WO1999001558A1 - Plant genes and polypeptides and uses thereof - Google Patents

Abstract

The invention relates to plant glycosyltransferase genes and polypeptides. Nucleic and amino acid sequences for plant glycosyltransferases are provided.

Description

PLANT GENES AND POLYPEPTIDES AND USES THEREOF

The present invention is based on the identification of plant genes encoding glycosyltransferases which in the plant are membrane associated. It relates to nucleic acid, both encoding sequences and oligonucleotide probes and primers useful in cloning and diagnostic work, glycosyltransferase enzymes, and methods and uses. Glycosyltransferases are involved in determining plant cell wall structure and composition and so their manipulation may be used to modify such structure and composition.

Many of the polysaccharide components of plant cell walls are synthesised in the Golgi apparatus, an organelle made of a stack of membrane cisternae (Northcote and Pickett-Heaps 1966) . The enzymes that carry out the synthesis transfer individual sugar residues from specific nucleotide-sugar precursors onto the growing oligosaccharide chain of polysaccharides and glycoproteins . Despite much effort directed towards identifying and purifying these enzymes and cloning the genes encoding these enzymes, there have been no published reports to date (Gibeaut and Carpita 1994) . Despite the fact that the plant cell wall has a structure and composition very different to that of fungi, we report here the unexpected discovery of plant genes encoding proteins with significant similarity in sequence and predicted membrane topology to enzymes involved in yeast cell wall synthesis . These plant genes define a new family of plant proteins indicated to be involved in normal plant cell wall synthesis . The family is present in both monocotyledonous and dicotyledonous plants. An alteration in the expression and/or properties of these enzymes may be used to produce plants with modified cell wall properties. The present invention may be applied in a wide range of contexts in which plants and their polysaccharides are relevant, including the food processing industry, agriculture and horticulture, and the industries that use plant fibres such as the cotton, paper and pulp industries.

Plant cell walls have important roles in plant growth and development and in mediating the interactions between the plant and its environment, including the response of a plant to infection by pathogens. The cell walls are composed predominantly of a variety of polysaccharides and glycoproteins which determine its strength and properties. Cell types vary in the proportions and types of polysaccharides and other components of the walls. Consequently the properties of cell walls range from those forming wood to the weaker, yielding walls of soft ripe fruit. The cell walls therefore influence the industrial utility of many plant products, including cotton and other fibres, animal and human food, and wood for pulp and paper. For example, the texture of fruit and vegetables influences their harvest, transport and the processing qualities.

Of the three major classes of polysaccharides in the cell wall, cellulose alone is synthesised at the plasma membrane (Moore et al . 1991) . The hemicelluloses and pectins are synthesised within the cells, in the Golgi apparatus, by polymerisation of specific sugars into the desired polysaccharide chains. The cell wall glycoproteins, including the arabinogalactan proteins, the hydroxyproline-rich cell wall proteins and extensions also receive extensive addition of sugars in the Golgi apparatus . These proteins are thought to be involved in cell growth, development and the generation and maintenance of the cell wall structure.

Synthesis of the non-cellulosic cell wall polysaccharides and glycosylation of the cell wall proteins requires the action of specific enzymes in the Golgi apparatus . The polymers are made by stepwise transfer of nucleotide sugar precursors to an oligosaccharide precursor. The enzymes that carry out this transfer are known as glycosyltransferases . Although it has been the subject of much research over the last 30 years, there are no published reports of the cloning of any genes that might encode such glycosyltransferase enzymes. The present inventors have now identified genes that encode such enzymes, via a surprising route, as discussed further in the experimental section below.

Brief Description of the Figures

Figure 1 shows the nucleotide sequence, including coding sequence, of an Arabidopsis thaliana glycosyltransferase termed gtl β , and the encoded amino acid sequence. The putative transmembrane domain is underlined, the glycosyltransferase family homology domain identified herein is in italics, and the two cysteines predicted to form a disulphide bridge are double-underlined.

Figure 2a shows an alignment of a translation of rice EST C20120 with gtl6, providing indication that related glycosyltransferases are expressed in cereals .

