WO2013151701A1 - Improvement of freeze and drought tolerance in plants - Google Patents

Abstract

The present invention provides methods for improving stress tolerance in plants of industrial interest by simultaneously regulating the expression of a gene encoding a C-repeat-binding factor (CBF), a gene encoding an anti-freeze protein and a gene encoding a galactinol synthase, while reducing undesirable effects associated with the expression of the desired genes.

Description

IMPROVEMENT OF FREEZE AND DROUGHT TOLERANCE IN

PLANTS

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of US Provisional Patent Application No. 61/620,852, filed on April 5, 2012, and US Provisional Patent Application No. 61/681,835, filed on August 10, 2012, the contents of which are hereby incorporated by reference herein in their entirety.

FIELD OF THE INVENTION

The present invention relates to the field of plant biotechnology and alteration of gene expression in transgenic plants. More specifically, this invention relates to methods of improving stress tolerance in plants of industrial interest by simultaneously regulating the expression of a gene encoding a C-repeat-binding factor (CBF), a gene encoding an anti-freeze protein and a gene encoding a galactinol synthase, while reducing undesirable effects associated with the expression of the desired genes.

BACKGROUND OF THE INVENTION

Plants vary greatly in their ability to withstand low temperature or water stress. Plants that originate in tropical regions, such as corn, rice and cassava, can be killed or severely damaged during a drought or when exposed to a low temperature, even if the temperature is above freezing. Plants that originate in temperate climates, on the other hand, are less susceptible to water stress or freezing temperatures.

The genus Eucalyptus comprises more than eight hundred species which grow in the tropical and temperate regions of the world. Eucalyptus species and hybrids thereof have a high growth rate, adapts to wide range of environments, and displays little susceptibility to insect damage. In addition to their exceptional growth properties, eucalyptus trees provide the largest source of fibers for the paper industry. Eucalyptus short fibers are the source of pulp and paper with desirable surface characteristics, including smoothness and brightness, and low tear or tensile strength. Eucalyptus timber is used for plywood and particleboard and the lumber is used in the furniture and flooring industries and provides a source of firewood and ornamental and construction materials. Wood chips from eucalyptus can be used in a variety of composite lumber products, such as oriented strand board (OSB) or medium density fiberboard (MDF). As a fast growing species, eucalyptus is used as a fuelwood, for charcoal, and for additional energy production applications such as a feedstock for biofuels and bioproducts manufacture.

Eucalyptus is grown to produce mulch and provide windbreaks for aesthetic and industrial applications. Essential oils from eucalyptus are also used for cleaning, cosmetic products, such as soaps and perfumes, as well as for medicinal purposes. Furthermore, eucalyptus plantations are considered valuable for production of carbon-neutral and renewable biofuel, which can be used as a substitute for costly fossil fuels. Eucalyptus biomass can be converted into building materials, paper, fuels, food and other products, such as plant-derived chemicals, like waxes and cleaners. Solid biomass may be also used to generate process heat and electric power. Biomass processing may additionally be used for biorefmery to produce fuels, chemicals, new bio-based materials, and electric power.

Eucalyptus is the most commonly planted hardwood in the world. However, most of the fastest growing Eucalyptus species are mostly confined to tropical areas because of their high sensitivity to low temperatures and their limited ability to withstand water stress. While some species of Eucalyptus are more tolerant than others to exposure to low temperature, sudden severe frosts pose a great threat to survival in most, if not all, Eucalyptus species. Induced tolerance to progressively lower temperatures, known as cold acclimation, may be obtained only after exposure to a hardening cold treatment accompanied by a decrease in light intensity and day length, but its success depends on several factors, including the species and its origin, duration of the hardening period and the health of the tree. The ability of most Eucalyptus species to resist water stress is also very limited. Excess water loss leads to a general decrease in growth and significant reductions in leaf area ratio, specific leaf area and leaf-to-root area ratio.

The inventors of the present application have already developed a method for enhancing freeze tolerance in eucalyptus and other plants by regulating the expression of genes encoding C- repeat-binding factors (CBFs). See US Patent Application No. 12/593,225, which is herein incorporated by reference in its entirety. CBFs are transcriptional activators that activate transcription by binding to a DNA regulatory element, the CRT (C-repeat)/DRE (dehydration- responsive element), which confers cold- and dehydration-responsive gene expression.

However, it has been found that transgenic eucalyptus trees transformed with the CBF genes are able to grow only in the coastal regions of southeastern United States. It is therefore desirable to further increase Eucalyptus tolerance, as well as other plant tolerance, to low temperatures, so that the transgenic trees can be planted and grown in both coastal and inland areas around the world.

Plants that are more resistant to stress, including low temperature stress and dehydration stress, could be grown in a larger range of geographical areas and their crops and products would be subject to fewer environmental risks.

Thus, there is a need to develop better technologies to improve stress tolerance, particularly low temperature and water loss tolerance, in commercially important trees and field crops, that allow modification of the expression of the genes involved in stress response and cold and water loss tolerance, and reduce undesirable effects associated with the complex

mechanisms that regulate stress tolerance in plants.

The present application answers to this need. SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide solutions to the

aforementioned deficiencies in the art.

To this end, the invention provides for methods of improving stress tolerance in plants of industrial interest by simultaneously regulating plant expression of multiple stress-related genes under the control of different promoters. Specifically, the invention provides enhanced and regulated expression of a C-repeat-binding factor (CBF), an anti-freeze protein and a galactinol synthase in targeted tissues in plants of industrial interest, by transforming the plants with DNA constructs in which a stress-inducible promoter, a short-day inducible and vascular tissue- preferred promoter, or a strong and constitutive promoter mediates the expression of each of the desired stress-related genes.

Thus, in one embodiment the invention provides a DNA construct comprising (a) a first polynucleotide comprising a functional sequence of a CBF2 gene operably linked to a promoter; (b) a second polynucleotide comprising a functional sequence of a carrot antifreeze protein gene operably linked to a promoter; and (c) a third polynucleotide comprising a functional sequence of a galactinol synthase 2 gene operably linked to a promoter. The first polynucleotide may be operably linked to a short-day-inducible and vascular tissue -preferred promoter, a constitutive promoter or a stress-inducible promoter. In a preferred aspect of the invention, the short-day- inducible and vascular tissue-preferred promoter is a poplar bark storage protein gene 1 (BSP1) promoter and the constitutive promoter is an Arabidopsis vegetative storage protein gene 2 (VSP2) promoter. In one aspect of the invention, the stress-inducible promoter is an Arabidopsis thaliana rd29A promoter. In one preferred aspect of the invention, the second polynucleotide or the third polynucleotide are operably linked to a short-day-inducible and vascular tissue- preferred promoter. In one embodiment, the second polynucleotide is operably linked to a short- day-inducible, vascular tissue-preferred promoter, and the third polynucleotide is operably linked to a constitutive promoter. In a different embodiment, the second polynucleotide is operably linked to a constitutive promoter, and the third polynucleotide is operably linked to a short-day- inducible, vascular tissue-preferred promoter. Preferably, the short-day-inducible, vascular tissue -preferred promoter is a poplar bark storage protein gene 1 (BSP1) promoter, and the constitutive promoter is an Arabidopsis vegetative storage protein gene 2 (VSP2) promoter.

In a further aspect, the invention provides isolated plant cells transformed with any of the DNA constructs described above.

In yet another aspect, the invention provides a transgenic plant comprising the isolated transformed plant cells. In one aspect of the invention, the transgenic plant is a transgenic ornamental plant or a transgenic field crop selected from the group consisting of corn, wheat, rice, carrots, broccoli, tomatoes and grape. In another aspect, the transgenic plant is Arabidopsis. In a different aspect of the invention, the transgenic plant is a transgenic tropical tree selected from the group consisting of eucalyptus, poplar, citrus, papaya, avocado, teak, acacia, nutmeg, pistachio, jatropha, pine, Cottonwood, 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. Preferably, the eucalyptus is a Eucalyptus species selected from the group consisting of

Eucalyptus amplifolia, Eucalyptus badjensis, 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.

In a further embodiment, the invention provides wood products of the transgenic tropical trees of the invention. The wood products are preferably wood, wood pellets, wood pulp, paper, lumber, veneer, charcoal, extractives, tall oil, biofuel and bioenergy. In a different embodiment, the invention provides a method for increasing stress tolerance in a plant comprising (i) transforming isolated plant cells with a DNA construct comprising (a) a first polynucleotide comprising a functional sequence of a CBF2 gene operably linked to a promoter; (b) a second polynucleotide comprising a functional sequence of a carrot antifreeze protein gene operably linked to a promoter; and (c) a third polynucleotide comprising a functional sequence of a galactinol synthase 2 gene operably linked to a promoter; and (ii) culturing the isolated plant cells under conditions that promote growth of a transgenic plant that expresses the DNA construct with no detrimental effects to the plant. Preferably, the first polynucleotide encodes a CBF polypeptide, the second polynucleotide encodes a carrot antifreeze protein, and the third polynucleotide encodes a galactinol synthase. In a preferred aspect of the invention, the expression of the protein encoded by the first, second and third polynucleotide in the plant is driven by the promoter to which each polynucleotide is operably linked, upon exposure of the plant to a stress condition. In a preferred aspect of the invention, the transgenic plant has increased stress tolerance and leaf retention compared to a plant of the same species which does not express the DNA construct. In a preferred embodiment, the first polynucleotide is operably linked to a short-day-inducible, vascular tissue-preferred promoter, a constitutive promoter or a stress-inducible promoter. In one aspect of the invention, the second polynucleotide is operably linked to a short-day-inducible, vascular tissue-preferred promoter, and the third polynucleotide is operably linked to a constitutive promoter. In a different aspect of the invention, the second polynucleotide is operably linked to a constitutive promoter, and the third polynucleotide is operably linked to a short-day-inducible, vascular tissue-preferred promoter. Preferably, the short-day-inducible, vascular tissue-preferred promoter is a poplar bark storage protein gene 1 BSP1 promoter, and the constitutive promoter is an Arabidopsis vegetative storage protein gene 2 VSP2 promoter. In one embodiment of the invention, the stress condition is the wilting point of the plant. In a different embodiment of the invention, the stress condition is a low temperature. In one aspect, the low temperature is a chilling temperature from about 0°C to about 12°C. In a different aspect, the low temperature is a freezing temperature from about 0°C to about -12°C. The exposure to the low temperature is for a period of time from about 2 hours to about 72 hours. In yet another embodiment, the method of the invention further comprises making a wood product from transgenic tropical trees. Preferably the wood product is wood, wood pellets, wood pulp, paper, lumber, veneer, charcoal, extractives, tall oil, biofuel and bioenergy.

