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WO1998055596A1 - Utilisation des genes codant la xylane-synthase pour la modification de la composition de la paroi cellulaire des vegetaux - Google Patents

Utilisation des genes codant la xylane-synthase pour la modification de la composition de la paroi cellulaire des vegetaux Download PDF

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WO1998055596A1
WO1998055596A1 PCT/US1998/011531 US9811531W WO9855596A1 WO 1998055596 A1 WO1998055596 A1 WO 1998055596A1 US 9811531 W US9811531 W US 9811531W WO 9855596 A1 WO9855596 A1 WO 9855596A1
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seq
plant
xylan
nucleic acid
nucleotide sequence
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Chris Somerville
Sean Cutler
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8245—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis
    • C12N15/8246—Non-starch polysaccharides, e.g. cellulose, fructans, levans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • 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.
  • the present invention describes methods and materials for increasing or decreasing the xylan content of plants.
  • 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.
  • xylans 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).
  • 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.
  • 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.
  • Table 1 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).
  • 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.
  • Plant cell walls are primarily composed of polysaccharides encompassing cellulose microfibrils and matrix components (McNeil et al., 1984; Bacic et al. , 1988).
  • Cellulose (j ⁇ l-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).
  • xyloglucan 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.
  • 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.
  • NDP nucleoside- diphospho
  • cyclic-di-GMP cyclic-di-GMP
  • cyclic-di-GMP 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).
  • Acetobacter xylinum AcsA shares a common domain structure with a group of bacterial and eukaryotic proteins that function in the synthesis of diverse ⁇ -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.
  • non- cellulosic plant cell wall polysaccharides e.g., xylan and others such as xyloglucan, glucomannan, galactomannan, callose, galactan and mannan
  • xylan and others such as xyloglucan, glucomannan, galactomannan, callose, galactan and mannan
  • beta-glycosidic linkages are synthesized by enzymes that exhibit structural similarity to plant and bacterial cellulose synthases.
  • 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.
  • 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.
  • 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-) ⁇ -D-glucopyranosyluronic acid residues, most frequently attached by 1-2 linkages to D-xylose units in the backbone; (b) single ⁇ -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.
  • xylans notably the 4-0- methylglucuronoxylans from hardwoods
  • xylans notably the 4-0- methylglucuronoxylans from hardwoods
  • 0-Acetyl groups are attached only to xylose residues.
  • Studies on partially acetylated 4-0-substituted ⁇ -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.
  • 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.
  • xylans are undesirable components of plant cell walls that are used for the production of cellulose fibers
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • enzymes with homologous sequences catalyze similar reactions rather than identical reactions.
  • 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.
  • 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.
  • 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.
  • 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.
  • nucleic acids e.g. , DNA, RNA, cDNA and genomic recombinant clones, expression vectors
  • this invention is directed to recombinant constructs which can provide for the transcription, or transcription and translation (expression) of the plant xylan synthase sequences.
  • 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.
  • this invention relates to the presence of such constructs in host cells, especially plant host cells which have an expressed plant xylan synthase therein.
  • 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.
  • 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.
  • 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.
  • 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).
  • FIG. 1 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 7 Diagram of plasmid used to produce antisense plants.
  • BNAF03353 to Arabidopsis Csl4 protein.
  • the two sequences were 95% similar and 86% identical.
  • GenBank nucleotide sequence of EST 160C16T7 1. Full-length nucleotide sequence of EST clone 160C16T7 obtained for this application.
  • 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.
  • nucleic acids e.g., DNA, RNA
  • 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.
  • xylan is meant any polysaccharide containing more than three D-xylose residues linked to each other in a 0-1-4 linkage.
  • 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.
  • xylan synthase are obtainable from the specific exemplified sequences provided herein.
  • the enzymes that synthesize xylan in trees and other plants are structurally similar to the xylan synthase of this invention.
  • 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.
  • the enzymes that produce galactans, mannans, and xyloglucans are structurally related to the xylan synthases of this invention.