Figure^' 2b shows the nucleotide sequence of rice EST C20120.

Figure 3 shows an alignment of three members from Arabidopsis thaliana of the gtl family of plant glycosyltransferases identified herein. The incomplete sequence of two members gtl4 and gtl 5 is aligned with that of the complete sequence of gtlβ . Dots represent spaces introduced to improve the alignment. The predicted catalytic domain with conserved sequence in the family is underlined. Conserved cysteines, 2 of which may form the typical disulphide bridge, are in bold.

Figure 4 shows partial sequences according to the present invention: Figure 4a shows the nucleotide and amino acid sequences of gtll .

Figure 4b shows the nucleotide and amino acid sequences of gtl2.

Figure 4c shows the nucleotide and amino acid sequences of gtl4 .

Figure 4d shows the nucleotide and amino acid sequences of gtl5.

Figure 5 shows an alignment of a highly conserved region of the gtl ORFs and the sequences of 5 yeast glycosyltransferases . 0 represents a hydrophobic amino acid. Sequences of gtll and gtl2 have not yet been completely determined.

All documents mentioned herein are incorporated by reference .

The present invention provides nucleic acid encoding plant glycosyltransferases . A plant glycosyltransferase may be obtainable or derived from a suitable sample of plant material. It may be capable of catalysing one or more steps in the biosynthesis of complex non-cellulosic plant cell wall polysaccharides. Similarly it may be capable of catalysing the glycosylation of plant cell wall protein and/or the transfer of saccharide moieties from nucleotide sugar precursors to oligosaccharide precursors. In such a catalysis reaction a plant glycosyltransferase may show greater activity than an equivalent non-plant glycosyltransferase.

Nucleic acid according to the present invention may include any sequence disclosed herein, such as of an Arabidopsis thaliana sequence as shown in any of the Figures, including coding and/or non-coding regions, or be a mutant, variant, derivative or allele of any such sequence or complementary sequence thereof. Preferred mutants, variants, derivatives and alleles are those which encode a product which retains a functional characteristic of the product encoded by the wild-type gene, especially glycosyltransferase activity. Homologues from other species are also provided. In various embodiments a mutant, variant, derivative or allele have altered, e.g. enhanced or diminished glycosyltransferase activity compared with wild-type.

Changes to a sequence, to produce a mutant, variant or derivative, may be by one or more of addition, insertion, deletion or substitution of one or more nucleotides in the nucleic acid, leading to the addition, insertion, deletion or substitution of one or more amino acids in the encoded polypeptide. Of course, changes to the nucleic acid which make no difference to the encoded amino acid sequence are included.

One nucleic acid sequence including a coding sequence according to the present invention is shown in Figure 1, along with the predicted amino acid sequence of a polypeptide according to the present invention.

A mutant, allele, variant or derivative amino acid sequence in accordance with the present invention may include within the sequence shown in Figure 1, a single amino acid change with respect to the sequence shown in Figure 1, or 2, 3, 4, 5, 6, 7, 8, or 9 changes, about 10, 15, 20, 30, 40 or 50 changes, or greater than about 50, 60, 70, 80 or 90 changes. In addition to one or more changes within the amino acid sequence shown in Figure 1 a mutant, allele, variant or derivative amino acid sequence may include additional amino acids at the C- terminus and/or N-terminus.

A sequence related to a sequence specifically disclosed herein shares homology with that sequence. Homology may be at the nucleotide sequence and/or amino acid sequence level. Preferably, the nucleic acid and/or amino acid sequence shares homology with the coding sequence or the sequence encoded by the nucleotide sequence of Figure 1, preferably at least about 50%, or 60%, or 70%, or 80% homology, most preferably at least about 90%, 95%, 96%, 97%, 98% or 99% homology.

