# Patent

## Title
Methods for modifying plant cell walls and modified plants produced thereby

## Abstract

The present invention provides methods for modulating cellulose, hemicellulose and lignin composition and deposition in secondary cell wall layers of plants to improve plant traits that are commercially desirable (e.g., enhanced digestibility of forage crops by animals, increased post-harvest processing of wood and crops for energy production and pulping, increase mechanical strength of plants, and others). The invention also provides methods for identifying genes encoding transcription factors that regulate the formation of secondary cell walls, polynucleotide sequences that encode key components of secondary cell walls, and transgenic plants comprising these sequences.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application No. 60/306,267, filed on Jul. 17, 2001, U.S. Provisional Application No. 60/306,327, filed on Jul. 17, 2001 and U.S. Provisional Application No. 60/345,397, filed on Nov. 9, 2001, which applications are incorporated herein by reference in their entirety.


FIELD OF THE INVENTION
This application relates to modification of cell walls in plants and, in particular, to the modification of secondary cell walls, including the modification of microfibril angles in S1 and/or S2 secondary cell wall layers, the modification of the relative thickness of the S1, S2 and/or S3 secondary cell wall layers, the modification of the degree of lignification of the S1, S2 and/or S3 secondary cell wall layers, the modification of the composition and/or relative amounts of lignin, lignin synthesis enzymes and intermediates, cellulose synthesis enzymes, including cellulose synthase and/or hemicellulose in the S1, S2 and/or S3 secondary cell walls, and plants having such modified cell walls. Trees, forage crops and other plants may be modified in accordance with the present invention.
BACKGROUND
Plant cells have rigid cell walls that determine the size, form and stability of the plant cell. These cell walls are comprised primarily of polymers of simple sugar monomers linked in a variety of linear or branched polymers known as polysaccharides. The most abundant simple sugar monomer is glucose, and the most abundant polymer is cellulose. Cellulose is a linear, unbranched polymer, comprised of β-1,4 linked glucose monomers. Other polysaccharides found in plant cell walls include hemicelluloses, which comprise a group of polysaccharides composed of β-1,4 linked glucose monomers having side chains which may include sugars other than glucose, including xylose, fucose, arabinose, and galactose. Hemicelluloses are a heterogeneous mixture of polysaccharides, the composition of which varies substantially for different plants. Hemicelluloses are defined, operationally, as that polymer fraction which may be extracted from the cell wall with alkali.
Pectins are another type of polysaccharide found in plant cell walls. Pectins are acidic polysaccharides, which are generally comprised primarily of galacturonic acid and rhamnose sugar monomers. Amylose, another common plant polysaccharide, is not a major component of cell walls, but instead acts primarily as a storage material for glucose, rather than as a structural polymer. Because amylose is composed primarily of α-1,4-linked glucose monomers, it is considered to be a related polymer from a biochemical and physiological perspective. The molecular structure of the cell wall and its biogenesis during growth are not completely understood.
The alignment of cellulose microfibrils in the cell wall changes during development of the plant cell. In isodiametric meristematic cells, the fibrils are oriented randomly in the plane of the wall. During the transition to extension growth, an increasingly parallel orientation of the newly deposited fibrils is observed. Cells that grow predominantly in one direction generally have parallel fibrils oriented generally perpendicular to the direction of growth. Plant cells having thick cell walls, such as epidermal and xylem cells, often have a multilayered microfibril structure. These walls may have thin layers of parallel fibrils, the direction of which changes from layer to layer by a substantially constant angle.
An important difference between the cell walls of trees and herbaceous plants is that tree cell walls have more complex xylem layers. The xylem types in cell walls of trees vary depending on the age of the tree and the position of the xylem in the tree. For example, young trees (less that eight years old for pine) or upper parts of the tree (with fewer than 6-8 growth rings) produce so-called juvenile wood xylem. Older parts of the tree produce so-called late wood xylem. Xylem cells have additional cellulose-rich secondary wall layers incorporated into the primary wall, which may become thickened and develop an increased tensile strength and resistance to pressure. The secondary cell wall comprises three additional layers, namely the S1, S2 and S3 layers. In mature wood and late wood (wood formed in autumn) the S2 layers are thicker and the cellulose fibrils have higher angles (both of which are commercially desirable traits), when compared to juvenile or early wood.
The secondary walls may comprise a considerable amount of lignin in addition to cellulose, pectins and hemicelluloses. The S1 layer is generally highly lignified, the S2 layer is lightly lignified, whereas the S3 layer is also highly lignified. Lignin is an insoluble polymer that is primarily responsible for the rigidity of plant stems. Specifically, lignin serves as a matrix around the polysaccharide components of some plant cell walls. In general, the higher the lignin content, the more rigid the plant. For example, tree species synthesize large quantities of lignin, with lignin constituting between 20% to 30% of the dry weight of wood. The lignin content of grasses ranges from 2-8% of dry weight and changes during the growing season. In addition to providing rigidity, lignin aids in water transport within plants by rendering cell walls hydrophobic and water impermeable. Lignin also plays a role in disease resistance of plants by impeding the penetration and propagation of pathogenic agents.
The presence and composition of lignin in plant cell walls is desirable for some applications and undesirable for others. In forestry trees, lignification reduces access by chemicals during pulping or during timber treatment. Similarly, in forage crops, the lignification reduces the digestibility of the forage crops for animals. Lignin is, however, an essential component of cell walls and provides structural support for the plant. Two major goals for the forestry industry are reduced rotation times and reduced costs of extracting pulp from wood. To reduce rotation times, young trees need to have enhanced growth characteristics, and have the wood characteristics of older trees. To reduce the costs of extracting pulp from wood, young trees need to have a reduced or modified lignin content. Similarly, for forage crops, an objective is to increase the digestibility and efficiency of the crop without adversely altering its growth and structural properties. By reducing lignin content of cereal stubble the time required to degrade the stubble in the soil will be greatly reduced. Furthermore by reducing the lignin content of high biomass producing cereals such as maize or sorghum the ability to utilize biomass for conversion to ethanol will be greatly enhanced.
