeria nivea), paper mulberry (Broussonetya papyrifera), New Zealand flax (Phormium tenax), dogbane (Apocynum cannabinum), Iris species (/.

douglasiana, I. macrosiphon and I. purdyi), milkweeds (Asclepia species), pineapple and banana.

DNA Constructs

[0085] In accordance with one aspect of the invention, a ERF, NF-YC or DOF gene or ERF, NF-YC or DOF homologous gene sequence is incorporated into a DNA construct that is suitable for plant transformation. Such a DNA construct can be used to modify ERF, NF- YC or DOF expression in plants, as described above.

[0086] Accordingly, DNA constructs are provided comprising a ERF, NF-YC or DOF gene sequence or ERF, NF-YC or DOF homologous gene sequence, under the control of a promoter, such as any of those mentioned above, so that the construct can generate RNA in a host plant cell.

[0087] Recombinant DNA constructs may be made using standard techniques. For example, the DNA sequence for transcription may be obtained by treating a vector containing said sequence with restriction enzymes to cut out the appropriate segment. The DNA 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 DNA sequence then is cloned into a vector containing upstream promoter and downstream terminator sequences.

[0088] The expression 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 35 S terminator and the nopaline synthase gene terminator. The expression vector may also contain enhancers, start codons, splicing signal sequences, and targeting sequences.

[0089] Expression vectors of the invention may also contain a selection marker by which transformed plant 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 plant cell containing the marker. Usually, 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.

[0090] 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'-0- phosphotranserase (kanamycin, neomycin and G418 resistance). These markers 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., 1987, EMBO J. 9: 2519-2523). Other suitable selection markers are known to the person skilled in the art.

[0091] 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.

[0092] Other DNA 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.

Plant Transformation

[0093] Constructs according to the invention may be used to transform any plant cell, using a suitable transformation technique. Both monocotyledon and dicotyledonous angiosperm or gymnosperm plant cells may be transformed in various ways known to the art. For example, see Klein et al., 1993, Biotechnology 4: 583-590; Bechtold et al., 1993, C. R. Acad. Sci. Paris 316: 1194-1199; Bent et al., 1986, Mol. Gen. Genet. 204: 383-396; Paszowski et al., 1984, EMBO J. 3: 2717-2722; Sagi et al., 1994, Plant Cell Rep. 13: 262-266.

[0094] Agrobacterium species such as A. tumefaciens and A. rhizogenes can be used, for example, in accordance with Nagel et al., 1990, Microbiol Lett 67: 325. Agrobacterium may be transformed with a plant expression vector via electroporation, followed by introduction of the Agrobacterium into plant cells via the well known leaf-disk method. Additional methods include, but are not limited to, particle gun bombardment, calcium phosphate precipitation, polyethylene glycol fusion, transfer into germinating pollen grains, direct transformation (Lorz et al., 1985, Mol. Genet. 199: 179-182), and other methods known to the art. Use of a selection marker, such as kanamycin resistance, allows quick identification of successfully transformed cells.

[0095] The Agrobacterium transformation methods discussed above are known to be useful for transforming dicots. 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 according to methods well known in the art.

Wood Density

[0096] Transgenic plants of the invention are characterized by increased wood yield and/or an increase in wood density, modulus of elasticity and modulus of rupture. Density is defined as a substance's mass per unit volume. Wood density varies with moisture content since moisture content can affect both mass and volume. Specific Gravity (SG) or basic density is the density of a substance relative to the density of water. The specific gravity of wood is calculated using its oven-dry mass and is the most important predictor of wood strength. Density affects the mechanical or strength properties of wood, which in turn have far-ranging impacts on the use of wood in many applications. Wood strength is measured by the modulus of rupture, which measures the ultimate strength of wood in bending, tension, or compression, and by the modulus of elasticity, which measures the bending strength of wood.

[0097] Specific examples are presented below of methods for obtaining DNA constructs comprising the ERF, NF-YC or DOF genes and homologous genes with similar expression patterns operably linked to promoters that drive their expression in trees, as well as for introducing the target genes, via Agrobacterium, to produce tree transformants with increased wood density. It is understood that additional promoters may be used in place of the promoters illustrated in the examples, to obtain targeted expression.

[0098] The present invention, thus generally described, will be understood more readily by reference to the following examples, which are provided by way of illustration and are not intended to be limiting to the present invention.

EXAMPLES

Example 1: Transgenic tree production and wood harvesting

[0099] cDNA sequences were isolated, inserted into DNA constructs and used for plant transformation, as described in US Patent No. 7,507,875, which is herein incorporated by reference in its entirety. After transformation with the DNA constructs, transgenic trees were grown in the field for approximately three years, at the end of which their wood was harvested and dried. The basic gravity of the lines was determined for chunks of wood weighing approximately 10-50 grams, as described in Simpson, W.T. 1993. Specific gravity, moisture content, and density relationship for wood. Gen. Tech. Rep. FPL-GTR-76.