As is well-understood, homology at the amino acid level is generally in terms of amino acid similarity or identity. Similarity allows for "conservative variation", i.e. substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine . Similarity may be as defined and determined by the TBLASTN program, of Altschul et al . (1990) J^". Mol . Biol . 215: 403-10, which is in standard use in the art, or, and this may be preferred, the standard program BestFit, which is part of the Wisconsin Package, Version 8, September 1994, (Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA, Wisconsin 53711) . BestFit makes an optimal alignment of the best segment of similarity between two sequences. Optimal alignments are found by inserting gaps to maximize the number of matches using the local homology algorithm of Smith and Waterman

Homology may be over the full-length of the relevant sequence shown herein, or may more preferably be over a contiguous sequence of about or greater than about 20, 25, 30, 33, 40, 50, 67, 133, 167, 200, 233, 267, 300, 333, 400, 450, 500, 550, 600 or more amino acids or codons, compared with the relevant amino acid sequence or nucleotide sequence as the case may be. Figures 2 and 3 provide partial sequences of homologues of the gene sequence shown in Figure 1. These sequences and full-length sequences including them are also provided as aspects of the present invention, as are mutants, variants, derivatives, alleles and homologues in equivalent terms as discussed for the sequence of Figure 1. Thus, for instance, the present invention provides nucleic acid encoding a polypeptide which includes the amino acid sequence shown in Figure 3 for gtl4 or gtl5 and which is a glycosyltransferase. The complete polypeptide which has glycosyltransferase activity may have a certain degree of homology with the sequence of Figure 1, as discussed in general terms. Similarly, other aspects relate to a glycosyltransferase including the sequence shown in Figure 2 for a rice homologue of the sequence of Figure 1, and methods and uses employing such glycosyltransferase or nucleic acid encoding it. Furthermore, Figure 5 shows an alignment of sequences obtained in the work described below, along with five yeast proteins, and indicates a consensus sequence. There are distinct sequence motifs which are characteristic of the general family of sequences such as shown in Figure 2 and also three uncharacterised S . pombe genes showing significant similarity to gmal2p. An alignment of the five members from Arabidopsis with five yeast proteins is shown in Figure 5. Among other homology domains, the homology includes a sequence closely related to the sequence of amino acids :

E- [FW] -hydrophobic-W-W-hydrophobic-D-x-D-A-hydrophobic- hydrophobic .

The present invention encompasses plant glycosyltransferases which include a sequence conforming to this consensus or having at least about 60% homology therewith (7/12), more preferably at least about 67% homology (8/12) , 75% homology (9/12) , 83% homology (10/12) or 92% homology (11/12) . This homology domain is part of a domain of the glycosyltransferase polypeptide which is responsible for catalysis ("the catalytic domain") . The catalytic domain is substantially at the C-terminal end of the plant glycosyltransferases disclosed herein. It comprises the amino acids involved in the formation of the enzyme's active site. Further 75% or greater of the amino acid residues in the catalytic domain of the plant glycosyltransferases according to the invention and exemplified herein are different to the equivalent amino acid residues of other known glycosyltransferases eg yeast glycosyltransferases gmal2 and mnnlO. Put another way 25% or fewer of the amino acid residues in the catalytic domain of said glycosyltransferases according to the invention and exemplified herein are the same as the equivalent amino acid residues of other known glycosyltransferases eg yeast glycosyltransferases gmal2 and mnnlO .

Also provided by an aspect of the present invention is nucleic acid including or consisting essentially of a sequence of nucleotides complementary to a nucleotide sequence hybridisable with any encoding sequence provided herein. Another way of looking at this would be for nucleic acid according to this aspect to be hybridisable with a nucleotide sequence complementary to any encoding sequence provided herein. Of course, DNA is generally double-stranded and blotting techniques such as Southern hybridisation are often performed following separation of the strands without a distinction being drawn between which of the strands is hybridising. Preferably the hybridisable nucleic acid or its complement encode a product able to influence glycosyltransferase activity in a plant. Preferred conditions for hybridisation are familiar to those skilled in the art, but are generally stringent enough for there to be positive hybridisation between the sequences of interest to the exclusion of other sequences.