Forage digestibility is affected by both lignin composition and concentration. Lignin is largely responsible for the digestibility, or lack thereof, of forage crops, with small increases in plant lignin content resulting in relatively high decreases (>10%) in digestibility (Buxton and Russell, Crop Sci. 28:538-558, 1988). For example, crops with reduced lignin content provide more efficient forage for cattle, with the yield of milk and meat being higher relative to the amount of forage crop consumed. During normal plant growth, an increase in the maturity of the plant stem is accompanied by a corresponding increase in lignin content and composition that causes a decrease in digestibility. This change in lignin composition is to one of increasing S:G ratio (syringyl/guaiacyl units). When deciding on the optimum time to harvest forage crops, farmers must therefore choose between a high yield of less digestible material and a lower yield of more digestible material.
As discussed in detail below, lignin is formed by polymerization of different monolignols that are synthesized in a multistep pathway, each step in the pathway being catalyzed by a different enzyme. It has been shown that manipulation of the number of copies of genes encoding certain enzymes, such as cinnamyl alcohol dehydrogenase (CAD) and caffeic acid 3-O-methyltransferase (COMT) results in modification of the amount of lignin produced; see, for example, U.S. Pat. No. 5,451,514 and PCT Publication No. WO 94/23044. Furthermore, it has been shown that antisense expression of sequences encoding CAD in poplar leads to the production of lignin having a modified composition (Grand et al., Planta (Berl.) 163:232-237, 1985). Quantitative and qualitative modifications in plant lignin content are known to be induced by external factors such as light stimulation, low calcium levels and mechanical stress. Synthesis of new types of lignins, sometimes in tissues not normally lignified, can also be induced by infection with pathogens.
Lignin is formed by polymerization of at least three different monolignols, primarily para-coumaryl alcohol, coniferyl alcohol and sinapyl alcohol. While these three types of lignin subunits are well known, it is likely that slightly different variants of these subunits may be involved in the lignin biosynthetic pathway in various plants. For example, studies suggest that both free monolignols and monolignol-p-coumarate esters may be substrates for lignin formation in grasses. The relative concentration of the monolignol residues in lignin varies among different plant species and within species. The composition of lignin may also vary among different tissues within a specific plant. The three monolignols are derived from phenylalanine or tyrosine in a multistep process and are believed to be polymerized into lignin by a free radical mechanism.
Coniferyl alcohol, para-coumaryl alcohol and sinapyl alcohol are synthesized by similar pathways. The first step in the lignin biosynthetic pathway is the deamination of phenylalanine or tyrosine by phenylalanine ammonia-lyase (PAL) or tyrosine ammonia-lyase (TAL), respectively. In maize, the PAL enzyme also has TAL activity (Rosier et al., Plant Physiol. 113: 175-179, 1997). The product of TAL activity on tyrosine is p-coumarate. The product of PAL activity on phenylalanine is trans-cinnamic acid which is then hydroxylated by cinnamate 4-hydroxylase (C4H) to form p-coumarate. p-Coumarate is believed to be hydroxylated by coumarate 3-hydroxylase (C3H) to give caffeate. The newly added hydroxyl group is then methylated by caffeic acid O-methyl transferase (COMT) to give ferulate. More recently, a caffeoyl-CoA O-methyl transferase (CAMT) enzyme has been hypothesized to play a role in the lignin biosynthetic pathway (Ye et al., Plant Physiol. 108:459-467, 1995).
Ferulate is conjugated to coenzyme A by 4-coumarate:CoA ligase (4CL) to form feruloyl-CoA. Reduction of feruloyl-CoA to coniferaldehyde is catalyzed by cinnamoyl-CoA reductase (CCR). Coniferaldehyde is further reduced by the action of cinnamyl alcohol dehydrogenase (CAD) to give coniferyl alcohol which is then converted into its glucosylated form for export from the cytoplasm to the cell wall by coniferol glucosyl transferase (CGT). Following export, the de-glucosylated form of coniferyl alcohol is obtained by the action of coniferin beta-glucosidase (CBG). Finally, polymerization of the three monolignols to provide lignin is catalyzed by phenolase (PNL), laccase (LAC) and peroxidase (PER). The formation of sinapyl alcohol involves an additional enzyme, ferulate-5-hydroxylase (F5H). For a more detailed review of the lignin biosynthetic pathway, see Whetton R and Sederoff R, The Plant Cell, 7:1001-1013, 1995 and Whetten R et al., Annu. Rev. Plant Physiol. Plant Mol. Biol. 49:585-609, 1998.
Cellulose Synthesis
The major source of dietary fiber for grazing animals comes from plant cell walls. Mammals possess no enzymes capable for breaking down the polysaccharides in plant cell walls. Instead animals such as ruminants depend on microbial breakdown of plant cell walls through fermentation in either the rumen or large intestine.
Fiber in plants is measured using the Neutral Detergent Fiber (NDF) technique in which plant samples are boiled in a solution containing sodium lauryl sulfate (van Soest in “Nutritional Ecology of the Ruminant”. Cornell University Press, Ithaca, N.Y., 1994). This detergent extracts water-soluble components such as sugars, lipids and organic acids. The remaining insoluble residue (fiber) is termed NDF and consists predominantly of plant cell wall components such as cellulose, hemicellulose, and lignin. The amount of cellulose and lignin in cell walls can be determined using the Acid Detergent Fiber (ADF) method where plant samples are boiled in sulfuric acid and sodium lauryl sulfate. The difference between NDF and ADF for a plant sample is normally considered to be the amount of hemicellulose (van Soest in “Nutritional Ecology of the Ruminant”. Cornell University Press, Ithaca, N.Y., 1994).
Stems of most forage species have greater NDF content than leaves. For example, for a temperate C3 grass in mid-flowering such as tall fescue (Festuca arundinacea), NDF content of leaves and stems is 50 and 70%, respectively (Buxton and Redfearn, J. Nutrition 127:S814-S818, 1997). In contrast, for a C4 tropical grass such as switchgrass (Panicum virgatum L.) the NDF content of leaves and stems is 70 and 85%, respectively. The digestibility of a forage is determined by cell wall content, so that legumes are more digestible than grasses because they contain less NDF. The NDF of a legume, however, is generally less digestible than that of grasses because a higher proportion of the NDF is made up by lignin. The increase of lignin as a component of NDF is also responsible for the decrease in digestibility of grasses at the time of flowering. In fact, ruminants can digest only 40-50% of NDF in legumes compared to 60-70% for grass NDF (Buxton and Redfearn, J. Nutrition 127:S814-S818, 1997). Digestibility of cellulose by ruminants is therefore directly related to the extent of lignification. Generally hemicellulose is more digestible than cellulose.
