WO1998055596A1 - Use of genes encoding xylan synthase to modify plant cell wall composition - Google Patents

Abstract

This invention relates to plant xylan synthases. The isolation of genes, or fragments thereof, for xylan synthases from Arabidopsis thaliana is described. Also described is the use of cDNA clones encoding plant xylan synthases to alter the amount of xylan in transgenic plants. Also described is the use of sequence information derived from the xylan synthase gene to identify genes for other plant xylan synthases.

Description

USE OF GENES ENCODING XYLAN SYNTHASE TO MODIFY PLANT CELL WALL COMPOSITION

GOVERNMENT RIGHTS

The invention described herein was made in the course of work under grant number DE-FG02-94ER20133 from the U.S. Department of Energy. The U.S. Government may retain certain rights in this invention.

TECHNICAL FIELD

The present invention concerns the identification of nucleotide sequences and nucleic acid constructs, and methods related thereto, and the use of these sequences and constructs to produce genetically modified plants for the purpose of altering the polysaccharide composition of plant cell walls. In particular, the present invention describes methods and materials for increasing or decreasing the xylan content of plants.

DEFINITIONS The subject of the present invention is a class of enzymes, herein referred to as xylan synthases, that polymerize sugars into polysaccharides known as xylans. For the present specification, we define xylans as polysaccharides that contain a backbone of 31-4-linked xylose residues. The xylose residues may be modified by the attachment of carbohydrate residues, acetyl groups or other modifications. The enzyme that catalyzes the synthesis of the 31-4-linked xylose residues is herein referred to as xylan synthase. This enzyme is also referred to as xylosyltransferase in the scientific literature (e.g., Baydoun et al. , 1989). BACKGROUND

The ability to genetically control the chemical composition and molecular organization of the polysaccharide component of plant cell walls has many useful applications. Plant cell walls comprise the principal component of wood, and the chemical composition and molecular organization of the polysaccharides in wood is thought to have major effects on the physical properties of wood. Plant cell walls are also the principal component of plant derived fibers such as those used for the production of paper by the pulp and paper industry. Plant fibers such as cotton, ramie, linen, jute a^"nd sisal are also primarily composed of plant cell walls.

The cell walls of higher plants also comprise a major component of the biomass used to feed ruminant animals. There is evidence that the chemical composition and molecular structure of plant cell walls has a major effect on the digestibility of plant cell walls by ruminants. Plant cell walls also comprise a significant component of the dietary fiber in human and animal foods and it is, therefore, of interest to be able to control the amount and quality of this dietary fiber.

A list of some of the important softwood species that are used for production of wood or plant fibers are presented in Table 1. A list of some of the important hardwood species that are used for production of wood or plant fibers is presented in Table 2. A list of some of the non-wood species that are used for the production of plant fibers is presented in Table 3. The invention described herein is useful for the modification of cell walls in most plant species but particularly applicable to all of the species listed in Tables 1-3.

Plant cell walls are complex structures that contain a number of chemically distinct polysaccharides. Imaginative models for the molecular organization of cell walls have been proposed by several authors who have attempted to interpret the available information into coherent models (reviewed by Carpita and Gibeaut, 1993; McCann and Roberts, 1991; and Reiter, 1994). However, the mechanisms involved in the synthesis of the polysaccharides and the factors that control the amounts of particular polysaccharides, or the degree of polymerization or modification were not well understood. Growing plant cells expand by insertion of cell wall material into primary walls which yield to the turgor pressure of the protoplasts. It was not known how the synthesis of new wall material and expansion of the existing wall are regulated, or how neighboring cells in a growing tissue coordinate the synthesis of their respective walls which are firmly connected to each other via the middle lamella. It was also not known how synthesis of the primary cell wall is terminated, or how some cell types switch to the production of a thick secondary wall after they have reached their final size.

Plant cell walls are primarily composed of polysaccharides encompassing cellulose microfibrils and matrix components (McNeil et al., 1984; Bacic et al. , 1988). Cellulose (jβl-4-D-glucan) is thought to be synthesized at the plasma membrane from UDP-D-glucose, and released into the apoplasm where it associates with other cell wall components (Delmer and Amor, 1995). The matrix polysaccharides can be further subdivided into hemicelluloses, including xylans, which bind to the cellulose microfibrils, and pectic material which is highly negatively charged, and tends to form gel-like structures in vitro (Jarvis, 1984). A prominent hemicellulose of most plant cell walls is xyloglucan which consists of a /31-4-D-glucan backbone heavily substituted by mono-, di-, or trisaccharide side chains (Hayashi, 1989). Pectic polysaccharides are usually classified as homogalacturonans , rhamnogalacturonan-I (RG-I) and rhamnogalacturonan-II (RG-II) (Aspinall, 1980). The latter two polysaccharides are structurally highly complex, and appear to be covalently connected to homogalacturonans (Bacic et al. , 1988); however, the precise nature of the linkages between pectic components remained a subject of interest. Unlike bacterial polysaccharides, plant cell wall glycans are not composed of strictly defined repeat units leading to some ambiguity in the structure of individual building blocks. Arabidopsis has a type I wall typical of most higher plants (Zablackis et al., 1995) making it a good genetic model for understanding cell wall synthesis, structure and function.

All matrix polysaccharides are synthesized from nucleoside- diphospho (NDP) sugars at the endomembrane system (Darvill et al. , 1980). The mechanisms of translocation of NDP-sugars across the ER or Golgi membranes, and the polymerization of NDP-sugars into cell wall polysaccharides are subjects of interest. But, with one possible exception, genes encoding membrane carriers for cell wall precursors or for glycosyl- transferases in cell wall synthesis have not been described. The single exception is a recent report of the isolation of two genes from cotton that are thought to encode the enzyme cellulose synthase (Pear et al., 1996). Cellulose synthase from plants has not been characterized to any extent because the enzyme loses activity when cells are disrupted. This could reflect the loss, by dilution, of a critical cofactor, disruption of a complex, loss of membrane potential or a host of other possibilities. Therefore, efforts have been directed towards understanding cellulose biosynthesis in several species of bacteria which produce cellulose as an extracellular ribbon that facilitates adhesion to other cells. Using the bacterium Acetobacter xylinum, cell-free extracts capable of synthesizing cellulose were reported as early as the 1950' s (Glasser, 1958). Following the discovery that a novel regulator, cyclic-di-GMP, is required as an activator of Acetobacter xylinum cellulose synthase, the enzyme was purified to homogeneity. The catalytic subunit of Acetobacter xylinum cellulose synthase, AcsA, was defined biochemically by its presence in purified cellulose synthesizing fractions and its ability to bind the substrate of cellulose biosynthesis, UDP-glucose (Lin et al. , 1990). It has been observed that Acetobacter xylinum AcsA shares a common domain structure with a group of bacterial and eukaryotic proteins that function in the synthesis of diverse β-linked polysaccharides (Saxena et al., 1995). The sugars incorporated into polysaccharides by this family are diverse and include glucuronic acid, N-acetyl-glucosamine, mannuronic acid, galactose, glucose and mannose.

A major discovery associated with the present invention is that non- cellulosic plant cell wall polysaccharides (e.g., xylan and others such as xyloglucan, glucomannan, galactomannan, callose, galactan and mannan) that are constructed with beta-glycosidic linkages are synthesized by enzymes that exhibit structural similarity to plant and bacterial cellulose synthases.

Attempts to use antibodies or nucleic acid hybridization probes based on the bacterial cellulose synthase genes failed to identify corresponding plant proteins or genes. To circumvent these difficulties, Pear et al. (1996) exploited the fact that computer comparisons of predicted protein sequences based on gene sequences can detect similarities between distantly related proteins with much higher sensitivity than direct hybridization experiments. In an effort to find the plant homologs of bacterial cellulose synthase, Pear et al. (1996) carried out an analysis of 250 anonymous cDNA partial sequences from a cotton fiber cDNA library. Utilizing this approach, they identified two cotton cDNAs, designated CELA1 and CELA2, encoding proteins with approximately 30% sequence similarity to the Acetobacter xylinum cellulose synthase. The genes encode polypeptides of 110 kDa with eight putative membrane-spanning helices. Importantly, several residues predicted to be important in catalyzing the formation of /3-glycosidic linkages are conserved between NCSA and the CELA genes. Both CELA1 and CELN2 are expressed abundantly and specifically at the onset of secondary cell wall cellulose synthesis in cotton fibers. Additionally, a protein fragment of CelAl expressed in E. coli binds UDP-glucose in vitro. This binding is dependent on a region of the protein predicted to contain a UDP-glucose binding domain and was inhibited by calcium. Thus, plants contain homologs of bacterial cellulose synthases. These homologs are expressed at the right time to be cellulose synthases and they bind the presumed substrate of the enzyme. Pear et al. (1996) prdicted that the identified genes encoded cellulose synthase. Xylans are the most abundant non-cellulosic polysaccharides in the majority of angiosperms where they account for 20-30% of the dry weight of woody tissues (reviewed by Aspinall, 1980). They are mainly secondary wall components but, in monocotyledonous plants, they are also found in the primary walls of suspension cultured cells. In gymnosperms, where glucomannans and galactomannans form the major hemicelluloses, xylans are less abundant.

The xylans form a family of polysaccharides with essentially linear 3-linked D-xylan backbones although a low degree of branching has been observed in some samples. Side chains of other sugar residues are short and are of three types: (a) single (4-0-methyl-) α-D-glucopyranosyluronic acid residues, most frequently attached by 1-2 linkages to D-xylose units in the backbone; (b) single α-L-arabinofuranose residues, most frequently attached by 1-3 linkages but with double branching (1-3) and (1-2) on xylose units in the more highly substituted arabinoxylans; and (c) more extended side chains in which L-arabinofuranose residues carry additional substituents (see Aspinall, 1980).

Typical xylans from gymnosperms contain the highest proportions (14-18%) of 4-0-methyl-D-glucuronic acid residues but lower and variable (usually < 8%) of L-arabinofuranose side chains. The xylans from dicotyledonous plants contain approximately 10% of 4-0-methyl-D-glucuronic acid units and only infrequently L-arabinose units. Some of these xylans, especially those from deciduous woods, are partially acetylated on D-xylose residues and may contain up to nine 0-acetyl groups per 10 xylose units. Xylans from monocotyledonous plants are of two types, highly branched arabinoxylans devoid of uronic acid units from cereal endosperms which are major components of primary walls in suspension-cultured cells, and rather less branched arabinoxylans with additional uronic acid and galactose units in a diversity of side chains types. These latter polysaccharides are usually isolated from more highly lignified tissues (presumably secondary wall components) such as grasses, maize cobs, and barley husks. A general structure is shown in Figure 1, and Table 4 summarizes the side chains encountered in various xylans.

The modes of attachment of single unit (4-0-methyl-) D-glucuronic acid and L-arabinofuranose residues are well established for many xylans, but the lengths and exact structures of the more extended side chains remain to be established. Table 4 lists oligosaccharide units for which direct evidence

(usually isolation on partial hydrolysis) has been obtained.

Some progress has been made in assessing the distribution of side chains in xylans. The highly branched arabinoxylans from rye and wheat flour which contain 35-40% of arabinofuranose residues do not show a regular pattern of attachment. The less frequent distribution of uronic acid units in

4-0-methyl-glucuronoxylans is not easily shown but a study of birch xylan involving eliminative degradation of uronic acids from the methylated polysaccharide, followed by specific cleavage of the xylan backbone at the original branch, points to a random distribution. However, despite the low frequency, the isolation on partial acid hydrolysis of an oligosaccharide carrying two uronic acid units has shown that both birch and larch xylans contain regions in which adjacent xylose units carry 4-0-methyl-D-glucuronic acid residues (Aspinal, 1980).

One important group of xylans, notably the 4-0- methylglucuronoxylans from hardwoods, occurs in partially acetylated form. 0-Acetyl groups are attached only to xylose residues. Studies on partially acetylated 4-0-substituted β-D-xylopyranosides as model compounds have shown that the 2-0-acetyl and 3-O-acetyl derivatives undergo redistribution to give an equilibrium mixture. The finding that 2-0- and 3-0-acetyl groups in birch xylan are attached in nonequilibrium proportions therefore seems to preclude exclusive biological acetylation at one position. Relatively little was known about the biosynthesis of xylans prior to the present invention. Several authors have reported the preparation of enzyme extracts from plant tissues that incorporate D-[¹⁴C]-xylose from UDP- D-[¹⁴C]xylose into j31-4-xy Ian (e.g., Baydon et al., 1989; Hobbs et al. , 1991). This enzymatic activity was thought to represent the activity of xylan synthase. Separation of the membrane fractions from extracts of pea epicotyls on sucrose gradients indicated that xylan synthase activity comigrated with the Golgi fraction (Waldron and Brett, 1987). Thus, it was generally thought that xylan synthase was located on Golgi membranes (Hobbs et al., 1991). It was further thought that after further modification in the Golgi, the xylan polymer is transported to the cell surface in secretory vesicles. There was no direct information on how the polymer subsequently becomes incorporated into the cell wall, and nothing was known about the physical properties of xylan synthase.

The functional significance of xylans to plant growth and development was not previously known. We now envision that the amount and chemical structure of xylans in plant cell walls exerts effects on the physical properties of the walls. The observations, noted above, indicating substantial interspecies variation in xylan content and chemical structure suggest that altering the xylan content of plants by genetic modification is not necessarily deleterious to the growth and development of plants. Whatever the precise reasons for the presence of xylans in plants, because xylans comprise a large proportion of the polysaccharide composition of plant cell walls, it is useful to be able to modify the xylan content by genetic methods. Of particular interest, because xylans are undesirable components of plant cell walls that are used for the production of cellulose fibers, we envision that the genes encoding xylan synthases of the present invention can be used to decrease the amount of xylan in plant tissues so that they are better suited for the production of cellulosic fibers. We also envision that in some instances, where plant material is used for fiber or structural materials such as wood, it will be useful to alter the structural properties of plant tissues by increasing the xylan content. We also envision that since xylans comprise an important component of the dietary fiber in many plant foodstuffs for both humans and animals, it will be useful to increase or decrease the xylan content by the use of the invention described herein. We also envision that since xylans contribute to the physical properties of plants, it will be useful to modify the xylan content of plants so that they exhibit altered growth and development which may be useful in providing resistance to various natural stresses or supporting desirable changes in the architecture of the plants made by genetic modification of other traits. We also envision that by expression of the xylan synthase genes of the present invention in microbial hosts, such as fungi and bacteria, it will be possible to produce xylans by the large scale culture of these microbial hosts.

The genes which are described in the present invention were obtained by screening for sequences that were homologous to bacterial cellulose synthase. The conceptual basis for this approach is that homologous sequences often have identical functions. However, in some cases, enzymes with homologous sequences catalyze similar reactions rather than identical reactions. In plants, for example, the enzymes which desaturate membrane lip ids form a large gene family. All members of the desaturase gene family are homologous to one another (they share greater than 25 % amino acid sequence identity), however, many of them catalyze different chemical reactions in that they introduce the double bond at different positions in the acyl chains. More importantly, it has been shown that certain fatty acyl hydroxylases have very high levels of sequence identity to fatty acyl desaturases (van de Loo et al., 1995). This and other examples support the notion that gene families are often composed of homologous sequences with similar, but not identical, functions. Thus, the identification of a novel gene family in an organism of interest may lead to the hypothesis that members of the gene family perform similar functions. The enzymes of the present invention show significant sequence similarity to cellulose synthase, and are similar to cellulose synthase in that they are jSl-4-glycan synthases, but they differ in that they utilize a nucleotide derivative of xylose rather than glucose as the substrate.

SUMMARY OF THE INVENTION

The present invention relates to plant xylan synthases. Methods to use amino acid or nucleotide sequences of conserved regions, or antibodies against a conserved epitope to obtain plant genes for xylan synthases are described. Also described is the use of nucleic acids (e.g. , DNA, RNA, cDNA and genomic recombinant clones, expression vectors) encoding plant xylan synthases to increase or decrease the amount of xylans in transgenic plants.

In one embodiment, this invention is directed to recombinant constructs which can provide for the transcription, or transcription and translation (expression) of the plant xylan synthase sequences. In particular, constructs which are capable of transcription, or transcription and translation in plant host cells are preferred. Such constructs may contain a variety of regulatory regions including transcriptional initiation regions obtained from genes preferentially expressed in various tissues of the plant depending on the intended utility of the modified xylan content. In a second aspect, this invention relates to the presence of such constructs in host cells, especially plant host cells which have an expressed plant xylan synthase therein.

In another embodiment, this invention relates to methods of using a nucleic acid encoding a plant xylan synthase for the modification of the polysaccharide composition of the plant's cell wall, wood, fiber, or a combination thereof. A preferred method of using the nucleic acid is by making a transgenic plant or transfecting a plant cell. The nucleic acid may cause decreased xylan synthase activity (e.g., antisense, sense or ribozyme suppression of gene expression, loss-of-function mutant protein) or increased xylan synthase activity (e.g. , extra gene copies, regulated transcription, gain- of- function mutant protein).

Transgenic plants and transfected plant cells having an altered cell wall composition are also contemplated herein. In particular, isolated components of a plant with an altered level of xylan are an object of this invention (e.g., seed, cell wall, wood, fiber); such components may be partially purified and/or specially prepared for shipment, storage or commercial processing.

In a further aspect of this invention, plant xylan synthase enzymes and genes which are related thereto, including amino acid sequences of xylan synthase proteins and nucleotide sequences of nucleic acids encoding xylan synthase, are contemplated. Plant xylan synthases exemplified herein include three Arabidopsis thaliana xylan synthases and a Brassica napus xylan synthase. These exemplified xylan synthases may be used to obtain other plant xylan synthases of this invention, preferably from nucleic acids or proteins of a plant species containing xylan.

By using the degeneracy of the genetic code, variants of SEQ ID NO:20 may be generated that are translated into SEQ ID NO:21 and, thus, encode xylan synthase. Such nucleotide variants may be used instead of the coding sequence of the natural xylan synthase gene because, for example, the host cell used for expressing the nucleotide variant has a different codon preference than the plant from which the xylan synthase gene was derived; in this manner, variant nucleotide sequences may be selected for expression by considering the frequency of codon usage, GC richness, or the species specificity of regulatory regions in the host cell or plant.

A functional equivalent of plant xylan synthase is a protein or nucleic acid with sequence similarity, and either xylan synthase activity or encoding xylan synthase activity, respectively; such are considered within the scope of the present invention. Functional equivalents may be generated by making minor sequence variations in SEQ ID NO:20 by point mutation (e.g. , transition, transversion), deletion, insertion, or a combination thereof, and measuring xylan synthase activity of the translated variant protein; similarly, amino acid substitutions may be made in SEQ ID NO:21 that conserve structure and/or function of xylan synthase (see generally, Creighton, 1983; Creighton, 1992). The degree of functional equivalency may be assessed by comparing xylan synthase activity among proteins or genes with similar sequences. Such comparison of enzymatic activity and/or determination of the product of the enzyme product may lead to variants of the disclosed plant xylan synthase which are quantitatively or qualitatively different from the enzymes found in nature. For example, a transgenic plant containing the variant may contain an altered amount of xylans or a product similar, but not identical, to xylans.

Nucleic acids which share nucleotide sequence with SEQ ID NO:20, or proteins which share amino acid sequence with SEQ ID NO:21 are also an object of this invention. Sequence identity is preferably 60% or greater, more preferably 80% or greater, and most preferably 100% . Besides computer- assisted comparison of sequences using algorithms well-known in the art (see and references cited therein), the degree of nucleotide sequence variation may be assessed by low or high stringency hybridization with a target sequence of SEQ ID NO:20 (see generally, Hames and Higgins, 1985), or by reference to substitution of codons for chemically similar amino acid residues (e.g. , charged vs. uncharged, polar vs. nonpolar, hydrophilic vs. hydrophobic; see generally, Dickerson and Geis, 1969; Branden and Tooze, 1991).

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. General structure of a fragment of a plant xylan (reproduced from

Aspinall, 1980).

Figure 2. Similarity of Arabidopsis EST 160C16T7 to bacterial cellulose synthases as determined by the BLASTX search algorithm. Figure 3. BESTFIT alignment of Agrobacterium CELA gene product to predicted product from 160C16T7. The two sequences show 53.7% similarity and 24.6% identity.

Figure 4. Alignment of deduced amino acid sequences from partial or complete nucleotide sequences of CSL1, CSL2, CSL3, CSL4, CSL5, CSL6, and CSL7 genes of Arabidopsis. Regions of sequence that are highly conserved in xylan synthases are underlined with asterisks.

Figure 5. Nucleic acid and amino acid sequence of cDNA clone of CSL4 gene from Arabidopsis.

Figure 6. BESTFIT alignment of Csl4 to CelAl . The two sequences were 46% similar and 17% identical.

Figure 7. Diagram of plasmid used to produce antisense plants.

Figure 8. BESTFIT alignment of open reading frame from B. rapa EST clone

BNAF03353 to Arabidopsis Csl4 protein. The two sequences were 95% similar and 86% identical.

SUMMARY OF THE SEQUENCE LISTING

1. GenBank nucleotide sequence of EST 160C16T7. 2. Full-length nucleotide sequence of EST clone 160C16T7 obtained for this application.

3. Amino acid sequence of longest open reading frame in ID2.

4. Nucleotide sequence of EST clone FAFL51 obtained for this application.

5. Longest available nucleotide sequence of EST clone 92K11T7 obtained for this application.

6. Longest available nucleotide sequence of EST clone 178H9T7 obtained for this application. 7. Longest available nucleotide sequence of EST clone 133A23T7 obtained for this application.

8. Full-length nucleotide sequence of EST clone 210A22T7 obtained for this application.

9. Amino acid sequence of longest open reading frame in ID4. 10. Amino acid sequence of longest open reading frame in ID5.

1 1. Amino acid sequence of longest open reading frame in ID6.