The nucleic acid, which may contain for example DNA encoding the amino acid sequence of Figure 1, as genomic or cDNA, may be in the form of a recombinant and preferably replicable vector, for example a plasmid, cosmid, phage or Agrobacterium binary vector. The nucleic acid may be under the control of an appropriate promoter or other regulatory elements for expression in a host cell such as a microbial, e.g. bacterial, or plant cell . In the case of genomic DNA, this may contain its own promoter or other regulatory elements and in the case of cDNA this may be under the control of an appropriate promoter or other regulatory elements for expression in the host cell.

A vector including nucleic acid according to the present invention need not include a promoter or other regulatory sequence, particularly if the vector is to be used to introduce the nucleic acid intc cells for recombination into the genome.

Those skilled in the art are well able to construct vectors and design protocols for recombinant gene expression. Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate . Suitable promoter sequences may include inducible promoters . For further details see, for example, Mol ecular Cloning: a Laboratory Manual : 2nd edition, Sambrook et al , 1989, Cold Spring Harbor Laboratory Press. Many known techniques and protocols for manipulation of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Current Protocols in Molecular Biology, Second Edition, Ausubel et al . eds . , John Wiley & Sons, 1992. The disclosures of Sambrook et al . and Ausubel et al . are incorporated herein by reference. Specific procedures and vectors previously used with wide success upon plants are described by Bevan (Nucl. Acids Res. 12, 8711-8721 (1984)) and Guerineau and Mullineaux (1993) (Plant transformation and expression vectors. In: Plant Molecular Biology Labfax (Croy RRD ed) Oxford, BIOS Scientific Publishers, pp 121-148) . Selectable genetic markers may be used consisting of chimaeric genes that confer selectable phenotypes such as resistance to antibiotics or herbicides, such as kanamycin, hygromycin, phosphinotricin, chlorsulfuron, methotrexate, gentamycin, spectinomycir. , imidazolinones and glyphosate.

Nucleic acid molecules and vectors according to the present invention may be provided isolated and/or purified from their natural environment, in substantially pure or homogeneous form, or free or substantially free of nucleic acid or genes of the species of interest or origin other than the sequence encoding a polypeptide with the required function. Nucleic acid according to the present invention may include cDNA, RNA, genomic DNA and may be wholly or partially synthetic. The term "isolate" encompasses all these possibilities. Where a DNA sequence is specified, e.g. with reference to a figure, unless context requires otherwise the RNA equivalent, with U substituted for T where it occurs, is encompassed.

The present invention also encompasses the expression product of any of the nucleic acid sequences disclosed and methods of making the expression product by expression from encoding nucleic acid therefore under suitable conditions, which may be in suitable host cells. Following expression, the product may be isolated from the expression system and may be used as desired, for instance in formulation of a composition including at least one additional component.

Thus, a further aspect of the present invention provides a polypeptide which includes an amino acid sequence as disclosed herein, which may be in isolated and/or purified form, free or substantially free of material with which it is naturally associated, such as other polypeptides or such as plant polypeptides other than the polypeptide of interest or (for example if produced by expression in a prokaryotic cell) lacking in native glycosylation, e.g. unglycosylated.

Generally, such a substance according to the present invention is provided in an isolated and/or purified form, i.e. substantially pure. This may include being in a composition where it represents at least about 90% active ingredient, more preferably at least about 95%, more preferably at least about 98%. Such a composition may, however, include inert carrier materials.

Purified protein, or a fragment, mutant, derivative or variant thereof, e.g. produced recombinantly by expression from encoding nucleic acid therefor, may be used to raise antibodies employing techniques which are standard in the art. Antibodies and polypeptides including antigen-binding fragments of antibodies may be used in identifying homologues from other species as discussed further below.

Methods of producing antibodies include immunising a mammal (e.g. human, mouse, rat, rabbit, horse, goat, sheep or monkey) with the protein or a fragment thereof. Antibodies may be obtained from immunised animals using any of a variety of techniques known in the art, and might be screened, preferably using binding of antibody to antigen of interest. For instance, Western blotting techniques or i munoprecipitation may be used (Armitage et al, 1992, Nature 357: 80-82) . Antibodies may be polyclonal or monoclonal .