Madison, WI: U.S. Department of Agriculture, Forest Service, Forest Products Laboratory. 13 pp. Basic gravity was calculated from the weight of oven-dried wood and the weight of water displaced by the same piece of wood when saturated with water. Percentage gain was calculated for each transgenic line as the increase in basic gravity relative to the basic gravity average for control plants transformed with DNA constructs containing the GUS gene. Gains or losses are not shown for the controls, although variation within 5% of the average was common. Transgenic lines showing less than 5% gain in basic gravity were considered as having essentially normal density. Example 2: Effects of DOF Overexpression in Cottonwood

[0100] The table and graphs below show a mild effect of DOF gene transformation on wood density, as measured by basic gravity.

Avg Height (Ft) By Basic Gravity Avg Volume (Ft^A3) By Basic Gravity

Basic Gravity Basic Gravity

[0101] The small points in the graphs represent wild control plants, empty-vector- transformed control plant and GUS-transformed control plants. The large squares represent transformation with the pOX32 DNA construct containing the DOF gene operably linked to the pine 4CL promoter. The data show that a moderate increase in density was achieved in multiple lines with no effect seen on height or volume growth. Example 3: Effects of ERF Overexpression in Pine

[0102] The table and graphs below show a strong effect of ERF gene transformation on wood density, as measured by basic gravity, in pine.

Plasmid Gene Line ID Basic Gravity % Gain

None None 98GE0808 0.411 -- pWVK147 None TTAO 12087 0.402 -- pWVK147 None TTAO 12089 0.376 -- pWVK147 None TTAO 12091 0.413 -- pWVK147 None TTAO 12098 0.302 -- pWVK147 None TTA012110 0.352 -- pWVK147 None TTAO 12591 0.417 -- pWVK147 None TTA520152 0.424 -- pWVK147 None TTA520156 0.427 -- pWVK147 None TTA520767 0.406 -- pOX63 4CL::ERF TTAO 12642 0.493 25 pOX63 4CL::ERF TTAO 12643 0.500 27 pOX63 4CL::ERF TTAO 12646 0.478 22 pOX63 4CL::ERF TTAO 12651 0.524 33 pOX63 4CL::ERF TTAO 12654 0.618 57 pOX63 4CL::ERF TTA012657 0.528 34 pOX63 4CL::ERF TTAO 12659 0.452 15 pOX63 4CL::ERF TTAO 12666 0.406 3 pOX73 SUB::ERF TTAO 12600 0.413 6 pOX73 SUB::ERF TTA012608 0.420 7 pOX73 SUB::ERF TTAO 12610 0.431 10 pOX73 SUB::ERF TTAO 12613 0.411 5 pOX73 SUB::ERF TTAO 12614 0.433 11

Avg Height (Ft) By Basic Gravity Avg Volume (Ft^A3) By Basic Gravity

[0103] The small points in the graphs represent wild control plants as well as empty- vector- transformed control plants. The open squares represent transformation with the pOX63 DNA construct containing the ERF gene operably linked to the pine 4CL promoter. The open circles represent transformation with the pOX73 DNA construct containing the ERF gene operably linked to the pine superubiquitin promoter. The results show minimal effects on tree height. Although most transgenic lines showed some decrease in volume, it was possible to identify a line with increased wood density and no growth loss.

Example 4: Effects of NF-YC Overexpression in Pine

[0104] The table and graphs below show a moderate effect of NF-YC gene transformation on wood density, as measured by basic gravity, in pine.

Plasmid Gene Line ID Basic Gravity % Gain

None None GE0808 0.403 -- pWVK147 None TTA012386 0.371 -- pWVK147 None TTA012387 0.391 -- pWVK147 None TTA012368 0.394 -- pWV 31 GUS TTA012359 0.384 -- pWVR31 GUS TTA012362 0.394 -- pWVR31 GUS TTA012365 0.400 -- pOX41 4CL::NF-YC TTA518939 0.404 3 pOX41 4CL::NF-YC TTA520322 0.412 6 pOX41 4CL::NF-YC TTA520323 0.423 8 pOX41 4CL::NF-YC TTA520340 0.436 12 pOX41 4CL::NF-YC TTA518942 0.441 13 pOX41 4CL::NF-YC TTA518941 0.446 14 pOX41 4CL::NF-YC TTA520328 0.455 17 pOX41 4CL::NF-YC TTA518943 0.469 20 pOX55 SUB::NF-YC TTA012284 0.411 5 pOX55 SUB::NF-YC TTA520348 0.415 6 pOX55 SUB::NF-YC TTA012285 0.433 11 pOX55 SUB::NF-YC TTA520359 0.455 17 pOX55 SUB::NF-YC TTA520350 0.480 23 pOX55 SUB::NF-YC TTA520353 0.518 33