Cellulose is the most abundant carbohydrate in forage making up to 20-40% of dry matter (van Soest in “Nutritional Ecology of the Ruminant”. Cornell University Press, Ithaca, N.Y., 1994). The cellulose in forages consists predominantly of β1-4 glucan (85%) and smaller amounts of pentosans (e.g. xylose and arabinose; 15%). There appear to be two pools of cellulose within the plant cell wall, the difference being one is lignified and the other is not (van Soest in “Nutritional Ecology of the Ruminant”. Cornell University Press, Ithaca, N.Y., 1994). The lignified cellulose is mostly found in the primary cell wall and in the S1 outer layer of the secondary cell wall. Independent of lignification, it appears that cellulose possesses variability in nutritive quality (van Soest in “Nutritional Ecology of the Ruminant”. Cornell University Press, Ithaca, N.Y., 1994). This indicates that it is possible to alter the rate of cellulose digestibility by modifying the chemical composition of cellulose. This could be achieved through manipulating the actions of the cellulose synthesis and cellulose synthesis-like enzymes found in plant cells. One method to increase digestibility in this way is to increase the activity of the cellulose synthesis and cellulose synthesis-like enzymes responsible for synthesizing hemicellulose or to down regulate the cellulose synthesis and cellulose synthesis-like enzymes making cellulose. Hemicellulose is much more digestible than cellulose and is less likely to become lignified. Another way of manipulating cell wall composition is through modifying the rate and supply of primary components required for cellulose synthesis, i.e. of β1-4 glucan and pentosans such as xylose and arabinose. One way to achieve this is to modify the actions of soluble sucrose synthase and UDP glucose pyrophosphorylase enzymes that produce the UDP-glucose required for cellulose synthesis. This may be further modified by manipulating the actions of the large and small subunits of ADP-glucose pyrophosphorylase, the two enzymes that are rate-limiting steps in starch synthesis (Smith et al., Ann. Rev. Plant Phys. Plant Mol. Biol. 48:67-87, 1997).
Cellulose synthases are found in all tissues and cell types of plants and are involved in both primary and secondary cell wall biosynthesis. Cellulose synthase (cel or cesA) is a glycosyltransferase that utilizes UDP-glucose as a substrate in the polymerization of glucose residues to form 1,4-β-D-glucans (Richmond, Genome Biology I: reviews 3001.1-3001.6, 2000), thereby catalyzing the synthesis of cellulose (an aggregate of β-1,4-linked glucose residues as unbranched polymers). The CesA protein contains putative transmembrane domains and is thought to span the plasma membrane, where this catalytic component may interact with other proteins to form a cellulose synthase ‘complex’. In plants, cesA proteins are encoded by a multi-gene family, of which ten have been identified from Arabidopsis thaliana, nine from maize (Zea Mays) and eight from rice (Oryza sativa) (Arioli et al., Science 279:717-720, 1998; Holland et al., Plant Physiol. 123:1313-1324, 2000). Differential expression of the Arabidopsis CesA genes suggests these genes have different functions within the plant.
In addition to the cellulose synthase genes described above, plants have a superfamily of cellulose synthase-like (CSL) genes, whose amino acid sequences are related to the CesA genes (Richmond and Somerville, Plant Physiol. 124:495-498, 2000; Richmond and Somerville, Plant Mol. Biol. 47: 131-143, 2001; Hazen et al., Plant Physiol. 128: 336-340, 2002). The CSL proteins are predicted to be integral membrane proteins and contain a highly conserved motif that is characteristics of glycosyl transferases. This family of proteins synthesizes repeating β-glycosyl subunits and the CSLs may be involved in the biosynthesis of plant cell wall components, for example hemicellulose. Using sequence data, the CSL superfamily can be divided into several distinct families, for example Arabidopsis has six CSL families (with 40+ members), whereas maize lacks one of these families but has a further two families.
Manipulating expression of genes in the cel/CSL superfamily would alter the chemical composition of plant cell walls in forage plants. Altering cell wall biosynthesis therefore provides an opportunity to increase digestibility of the plant dry matter. This may be achieved by increasing the amount of carbon in the plant allocated to cellulose biosynthesis at the expense of lignin biosynthesis. Alternatively, decreasing the amount of cellulose biosynthesis and increasing the amount of water-soluble carbohydrates would have a similar effect. Furthermore, specifically increasing hemicellulose levels in the plant cell walls at expense of cellulose would also increase forage digestibility. By utilizing specific promoters in combination with the cel and CSL genes it is possible to increase or decrease cellulose and hemicellulose levels in the leaf or stem.
SUMMARY OF THE INVENTION
The present invention provides methods for modulating cellulose, hemicellulose and lignin composition and deposition in secondary cell wall layers of plants to improve plant traits that are commercially desirable (e.g., enhanced digestibility of forage crops by animals, increased post-harvest processing of wood and crops for energy production and pulping, increase mechanical strength of plants, and others). In particular, the invention provides methods for use in (i) modulating microfibril angles in the S1, S2 and/or S3 layers of secondary cell walls; (ii) modifying the composition and deposition of lignin and/or cellulose and/or hemicellulose in S1, S2 and/or S3 secondary cell wall layers; (iii) modifying the thickness of S1, S2 and/or S3 secondary cell wall layers; (iv) identifying genes encoding transcription factors that regulate the modifications described above and the process of development of secondary cell walls; and (v) screening for inducers, repressors and modulators that effect the modifications described above. Preferably, these methods will use transcription factors that can be upregulated or downregulated in a tissue- and time-specific manner to restrict lignification in angiosperms to cell types that require lignin for structural support (e.g., vessel elements, extraxylary fibers), to eliminate or reduce lignification (and increase cellulose deposition) of tissues such as xylary fibers, fiber tracheids and tracheids and to increase the volume of the S2 layer in xylary fibers, fiber tracheids and tracheids (e.g., by repressing activation of the S3 layer or by preventing termination of cellulose deposition in the S2 layer). Likewise, in conifers, transcription factors will preferably be used to restrict lignification to latewood tracheids without affecting lignification in early wood, and more preferably, to restrict lignification in mid season wood, but not in early or late wood zones. Similarly, in forage crops and grasses, transcription factors will be preferably used to reduce the extent and type of lignification of stems to increase the digestibility for animals, reduce the time required for cereal stubble to degrade in the soil, and to improve the efficiency by which plant biomass is converted into ethanol.
The invention also encompasses modified plants that display modified microfibril angles in the S1, S2 and/or S3 secondary cell wall layers compared with unmodified native plants; modified plants that display modified composition and deposition of lignin and/or cellulose and/or hemicellulose in the S1, S2 and/or S3 secondary cell wall layers compared with unmodified native plants; modified plants that have modified thickness of S1, S2 and/or S3 secondary cell wall layers compared with unmodified native plants; and modified plants that have modified regulatory elements for modulating any of the above properties.
The invention also provides polynucleotide sequences, preferably from Cucurbitaceae (e.g., pumpkin and cucumber), but also from Arabidopsis, rice and from pine and Eucalyptus that encode enzymes that are involved in the synthesis of cellulose, hemicellulose, and lignin, transcription factors that regulate enzymes in the lignin biosynthetic pathway, and sequences encoding other cell wall polysaccharides, biosynthetic enzymes and cell wall proteins. The promoters of these sequences are isolated and used in DNA constructs for expressing transcription factors in desired cell types in plants or wood zones in trees. The polynucleotide sequences identified herein encode cellulose synthase (SEQ ID NOs 1-20), LIM Transcription Factors (SEQ ID NOs 21-25) caffeic acid 3-O-methyltransferase (cOMT) (SEQ ID NOs 26-33), 4-Coumarate: CoA ligase (4CL) (SEQ ID NOs 34-44), F5H (SEQ ID NO: 45) and caffeoyl-CoA O-Methyltransferase (ccOMT) (SEQ ID NOs 46-48). The predicted polypeptide sequences encoded by SEQ ID NOs 1-48 are listed in the Sequence Listing as SEQ ID NOs 49-96.