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

Figure 1 depicts the plasmid map of the construct pAGSM379.

Figure 2 shows the effects of freezing temperature stress on potted trees of transgenic eucalyptus lines transformed with the pAGSM379 construct containing the CBF2, carrot AFP, and galactinol synthase genes as compared to the freezing temperature stress on the control eucalyptus elite line TUH000427, transformed with the construct of pABCTEOl, which carries the rd29A promoter driving the expression of the CBF2 gene (positive control). The plants were exposed to a freezing stress temperature between -4.5° and -7.0° C for 24 hours and then allowed to recover at 4° C for 8 hours before being transferred to the greenhouse . The photos were taken in a greenhouse 10 days after exposure to the freezing temperature stress. The transgenic pAGSM379 eucalyptus line in the pot on the left showed freeze tolerance similar to the positive control. In contrast, the transgenic pAGSM379 eucalyptus line in the pot on the right did not show significant freeze tolerance; indicating that individual transgenic lines transformed with the pAGSM379 construct can show different freeze tolerance performance.

Figure 3 shows the effects of freezing temperature stress on leaf retention in potted trees of transgenic eucalyptus lines transformed with the pAGSM379 construct as compared to the effects of freezing temperature stress on leaf retention in the control wild-type eucalyptus lines and in the positive control Eucalyptus elite line TUH000427 transformed with the construct pABCTEOl carrying the rd29A promoter driving the expression of the CBF2 gene. The plants were exposed to a freezing stress temperature between -4.5° to -7.0° C for 24 hours and then allowed to recover at 4° C for 8 hours before being transferred to the greenhouse. Leaf retention after freezing stress was improved in the pAGSM379 lines.

Figure 4 shows the results of GUS staining of transgenic Arabidopsis lines carrying the pEIBSPlGUSara construct comprising the bark storage protein 1 (BSP1) promoter fused to the GUS gene. The GUS staining was performed at 37°C overnight. The plants were de-stained in ethanol before photographing. Most lines showed vascular tissue-preferred staining, with GUS staining only in veins of the leaves and cotyledons (left and middle panels). Some lines showed strong staining with no tissue specificity (right panel).

Figure 5 shows the results of GUS staining of transgenic Arabidopsis lines carrying the pEIBSP2GUSara construct comprising the bark storage protein 2 (BSP2) promoter fused to the GUS gene (left panel), or the pEIVSP2GUSara construct comprising the vegetative storage protein (VSP2) gene promoter fused to the GUS gene (right panel). The GUS staining was performed at 37°C overnight. The plants were de-stained in ethanol before photographing. Most lines showed uneven mild staining of the leaves.

Figure 6 depicts the pABCTElO construct providing short-day inducible, vascular tissue- preferred expression of the CBF2 gene. In this construct, the CBF2 gene is operably linked to the BSP1 promoter for expression in the vascular tissues.

Figure 7 depicts the pABCTEl 1 construct providing strong expression of the CBF2 gene in all vegetative tissues, and especially in the young growing shoots. In this construct, the CBF2 gene is operably linked to the VSP2 promoter for expression in the vegetative tissues.

Figure 8 depicts the pABCTE12 construct providing stress-induced expression of the CBF2 gene, short-day inducible and vascular tissue-preferred expression of the anti- freeze protein (AFP). In this construct, the CBF2 gene is operably linked to the RD29A promoter for stress-induced expression, and the AFP gene is operably linked to the BSP1 promoter for expression in the vascular tissues.

Figure 9 depicts the pABCTE13 construct providing stress-induced expression of the CBF2 gene, short-day inducible and vascular tissue-preferred expression of galactinol synthase inside cells. In this construct, the CBF2 gene is operably linked to the RD29A promoter for stress-induced expression, and the galactynol synthase gene is operably linked to the BSP1 promoter for expression in the vascular tissues.

Figure 10 depicts the pABCTE14b construct providing stress-induced expression of the CBF2 gene, short-day inducible and vascular tissue-preferred expression of the anti-freeze protein (AFP), and constitutive expression of galactinol and raffmose in all vegetative tissues. In this construct, the CBF2 gene is operably linked to the RD29A promoter for stress-induced expression, the AFP gene is operably linked to the BSP1 promoter for expression in the vascular tissues, and the galactinol synthase gene is operably linked to the VSP2 promoter for expression in the vegetative tissues.

Figure 11 depicts the pABCTE15b construct providing stress-induced expression of the CBF2 gene, short-day inducible and vascular tissue-preferred expression of galactinol and raffmose in all vegetative tissues, and constitutive expression of the anti-freeze protein (AFP). In this construct, the CBF2 gene is operably linked to the RD29A promoter for stress-induced expression, the AFP gene is operably linked to the VSP2 promoter for expression in the vegetative tissues, and the galactinol synthase gene is operably linked to the BSP1 promoter for expression in the vascular tissues.

Figure 12 depicts the pABCTE16b construct providing stress-induced expression of the CBF2 gene, short-day inducible and vascular tissue-preferred expression of the anti-freeze protein (AFP), and constitutive expression of galactinol and raffmose in all vegetative tissues. In this construct, the CBF2 gene is operably linked to the RD29A promoter for stress-induced expression, the AFP gene is operably linked to the BSP1 promoter for expression in the vascular tissues, and the galactinol synthase gene is operably linked to the VSP2 promoter for expression in the vegetative tissues.

Figure 13 shows the effects of freezing temperature stress on freeze tolerance in potted trees of transgenic TGU567248 eucalyptus lines transformed with the pABCTE14b construct as compared to the effects of freezing temperature stress on freeze tolerance in positive control AGEH427 eucalyptus lines transformed with the pABCTEOl construct carrying the CBF2 gene operably linked to the rd29A promoter, and in negative control TGU567205 eucalyptus lines transformed with the pABCTEl 1 construct carrying the CBF2 gene operably linked to the VSP2 promoter. The plants were exposed to a freezing stress temperature between -4.5° and -7.2° C for 24 hours and then allowed to recover at 4° C for 16 hours before being transferred to the greenhouse. The results show that freeze tolerance following freezing stress was significantly high in the TGU567248 transgenic lines, as compared to freeze tolerance in the positive control AGEH427 transgenic lines.

Figure 14 shows the results obtained from semi-quantitative PCR analysis using single- stranded cDNA synthesized from total RNA extracted from the leaves and stems of transgenic TGU567248 eucalyptus lines transformed with the pABCTE14b construct. Lines 1 and 5: young stem from greenhouse- grown transgenic TGU567210 lines containing the rd29A::CBF2 cassette and the BSPl ::AFP cassette. Lines 2, 6, and 10: young stem from greenhouse-grown transgenic TGU567248 lines carrying the pABCTE14b construct. Lines 3, 7 and 11 : young leaves from outdoors-grown transgenic TGU567248 lines carrying the pABCTE14b construct. Line 8:

young leaves from oudoors-grown positive control transgenic AGEH427 lines carrying the pABCTEOl construct. Lines 4, 9 and 12: young leaves from greenhouse-grown transgenic TGU567248 lines carrying the pABCTE14b construct. Lines 13, 14 and 15: pABCTE14b plasmid DNA as positive control. DNA marker size is shown on the left side of the DNA gel. The numbers in the table correspond to the lane numbers at the top of the DNA gel. The PCR primer pair AFPex5/AFPex3 amplifies a 490 bp of the AFP coding sequencing; the primer pair CBF2ex5/CBF2ex3 amplifies a 438 bp of the CBF2 coding sequence, and the primer pair GolS2ex5/GolS2ex3 amplifies a 745 bp of the GolS2 coding sequence.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to multiple gene plant transformation and simultaneous expression of multiple genes under the control of promoters with different characteristics in the production of commercially-important transgenic plants with increased resistance or tolerance to stress conditions which normally occur in nature, such as water stress, high salt conditions and cold temperature stress. Further, the invention provides strategies to grow the desired plant species in geographical areas where normally they would not be able to adapt because of the stress conditions. The identification of stress-regulated genes and their functions in the mechanisms responsible for stress tolerance in plants is of uttermost importance. The genes that are induced upon cold treatment in plants are collectively named Cold-Regulated Genes (COR). In

Arabidopsis, cold acclimation is associated with the induction of COR genes mediated by the cold- and dehydration-responsive DNA regulatory element designated the CRT (C-repeat)/DRE (dehydration responsive element) DNA regulatory element. A small family of cold-responsive transcriptional activators known as CBF1, CBF2 and CBF3 or DREBlb, DREBlc and DREBla, respectively, recognizes the CRT (C-repeat)/DRE sequence present in the promoter regions of cold and dehydration responsive genes. Increased expression of Arabidopsis CBF1, a transcriptional activator that binds to the CRT/DRE sequence, induces COR gene expression and increases freeze tolerance in non-acclimated Arabidopsis plants. CBF genes are induced within 15 min after exposure of the plants to a low, nonfreezing temperature and, within two hours, induction of cold-regulated genes that contain the CRT/DRE -regulatory element, known as the "CBF regulon", takes place, leading to an increase in plant freeze tolerance over the next few days (Jaglo-Ottosen et al, 1998). CBF-regulon expression is also known to increase tolerance to drought and high salinity stress. (Stockinger et al., 1997; Fowler and Thomashow, 2002; Kasuga et al, 1999; Haake et al, 2002).

CBF genes have been found in several crop species, including corn, soybean, wheat, rice, barley, tomato, alfalfa, canola, as well as in vegetables, such as Brassica napus, and trees.

However, targeted expression of Arabidopsis CBF1 in transgenic plants, while improving tolerance to cold, drought and salt loading, has been accompanied by negative effects on vegetative growth and on the yield of the plants under normal growth conditions. Thus, expression of CBF/DREBl has been reported to produce dwarfism in Arabidopsis and tomato (Gilmour et al, 2000; Hsieh et al, 2002b; Kasuga et al, 1999; Liu et al, 1998). The

introduction of a wheat DREB2A homologue gene into rice plants also results in dwarfism (Shen et al, 2003).