As an alternative or supplement to immunising a mammal, antibodies with appropriate binding specificity may be obtained from a recombinantly produced library of expressed immunoglobulin variable domains, e.g. using lambda bacteriophage or filamentous bacteriophage which display functional immunoglobulin binding domains on their surfaces; for instance see WO92/01047.

Antibodies raised to a polypeptide or peptide can be used in the identification and/or isolation of homologous polypeptides, and then the encoding genes. Furthermore, many of the polysaccharides are synthesised by multi enzyme complexes, probably made of several glycosyltransferase polypeptides such as those described here. It is therefore possible now to use the proteins encoded by the genes according to the present invention for which sequence information is included herein to isolate other glycosyltransferases, unrelated in sequence. Using antibodies against these proteins, such enzymes may be isolated by co-immunoprecipitation with the gtl glycosyltransferases from detergent-solubilised Golgi-enriched membranes. An alternative is to generate transgenic plants that express an epitope tagged fusion protein, such as a myc- tagged gtl6 (see experimental section) . The antibody recognising the myc epitope may then be used to isolate the protein together with other interacting proteins. By crosslinking of neighbouring proteins in the isolated membranes, the efficiency of co-immunoprecipitation may be improved. The co- immunoprecipitating proteins may be identified by amino acid sequencing or mass-spectrometric methods, and the corresponding genes isolated. These interacting enzymes, which may or may not be related in amino acid sequence to the gtl family but which should have the defining glycosyltransferase topology, will be equally useful for the generation of plants with altered glycosylation and thus cell wall properties.

Thus, the present invention provides a method of identifying or isolating a polypeptide with glycosyltransferase activity, including screening candidate polypeptides with a polypeptide including the antigen-binding domain of an antibody (for example whole antibody or a fragment thereof) which is able to bind a polypeptide or fragment, variant or derivative thereof in accordance with the present invention or preferably has binding specificity for such a polypeptide. Specific binding members such as antibodies and polypeptides including antigen binding domains of antibodies that bind and are preferably specific for a polypeptide or mutant, variant or derivative thereof in accordance with the present invention represent further aspects of the present invention, as do their use and methods which employ them.

Candidate polypeptides for screening may for instance be the products of an expression library created using nucleic acid derived from an plant of interest, or may be the product of a purification process from a natural source. A polypeptide found to bind the antibody may be isolated and then may be subject to amino acid sequencing. Any suitable technique may be used to sequence the polypeptide either wholly or partially (for instance a fragment of the polypeptide may be sequenced) . Amino acid sequence information may be used in obtaining nucleic acid encoding the polypeptide, for instance by designing one or more oligonucleotides (e.g. a degenerate pool of oligonucleotides) for use as probes or primers in hybridization to candidate nucleic acid, or by searching computer sequence databases, as discussed further below. Homologues of the genes for which sequence information is provided herein exist in monocotyledonous and dicotyledonous plants (Figure 2) . This means that corresponding properties may be altered across the whole range of crops from cereals to legumes. The corresponding genes from other plants may be identified by means of their having regions of sequence similarity including the defining motif of Figure 5. This may be used to find the homologues by for example, sequence similarity searches of genome sequencing projects, by low-stringency hybridisation screening of cDNA libraries, or by designing degenerate oligonucleotides for PCR priming.

A further aspect of the present invention provides a method of identifying and cloning homologues of the gene sequences provided herein from plant species other than Arabidopsis thaliana, which method employs a nucleotide sequence derived from any shown herein. Sequences derived from these may themselves be used in identifying and in cloning other sequences. The nucleotide sequence information provided herein, or any part thereof, may be used in a data-base search to find homologous sequences, expression products of which can be tested for glycosyltransferase activity. Alternatively, nucleic acid libraries may be screened using techniques well known to those skilled in the art and homologous sequences thereby identified then tested. Thus, a method of obtaining nucleic acid encoding a plant glycosyltransferase is provided, including hybridisation of an oligonucleotide or a nucleic acid molecule including such an oligonucleotide to target/candidate nucleic acid. Target or candidate nucleic acid may, for example, include a genomic or cDNA library obtainable from an organism known to contain or suspected of containing such nucleic acid. Successful hybridisation may be identified and target/candidate nucleic acid isolated for further investigation and/or use.