BRIEF DESCRIPTION OF FIGURES
FIG. 1. Amino acid sequence of SEQ ID NO: 1 showing conserved Cys residues of the N-terminal CxxC motif (boxed) and other conserved residues (boxed and in bold).



DETAILED DESCRIPTION
The present invention provides methods for modulating the composition and properties of secondary cell walls during plant growth and development. The methods comprise: identifying transcription factors in a plant species of interest that are candidates for use in modulating the expression of genes involved in the formation of secondary cell walls (“candidate transcription factors”); identifying genes involved in the formation of key components of secondary cell walls; and isolating the promoters of these genes for use in DNA constructs to increase or reduce the expression of candidate transcription factors in desired cell types in plants or wood zones in trees, thereby controlling the composition and deposition of lignin and/or cellulose and/or hemicellulose in S1, S2 and/or S3 secondary cell wall layers.
Polynucleotide sequences that encode: transcriptional activators or repressors of secondary cell wall biogenesis; enzymes involved in the synthesis, modification and/or turnover of cell wall components; and cortical microtubules (and associated proteins) of differentiating xylem cells are identified in a plant species of interest (e.g., model plant systems such as Arabidopsis, cucumber, pumpkin; forage crops and forestry trees). Expression constructs comprising sense and antisense orientations of selected polynucleotide sequences are introduced into plants for modulating the formation and composition of secondary cell walls. The resulting transgenic plants are analyzed for differences in secondary cell wall morphology and composition and plants with appropriate traits are selected for further modification and propagation. The biogenesis of secondary cell walls is a multistep, multigene process. Consequently, it is expected that improved cell wall qualities may require the expression or upregulation of combinations of genes that are not normally expressed in a particular plant species, or are expressed in that species, but not in combination. In addition, the repression or downregulation of particular genes or combinations of genes may also be required. A preferred approach, which has been mentioned above and is described in further detail below, is to identify transcription factors and use these to modulate key processes involved in secondary cell wall formation. Other useful approaches for modulation of multiple genes include crossing plants comprising different transgenes, concurrently introducing and expressing multiple genes into a plant (see, e.g., WO127241A2), and using gene silencing vectors comprising multiple sequences to reduce the expression of one or multiple genes (see, e.g., WO 99/49029, WO 99/53050, and WO 98/36083).
Identification of Candidate Genes
1. Candidate Transcription Factors
Transcription factors are initially selected from Arabidopsis databases. The transcription factors of interest may be rare and tightly regulated and or common and constitutively regulated.
The tightly regulated transcription factors are identified as “candidate transcription factors” in a functional assay for identifying heterologous plant genes, including transcription factors, that are involved in xylogenesis. The assay uses the Zinnia elegans mesophyll cell culture system and is described in U.S. Provisional Application 60/345,397. Briefly, the assay comprises culturing Zinnia mesophyll cells in tracheary element (TE)-inducing and non-inducing medium, transforming control cells with sham constructs (i.e., constructs that do not contain expressable heterologous polynucleotide sequences) and test cells with DNA constructs comprising promoters that are active during xylogenesis operably linked to coding regions of heterologous polynucleotide sequences to be tested, or reporter genes, and comparing xylogenesis in the test and control cells. Genes that encode transcriptional activators and repressors are assessed by cotransforming cells with sham- or test-genes and reporter genes and assessing the effect of test gene expression on the reporter gene signal. Genes that affect the morphology, architecture and composition of secondary cell walls can be identified based on analysis of tracheary element morphology, architecture, composition of secondary cell walls using microscopic techniques, flow cytometry, GC-MS, FTIR and other standard well-known chemical/biochemical/immunochemical methods. For the present purposes, promoter-reporter gene constructs can be used to determine whether promoters of candidate transcription factors are activated under TE-inducing conditions.
In addition, candidate transcription factors are identified directly by overexpressing the transcription factors in transgenic plants under a constitutive promoter such as the Super Ubiquitin promoter (see U.S. Pat. No. 6,380,459 and Example 6 below), or a xylem- or vascular tissue-specific promoter from Arabidopsis, rice and cucurbits (e.g., pumpkin, cucumber), for example. Cucurbit promoters have the advantage that they are not highly responsive to bending and can be used to identify transcription factors that activate processes involved in normal secondary cell wall formation, rather than secondary cell wall processes that are also influenced by bending stress.
2. Secondary Cell Wall Related Genes
Transgenic plants showing altered phenotypes are analyzed further to determine the genes that are regulated by the transcription factors. This analysis includes the use of microarrays comprising sequences of secondary cell wall related genes for transcriptional profiling and analysis of plant tissues for expression of reporter genes driven by promoters of secondary cell wall related genes using histochemical stains, transmission electron microscopy, confocal microscopy.
Microarray transcriptional profiling is performed on individual plants to identify which combinations of cell wall gene products are present during xylogenesis. The technique is well known to those of ordinary skill in the art and is also described briefly in Example 2. Transcription from various tissues of a plant or from plants that exhibit secondary cell wall defects may be analyzed to facilitate the identification of changes in expression of genes that are likely to be related to secondary, rather than primary, cell wall development. Microarray data analysis software is used for cluster analysis to identify transcripts that are specific to xylem development. Results of microarray transcriptional profiling are confirmed using real time RT-PCR.
In the present invention, the particular genes of interest for use in modulating plant quality include: genes involved in cellulose production, particularly those that are responsible for increasing the cellulose content of the S2 layer in the secondary cell wall; genes that control the amount and type of lignin, microtubule genes and genes for microtubule associated proteins which are expected to influence the orientation of cortical microtubules in differentiating secondary xylem cells, and transcription factors that are preferentially expressed during xylogenesis and may function as activators, repressors or coactivators of single or multiple genes involved in cellulose synthesis, hemicellulose synthesis, monolignol synthesis, lignification processes and other processes that determine plant quality. Full-length sequences of selected genes are obtained, and the genes are cloned into viral/binary vectors for in vitro and in planta expression and function testing, using art-recognized methods several of which are described in the Examples below.
EST libraries and databases for forestry and non-forestry plant species have been established from tissues that undergo secondary cell wall formation, as illustrated below in Example 1. Comparisons of the EST sequences in these libraries with sequences in other plant databases, including Arabidopsis, pumpkin and cucumber have identified homologs of xylem-specific transcription factors, cell cycle genes, genes for cell wall polysaccharides, cellulose synthases and cellulose synthase-like genes, and genes involved in lignin formation, among others.
Sequences of secondary cell wall related genes for use in the inventive methods are disclosed in SEQ ID NOs: 1-48 of the Sequence Listing. Representative examples of genes of interest are shown below.