Cold-induced antifreeze proteins isolated from the tap root of cold-acclimated carrot (Daucus carota) plants (Smallwood et al. 1999 Biochem J. 340(Pt 2): 385-391) or from fish (Duman JG and de Vries AL 1976 Comp. Biochem. Physiol. B 54 (3): 375-80) are known to inhibit ice re-crystallization in cells. However, expression of the fish antifreeze protein in transgenic tobacco did not lead to an increase in frost tolerance (Kenward et al. 1999 Transgenic Research 8: 105-117).

Raffinose and galactinol, which accumulate in plants during seed development, are involved in tolerance to drought, high salinity and cold stress in Arabidopsis (Taji et al. 2002 Plant J. 29 (4) All -26). Galactinol synthase (GolS) catalyzes the first step in the biosynthesis of raffinose oligosaccharides from UDP-galactose.

The inventors of the present application have surprisingly and unexpectedly devised DNA constructs comprising the Arabidopsis CBF2 gene, the carrot antifreeze protein (AFP) gene and the Arabidopsis galactinol synthase 2 gene under the control of stress-inducible, short- day inducible, tissue-preferred or constitutive promoters that, when expressed simultaneously in plants, confer targeted stress tolerance without causing the negative side effects that normally accompany the expression of these genes. In particular, the inventors of the present application have devised methods of producing ornamental plants and economically-valuable trees with increased tolerance to low temperature, water stress and salt conditions, without detrimentally affecting vegetative growth. Accordingly, the invention provides transgenic plants and transgenic trees with enhanced stress tolerance and methods for producing transgenic plants and transgenic trees with enhanced stress tolerance. Further, the present invention provides for wood products obtained from transgenic trees with enhanced stress tolerance and methods of producing wood products.

Transgenic plants may be gymnosperms, dicotyledonous or monocotyledon plants.

Preferably, the transgenic plants are conifers or angiosperm plants. More preferably, the transgenic plants are hardwood tropical trees, including eucalyptus, poplar, citrus, papaya, avocado, teak, acacia, nutmeg, pistachio, jatropha, pine, Cottonwood, 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. Organs of transgenic plants, comprising leaves, stems, flowers, ovaries, fruits, seeds and calluses, are also included in the embodiments of the invention. Definitions

The present invention uses terms and phrases that are well known to those practicing the art. 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. See, e.g., Sambrook & Russel, MOLECULAR CLONING: A LABORATORY MANUAL, 3.sup.rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001.

Agrobacterium: Agrobacteria that are used for transforming plant cells are disarmed and virulent derivatives of, usually, Agrobacterium tumefaciens or Agrobacterium rhizogenes that contain a vector. The vector typically contains a desired polynucleotide that is located between the borders of a T-DNA.

Angiosperms: Angiosperms are vascular plants having seeds enclosed in an ovary. Angiosperms are seed plants that produce flowers that bear fruits. Angiosperms are divided into dicotyledonous and monocotyledonous plants.

C-Repeat Binding Factor: a gene that encodes a transcriptional activator that binds to the CRT (C-repeat)/DRE (dehydration responsive element) DNA regulatory element present in the promoters of many cold- and drought-inducible genes, including those designated COR (cold-regulated). The phrase "homologous CBF gene" refers to a gene that shares a high sequence identity or similarity with the CBF gene and has CBF function.

In this description, the phrases "CBF gene sequence" and "CBF homologous gene sequence" denote any nucleic acid, gene, polynucleotide, DNA, RNA, mRNA, or cDNA molecule that confers stress-related plant C-repeat binding factor (CBF) activity. A CBF or homologous CBF polynucleotide sequence suitable for the present invention may be identified from a myriad of plants characterized by the presence of a CBF gene. A CBF DNA sequence may be isolated as cDNA or genomic DNA from any suitable plant species using oligonucleotide primers or probes based on DNA or protein sequences disclosed herein. Specific examples of plant species from which CBF genes may be isolated include dicotyledons, such as

Cucurbitaceae, Solanaceae, Brassicaceae, Rutaceae, Papilionaceae, such as alfalfa and Vigna unguiculata, Malvaceae, Asteraceae, Malpighiaceae such as Populus, Myrtaceae such as Eucalyptus; and monocotyledons, such as Gramineae, including wheat, barley, and corn. For the purposes of the present invention, a CBF gene is preferably isolated from Arabidopsis thaliana, and a CBF homologous gene is preferably isolated from Eucalyptus.

Desired Polynucleotide: a desired polynucleotide of the present invention is a genetic element, such as a promoter, enhancer, or terminator, or gene or polynucleotide that is to be transcribed and/or translated in a transformed cell that comprises the desired polynucleotide in its genome. If the desired polynucleotide comprises a sequence encoding a protein product, the coding region may be operably linked to regulatory elements, such as to a promoter and a terminator, that bring about expression of an associated messenger RNA transcript and/or a protein product encoded by the desired polynucleotide. Thus, a "desired polynucleotide" may comprise a gene that is operably linked in the 5'- to 3 '-orientation, a promoter, a gene that encodes a protein, and a terminator. Alternatively, the desired polynucleotide may comprise a gene or fragment thereof in an "antisense" orientation, the transcription of which produces nucleic acids that may form secondary structures that affect expression of an endogenous gene in the plant cell. A desired polynucleotide may also yield a double-stranded RNA product upon transcription that initiates RNA interference of a gene to which the desired polynucleotide is associated. A desired polynucleotide of the present invention may be positioned within a T- DNA, such that the left and right T-DNA border sequences flank or are on either side of the desired polynucleotide. The present invention envisions the stable integration of one or more desired polynucleotides into the genome of at least one plant cell. A desired polynucleotide may be mutated or may be a variant of its wild-type sequence. It is understood that all or part of the desired polynucleotide can be integrated into the genome of a plant. It also is understood that the term "desired polynucleotide" encompasses one or more of such polynucleotides. Thus, a T- DNA of the present invention may comprise one, two, three, four, five, six, seven, eight, nine, ten, or more desired polynucleotides.

Detrimental or Undesirable effects are negative effects associated with foreign gene expression, including, but not limited to, negative pleiotropic effects on plant growth and development, slow plant growth, plant growth retardation, reduced plant stature, plant dwarfism, aberrant root development, delayed flowering, short petals, abnormal stamens and reduced seed production.

Dicotyledonous plant (dicot): a flowering plant whose embryos have two seed halves or cotyledons, branching leaf veins, and flower parts in multiples of four or five. Examples of dicots include but are not limited to, Eucalyptus spp, Populus spp., Liquidambar spp., Salix spp., Acacia spp., Tectona spp., Swietenia spp., Quercus spp., Acer spp., Juglans spp., Persea americana, Gossypium spp., Nicotiana spp., Arabidopsis, Solarium spp., Beta spp., Brassica spp., Manihot esculenta, Ipomoea batatas, Euphorbia spp., Glycine spp., Phaseolus spp.

Medicago spp., Daucus spp., Fragaria spp., Lactuca spp., Rosa spp., Mentha spp., Cucurbita spp., Chrysanthemum spp., Pelargonium spp., Opuntia spp., Linum spp., Heliothus spp., Arachis spp., Jatropha curcas and Dichondra spp.

Encoding: a 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.

Endogenous: refers to a gene that is native to a plant genome.

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

Fiber Quality: as used herein, fiber quality refers to a trait that can be modified to change the structure, appearance, or use of fiber. Traits that determine fiber quality include but are not limited to chemical composition, fiber length, coarseness, strength, color, cross-sectional, width, and fiber density. For example, it is known that fiber length imparts strength, whereas fiber coarseness determines texture and flexibility.

Foreign: "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 inter-fertile with the plant to be

transformed, or does not belong to the species of the target plant. According to the present invention, foreign DNA or RNA may include 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.

Gene: a gene is a segment of a DNA molecule that contains all the information required for synthesis of a product, polypeptide chain or RNA molecule, and includes both coding and non-coding sequences. Genetic element: a "genetic element" is any discreet nucleotide sequence including, but not limited to, a promoter, a gene, a terminator, an intron, an enhancer, a spacer, a 5 '-untranslated region, a 3 '-untranslated region, or a recombinase recognition site.

Genetic modification: stable introduction of DNA into the genome of certain organisms by applying methods in molecular and cell biology.

Gymnosperm: as used herein, refers to a seed plant that bears seed without ovaries. Examples of gymnosperms include conifers, cycads, ginkgos, and ephedras. In gymnosperms, reproductive shoot primordia develop into either male cones (staminate cones) or female cones (ovulate cones).

Introduction: as used herein, refers to the insertion of a nucleic acid sequence into a cell, by methods including infection, transfection, transformation or transduction.

Lignin: as used herein, refers to a polymeric composition composed of phenylpropanoid units, including polymerized derivatives of monolignols coniferyl, coumaryl, and sinapyl alcohol. Lignin quality refers to the ability of a lignin composition to impart strength to cell wall matrices, assist in the transport of water, and/or impede degradation of cell wall polysaccharides. Lignin composition or lignin structure may be changed by altering the relative amounts of each of monolignols or by altering the type of lignin. For example, guaiacyl lignins (derived from ferulic acid) are prominent in softwood or coniferous species, whereas guaiacyl-syringyl lignins (derived from ferulic acid and sinapic acid) are characteristic of hardwood or angiosperm species. The degradation of lignin from softwoods, such as pine, requires substantially more alkali and longer incubations, compared with the removal of lignin from hardwoods. Lignin composition may be regulated by either up-regulation or down-regulation of enzymes involved lignin biosynthesis. For example, key lignin biosynthsesis enzymes include, but are not limited to, 4-coumaric acid: coenzyme A ligase (4CL), Cinnamyl Alcohol dehydrogenase (CAD), and Sinapyl Alcohol Dehydrogenase (SAD). Monocotyledonous plant (monocot): a flowering plant having embryos with one cotyledon or seed leaf, parallel leaf veins, and flower parts in multiples of three. Examples of monocots include, but are not limited to, maize, rice, oat, wheat, barley, sorghum, orchid, iris, lily, onion, palm, turf grasses, and bioenergy grasses. Examples of turf grasses include, but are not limited to, Agrostis spp. (bentgrass species including colonial bentgrass and creeping bentgrasses), Poa pratensis (kentucky bluegrass), Lolium spp. (ryegrass species including annual ryegrass and perennial ryegrass), Festuca arundinacea (tall fescue), Festuca rubra commutata (fine fescue), Cynodon dactylon (common bermudagrass varieties including Tifgreen, Tifway II, and Santa Ana, as well as hybrids thereof); Pennisetum clandestinum (kikuyugrass),

Stenotaphrum secundatum (St. Augustine grass), and Zoysia japonica (zoysiagrass). Examples of bioenergy grasses include Saccharum spp., including S. officinarum (sugar cane), Miscanthus spp. and Panicum virgatum (switchgrass).