Avg Height (Ft) By Basic Gravity Avg Volume (Ft^A3) By Basic Gravity

Basic gravity

[0105] The small points in the graphs represent wild control plants empty- vector- transformed control plants and GUS -transformed control plants, with * being the average of all controls. The large squares represent transformation with the pOX41 DNA construct containing the NF-YC gene operably linked to the pine 4CL promoter, and the circles represent transformation with the pOX55 DNA construct containing the NF-YC gene operably linked to the constitutive pine superubiquitm promoter. The results indicate that a moderate density increase can be achieved without a loss in growth in transgenic conifer trees transformed with a DNA construct containing the NF-YC gene operably linked to a xylem- preferred promoter, such as the pine 4CL promoter.

Example 5: Effects of NF-YC Overexpression in Cottonwood

[0106] The graphs below show a moderate effect of NF-YC gene transformation on wood density, as measured by basic gravity, in cottonwood. Avg Height (Ft) By Basic Gravity Avg Volume (Ft^A3) By Basic Gravity

Mean Basic Gravity Mean Basic Gravity

[0107] The small points in the graphs represent GUS-transformed control plants, with * being the average of all controls. The large squares represent transformation with the pOX41 DNA construct containing the NF-YC gene operably linked to the pine 4CL promoter, and the circles represent transformation with the pOX55 DNA construct containing the NF-YC gene operably linked to the constitutive pine superubiquitin promoter. The two filled symbols in each graph indicate lines where expression was very low or undetectable, such that the basic gravity of these transgenic lines was essentially similar to the basic gravity of the control plants. The results indicate that a moderate density increase can be achieved without a loss in growth in transgenic hardwood trees transformed with a DNA construct containing the NF-YC gene operably linked to a xylem-preferred promoter, such as the pine 4CL promoter.

Example 6: Improvement of pine wood strength through NF-YC Overexpression

[0108] Pine trees of approximately three years of age were harvested and dried. Wood from the base of the main stem was cut to multiple cam x 1 cm x 16 cm samples for each line. Modulus of elasticity (MOE) and modulus of rupture (MOR) were determined using an MTS Alliance electromechanical test apparatus. Specimens were loaded on the tangential face on the side nearest the pith with a span of 14 cm and a speed of 0.125 inches per minute so that the specimens would break in about three to five minutes each.

[0109] The table below shows the samples ordered as in Example 5 above, with increasing basic density within each plasmid set. The Table shows that the lines with higher density have higher strength (higher MOE and MOR). Plasmid Gene Line ID MOE % Gain MOR % Gain pWV 31 GUS TTA012359 298.5 - 5510 - pWVR31 GUS TTA012362 302.9 - 5838 - pWVR31 GUS TTA012365 321.9 - 6028 - pOX41 4CL::NF-YC TTA518939 320.0 4 6208 7 pOX41 4CL::NF-YC TTA520322 307.8 0 5964 3 pOX41 4CL::NF-YC TTA520323 305.7 -1 5904 2 pOX41 4CL::NF-YC TTA520340 366.6 19 6693 16 pOX41 4CL::NF-YC TTA518942 315.6 3 6163 6 pOX41 4CL::NF-YC TTA518941 344.2 12 6857 18 pOX41 4CL::NF-YC TTA520328 332.2 8 6524 13 pOX41 4CL::NF-YC TTA518943 400.0 30 7164 24 pOX55 SUB::NF-YC TTAO 12284 335.0 9 6185 7 pOX55 SUB::NF-YC TTA520348 326.0 6 6382 10 pOX55 SUB::NF-YC TTAO 12285 392.7 28 7100 23 pOX55 SUB::NF-YC TTA520359 336.4 9 6336 9 pOX55 SUB::NF-YC TTA520350 368.4 20 6339 9 pOX55 SUB::NF-YC TTA520353 410.8 33 7971 38

[0110] The data shown in the Table above are further illustrated in the following graphs, where pOX41 lines average greater MOE and MOR than the controls, and the pOX55 lines average even greater MOE and MOR (horizontal lines indicate group means).

[0111] The examples shown above demonstrate that optimal overexpression or ectopical expression of transcription factors affecting wood density in trees may be achieved by driving the expression with xylem-preferred promoters. The results also indicate that, in order to increase wood density without affecting growth, a moderate level of transcription factor expression should be maintained.