Component
Representative Examples









CELLULOSE
Cellulose synthase




Hexose pyrophosphorylase




Sucrose synthase



HEMICELLULOSE
Beta glucosidase




Hexose pyrophosphorylase




Sucrose phosphate synthase




Xyloglucan Endotransglycosylase




Arabinan synthase




Xylan synthase



LIGNIN
4-coumarate CoA ligase




Cinnamoyl-CoA reductase




Coniferin beta-glucosidase




Coniferyl alcohol dehydrogenase




Mannitol dehydrogenase




Coniferyl-alcohol Glucosyltransferase




Laccase




Ferrulate 5-monooxygenase




para-Coumarate 3-monooxygenase




Trans-cinnamate 4-monooxygenase




Caffeate O-methyltransferase




Caffeoyl-CoA O-methyltransferase




Peroxidase



PECTIN
Hexose pyrophosphorylase




Pectinesterase




Polygalacturonase



STARCH
Alpha amylase




Hexose pyrophosphorylase




Starch branching enzyme




Starch synthase




Phenylalanine ammonia-lyase



TERPENOIDS
Terpenoid Synthases




Oxidosqualene



CELL WALL PROTEINS
Dirigent




Expansin




Arabinogalactan




Extensin




Yieldin










Analysis of Functions of Candidate Genes

Genes that are candidates for enzymes involved in cell wall synthesis and lignification are expressed in vitro, and assayed for enzymatic activity. Cellulose deposition assays are carried out on cell lines stably transformed with cellulose biosynthesis genes. High throughput gene function assays are routinely used in the applicants' laboratories and include protoplast transformation assays, cell growth assays, promoter-reporter gene assays, tracheary element assays, and assays for cellulose and lignin synthesis.
Candidate genes can also be overexpressed in a model plant system such as Arabidopsis or tobacco plants to screen for genes that are involved in the control of formation of S1, S2 and S3 layers (e.g., homeobox genes and other transcriptional regulators). Transcription factors isolated from the xylem of plants used for wood production or as forage crops can be assayed in a model plant system, such as Arabidopsis or tobacco plants, by transforming and expressing wild-type and mutated forms of the factors and examining the resultant phenotypes. Gene silencing constructs and/or antisense constructs can be used to produce loss of function phenotypes which can be compared with phenotypes of wild-type or unmodified plants. The functional characterization of candidate genes includes the use of reverse genetic techniques to generate transgenic plants with loss of function mutations (e.g., T-DNA insertional mutagenesis, RNAi, Tilling, VIGS, and homologous recombination) and gain of function mutations (e.g., activation tagging). A transcription factor can be made constitutively active, for example, by translational fusion of the transcription factor to a VP16 activation domain. Similarly, a transcription factor can be made conditionally active, for example, by translationally fusing the transcription factor to a heterologous glucocorticoid receptor. Such techniques have been employed in the analysis of transcription factors in Arabidopsis (see, e.g., Riechmann and Ratcliffe, Curr. Opin. Plant Biol. 3:423-434 (2000)).
The temporal and spatial expression of genes of interest is examined by cloning the promoters specific for these genes, creating promoter-reporter fusion gene constructs and transforming plants with these constructs. Methods for cloning promoters, making promoter-reporter gene constructs, and transforming plants with these constructs are well known in the art. Promoter prediction programs are also well known (for examples, see Mount, “Bioinformatics”, Cold Spring Harbor Laboratory Press, 2001, Chapter 8). The expression of the reporter protein, which is preferably GFP or another fluorescent protein, is monitored using epifluorescence microscopy on tissue sections. Localization of RNA transcripts in sectioned tissues may be performed on paraffin-embedded sectioned tissues essentially as described by Eshed et al., Cell 99:199-209 (1999) and cited references, and in fresh cut sections according to the methods of Xoconostle-Cazares et al., Science 283:94-98 (1999), Ruiz-Medrano et al., Development 126:4405-4419 (1999) and Koltai and Bird, Plant Physiol. 123:1203-1212 (2000).
Modulation of Expression in Transgenic Plants
Various constructs can be used to modulate lignin and/or cellulose content in developing secondary cell walls of plants to produce plants with improved properties. For example, constructs that comprise sense orientations of one or more genes selected from cellulose synthase genes, UDPG binding domains and UDPGPase, antisense orientations of selected lignin genes 4CL, LIM, and/or sense orientations of lignin genes Cald5H/F5H, CCoAOMT/AEOMT are useful for modifying the polysaccharide and lignin content of cell walls. The 4CL or Super Ubiquitin (with intron) promoters optionally including modified enhancer elements are useful for directing the expression of genes in these constructs. For producing stronger wood with an increased amount of crystalline cellulose in forestry plants, it is advantageous to express transcription factors isolated from tension wood and cellulose-producing genes from this wood type. As described previously, constructs that comprise genes for modulating matrix composition (i.e., hemicelluloses and specific cell wall proteins) and constructs for use in coregulating the expression of more than one gene are also within the scope of the present invention.
METHODS AND EXAMPLES