Nucleic Acids

By "isolated" nucleic acid molecule(s) is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment. For example, recombinant DNA molecules contained in a vector are considered isolated for the purposes of the present invention. Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution. Isolated RNA molecules include in vitro RNA transcripts of the DNA molecules of the present invention. Isolated nucleic acid molecules, according to the present invention, further include such molecules produced synthetically. Nucleic acid molecules of the present invention may be in the form of RNA, such as mRNA, or in the form of DNA, including, for instance, cDNA and genomic DNA obtained by cloning or produced synthetically. The DNA or RNA may be double- stranded or single-stranded. Single-stranded DNA may be the coding strand, also known as the sense strand, or it may be the non-coding strand, also referred to as the anti-sense strand. Unless otherwise indicated, all nucleotide sequences determined by sequencing a DNA molecule herein were determined using an automated DNA sequencer (such as the Model 3700 from Applied Biosystems, Inc.), and all amino acid sequences of polypeptides encoded by DNA molecules determined herein were predicted by translation of a DNA sequence determined as above.

Therefore, as is known in the art for any DNA sequence determined by this automated approach, any nucleotide sequence determined herein may contain some errors. Nucleotide sequences determined by automation are typically at least about 95% identical, more typically at least about 96%o to at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule. The actual sequence can be more precisely determined by other approaches including manual DNA sequencing methods well known in the art. As is also known in the art, a single insertion or deletion in a determined nucleotide sequence compared to the actual sequence will cause a frame shift in translation of the nucleotide sequence such that the predicted amino acid sequence encoded by a determined nucleotide sequence may be completely different from the amino acid sequence actually encoded by the sequenced DNA molecule, beginning at the point of such an insertion or deletion.

Unless otherwise indicated, each "nucleotide sequence" set forth herein is presented as a sequence of deoxyribonucleotides (abbreviated A, G, C and T). However, by "nucleotide sequence" of a nucleic acid molecule or polynucleotide is intended, for a DNA molecule or polynucleotide, a sequence of deoxyribonucleotides, and for an RNA molecule or

polynucleotide, the corresponding sequence of ribonucleotides (A, G, C and U) where each thymidine deoxynucleotide (T) in the specified deoxynucleotide sequence in is replaced by the ribonucleotide uridine (U). For instance, reference to an RNA molecule having the sequence of SEQ ID NO: 1 set forth using deoxyribonucleotide abbreviations is intended to indicate an RNA molecule having a sequence in which each deoxynucleotide A, G or C of SEQ ID NO: 1 has been replaced by the corresponding ribonucleotide A, G or C, and each deoxynucleotide T has been replaced by a ribonucleotide U.

The present invention is also directed to fragments of the isolated nucleic acid molecules described herein. By a fragment of an isolated DNA molecule having the nucleotide sequences disclosed herein is intended DNA fragments at least 15 nucleotides, at least 20 nucleotides, at least 30 nucleotides in length, which are useful as diagnostic probes and primers is discussed in more detail below. Of course larger nucleic acid fragments of up to the entire length of the nucleic acid molecules of the present invention are also useful diagnostically as probes, according to conventional hybridization techniques, or as primers for amplification of a target sequence by the polymerase chain reaction (PCR), as described, for instance, in Molecular Cloning, A Laboratory Manual, 3rd. edition, edited by Sambrook, J and Russel, D. W., (2001), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., the entire disclosure of which is hereby incorporated herein by reference.

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.

Phenotype: phenotype is a distinguishing feature or characteristic of a plant, which may be altered according to the present invention by integrating one or more "desired

polynucleotides" and/or screenable/selectable markers into the genome of at least one plant cell of a transformed plant. The "desired polynucleotide(s)" and/or markers may confer a change in the phenotype of a tranformed plant by modifying any one of a number of genetic, molecular, biochemical, physiological, morphological, or agronomic characteristics or properties of the transformed plant cell or plant as a whole. Thus, expression of one or more, stably integrated desired polynucleotide(s) in a plant genome may yield a phenotype selected from the group consisting of, for example, increased drought tolerance, enhanced cold and frost tolerance, improved vigor, enhanced color, enhanced health and nutritional characteristics, improved storage, enhanced yield, enhanced salt tolerance, enhanced heavy metal tolerance, increased disease tolerance, increased insect tolerance, increased water-stress tolerance, enhanced sweetness, improved vigor, improved taste, improved texture, decreased phosphate content, increased germination, increased micronutrient uptake, improved starch composition, and improved flower longevity.

Plant tissue: a part of a plant, i.e., a "plant tissue" may be transformed according to the methods of the present invention to produce a transgenic plant. Many suitable plant tissues can be transformed according to the present invention and include, but are not limited to, somatic embryos, pollen, leaves, stems, calli, stolons, microtubers, and shoots. Thus, the present invention envisions the transformation of angiosperm and gymnosperm plants such as turfgrass, wheat, maize, rice, barley, oat, sugar beet, potato, tomato, tobacco, alfalfa, lettuce, carrot, strawberry, cassava, sweet potato, geranium, soybean, oak, apple, grape, pine, fir, acacia, eucalyptus, walnut, teak and palm. According to the present invention "plant tissue" also encompasses plant cells. Plant cells include suspension cultures, callus, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, seeds and microspores. Plant tissues 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. A plant tissue also refers to any clone of such a plant, seed, progeny, propagule whether generated sexually or asexually, and descendents of any of these, such as cuttings or seed. Of particular interest are conifers such as pine, fir, and spruce, monocots such as Kentucky bluegrass, creeping bentgrass, maize, and wheat, and dicots such as cotton, tomato, lettuce, Arabidopsis, tobacco, apple and geranium. 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.

Plants that can be engineered in accordance with the invention include, but are not limited to, trees, such as Eucalyptus species and hybrids thereof (E. alba, E. albens, E.

amplifolia, E. amygdalina, E. aromaphloia, E. baileyana, E. balladoniensis, E. badjensis, 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); Populus species and hybrids thereof (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); Conifers 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); citrus species, including C. medica, C. aurantifolia, C. latipes, C. limon; C. reticulata, C. sinensis, C. paradisi, C. aurantium, C.jambhiri, C. grandis, C. indica, C. ichangensis, C. tachibana, C. micrantha, and citrus hybrids, including, Palestine sweet lime, bergamot and Volkamer lemon, Rangpur lime and Rough lemon; avocado (Persea americana Mill), papaya (Carica papaya), nutmeg (Myristica insipida), pistachio (Pistacio vera), kiwi (Actinidia deliciosa A. Chev.), and jojoba (Simmondsia chinensis), rubber tree (Hevea brasiliensis)

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), lime 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.

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 RNA 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 R₀ plants and Ri and R₂ progenies. The invention also contemplates production of hybrids using an Ro, Ri or R₂ plant as a parent.

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.

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. Such methods are well known to the skilled artisan.

Polynucleotide is a nucleotide sequence comprising a gene coding sequence or a fragment thereof (comprising at least 15 consecutive nucleotides, at least 30 consecutive nucleotides, or at least 50 consecutive nucleotides), a promoter, an intron, an enhancer region, a polyadenylation site, a translation initiation site, 5' or 3' untranslated regions, a reporter gene, a selectable marker or the like. The polynucleotide may comprise single stranded or double stranded DNA or RNA. The polynucleotide may comprise modified bases or a modified backbone. The polynucleotide may be genomic, an RNA transcript (such as an mRNA) or a processed nucleotide sequence (such as a cDNA). The polynucleotide may comprise a sequence in either sense or antisense orientations. An isolated polynucleotide is a polynucleotide sequence that is not in its native state, e.g., the polynucleotide is comprised of a nucleotide sequence not found in nature, or the polynucleotide is separated from nucleotide sequences to which it typically is in proximity, or is in proximity to nucleotide sequences with which it typically is not in proximity.

Progeny: a "progeny" of a transgenic plant that is born of, begotten by, or derived from a transgenic plant. Thus, a "progeny" plant, i.e., an "Fl" generation plant is an offspring or a descendant of the transgenic plant produced by the inventive methods. A progeny of a transgenic plant may contain in at least one, some, or all of its cell genomes, the desired polynucleotide that was integrated into a cell of the parent transgenic plant by the methods described herein. Thus, the desired polynucleotide is "transmitted" or "inherited" by the progeny plant. The desired polynucleotide that is so inherited in the progeny plant may reside within a T-DNA construct, which also is inherited by the progeny plant from its parent. The term "progeny" as used herein also may be considered to be the offspring or descendants of a group of plants.

Promoter: a nucleic acid, preferably DNA, that binds RNA polymerase and/or other transcription regulatory elements. As with any promoter, the promoter sequences of the current present invention will facilitate or control the transcription of DNA or RNA to generate an mRNA molecule from a nucleic acid molecule that is operably linked to the promoter. The RNA generated may code for a protein or polypeptide or may code for an RNA interfering, or antisense molecule. A promoter, as used herein, may also include regulatory elements.