12. Amino acid sequence of longest open reading frame in ID7.

13. Amino acid sequence of longest open reading frame in ID8.

14. Longest available nucleotide sequence of EST clone 119C22T7 obtained for this application.

15. Amino acid sequence of longest open reading frame in ID 14.

16. Nucleotide sequence of oligonucleotide PI .

17. Nucleotide sequence of oligonucleotide P2.

18. Nucleotide sequence of oligonucleotide P3. 19. Nucleotide sequence of oligonucleotide P4.

20. Full-length nucleotide sequence of cDNA insert in clone pCSL4.

21. Amino acid sequence of longest open reading frame in ID20.

22. Nucleotide sequence of oligonucleotide P5.

23. Nucleotide sequence of oligonucleotide P6. 24. GenBank nucleotide sequence of EST BNAF03353.

25. Amino acid sequence of longest open reading frame in ID24.

DETAILED DESCRIPTION OF THE INVENTION

A genetically transformed plant of this invention which accumulates altered amounts of xylans can be obtained by expressing the nucleic acids (e.g., DNA, RNA) envisioned in this application.

A plant xylan synthase of this invention includes any sequence of amino acids, such as a protein, polypeptide or peptide fragment, or nucleotide sequences encoding such obtainable from a plant source which has the ability to catalyze the synthesis of xylan. By xylan is meant any polysaccharide containing more than three D-xylose residues linked to each other in a 0-1-4 linkage.

Although the substrate of the A. thaliana xylan synthase is not known precisely, it is thought to be UDP-D-xylose. However, it is also possible, although unlikely, that UDP-xylose is converted to another compound before being utilized by the xylan synthase of this invention.

Other plant xylan synthase are obtainable from the specific exemplified sequences provided herein. Thus, we envision that the enzymes that synthesize xylan in trees and other plants are structurally similar to the xylan synthase of this invention. We envision that genes encoding the xylan synthases of this invention can be isolated by designing PCR primers or hybridization probes based on conserved sequences from the xylan synthases described herein. Similarly, we envision that the enzymes that produce galactans, mannans, and xyloglucans are structurally related to the xylan synthases of this invention. Furthermore, it will be apparent that one can obtain natural and synthetic plant xylan synthases including modified amino acid sequences and starting materials for synthetic-protein modeling from the exemplified plant xylan synthases and from plant xylan synthases which are obtained through the use of such exemplified sequences. Modified amino acid sequences include sequences which have been mutated, truncated, elongated or the like, whether such sequences were partially or wholly synthesized. Sequences which are actually isolated from plant preparations or are identical or encode identical proteins thereto, regardless of the method used to obtain the protein or sequence, are equally considered naturally derived.

Thus, one skilled in the art will readily recognize that antibody preparations, nucleic acid probes (DNA and RNA) or the like may be prepared and used to screen and recover "homologous" or "related" xylan synthases from a variety of plant sources. Typically, nucleic acid probes are labeled to allow detection, preferably with radioactivity although enzymes or other methods may also be used. For immunological screening methods, antibody preparations, either monoclonal or polyclonal, may be utilized. Polyclonal antibodies, although less specific, typically are more useful in gene isolation. For detection, the antibody is labeled using radioactivity or any one of a variety of second antibody /enzyme conjugate systems that are commercially available.

Homologous sequences are found when there is some degree of identity or similarity of sequence above that expected by chance alone and this may be determined by comparison of sequence information, nucleotide or amino acid, by using computer programs such as FASTA or through hybridization reactions between a known xylan synthase and a candidate source. Conservative changes, such as Glu/ Asp, Val/Ile, Ser/Thr, Arg/Lys and Gin/ Asn may also be considered in determining sequence similarity. Typically, a lengthy nucleotide sequence may show as little as about 50-60% sequence identity, and more preferably at least about 70% sequence identity, between the target sequence and the given plant xylan synthase of interest excluding any deletions or additions which may be present, and still be considered related. Amino acid sequences are considered to be homologous with as little as 25 % sequence identity between the two complete mature proteins (see generally, Doolittle, 1986).

Homologous sequences often have identical functions, however this is not always true. It is generally true, however, that similar sequences have similar functions. In plants, for example, the enzymes which desaturate membrane lipids form a large gene family. All members of the desaturase gene family are homologous to one another (they share greater than 25% amino acid sequence identity), however, many of them catalyze different chemical reactions. The desaturases are similar in that they all perform lipid desamrations, they differ however in their substrate specificities, the specific positions of carbon-carbon bonds they desaturate and their subcellular places of action. The desaturases share conserved sequence motifs, histidine boxes, predicted to be important for the catalysis of lipid desaturation. Presumably, over the course of plant evolution gene duplication generated multiple copies of desaturases which subsequently evolved the abilities to desaturate different lipid substrates at different positions along the lipid chain and in different sub-cellular compartments. This example, and others similar to it support the notion that gene families are often composed of homologous sequences with similar, but not identical, functions. Thus, the identification of a novel gene family in an organism of interest may lead to the hypothesis that members of the gene family perform similar functions.

A genomic or other appropriate library prepared from the candidate plant source of interest may be probed with conserved sequences from the plant xylan synthase to identify homologously related sequences. Use of an entire cDNA or other sequence may be employed if shorter probe sequences are not identified. Positive clones are then analyzed by obtaining the nucleotide sequence and related methods. When a genomic library is used, one or more sequences may be identified providing both the coding region, as well as the transcriptional regulatory elements of the xylan synthase gene from such plant sources. Probes can also be considerably shorter than the entire sequence. Oligonucleotides may be used, for example, but should be at least about 10, preferably at least about 15, more preferably at least 20 nucleotides in length. When shorter length regions are used for comparison, a higher degree of sequence identity is required than for longer sequences. Shorter probes are often particularly useful for polymerase chain reactions (PCR), especially when highly conserved sequences can be identified (see Gould et al., 1989 for examples of the use of PCR to isolate homologous genes from taxonomically diverse species). When longer nucleic acid fragments are employed ( > 100 bp) as probes, especially when using complete or large cDNA sequences, one would screen with low stringencies (for example, 40-50°C below the melting temperature of the probe) in order to obtain signal from the target sample with 20-50% deviation, i.e. , homologous sequences (Beftz et al., 1983). In a preferred embodiment, a plant xylan synthase of this invention will have at least 60% , and preferably at least 75 %, overall amino acid sequence similarity with the exemplified plant xylan synthases. In particular, xylan synthases which are obtainable from the use of an amino acid or nucleotide sequence of an A. thaliana xylan synthase by the methods exemplified herein are especially preferred. Xylans produced by the xylan synthases of this invention may be subject to further enzymatic modification by other enzymes which are normally present or are introduced by genetic engineering methods. For example, the 01-4-xylose backbone of many xylans contains 4-O-methyl-i3-D-glucopyranosyluronic acid residues attached by 1-2 linkages to D-xylose units in the backbone. We also envision that as genes become available for the enzymes that catalyze modification of xylans, many different xylans will be produced in transgenic plants.

Again, not only can gene clones and materials derived therefrom be used to identify homologous plant xylan synthases, but the resulting sequences obtained therefrom may also provide a further method to obtain plant xylan synthase from other plant sources. In particular, PCR may be a useful technique to obtain related xylan synthases from sequence data provided herein. One skilled in the art will be able to design oligonucleotide probes based upon sequence comparisons or regions of typically highly conserved sequence. Of special interest are polymerase chain reaction primers based on the conserved regions of amino acid sequence between the xylan synthase of this invention and cellulose synthases. Details relating to the design and methods for a PCR reaction using these probes are described more fully in the examples. It should also be noted that the xylan synthases of a variety of sources can be used to investigate xylan synthesis in a wide variety of plant and in vivo applications. Because all plants are thought to synthesize xylans via a common metabolic pathway, the study and/or application of one plant xylan synthase to a heterologous plant host may be readily achieved in a variety of species. Once the nucleotide sequence is obtained, the transcription, or transcription and translation (expression), of the plant xylan synthase in a host cell is desired to produce a ready source of the enzyme and/or to increase the composition of xylans found associated with the cells, typically in the cell walls. Other useful applications may be found when the host cell is a plant host cell, in vitro or in vivo. For example, by increasing the amount of a xylan synthase available to the plant, an increased percentage of xylan may be provided. Conversely, by decreasing the amount of xylan synthase activity through antisense, cosuppression or by identification of mutations, plants with decreased amounts of xylan may be obtained. Plants having significant amounts of xylan are preferred candidates to obtain naturally -derived xylan synthases. In addition, a comparison between xylan synthases and cellulose synthase or other polysaccharide synthases (e.g. , arabinan synthase, mannan synthase, xyloglucan synthase, callose synthase) may yield insights for protein modeling or other modifications to create synthetic xylan synthases.

Genetic Engineering Applications

As is well known in the art, once a cDNA clone encoding a xylan synthase is obtained, it may be used to obtain its corresponding genomic nucleic acids.

The nucleotide sequences which encode plant xylan synthases may be used in various constructs. For example, as probes to obtain further nucleic acids from the same or other species. Alternatively, these sequences may be used in conjunction with appropriate regulatory sequences to increase levels of the respective xylan synthase of interest in a host cell for the production of xylans or study of the enzyme in vitro or in vivo, or to decrease or increase levels of the respective xylan synthase of interest for some applications when the host cell is a plant entity, including plant cells, plant parts (including, but not limited to, seeds, stems, cambial tissues, cuttings, and tissues), and plants.

A nucleotide sequence encoding a xylan synthase of tis invention may include genomic, cDNA or mRNA derived sequences. By "encoding" is meant that the sequence corresponds to a particular amino acid sequence either in a sense or anti-sense orientation. By "recombinant" is meant that the sequence contains a genetically engineered modification through manipulation via mutagenesis, nucleic acid modifying enzymes, or the like. A cDNA sequence may or may not encode pre-processing sequences, such as transit or signal peptide sequences. Transit or signal peptide sequences facilitate the delivery of the protein to a given organelle and are frequently cleaved from the polypeptide upon entry into the organelle, releasing the "mature" sequence. The use of the precursor DNA sequence is preferred in plant cell expression cassettes.

Furthermore, as discussed above, the complete genomic sequence of the plant xylan synthase may be obtained by the screening of a genomic library with a probe, such as a cDNA probe, and isolating those sequences which regulate expression of the gene. In this manner, the transcription and translation initiation regions, introns, and/or transcript termination regions of the plant xylan synthase may be obtained for use in a variety of nucleic acid constructs, with or without the xylan synthase structural gene. Thus, nucleotide sequences corresponding to the plant xylan synthase of this invention may also provide signal sequences useful to direct transport into an organelle such as the Golgi, 5' upstream non-coding regulatory regions (promoters) having useful tissue and timing profiles, 3' downstream non-coding regulatory region useful as transcriptional and/or translational regulatory regions, or may lend insight into other features of the gene.

Once the desired plant xylan synthase nucleotide sequence is obtained, it may be manipulated in a variety of ways. Where the sequence involves non- coding flanking regions, the flanking regions may be subjected to resection, mutagenesis, etc. Thus, point mutations (e.g. , transition, transversion), deletions, and insertions may be performed on the naturally occurring sequence. In addition, all or part of the sequence may be synthesized. In the structural gene, one or more codons may be modified to provide for a modified amino acid sequence, or one or more codon mutations may be introduced to provide for a convenient restriction site, or other purposes involved with construction or expression. The structural gene may be further modified by employing synthetic adapters, linkers to introduce one or more convenient restriction sites, or the like.

The nucleotide or amino acid sequences encoding a plant xylan synthase of this invention may be combined with other non-native, or

"heterologous", sequences in a variety of ways. By "heterologous" sequences is meant any sequence which is not naturally found joined to the plant xylan synthase, including, for example, combination of nucleotide sequences from the same plant which are not naturally found joined together. The DNA sequence encoding a plant xylan synthase of this invention may be employed in conjunction with all or part of the gene sequences normally associated with the xylan synthase. In its component parts, a DNA sequence encoding xylan synthase is combined in a DNA. construct having, in the 5' to 3' direction of transcription, a transcription initiation control region capable of promoting transcription and translation in a host cell, the DNA sequence encoding plant xylan synthase, and transcription and translation termination regions.

Potential host cells include both prokaryotic and eukaryotic cells. A host cell may be unicellular or found in a multicellular differentiated or undifferentiated organism depending upon the intended use. Cells of this invention may be distinguished by having a plant xylan synthase foreign to the wild-type cell present therein, for example, by having a recombinant nucleic acid construct encoding a plant xylan synthase therein. Depending upon the host, the regulatory regions will vary, including regions from viral, plasmid or chromosomal genes, or the like. For expression in prokaryotic or eukaryotic microorganisms, particularly unicellular hosts, a wide variety of constitutive or regulatable promoters may be employed. Expression in a microorganism can provide a ready source of the plant enzyme. Among transcriptional initiation regions which have been described are regions from bacterial and yeast hosts, such as E. coli, B. subtilis, Saccharomyces cerevisiae, including promoters such as lacUV5 or a derivative such as trc; bacteriophage T3, T7 or SP6 promoters; trpE; ADC1 , Gall, GallO, PHO5 or the like (see generally, Goeddell, 1990). For the most part, the constructs will involve regulatory regions functional in plants which provide for modified production of plant xylan synthase with resulting modification of the cell wall polysaccharide composition. To obtain increased expression, the open reading frame, coding for the plant xylan synthase or a functional fragment thereof will be joined at its 5' end to a transcription initiation regulatory region such as the wild-type sequence naturally found 5' upstream to the xylan synthase structural gene. For decreased expression via cosuppression or gene silencing, similar constructs to those that produce overexpression are used. Plants which exhibit cosuppression are identified by screening the transgenic plants produced. For decreased expression of xylan synthase via antisense, constructs are used in which part or all of the gene is placed under transcriptional control of a promoter in such an orientation so that the resulting transcript is complementary to the normal sense transcript. Numerous transcription initiation regions are available which provide for a wide variety of constitutive or regulatable (e.g., inducible) transcription of the structural gene. Among transcriptional initiation regions used for plants are such regions associated with the structural genes such as for nopaline and mannopine synthases, or with napin, the cauliflower mosaic virus 35S promoters, or the like. The transcription/translation initiation regions corresponding to such structural genes are found immediately 5' upstream to the respective start codons. In embodiments wherein the expression of the xylan synthase protein is desired in a plant host, the use of all or part of the complete xylan synthase gene is desired, namely all or part of the 5' upstream non-coding regions (promoter) together with the structural gene sequence and 3' downstream non-coding regions may be employed. If a different promoter is desired, such as a promoter native to the plant host of interest or a modified promoter, i.e., having transcription initiation regions derived from one gene source and translation initiation regions derived from a different gene source, including the sequence encoding the xylan synthase of interest, or enhanced promoters, such as double 35S CaMV promoters, the sequences may be joined together using standard techniques.

Regulatory transcription termination regions may be provided in DNA constructs of this invention as well. Transcription termination regions may be provided by the DNA sequence encoding the plant xylan synthase or a convenient transcription termination region derived from a different gene source, for example, the transcription termination region which is naturally associated with the transcription initiation region. Where the transcription termination region is from a different gene source, it will contain at least about 0.5 kb, preferably about 1 to about 3 kb of sequence 3' to the structural gene from which the termination region is derived.

Plant expression or transcription constructs having a plant xylan synthase as the DNA sequence of interest for increased or decreased expression thereof may be employed with a wide variety of plant life, particularly, plant life involved in the production of cellulose or other natural fibers or plants used for forage by ruminant animals. Most especially preferred are various trees used for pulp and paper production, or lumber. Also preferred are non-wood species such as those listed in Table 3 that are used for production of fiber, and forage grasses or other plants such as silage varieties of maize that are used to feed ruminant animals.

Depending on the method for introducing the recombinant constructs into the host cell, other DNA sequences may be required. Importantly, this invention is applicable to any transformable plant species and will be readily applicable to new and/or improved transformation and regulation techniques. The method of transformation is not critical to this invention: various methods of plant transformation are currently available. As newer methods are available to transform plants, they may be directly applied. For example, many plant species naturally susceptible to Agrobacterium infection may be successfully transformed via tripartite or binary vector methods of Agrobacterium mediated transformation. In addition, techniques of microinjection, DNA particle bombardment, electroporation have been developed which allow for the transformation of various plant species.

In developing the DNA construct, the various components of the construct or fragments thereof will normally be inserted into a convenient cloning vector which is capable of replication in a bacterial host, e.g., E. coli. Numerous vectors exist that have been described in the literature. After each cloning, the plasmid may be isolated and subjected to further manipulation, such as restriction, insertion of new fragments, ligation, deletion, insertion, resection, etc., so as 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.

Normally, included with the DNA construct will be a structural gene having the necessary regulatory regions for expression in a host and providing for selection of transformant cells. The gene may provide for resistance to a cytotoxic agent, e.g. , antibiotic, heavy metal, toxin, etc., complementation providing prototropy to an auxotrophic host, viral immunity, or the like. Depending upon the number of different host species into which the expression construct or components thereof are introduced, one or more markers may be employed, where different conditions for selection are used for the different hosts.

It is noted that the degeneracy of the DNA code provides that codon substitutions are permissible in the nucleotide sequence contained in nucleic acids of this invention without any corresponding modification of the amino acid sequence.

As mentioned above, the manner in which nucleic acids are introduced into the plant host is not critical to this invention. Any method which provides for efficient transformation may be employed. Various methods for plant cell transformation include the use of Ti- or Ri-plasmids, microinjection, electroporation, infiltration, imbibition, particle bombardment, liposome fusion, nucleic acid bombardment, or the like. In many instances, it will be desirable to have the recombinant construct bordered on one or both sides of the T-DNA, particularly having the left and right borders, more particularly the right border. This is particularly useful when the construct uses A. tumefaciens or A. rhizogenes as a mode for transformation, although the T- DNA borders may find use with other modes of transformation.

Where Agrobacterium is used for plant cell transformation, a vector may be used which may be introduced into the Agrobacterium host for homologous recombination with T-DNA or the Ti- or Ri-plasmid present in the Agrobacterium host. The Ti- or Ri-plasmid containing the T-DNA for recombination may be armed (capable of causing gall formation) or disarmed (incapable of causing gall), the latter being permissible, so long as the vir genes are present in the transformed Agrobacterium host. The armed plasmid can give a mixture of normal plant cells and gall.

In some instances where Agrobacterium is used as the vehicle for transforming plant cells, the expression construct bordered by the T-DNA border(s) will be inserted into a broad host spectrum vector, there being broad host spectrum vectors described in the literature. Commonly used is pRK2 or derivatives thereof. See, for example, Ditta et al. (1980). Included with the expression construct and the T-DNA will be one or more markers, which allow for selection of transformed Agrobacterium and transformed plant cells. A number of markers have been developed for use with plant cells, such as resistance to kanamycin, BASTA, chlorsulfuron, hygromycin, or the like. The particular marker employed is not essential to this invention, one or another marker being preferred depending on the particular host and the manner of construction.

For transformation of plant cells using Agrobacterium, explants may be combined and incubated with the transformed Agrobacterium for sufficient time for transformation, the bacteria killed, and the plant cells cultured in an appropriate selective medium. Once callus forms, shoot formation can be encouraged by employing the appropriate plant hormones in accordance with known methods and the shoots transferred to rooting medium for regeneration of plants. The plants may then be grown to seed, and the seed used to establish repetitive generations.

Expression of xylan synthase may be monitored by gene or protein fusions with a polypeptide whose enzymatic activity is easily assayed such as, for example, alkaline phosphatase, beta galactosidase, chloramphenicol acetyltransferase, luciferase, green fluorescent protein, beta glucoronidase, or derivatives thereof.

Polypeptides with xylan synthase activity may be isolated using the identified nucleic acid sequence. The polypeptide may be isolated from natural sources (i.e. , plants) or from host cells expressing recombinant xylan synthase sequences. Polypeptides may be purified using centrifugation, precipitation, specific binding, electrophoresis, and/or chromatography. Separation may be facilitated using enzyme substrates, antibody and/or attachment of a fusion peptide (e.g. , avidin, glutathione S-transferase, poly-His, maltose binding protein, myc 9E10-epitope, protein A7G, SV40 T antigen).

In addition to direct uses of the genes for xylan synthases of this invention to increase or decrease xylan content, these genes may also be used to identify mutations in which the activity of a xylan synthase has been reduced or eliminated by a change in the nucleotide sequence of the gene. One way in which this may be accomplished is to screen populations of plants for major changes in the structure of xylan synthase genes caused by insertions or deletions. Alternatively, populations of mutagenized plants can be screened by PCR-based methods for single nucleotide changes that alter the function of the xylan synthases. This can be done by designing PCR primers based on the sequence of the xylan synthase of interest so that the PCR reaction produces fragments of less than about 300 nucleotides in length. The PCR products obtained by performing the PCR reaction on large numbers of individuals from a mutagenized population are then electrophoretically resolved on SSCP gels that permit the identification of mobility variants that are due to as few as one nucleotide changes.

The present invention now being generally described, it will be more readily understood by reference to the following examples which are included for purposes of illustration only and are not intended to limit this invention.

Although Arabidopsis is not a commercially important plant species, it is widely accepted by plant biologists as a model for higher plants. Therefore, the inclusion of examples based on Arabidopsis is intended to demonstrate the general utility of the present invention described here to the modification of cell wall polysaccharide composition in higher plants.

In the examples which follow, all temperatures are given in degrees celsius (°C), weights are given in grams (g), milligram (mg) or micrograms (μg), concentrations are given as molar (M), millimolar (mM) or micromolar (μM) and all volumes are given in liters (1), milliliters (ml) or microliters (μl), unless otherwise indicated. Where solutions contain a certain percentage of a compound the value is expressed as % weight/ volume. We distinguish between a gene and the protein product of that gene by capitalizing the gene name (e.g., CSL4) but only capitalizing the first letter of the name of the corresponding gene product (e.g. , Csl4).

EXAMPLE 1

ISOLATION OF ARABIDOPSIS GLYCAN SYNTHASE GENES Overview

An Arabidopsis EST clone, named 160C16T7, with deduced amino acid sequence similarity to bacterial cellulose synthases was identified by searching the public dbEST database of partially sequenced cDNA clones. This clone was used to search for other Arabidopsis clones with significant sequence similarity. Many clones were obtained and observed to form a family of seven genes. The CSL gene family is now the second family of polysaccharide synthase homologs described in higher plants. The CELA genes of cotton define the first gene family and are highly expressed during cotton fiber development and are likely to encode cellulose synthases. The CSL clones share low amino acid sequence identity to the CELA genes of cotton. Based on these observations, we envision that the CSL gene family contains genes which perform functions related to, but different from, cellulose biosynthesis. Since the catalytic event in cellulose biosynthesis is the formation of a 0-1 ,4 glucose linkage, we envision that the Csl proteins catalyze the formation of 0-1 ,4 linked cell wall polymers other than cellulose. Such polymers could include mannans, galactans, xyloglucans and/or xylans. The identity of several members of the

CSL gene family as xylan synthases was demonstrated by isolating a transposon- induced mutation in one of these genes, CSL . The mutant was isolated and found to be deficient in xylan content indicating that this gene does not encode cellulose synthase but rather, encodes xylan synthase. Transgenic plants that express sense RNA for CSL4 also have reduced levels of xylan, confirming the function of the gene. Additionally a gene from Brassica rapa has also been identified. Based on its similarity to the Arabidopsis xylan synthase Csl4, we conclude that it encodes a Brassica xylan synthase.