The following examples and methods are offered by way of illustration and are not intended to limit the scope of the claimed invention.
The isolation and functional expression of genes encoding lignin biosynthesis enzymes in P. radiata and E. grandis is described in pending U.S. patent application Ser. Nos. 09/169,789 and 09/615,192, each of which is incorporated herein by reference in its entirety.
Modification of lignin reactivity in gymnosperms by changing the syringyl/guaiacyl ratio is described in U.S. Pat. No. 6,252,135, which is incorporated herein by reference in its entirety.
Example 1
Library Construction
A. Eucalyptus grandis 
Eucalyptus grandis cDNA expression libraries (from various tissues, including flowers, leaves, phloem, roots, seeds, shoot buds and xylem) were constructed and screened as follows.
mRNA was extracted from the plant tissue using the protocol of Chang et al., Plant Mol. Biol. Rep. 11:113-116, 1993, with minor modifications. Specifically, samples were dissolved in CPC-RNAXB (100 mM Tris-Cl, pH 8,0; 25 mM EDTA; 2.0 M NaCl; 2% CTAB; 2% PVP and 0.05% Spermidine*3HCl) and extracted with chloroform:isoamyl alcohol, 24:1. mRNA was precipitated with ethanol and the total RNA preparate was purified using a Poly(A) Quik mRNA Isolation Kit (Stratagene, La Jolla, Calif.). A cDNA expression library was constructed from the purified mRNA by reverse transcriptase synthesis followed by insertion of the resulting cDNA clones in Lambda ZAP using a ZAP Express cDNA Synthesis Kit (Stratagene), according to the manufacturer's protocol. The resulting cDNAs were packaged using a Gigapack II Packaging Extract (Stratagene) employing 1 μl of sample DNA from the 5 μl ligation mix. Mass excision of the library was done using XL1-Blue MRF′ cells and XLOLR cells (Stratagene) with ExAssist helper phage (Stratagene). The excised phagemids were diluted with NZY broth (Gibco BRL, Gaithersburg, Md.) and plated out onto LB-kanamycin agar plates containing 5-bromo-4-chloro-3-indolyl-beta-D-galactoside (X-gal) and isopropylthio-beta-galactoside (IPTG).
Of the colonies plated and picked for DNA miniprep, 99% contained an insert suitable for sequencing. Positive colonies were cultured in NZY broth with kanamycin and cDNA was purified by means of alkaline lysis and polyethylene glycol (PEG) precipitation. Agarose gel at 1% was used to screen sequencing templates for chromosomal contamination. Dye primer sequences were prepared using a Turbo Catalyst 800 machine (Perkin Elmer/Applied Biosystems Division, Foster City, Calif.) according to the manufacturer's protocol.
DNA sequences for positive clones were obtained using a Perkin Elmer/Applied Biosystems Division Prism 377 sequencer. cDNA clones were sequenced first from the 5′ end and, in some cases, also from the 3′ end. For some clones, internal sequence was obtained using subcloned fragments. Subcloning was performed using standard procedures of restriction mapping and subcloning to pBluescript II SK+ vector.
B. Lolium perenne and Festuca arundinacea 
In like manner, L. perenne and F. arundinacea cDNA expression libraries were constructed and screened. The tissues were taken at different times of the year, specifically in winter and spring, and from different parts of the plants, including leaf blades, leaf base, pseudostems, roots and stems, and were snap-frozen for storage prior to isolation of RNA.
C. Cucurbits
In like manner, cDNA libraries were prepared from vascular tissues of pumpkin (e.g. (Cucurbita maxima), melon (Cucumis melo), and cucumber (e.g. (Cucumis sativus) and screened. The databases were screened using publicly available homology search programs to identify structural homologs to previously identified genes and polypeptides from public databases. For example, FIG. 1 of the present application shows the predicted amino acid sequence encoded by SEQ ID NO: 1 and identifies conserved sequence features characteristic of cellulose synthase (Richmond, Genome Biology 1: 3001.1-3001.6, 2000).
Example 2
Microarray Transcriptional Profiling
cDNA clones are arrayed onto glass slides as multiple replicas, with each location corresponding to a unique cDNA clone (as many as 5500 clones can be arrayed on a single slide, or chip). Each chip is hybridized with a pair of cDNA probes that are fluorescence-labeled with Cy3 and Cy5, respectively. RNA is prepared from stems or wood tissues sampled at multiple times during the growth and development of the plant or tree. Typically, 1 μg of polyA+ RNA is used to generate each cDNA probe. After hybridization, the chips are scanned and the fluorescence intensity recorded for both Cy3 and Cy5 channels. There are multiple built-in quality control steps. First, the probe quality is generally monitored using a panel of ubiquitously expressed genes. Secondly, the control plate also can include yeast DNA fragments of which complementary RNA may be spiked into the probe synthesis for measuring the quality of the probe and the sensitivity of the analysis. Currently, the technology offers a sensitivity of about 1 in 100,000 copies of mRNA. Finally, the reproducibility of this technology can be ensured by including duplicated control cDNA elements at different locations.
Example 3
Comparison of P. radiata, E. grandis and Arabidopsis thalinana cellulose Synthase and Cellulose Synthase-Like Genes
Comparison of EST sequences from P. radiata, E. grandis and Arabidopsis thaliana databases revealed both similarities (+) and differences (−), as shown in the Table below.