Conversely, a regulatory element may also be separate from a promoter. Regulatory elements confer a number of important characteristics upon a promoter region. Some elements bind transcription factors that enhance the rate of transcription of the operably linked nucleic acid. Other elements bind repressors that inhibit transcription activity. The effect of transcription factors on promoter activity may determine whether the promoter activity is high or low, i.e. whether the promoter is "strong" or "weak." A plant promoter is a promoter capable of initiating transcription in plant cells, whether or not its origin is a plant cell. Exemplary plant promoters include, but are not limited to, those that are obtained from plants, plant viruses, and bacteria such as Agrobacterium or Rhizobium which comprise genes expressed in plant cells. Examples of promoters under developmental control include promoters that preferentially initiate transcription in certain tissues, such as tapetum, xylem, leaves, roots, or seeds. Such promoters are referred to as tissue-preferred promoters. Promoters which initiate transcription only in certain tissues are referred to as tissue-preferred promoters. A cell type specific promoter primarily drives expression in certain cell types in one or more organs, for example, vascular cells in roots or leaves. An inducible or repressible promoter is a promoter which is under environmental control or is a stress-responsive promoter, such as the Arabidopsis thaliana rd29A promoter and dehydrin promoters. Examples of environmental conditions that may effect transcription by inducible promoters include anaerobic conditions, heat, or the presence of light. Tissue specific, tissue preferred, cell type specific, and inducible promoters constitute the class of non-constitutive promoters. A constitutive promoter, such as the cauliflower mosaic virus CaMV 35 S promoter, the maize Adhl -based pEmu promoter, the rice Actl promoter and the maize Ubi promoter, is a promoter which is active under most environmental conditions and in most of the plant tissues.

The present invention provides for DNA constructs wherein, upon insertion of the DNA constructs into a plant, promoters with different characteristics drive the simultaneous expression of the Arabidopsis CBF2 gene, the carrot antifreeze protein (AFP) and the Arabidopsis galactinol synthase 2, and thus lead to enhanced stress tolerance in the plant. The promoters used in the inventive constructs include, but are not limited to, the Arabidopsis rd29A promoter, the poplar bark storage protein gene 1 (BSP1) promoter, the Arabidopsis vegetative storage protein gene 2 (VSP2), the poplar soybean gene regulated by cold 2 (Src2) promoter, and the eucalyptus dehydrin (EdDeh) promoter. The rd29A promoter is stress (cold and drought)-inducible; the BSP1 promoter is short-day inducible and vascular-tissue preferred; the VSP2 promoter is a strong and constitutive promoter which drives expression in all tissues; the EdDeh promoter is cold-inducible and the Src2 promoter is induced by low temperatures. Regenerability: as used herein, refers to the ability of a plant to re-differentiate from a de-differentiated tissue.

Selectable/screenable marker: a gene that, if expressed in plants or plant tissues, makes it possible to distinguish them from other plants or plant tissues that do not express that gene. Screening procedures may require assays for expression of proteins encoded by the screenable marker gene. Examples of such markers include the beta glucuronidase (GUS) gene and the luciferase (LUX) gene. Examples of selectable markers include the neomycin

phosphotransferase (NPTII) 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 (BAR and/or PAT) coding for resistance to herbicides which act to inhibit the action of glutamine synthase such as phosphinothricin (Liberty or Basta), or other similar genes known in the art.

Sequence identity: as used herein, "sequence identity" or "identity" in the context of two nucleic acid or polypeptide sequences includes reference to the residues in the two sequences which are the same when aligned for maximum correspondence over a specified region.

As used herein, percentage of sequence identity means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

Transcription factor: a polypeptide sequence that regulates the expression of a gene or genes by either directly binding to one or more nucleotide sequences associated with a gene coding sequence or indirectly affecting the activity of another polypeptide(s) that bind directly to one or more nucleotide sequences associated with a gene coding sequence. A transcription factor may activate (up-regulate) or repress (down-regulate) expression of a gene or genes. A

transcription factor may contain a DNA binding domain, an activation domain, or a domain for protein-protein interactions. In the present invention, a transcription factor is capable of at least one of (1) binding to a nucleic acid sequence or (2) regulating expression of a gene in a plant.

Transcription and translation terminators: The DNA constructs of the present invention typically have a transcriptional termination region at the opposite end from the transcription initiation regulatory element. The transcriptional termination region may be selected for stability of the mRNA to enhance expression and/or for the addition of

polyadenylation tails added to the gene transcription product.

Transfer DNA (T-DNA): an Agrobacterium T-DNA is a genetic element that is capable of integrating a nucleotide sequence contained within its borders into another genome. In this respect, a T-DNA is flanked, typically, by two "border" sequences. A desired polynucleotide of the present invention and a selectable marker may be positioned between the left border-like sequence and the right border-like sequence of a T-DNA. The desired polynucleotide and selectable marker contained within the T-DNA may be operably linked to a variety of different, plant-specific (i.e., native), or foreign nucleic acids, like promoter and terminator regulatory elements that facilitate its expression, i.e., transcription and/or translation of the DNA sequence encoded by the desired polynucleotide or selectable marker.

Transformation of plant cells: A process by which a nucleic acid is stably inserted into the genome of a plant cell. 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-rnQdiated transformation protocols, viral infection, whiskers, electroporation, microinjection, polyethylene glycol-treatment, heat shock, lipofection and particle bombardment.

Transgenic plant: a transgenic plant of the present invention is one that comprises at least one cell genome in which an exogenous nucleic acid has been stably integrated. According to the present invention, a transgenic plant is a plant that may comprise only one genetically modified cell and cell genome, or it may comprise several or many genetically modified cells, or all of the cells may be genetically modified. A transgenic plant of the present invention may be one in which expression of the desired polynucleotide, i.e., the exogenous nucleic acid, occurs in only certain parts of the plant. Thus, a transgenic plant may contain only genetically modified cells in certain parts of its structure.

Vectors: The vectors of the present invention are Ti-plasmids derived from the A.

tumefaciens. The components of the construct or fragments thereof are normally inserted into a cloning vector that is capable of replication in a bacterial host, e.g., E. coli. After each cloning, the cloning vector with the desired insert may be isolated and subjected to further manipulation, such as restriction digestion, insertion of new fragments or nucleotides, ligation, deletion, mutation, resection, etc. to tailor the components of the desired sequence. Once the construct has been completed, it may then be transferred to an appropriate vector for further manipulation in accordance with the manner of transformation of the host cell. The vectors may include selectable markers, as described above.

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.

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 35S terminator and the nopaline synthase gene Tnos terminator. The expression vector may also contain enhancers, start codons, splicing signal sequences, and targeting sequences. 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 DNA sequences encoding additional functions may also be present in the vector, as is known in the art.

Vegetative growth: is the overall development of a plant. After reproduction, meristem cells differentiate into apical-, lateral meristems that ultimately develop into roots and shoots and, later, into leaves and flowers, for instance. Shoot and root architecture, branching patterns, development of stems, axillary buds, and primordial cells into leaves, petals, flowers, and fruit etc. are all considered "vegetative" and part of the "vegetative growth" cycle of a plant. The rate of development of such features depends on a variety of factors, such as the species of the plant, photosynthesis, availability of nutrients, and the general environment in which the plant is growing.

Wilting Point is defined as the minimal point of soil moisture a plant requires not to wilt. A decrease in moisture to the wilting point or below causes the plant to wilt and no longer recover its turgidity when placed in a saturated atmosphere for 12 hours.

Wood Extractives as used herein are non-cell wall small molecules that can be extracted from wood, bark or foliage with a solvent and include, but are not limited to, lipids, terpenoids, phenolics, alkanes, proteins and monosaccharides.

Wood Quality refers to a trait that can be modified to change the chemical makeup, structure, appearance, or use of wood. While not limiting, traits that determine wood quality include cell wall thickness, cell length, cell size, lumen size, cell density, microfibril angle, tensile strength, tear strength, wood color, cell wall chemistry/lignin modification, and length and frequency of cell division.

Wood pulp: refers to fiber generated from wood having varying degrees of purification. Wood pulp can be used for producing paper, paper board, and chemical products.

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.

DNA Constructs

The DNA constructs according to the invention may comprise a first cassette containing the Arabidopsis rd29A promoter operably linked to the Arabidopsis CBF2 gene; a second cassette containing the cold-inducible eucalyptus dehydrin promoter (EdDeh), the short-day inducible, vascular tissue-preferred Populus trichocarpa bark storage protein 1 (BSP1) promoter, or the constitutive Arabidopsis vegetative storage protein (AtVSP2) promoter operably linked to the carrot antifreeze protein (AFP) cDNA; and a third cassette containing the low temperature- inducible poplar Soybean Gene Regulated By Cold-2 (Src2) homolog promoter, the constitutive Arabidopsis vegetative storage protein (AtVSP2) promoter or the short-day inducible, vascular tissue-preferred Populus trichocarpa bark storage protein 1(BSP1) promoter operably linked to the Arabidopsis galactinol synthase2 cDNA (AtGolS2).

Preferred DNA constructs according to the invention include: The pAGSM379 construct, which comprises three gene cassettes (Figure 1) : the Arabidopsis rd29A promoter driving the Arabidopsis CBF2 gene, the eucalyptus dehydrin promoter (EdDeh) driving the carrot antifreeze protein (AFP) cDNA; and the poplar Src2 (Soybean Gene Regulated By Cold-2) homolog promoter driving the Arabidopsis galactinol synthase2 cDNA (AtGolS2). This construct, upon insertion into a plant, provides for the expression of the CBF2 gene, cold-inducible expression of the anti-freeze protein (AFP) and low temperature-inducible expression of galactinol and raffinose.

The pABCTE14b construct, which comprises a first cassette containing the Arabidopsis rd29A promoter operably linked to the Arabidopsis CBF2 gene; a second cassette containing the short-day inducible, vascular tissue-preferred Populus trichocarpa bark storage protein 1 (BSP1) promoter operably linked to the carrot antifreeze protein (AFP) cDNA; and a third cassette containing the constitutive Arabidopsis vegetative storage protein (AtVSP2) promoter operably linked to the Arabidopsis galactinol synthase2 cDNA (AtGolS2). This construct, upon insertion into a plant, provides for the expression of the CBF2 gene, short-day inducible, vascular tissue- specific expression of the anti-freeze protein (AFP) in the space between the cell membrane and the cell wall, and expression of galactinol and raffinose inside the cells in all vegetative tissues.