Hybridisation may involve probing nucleic acid and identifying positive hybridisation under suitably stringent conditions (in accordance with known techniques) and/or use of oligonucleotides as primers in a method of nucleic acid amplification, such as PCR. For probing, preferred conditions are those which are stringent enough for there to be a simple pattern with a small number of hybridisations identified as positive which can be investigated further. It is well known in the art to increase stringency of hybridisation gradually until only a few positive clones remain. As an alternative to probing, though still employing nucleic acid hybridisation, oligonucleotides designed to amplify DNA sequences may be used in PCR reactions or other methods involving amplification of nucleic acid, using routine procedures. See for instance "PCR protocols; A Guide to Methods and Applications", Eds. Innis et al , 1990, Academic Press, New York.

Preferred amino acid sequences suitable for use in the design of probes or PCR primers are sequences conserved (completely, substantially or partly) between at least two polypeptides according to the present invention, such as illustrated in the sequence alignment of Figure 3. On the basis of amino acid sequence information oligonucleotide probes or primers may be designed, taking into account the degeneracy of the genetic code, and, where appropriate, codon usage of the organism from the candidate nucleic acid is derived.

Preferably an oligonucleotide in accordance with the present invention, e.g. for use in nucleic acid amplification, has about 10 or fewer codons (e.g. 6, 7 or 8), i.e. is about 30 or fewer nucleotides in length (e.g. 18, 21 or 24) . Primers for amplifying a plant glycosyltransferase gene include a degenerate oligonucleotide, designed using the conserved sequence (E/K) -W-Hydrophobic-W-W-Hydrophobic-D, (A/G)A(A/G)TGGNTNTGGTGGNTNGA(T/C) where N could be a mixture of all four bases or inosine, and a second degenerate oligonucleotide, designed using a second conserved sequence F-L-Hydrophobic-R-N-X-Q-W, TT (T/C) (T/C) TN (A/G) GNAA (T/C) NNCA (A/G) TGG.

Assessment of whether or not a PCR product corresponds to a glycosyltransferase gene may be conducted in various ways . A PCR band from such a reaction might contain a complex mix of products. Individual products may be cloned and each one individually screened. They may be analysed by transformation to assess function on introduction into a plant of interest.

Glycosyltransferase activity may be determined by measuring specific incorporation by membrane preparations from (modified) plants of radiolabelled sugars from sugar nucleotides into large molecules, or measuring similarly specific incorporation into short oligosaccharides from radiolabelled sugar nucleotides catalysed by protein expressed under the control of the gene for example in bacteria or fungi. Alternatively, the activity may be assessed by measuring the sugar composition of the cell wall of modified or selected plants, measuring particularly the amount and sugar composition of various hemicelluloses, pectins or glycoproteins.

Polypeptides according to the present invention which possess glycosyltransferase activity may be used for the in vitro synthesis of polysaccharides such as complex non-cellulosic plant cell wall polysaccharides.

The present invention also extends to nucleic acid encoding a homologue obtained using a nucleotide sequence derived from that shown in any figure herein, and uses thereof.

The provision of sequence information herein for glycosyltransferases of Arabidopsis thaliana enables the obtention of homologous sequences from other plant species. In particular, those skilled in the art may isolate analogues from related, commercially important

Brassica species (e.g. Brassica nigra, Brassica napus and Brassica oleraceae) , and trees such as Loblolly pine, fibre crops such as flax, hemp and cotton, cereals such as wheat, barley, maize, fruits such as tomatoes, strawberries, apples, pears, and vegetables such as carrot, lettuce and onion.

Thus, included within the scope of the present invention are nucleic acid molecules which encode amino acid sequences which are homologues of an Arabidopsis thaliana sequence provided herein. Homology may be at the nucleic acid sequence or amino acid sequence level . Preferably, the nucleic acid or amino acid sequence shares homology with a sequence of Figure 1, preferably at least about 50%, or at least about 60% or at least about 70% or at least about 80% homology, most preferably at least about 90% homology from species other than Arabidopsis thaliana and the encoded polypeptide shares a property with the Arabidopsis thaliana gene, most preferably glycosyltransferase activity. "Homology" may be used to refer to identity.