  • 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.
  • nucleic acid probes DNA and RNA
  • nucleic acid probes are labeled to allow detection, preferably with radioactivity although enzymes or other methods may also be used.
  • antibody preparations either monoclonal or polyclonal, may be utilized.
  • Polyclonal antibodies although less specific, typically are more useful in gene isolation.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • genes become available for the enzymes that catalyze modification of xylans, many different xylans will be produced in transgenic plants.
  • 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.
  • polymerase chain reaction primers based on the conserved regions of amino acid sequence between the xylan synthase of this invention and cellulose synthases.
  • 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.
  • 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.
  • a comparison between xylan synthases and cellulose synthase or other polysaccharide synthases may yield insights for protein modeling or other modifications to create synthetic xylan synthases.
  • a cDNA clone encoding a xylan synthase may be used to obtain its corresponding genomic nucleic acids.
  • 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.
  • encoding is meant that the sequence corresponds to a particular amino acid sequence either in a sense or anti-sense orientation.
  • 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.
  • 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.
  • a probe such as a cDNA probe
  • 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.
  • 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.
  • 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.
  • the desired plant xylan synthase nucleotide sequence 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the regulatory regions will vary, including regions from viral, plasmid or chromosomal genes, or the like.
  • a wide variety of constitutive or regulatable promoters may be employed.
  • Expression in a microorganism can provide a ready source of the plant enzyme.
  • 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).
  • 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.
  • 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.
  • a transcription initiation regulatory region such as the wild-type sequence naturally found 5' upstream to the xylan synthase structural gene.
  • similar constructs to those that produce overexpression are used. Plants which exhibit cosuppression are identified by screening the transgenic plants produced.
  • 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.
  • transcription initiation regions are available which provide for a wide variety of constitutive or regulatable (e.g., inducible) transcription of the structural gene.
  • 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.
  • 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.
  • a different promoter 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.
  • 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.
  • 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.
  • 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.
  • a convenient cloning vector which is capable of replication in a bacterial host, e.g., E. coli.
  • 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.
  • the construct 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.
  • 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.
  • a cytotoxic agent e.g. , antibiotic, heavy metal, toxin, etc.
  • complementation providing prototropy to an auxotrophic host, viral immunity, or the like.
  • one or more markers may be employed, where different conditions for selection are used for the different hosts.
  • nucleic acids are introduced into the plant host. 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.
  • 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.
  • 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.
  • explants 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.
  • 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.
  • 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).
  • a fusion peptide e.g. , avidin, glutathione S-transferase, poly-His, maltose binding protein, myc 9E10-epitope, protein A7G, SV40 T antigen.
  • genes for xylan synthases of this invention 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.
  • populations of mutagenized plants can be screened by PCR-based methods for single nucleotide changes that alter the function of the xylan synthases.
  • 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.
  • 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.
  • 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.
  • 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.
  • 160C16T7 is presented in SEQ ID NO: l .
  • 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).
  • 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.
  • 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.
  • 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.
  • CELA1 and CELA2 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.
  • pCSL4 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 ⁇ ZAPII (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.
  • 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.
  • an oligonucleotide primer based on the sequence of Csl4 (oligonucleotide P3, SEQ ⁇ D 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).
  • 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.
  • cellulose Biosynthesis is the formation of a 0-1,4 glucose linkage