The various steps involved in this process are described in detail below. Unless otherwise indicated, routine methods for manipulating nucleic acids, bacteria, and phage were as described by Sambrook et al. (1989).

Identification of Arabidopsis Glvcan Synthases

An Arabidopsis EST, 160C16T7, with weak similarity to two bacterial cellulose synthase genes was observed within the public EST database collection (dbEST) housed at NCBI. The publicly available sequence of the 5' end of

160C16T7 is presented in SEQ ID NO: l . When this sequence was compared to a non-redundant protein database using the NCBI BLASTX search algorithm, 160C16T7 was observed to share low similarity with two bacterial cellulose synthases from Agrobacterium tumefaciens and Acetobacter xylinum (Figure 2). To determine if 160C16T7 was indeed a plant homolog of bacterial cellulose synthases, the clone was obtained from the Arabidopsis Stock center at Ohio State University (ABRC) and sequenced by conventional methods on an Applied Biosystems 310 automated sequencer according to the manufacturers instructions. The complete nucleotide sequence of the cDNA in the pl60C16T7 clone is shown in SEQ ID NO:2. The deduced amino acid sequence of the longest open reading frame of 160C16T7 is shown in SEQ ID NO: 3. An alignment of the predicted protein encoded by 160C16T7 to Agrobacterium cellulose synthase (celA) is presented in Figure 3. The alignment was generated using BESTFIT in the GCG software package, version 8.0. The two proteins share approximately 25% identity and 53% similarity over the lengths of their overlap. Based on this similarity, the gene coding for the 160C16T7 cDNA is considered a cellulose synthase homolog and designated Csll, for cellulose synthase like 1.

We sought to determine if other genes in the Arabidopsis genome encode polypeptides which have significant sequence similarity to Csll. Such genes could be part of a CSL gene family and have functions similar to Csll . The predicted protein sequence of Csll was used to search dbEST using the TBLASTN algorithm. This algorithm compares the six frame amino acid translations of nucleic acid sequences to a protein query to identify ESTs that encode proteins similar to the query sequence. Using TBLASTN, five new Arabidopsis ESTs encoding proteins similar to Csll were identified. Table 5 displays the TBLASTN scores, and their estimated significance, for each of these new ESTs when compared to the longest open reading frame encoded by the 160C16T7 cDNA. Each of these ESTs was obtained from the Arabidopsis stock center at Ohio State University and sequenced using conventional methods. The complete nucleotide sequences of the cDNAs FAFL51 , 92K11T7, 178H9T7, 133A23T7, and 210A22T7 are presented in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8 respectively. The longest open reading frames endcoded by each of these cDNAs are listed in SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, and SEQ ID NO: 13. The longest open reading frame encoded by the 210A22T7 cDNA was utilized in a TBLASTN search of the EST database housed at NCBI. This search re-identified all of the previously identified ESTs as well as a new family member, 119C22T7. Table 6 shows the TBLASTN scores, and their estimated significance, of these ESTs compared to the longest open reading frame encoded by the 210A22T7 cDNA. The newly identified clone, 119C22T7 was obtained from the ABRC stock center and sequenced using conventional methods. The nucleotide sequence of the 119C22T7 cDNA is listed in SEQ ID NO: 14, the longest open reading frame encoded by 119C22T7 is listed in SEQ ID NO: 15. This search identified all of the previously identified ESTs which encode polypeptides with significant similarity to Csll and additionally identified a new EST, 119C22T7.

To confirm that the seven cDNAs identified form a family of related sequences the GCG program PILEUP was utilized to perform a multiple sequence alignment of the predicted open reading frame of each of the seven cDNAs identified. This comparison is shown in Figure 4. Several residues are conserved among all the family members indicating that they are a gene family of homologous sequences. Based on this observation, each of the cDNAs was defined as a CSL gene family member. Table 7 lists the Csl name designation for each cDNA identified. It should be noted that the sequences form two clusters: one cluster of closely related sequences includes Csl4, Csl5 and Csl6, and the other cluster contains Csll, Csl2, Csl3 and Csl7.

Two cotton genes, CELA1 and CELA2 have been identified which share sequence similarity to bacterial cellulose synthases. The mRNAs encoded by these genes are expressed abundantly during cotton fiber development and probably encode cellulose synthases. Homologs of the cotton CELA genes have been found in many plant species including Arabidopsis where eight Arabidopsis ESTs homologous to CELA1 have been identified (Pear et al.. 1996) These ESTs were not identified as CSL gene family members in our previously described TBLASTN analyses, suggesting that the Csl polypeptides are not closely related to the products of the cotton and Arabidopsis CELA genes.

A full length cDNA clone, designated pCSL4, was isolated for CSL4 so that a complete comparison could be made between the cotton CelAl and Csl4 predicted proteins. Sequences derived from the 5' end of the insert in clone 210A22T7 were used to probe an Arabidopsis cDNA library (CD4-15) constructed in the cloning vector λZAPII (Kieber et al. , 1993). This cDNA library contains size selected inserts (2-3 kb ) prepared from mRNA isolated from etiolated seedlings as described elsewhere (Kieber et al., 1993) and was obtained from the ABRC. Several clones containing full-length or near full length cDNA sequences corresponding to CSL4 were isolated by probing nylon filter plaque- lifts of the library with digoxigenin-labelled insert from 210A22T7. To obtain labelled probe, an approximately 800 bp digoxigenin-labelled 210A22T7 fragment was synthesized by PCR using primers PI (SEQ ID NO: 16) and P2 (SEQ ID NO: 17) with 10 ng 210A22T7 cDNA as template. The digoxigenin labelling was done with a GENIUS kit supplied by Boehringer-Mannheim according to the manufacturers instructions. The reaction mix was standard in all respects except that it contained 100 μm each of dATP, dCTP, dGTP, 90 μM dTTP and 10 μM digoxigenin-dUTP. The labelling efficiency was estimated by a western blot of dilutions of the PCR product utilizing an anti- digoxigenin monoclonal antibody. A dilution series of a commercially prepared standard was included on the western blot to calibrate measurements. Filters containing CD4-15 phage were prehybridized for 2 hours at 65°C in hybridization solution and hybridized overnight in hybridization solution plus the 210A22T7 probe at a concentration of 25 ng per ml hybridization solution at 65 °C (GENIUS kit). The filters were sequentially washed at room temperature in solutions containing 2 X SSC, 1 X SSC, 0.5 X SSC in addition to 0.1 % SDS. Probe that had hybridized to phage was visualized by western blotting the washed filters with an anti-digoxigenin monoclonal antibody (Boehringer- Mannheim).

Plaques were picked from 96 positive phage into phage dilution buffer. To identify clones containing the full length inserts, an oligonucleotide primer based on the sequence of Csl4 (oligonucleotide P3, SEQ ΪD NO: 18) and a primer from the region of the vector flanking the cloning site (oligonucleotide P4, SEQ ID NO: 19) was used to prime PCR reactions using the 96 clones as templates. The clone producing the longest PCR product was retained as the best candidate for a full length clone and named XCSL4.

A clone encoding a full length cDNA was identified, and purified by two rounds of re-screening using the same methods described above for the first round of cDNA screening. The cDNA contained in this phage was excised into plasmid form by infecting the phage into appropriate bacterial strains, as recommended by the manufacturer (Stratagene). The excised plasmid was purified and designated pCSL4 . The sequence of this clone was determined on both strands using the dideoxy chain termination method using an automated sequencer. The nucleotide sequence of this cDNA clone is shown in Figure 5 (SEQ ID NO:20). There are stop codons in frame and upstream of the imtiating methionine indicating that the pCSL4 cDNA encodes a full length cDNA. The deduced amino acid sequence of the longest open reading frame in this clone is also shown in Figure 5 (SEQ ID NO:21). The open reading frame encodes a polypeptide of 673 amino acids corresponding to a molecular weight of 77,511 daltons.

To confirm that the CSL gene family is separate from the CELA gene family, the predicted Csl4 protein sequence was compared to the cotton CelAl in a pairwise alignment using the GCG program BESTFIT. The results of this alignment are shown in Figure 6. It can be deduced from this alignment that Csl4 and CelAl are not highly similar sequences. They share approximately 17% identity, making them less similar to one another than either are to the bacterial cellulose synthase genes they are homologs of. The CSL gene family thus forms a separate family of plant sequences similar to bacterial cellulose synthases. Because the cotton genes CELAl and CELA2 function to synthesize cellulose, it is likely that the Csl polypetides do not function in the biosynthesis of cellulose. Furthermore, since the catalytic event in cellulose Biosynthesis is the formation of a 0-1,4 glucose linkage, it is likely that the members of the Csl protein family catalyze the formation of 0-1,4 linked cell wall polymers other than cellulose. Such polymers could include mannans, galactans, xyloglucans, glucomannans, and/or xylans.

We envision that it will be possible to identify the functions of all of these disclosed CSL genes. In particular, these genes can be used to produce transgenic plants in which the expression of the genes has been reduced by antisense suppression or by cosuppression. In addition, oligonucleotides based on the sequences of these genes can be used to screen collections of plants with random DNA insertions for mutants caused by insertions. The functions of the genes can then be determined by measuring the composition of the cell wall polysaccharides as described in the following examples.

EXAMPLE 2

DETERMINATION THAT CSL4 IS A XYLAN SYNTHASE

A generally useful method of determining the function of a plant gene is to identify a mutation that inactivates the gene product or prevents expression of a functional gene product. Transposons such as the Ac element of Zea mays have been very useful in this respect. We used this method to demonstrate the function of the CSL4 gene product. A collection of transgenic plants in which an introduced Ac element had transposed from the original site of insertion was made available for our use by Francois Belzile (Laval University). In order to identify a plant line containing an Ac insertion in the CSL4 gene, oligonucleotide primers based on the sequence of the CSL4 gene and the Ac element were used to test the various lines. Oligonucleotide P5 (SEQ ID NO:22) is derived from the sequence of the maize Ac element. Oligonucleotide P6 (SEQ ID NO:23) is derived from the CSL4 gene. The conceptual basis for this test is that if DNA is prepared from a plant in which the Ac element is near or within the CSL4 gene, a PCR reaction using this DNA and primed with oligonucleotides P5 and P6 will produce a PCR product that contains at least some sequence of the CSL4 gene. In contrast, if the Ac element is farther than about 5 kb from the CSL4 gene, no PCR product containing sequences from the CSL4 gene will be obtained. Furthermore, in practice, this method can be performed on pooled DNA from many plants so that it is possible to test whether any of the plants in the collection contain an Ac element inserted in the genome near the CSL4 gene.

This method was used to identify a mutant line of Arabidopsis designated Ac39-2. To confirm that the PCR product was derived from the amplification of an AC insertion into the CSL4 gene and not a PCR artifact, the PCR product was sequenced using conventional methods. The PCR product was observed to contain AC sequence, CSL4 sequence and intervening intron sequence, confirming that the PCR product truly reflected the presence of an AC insertion into the CSL4 gene.

The phenotypic consequences of the AC insertion in line Ac39-2 were evaluated by comparing the amounts of the various sugars in the cell wall polysaccharides of stems from the mutant and the wild type using gas chromatography (GC).

Plants were grown at approximately 23 °C under natural light in a glasshouse until ten days after bolting. Twenty milligrams of stem material was taken from each plant and extracted twice with 2 mL of 70% ethanol for 1 hour at 70°C yielding a cell wall residue.

Hydrolysis of cell wall residues to yield cell wall monosaccharides and quantitation of the cell wall monosaccharides via gas-liquid chromatography of alditol acetates was carried out essentially as described previously

(Reiter et al. , 1993). For calibration purposes, a mixture of 50 μg each of L-rhamnose, L-arabinose, D-xylose, D-mannose, D-galactose, and D-glucose was autoclaved in 250 μL of 1 M H₂SO₄. In brief, to determine the monosaccharide composition of cell wall matrix components, cell wall residues were hydrolyzed for one hour at 121 °C in 250 μL each of 1 M H₂SO₄.

Cellulose microfibrils are expected to remain essentially intact under these conditions (Fry, 1988) and, thus, are not hydrolyzed. The supernatants were removed and derivatized for monosaccharide quantitation by GC. The sugars in each of the hydrolysates were reduced to alditols by neutralization with 100 μl of 9M NH₃ followed by reaction with 1 ml of 2% NaBH₄ in DMSO. The reduction was carried out for 90 min at 40°C. 250 μl of acetic acid was added to each reaction to destroy remaining borohydride. The alditols were next acetylated by the addition of 4 ml acetic anhydride and 250 μl methylimmidazole to each reaction. Methylimidizole acts as an acetylation catalyst. The remaining acetic anhydride in each reaction was destroyed by the addition of 8 ml H₂O to each reaction mix. The alditol acetates were extracted from each reaction mix by the addition of 1.5 ml CH₂C1₂. The organic phase was collected and transferred to a fresh tube. The CH₂C1₂ was evaporated off at 55 °C in a water bath. Hydrophilic contaminants were extracted from the remaining residue by the addition of 1 ml H₂O. The organic residue from each reaction was extracted into 250 ul CH₂C1₂, transferred to GC vials and analyzed by GC using flame ionization detector. The injector and detector were set at 300°C. The column was a Supelco SP-2330 30 meter glass capillary column (0.75 mm inner diameter, 0.2 μM film thickness). The temperamre profile was 160°C for 2 min, increased to 200°C at 20°C/min, hold at 200°C for 5 min, increased to 245 °C at 20°C/min, hold for 8 min at 245 °C, and decreased back to 160°C at 25°C/min.

The monosaccharide contents of polysaccharides from the stem of wild type and mutant A. thaliana plants are shown in Table 8. Comparison of the relative amounts of xylose in the wild type and mutant stems indicates that the mutant has a greatly reduced amount of xylose. Since xylans are the major xylose-containing constituent of stems of dicotyledenous plants such as A. thaliana, we conclude that the mutant has a reduction in xylan content. This in turn indicates, in conjunction with the other evidence indicating that Csl4 is a glycan synthase, that the CSL4 gene encodes xylan synthase.

Additional evidence indicating that Csl4 is a xylan synthase will be obtained by performing a methylation analysis of polysaccharides from stems of the mutant and the wild type. The concept of this test is that in xylans, the xylose residues are linked through 1,4-linkages. Thus, when the free hydroxyls of xylans are chemically methylated in vitro, followed by hydrolysis of the methylated polysaccharide to free sugars, a large proportion of the partially methylated xylose residues will have free hydroxyls on carbons 1 and 4 (since these were not susceptible to methylation before hydrolysis of the polymer). We will perform this analysis on polysaccharides from stems of the mutant and wild type as follows: bulk cell wall material will be isolated from the stems of wild type and mutant plants. Cell wall material will be ethanol extracted and subsequently lyophilized. 1.5 g of cell wall material will be fractionated into pectic and hemicellulosic fractions, and each will be analyzed by the methods described in the following paragraph. To fractionate pectic materials from hemicellulosic materials, 1.5 g of cell wall material will be extracted sequentially with EDTA, ammonium oxalate, and 0.1 M KOH. The supernatants from each of these extractions will be combined and designated the pectic fraction. The remaining insoluble cell wall material will be extracted with 4M KOH. This treatment extracts hemicellulosic material from cellulose. The material solubilized by this process will be designated the hemicellulosic fraction. Hemicellulosic and pectic fractioas will be neutralized and dialyzed overnight against water at 4°C. Approximately 3 mg each of the pectic and hemicellulosic fractions will be suspended in 1 ml anhydrous DMSO in 15 ml corex tubes capped by serum sleeve stoppers. The tubes will be evacuated of oxygen and sonicated at 50°C to disperse the polysaccharides. The free sugar hydroxyl groups will be converted to lithium salts by the addition of 250 μl of 2.5 M n-butyl lithium (dissolved in hexane) to each tube. This reaction will be allowed to proceed for four hours under continuous Ar₂ flow. The sugar lithium salts will be methylated by the addition of 500 μl CH₃I to each tube. This reaction will be allowed to proceed overnight. The organic layers from each reaction mix will be transferred to fresh bes and evaporated to dryness under a stream of N₂. The methylated polysaccharides will be hydrolyzed, acetylated and prepared for GC as previously described. Linkages will be deduced by GC- MS analysis of the partially methylated and acetylated alditol acetates. We expect to see a reduction in the content of 1 ,4 linked xylose residues in the hemicellulosic fraction of Ac39-2 mutant plants.

EXAMPLE 3 USE OF THE CSL4 GENE TO PRODUCE SENSE-SUPPRESSION TRANSGENIC PLANTS WITH REDUCED XYLAN CONTENT

The cloning of the CSL4 gene also provides a tool to decrease the levels of xylans via the mechanism of cosuppression. The molecular mechanism of cosuppression occurs when plants are transformed with a gene that is identical or highly homologous to an allele found in the plants genome (Matzke and Matzke, 1995). There are several examples where expression of a chimeric gene in plants can result in a reduction of the gene product from both the chimeric gene and the endogenous gene(s). Therefore the CSL gene product of A. thaliana may be reduced by transformation of A. thaliana with all or a portion of the CSL4 cDNA which has been isolated. The resulting plant has reduced xylan synthase activity in tissues where the chimeric gene is expressed. The phenotype of reducing the xylan synthase activity is a reduction in the levels of xylans. The mechanism of cosuppression could be applied to any plant species from which the CSL4 genes, or members of the CSL4 gene family, are cloned and the plant species is transformed with one or more members of the CSL4 gene family, or a part of the gene which is adequate to cause the effect, in a sense orientation. There are a wide variety of plant promoter sequences which may be used to cause tissue-specific expression of cloned genes in transgenic plants. Thus, although we describe the use of the cauliflower mosaic virus promoter in the example described here, other promoters which lead to tissue-specific expression may also be employed for the production of modified xylan composition. Such modifications of the present invention described here will be obvious to one skilled in the art.

In order to decrease the xylan synthase activity by genetic engineering methodology, a fragment of the CSL4 cDNA was cloned into the plant expression vector pBIMC in a sense orientation. pBIMC is a derivative of the plant expression vector pBI121, in which the 0-glucuronidase gene has been replaced by a multicloning-site polylinker. pBIMC was constructed by Deane Falcone (University of Kentucky). An approximately 700 bp Xhol + Smal restriction fragment of p210A22T7 was used to construct the cosuppression expression vector. This fragment contains 667 bp of CSL4 cDNA sequence and approximately 30 bp of vector polylinker derived from the pZLl vector that the 210A22T7 cDNA was cloned into (BRL-Gibco, Gaithersburg, MD). The Xhol to Smal fragment extends from 520 nucleotides downstream of the initiating methionine codon of the cDNA to an Xhol restriction site that is located 1154 nucleotides downstream of the initiating methionine; 1384 nucleotides of the coding region are excluded from this fragment. An expression cassette in which the a sense-oriented fragment of 210A22T7 was constimtively expressed in most or all tissues of the plant was constructed by insertion of the Smal - Xhol fragment of 210A22T7 in an sense orientation behind the cauliflower mosaic virus promoter (35S promoter) in plasmid pBIMC (provided by Deanne Falconne). The Smal - Xhol fragment from 210A22T7 was prepared by digestion with Smal and Xhol for 2 hrs at 37 °C. The approximately 700bp Smal - Xhol 210A22T7 restriction fragment was separated from vector DNA in an agarose gel. The approximately 700 bp Smal - Xhol fragment was excised from the gel using a sterile scalpel blade and transferred to an eppendorf tube. The fragment was purified from the agarose matrix using an agarose gel purification system according to manufacturers instructions (Qiagen). The vector pBIMC was digested with Xhol and Smal for 2 hrs at 37 °C and gel purified in low melting point agarose. Fifty to 200 ng of the purified fragment from 210A22T7 and 200 ng of digested pBIMC was ligated under conditions suggested by the manufacturer of the ligase (Promega, Madison, WI) for one hour at room temperamre followed by transformation into the E. coli strain DH5c_. Resulting transformant colonies were used for plasmid preparation and restriction digestion analysis. Two diagnostic digests were performed to confirm that the clone was constructed properly; one with Xhol and Smal and a second using Xhol and Hindll. One clone was designated as correct and named pSCl. A map of this clone is presented as Figure 7.

In preparation for transforming A. thaliana cells, pSCl was transformed into Agrobacterium tumefaciens strain GV3101 by electroporation. Strain GV3101 (Koncz and Schell, 1986) contains a disarmed Ti plasmid. Cells for electroporation were prepared as follows. GV3101 is grown in LB medium with reduced NaCI (5 g/1). A 250 ml culture is grown to OD^ = 0.6, then centrifuged at 4000 rpm (Sorvall GS-A rotor) for 15 min. The supernatant is aspirated immediately from the loose pellet, which is gently resuspended in 500 ml ice-cold water. The cells were centrifuged as before, resuspended in 30 ml ice-cold water, transferred to a 30 ml tube, and centrifuged at 5000 rpm (Sorvall SS-34 rotor) for 5 min. This was repeated three times, resuspending the cells consecutively in 30 ml ice-cold water, 30 ml ice-cold 10% glycerol. and finally in 0.75 ml ice-cold 10% glycerol. These cells were aliquoted, frozen in liquid nitrogen, and stored at -80°C. Electroporation employed a Biorad Gene Pulser instrument using cold 2 mm gap cuvettes containing 40 μl cells and 1 μl of DNA in water, at a voltage of 2.5 KV, and 200 ohms resistance. The electroporated cells were diluted with 1 ml SOC medium (Sambrook et al., 1989, page A2) and incubated at 28°C for 2-4 h, before plating on LB medium containing kanamycin (50 mg/1).