Probes and primers may also be used in accordance with the present invention to determine the presence in a plant of interest the presence or absence of a particular gene or allele. In plant breeding it is useful to be able to determine the presence in a plant of a particular allele or gene. This could be achieved, for example, by a nucleic acid amplification technique such as PCR, using primers derived from non-conserved regions unique to the particular allele or gene according to the present invention. The presence of specific amplification products would indicate the presence of the particular allele or gene.

As noted, plant glycosyltransferase expression may be used to alter the properties and/or composition of the cell wall. Modifying sugar properties has implications for the different uses to which a plant may be put, e.g. in baking different foodstuffs. It is useful to the plant breeder to be able to screen for one or more markers (e.g. RFLP markers) indicative of the presence or absence of a gene or allele in a plant, allowing for a much faster and thorough through-put of plants which would be possible if the sugar composition or baking properties had to be determined in each case.

Also according to the present invention there is provided a plant cell having incorporated into its genome a heterologous sequence of nucleotides as provided by the present invention, under operative control of a regulatory sequence for control of expression. A further aspect of the present invention provides a method of making such a plant cell involving introduction of a vector including the sequence of nucleotides into a plant cell and causing or allowing recombination between the vector and the plant cell genome to introduce the sequence of nucleotides into the genome. A plant may be regenerated from one or more transformed plant cells. When introducing a chosen gene construct into a cell, certain considerations must be taken into account, well known to those skilled in the art . The nucleic acid to be inserted should be assembled within a construct which contains effective regulatory elements which will drive transcription. There must be available a method of transporting the construct into the cell . Suitable methods of preparing constructs and transforming plant cells are described in Gene Cloning and Manipulation' CJ Howe, Cambridge University Press 1995. Once the construct is within the cell membrane, integration into the endogenous chromosomal material either will or will not occur. Finally, as far as plants are concerned the target cell type must be such that cells can be regenerated into whole plants.

Plants transformed with the DNA segment containing the sequence may be produced by standard techniques which are already known for the genetic manipulation of plants. DNA can be transformed into plant cells using any suitable technology, such as a disarmed Ti-plasmid vector carried by Agrobacterium exploiting its natural gene transfer ability (EP-A-270355 , EP-A-0116718 , NAR 12(22) 8711 - 87215 1984) , particle or microprojectile bombardment (US 5100792, EP-A-444882, EP-A-434616) microinjection (WO 92/09696, WO 94/00583, EP 331083, EP 175966, Green et al . (1987) Plant Tissue and Cell Cul ture, Academic Press) , electroporation (EP 290395, WO 8706614 Gelvin Debeyser - see attached) other forms of direct DNA uptake (DE 4005152, WO 9012096, US 4684611), liposome mediated DNA uptake (e.g. Freeman et al . Plant Cell Physiol . 29: 1353 (1984)), or the vortexing method (e.g. Kindle, PNAS U. S.A . 87: 1228 (1990d) Physical methods for the transformation of plant cells are reviewed in Oard, 1991, Biotech . Adv. 9 : 1-11. Agrobacterium transformation is widely used by those skilled in the art to transform dicotyledonous species. Recently, there has been substantial progress towards the routine production of stable, fertile transgenic plants in almost all economically relevant monocot plants (Toriyama, et al . (1988) Bio/Technology 6, 1072-1074; Zhang, et al . (1988) Plant Cell Rep . 7, 379-384; Zhang, et al. (1988) Theor Appl Genet 76, 835-840; Shimamoto, et al . (1989) Na ture 338, 274-276; Datta, et al . (1990) Bio/Technology 8, 736-740; Christou, et al . (1991) Bio/Technology 9, 957-962; Peng, et al . (1991) International Rice Research Institute, Manila, Philippines 563-574; Cao, et al . (1992) Plant Cell Rep . 11, 585-591; Li, et al . (1993) Plant Cell Rep . 12, 250- 255; Rathore, et al . (1993) Plant Molecular Biology 21, 871-884; Fromm, et al . (1990) Bio/Technology 8, 833-839; Gordon-Kamm, et al . (1990) Plant Cell 2, 603-618; D'Halluin, et al . (1992) Plant Cell 4, 1495-1505;