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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 ⁇ l of 9M NH 3 followed by reaction with 1 ml of 2% NaBH 4 in DMSO. The reduction was carried out for 90 min at 40°C. 250 ⁇ l 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 ⁇ l 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 H 2 O to each reaction mix. The alditol acetates were extracted from each reaction mix by the addition of 1.5 ml CH 2 C1 2 . The organic phase was collected and transferred to a fresh tube. The CH 2 C1 2 was evaporated off at 55 °C in a water bath. Hydrophilic contaminants were extracted from the remaining residue by the addition of 1 ml H 2 O.
  • the organic residue from each reaction was extracted into 250 ul CH 2 C1 2 , 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 ⁇ M 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.
  • 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.
  • 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).
  • 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 ⁇ l 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 Ar 2 flow.
  • the sugar lithium salts will be methylated by the addition of 500 ⁇ l CH 3 I 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 N 2 .
  • 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.
  • 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).
  • 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.
  • plant promoter sequences which may be used to cause tissue-specific expression of cloned genes in transgenic plants.
  • 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.
  • 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_.
  • pSCl was transformed into Agrobacterium tumefaciens strain GV3101 by electroporation.
  • Strain GV3101 (Koncz and Schell, 1986) contains a disarmed Ti plasmid.
  • 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 ⁇ l cells and 1 ⁇ l 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).
  • 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 ⁇ g/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).
  • 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 ⁇ mol 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.
  • a mixture of 50 ⁇ g each of L-rhamnose, L-arabinose, D-xylose, D-mannose, D-galactose, and D-glucose was autoclaved in 250 ⁇ L of 1 M H 2 SO 4 .
  • cell wall residues were hydrolyzed for one hour at 121 °C in 250 ⁇ l each of 1 M H 2 SO 4 .
  • 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 ⁇ l of 9M NH 3 . The reduction was carried out for one hour at 40°C. 250 ⁇ l 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 ⁇ l 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 H 2 O to each reaction mix.
  • the alditol acetates were extracted from each reaction mix by the addition of 1.5 ml CH 2 C1 2 .
  • the organic ' phase was collected and transferred to a fresh mbe.
  • the CH 2 C1 2 was evaporated off at 55 °C in a water bath. Hydrophilic contaminants were extracted from the remaining residue by the addition of 1 ml H 2 O.
  • the organic residue from each reaction was extracted into 250 ul CH 2 C1 2 , 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
  • an Arabidopsis genomic library made from ecotype Columbia in the vector ⁇ EMBL4 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.
  • 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.
  • 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.
  • 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?].
  • a non-specific promoter such as the CaMV 35S promoter or the ubiquitin promoter will be adequate.
  • 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.
  • 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.
  • 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.
  • a non-specific promoter such as the CaMV 35S promoter or the ubiquitin promoter will be adequate.
  • 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.
  • 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.
  • 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.
  • 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 Csl ⁇ 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 Csl ⁇ .
  • 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).
  • the region corresponds to amino acid 63 to 102 in the partial sequence presented in SEQ ID NO: 12.
  • Csl ⁇ the region corresponds to amino acid 20 to 55 in the partial sequence presented in SEQ ID NO: 15.
  • Arabidopsis contains several genes which are closely related to CSL4 and perform the same enzymatic function.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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 ⁇ M of dNTPs, 50 mM KCl, 10 mM TrisHCl (pH 9), 0.1 %
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 of 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 polymo ⁇ him 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 polymo ⁇ hism, another 1000 mutagenized plants are screened by the same method until 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.
  • 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.
  • Liriodendron tulipifera Yellow-poplar Magnolia acu inata Cucumbertree
  • Salix alba White willow S . nigra Black willow
  • Table 7 ESTs Identified as CSL gene family members and their defined Csl names.
  • AAAAAAAA 1488 INFORMATION FOR SEQ ID NO: 3:
  • Lys Met Thr Met Glu lie lie Phe Asn Lys Arg Val 255 260
  • 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
  • Pro Lys Lys lie Leu Leu Ser Lys Ser Glu Phe Gin 385 390 395
  • TATCCTTAAC GCAATCGCTA CACCTCGATC ACTCCATCTT 160 CTTGTCTTTT GGATCTTATT CGAGAATGTA ATGTCGATGC 200
  • Trp Arg lie Ala Ala
  • AAAAGGCGTA AACATAATTT ACAGGCATAG GTTGATCAGA 1080
  • CACGAGGCTA CAAAACATAA ACTTATGTTT CCACTTCGAA 1320 GTAGAACAAC AAGTGAACGG TGTGTTTCTC AATTTCTTCG 1360