Production of Transgenic Plants

A variety of methods have been developed to insert a DNA sequence of interest into the genome of a plant host to obtain the transcription and translation of the sequence to effect phenotypic changes. The following methods represent only one of many equivalent means of producing transgenic plants and causing expression of the xylan synthase gene of the present invention. Arabidopsis plants were transformed, by Agrobacterium-mediated transformation, with the xylan synthase gene carried on binary Ti plasmid pSCl.

Inoculums of Agrobacterium tumefaciens strain GV3101 containing binary Ti plasmid pSC 1 were plated on L-broth plates containing 50 μg/ml kanamycin and incubated for 2 days at 30°C. Single colonies were used to inoculate large liquid cultures (L-broth medium with 50 mg/1 rifampicin, 25 mg/1 gentamycin and 50 mg/1 kanamycin) and used for the transformation of Arabidopsis plants.

Arabidopsis plants were transformed by the in planta transformation procedure essentially as described by Bechtold et al. (1993). Cells of A. tumefaciens GV3101(pSCl) were harvested from liquid cultures by centrifugation, then resuspended in infiltration medium at OD^ = 0.8 (infiltration medium will be Murashige and Skoog macro and micronutrient medium (Sigma, St. Louis, MO) containing 10 mg/1 6-benzylaminopurine and 5% glucose). Batches of 12-15 plants were grown for 3 to 4 weeks in natural light at a mean daily temperamre of approximately 25 °C in 3.5 inch pots containing soil. The intact plants were immersed in the bacterial suspension, then transferred to a vacuum chamber, and placed under 600 mm Hg of vacuum produced by a laboratory vacuum pump until tissues appeared uniformly water-soaked (approximately 10 min). The plants were grown at 25 °C under continuous light (100 μmol m^'2 s ^l irradiation in the 400 to 700 nm range) for four weeks. The seeds obtained from all the plants in a pot were harvested as one batch. The seeds were sterilized by sequential treatment for 10 min in a mixture of household bleach (Chlorox), water, and Tween-80 (33%, 66%, 0.05%) then rinsed thoroughly with sterile water. The seeds were plated at high density (2000 to 4000 per plate) onto agar-solidified medium in 100 mm petri plates containing 1/2 X Murashige and Skoog salts medium enriched with B5 vitamins (Sigma, St. Louis MO) and containing kanamycin at 50 mg/1. After incubation for 48 h at 4°C to stimulate germination, seedlings were grown for a period of 14 days until transformants are clearly identifiable as healthy green seedlings against a background of chlorotic kanamycin-sensitive seedlings. The transformants were transferred to soil for two weeks before stem tissue was used for cell wall analysis. More than 80 transformants were obtained.

Analysis of Transgenic Plants Plants were grown at approximately 23 °C under natural light in a glasshouse until ten days after bolting. Twenty milligrams of stem material was taken from each plant and extracted twice with 2 ml of 70% ethanol for 1 hour at 70°C yielding a cell wall residue. Hydrolysis of cell wall residues to yield cell wall monosaccharides and quantitation of the cell wall monosaccharides via gas-liquid chromatography of alditol acetates was carried out essentially as described previously (Reiter et al. _, 1993). For calibration purposes, a mixture of 50 μg each of L-rhamnose, L-arabinose, D-xylose, D-mannose, D-galactose, and D-glucose was autoclaved in 250 μL of 1 M H₂SO₄. In brief, to determine the monosaccharide composition of cell wall matrix components, cell wall residues were hydrolyzed for one hour at 121 °C in 250 μl each of 1 M H₂SO₄. Cellulose microfibrils are expected to remain essentially intact under these conditions (Fry 7 1988) and thus are not hydrolyzed. The supernatants were removed and prepared for monosaccharide quantitation. The sugars in each of the hydroly sates were reduced to alditols by reaction with 1 ml of 2% NaBH4 in DMSO and 100 μl of 9M NH₃. The reduction was carried out for one hour at 40°C. 250 μl of acetic acid was added to each reaction to destroy remaining borohydride. The alditols were next acetylated by the addition of 4 ml acetic anhydride and 250 μl of methyl immidazole to each reaction. Methylimidizole acts as an acetyation catalyst. The remaining acetic anhydride in each reaction was destroyed by the addition of 8 ml H₂O to each reaction mix. The alditol acetates were extracted from each reaction mix by the addition of 1.5 ml CH₂C1₂. The organic^'phase was collected and transferred to a fresh mbe. The CH₂C1₂ was evaporated off at 55 °C in a water bath. Hydrophilic contaminants were extracted from the remaining residue by the addition of 1 ml H₂O. The organic residue from each reaction was extracted into 250 ul CH₂C1₂, transferred to GC vials, and analyzed by GC using flame ionization detection. The injector and detector were set at 300 °C. The column was a Supelco SP-2330 30 meter glass capillary column

(0.75 mm inner diameter, 0.2 μM film thickness). The temperature profile was 160°C for 2 min, increased to 200°C at 20°C/min, hold at 200°C for 5 min, increased to 245 °C at 20°C/min, hold for 8 min at 245 °C, and decreased back to 160°C at 25°C/min. The results of this analysis are shown in Table 9. It can be seen that in lines Cl and C4, the amount of xylose was strongly reduced by the DNA construct in these lines. Measurement of the amount of mRNA for the CSL4 gene in these lines may reveal that the amount of mRNA has been strongly decreased due to cosuppression.

EXAMPLE 5

CHARACTERIZATION OF A GENOMIC CLONE OF THE XYLAN

SYNTHASE

To isolate a genomic clone for the CSL4 gene, an Arabidopsis genomic library made from ecotype Columbia in the vector λEMBL4 was screened. Approximately 60,000 lambda phage, immobilized on nylon filters, were screened utilizing the digoxigenin-labelled CSL4 probe described previously. Several clones carrying genomic sequences corresponding to the A. thaliana xylan synthase have been isolated. DNA will be prepared from these positive plaques and the regions of DNA that contain the coding sequence of the xylan synthase gene will be identified by probing Southern blots containing restriction enzyme digests of the phage DNA with the insert from pCSL4. Fragments that hybridize to the insert in pCSL4 will be subcloned into a plasmid vector such as pBluescript and the nucleotide sequence determined by the chain termination method as described above. In addition, approximately 2000 bp of nucleotide sequence immediately upstream of the coding sequence will be determined in order to facilitate subsequent investigation of the properties of the promoter that normally controls transcription of the CSL4 gene in Arabidopsis. The identity of the gene as the genomic clone corresponding to the insert in pCSL4 will be evident from the sequence identity of regions of the genomic clone to the cDNA sequence except where the genomic clone is interrupted by introns. EXAMPLE 6

ANTI-SENSE EXPRESSION OF CSL4 TO OBTAIN REDUCED LEVELS OF

XYLANS

The cloning of the CSL4 cDNA provides materials with which one skilled in the art could construct antisense construct vectors to specifically reduce plant xylan levels by the introduction of these vectors into plant cells. In order to reduce levels of xylan by the mechanism of anti-sense, a plant transformation construct is assembled with part or all of the CSL4 gene or cDNA in antisense orientation. The entire clone or a portion thereof is placed downstream of a promoter sequence in anti-sense orientation. Suitable promoters include any promoter that has the property that it causes adequate levels of gene expression in the tissue in which it is desired to reduce the xylan content, and the amount of transcripts produced by the promoter are high enough to cause the cosuppression effect [DO YOU MEAN COSUPPRESSION?]. For many applications, a non-specific promoter such as the CaMV 35S promoter or the ubiquitin promoter will be adequate. In some cases, it may be desirable to use the promoter of the CSL4 gene from the species that is being transformed so that the cosuppression effect is specifically directed to the cells where the CSL4 gene is normally expressed. An appropriate 3' non-translated region is placed downstream of the CSL4 gene to allow for transcription termination and for the addition of polyadenylated nucleotides to the 3' end of the RNA sequence. The region from the 3' end of the nopaline synthase gene of Agrobacterium tumefaciens is commonly used for this purpose. This expression cassette is then combined with a selectable marker gene and plant cells are transformed by one of the many available methods of plant transformation. Plants recovered are allowed to set seed and mature seed are used for the production of plants which are then analyzed as described above for modified xylan content. Plants which exhibit a desirable level of xylan are then used for the production of cell walls for whatever particular purpose is appropriate to the species in question.

EXAMPLE 7

USE OF CSL4 TO OBTAIN INCREASED LEVELS OF XYLANS The cloning of the CSL4 gene also provides a tool to increase the levels of xylans by increasing the amount of xylan synthase activity via increased levels of accumulation of the xylan synthase mRNA.

In order to increase levels of xylan by increased expression of the xylan synthase gene, a plant transformation construct is assembled containing all of the coding sequence of the CSL4 gene from Arabidopsis or another plant species, or the corresponding cDNA, in a sense orientation downstream of a promoter sequence. Suitable promoters include any promoter that has the property that it causes adequate levels of gene expression in the tissue in which it is desired to increase the xylan content. For many applications, a non-specific promoter such as the CaMV 35S promoter or the ubiquitin promoter will be adequate. In some cases it may be desirable to use the promoter of the CSL4 gene from the species that is being transformed so that the enhanced expression is specifically directed to the cells where the CSL4 gene is normally expressed. An appropriate 3' non-translated region is placed downstream of the CSL4 gene to allow for transcription termination and for the addition of polyadenylated nucleotides to the 3' end of the RNA sequence. This expression cassette is then combined with a selectable marker gene and plant cells are transformed by an Agrobacterium based method of plant transformation. Transformed plants are then analyzed as described above for modified xylan content. Plants which exhibit a desirable level of xylan are propagated by appropriate means and used for the production of cell walls for whatever particular purpose is appropriate to the species in question. As noted in a later example, Arabidopsis has at least three xylan synthase genes. Any of the Arabidopsis xylan synthase genes or their homologs from other plants would be suitable for the purpose of causing increased activity in transgenic plants. In some cases it may be preferable to use a xylan synthase gene from a different species for this purpose to minimize the possibility of cosuppression or gene silencing.

EXAMPLE 8

ISOLATION OF ADDITIONAL XYLAN SYNTHASE GENES FROM A. THALIANA Random cDNA sequencing generates a large number of sequenced clones but frequently provides no conclusive information about the function of the encoded proteins. Sequence similarity to known proteins is a facile method for identifying the putative protein function encoded in the sequenced cDNA. However, the results of similarity searches are informative only when a high degree of sequence similarity with a previously characterized protein are found. Also, cDNA sequence that do not exhibit sequence similarity to any known protein remain in the unknown function category. Thus, the results of functionally identifying the xylan synthases by sequence and by their ability to complement mutations in plant xylan synthase now provides a method for identifying the function and identity of random cDNA clones by their sequence similarity to the xylan synthases.

Our previously described TBLASTN searches of dbEST database revealed several EST clones which encoded polypeptides highly similar to Csl4. In particular, the deduced amino acid sequence of EST clones 119C22T7 (SEQ ID NO: 15) and clone 133A23T7 (SEQ ID NO: 12) are significantly similar to the xylan synthase encoded by CSL4 (Table 6). The inserts in these clones are significantly smaller than the insert in ρCSL4 indicating that these clones are not full length. However, full-length clones may be obtained by screening for additional cDNA clones from the existing cDNA library or other libraries prepared from other tissues.

The deduced partial amino acid sequences of Csl5 and Csl6 exhibited 71.8% and 58.8% sequence identity with the corresponding region of Csl4, respectively. The strong sequence similarity with Csl4 indicates that the Csl5 and Csl6 proteins are also xylan synthases. It can be seen from the alignment shown in Figure 4 that Csl4, Csl5 and Cslό also show significant sequence similarity to at least four other gene products from Arabidopsis. However, the three xylan synthases are distinguished from the sequences of Csll , Csl2, Csl3, and Csl7 by the presence of an additional stretch of sequence and several amino acid residues unique to Csl4, Csl5 and Cslό. Thus, we envision that the xylan synthases of this invention can be uniquely distinguished from cellulose synthase and other glycan synthases such as mannan synthase and xyloglucan synthase by the presence of a region of amino acid sequence that is not present in the other known glycan synthases. This region corresponds to amino acid 573 to about 617 in Csl4 (Figure 4). In the available sequence of Csl5, the region corresponds to amino acid 63 to 102 in the partial sequence presented in SEQ ID NO: 12. In Cslό, the region corresponds to amino acid 20 to 55 in the partial sequence presented in SEQ ID NO: 15. Full-length clones of the CSL5 and CSL6 genes will be obtained by standard methods, inserted into plant gene expression and transformation vectors, and transformed into the Ac39-2 mutant of Arabidopsis to confirm the identity of the xylan synthase by genetic complemention as described in the proceeding examples. We envision that any of a variety of promoters, such as the CaMv 35S promoter, will be suitable promoters to cause adequate accumulation of the xylan synthases encoded by these genes to prevent the mutant phenotype.

Thus, Arabidopsis contains several genes which are closely related to CSL4 and perform the same enzymatic function. We envision that most or all xylan-containing plants also contain multiple copies of the xylan synthase genes and that the methods used here to identify and characterize the other members of this gene family in Arabidopsis will permit the isolation and characterization of the xylan synthase gene family in these other plants also. The observation that Arabidopsis xylan synthases are only about 50% identical illustrates that xylan synthases from different plant species can have as little as 50% sequence identity and still perform the same enzymatic function.

EXAMPLE 9

IDENTIFICATION OF A XYLAN SYNTHASE FROM BRASSICA RAPA The nucleotide sequence of the xylan synthase encoded by pCSL4 was compared against all nucleotide sequences in the public databases maintained by NCBI using the BLASTN program implemented at NCBI. Similarly, the deduced amino acid sequence of the xylan synthase encoded by pCSL4 was compared against all amino acid sequences in the public databases maintained by NCBI using the BLASTP program implemented at NCBI and against the concepmal translation of all the nucleotide sequences in the public database dbEST using the program TBLASTN . This search revealed the existence of a highly homologous B. rapa EST clone designated BNAF0335E (Genbank Accession # L38040). The nucleotide sequence of BNAF03353 is presented in SEQ ID NO:24. The concepmal translation of the longest open reading frame encoded by this EST is provided in SEQ ID NO:25. A comparison of the amino acid sequence of the Arabidopsis xylan synthase Csl4 and the deduced amino acid sequence of the B. rapa EST is shown in Figure 8. The overall sequence similarity of the sequences is 85% identical and 96% similar. Because of the high degree of sequence similarity we conclude that the B. rapa EST encodes a xylan synthase.

To confirm that the Brassica gene is a xylan synthase, we will complement the Ac39-2 mutant with a full-length cDNA copy of the Brassica CSL4 gene identified by BNAF03353. The concepmal basis of this test is that if the Brassica gene is indeed a xylan synthase, it will restore the reduced xylan content of the Ac39-2 mutant to approximately the same levels observed in wildtype. To perform this genetic complementation test, a full-length copy of the Brassica CSL4 cDNA will be isolated, constructs which put the Brassica cDNA under the control of appropriate promoters will be made, and these constructs will be introduced into Ac39-2 mutant plants. To isolate a full-length CSL cDNA, the cDNA insert in the BNAF03353 clone will be labelled non- radioactively using procedures similar to those used in the identification of the Arabidopsis CSL4 cDNA. The probe generated by this process will be used to screen a Brassica rapa cDNA library, and full-length clones will be identified using methods similar to those employed for the full-length Arabidopsis CSL4 cDNA. Once obtained, the Brassica CSL4 cDNA will be cloned into a vector for plant transformation, such as the pBIMC vector utilized successfully in previously described experiments. The endogenous Arabidopsis CSL4 promoter, isolated by the methods described in preceding examples, will be fused to the Brassica CSL4 cDNA. This construct is defined as a Brassica CSL4 mini-gene. The 35S promoter of pBIMC will be removed, the Brassica CSL4 'mini-gene' will be placed into pBIMC, and introduced into the Arabidopsis Ac39-2 mutant. If the Brassica Csl4 homolog is a xylan synthase, the levels of xylan observed in transgenic plants should be near those of wild type plants, or elevated significantly above the levels of the Ac39-2 mutant. Xylan levels in mutant and transgenic plants will be determined by cell wall sugar analysis, as described in previous examples. It should be noted that while this example provides a specific method for establishing whether the Brassica Csl4 homolog is a xylan synthase, the method described is applicable to any gene from any plant species. This example therefore provides a general method for determining if a gene is a xylan synthase by the experimental construction of a mini-gene. EXAMPLE 10

ISOLATION OF XYLAN SYNTHASE GENES FROM OTHER PLANT

SPECIES

The deduced amino acid sequence information available for the three xylan synthase genes from Arabidopsis can be used to design probes and procedures that will permit the isolation of xylan synthase genes from most or all higher plant species such as those listed in Tables 1-3. A variety of methods can be used to exploit knowledge of regions of conserved amino acid sequence similarity to isolate genes encoding such conserved sequences from distantly related species. These include low stringency probing of libraries with the Arabidopsis genes or cDNAs or oligonucleotides based on the amino acid or nucleotide sequences from conserved regions, sequence gazing of nucleotide or deduced amino acid sequences in databases or obtained by partial or complete sequencing of random cDNA clones, or the production of antibodies against conserved epitopes and the use of the antibodies to screen expression libraries. However, the currently most powerful method is to use mixed oligonucleotides based on the regions of conserved amino acid sequence to prime the PCR reaction. In an early demonstration of the power of this approach, Gould et al. (1989) showed that even when one of the primers was present in a pool of oligonucleotides representing more than 100,000 different sequences, the method could produce the desired PCR product. Since the size of the PCR product expected for the xylan synthase of this invention is known when the reaction uses cDNA as a template, it is possible to avoid many spurious products. Production of the correct PCR product can be verified by cloning and sequencing the product to show that it has a sequence with a high degree of similarity to the known xylan synthases. This, in turn, can be used to probe a cDNA or genomic library to obtain the corresponding full-length sequence by the methods described in the foregoing examples. The most highly conserved peptide regions in the Arabidopsis xylan synthases were chosen as regions likely to be conserved in xylan synthases from other species. These three conserved regions are shown in Figure 4. These regions were chosen because they have areas highly conserved between the three Arabidopsis xylan synthases, with at least five identical amino acids over a six amino acid span.

Several peptide end points in each conserved area were chosen as the basis to subsequently design oligonucleotide probes for the xylan synthase genes. The peptide endpoints were chosen to be between about five and about nine amino acids in length. The peptide end points were chosen to end on the conserved (identical) amino acids, and most often to begin on conserved amino acids. The rationale is that within the larger conserved area, some amino acid portions are more highly conserved than others, that about 15 to about 27 (about 5 to about 9 amino acids) nucleotides is a good primer size for PCR, and that for PCR it is important that the 3' end of the primer matches the target, with the conserved (identical) amino acids the most likely to be present in the xylan synthases. These 3 "xylan synthase" peptide targets (Figure 4) are the basis for oligonucleotides that are designed to hybridize to the xylan synthase cDNA sequences to identify and isolate homologous xylan synthase cDNA clones. Several possible methods for designing oligonucleotides and isolating the genes encoding the target peptide regions are known. For a discussion of designing degenerate oligonucleotides, see PCR Protocols - A Guide to Methods and Applications, Eds. M.A. Innis, D.H. Gelfand, J.J. Sinsky and T.I. White, Academic Press, San Diego, 1990. The two most common screening methods using oligonucleotides are screening cDNA libraries and PCR amplification of specific cDNAs. The method for using degenerate oligonucleotides to screen a cDNA library are briefly as follows. A tissue that is actively synthesizing xylans, such as developing stems of herbaceous plants such as flax or cambial tissue of woody plants such as aspen or wattle is used as the source of mRNA for making cDNA. First strand cDNA is made from the isolated mRNA. The cDNA is used for PCR reactions. For 5' RACE, see below, a poly A tail is added to the first strand cDNA 3' end. A method that can readily evaluate a number of degenerate oligonucleotides probes is degenerate PCR (See chapters by Compton and by Lee and Caskey in PCR Protocols, cited above). In this method a degenerate set of oligonucleotides encompassing all the possible codon choices for the target peptide is synthesized; such degenerate targets (Table 10) are the basis for oligonucleotides that are designed for hybridizing to the xylan synthase cDNA sequences to identify and isolate the xylan synthase cDNA clone.

Table 10 shows three of the useful peptide targets from the four conserved regions, and the 13 degenerate oligonucleotides derived from the peptide sequences that are suitable primers for PCR. Additional probes could be designed from these sequences but the method is adequately illustrated by the examples presented here. The PCR products resulting from the use of these primer pairs on cDNA from other plant species is expected to produce products of approximately 200 to 400 bp in length, depending on the primers used and the target xylan synthases (i.e., the various xylan synthases from Arabidopsis have variable numbers of amino acids in the region that would be amplified by the primers in Table 10). DNA will be extracted from a representative aliquot of a cDNA library, or reverse transcribed mRNA will be used directly as the template for PCR reactions. PCR will be performed using the following conditions. Approximately 100 ng of DNA from the library will be added to a solution containing 25 pmol of each primer, 1.5 U Taq polymerase (Boehringer Manheim), 200 μM of dNTPs, 50 mM KCl, 10 mM TrisHCl (pH 9), 0.1 %

(v/v) Triton X-100, 1.5 mM MgCl₂, 3% (v/v) formamide, to a final volume of 50 μl. Amplifications conditions will be a 4 min denaturation step at 94°C, followed by 30 cycles of 92°C for 1 min, 55°C for 1 min, and 72°C for 2 min. A final extension step will close the program at 72°C for 5 min. A PCR product of approximately 200-400 nucleotides will be observed following electrophoretic separation of the products of the PCR reaction in agarose gels. This fragment will be cloned into pGEM-T (Promega, Madison, WI), a vector that facilitates cloning of PCR products. Approximately 50 colonies will be obtained and a single run of nucleotide sequence will be obtained from each plasmid using the T7 primer to prime the nucleotide sequencing reactions by the chain termination method. A number of different clones will be identified by this method. The identity of these clones as xylan synthases will be determined by first comparing the sequences to the known Arabidopsis xylan synthase sequences. The most highly homologous clones will be used as probes to identify full-length clones and the complete nucleotide sequence of the clones will be determined. Those clones that exhibit the characteristic insertion of sequence corresponding to the region between about amino acid 573 to about amino acid 617 in Csl4 will be considered possible xylan synthases.