Arabidopsis




Ath group
thaliana
Pinus radiata
E. grandis







AtCesA01(RSW1)
+
−
+


AtCesA02,05,06,09
+
−
+


AtCesA03
+
+
+


AtCesA07(IRX3)
+
+
+


AtCesA08(IRX1)
+
+
+


Cs1A
+
+
−


Cs1B
+
−
−


Cs1C
+
+
+


Cs1D
+
−
−


Cs1E
+
+
−


Cs1G
+
−
+









Example 4
Use of an O-methyltransferase (OMT) Gene to Modify Lignin Biosynthesis
a) Transformation of tobacco plants with a Pinus radiata OMT gene
Sense and anti-sense constructs containing a polynucleotide including the coding region of OMT from Pinus radiata were inserted into Agrobacterium tumefaciens LBA4301 (provided as a gift by Dr. C. Kado, University of California, Davis, Calif.) by direct transformation using published methods (see, An G, Ebert P R, Mitra A, Ha SB: Binary Vectors. In: Gelvin S B, Schilperoort R A (eds) Plant Molecular Biology Manual, Kluwer Academic Publishers, Dordrecht (1988)). The presence and integrity of the transgenic constructs were verified by restriction digestion and DNA sequencing.
Tobacco (Nicotiana tabacum cv. Samsun) leaf sections were transformed using the method of Horsch et al., Science 227:1229-1231, 1985. Five independent transformed plant lines were established for the sense construct and eight independent transformed plant lines were established for the anti-sense construct for OMT. Transformed plants containing the appropriate lignin gene construct were verified using Southern blot experiments. A “+” in the column labeled “Southern” in Table 1 below indicates that the transformed plant lines were confirmed as independent transformed lines.
b) Expression of Pinus OMT in Transformed Plants
Total RNA was isolated from each independent transformed plant line created with the OMT sense and anti-sense constructs. The RNA samples were analyzed in Northern blot experiments to determine the level of expression of the transgene in each transformed line. The data shown in the column labeled “Northern” in Table 1 shows that the transformed plant lines containing the sense and anti-sense constructs for OMT all exhibited high levels of expression, relative to the background on the Northern blots. OMT expression in sense plant line number 2 was not measured because the RNA sample showed signs of degradation. There was no detectable hybridization to RNA samples from empty vector-transformed control plants.
c) Modulation of OMT Enzyme Activity in Transformed Plants
The total activity of OMT enzyme, encoded by the Pinus OMT gene and by the endogenous tobacco OMT gene, in transformed tobacco plants was analyzed for each transformed plant line created with the OMT sense and anti-sense constructs. Crude protein extracts were prepared from each transformed plant and assayed using the method of Zhang et al., Plant Physiol. 113:65-74, 1997). The data contained in the column labeled “Enzyme” in Table 1 shows that the transformed plant lines containing the OMT sense construct generally had elevated OMT enzyme activity, with a maximum of 199%, whereas the transformed plant lines containing the OMT anti-sense construct generally had reduced OMT enzyme activity, with a minimum of 35%, relative to empty vector-transformed control plants. OMT enzyme activity was not estimated in sense plant line number 3.
d) Effects of Pinus OMT on Lignin Concentration in Transformed Plants
The concentration of lignin in the transformed tobacco plants was determined using the well-established procedure of thioglycolic acid extraction (see, Freudenberg et al., in Constitution and Biosynthesis of Lignin, Springer-Verlag, Berlin, 1968). Briefly, whole tobacco plants, of an average age of 38 days, were frozen in liquid nitrogen and ground to a fine powder in a mortar and pestle. 100 mg of frozen powder from one empty vector-transformed control plant line, the five independent transformed plant lines containing the sense construct for OMT and the eight independent transformed plant lines containing the anti-sense construct for OMT were extracted individually with methanol, followed by 10% thioglycolic acid and finally dissolved in 1 M NaOH. The final extracts were assayed for absorbance at 280 nm. The data shown in the column labeled “TGA” in Table 1 shows that the transformed plant lines containing the sense and the anti-sense OMT gene constructs all exhibited significantly decreased levels of lignin, relative to the empty vector-transformed control plant lines.













TABLE 1





Plant








line
Transgene
Orientation
Southern
Northern
Enzyme
TGA





















1
control
na
+
blank
100
104


1
OMT
sense
+
2.9E+6
86
55


2
OMT
sense
+
na
162
58


3
OMT
sense
+
4.1E+6
na
63


4
OMT
sense
+
2.3E+6
142
66


5
OMT
sense
+
3.6E+5
199
75


1
OMT
anti-sense
+
1.6E+4
189
66


2
OMT
anti-sense
+
5.7E+3
35
70


3
OMT
anti-sense
+
8.0E+3
105
73


4
OMT
anti-sense
+
1.4E+4
109
74


5
OMT
anti-sense
+
2.5E+4
87
78


6
OMT
anti-sense
+
2.5E+4
58
84


7
OMT
anti-sense
+
2.5E+4
97
92


8
OMT
anti-sense
+
1.1E+4
151
94









Example 5
Use of a 4-Coumarate:CoA Ligase (4CL) Gene to Modify Lignin Biosynthesis
a) Transformation of Tobacco Plants with a Pinus radiata 4CL Gene
Sense and anti-sense constructs containing a Polynucleotide including the coding region of 4CL from Pinus radiata were inserted into Agrobacterium tumefaciens LBA4301 by direct transformation as described above. The presence and integrity of the transgenic constructs were verified by restriction digestion and DNA sequencing.
Tobacco (Nicotiana tabacum cv. Samsun) leaf sections were transformed as described above. Five independent transformed plant lines were established for the sense construct and eight independent transformed plant lines were established for the anti-sense construct for 4CL. Transformed plants containing the appropriate lignin gene construct were verified using Southern blot experiments. A “+” in the column labeled “Southern” in Table 2 indicates that the transformed plant lines listed were confirmed as independent transformed lines.
b) Expression of Pinus 4CL in Transformed Plants
Total RNA was isolated from each independent transformed plant line created with the 4CL sense and anti-sense constructs. The RNA samples were analyzed in Northern blot experiments to determine the level of expression of the transgene in each transformed line. The data shown in the column labeled “Northern” in Table 2 below shows that the transformed plant lines containing the sense and anti-sense constructs for 4CL all exhibit high levels of expression, relative to the background on the Northern blots. 4CL expression in anti-sense plant line number 1 was not measured because the RNA was not available at the time of the experiment. There was no detectable hybridization to RNA samples from empty vector-transformed control plants.
c) Modulation of 4CL Enzyme Activity in Transformed Plants
The total activity of 4CL enzyme, encoded by the Pinus 4CL gene and by the endogenous tobacco 4CL gene, in transformed tobacco plants was analyzed for each transformed plant line created with the 4CL sense and anti-sense constructs. Crude protein extracts were prepared from each transformed plant and assayed using the method of Zhang et al., Plant Physiol. 113:65-74, 1997). The data contained in the column labeled “Enzyme” in Table 2 shows that the transformed plant lines containing the 4CL sense construct had elevated 4CL enzyme activity, with a maximum of 258%, and the transformed plant lines containing the 4CL anti-sense construct had reduced 4CL enzyme activity, with a minimum of 59%, relative to empty vector-transformed control plants.
d) Effects of Pinus 4CL on Lignin Concentration in Transformed Plants
The concentration of lignin in samples of transformed plant material was determined as described in Example 3. The data shown in the column labeled “TGA” in Table 2 shows that the transformed plant lines containing the sense and the anti-sense 4CL gene constructs all exhibited significantly decreased levels of lignin, relative to the empty vector-transformed control plant lines. These data clearly indicate that lignin concentration, as measured by the TGA assay, can be directly manipulated by either sense or anti-sense expression of a lignin biosynthetic gene such as 4CL.