The pABCTE15b construct, which comprises a first cassette containing the Arabidopsis rd29A promoter operably linked to the Arabidopsis CBF2 gene; a second cassette containing the constitutive Arabidopsis vegetative storage protein (AtVSP2) promoter operably linked to the carrot antifreeze protein (AFP) cDNA; and a third cassette containing the short-day inducible, vascular tissue-preferred Populus trichocarpa bark storage protein 1(BSP1) promoter operably linked to the Arabidopsis galactinol synthase2 cDNA (AtGolS2). This construct, upon insertion into a plant, provides for the expression of the CBF2 gene, short-day inducible, vascular tissue- specific expression of galactinol and raffinose in cells, and expression of the anti-freeze protein (AFP) in all vegetative tissues in the space between the cell membrane and the cell wall. The pABCTE16b construct, which comprises a first cassette containing the Arabidopsis rd29A promoter operably linked to the Arabidopsis CBF2 gene; a second cassette containing the short-day inducible, vascular tissue-preferred Populus trichocarpa bark storage protein 1 (BSP1) promoter operably linked to the carrot antifreeze protein (AFP) cDNA; and a third cassette containing the constitutive Arabidopsis vegetative storage protein (AtVSP2) promoter operably linked to the Arabidopsis galactinol synthase2 cDNA (AtGolS2). This construct, upon insertion into a plant, provides for the expression of the CBF2 gene, short-day inducible, vascular tissue- preferred expression of the anti-freeze protein (AFP) between the cell membrane and the cell wall, and expression of galactinol and raffinose in all vegetative tissues.

Such DNA constructs can be used to modify, improve or enhance stress tolerance in plants, as described above.

Plant Transformation

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. 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.

Stress Tolerance

The transgenic plants of the invention are characterized by modified, increased or enhanced stress tolerance. The phrase "increased stress tolerance" refers to a transgenic plant that survives exposure to wilting point, salt stress or low temperature stress and maintains its normal phenotype after survival, when compared to a wild-type or non-transformed plant of the same species that does not survive the wilting point or low temperature stress, or shows significant water loss or low temperature damage. The phrase "low temperature" refers to a chilling temperature between 0°C and 12°C or, alternatively, to a freezing temperature between 0°C and -12°C. The terms "hardening" or "acclimatization" refer to a plant grown under conditions of suboptimal water supply. The terms "cold acclimation" or "cold acclimated" refer to a plant exposed for 5 to 25 days to a cold hardening treatment that consists in exposing the plant to a low, above-freezing temperature, while decreasing light intensity and day length. The phrase "water stress" indicates exposure of a non-hardened plant to dry conditions (lack of water) for one to ten days or up to the wilting point, followed by watering and a recovery period of 24 hours at room temperature, before transfer into the greenhouse at 22° C. The phrases "dry conditions" or "lack of water" refer to conditions that may cause incipient, temporary or permanent wilting of leaves, without causing irreversible wilting. The phrase "incipient wilting" refers to a stage of wilting of leaves that is not readily noticeable. The phrase "temporary wilting" refers to a stage of wilting which is characterized by visible drooping of the leaves during the day, from which the plant recovers at night. The phrase "permanent wilting" refers to a stage of wilting, where the plant does not recover during the overnight period. Permanently wilted plants may recover when water is added to the soil. In addition to wilting, leaves may curl or warp, become crinkly, turn brown along the edges (scorch), turn yellow, turn brown, and/or fall from the tree. The phrase "prolonged permanent wilting" refers to a stage where the plant has reached the wilting point and does not recover after addition of water. The term "wilting point" refers to the minimal point of soil moisture that the plant requires not to irreversibly wilt and indicates the limit of moisture decrease at which or under which a plant wilts and can no longer recover its turgidity when placed in a saturated atmosphere for 12 hours. Increase in water stress tolerance is assessed by scoring the number of transgenic plants surviving the water stress after 10 days in the greenhouse, compared to the number of wild-type or non-transformed plants of the same species. Increase in water stress tolerance can also be assessed by scoring the degree of wilting of shoots and leaves after exposure to water stress.

The phrase "freezing stress" indicates exposure of a cold acclimated plant to a

temperature in a range between 0° and -30°C for 2 to 72 hours, followed by a 4 to 8 hour recovery period at 4°C, before transfer into the greenhouse at 22°C. Increase in cold tolerance is assessed by scoring the number of transgenic plants surviving the freezing stress after 1 to 5 days in the greenhouse, compared to the number of wild-type or non-transformed plants of the same species. Increase in cold tolerance can also be assessed by scoring the freezing damage to leaves and shoots after exposure to freezing stress.

Those of skill in the art will recognize that numerous modifications and changes may be made to the exemplary designs and embodiments described herein and that the invention is limited only by the claims that follow and any equivalents thereof.

The invention is further represented by the following examples. The examples are representative only and are not intended to limit the invention to the particular embodiments described therein.

EXAMPLES

Example 1: The pAGSM379 Construct

The DNA construct pAGSM379 was devised for conferring freeze tolerance to

Eucalyptus trees. The construct contained three gene cassettes (Figure 1) : the Arabidopsis rd29A promoter driving the Arabidopsis CBF2 gene, the eucalyptus dehydrin promoter (EdDeh) driving the carrot antifreeze protein (AFP) cDNA; and the poplar Src2 (Soybean Gene Regulated By Cold-2) homolog promoter driving the Arabidopsis galactinol synthase2 cDNA (AtGolS2). The EdDeh promoter is cold-inducible in Arabidopsis and eucalyptus (data not shown), and SRC2 is induced by low temperatures in soybean (Takahashi and Shimosaka, 1997).

Example 2: Effects of Eucalyptus Transformation with the pAGSM379

Construct on Stress Tolerance and Vegetative Growth

The pAGSM 379 construct was tested for its ability to further protect eucalyptus trees from the cold in addition to the CBF2 effects, by cold-regulating the expression of AFP and AtGolS2 and increasing the content of the AFP and galactinol/raffinoase in the plants. Table 1 below presents the results of chamber tests of potted trees of 36 IP-B1 lines carrying the pAGSM379 construct. Two eucalyptus plants were grown in one 1 -gallon pot, with one being transformed with the pAGSM319 construct and the other being an elite line transformed with the TUH000427 construct, which contained the rd29A promoter operably linked to the CBF2 gene, as the positive control in the study. The plants were acclimated in the transgenic fence area (TFA) for 25 days before the chamber test. For the chamber test, the pots were wrapped in a plastic bag to prevent desiccation and placed into the Precision Low Temperature Growth Chamber. The freezing stress temperatures were set between -4.5° and -7.0° C, depending on the level of plant acclimation. Exposure to freezing stress lasted 24 hours. Following freezing temperature stress, the plants were allowed to recover at 4° C for 8 hours before being transferred to the greenhouse (GH). At the end of 15 days in the GH, the dieback of the main stems of the plants was measured and recorded.

The chamber test conditions described above resulted in 100% dieback of potted non- transformed IP-B1 trees. The chamber tests indicated that 10 of the 36 pAGSM379 lines, including two dwarf lines, reached a freeze tolerance level similar to the freeze tolerance level seen in the positive control TUH000427 lines (Table 1 and Figure 2 on the left). In these lines, the dieback percentage was between 0% and 12.8%. Six pAGSM379 lines showed a dieback percentage between 26 to 88% whereas the TUH000427 plants in the same pot showed 0% dieback. These results suggested no improvement in freeze tolerance in these pAGSM379 lines. The remaining 20 pAGSM379 lines showed a dieback between 92 to 100%. These results suggested that these lines had little or no enhanced freeze tolerance (Figure 2, right panel).

Most pAGSM379 lines showed a better growth rate than the TUH000427 control plants grown in the same pots. The average height of the 34 pAGSM379 lines, excluding the two dwarf lines, was 21.1 inches, compared to the average height of 16.0 inches in the 34

THU000427 control plants grown in the same pots. Furthermore, the average height of the eight pAGSM379 lines (excluding the two dwarf lines) with freeze tolerance similar to the freeze tolerance in the TUH000427 lines was 18.9 inches compared to the average height of 16.4 inches in the eight THU000427 plats grown in the same pots. These results demonstrated that the presence of the two additional cassettes, the dunnii Deh-AFP cassette and the Src2-AtGolS2 cassette, in addition to the rd29A-CBF2 cassette, in the pAGSM379 construct provides for enhanced freeze tolerance in eucalyptus, and minimizes the negative effects of CBF2 expression on vegetative growth, thus enabling improved growth.

Table 1. Chamber test of IP-Bl carrying the pAGSM379 construct. The dieback percentage was calculated by the dead portion of main stem against the total height of the plants and compared to the positive control TUH000427 line transformed with a construct containing only the CBF2 gene operably linked to the rd29A promoter.

TGU535849 131 23 22 95.7

TUH000427-20 132 16 0 0

TGU535863 132 25 23 92

TUH000427-12 133 15 0 0

TGU535852 133 14.5 14.5 100

TUH000427-16 134 17.5 0 0

TGU535857 134 21.5 0 100

TUH000427-18 141 15 0 0

TGU535859 141 24 24 100

TUH000427-8 142 14 0 0

TGU535847 142 19.5 2.5 12.8

TUH000427-35 143 18.5 0 0

TGU535887 143 22.5 22.5 100

TUH000427-10 144 16.5 0 0

TGU535850 144 24 10 41.7

TUH000427-25 145 15 0 0

TGU535868 145 19 19 100

TUH000427-2 146 15 0 0

TGU535839 146 26.5 23.5 88.7

TUH000427-24 147 17 0 0

TGU535867 147 20.5 0 0

TUH000427-29 148 15 1.5 10

TGU535877 148 13 13 100

TUH000427-5 149 17 0 0

TGU535843 149 23 23 100

TUH000427-9 150 15 0 0

TGU535848 150 21 5.5 26.2

TUH000427-14 151 14.5 0 0 TGU535855 151 16 0 0

TUH000427-15 152 16 0 0

TGU535856 152 25 25 100

TUH000427-17 153 19 0 0

TGU535858 153 22 15 68.1

TUH000427-4 154 17 0 0

TGU535842 154 20.5 0 0

TUH000427-31 155 16 0 0

TGU535882 155 23 23 100

TUH000427-30 156 11.5 0 0

TGU535881 156 18 16 88.9

TUH000427-13 157 16 0 0

TGU535854 157 20 0 0

TUH000427-7 158 15 0 0

TGU535846 158 23.5 23.5 100

TUH000427-21 159 19 0 0

TGU535864 159 10 0 0 dwarfing

TUH000427-22 160 17.5 0 0

TGU535865 160 22 0 0

TUH000427-30 161 15 0 0

TGU535840 161 21 21 100

TUH000427-33 162 18 0 0

TGU535884 162 7 0 0 dwarfing

TUH000427-23 163 15.5 4.5 29

TGU535866 163 21 17.5 83.3

TUH000427-11 164 17 0 0

TGU535851 164 19 19 100 Example 3: Effects of Eucalyptus Transformation with the pAGSM379 Construct on Leaf Retention

11 pAGSM379 lines were selected for further tests using potted trees in the transgenic fenced area (TFA). Six ramets from each of the pAGSM379 lines and six ramets from each of the TUH000427 and TUH000435 lines were transplanted into 3 -gallon pots (Table 2). Six control non-transformed IP-B1 plants were also planted in 3-gallon pots. The pots were maintained in TFA for two months and the height of the potted plants was then measured. The height data were recorded as the height of the plants at the end of the growing season or the beginning of the winter season (Table 2). The potted plants were visually observed periodically for the next four months before measuring the height of the plants again. The height data were recorded as the height of the plants at the end of the winter season (Table 2).