The identity of the candidate clones as xylan synthases will be determined by cloning the full length cDNA clones into a suitable binary Ti plasmid and using the construct to produce transgenic plants of the Ac39-2 mutant of Arabidopsis. Complementation of the Ac39-2 defect will be considered as proof that the clones encode xylan synthase.

EXAMPLE 11

MODIFICATION OF XYLAN LEVELS IN ASPEN

The isolation of the xylan synthase gene from A. thaliana provides a tool to those with ordinary skill in the art to isolate the corresponding gene or cDNA from other plant species. There are many examples in which genes from one plant species have been used to isolate the homologous genes from another plant species. One such plant which could be improved upon by the modification of the level of xylan is aspen, e.g. , hybrid aspen (Populus tremula times Populus tremuloides. Aspen wood typically contains a high proportion of the total polysaccharide content as xylans. This level is undesirable because xylans are not useful in the production of cellulose fiber and, therefore, the xylans are a wasteproduct of cellulose production that must be removed and disposed of. Also, because xylose cannot be efficiently converted to ethanol by fermentation using commonly used microbial strains, the high xylan content of some woody species such as aspen prevents efficient use of woody biomass for ethanol production from biomass by fermentation. We envision that the levels of xylans can be lowered by the expression of the aspen xylan synthases genes or cDNAs in an antisense orientation, or by cosuppression, in the developing wood tissues. The following example describes one method for the isolation and use of a xylan synthase cDNA from aspen. However, similar procedures could be followed to isolate a genomic clone which could also be used to decrease the level of xylan synthase activity by antisense expression of a portion or all of the gene. Sequence information from the xylan synthase genes from A. thaliana will be used to prepare probes to screen a cDNA library constructed from aspen mRNA. In order to isolate a cDNA to be used in decreasing xylans in woody tissues, the optimal tissue to use for the isolation of mRNA is cambial tissues. There is, however, flexibility in the choice of methods and vectors which can be used in the construction and analysis of cDNA libraries (Sambrook et al. , 1989). Procedures for the construction of cDNA libraries are available from manufacturers of cloning materials or from laboratory handbooks such as Sambrook et al. (1989). Once a suitable cDNA library has been constructed from aspen, the CSL4 homologs from aspen will be isolated as described for Arabidopsis genes in the foregoing Examples. Clones are concluded to be aspen CSL4 homologs based upon comparison of the deduced amino acid sequences or by their ability to complement the Ac39-2 mutant of Arabidopsis. The criteria for considering a gene to be a xylan synthase is that it should have at least 60% amino acid sequence similarity to one of the Arabidopsis xylan synthase clones and should contain at least 20 amino acids in the region of the protein corresponding to the "insertion" shown in the xylan synthases shown in Figure 4.

To make the antisense, or cosuppressed, aspen plants, the tissue specificity and abundance of the mRNA corresponding to each of the xylan synthase clones would first be determined by northern blotting or a related method of mRNA quantitation. Genes which are expressed in the cell types in which it is desired to reduce xylan content would be targeted for reduction in expression by one of the methods described in the foregoing examples. For instance, to use antisense to reduce expression, the entire clone of the aspen

CSL4 gene or genes, or a portion thereof, is placed downstream of a promoter sequence in an antisense orientation. Suitable promoters include stem-specific promoters, or promoters that lack substantial tissue specificity. An appropriate 3' non-translated region, such as the nopaline synthase 3' region, is placed downstream of the antisense cDNA to allow for transcription termination and for the addition of polyadenylated nucleotides to the end of the RNA sequence. This expression cassette is then combined with a selectable or scorable marker gene and aspen cells are transformed. Methods for production of transgenic aspen trees have previously been described (Weigel and Nilsson, 1995). Plants recovered are then analyzed for xylan content by the procedures outlined above and clones with useful reduction in xylan content are propagated.

EXAMPLE 12

OBTAINING OTHER PLANT GLYCAN SYNTHASES

The present invention teaches that some plant xylan synthases are structurally related to cellulose synthases from plants and bacteria. In view of the sequence similarity between these enzymes that catalyze different reactions, we envision that other polysaccharide synthases or gylcosyl transferases are also encoded by other members of the multigene family described herein. The xylan synthases of this invention are the first plant xylan synthases characterized whose proteins enzymatically catalyze the synthesis of a xylan. The reaction that xylan synthase performs and the cofactors it uses are likely to be very similar for several other glycan synthases such as callose synthase, xyloglucan synthase, glucomannan synthase, galactan and mannan synthase. Given the similar reactions, similar substrates and probably similar cofactors, we envision that the xyloglucan synthase, galactan synthase and mannan synthase and other glycan synthase genes and proteins have sequence similarity to the xylan synthase genes and proteins. This proposal is supported by the finding that additional structurally related genes are present in the Arabidopsis genome. In particular, we have identified a family of genes exemplified by clones corresponding to the Csll, Csl2, Csl3 and Csl7 (Table 7) that exhibit significantly higher sequence identity to the xylan synthase family (Csl4, Csl5 and Cslό) than to the putative cellulose synthase gene family exemplified by the CELA genes of cotton or Arabidopsis CelA homologs represented by EST clones in GenBank having the accession numbers T45303, T45414, H76149, H36985, Z30729, H36425, T45311, and A35212 (Pear et al. , 1996).

We envision that the precise function of the Csll, Csl2, Csl3 and Csl7 proteins and corresponding genes can be determined by using antisense or cosuppression methods as described herein for the CSL4 gene in the foregoing Examples. Alternatively, the function of these proteins may be determined by isolation a mutant of Arabidopsis or another plant by the general method described here for isolation and characterization of the Ac39-2 mutant.

EXAMPLE 13 USE OF XYLAN SYNTHASE OR OTHER GENES TO ISOLATE MUTATIONS THAT INACTIVATE GENE FUNCTION

Under certain circumstances it may be desirable to reduce the amount of xylan by non-transgenic methods. A method for accomplishing this is to use the cloned xylan synthase gene to screen for mutations as follows. Pairs of oligonucleotide primers based on the coding sequence of the xylan synthase gene are designed so that each pair of primers amplifies a 200 to 300 nucleotide long fragment of the xylan synthase gene when genomic DNA from the source of the xylan synthase gene is used as the template. For maximal efficiency, it would be preferable to design sets of oligonucleotide primers so that the entire coding sequence of the gene was spanned by a series of adjacent nonoverlapping 300 nucleotide fragments.

Genomic DNA is prepared from each of approximately 1000 heavily mutagenized M2 plants. The exact number of plants required for this purpose will vary with the efficiency of mutagenesis. An M2 plant is the progeny of a plant that has been treated with a mutagen and is, therefore, nonchimeric for induced mutations. Any effective mutagen such as ethylmethane sulfonate, fast neutrons, nitrosomethylurea, x-rays or gamma rays are suitable mutagens for this purpose. To begin the screen for a mutation, a pair of PCR primers as described above is used to prime PCR reactions on each of the 1000 DNA preparations, and the products of the reactions are electrophoresed or chromatographed under conditions that permit the separation of DNA fragments that differ by as little as one nucleotide. A typical method of this kind that is now in widespread use is the SSCP (Single Strand Conformational

Polymorphism) method. A plant with a mutation in the xylan synthase gene will be apparent by the presence of an SSCP polymorphism in one or more of the samples. If no polymoφhim is apparent with one pair of primers, the process is repeated with another set of primers. If none of the pairs of primers produce a polymoφhism, another 1000 mutagenized plants are screened by the same method until a mutation is identified.

Once a mutation is identified, the nature of the mutation can be determined by sequencing the mutant allele. In some cases, such as when the mutation causes a stop codon, the effect of the mutation may be inferred directly from the sequence of the mutant allele. In other cases, it will be necessary to test the biological function of the mutation by a direct test of the xylan content of the homozygous mutant plant

This method is of general utility and can be used to identify mutations in any gene. Modifications of the method that increase the efficiency may include the use of gene microarrays or gene chips that permit the facile identification of nucleotide sequence variants.

CONCLUDING REMARKS

By the above examples, demonstration of critical factors in the production of modified cell wall polysaccharide composition by expression of a xylan synthase gene from Arabidopsis thaliana in transgenic plants is described. Through the present invention, one can obtain amino acid and nucleotide sequences which encode plant xylan synthases from a variety of sources and for a variety of applications. All publications (e.g. , patents, articles, books) mentioned in this specification are indicative of the level of skill of those skilled in the art to which the present invention pertains, and are herein incoφorated by reference to the same extent as if each individual publication was specifically and individually indicated to be incoφorated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for puφoses of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

REFERENCES Aspinall, G.O. (1980) Chemistry of cell wall polysaccharides. In The Biochemistry of Plants: A Comprehensive Treatise, Vol. 3 (Smmpf, P.K. and Conn, E.E. , eds.) Academic Press, New York, pp. 473-500.

Bacic, A., Harris, P.J. and Stone, B.A. (1988) Strucmre and function of plant cell walls. In 77_e Biochemistry of Plants: A Comprehensive Treatise, Vol. 14 (Smmpf, P.K. and Conn, E.E. , eds.) Academic Press, New York, pp. 297-371.

Baydoun, E.A. , Waldron, K.W. , Brett, C.T. (1989) The interaction of xylosyltransferase and glucuronyltransferase involved in glucuronoxylan synthesis in pea (Pisum sativum) epicotyls. Biochem. J. 257,853-858.

Bechtold, N. , Ellis, J. , Pelletier, G. (1993) In planta Agrobacterium mediated gene transfer by infiltration of adult Arabidopsis thaliana plants. C.R. Acad. Sci. Paris 316, 1194-1199.

Beltz, G.A. , Jacobs, K.A., Eickbuch,. T.H., Cherbas, P.T., Kafatos, F.C. (1983) Isolation of multigene families and determination of homologies by filter hybridization methods. Meth. Enzymol. 100, 266-285.

Branden, C. and Tooze, J. (1991) Introduction to Protein Structure. Garland, New York.

Bray, E.A., Naito, S. , Pan, N.S., Anderson, E., Dube, P., Beachy, R.N. (1987) Expression of the 0-subunit of 0-conglycinin in seeds of transgenic plants. Planta 172, 364-370.

Brown R.M. , Saxena, I.M. , Kudlicka, K. (1996) Cellulose biosynthesis in higher plants. Trends Plant Sci. 1, 149-156. Caφita, N.C. and Gibeaut, D.M. (1993) Strucmral models of primary cell walls in flowering plants: consistency of molecular strucmre with the physical properties of the walls during growth. Plant J. 3, 1-30.

Creighton, T.E. (1983) Proteins: Structures and Molecular Properties. Freeman, New York.

Creighton, T.E. (1992) Protein Folding. Freeman, New York.

Darvill, A. , McNeil, M. , Albersheim, P. , and Delmer, D.P. (1980) The primary cell walls of flowering plants. In 77ιe Biochemistry of Plants (Smmpf, P.K. and Conn, E . eds.), Vol. 1, pp. 91-162, Academic Press, New York.

Delmer, D.P. and Amor, Y. (1995) Cellulose biosynthesis. Plant Cell 7, 987-1000.

Dickerson, R.E. and Geis, I. (1969) 77ιe Structure and Action of Proteins. Haφer & Row, New York.

Ditta, G. , Stanfield, S. , Corbin, D., Helinski, D.R. (1980) Broad host range DNA cloning system for gram-negative bacteria: Construction of a gene bank of Rhizobium meliloti. Proc. Natl. Acad. Sci. USA 77, 7347-7351.

Doolittle, R.F. (1986) Of URFS and ORFS. University Science Books, CA.

Fry, S.C. (1988) 77ze Growing Plant Cell Wall: Chemical and Metabolic Analysis. John Wiley, New York. Glasser, L. (1958) The synthesis of cellulose in cell-free extracts of Acetobacter xylinum. J. Biol. Chem. 232, 627-637.

Goeddell, D.V. (1990) Gene Expression Technology, Academic Press, San Diego.

Gould, S.J., Subramani, S. , Scheffler, I.E. (1989) Use of the DNA polymerase chain reaction for homology probing. Proc. Natl. Acad. Sci. USA 86, 1934- 1938.

Hames, B.D. and Higgins, SJ. (1985) Nucleic Acid Hybridisation. IRL Press, Oxford.

Hayashi, T. (1989) Xyloglucans in the primary cell wall. Annu. Rev. Plant Physiol. Plant Mol. Biol. 40, 139-168.

Hobbs, M.C., Delarge, M.H. P., Baydoun, E.A.H., Brett, C.T. (1991) Differential distribution of a glucuronyl transferase involved in glucuronoxylan synthesis within the Golgi apparatus of pea (Pisum sativum var Alaska). Biochem. J. 277,653-658.

Iba, K. , Gibson, S. , Ntshiuchi, T., Fuse, T., Nishimura, M. , Arondel, V. , Hugly, S., Somerville, C. (1993) A gene encoding a chloroplast omega-3 fatty acid desamrase complements alterations in fatty acid desamration and chloroplast copy number of the fad7 mutant of Arabidopsis thaliana. J. Biol. Chem. 268, 24099-24105.

Jarvis, M.C. (1984) Strucmre and properties of pectin gels in plant cell walls. Plant Cell Env. 7, 153-164. Kieber, J.J. , Rothenberg, M., Roman, G. , Feldman, K.A. , Ecker, J.R. (1993) CTR1 , a negative regulator of the ethylene response pathway in Arabidopsis encodes a member of the RAF family of protein kinases. Cell 72, 427-441.

Koncz, C, Schell, J. (1986) The promoter of T_L-DNA gene 5 controls the tissue-specific expression of chimeric genes carried by a novel type of Agrobacterium binary vector. Mol. Gen. Genet. 204, 383-396.

Lin, F.C. , Brown, R.M. Jr., Drake, R.R. Jr., Haley, B.E. (1990) Identification of the uridine 5'-diphosphoglucose (UDP-Glc)binding subunit of cellulose synthase in Acetobacter xylinum using the photoaffinity probe 5-azido-UDP-Glc. J. Biol. Chem. 265, 4782-4784.

Matzke, M.A., Matzke, A. J.M. (1995) How and why do plants inactivate homologous (trans)genes? Plant Physiol. 107, 679-685.

McCann, M., Roberts, K. (1991) Architecture of the primary cell wall. In 77je Cytoskeletal Basis of Plant Growth and Form (Lloyd, C.W. , ed.). Academic Press, London, pp. 109-129.

McNeil, M., Darvill, A.G., Fry, S.C, and Albersheim, P. (1984) Strucmre and function of the primary cell walls of plants. Annu. Rev. Biochem. 53, 625-663.

Matthysse, A.G., White, S., Lightfoot, R. , (1995) Genes required for cellulose synthesis in Agrobacterium tumefaciens. J. Bacteriol. 177, 1069-1075.

Murray, M.G. , Thompson, W.F. (1980) Rapid isolation of high molecular weight plant DNA. Nucl. Acid Res. 8, 4321-4325. Pear J.P. , Kawagoe, Y. , Schreckengost, W.E. , Delmer, D.P., Stalker, D.M. (1996) Higher plants contain homologs of the bacterial celA genes encoding the catalytic subunit of cellulose synthase. Proc. Natl. Acad. Sci. USA 93, 12637- 12642.

Reiter, W.-D., Chappie, C.C.S., Somerville, C.R. (1993) Altered growth and cell walls in a fucose-deficient mutant of Arabidopsis . Science 261, 1032-1035.

Reiter, W.-D. (1994) Strucmre, synthesis, and function of the plant cell wall. In Arabidopsis (Somerville, C.R. and Meyerowitz, E.M. , eds.), CSHL Press, Cold Spring Harbor, NY, pp. 955-988.

Ross, P, Weinhouse, H, Aloni, Y, Michaeli, D, Weinberger-Ohana, P, Mayer, R, Braun, S, de Vroom, E, van der Marel, GA, van Broom, JH, Benziman, M. (1987) Regulation of cellulose synthesis in Acetobacter xylinum by cyclic diguanylic acid. Nature 325, 279-281.

Sambrook, J., Fritsch, E.F. , Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., CSHL Press, Cold Spring Harbor, NY.

Saxena, I.M. , Kudlicka, K., Okuda, K., and Brown, R.M. (1994) Characterization of genes in the cellulose -synthesizing operon (acs operon) of Acetobacter xylinum: implications for cellulose crystallization. J. Bacteriol. 176, 5735-5752.

Saxena, I.M. , Brown, R.M. Jr., Fevre, M. , Geremia, R.A. , Henrissat, B.

(1995) Multidomain architecture of beta-glycosyl transferases: Implications for mechanism of action. J. Bacteriol. 177, 1419-1424. Waldron, K.W., Brett, C.T. (1987) The subcellular localisation of a glucuronytransferase involved in glucuronoxylan biosynthesis in pea (Pisum sativum) epicotyls. Plant Science 49, 1-8.

Weigel, D. , Nilsson, O. (1995) A developmental switch sufficient for flower initiation in diverse plants. Nature 377, 495-500

Zablackis, E., Huang, J., Muller, B. , Darvill, A.G., and Albersheim, P. (1995) Characterization of the cell wall polysaccharides of Arabidopsis Thaliana leaves. Plant Physiol. 107, 1129-1138.

Table 1. Examples of softwood species used for production of plant fibers

Scientific name Common name

Abies alba Silver fir A . balsa ea Balsam fir

A . firma Japanese fir A . sibirica Siberian silver fir Araucaria angustifolia Parana-pine

Chamaecyparis obtusa Japanese cypress Cryptomeria japonica Sugi

Larix decidua European larch

L . occiden talis Western larch

L . sibirica Siberian larch

Picea abies European spruce P. glauca White spruce

P. si tchensis Sitka spruce

Pinus banksiana Jack pine

P. caribaea Caribbean pine

P. contorta Contorta pine P. dens i flora Japanese red pine

P. echina ta Shortleaf pine

P. elliottii Slash pine

P. halepensis Aleppo pine

P. kesiya Kesiya pine P. lambertiana Sugar pine

P. erkusii Merkus pine

P. monticola Western white pine

P . nigra European black pine

P. palus tris Longleaf pine P. patula Patula pine

P. pinaster Seaside pine

P. ponde osa Ponderosa pine

P . radiata Radiata pine

P. resinosa Red pine P. rigida Pitch pine . s trobus Eastern white pine

P. sylvestris Scots pine

P. taeda Loblolly pine

Podocarpus spica tus Matai Pseudotsuga menziesii Douglas-fir

Sequoia sempervirens Sequoia

Taxodium dis tichuni Baldcypress

Thuja plica ta Western redcedar

Tsuga canadensis Eastern hemlock T. heterophylla Western hemlock Table 2. Examples of hardwood species used for production of plant fibers.

Scientific name Common name

Acacia auriculiformis Papuan wattle Acer pla tanoides Norway maple A. pseudopla tanus Great maple A . rubru Red maple A . saccharum Sugar maple Albizzia falcataria White albizzia Alnus glu tinosa Black alder

A . rubra Red alder

Anthocephalus chinensis Anthocephalus_ Betula maximowicziana Japanese birch

B . papyri fera Paper birch B . verrucosa European white birch

Carpinus betulus European hornbeam Carya ova ta Shagbark hickory Castanea sativa Sweet chestnut Eucalyptus globulus Southern blue gum E. regnans Mountain ash

E. saligna Saligna Fagus syl va tica European beech Fraxinus excel sior European ash

F. mandshurica Japanese ash Gmelina arbor ea Gmelina

Juglans nigra Black walnut

J. regia European walnut

Liquidambar s tyraci flua Sweetgum

Liriodendron tulipifera Yellow-poplar Magnolia acu inata Cucumbertree

M. grandiflora Southern magnolia

Musanga smi thi i Aga umbrella tree

Nys sa syl vatica Black tupelo

Populus del toides Eastern cottonwood P. tremula European aspen

P. tremuloides Quaking aspen

Quercus alba American white oak

Q . robur English oak

Salix alba White willow S . nigra Black willow

Shorea polysperwa Red lauan

Tilia cor data European small-leaf lime

Ulmus americana American elm

U. glabra Mountain elm Table 3. Examples of Non-wood Plant Fibers

Scientific name Common name

Grasses, papyrus, and palms

Triticum sativum Wheat

Zea mays Corn

Saccharum officinarum Sugar cane

Phragmi tes cowmunis Common reed

Dendrocalamus strictus Bamboo

Eulaliopsis binata Sabal

Oryza sativa Rice

Lygeum spar turn Albardine

Stipa tenacissima Esparto

Cyperus papyrus Papyrus

Elaeis guineensis Oil palm

Raphia hookeri Raphia

Bast fibers

Linum usi ta tissimum Flax

Cannabis sativa Hemp

Crotalaria juncea Sunn

Hibiscus cannabinus Kenaf

Cor chorus capsular is Jute

Boehmeria nivea Ramie

Broussonetia papyrifera Paper-mulberry

Wikstroemia canescens Gam i

Edgeworthia papyrifera Mitsumata

Leaf Fibers

Musa textilis Abaca

Agava sisalana Sisal Fruit fibers Gossypium s . Cotton Ceiba pentandra Ceiba (kapok tree)

Table 4. Structures of side chains in xylans ⁽reproduced from Aspinall, 1980)

Side chains

α-L-Araf- (1-3) - ₄-Me-c_.-D-GlcpA- (1-2) - α-D-GlcpA- (1-2) - α-D-Xy lp- (1-3) -L-Araf ⁽1-

0-D-Xy lp- (1-2) -L-Araf ⁽1-3⁾-

0-D-Galp- (1-5) -L-Araf^{" (}1-

0-D-Galp- (1-4) -D-Xylp (1-

D-[or L-] Galp- ₍₁-₄) -D-Xylp- (1-2) -L-Araf- ⁽1- [4-Me] -α-D-GlcpA- (1-4) -D-Xylp- ⁽1-4⁾ -D-Galp- ⁽1-

Table 5. TBLAS_N scores and estimated significance of open reading frames of newly identified ESTs compared to Csll.

TBLASTN Smallest

Score Sum

GenBank against Probability Ace . # F.ST name : Csll P.NΪ

Z34860 FAFL51 426 l.le-52

T20778 92K11T7 368 2.3e-44

H36778 178H9T7 229 2.7e-36

N37334 210A22T7 219 3.6e-23

T45861 133A23T7 86 0.010 Table 6. TBLASTN search of dbEST with the predicted coding sequence of 210A22T7.