Claims

WHAT IS CLAIMED IS:

1. A DNA construct comprising a sequence selected from the group consisting of SEQ ID NO: 3; SEQ ID NO: 9; SEQ ID NO: 13 and SEQ ID NO: 14.

2. A DNA construct selected from the group consisting of SEQ ID NO: 4; SEQ ID NO: 10; SEQ ID NO: 15 and SEQ ID NO: 16.

3. Isolated tree cells comprising a DNA construct of claim 2.

4. A transgenic tree comprising the tree cells of claim 3.

5. The transgenic tree of claim 4, wherein the tree is a hardwood tree or a softwood tree.

6. The transgenic tree of claim 5, wherein the tree is selected from the group consisting of eucalyptus, pine, Cottonwood, poplar, citrus, papaya, avocado, nutmeg, pistachio, acacia, teak, spruce, cedar, fir, larch, douglas-fir, hemlock, cypress, redwood, yew, mahogany, walnut, oak, ash, elm, aspen, birch, maple, palm, cherry, magnolia, hickory, balsa, buckeye, butternut, chestnut, alder, basswood, beech, boxelder, catalpa, corkwood, dogwood, gum, hornbeam, ironwood, laurel, locust, sassafras, sycamore and willow.

7. The transgenic tree of claim 6, wherein the eucalyptus is a Eucalyptus species selected from the group consisting of Eucalyptus amplifolia, Eucalyptus benjensis,

Eucalyptus benthamii, Eucalyptus calmaldulensis, Eucalyptus dorrigoensis, Eucalyptus dunnii, Eucalyptus globulus, Eucalyptus grandis, Eucalyptus gunnii, Eucalyptus macarthurii, Eucalyptus nitens, Eucalyptus urophylla, Eucalyptus viminalis and hybrids thereof.

8. A wood product of the transgenic tree of claim 5 selected from the group consisting of wood, wood pulp, wood pellets, paper, lumber, veneer, charcoal, extractives, tall oil, biofuel and bioenergy.

9. A method for increasing wood density, modulus of elasticity or modulus of rupture, in a tree comprising (i) isolating cells from a tree; (ii) transforming the isolated tree cells with a DNA construct comprising a polynucleotide comprising a gene selected from the group consisting of the DOF gene of SEQ ID NO: 1, the ERF gene of SEQ ID NO: 5 and the NF-YC gene of SEQ ID NO: 11, operably linked to a promoter; and (iii) culturing the isolated plant cells under conditions that promote growth of a transgenic tree that expresses the DNA construct; wherein the tree has increased wood density, modulus of elasticity or modulus of rupture, compared to a tree of the same age and/or genotype which does not express the DNA construct, and wherein the tree has no reduced growth compared to a tree of the same age and/or genotype which does not express the DNA construct.

10. The method of claim 9, wherein the promoter is the pine 4CL promoter or the pine polyubiquitin promoter.

11. The method of claim 9, wherein the tree is a hardwood tree or a softwood tree.

12. The method of claim 17, wherein the tree is selected from the group consisting of eucalyptus, pine, Cottonwood, poplar, citrus, papaya, avocado, nutmeg, pistachio, acacia, teak, spruce, cedar, fir, larch, douglas-fir, hemlock, cypress, redwood, yew, mahogany, walnut, oak, ash, elm, aspen, birch, maple, palm, cherry, magnolia, hickory, balsa, buckeye, butternut, chestnut, alder, basswood, beech, boxelder, catalpa, corkwood, dogwood, gum, hornbeam, ironwood, laurel, locust, sassafras, sycamore and willow.

13. The method of claim 12, wherein the eucalyptus is a Eucalyptus species selected from the group consisting of Eucalyptus amplifolia, Eucalyptus benjensis,

Eucalyptus benthamii, Eucalyptus calmaldulensis, Eucalyptus dorrigoensis, Eucalyptus dunnii, Eucalyptus globulus, Eucalyptus grandis, Eucalyptus gunnii, Eucalyptus macarthurii, Eucalyptus nitens, Eucalyptus urophylla, Eucalyptus viminalis and hybrids thereof.

14. A method of making a wood product with increased density, modulus of elasticity or modulus of rupture, comprising obtaining wood from the transgenic tree of claim 6 and making a wood product selected from the group consisting of wood, wood pulp, wood pellets, paper, lumber, veneer, charcoal, extractives, tall oil, biofuel and bioenergy from the wood.

15. A method to identify a gene involved in wood density comprising (i) isolating cells from a tree; (ii) transforming the isolated tree cells with the DNA construct of claim 2; (iii) culturing the isolated plant cells under conditions that promote growth of a transgenic tree that expresses the DNA construct; and (iv) identifying a gene whose expression is upregulated or downregulated compared to its expression in a tree of the same age and/or genotype which does not express the DNA construct.