The results obtained from the potted tree test in TFA largely confirmed the results obtained from the chamber tests. Two pAGSM379 lines, the TGU535858 and the TGU535850 lines, showed little improvement in freeze tolerance (Table 2).

Table 2. Potted Tree Test in Transgenic Fenced Area Exposed to Natural Winter Temperatures

TGU535850 2.0 1.7 0.3 15.0

TGU535855 2.3 2.2 0.1 4.3*

TUH000435 2.1 1.9 0.2 9.5

TUH000427 2.5 2.4 0.1 4.0

IP-B1 2.7 0.0 2.7 100

* Value insignificantly different from the value for the positive control TUH000427 line transformed with a construct containing the CBF2 gene operably linked to the rd29A promoter at 95% confidence.

Most pAGSM379 plants showed a higher degree of leaf retention over the winter time as indicated by the presence of more green and/or purple leaves in the pAGSM379 plants than in the TUH000427 plants (Figure 3). These results demonstrated that the presence of the two additional cassettes, the E. dunnii Deh-AFP cassette and the Src2-AtGolS2 cassette, in addition to the rd29A-CBF2 cassette, in the pAGSM379 construct provides for the expression of the antifreeze protein and the GolS2 transgene in the leaves and thus enhances freeze tolerance in eucalyptus.

Example 4: The Populus trichocarpa Vascular Tissue-Preferred, Short Day- Inducible Bark Storage Protein Gene (BSP1) Promoter

A cold-inducible promoter, such as the rd29A promoter, does not respond to short days associated with the fall and winter seasons. In many subtropical geographies, including

Southeastern United States, temperatures may vary widely in the winter, such that plants may be alternatively exposed to low temperatures and relatively mild or high temperatures during the season. Mild temperatures would reduce the activity of the rd29A promoter with consequent reduction in CBF2 expression and low temperature tolerance in transgenic plants. The inventors thought that a short day-inducible promoter would be advantageous, as it would drive gene expression in the fall and winter, independent of the ambient temperature. The use of a promoter inducible not only by low temperatures, but also by short-day length, would lead to improved stress tolerance in plants. In addition, since protection of the stem from cold damage is essential to ensure leaf regeneration at the end of the winter in many plants, including eucalyptus, a useful promoter for increasing tolerance to low temperatures in plants is a vascular tissue-preferred promoter.

The Populus deltoides bark storage protein gene (Bspa) promoter has been reported to be induced or activated by short-days in winter and is vascular tissue-preferred (Zhu and Coleman, 2001a, 2001b; Coleman et al. 1993). The search of the whole genome sequence of Populus trichocarpa revealed that Populus trichocarpa has two bark storage protein (BSP) genes, BSPl and BSP2. An ~4 kb genomic DNA sequence at the 5' of the translation ATG was obtained from both BSPl and BSP 2. The two promoters share 85% identity in DNA sequence, but the gene structures of Populus trichocarpa BSPl are more similar to the gene structures of Populus deltoides BspA gene. Both promoters (BSPl and BSP2) were fused with GUS and the resulting constructs were introduced into Arabidopsis. The Arabidopsis vegetative storage protein (VSP2) gene promoter was also cloned and fused it with GUS. Twenty-two transgenic

Arabidopsis lines carrying BSPl -GUS, 22 Arabidopsis lines carrying BSP2-GUS and 26 Arabidopsis lines carrying VSP2-GUS were obtained. Fourteen of the 22 BSPl -GUS lines showed vascular tissue specific staining (Figure 4, left and middle panels). Six of the BSP1- GUS 22 lines showed very strong staining with no tissue specificity (Figure 4, right panel) and the remaining two lines showed no staining (data not shown). In contrast, most of the 22 BSP2- GUS lines showed uneven, mild staining of the leaves (Figure 5, left panel). Most of the 26 VSP2-GUS lines showed strong staining in the leaf growing point with no tissue-specificity (Figure 5, right panel).

Example 5: Inventive Constructs

Seven new constructs for enhancing freeze tolerance in trees and field crops were devised (Figures 6-12). The pABCTElO construct provides short-day inducible, vascular tissue- preferred expression of the CBF2 gene. The pABCTEl 1 construct provides strong expression of the CBF2 gene in all vegetative tissues, and especially in the young growing shoots. The pABCTE12 construct provides stress-inducible expression of the CBF2 gene and short-day- inducible plus vascular tissue-preferred expression of the anti-freeze protein (AFP), particularly in the stem cells. The pABCTE13 construct provides stress-inducible expression of the CBF2 gene, and short-day-inducible, as well as vascular tissue-preferred expression of galactinol and raffinose, in all cells. The pABCTE14b and pABCTE16b constructs provide the stress-inducible expression of the CBF2 gene, short-day inducible and vascular tissue-preferred expression of the anti-freeze protein (AFP), and expression of galactinol and raffinose in all vegetative tissues. The pABCTE15b construct provides stress-inducible expression of the CBF2 gene, short-day inducible, vascular tissue-preferred expression of galactinol and raffinose , and expression of the anti-freeze protein (AFP) in all vegetative tissues.

Example 6: Effects of Eucalyptus Transformation with the pABCTE14b Construct on Low Temperature Tolerance

The ABCTE14b construct was tested for its ability to protect eucalyptus trees from freezing stress. The pABCTE14b construct comprises a first cassette containing the Arabidopsis rd29A promoter operably linked to the Arabidopsis CBF2 gene; a second cassette containing the short-day inducible, vascular tissue-preferred Populus trichocarpa bark storage protein 1 (BSP1) promoter operably linked to the carrot antifreeze protein (AFP) cDNA; and a third cassette containing the constitutive Arabidopsis vegetative storage protein (AtVSP2) promoter operably linked to the Arabidopsis galactinol synthase2 cDNA (AtGolS2). This construct, upon successful insertion into a plant, provides for the expression of the CBF2 gene, short-day inducible, vascular tissue-specific expression of the anti-freeze protein (AFP), and expression of galactinol and raffinose in all vegetative tissues.

The effects of transformation with the ABCTE14b construct on transgenic eucalyptus TGU567248 lines were compared to the effects of transformation with the pABCTEOl construct in the positive control transgenic eucalyptus AGEH427 lines, and to the effects of transformation with the pABCTEl 1 construct in negative control transgenic eucalyptus TGU567205 lines. In the pABCTEOl construct the CBF2 gene is operably linked to the cold-inducible rd29A promoter, and in the pABCTEl 1 construct the CBF2 gene is operably linked to the constitutive VSP2 promoter. Figure 13 presents the results of chamber tests of potted trees of TGU567248 lines carrying the pABCTE14b construct, as compared to potted trees of the positive control

AGEH427 lines carrying the pABCTEOl construct, and potted trees of the negative control TGU567205 lines carrying the pABCTEl 1 construct. Two eucalyptus plants were grown in each one 1 -gallon pot. Figure 13 shows three pots in which one TGU567248 eucalyptus plant transformed with the pABCTE14b construct (on the left) and one AGEH427 eucalyptus elite positive control plant transformed with the pABCTEOl construct (on the right) were planted. In the fourth pot one AGEH427 eucalyptus positive control plant transformed with the pABCTEOl construct (on the right) was co-planted and compared to one negative control TGU567205 plant carrying the pABCTEl 1 construct (on the left).

The plants were kept in the greenhouse for two weeks and then acclimated in the transgenic fence area (TFA) before the chamber test. For the chamber test, the pots were wrapped in a plastic bag to prevent desiccation and placed into the Precision Low Temperature Growth Chamber.

The trees in pot No. 1 and No. 2 were acclimated in the TFA for 30 and 45 days respectively, and then exposed to a temperature of -6°C for 24 hours, whereas the trees in pot No. 3 were acclimated in the TFA for 51 days and then exposed to a temperature of -7.2°C for 24 hours, and the trees in pot No. 4 were acclimated in the TFA for 51 days and then exposed to a temperature of -4.5°C for 24 hours. Following exposure to freezing temperature stress, the plants were allowed to recover at 4° C for 16 hours before being transferred to the greenhouse (GH). The dieback of the main stems of the plants was measured and recorded after recovery in the GH. At the time the photograph was taken, the plants in pot No. 1 had been in the GH for 26 days; the plants in pot No. 2 had been in the GH for 11 days; and the plants in pots No. 3 and No. 4 had been in the GH for 6 days. In pot No. 1, the TGU567248 transgenic line suffered 50% dieback and the AGEH427 positive control transgenic line in the same pot showed 100% dieback. In pot No. 2, the TGU567248 transgenic line suffered 14% dieback compared to 98% dieback in the AGEH427 positive control transgenic line. In pot No. 3, the TGU567248 trangenic line suffered 39% dieback compared to 90% dieback in the AGEH427 positive control transgenic line. Finally, the TGU567205 negative control transgenic line in pot No. 4 showed significant damage compared to the AGEH427 positive control transgenic line as a consequence of exposure to a temperature of -4.5°C, similar to the damage commonly found in non- transformed plants.

These results clearly showed that the TGU567248 plants exhibited increased freeze tolerance compared to the AGEH427 positive control transgenic lines.

Molecular Characterization

Expression analysis of the AFP, CBF2 and GolS2 genes in the transgenic TGU567248 lines was performed by semi-quantitative PCR and quantitative PCR. Semi-quantitative PCR and quantitative PCR analyses were conducted using single-stranded cDNA synthesized from total RNA extracted from the leaves and stems of transgenic TGU567248 eucalyptus lines transformed with the pABCTE14b construct, and compared to the data obtained from the leaves of positive control transgenic AGEH427 lines carrying the pABCTEOl construct.