TABLE 2





plant








line
transgene
orientation
Southern
Northern
Enzyme
TGA





















1
control
na
+
blank
100
92


2
control
na
+
blank
100
104


1
4CL
sense
+
2.3E+4
169
64


2
4CL
sense
+
4.5E+4
258
73


3
4CL
sense
+
3.1E+4
174
77


4
4CL
sense
+
1.7E+4
164
80


5
4CL
sense
+
1.6E+4
184
92


1
4CL
anti-sense
+
na
59
75


2
4CL
anti-sense
+
1.0E+4
70
75


3
4CL
anti-sense
+
9.6E+3
81
80


4
4CL
anti-sense
+
1.2E+4
90
83


5
4CL
anti-sense
+
4.7E+3
101
88


6
4CL
anti-sense
+
3.9E+3
116
89


7
4CL
anti-sense
+
1.8E+3
125
94


8
4CL
anti-sense
+
1.7E+4
106
97









Example 6
Isolation, Cloning and Expression of Promoters for Use in Genetic Constructs of the Present Invention
A. Isolation and Cloning of a Superubiquitin Promoter from Pinus radiata 
Pinus radiata cDNA expression libraries were constructed and screened by the method described in Example 1.
Isolation of cDNA Clones Containing the Ubiquitin Gene:
Sequences of cDNA clones with homology to the ubiquitin gene were obtained from high-throughput cDNA sequencing as described above. Sequences from several independent clones were assembled in a contig and a consensus sequence was generated from overlapping clones.
Cloning of the Super Ubiquitin Promoter:
Fragments of the Super Ubiquitin promoter were cloned by two different PCR-based approaches.
Method 1: Long Distance Gene Walking PCR
Using “Long Distance Gene Walking” PCR (Min & Powell, Biotechniques 24:398-400, 1998), a 2 kb fragment was obtained that contained the entire coding region of the ubiquitin gene, a 900 bp intron in the 5′ UTR and approximately 100 bp of the promoter.
To generate this fragment, 2 nested primers were designed from the 3′ UTR of the Super Ubiquitin cDNA sequence isolated from pine. Generally, the 5′ UTR is used for primer design to amplify upstream sequence. However, the available 5′ UTR of Super Ubiquitin was very short, and two initial primers derived from this region failed to amplify any fragments. Therefore, specific primers were designed from the 3′ UTR.
The final PCR product contained fragments of different sizes. These fragments were separated by electrophoresis and the largest were purified from the gel, digested with restriction endonuclease NotI and cloned in the NotI site of expression vector pBK-CMV (Stratagene, La Jolla, Calif.). The largest of these clones contained the complete coding region of the gene (no introns were found in the coding sequence) and a 5′ UTR which contained a 900 bp intron.
Method 2: “Genome Walker” Kit
The Super Ubiquitin gene promoter was cloned using a “Genome Walker” kit (Clontech, Palo Alto, Calif.). This is also a PCR-based method, which requires 2 PCR primers to be constructed, one of which must be gene-specific. Although the ubiquitin coding region is highly conserved, the 5′ UTR from different ubiquitin genes is not conserved and could therefore be used to design a gene-specific primer. A 2.2 kb fragment was amplified and subcloned in pGEM-T-easy (Promega, Madison, Wis.). Analysis by PCR and DNA sequencing showed that the clone contained 5′ UTR sequence of the Super Ubiquitin gene, including the 900 bp intron and approximately 1 kb of putative promoter region. An intron in the 5′ UTR is a common feature of plant polyubiquitin genes and may be involved in determining gene expression levels.
Expression of Super Ubiquitin:
Using primers derived from the gene-specific 5′ and 3′ UTR sequences, expression levels of Super Ubiquitin in different plant tissues was examined by means of RT-PCR. Super Ubiquitin was found to be expressed in all plant tissues examined, including branch phloem and xylem, feeder roots, fertilized cones, needles, one year old cones, pollen sacs, pollinated cones, root xylem, shoot buds, structural roots, trunk phloem and trunk. Expression of Super Ubiquitin in plant tissues was also demonstrated in a Northern blot assay using a PCR probe prepared from the 5′UTR.
Functional Analysis of the Super Ubiquitin Promoter:
To test the function of the Super Ubiquitin promoter in plants, Arabidopsis thaliana was transformed with constructs containing the reporter gene for Green Fluorescent Protein (GFP) operably linked to the Super Ubiquitin promoter either with or without the intron. Constructs lacking a promoter were used as a negative control, with a plant T-DNA vector carrying a CaMV 35S promoter cloned in front of GFP being used as a positive control. The constructs were introduced into Arabidopsis via Agrobacterium-mediated transformation.
All the plant culture media were according to the protocol of Valvekens and Van Montagu, Proc. Natl. Acad. Sci. USA 85:5536-5540, 1998 with minor modifications. For root transformation, sterilized seeds were placed in a line on the surface of germination medium, the plates were placed on their sides to facilitate root harvesting, and the seeds were grown for two weeks at 24° C. with 16 h photoperiod.
Expression of the constructs was measured by determining expression levels of the reporter gene for Green Fluorescent Protein (GFP). Preliminary GFP expression (transient) was detected in early transgenic roots during T-DNA transfer. Transgenic roots that developed green callus, growing on shoot-inducing medium containing 50 μg/ml Kanamycin and 100 μg/ml Timentin, were further tested for GFP expression. After several weeks of stringent selection on Kanamycin medium, several independent transgenic Arabidopsis lines were engineered and tested for GFP expression.
Expression was seen both with the Super Ubiquitin promoter including intron and the Super Ubiquitin promoter without the intron. However, preliminary results indicated that the levels of expression obtained with the Super Ubiquitin intron-less promoter construct were significantly higher than those seen with the promoter including intron, suggesting that the intron may contain a repressor.
Expression Levels of Promoter in Different Plant Tissues
Expression levels of Super Ubiquitin in different plant tissues was examined by means of RT-PCR. Super Ubiquitin was found to be expressed in all plant tissues examined, including branch phloem and xylem, feeder roots, fertilized cones, needles, one-year old cones, pollen sacs, pollinated cones, root xylem, shoot buds, structural roots, trunk phloem and trunk. Expression of Super Ubiquitin in plant tissues was also demonstrated in a Northern blot assay using a PCR probe prepared from the 5′UTR.
B. Isolation and Cloning of Promoters for Genes Involved in Secondary Cell Wall Formation
In like manner, gene-specific promoters are isolated, cloned and tested.
While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, method, method step or steps, for use in practicing the present invention. All such modifications are intended to be within the scope of the claims appended hereto.
All of the publications, patent applications and patents cited in this application are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent application or patent was specifically and individually indicated to be incorporated by reference in its entirety.

## Claims

1. A method for modulating gene expression involved in secondary cell wall formation comprising:
(a) transforming a plant cell with a DNA construct comprising a promoter sequence operably linked to a polynucleotide encoding a candidate transcription factor;
(b) regenerating the plant cell to provide a transgenic plant;
(c) expressing the DNA construct in the cells of the transgenic plant and selecting a transgenic plant with an altered phenotype compared to the phenotype of a nontransformed plant; and
(d) determining whether gene expression involved in secondary cell wall formation is modulated,
wherein the candidate transcription factor is a LIM Transcription Factor having SEQ ID NO: 21.

2. The method of claim 1, wherein the candidate transcription factor is identified by determining that the promoter of the transcription factor is active during secondary cell wall formation.

3. The method of claim 1, wherein the candidate transcription factor is identified by expressing the transcription factor in a transgenic plant and detecting an altered phenotype that is associated with changes in secondary cell wall formation.