TBLASTN

GenBank Score

Accession against

Number EST Name 210A22T7 P(N)

T88271 160C16T7 340 7.5e- 40

Z34860 FAFL51 322 3.9e-37

T45861 133A23T7 261 2.0e-28

H36778 178H9T7 226 9.8e-28

T20778 92K11T7 211 1.7e-21

T44297 119C22T7 97 1.5e-_D8

Table 7 : ESTs Identified as CSL gene family members and their defined Csl names.

EST Name GenBank Ace C .si desiqnation

160C16T7 T88271 Csll

FAFL51 Z34860 Csl2

92K11T7 T20778 Csl3

210A22T7 N37334 Csl4

133A23T7 T45861 Csl5

119C22T7 T44297 Cslδ

178H9T7 H36778 Csl7

Table 8. Monosaccharide content of cell wall polysaccharides from wild type Arabidopsis and Ac39-2 mutant (not all sugars are shown) .

Sugar WT Ac39-2 mutant mol% mol%

rhamnose 7.1 9.5 arabinose 7.4 16.3 xylose 50.8 23.1 mannose 8.5 7.5

Table 9. Monosaccharide content of cell wall polysaccharides from wild type Arabidopsis and cosuppression lines.

Sugar WT Cl C2 C3 C4 C5 mol% mol% mol% mol% mol% mol%

rhamnose 6.8 7.6 7.1 8.0 9.0 5.5 arabinose 4.2 9.7 10.8 8.5 16.7 7.9 mannose 8.4 7.0 7.6 7.3 5.7 7.9 xylose 47.0 27.6 38.2 39.5 24.5 40.5

Table 10. Conserved regions of amino acid sequence of Arabidopβia xylan synthases and corresponding oligonucleotide primers that can be used to amplify the conserved region from other plant species. Nucleotide ambiguities are indicated by the following symbols: N=A/T/G/C; H=T/C/A; R=A/G; Y=C/T. An ambiguity in the peptide VTKK(ATL)G is indicated by X to indicate that at that position any amino acid may occur.

Peptide Primer VTKKXG SEQ ID NO:

GTNACNAARAARNNNGG SEQ ID NO:

HFYFLLFQG SEQ ID NO

CAYTTYTAYTTYYTNYTNTTYCARGG SEQ ID NO

CAYTTYTAYTTYYTNYTNTTYCA SEQ ID NO CAYTTYTAYTTYYTNYTNTT SEQ ID NO

CAYTTYTAYTTYYTNYT SEQ ID NO

TTYTAYTTYYTNYTNTTYCARGG SEQ ID NO

TAYTTYYTNYTNTTYCARGG SEQ ID NO

TTYYTNYTNTTYCARGG SEQ ID NO VGLDLIGEQ SEQ ID NO

GGNYTNGAYYTNATHGGNGARCA SEQ ID NO

GGNYTNGAYYTNATHGGNGA SEQ ID NO

GGNYTNGAYYTNATHGG SEQ ID NO

YTNGAYYTNATHGGNGARCA SEQ ID NO GAYYTNATHGGNGARCA SEQ ID NO

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Somerville, Chris

Cutler, Sean

(ii) TITLE OF INVENTION: USE OF GENES ENCODING XYLAN SYNTHASE TO

MODIFY PLANT CELL WALL COMPOSITION OF PLANTS

(iii) NUMBER OF SEQUENCES: 25

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: PILLSBURY MADISON & SUTRO. LLP

(B) STREET: 1100 New York Avenue, N.W.

(C) CITY: Washington

(D) STATE: D.C

(E) COUNTRY: USA

(F) ZIP: 20005-3918

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Diskette

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: MS-DOS/PC-DOS

(D) SOFTWARE: WordPerfect 5.1

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 466 nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 :

AGAAGATGTT GAGGTTGGAC CCGATAACTA CCCAATGGTT 40

CTTATCCAAA TACCAATGTA CAATGAAAAA GAGGTCTTTC 80

AATTATCTAT AGCAGCAATA TGTAGTTTGG TCTGGCCATC 120

GAGCCGTCTA GTAGTTCAAG TTGTAGATGA TTCTACGGNT 160

CCGGCCGTAA GGGAAGGTGT GGACGTAGAG ATTGCAAAAT 200

GGCAAAGCCA AGGCATAAAC ATAAGGTGTG AAAGGAGAGA 240

TAACAGGAAC GGCTACAAAG CCGGAGCTAT GAAAGANGCT 280

CTTACGCAGA GCTACGTCAA GCAATGCGAC TTCGTAGCAG 320

TCTTCGNTGC TGATTTCCAA CCCGAGCCCG ATTATCTNAT 360

CCGCGNTGTC CCTTTCCTTG TCCACANCCC TGACGTTGCT 400 CTNGTTCAAG CCCGATGNNT ATTTNGTTAN CGNGAACANN 440

TGCTTGTNGT CGNGGNTGCA AGTGNT 466 ( 2 ) INFORMATION FOR SEQ ID NO : 2 :

( i ) SEQUENCE CHARACTERISTICS :

(A) LENGTH : 1488 nucleotides

(B) TYPE : nucleic acid

(C) STRANDEDNESS : single

(D) TOPOLOGY : linear

(xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 2 :

AGAAGATGTT GAGGTTGGAC CCGATAACTA CCCAATGGTT 40

CTTATCCAAA TACCAATGTA CAATGAAAAA GAGGTCTTTC 80

AATTATCTAT AGCAGCAATA TG AGTTTGG TCTGGCCATC 120

GAGCCGTCTA GTAGTTCAAG TTGTAGATGA TTCTACGGAT 160

CCGGCCGTAA GGGAAGGTGT GGACGTAGAG ATTGCAAAAT 200

GGCAAAGCCA AGGCATAAAC ATAAGGTGTG AAAGGAGAGA 240

TAACAGGAAC GGCTACAAAG CCGGAGCTAT GAAAGAAGCT 280

CTTACGCAGA GCTACGTCAA GCAATGCGAC TTCGTAGCAG 320

TCTTCGATGC TGATTTCCAA CCCGAGCCCG ATTATCTCAT 360

CCGCGCTGTC CCTTTCCTTG TCCACAACCC TGACGTTGCT 400

CTAGTTCAAG CCCGATGGAT ATTTGTTAAC GCGAACAAAT 440

GCTTGATGAC GAGGATGCAA GAGATGTCTC TCAACTATCA 480

TTTCAAAGTG GAACAAGAAT CAGGGTCGAC TAGACATGCT 520

TTCTTCGGGT TTAATGGAAC CGCGGGTGTA TGGAGAATAT 560

CGGCAATGGA AGCAGCAGGA GGATGGAAAT CAAGGACCAC 600

AGTAGAGGAC ATGGACTTGG CTGTTCGTGT TGGTCTTCAT 640

GGCTGGAAAT TTGTCTACCT TAACGACCTC ACGGTGAGAA 680

ACGAGCTTCC AAGCAAATTT AAGGCCTACA GATTCCAGCA 720

ACATAGGTGG TCCTGTGGAC CGGCGAATCT ATTTAGAAAA 760

ATGACGATGG AGATCATTTT CAATAAGAGA GTATCAATTT 800

GGAAGAAGTT TTATGTGATC TACAGCTTTT TCTTCGTAAG 840

GAAAGTGGCG GTACACTTCT TGACATTCTT CTTCTACTGT 880

ATAATTGTGC CAACAAGTGT CTTCTTCCCT GAAATCCACA 920 TCCCATCTTG GTCTACCATT TACGTTCCCT CTTTGATCAG 960

TATCTTCCAC ACCCTGGCAA CTCCAAGATC CTTCTACCTC 1000

GTGATATTTT GGGTCTTGTT CGAGAATGTA ATGGCTATGC 1040

ATCGAACCAA AGGTACGTGC ATTGGCCTAC TTGAAGGAGG 1080

AAGAGTAAAC GAATGGGTTG TGACCGAAAA ACTAGGAGAT 11 0

GCTTTGAAGA GTAAGCTACT CTCTCGGGTA GTCCCAAAGA 1160

AAATCTTGTT ATCAAAGAGT GAATTCCAAG GAAGTGATGG 1200

TGGGGGTATA CATATTAGGA TGTGCACTCT ATGGCCTGAT 1240

CTATGGGCAC ACATGGTTAC ATTTCTATCT TTTTCTTCAG 1280

GCCACAGCCT TTTTCGTCTC CGGTTTTGGT TTTGTCGGAA 1320

CGGCCTAAGA ACCTTCCCTG CCCATTATTT TTAGTCACCA 1360

AATAAATTCT CCATGTTTTA GTTCTTATTT ACACTTTTAT 1400

TTATTTTGAC ACCATTGTAC GGTTTGGACC CCATATCATC 1440

ATGTTGTATA AGTATAACGA ATAATGATTA AAAAAAAAAA 1480

AAAAAAAA 1488 (2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 450 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

Glu Asp Val Glu Val Gly Pro Asp Asn Tyr Pro Met 1 5 10

Val Leu lie Gin lie Pro Met Tyr Asn Glu Lys Glu 15 20

Val Phe Gin Leu Ser lie Ala Ala lie Cys Ser Leu 25 30 35

Val Trp Pro Ser Ser Arg Leu Val Val Gin Val Val 40 45

Asp Asp Ser Thr Asp Pro Ala Val Arg Glu Gly Val 50 55 60

Asp Val Glu lie Ala Lys Trp Gin Ser Gin Gly lie 65 70

Asn lie Arg Cys Glu Arg Arg Asp Asn Arg Asn Gly 75 80 Tyr Lys Ala Gly Ala Met Lys Glu Ala Leu Thr Gin 85 90 95

Ser Tyr Val Lys Gin Cys Asp Phe Val Ala Val Phe 100 105

Asp Ala Asp Phe Gin Pro Glu Pro Asp Tyr Leu lie 110 115 120

Arg Ala Val Pro Phe Leu Val His Asn Pro Asp Val 125 130

Ala Leu Val Gin Ala Arg Trp lie Phe Val Asn Ala 135 140

Asn Lys Cys Leu Met Thr Arg Met Gin Glu Met Ser 145 150 155

Leu Asn Tyr His Phe Lys Val Glu Gin Glu Ser Gly 160 165

Ser Thr Arg His Ala Phe Phe Gly Phe Asn Gly Thr 170 175 180

Ala Gly Val Trp Arg lie Ser Ala Met Glu Ala Ala 185 190

Gly Gly Trp Lys Ser Arg Thr Thr Val Glu Asp Met 195 200

Asp Leu Ala Val Arg Val Gly Leu His Gly Trp Lys 205 210 215

Phe Val Tyr Leu Asn Asp Leu Thr Val Arg Asn Glu 220 225

Leu Pro Ser Lys Phe Lys Ala Tyr Arg Phe Gin Gin 230 235 240

His Arg Trp Ser Cys Gly Pro Ala Asn Leu Phe Arg 245 250

Lys Met Thr Met Glu lie lie Phe Asn Lys Arg Val 255 260

Ser lie Trp Lys Lys Phe Tyr Val lie Tyr Ser Phe 265 270 275

Phe Phe Val Arg Lys Val Ala Val His Phe Leu Thr 280 285

Phe Phe Phe Tyr Cys lie lie Val Pro Thr Ser Val 290 295 300

Phe Phe Pro Glu lie His lie Pro Ser Trp Ser Thr 305 310 lie Tyr Val Pro Ser Leu lie Ser lie Phe His Thr 315 320 Leu Ala Thr Pro Arg Ser Phe Tyr Leu Val lie Phe 325 330 335

Trp Val Leu Phe Glu Asn Val Met Ala Met His Arg 340 345

Thr Lys Gly Thr Cys lie Gly Leu Leu Glu Gly Gly 3S0 355 360

Arg Val Asn Glu Trp Val Val Thr Glu Lys Leu Gly 365 370

Asp Ala Leu Lys Ser Lys Leu Leu Ser Arg Val Val 375 , 380

Pro Lys Lys lie Leu Leu Ser Lys Ser Glu Phe Gin 385 390 395

Gly Ser Asp Gly Gly Gly lie His lie Arg Met Cys 400 405

Thr Leu Trp Pro Asp Leu Trp Ala His Met Val Thr 410 415 420

Phe Leu Ser Phe Ser Ser Gly His Ser Leu Phe Arg 425 430

Leu Arg Phe Trp Phe Cys Arg Asn Gly Leu Arg Thr 435 440

Phe Pro Ala His Tyr Phe 445 450

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1231 nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS : s ingle

(D) TOPOLOGY : l inear

(xi) SEQUENCE DESCRIPTION : SEQ ID NO : 4 :

GATTTTCTTC GCCGTAGCAT TCCTTTCCTC ATGCACAATC 40

CCAACATTGC CTTGGTTCAG GCTCGATGGC GGTTCGTAAA 80

TTCTGATGAG TGCTTATTGA CGAGAATGCA AGAAATGTCA 120

TTGGATTACC ATTTCACTGT TGAGCAAGAA GTGGGTTCAT 160

CAACACATGC TTTTTTCGGC TTCAACGGAA CCGCCGGAAT 200

ATGGAGAATA GCGGCGATAA ATGAAGCTGG TGGGTGGAAA 240

GATCGGACCA CCGTGGAAGA TATGGATCTC GCCGTCCGAG 280

CAAGTCTTCG CGGCTGGAAA TTTCTCTACC TCGGTGACCT 320

TCAGGTGAAA AGTGAGCTTC CAAGTACTTT TAGAGCCTTC 360 CGTTTTCAGC AACATAGATG GTCTTGTGGA CCTGCAAATC 400

TCTTTAGGAA AATGGTTATG GAGATCGTAA GAAACAAGAA 440

AGTGAGATTC TGGAAGAAAG TGTACGTGAT ATACCGCTTC 480

TTCTTTGTGA GGAAAATCAT TGCACATTGG GTCACATTTC 520

GTTTCTACTG CGTTGTTCTT CCTCTCACAA TTCTCGTCCC 560

GGAGGTTAAA GTTCCGATGT GGGGTTCGGT TTATATCCCA 600

TCCATCATCA CTATCCTCAA TTCCGTCGGT ACTCCAAGGT 640

CAATTCATCT GCTGTTCTAT TGGATTCTAT TCGAGAATGT 680

GATGTCGCTG CACCGGACAA AGGCCACTCT CATTGGTCTG 720

TTTGAGGCAG GAAGGGCTAA CGAGTGCGTA GTGACTGCTA 760

AGCTTGGAAG CGGTCAGAGC GGTAAAGGAA ACAATAAAGG 800

GATCAAAAGG TTCCCAAGAA TCTTCAAATT GCCTGATCGA 840

TTGAATACAT TGGAGCTTGG ATTTGCGGCT TTCTTGTTCG 880

TGTGCGGATG CTATGACTTT GTGCACGGGA AGAACAATTA 920

CTTCATCTAC CTTTTTCTTC AGACAATGTC TTTCTTCATC 960

AGTGGGCTGG GCTGGATCGG GACTTATGTC CCGAGTTAGT 1000

AGTTGTGTTG TTTCAGAGAG AAAAGAGAAT GT ATTAATT 1040

TTCTTGAGAA ATAAAGACAA TTTTCATTGA AATGACAAAG 1080

GAAAATTGAT AGGGGAGATA GAGACGTACC GGTAACAAC 1120

AGTAGGAAGA AAGGAGAAGA CTTTTATCAA AGACCAGGAA 1160

AGAGGAGAAG CTCCAAGGGT TCTTTTGATT TATTTTTATT 1200

TTTTCTGTGT TTTATTTATA TAGCTATGGG T 1231

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 658 nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 5 :

GAAGATTGTA GTACACATCT TCACATTTGT CTTCTACTGT 40

CTGATATTAC CAACAACTGT GCTATTCCCT GAGCTCCAAG 80

TTCCTAAATG GGCAACTGTT TATTTTCCTA CTACAATCAC 120

TATCCTTAAC GCAATCGCTA CACCTCGATC ACTCCATCTT 160 CTTGTCTTTT GGATCTTATT CGAGAATGTA ATGTCGATGC 200

ATCGCACAAA AGCGACATTC ATCGGGTTAC TAGAGGCAGG 240

ACGGGTTAAC GAATGGGTTG TTACTGAAAA ATTAGGTGAC 280

ACTCTCAAGT CTAAGTTAAT AGGTAAAGCT ACAACTAAGC 320

TTTATACCAG ATTTGGACAA AGACTCAACT GGAGAGAACT 360

CGTTGTTGGA TTATATATAT TCTTCTGCGG ATGTTACGAT 400

TTCGCATATG GAGGATCATA CTTTTATGTT TATCTGTTTT 440

TACAGTCTTG TGCATTTTTT GTTGCTGGAG TTGGTTATAT 480

TGGCACATTT TGTTCCAACT GTTTAGATTA GACTTTGGTA 520

TTTGAGAGCA AATGTGTTTT TGCTATTTGA TTAGACCAAA 560

TTAGAGATTG GATCTTGGTT ACTTAAACCA AGTTAACTCT 600

TCCAATATTC ATACTCCAAA TGTAATTTTT TAAAAAAAAA 640

AAAAAAAAAA AAAAAAAA 658 ( 2 ) INFORMATION FOR SEQ ID NO : 6 :

( i ) SEQUENCE CHARACTERISTICS :

(A) LENGTH : 995 nucleotides

(B ) TYPE : nucleic acid

( C ) STRANDEDNESS : single

(D) TOPOLOGY : linear

(xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 6 :

TTCTTTGGGT TCAACGGAAC TGCTGGCGTC TGGAGAATCT 40

TCAGCATTGA ATGAGTCCGG GGGATGGAAT GACCAGACAA 80

CAGTAGAAGA CATGGATTTA GCTGTACGAG CTACACTGAG 120

AGGCTGGAAG TTTCTATATA TCGATGATCT CAAGGTAAAA 160

AGTGAGTTAC CTTGTTCCTT CAAAGCTCTG CGTAGCCAGC 200

AGCATCGATG GACTTGCGGC CCTGCCAATC TTTTAAGGAA 240

AATGGCTGGA CAAATAATAA GAAGCGAGAA TGTATCTCTA 280

TGGAAGAAGT GGTATATGTT GTACAGCTTC TTCTTCATGC 320

GCAAGATCGT GGCTCACATC CTCACATTCT GCTTCTACTG 360

TGTTATCTTG CCAGCAACTG TATTGTTCCC AGAAGTCACA 400

GTTCCGAAAT GGGCTGCATT TTATCTCCCT TCTTTGATCA 440

CTCTCTTAAT CGCAATTGGT AGACTAAGAT CAATCCACCT 480 TTTGGCTTTC TGGGTTCTAT TTGAGAATGC AATGTCCCTG 520

CTAAGAGCAA AGGCTCTGGT CATGGGCTTG TTCGAAACAG 560

GAAGAGTACA AGAATGGGTT GTGACGGAGA AGTTAGGTGA 600

TACTCTCAAG ACGAAGCTGA TTCCACAAGT TCCTAACGTC 640

AGATTCAGAG AGAGGGTGCA TTTGCTGGAG CTATTGGTTG 680

GAGCGTACCT GTTATTCTGC GGAATCTACG ACATCGTCTA 720

TGGGAAGAAC ACACTCTATG TGTACCTTCT GTTTCAGTCA 760

GTGGCCTTCT TCGTTGTTGG TTTTGGATTT GTAGGAAATA 800

TGTTCCTGCT TCCTCTTACT TAGCATAGGA TAGATCATAG 840

TGTCTCAATA TGTTTCAGAG GTTTTTTGTT TGATGTCATA 880

TTGAGAACAA TGCCTATATA CTACTCTCCT TTGGTATTAA 920

CACTTGGGTA TCGGTAAACA ACGAAGCTCA AATGGGACCA 960

ATATTATTTT TTTAACAGTA AAAAAAAAAA AAAAA 995 (2) INFORMATION FOR SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 757 nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 7 :

ACCTTCTTCC ATCTCCAAAA TCGTTTCCTT TCATCGTTCC 40

TTATCTCTTA TTCGAGAACA CAATGTCTGT CACCAAGTTC 80

AATGCGATGG TGTCCGGATT ATTCCAATTG GGTAGCTCTT 120

ACGAGTGGAT TGTCACAAAG AAAGCTGGAA GATCATCAGA 160

GTCAGATCTT TTATCTATCA CCGAGAAAGA GACACCAACC 200

AAGAAAAGTC AATTGCTTAG GGGAGTTTCC GACAGCGAAC 240

TTTTGGAGCT GAGCCAACTT GAAGAACAGA AACAAGCGGT 280

TTCAAAGAAA CCTGTCAAGA AAACCAACAA GATATACCAC 320

AAAGAGCTCG CATTGGCCTT TCTTTTGCTC ACTGCAGCGC 360

TTAGGAGTCT CTTGGCAGCA CAAGGAGTGC ATTTCTACTT 400

CCTGTTATTC CAAGGTGTTA CCTTTCTTCT TGTGGGTCTC 440

GATCTCATAG GCGAGCAGAT GAGCTGAGAA GAACAAAAGA 480 AGATTCATAA CTCGGGTCAA TAAATCAGTG AGTTTGAATT 520

TCCACTTCCT CTATGTTCAG TGGTATCCTC TGTAATGAGA 530

TTGTTGGAAC CATAGTGGTA GGCATTAATT GATTTTCCGT 600

TTCGTTTGTT TGTTTGTTAT GTTGGTTTGA TTCCTCCATT 640

GTTAGGGATA GAGTGTGAGC CAGAGATTCA TACATGTATA 680

CAATATCTGT AAAGTTGGAA CATGTTGTTG GCAGTTAAAA 720

GAGCTTCTGT CTTGAACTAA AAAAAAAAAA AAAAAAA 757 (2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1695 nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 8 :