Total RNAs were isolated from the leaves or young stems collected from potted trees in the greenhouse, as well as from potted trees grown outdoors in December 2012. The leaf samples from plants grown outdoors were exposed to short-days and cold and therefore considered acclimated samples. Samples collected from the greenhouse were exposed to a minimum temperature of 18° C and 14 hour day length, and therefore considered non-acclimated samples. mRNA was extracted from the corresponding total RNA using oligo-dT magnetic beads (Anbiom) and single-stranded cDNA (sscDNA) pools were synthesized from each of the mRNA samples using the Smarter cDNA Synthesis kit (Clontech). Similar amounts of sscDNA were used in the PCR. The results are presented in Figure 14, with the primers specified in the table below the figure. The samples were loaded as follows: Lines 1 and 5: young stem from greenhouse- grown transgenic TGU567210 lines containing the rd29A::CBF2 cassette and the BSP1 ::AFP cassette. Lines 2, 6, and 10: young stem from greenhouse-grown transgenic TGU567248 lines carrying the pABCTE14b construct. Lines 3, 7 and 11 : young leaves from outdoors-grown transgenic TGU567248 lines carrying the pABCTE14b construct. Line 8: young leaves from oudoors-grown positive control transgenic AGE427 lines carrying the pABCTEOl construct. Lines 4, 9 and 12: young leaves from greenhouse-grown transgenic TGU567248 lines carrying the pABCTE14b construct. Lines 13, 14 and 15: pABCTE14b plasmid DNA as positive control. Size of the DNA marker is shown on the left side of the DNA gel. The numbers in the table correspond to the lane numbers at the top of the DNA gel. The PCR primer pair AFPex5/AFPex3 amplifies a 490 bp of the AFP coding sequencing; the primer pair CBF2ex5/CBF2ex3 amplifies a 438 bp of the CBF2 coding sequence, and the primer pair GolS2ex5/GolS2ex3 amplifies a 745 bp of the GolS2 coding sequence. Analysis of the genomic DNA extracted from the leaves of the transgenic TGU567248 line indicated that this line contains two copies of the transgenes with no vector backbone sequences.

The results showed that AFP expression (lines 1-4 and 13) was induced in the transgenic TGU567248 line in leaf samples from plants grown outdoors, but not in the stem and leaf samples obtained from greenhouse-grown plants, indicating the AFP expression is induced by exposure to a short-day and/or low temperature. Similarly, CBF2 expression (lanes 5-9 and 14) was induced in the transgenic TGU567248 line and in the positive control transgenic AGEH427 line in leaf samples from plants grown outdoors, but not in the stem and leaf samples obtained from greenhouse-grown plants, indicating that CBF2 expression is induced by exposure to a short-day and/or low temperature. In contrast, GolS2 expression in the transgenic TGU567248 line (lanes 10, 11, and 12) was low and was not induced by acclimation.

The single stranded cDNA samples described above were used for PCR quantification. The results are shown in Table 3 below as Ct values. Low Ct values correspond to high gene- specific amplification. 'No Ct' corresponds to a lack of PCR amplification and therefore to the absence of gene expression. A '+' or '-' in the table corresponds to the presence or absence of gene-specific amplification in quantitative PCR, as determined by the disassociation kinetic curves of the PCR products.

Table 3. Quantitative PCR Analysis of Single-Stranded cDNA samples

Stem 248 24.49 + 30.59 + n/a -

TFA 248 17.57 + 21.51 + 28.49 +

TFA 427 23.76 + No Ct n/a -

L GH 248 26.71 + 29.99 + n/a -

JP-B1 Euc none No Ct - No Ct No Ct -

Control pDNA 21.9 + i 21.38 +

The quantitative PCR analysis confirmed the results obtained from the semi-quantitative PCR analysis shown above. Specifically, AFP expression was induced in the transgenic TGU567248 line in leaf samples from plants grown outdoors, but not in the stem and leaf samples obtained from greenhouse-grown plants, indicating the AFP expression is induced by exposure to a short-day and/or freezing temperature. Likewise, CBF2 expression was higher in the acclimated transgenic TGU567248 line as compared to the acclimated positive control transgenic AGEH427 line. Finally, GolS2 expression was slightly induced only in leaf samples from outdoor grown transgenic TGU567248 plants.

These results clearly showed that AFP expression and CBF2 expression are induced in transgenic eucalyptus TGU567248 plants by exposure to a short-day and/or freezing temperature stress and confirmed that the transgenic eucalyptus TGU567248 plants have improved freeze tolerance compared to positive control transgenic lines and wild-type plants.

Claims

1. A DNA construct comprising (a) a first polynucleotide comprising a functional sequence of a CBF2 gene operably linked to a promoter; (b) a second polynucleotide comprising a functional sequence of a carrot antifreeze gene operably linked to a promoter; and (c) a third polynucleotide comprising a functional sequence of a Arabidopsis galactinol synthase 2 gene operably linked to a promoter.

2. The DNA construct of claim 1, wherein the first polynucleotide is operably linked to a short-day-inducible, vascular tissue-preferred promoter, a constitutive promoter or a stress- inducible promoter.

3. The DNA construct of claim 2, wherein the short-day-inducible, vascular tissue- preferred promoter is a poplar bark storage protein gene 1 BSP1 promoter; the constitutive promoter is an Arabidopsis vegetative storage protein gene 2 VSP2 promoter; and the stress- inducible promoter is an Arabidopsis thaliana rd29A promoter.

4. The DNA construct of claim 3, wherein the second or the third polynucleotide is operably linked to a short-day-inducible, vascular tissue-preferred promoter.

5. The DNA construct of claim 4, wherein the short-day-inducible, vascular tissue- preferred promoter is a poplar bark storage protein gene BSP1 promoter.

6. The DNA construct of claim 3, wherein the second polynucleotide is operably linked to a short-day-inducible, vascular tissue-preferred promoter, and the third polynucleotide is operably linked to a constitutive promoter; or wherein the second polynucleotide is operably linked to a constitutive promoter, and the third polynucleotide is operably linked to a short-day- inducible, vascular tissue-preferred promoter.

7. The DNA construct of claim 6, wherein the short-day-inducible, vascular tissue- preferred promoter is a poplar bark storage protein gene BSP1 promoter, and the constitutive promoter is an Arabidopsis vegetative storage protein gene 2 VSP2 promoter.

8. Isolated plant cells transformed with the DNA construct of claim 7.

9. A transgenic plant comprising the isolated plant cells of claim 8.

10. The transgenic plant of claim 9, wherein the transgenic plant is a transgenic Arabidopsis or a transgenic field crop selected from the group consisting of corn, wheat, rice, carrot, broccoli, tomato and grape.

11. The transgenic plant of claim 9, wherein the transgenic plant is a transgenic tropical tree selected from the group consisting of eucalyptus, poplar, citrus, papaya, avocado, teak, acacia, nutmeg, pistachio, jatropha, pine, Cottonwood, 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.

12. The transgenic plant of claim 11, wherein the eucalyptus is an eucalyptus species selected from the group consisting of Eucalyptus amplifolia, Eucalyptus badjensis, 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.

13. A wood product of the transgenic plant of claim 11 selected from the group consisting of wood, wood pellets, wood pulp, paper, lumber, veneer, charcoal, extractives, tall oil, biofuel and bioenergy.

14. A method for increasing stress tolerance in a plant comprising (i) transforming isolated plant cells with a DNA construct comprising (a) a first polynucleotide comprising a functional sequence of a CBF2 gene operably linked to a promoter; (b) a second polynucleotide comprising a functional sequence of a carrot antifreeze gene operably linked to a promoter; and (c) a third polynucleotide comprising a functional sequence of a galactinol synthase 2 gene operably linked to a promoter; and (ii) culturing the isolated plant cells under conditions that promote growth of a transgenic plant that expresses the DNA construct with no detrimental effects on the plant,

wherein the first polynucleotide encodes a CBF polypeptide, the second polynucleotide encodes a carrot antifreeze protein, and the third polynucleotide encodes a galactinol synthase;

wherein the expression of the protein encoded by the first, second and third polynucleotide in the plant is driven by the promoter to which the first, second and third polynucleotide is operably linked, upon exposure of the plant to a stress condition; and wherein the plant has increased stress tolerance and leaf retention compared to a plant of the same species which does not express the DNA construct.

15. The method of claim 14, wherein the first polynucleotide is operably linked to a short-day-inducible, vascular tissue-preferred promoter, a constitutive promoter or a stress- inducible promoter; and wherein the second polynucleotide is operably linked to a short-day- inducible, vascular tissue-preferred promoter, and the third polynucleotide is operably linked to a constitutive promoter; or wherein the second polynucleotide is operably linked to a constitutive promoter, and the third polynucleotide is operably linked to a short-day-inducible, vascular tissue -preferred promoter.

16. The method of claim 15, wherein the short-day-inducible, vascular tissue- preferred promoter is a poplar bark storage protein gene BSP1 promoter, and the constitutive promoter is an Arabidopsis vegetative storage protein gene 2 VSP2 promoter.

17. The method of claim 14, wherein the stress condition is the plant's wilting point or a low temperature.

18. The method of claim 17, wherein the low temperature is a chilling temperature from about 0°C to about 12°C or a freezing temperature from about 0°C to about -12°C and the exposure to the low temperature is for a period of time from about 2 hours to about 72 hours.

19. The method of claim 18, wherein the transgenic plant is a transgenic Arabidopsis, a transgenic field crop selected from the group consisting of corn, wheat, rice, carrot, broccoli, tomato and grape, or a transgenic tropical tree selected from the group consisting of eucalyptus, poplar, citrus, papaya, avocado, teak, acacia, nutmeg, pistachio, jatropha, pine, Cottonwood, 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.

20. The method of claim 19, wherein the eucalyptus is an eucalyptus species selected from the group consisting of Eucalyptus amplifolia, Eucalyptus badjensis, 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.

21. The method of claim 19, further comprising making a wood product from the transgenic tropical tree, wherein the wood product is selected from the group consisting of wood, wood pellets, wood pulp, paper, lumber, veneer, charcoal, extractives, tall oil, biofuel and bioenergy.