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Abstract

La présente invention se rapporte à des xylane-synthases végétales, à l'isolation des gènes, ou des fragments de gènes, pour des xylane-synthases tirées de Arabidopsis thaliana. Cette invention concerne également l'utilisation des clones d'ADNc codant les xylane-synthases végétales pour modifier la quantité de xylane dans les plantes transgéniques, ainsi que l'utilisation des informations de séquence obtenues à partir du gène de xylane-synthase pour identifier les gènes pour d'autres xylane-synthases végétales.
PCT/US1998/011531 1997-06-03 1998-06-01 Utilisation des genes codant la xylane-synthase pour la modification de la composition de la paroi cellulaire des vegetaux Ceased WO1998055596A1 (fr)

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Cited By (8)

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WO2005071086A1 (fr) * 2004-01-22 2005-08-04 Suzano Papel E Celulose S.A. Methode utilisant des cassettes d'expression genetique pour moduler genetiquement la biosynthese d'hemicelluloses, de cellulose et d'acides uroniques presents dans des cellules vegetales
WO2010102220A1 (fr) 2009-03-05 2010-09-10 Metabolix, Inc. Multiplication de plantes transgéniques
WO2014019028A1 (fr) * 2012-08-03 2014-02-06 Adelaide Research & Innovation Pty Ltd Polysaccharide synthases (x)
WO2014198885A1 (fr) * 2013-06-14 2014-12-18 Bayer Cropscience Nv Fibres végétales à propriétés de coloration améliorées
US8993842B2 (en) 2007-09-21 2015-03-31 Cambridge Enterprise Limited Modified xylan production
CN109220800A (zh) * 2018-10-18 2019-01-18 楚雄师范学院 一种有效提高杂交构树组培苗生根率的方法
CN110846297A (zh) * 2019-11-26 2020-02-28 温氏食品集团股份有限公司 一种多功能融合酶和多功能融合酶真核表达载体及其构建方法
CN112143722A (zh) * 2020-09-29 2020-12-29 江南大学 一种提高4,6-α-葡萄糖基转移酶可溶性表达量的方法

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NEWMAN T., ET AL.: "GENES GALORE: A SUMMARY OF METHODS FOR ACCESSING RESULTS FROM LARGE-SCALE PARTIAL SEQUENCING OF ANONYMOUS ARABIDOPSIS CDNA CLONES", PLANT PHYSIOLOGY., AMERICAN SOCIETY OF PLANT PHYSIOLOGISTS, ROCKVILLE, MD., US, vol. 106., 1 January 1994 (1994-01-01), US, pages 1241 - 1255., XP002913698, ISSN: 0032-0889, DOI: 10.1104/pp.106.4.1241 *
VALVEKENS D., MONTAGU VAN M., LIJSEBETTENS VAN M.: "AGROBACTERIUM TUMEFACIENS-MEDIATED TRANSFORMATION OF ARABIDOPSIS THALIANA ROOT EXPLANTS BY USING KANAMYCIN SELECTION.", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, NATIONAL ACADEMY OF SCIENCES, US, vol. 85., 1 August 1988 (1988-08-01), US, pages 5536 - 5540., XP002913699, ISSN: 0027-8424, DOI: 10.1073/pnas.85.15.5536 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005071086A1 (fr) * 2004-01-22 2005-08-04 Suzano Papel E Celulose S.A. Methode utilisant des cassettes d'expression genetique pour moduler genetiquement la biosynthese d'hemicelluloses, de cellulose et d'acides uroniques presents dans des cellules vegetales
US8993842B2 (en) 2007-09-21 2015-03-31 Cambridge Enterprise Limited Modified xylan production
WO2010102220A1 (fr) 2009-03-05 2010-09-10 Metabolix, Inc. Multiplication de plantes transgéniques
WO2014019028A1 (fr) * 2012-08-03 2014-02-06 Adelaide Research & Innovation Pty Ltd Polysaccharide synthases (x)
WO2014198885A1 (fr) * 2013-06-14 2014-12-18 Bayer Cropscience Nv Fibres végétales à propriétés de coloration améliorées
CN109220800A (zh) * 2018-10-18 2019-01-18 楚雄师范学院 一种有效提高杂交构树组培苗生根率的方法
CN110846297A (zh) * 2019-11-26 2020-02-28 温氏食品集团股份有限公司 一种多功能融合酶和多功能融合酶真核表达载体及其构建方法
CN110846297B (zh) * 2019-11-26 2023-04-07 温氏食品集团股份有限公司 一种多功能融合酶和多功能融合酶真核表达载体及其构建方法
CN112143722A (zh) * 2020-09-29 2020-12-29 江南大学 一种提高4,6-α-葡萄糖基转移酶可溶性表达量的方法

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