TTGGATCAAA TTTAAAAAGA TCGAACCTAA GCTCACCGAA 40

GAATCTATCG ATTTAGAAGA CCCCTCCAGT TTCCCAATGG 80

TCCTTATTCA GATCCCAATG TGCAACGAAC GAGAGGTGTA 120

TGAACAATCA ATAGGAGCAG CTTCACAACT TGATTGGCCA 160

AAAGATAGGA TCTTAATTCA AGTATTAGAT GATTCAGACG 200

ATCCAAATTT ACAGCTTTTG ATCAAAGAAG AAGTATCGGT 240

TTGGGCCGAA AAAGGCGTAA ACATAATTTA CAGGCATAGG 280

TTGATCAGAA CTGGTTACAA AGCTGGCAAT TTGAAATCAG 320

CCATGACTTG TGATTACGTT AAAGATTACG AGTTTGTGAC 360

TATCTTCGAC GCAGATTTCA CACCAAATCC TGATTTCCTC 400

AAGAAGACTG TTCCTCATTT CAAGGGTAAT CCAGAGCTAG 440

GGTTGGTCCA AGCAAGGTGG TCATTTGTGA ACAAAGATGA 480

GAATCTCCTC ACGAGGCTAC AAAACATAAA CTTATGTTTC 520

CACTTCGAAG TAGAACAACA AGTGAACGGT GTGTTTCTCA 560

ATTTCTTCGG ATTCAATGGA ACCGCTGGAG TATGGAGGAT 600

CAAGGCATTG GAAGAATCCG GCGGATGGCT CGAGAGAACC 640

ACCGTGGAAG ATATGGATAT CGCGGTTAGA GCGCATCTCA 680

ACGGCTGGAA GTTTATTTAC CTTAATGATG TTGAAGTCAC 720 TTGCGAGTTG CCAGAGTCTT ATGAAGCTTA CAAGAAGCAA 760

CAACATCGTT GGCATTCCGG TCCTATGCAG CTGTTCCGGT 800

TATGCCTTCC TTCAATTATC AAATCAAAGA TATCGGTTTG 840

GAAGAAGGCG AATTTGATCT TCCTTTTCTT TCTTCTAAGG 880

AAGCTTATTC TACCATTTTA CTCATTCACA CTCTTTTGCA 920

TTATACTTCC ATTGACAATG TTCATACCCG AAGCCGAGCT 960

TCCGTTGTGG ATCATCTGCT ATGTTCCTAT CTTCATTTCG 1000

CTTCTCAACA TTCTCCCGTC ACCTAAATCT TTCCCTTTCT 1040

TAGTCCCTTA CCTTCTTTTC GAAAACACAA TGTCAATAAC 1080

CAAGTTCAAC GCCATGATCT CCGGGCTGTT TCAGTTTGGA 1120

TCGGCTTACG AGTGGGTTGT GACGAAGAAA ACCGGGAGAT 1160

CATCGGAGTC CGATTTGCTA GCGTTTGCTG AAAAGGAAGA 1200

GAAGTTGCAT AGGAGAAACT CGGAGTCAGG TTTGGAGCTT 1240

CTGAGCAAAC TTAAGGAGCA AGAGACAAAT CTTGTAGGGC 1280

AAGAAACCGT GAAGAAGAGC CTTGGAGGGC TAATGAGGCC 1380

GAAGAACAAG AAGAAGACGA ACATGGTGTT CAAGAAAGAG 1360

CTCGGGCTTG CGTTCTTGCT GCTAACCGCA GCTGCAAGGA 1400

GCTTTCTATC GGCGCACGGT CTTCACTTCT ACTTTTTGTT 1440

GTTTCAGGGA CTGTCTTTCT TGGTTGTAGG GTTGGATTTG 1480

ATCGGAGAAC AGATCAGCTA GACACACACA AAACCAAGAC 1520

AGAGAAC AG GAAAAAAATA CCGTTTATAT TCACTATTTT 1560

TTTGTTTGTT TTTTAAATTC TTTTCCCTTT TTATATGTGG 1600

GTTTTATACA TAATTTTCAA GAACTTATAA TGTCCAGTTT 1640

CAGATAAATG AATTGAAACG AATTTGTTAT AAAAAAAAAA 1680

AAAAAAAAAA AAAAA 1695 (2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 332 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 9 :

10

Asp Phe Leu Arg Arg Ser lie Pro Phe Leu Met His 20 Asn Pro Asn lie Ala Leu Val Gin Ala Arg Trp Arg

30 Phe Val Asn Ser Asp Glu Cys Leu Leu Thr Arg Met

40 Gin Glu Met Ser Leu Asp Tyr His Phe Thr Val Glu 50 60

Gin Glu Val Gly Ser Ser Thr His Ala Phe Phe Gl

70 Phe Asn Gly Thr Ala Gly lie Trp Arg lie Ala Ala

80 lie Asn Glu Ala Gly Gly Trp Lys Asp Arg Thr Thr

90 Val Glu Asp Met Asp Leu Ala Val Arg Ala Ser Leu

100 Arg Gly Trp Lys Phe Leu Tyr Leu Gly Asp Leu Gin 110 120

Val Lys Ser Glu Leu Pro Ser Thr Phe Arg Ala Phe

130 Arg Phe Gin Gin His Arg Trp Ser Cys Gly Pro Ala

140 Asn Leu Phe Arg Lys Met Val Met Glu lie Val Arg

150 Asn Lys Lys Val Arg Phe Trp Lys Lys Val Tyr Val

160 •lie Tyr Arg Phe Phe Phe Val Arg Lys lie lie Ala 170 180

His Trp Val Thr Phe Arg Phe Tyr Cys Val Val Leu

190 Pro Leu Thr lie Leu Val Pro Glu Val Lys Val Pro

200 Met Trp Gly Ser Val Tyr lie Pro Ser lie lie Thr

210 lie Leu Asn Ser Val Gly Thr Pro Arg Ser lie His

220 Leu Leu Phe Tyr Trp lie Leu Phe Glu Asn Val Met 230 240

Ser Leu His Arg Thr Lys Ala Thr Leu lie Gly Leu

250 Phe Glu Ala Gly Arg Ala Asn Glu Cys Val Val Thr

260 Ala Lys Leu Gly Ser Gly Gin Ser Gly Lys Gly Asn

270 Asn Lys Gly lie Lys Arg Phe Pro Arg lie Phe Lys

280 Leu Pro Asp Arg Leu Asn Thr Leu Glu Leu Gly Phe 290 300

Ala Ala Phe Leu Phe Val Cys Gly Cys Tyr Asp Phe

310 Val His Gly Lys Asn Asn Tyr Phe lie Tyr Leu Phe

320 Leu Gin Thr Met Ser Phe Phe lie Ser Gly Leu Gly

330 Trp lie Gly Thr Tyr Val Pro Ser

(2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 169 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

10 Lys lie Val Val His lie Phe Thr Phe Val Phe Tyr

20 Cys Leu lie Leu Pro Thr Thr Val Leu Phe Pro Glu

30 Leu Gin Val Pro Lys Trp Ala Thr Val Tyr Phe Pro

40 Thr Thr lie Thr lie Leu Asn Ala lie Ala Thr Pro 50 60

Arg Ser Leu His Leu Leu Val Phe Trp lie Leu Phe

70 Glu Asn Val Met Ser Met His Arg Thr Lys Ala Thr

80 Phe lie Gly Leu Leu Glu Ala Gly Arg Val Asn Glu

90 Trp Val Val Thr Glu Lys Leu Gly Asp Thr Leu Lys

100 Ser Lys Leu lie Gly Lys Ala Thr Thr Lys Leu Tyr 110 120

Thr Arg Phe Gly Gin Arg Leu Asn Trp Arg Glu Leu

130 Val Val Gly Leu Tyr He Phe Phe Cys Gly Cys Tyr

140 Asp Phe Ala Tyr Gly Gly Ser Tyr Phe Tyr Val Tyr

150 Leu Phe Leu Gin Ser Cys Ala Phe Phe Val Ala Gly

160 Val Gly Tyr He Gly Thr Phe Cys Ser Asn Cys Leu

Asp

(2) INFORMATION FOR SEQ ID NO: 11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 273 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

10 Ser Leu Gly Ser Thr Glu Leu Leu Ala Ser Gly Glu

20 Ser Ser Ala Leu Asn Glu Ser Gly Gly Trp Asn Asp

30 Gin Thr Thr Val Glu Asp Met Asp Leu Ala Val Arg

40 Ala Thr Leu Arg Gly Trp Lys Phe Leu Tyr He Asp 50 60

Asp Leu Lys Val Lys Ser Glu Leu Pro Cys Ser Phe

70 Lys Ala Leu Arg Ser Gin Gin His Arg Trp Thr Cys

80 Gly Pro Ala Asn Leu Leu Arg Lys Met Ala Gly Gin

90 He He Arg Ser Glu Asn Val Ser Leu Trp Lys Lys 100 Trp Tyr Met Leu Tyr Ser Phe Phe Phe Met Arg Lys 110 120

He Val Ala His He Leu Thr Phe Cys Phe Tyr Cys

130 Val He Leu Pro Ala Thr Val Leu Phe Pro Glu Val

140 Thr Val Pro Lys Trp Ala Ala Phe Tyr Leu Pro Ser

150 Leu He Thr Leu Leu He Ala He Gly Arg Leu Arg

160 Ser He His Leu Leu Ala Phe Trp Val Leu Phe Glu 170 180

Asn Ala Met Ser Leu Leu Arg Ala Lys Ala Leu Val

190 Met Gly Leu Phe Glu Thr Gly Arg Val Gin Glu Trp

200 Val Val Thr Glu Lys Leu Gly Asp Thr Leu Lys Thr

210 Lys Leu He Pro Gin Val Pro Asn Val Arg Phe Arg

220 Glu Arg Val His Leu Leu Glu Leu Leu Val Gly Ala 230 240

Tyr Leu Leu Phe Cys Gly He Tyr Asp He Val Tyr

250 Gly Lys Asn Thr Leu Tyr Val Tyr Leu Leu Phe Gin

260 Ser Val Ala Phe Phe Val Val Gly Phe Gly Phe Val

270 Gly Asn Met Phe Leu Leu Pro Leu Thr

(2) INFORMATION FOR SEQ ID NO: 12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 154 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

10 Leu Leu Pro Ser Pro Lys Ser Phe Pro Phe He Val

20 Pro Tyr Leu Leu Phe Glu Asn Thr Met Ser Val Thr

30 Lys Phe Asn Ala Met Val Ser Gly Leu Phe Gin Leu

40 Gly Ser Ser Tyr Glu Trp He Val Thr Lys Lys Ala 50 60

Gly Arg Ser Ser Glu Ser Asp Leu Leu Ser He Thr

70 Glu Lys Glu Thr Pro Thr Lys Lys Ser Gin Leu Leu

80 Arg Gly Val Ser Asp Ser Glu Leu Leu Glu Leu Ser

90 Gin Leu Glu Glu Gin Lys Gin Ala Val Ser Lys Lys

100 Pro Val Lys Lys Thr Asn Lys He Tyr His Lys Glu 110 120

Leu Ala Leu Ala Phe Leu Leu Leu Thr Ala Ala Leu 130 Arg Ser Leu Leu Ala Ala Gin Gly Val His Phe Tyr

140 Phe Leu Leu Phe Gin Gly Val Thr Phe Leu Leu Val

150 Gly Leu Asp Leu He Gly Glu Gin Met Ser

(2) INFORMATION. FOR SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 499 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

10 N- Trp He Lys Phe Lys Lys He Glu Pro Lys Leu Thr

20 Glu Glu Ser He Asp Leu Glu Asp Pro Ser Ser Phe

30 Pro Met Val Leu He Gin He Pro Met Cys Asn Glu

40 Arg Glu Val Tyr Glu Gin Ser He Gly Ala Ala Ser 50 60

Gin Leu Asp Trp Pro Lys Asp Arg He Leu He Gin

70 Val Leu Asp Asp Ser Asp Asp Pro Asn Leu Gin Leu

80 Leu He Lys Glu Glu Val Ser Val Trp Ala Glu Lys

90 Gly Val Asn He He Tyr Arg His Arg Leu He Arg

100 Thr Gly Tyr Lys Ala Gly Asn Leu Lys Ser Ala Met 110 120

Thr Cys Asp Tyr Val Lys Asp Tyr Glu Phe Val Thr

130 He Phe Asp Ala Asp Phe Thr Pro Asn Pro Asp Phe

140 Leu Lys Lys Thr Val Pro His Phe Lys Gly Asn Pro

150 Glu Leu Gly Leu Val Gin Ala Arg Trp Ser Phe Val

160 Asn Lys Asp Glu Asn Leu Leu Thr Arg Leu Gin Asn 170 180

He Asn Leu Cys Phe His Phe Glu Val Glu Gin Gin

190 Val Asn Gly Val Phe Leu Asn Phe Phe Gly Phe Asn

200 Gly Thr Ala Gly Val Trp Arg He Lys Ala Leu Glu

210 Glu Ser Gly Gly Trp Leu Glu Arg Thr Thr Val Glu

220 Asp Met Asp He Ala Val Arg Ala His Leu Asn Gly 230 240

Trp Lys Phe He Tyr Leu Asn Asp Val Glu Val Thr

250 Cys Glu Leu Pro Glu Ser Tyr Glu Ala Tyr Lys Lys

260 Gin Gin His Arg Trp His Ser Gly Pro Met Gin Leu 270 Phe Arg Leu Cys Leu Pro Ser He He Lys Ser Lys

280 He Ser Val Trp Lys Lys Ala Asn Leu He Phe Leu 290 300

Phe Phe Leu Leu Arg Lys Leu He Leu Pro Phe Tyr

310 Ser Phe Thr Leu Phe Cys He He Leu Pro Leu Thr

320 Met Phe He Pro Glu Ala Glu Leu Pro Leu Trp He

330 He Cys Tyr Val Pro He Phe He Ser Leu Leu Asn

340 He Leu Pro Ser Pro Lys Ser Phe Pro Phe Leu Val 350 360

Pro Tyr Leu Leu Phe Glu Asn Thr Met Ser He Thr

370 Lys Phe Asn Ala Met He Ser Gly Leu Phe Gin Phe

380 Gly Ser Ala Tyr Glu Trp Val Val Thr Lys Lys Thr

390 Gly Arg Ser Ser Glu Ser Asp Leu Leu Ala Phe Ala

400 Glu Lys Glu Glu Lys Leu His Arg Arg Asn Ser Glu 410 420

Ser Gly Leu Glu Leu Leu Ser Lys Leu Lys Glu Gin

430 Glu Thr Asn Leu Val Gly Gin Glu Thr Val Lys Lys

440 Ser Leu Gly Gly Leu Met Arg Pro Lys Asn Lys Lys

450 Lys Thr Asn Met Val Phe Lys Lys Glu Leu Gly Leu

460 Ala Phe Leu Leu Leu Thr Ala Ala Ala Arg Ser Phe 470 480

Leu Ser Ala His Gly Leu His Phe Tyr Phe Leu Leu

490 Phe Gin Gly Leu Ser Phe Leu Val Val Gly Leu Asp

Leu He Gly Glu Gin He Ser

(2) INFORMATION FOR SEQ ID NO: 14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 726 nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:

AGTCACCAAA AAGCTAGGGA GATCCTCTGA GGCGGATCTG 40

GTTGCATACG CAGAGTCCGG CTCTTTGGTT GAGTCCACAA 80

CCATCCAACG ATCATCCTCT GATTCAGGTC TGACCGAGCT 120

TAGCAAACTA GGAGCAGCAA AGAAAGCTGG CAAAACCAAA 160

AGAAACCGTC TGTACAGAAC GGAAATCGCA CTCGCGTTTA 200 TCCTCTTGGC AGCCTCGGTG AGAAGCTTGT TGTCTGCGCA 240

AGGGATCCAT TTCTATTTCC TCTTGTTCCA AGGAATCACG 280

TTCGTTATTG TCGGTCTAGA TTTGATCGGG GAACAGGTCA 320

GTTAGTTTAT ATAACCCTTT TTCTCAGTTT AAAACTACAT 360

TGAACCATAC CACCTGAAGA ACGAACAAGA CCAAAAGATC 400

AAGTCAGGAA GGAACGAACC AACTCACTCG CAGGTCTTTT 440

CGTTACAGAT AGATACATAA AAACAATTCA TTTAAAGTTT 480

CTGTCTTGGG AACATGATTT TATACTCTAC TTGGGTTGGT 520

TTAGATTCTT CCTTTTTTTA CGGATAGTAT TAGAGAATGG 560

CAGAAACAAC CGTCGGAGAG GATTCCAAAG ACTTGCCTAC 600

CTGTCTTTGT TGTGGTATTA AGCCAAACCT TGGTTTTTTT 640

GTTGTGCAAA AATTGTTGTT AGGGTGTTGG TTTTTTGTTT 680

CGGATGATGT GGCAGAAACA TATTTTAAGC TTTGCTCTAA 720

TGCCACTCTC AAATTCTTTG TTAAAAAAAA AAAAAAAAAA 760

AA 762 (2) INFORMATION FOR SEQ ID NO: 15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 107 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:

10 Val Thr Lys Lys Leu Gly Arg Ser Ser Glu Ala Asp

20 Leu Val Ala Tyr Ala Glu Ser Gly Ser Leu Val Glu

30 Ser Thr Thr He Gin Arg Ser Ser Ser Asp Ser Gly

40 Leu Thr Glu Leu Ser Lys Leu Gly Ala Ala Lys Lys 50 60

Ala Gly Lys Thr Lys Arg Asn Arg Leu Tyr Arg Thr

70 Glu He Ala Leu Ala Phe He Leu Leu Ala Ala Ser

80 Val Arg Ser Leu Leu Ser Ala Gin Gly He His Phe

90 Tyr Phe Leu Leu Phe Gin Gly He Thr Phe Val He

100 Val Gly Leu Asp Leu He Gly Glu Gin Val Ser

(2) INFORMATION FOR SEQ ID NO: 16: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:

CGAACCTAAGCTCACCGAAGA 21

(2) INFORMATION FOR SEQ ID NO: 17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: TTCGCCTTCTTCCAAACC 18

(2) INFORMATION FOR SEQ ID NO: 18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:

GCCAGCTTTGTAACCAGTTCT 1

(2) INFORMATION FOR SEQ ID NO: 19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:

ATTAACCCTCACTAAAG 17

(2) INFORMATION FOR SEQ ID NO: 20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2480 nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:

TTCAGCACAT GATCCTCGTC TCTCTTTCTT TCTCTCTCTC 40 AATCACAAGT TTCAGTTACA CAGCTGAACC CAAAGTATCT 80 CACATTCTGA TCTAATCAGA ACACTGTTTT GTCTGTGTAA 120

CATAAACAAA CGATCCTGTT TTTATGGAAA ATTTAATGAA 160

ACAGTGACGA TTCTTCTACA CTTCTTTATA ATGACAAACA 200

TTTCATAGTA CCAAAATACA CAAATACCTT TTCTTTATTC 240

TTCTCACCCT TCTCTGTTTC TTTATCATTC ATGGCTCCAA 280

ATTCAGTAGC AGTGACAATG GAGAAGCCAG ACAACTTCTC 320

TTTACTAGAG ATCAACGGCT CAGATCCATC CTCATTCCCC 360

GACAAACGTA AATCCATCAG TCCAAAACAA TTCTCATGGT 400

TCCTTCTTCT CAAAGCTCAT AGACTCATCT CGTGTCTCTC 440

GTGGCTAGTC TCTTCGGTTA AAAAGCGAAT CGCGTTCTCC 480

GCGAAGAACA TTAACGAAGA AGAAGATCCT AAAAGCAGAG 520

GAAAACAAAT GTACAGATTC ATCAAAGCTT GTCTTGTCAT 560

CTCCATTATT GCCTTGTCCA TAGAAATCGT TGCACATTTC 600

AAGAAATGGA ATCTTGATCT CATTAACCGA CCGTCTTGGG 640

AGGTTTACGG GCTTGTCGAA TGGTCGTACA TGGCTTGGCT 680

CTCGTTTCGA TCCGATTACA TCGCTCCTCT TGTCATCAGT 720

CTCTCCAGAT TCTGCACTGT ACTCTTTTTG ATTCAGTCTC 760

TTGATCGGTT AGTCCTCTGT CTCGGTTGCT TTTGGATCAA 800

ATTTAAAAAG ATCGAACCTA AGCTCACCGA AGAATCTATC 840

GATTTAGAAG ACCCCTCCAG TTTCCCAATG GTCCTTATTC 880

AGATCCCAAT GTGCAACGAA CGAGAGGTGT ATGAACAATC 920

AATAGGAGCA GCTTCACAAC TTGATTGGCC AAAAGATAGG 960

ATCTTAATTC AAGTATTAGA TGATTCAGAC GATCCAAATT 1000

TACAGCTTTT GATCAAAGAA GAAGTATCGG TTTGGGCCGA 1040

AAAAGGCGTA AACATAATTT ACAGGCATAG GTTGATCAGA 1080

ACTGGTTACA AAGCTGGCAA TTTGAAATCA GCCATGACTT 1120

GTGATTACGT TAAAGATTAC GAGTTTGTGA CTATCTTCGA 1160

CGCAGATTTC ACACCAAATC CTGATTTCCT CAAGAAGACT 1200

GTTCCTCATT TCAAGGGTAA TCCAGAGCTA GGGTTGGTCC 1240

AAGCAAGGTG GTCATTTGTG AACAAAGATG AGAATCTCCT 1280

CACGAGGCTA CAAAACATAA ACTTATGTTT CCACTTCGAA 1320 GTAGAACAAC AAGTGAACGG TGTGTTTCTC AATTTCTTCG 1360

GATTCAATGG AACCGCTGGA GTATGGAGGA TCAAGGCATT 1400

GGAAGAATCC GGCGGATGGC TCGAGAGAAC CACCGTGGAA 1440

GATATGGATA TCGCGGTTAG AGCGCATCTC AACGGCTGGA 1480

AGTTTATTTA CCTTAATGAT GTTGAAGTCA CTTGCGAGTT 1520

GCCAGAGTCT TATGAAGCTT ACAAGAAGCA ACAACATCGT 1560

TGGCATTCCG GTCCTATGCA GCTGTTCCGG TTATGCCTTC 1600

CTTCAATTAT CAAATCAAAG ATATCGGTTT GGAAGAAGGC 1640

GAATTTGATC TTCCTTTTCT TTCTTCTAAG GAAGCTTATT 1680

CTACCATTTT ACTCATTCAC ACTCTTTTGC ATTATACTTC 1720

CATTGACAAT GTTCATACCC GAAGCCGAGC TTCCGTTGTG 1760

GATCATCTGC TATGTTCCTA TCTTCATTTC GCTTCTCAAC 1800

ATTCTCCCGT CACCTAAATC TTTCCCTTTC TTAGTCCCTT 1840

ACCTTCTTTT CGAAAACACA ATGTCAATAA CCAAGTTCAA 1880

CGCCATGATC TCCGGGCTGT TTCAGTTTGG ATCGGCTTAC 1920

GAGTGGGTTG TGACGAAGAA AACCGGGAGA TCATCGGAGT 1960

CCGATTTGCT AGCGTTTGCT GAAAAGGAAG AGAAGTTGCA 2000

TAGGAGAAAC TCGGAGTCAG GTTTGGAGCT TCTGAGCAAA 2040

CTTAAGGAGC AAGAGACAAA TCTTGTAGGG CAAGAAACCG 2080

TGAAGAAGAG CCTTGGAGGG CTAATGAGGC CGAAGAACAA 2120

GAAGAAGACG AACATGGTGT TCAAGAAAGA GCTCGGGCTT 2160

GCGTTCTTGC TGCTAACCGC AGCTGCAAGG AGCTTTCTAT 2200

CGGCGCACGG TCTTCACTTC TACTTTTTGT TGTTTCAGGG 2240

ACTGTCTTTC TTGGTTGTAG GGTTGGATTT GATCGGAGAA 2280

CAGATCAGCT AGACACACAC AAAACCAAGA CAGAGAACAA 2320

GGAAAAAAAT ACCGTTTATA TTCACTATTT TTTTGTTTGT 2360

TTTTTAAATT CTTTTCCCTT TTTATATGTG GGTTTTATAC 2400

ATAATTTTCA AGAACTTATA ATGTCCAGTT TCAGATAAAT 2440

GAATTGAAAC GAATTTGTTA TAAAAAAAAA AAAAAAAAAA 2480 (2) INFORMATION FOR SEQ ID NO: 21: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 673 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:

Met Ala Pro Asn Ser Val Ala Val Thr Met Glu Lys

20 Pro Asp Asn Phe Ser Leu Leu Glu He Asn Gly Ser

30 Asp Pro Ser Ser Phe Pro Asp Lys Arg Lys Ser He

40 Ser Pro Lys Gin Phe Ser Trp Phe Leu Leu Leu Lys 50 60

Ala His Arg Leu He Ser Cys Leu Ser Trp Leu Val

70 Ser Ser Val Lys Lys Arg He Ala Phe Ser Ala Lys

80 Asn He Asn Glu Glu Glu Asp Pro Lys Ser Arg Gly

90 Lys Gin Met Tyr Arg Phe He Lys Ala Cys Leu Val

100 He Ser He He Ala Leu Ser He Glu He Val Ala 110 120

His Phe Lys Lys Trp Asn Leu Asp Leu He Asn Arg

130 Pro Ser Trp Glu Val Tyr Gly Leu Val Glu Trp Ser

140 Tyr Met Ala Trp Leu Ser Phe Arg Ser Asp Tyr He

150' Ala Pro Leu Val He Ser Leu Ser Arg Phe Cys Thr

160 Val Leu Phe Leu He Gin Ser Leu Asp Arg Leu Val 170 180

Leu Cys Leu Gly Cys Phe Trp He Lys Phe Lys Lys

190 He Glu Pro Lys Leu Thr Glu Glu Ser He Asp Leu

200 Glu Asp Pro Ser Ser Phe Pro Met Val Leu He Gin

210 He Pro Met Cys Asn Glu Arg Glu Val Tyr Glu Gin

220 Ser He Gly Ala Ala Ser Gin Leu Asp Trp Pro Lys 230 240

Asp Arg He Leu He Gin Val Leu Asp Asp Ser Asp

250 Asp Pro Asn Leu Gin Leu Leu He Lys Glu Glu Val

260 Ser Val Trp Ala Glu Lys Gly Val Asn He He Tyr

270 Arg His Arg Leu He Arg Thr Gly Tyr Lys Ala Gly

280 Asn Leu Lys Ser Ala Met Thr Cys Asp Tyr Val Lys 290 300

Asp Tyr Glu Phe Val Thr He Phe Asp Ala Asp Phe

310 Thr Pro Asn Pro Asp Phe Leu Lys Lys Thr Val Pro

320 His Phe Lys Gly Asn Pro Glu Leu Gly Leu Val Gin 330

Ala Arg Trp Ser Phe Val Asn Lys Asp Glu Asn Leu

340 Leu Thr Arg Leu Gin Asn He Asn Leu Cys Phe His 350 360

Phe Glu Val Glu Gin Gin Val Asn Gly Val Phe Leu

370 Asn Phe Phe Gly Phe Asn Giy Thr Ala Gly Val Trp

380 Arg He Lys Ala Leu Glu Glu Ser Gly Gly Trp Leu

390 Glu Arg Thr Thr Val Glu Asp Met Asp He Ala Val

400 Arg Ala His Leu Asn Gly Trp Lys Phe He Tyr Leu 410 420

Asn Asp Val Glu Val Thr Cys Glu Leu Pro Glu Ser

430 Tyr Glu Ala Tyr Lys Lys Gin Gin His Arg Trp His

440 Ser Gly Pro Met Gin Leu Phe Arg Leu Cys Leu Pro

450 Ser He He Lys Ser Lys He Ser Val Trp Lys Lys

460 Ala Asn Leu He Phe Leu Phe Phe Leu Leu Arg Lys 470 480

Leu He Leu Pro Phe Tyr Ser Phe Thr Leu Phe Cys

490 He He Leu Pro Leu Thr Met Phe He Pro Glu Ala

500 Glu Leu Pro Leu Trp He He Cys Tyr Val Pro He

510 Phe He Ser Leu Leu Asn He Leu Pro Ser Pro Lys

520 Ser Phe Pro Phe Leu Val Pro Tyr Leu Leu Phe Glu 530 540

Asn Thr Met Ser He Thr Lys Phe Asn Ala Met He

550 Ser Gly Leu Phe Gin Phe Gly Ser Ala Tyr Glu Trp

560 Val Val Thr Lys Lys Thr Gly Arg Ser Ser Glu Ser

570 Asp Leu Leu Ala Phe Ala Glu Lys Glu Glu Lys Leu

580 His Arg Arg Asn Ser Glu Ser Gly Leu Glu Leu Leu 590 600

Ser Lys Leu Lys Glu Gin Glu Thr Asn Leu Val Gly

610 Gin Glu Thr Val Lys Lys Ser Leu Gly Gly Leu Met

620 Arg Pro Lys Asn Lys Lys Lys Thr Asn Met Val Phe

630 Lys Lys Glu Leu Gly Leu Ala Phe Leu Leu Leu Thr

640 Ala Ala Ala Arg Ser Phe Leu Ser Ala His Gly Leu 650 660

His Phe Tyr Phe Leu Leu Phe Gin Gly Leu Ser Phe

670 Leu Val Val Gly Leu Asp Leu He Gly Glu Gin He

Ser (2) INFORMATION FOR SEQ ID NO: 22:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22: ATAACGGTCGGTACGGGATTTTCC 24

(2) INFORMATION FOR SEQ ID NO:23:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:

CGCAAGTGACTTCAACATCAT 21

(2) INFORMATION FOR SEQ ID NO: 24:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 368 nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24

AGAACAACTG TTGAGGATAT GGATATAGCA GTCCGTGCGC 40

ATCTCCATGG ATGGAAGTTC ATTTATCTTA ATGATGTCAA 80

GGTCCTTTGT GAAGTTCCTG AGTCCTATGA AGCATATAAG 120

AAGCAGCAAC ACCGTTGGCA TTCAGGACCT ATGCAGCTTT 160

TTCGCCTTTG TCTTGGTGCA ATCTTGACCT CTAAGATAGC 200

AATATGGAAG AAAGCGAATC TAATACTACT CTTCTTCCTT 240

CTAAGGAAAC TCATACTTCC TTTCTACTCC TTCACACTGT 280

TCTGCATAAT CCTTCCTCTC ACCATGTTTG TACCAGAAGC 320

TGAGCTCCCC GTTTGGGTCA TATGCTACAT ACCTGTCTTC 360 ATGTCATT 368

(2) INFORMATION FOR SEQ ID NO: 25:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 122 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:

10 Arg Thr Thr Val Glu Asp Met Asp He Ala Val Arg

20 Ala His Leu His Gly Trp Lys Phe He Tyr Leu Asn

30 Asp Val Lys Val Leu Cys Glu Val Pro Glu Ser Tyr

40 Glu Ala Tyr Lys Lys Gin Gin His Arg Trp His Ser 50 60

Gly Pro Met Gin Leu Phe Arg Leu Cys Leu Gly Ala

70 He Leu Thr Ser Lys He Ala He Trp Lys Lys Ala

80 Asn Leu He Leu Leu Phe Phe Leu Leu Arg Lys Leu

90 He Leu Pro Phe Tyr Ser Phe Thr Leu Phe Cys He

100 He Leu Pro Leu Thr Met Phe Val Pro Glu Ala Glu 110 120

Leu Pro Val Trp Val He Cys Tyr He Pro Val Phe

Met Ser

Claims

What is claimed is:

1. An isolated nucleic acid comprising a nucleotide sequence encoding a xylan synthase with an amino acid sequence identity to SEQ ID NO:21 of 60% or greater.

2. The isolated nucleic acid of Claim 1, wherein the amino acid sequence identity to SEQ ID NO:21 is 80% or greater.

3. An isolated nucleic acid comprising a nucleotide sequence encoding an amino acid sequence of SEQ ID NO:21.

4. An isolated nucleic acid comprising a nucleotide sequence encoding a xylan synthase with a nucleotide sequence identity to SEQ ID NO:20 of 60% or greater.

5. The isolated nucleic acid of Claim 4, wherein the nucleotide sequence identity to SEQ ID NO:20 is 80% or greater.

6. An isolated nucleic acid comprising base 271 to base 2289 of SEQ ID NO:20.

7. An isolated nucleic acid comprising a nucleotide sequence encoding a plant xylan synthase, wherein said nucleotide sequence is obtained from a plant species containing xylan.

8. An isolated nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:7 and SEQ ID NO: 14.

9. An isolated nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

10. An isolated nucleic acid encoding a plant protein selected from the group consisting of Csll, Csl2, Csl3, Csl4, Csl5, Csl6 and Csl7.

11. The isolated nucleic acid of Claim 10, wherein the plant protein is selected from the group consisting of CSL1, CSL2, CSL3, CSL4, CSL5, CSL6 and CSL7.

12. A chimeric nucleic acid comprising a nucleotide sequence encoding a plant xylan synthase operably linked to a suitable regulatory sequence that transcribes plant xylan synthase in a host cell.

13. A chimeric nucleic acid comprising a nucleotide sequence of SEQ ID NO:20 operably linked to a suitable regulatory sequence that transcribes plant xylan synthase in a host cell.

14. A chimeric nucleic acid comprising a nucleotide sequence encoding an amino acid sequence of SEQ ID NO:21 operably linked to a suitable regulatory sequence that transcribes plant xylan synthase in a host cell.

15. A chimeric nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:7 and SEQ ID NO: 14 operably linked to a suitable regulatory sequence that transcribes the nucleotide sequence in a host cell.

16. A chimeric nucleic acid comprising a nucleotide sequence encoding a plant protein selected from the group consisting of Csll, Csl2, Csl3, Csl4, Csl5, Cslό and Csl7 operably linked to a suitable regulatory sequence that transcribes the nucleotide sequence in a host cell.

17. A chimeric nucleic acid comprising a nucleotide sequence selected from the group consisting of CSLl , CSL2, CSL3, CSL4, CSL5, CSL6 and CSL7 operably linked to a suitable regulatory sequence that transcribes the nucleotide sequence in a host cell.

18. A microbe containing the chimeric nucleic acid as in any one of Claims 10-17.

19. A plant containing the chimeric nucleic acid as in any one of Claims 10-17.

20. A seed obtained from the plant of Claim 19.

21. Cell wall obtained from the plant of Claim 19.

22. Fiber or wood obtained from the plant of Claim 19.

23. A chimeric nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 operably linked to a suitable regulatory sequence that transcribes the nucleotide sequence in a host cell.

24. A plant containing the chimeric nucleic acid of Claim 23.

25. A seed obtained from the plant of Claim 24.

26. Cell wall obtained from the plant of Claim 24.

27. Fiber or wood obtained from the plant of Claim 24.

28. A polypeptide which is a xylan synthase, wherein said xylan synthase is present in a plant species containing xylan.

29. A polypeptide comprising an amino acid sequence of SEQ ID NO:21.

30. A polypeptide with xylan synthase activity comprising an amino acid sequence of SEQ ID NO:21 or a functional derivative thereof.

31. A polypeptide selected from the group consisting of Csll, Csl2, Csl3, Csl4, Csl5, Csl6 and Csl7.

32. A polypeptide encoded by a plant gene selected from the group consisting of CSLl, CSL2, CSL3, CSL4, CSL5, CSL6 and CSL7.

33. A composition comprising the polypeptide as in any one of Claims 28-32.

34. A composition consisting essentially of the polypeptide as in any one of Claims 28- 32.

35. An expression construct which is capable of increasing xylan synthase activity in a host cell.

36. An expression construct which is capable of decreasing xylan synthase activity in a host cell.

37. An expression construct which is capable of increasing transcription of a nucleotide sequence of SEQ ID NO:20 in a host cell.

38. An expression construct which is capable of decreasing transcription of a nucleotide sequence of SEQ ID NO:20 in a host cell.

39. An expression construct which is capable of increasing translation of an amino acid sequence of SEQ ID NO:21 in a host cell.

40. An expression construct which is capable of decreasing translation of an amino acid sequence of SEQ ID NO:21 in a host cell.

41. An expression construct which is capable of increasing transcription of a nucleotide sequence selected from the group consisting of SEQ ID NO:7 and SEQ ID NO: 14 in a host cell.

42. An expression construct which is capable of decreasing transcription of a nucleotide sequence selected from the group consisting of SEQ ID NO:7 and SEQ ID NO: 14 in a host cell.

43. An expression construct which is capable of increasing expression of a plant protein selected from the group consisting of Csll , Csl2, Csl3, Csl4, Csl5, Cslό and Csl7 in a host cell.

44. An expression construct which is capable of decreasing expression of a plant protein selected from the group consisting of Csll, Csl2, Csl3, Csl4, Csl5, Cslό and Csl7 in a host cell.

45. An expression construct which is capable of increasing expression of a plant gene selected from the group consisting of CSLl, CSL2, CSL3, CSL4, CSL5, CSL6 and CSL7 in a host cell.

46. An expression construct which is capable of decreasing expression of a plant gene selected from the group consisting of CSLl , CSL2, CSL3, CSL4, CSL5, CSL6 and CSL7 in a host cell.

47. A microbe containing the expression vector as in any one of Claims 35-46.

48. A plant containing the expression vector as in any one of Claims 35-46.

49. A seed obtained from the plant of Claim 48.

50. Cell wall obtained from the plant of Claim 48.

51. Fiber or wood obtained from the plant of Claim 48.

52. An expression construct which is capable of increasing transcription of a nucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 in a host cell.

53. An expression construct which is capable of decreasing transcription of a nucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 in a host cell.

54. A microbe containing the expression vector as in any one of Claims 52-53.

55. A plant containing the expression vector as in any one of Claims 52-53.

56. A seed obtained from the plant of Claim 55.

57. Cell wall obtained from the plant of Claim 55.

58. Fiber or wood obtained from the plant of Claim 55.

59. A method of altering an amount of xylan in a plant, comprising: (a) transforming a plant cell with a nucleic acid containing a sufficient region of sequence which encodes a xylan synthase to cause increased production of xylan synthase, or reduced production of xylan synthase due to antisense or sense suppression of a corresponding endogenous gene;

(b) growing a plant from the transformed plant cell of step (a); and

(c) identifying a desired plant with an altered amount of xylan by measuring polysaccharide composition of a cell wall from the plant produced as in step (b).

60. The method of Claim 59, wherein said sequence is obtained from a plant selected from the group consisting of plants listed in Tables 1-3.

61. The method of Claim 59, wherein said xylan synthase is encoded by a nucleotide sequence comprising base 271 to base 2289 of SEQ ID NO:20.

62. The method of Claim 59, wherein said xylan synthase is encoded by a nucleotide sequence of SEQ ID NO: 14.

63. The method of Claim 59, wherein said xylan synthase is encoded by a nucleotide sequence of SEQ ID NO:7.

64. A seed of a plant produced as in any one of Claims 59-63.

65. Cell wall of a plant produced as in any one of Claims 59-63.

66. Fiber or wood of a plant produced as in any one of Claims 59-63.

67. A plant selected from the group consisting of those listed in Tables 1-3, wherein the plant contains an integrated expression vector as in any one of Claims 35-46.

68. The plant of Claim 67, wherein the expression vector comprises base 271 to base 2289 of SEQ ID NO:20.

69. The plant of Claim 67, wherein the expression vector comprises SEQ ID NO: 7 or SEQ ID NO: 14.

70. A seed from a plant selected from the group consisting of those listed in Tables 1-3, wherein the plant contains an integrated expression vector as in any one of Claims 35-46.

71. The seed of Claim 70, wherein the expression vector comprises base 271 to base 2289 of SEQ ID NO:20.

72. The seed of Claim 70, wherein the expression vector comprises SEQ ID NO:7 or SEQ ID NO: 14.

73. Cell wall from a plant selected from the group consisting of those listed in Tables 1- 3, wherein the plant contains an integrated expression vector as in any one of Claims 35-46.

74. The cell wall of Claim 70, wherein the expression vector comprises base 271 to base 2289 of SEQ ID NO:20.

75. The cell wall of Claim 70, wherein the expression vector comprises SEQ ID NO: 7 or SEQ ID NO: 14.

76. Fiber or wood from a plant selected from the group consisting of those listed in Tables 1-3, wherein the plant contains an integrated expression vector as in any one of Claims 35-46.

77. The fiber or wood of Claim 70, wherein the expression vector comprises base 271 to base 2289 of SEQ ID NO:20.

78. The fiber or wood of Claim 70, wherein the expression vector comprises SEQ ID NO:7 or SEQ ID NO: 14.

79. A plant selected from the group consisting of those listed in Tables 1-3, wherein the plant contains an integrated expression vector as in any one of Claims 52-53.

80. The plant of Claim 79, wherein the expression vector comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

81. A seed from a plant selected from the group consisting of those listed in Tables 1-3, wherein the plant contains an integrated expression vector as in any one of Claims 52-53.

82. The seed of Claim 81, wherein the expression vector comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

83. Cell wall from a plant selected from the group consisting of those listed in Tables 1- 3, wherein the plant contains an integrated expression vector as in any one of Claims 52-53.

84. The cell wall of Claim 83, wherein the expression vector comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

85. Fiber or wood from a plant selected from the group consisting of those listed in Tables 1-3, wherein the plant contains an integrated expression vector as in any one of Claims 52-53.

86. The fiber or wood of Claim 85, wherein the expression vector comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

87. A method to identify a nucleic acid encoding plant xylan synthase comprising:

(a) identifying conserved region(s) in a sequence alignment of xylan synthases;

(b) synthesizing a probe or primer based on the conserved region(s); and

(c) identifying the nucleic acid encoding plant xylan synthase by hybridization with the probe to a collection of plant-derived nucleic acids or amplification with the primer of a collection of plant-derived nucleic acids.

88. A method to identify a nucleic acid encoding plant xylan synthase comprising

(a) hybridizing a collection of plant-derived nucleic acids with a probe capable of specific binding to a gene selected from the group consisting of CSLl, CSL2, CSL3, CSL4, CSL5, CLS6 and CSL7; and

(b) identifying the nucleic acid encoding plant xylan synthase by a positive hybridization signal.

89. The method of any one of Claims 87-88, wherein the collection of nucleic acids is derived from a plant selected from the group of those listed in Tables 1-3.

90. The method of any one of Claims 87-88 further comprising isolating the identified nucleic acid.

91. The method of Claim 90 further comprising obtaining a full-length cDNA or a genomic clone corresponding to the isolated nucleic acid.

92. The method of Claim 90 further comprising confirmation that the isolated nucleic acid encodes xylan synthase by:

(i) producing a plant containing a chimeric nucleic acid or an expression vector comprised of a sequence of the isolated nucleic acid, and

(ii) detecting an alteration of xylan synthase activity in the plant of step (i).

93. An isolated nucleic acid containing a nucleotide sequence encoding a xylan synthase with an amino acid sequence identity to SEQ ID NO: 15 of 60% or greater.

94. The isolated nucleic acid of Claim 93, wherein the amino acid sequence identity of the encoded polypeptide is 80% or greater.

95. The isolated nucleic acid of Claim 93, wherein the amino acid identity is 100%.

96. An isolated nucleic acid containing a nucleotide sequence encoding a xylan synthase with an amino acid sequence identity to SEQ ID NO: 12 of 60% or greater.

97. The isolated nucleic acid of Claim 96, wherein the amino acid sequence identity of the encoded polypeptide is 80% or greater.

98. The isolated nucleic acid of Claim 96, wherein the amino acid identity is 100% .

99. An isolated nucleic acid containing a nucleotide sequence encoding a xylan synthase with a nucleotide identity to SEQ ID NO: 14 is 75% or greater.

100. The isolated nucleic acid of Claim 99, wherein the nucleotide sequence is SEQ ID NO: 14.

101. An isolated nucleic acid containing a nucleotide sequence encoding a xylan synthase with a nucleotide identity to SEQ ID NO:7 is 75% or greater.

102. The isolated nucleic acid of Claim 101, wherein the nucleotide sequence is SEQ ID NO:7.

103. An isolated nucleic acid containing a nucleotide sequence encoding a glycan synthase with nucleotide sequence identity of 60% or greater to a nucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

104. The isolated nucleic acid of Claim 103, wherein amino acid sequence identity of the encoded glycan synthase is 60% or greater to an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO: 10, and SEQ ID NO: l l .

105. An isolated nucleic acid containing a nucleotide sequence encoding a glycan synthase with nucleotide sequence identity of 80% or greater to a nucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

106. The isolated nucleic acid of Claim 105, wherein amino acid sequence identity of the encoded glycan synthase is 80% or greater to an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO: l l.