Walters, et al . (1992) Plant Molecular Biology 18, 189- 200; Koziel, et al . (1993) Biotechnology 11, 194-200; Vasil, I. K. (1994) Plant Molecular Biology 25, 925-937; Weeks, et al . (1993) Plant Physiology 102, 1077-1084; Somers, et al . (1992) Bio/Technology 10, 1589-1594; W092/14828) . In particular, Agrobacterium mediated transformation is now emerging also as an highly efficient alternative transformation method in monocots (Hiei et al . (1994) The Plant Journal 6, 271-282). The generation of fertile transgenic plants has been achieved in the cereals rice, maize, wheat, oat, and barley (reviewed in Shimamoto, K. (1994) Current Opinion in Biotechnology 5, 158-162.; Vasil, et al . (1992) Bio/Technology 10, 667-674; Vain et al . , 1995, Biotechnology Advances 13 (4) : 653-671; Vasil, 1996, Nature Biotechnology 14 page 702) .

Microprojectile bombardment, electroporaticn and direct DΝA uptake are preferred where Agrobacterium is inefficient or ineffective. Alternatively, a combination of different techniques may be employed to enhance the efficiency of the transformation process, eg bombardment with Agrobacterium coated microparticles (EP-A-436234 ) or microprojectile bombardment to induce wounding followed by co-cultivation with Agrobacterium (EP-A-486233 ) .

Following transformation, a plant may be regenerated, e.g. from single cells, callus tissue or leaf discs, as is standard in the art. Almost any plant can be entirely regenerated from cells, tissues and organs of the plant . Available techniques are reviewed in Vasil et al . , Cell Cul ture and Somatic Cell Genetics of Plants, Vol I, II and III, Laboratory Procedures and Their Applica tions, Academic Press, 1984, and Weissbach and Weissbach, Methods for Plant Molecular Biology, Academic Press, 1989.

The particular choice of a transformation technology will be determined by its efficiency to transform certain plant species as well as the experience and preference of the person practising the invention with a particular methodology of choice. It will be apparent to the skilled person that the particular choice of a transformation system to introduce nucleic acid into plant cells is not essential to or a limitation of the invention, nor is the choice of technique for plant regeneration.

Nucleic acid provided herein may be used to affect a physical characteristic of a plant, particularly of cell walls. For this purpose nucleic acid such as a vector as described herein may be used for the production of a transgenic plant. Such a plant may possess an altered phenotype, particular in cell wall structure and/or composition compared with wild-type (that is to say a plant that is wild-type for the relevant glycosyltransferase gene) .

The invention further encompasses a host cell transformed with nucleic acid or a vector according to the present invention, especially a plant or a microbial cell. Thus, a host cell, such as a plant cell, including heterologous nucleic acid according to the present invention is provided. Within the cell, the nucleic acid may be incorporated within the chromosome . There may be more than one heterologous nucleotide sequence per haploid genome . Also according to the invention there is provided a plant cell having incorporated into its genome nucleic acid, particularly heterologous nucleic acid, as provided by the present invention, under operative control of a regulatory sequence for control of expression. The coding sequence may be operably linked to one or more regulatory sequences which may be heterologous or foreign to the gene, such as not naturally associated with the gene for its expression. The nucleic acid according to the invention may be placed under the control of an externally inducible gene promoter to place expression under the control of the user.

A suitable inducible promoter is the GST-II-27 gene promoter which has been shown to be induced by certain chemical compounds which can be applied to growing plants. The promoter is functional in both monocotyledons and dicotyledons. It can therefore be used to control gene expression in a variety of genetically modified plants, including field crops such as canola, sunflower, tobacco, sugarbeet, cotton; cereals such as wheat, barley, rice, maize, sorghum; fruit such as tomatoes, mangoes, peaches, apples, pears, strawberries, bananas, and melons; and vegetables such as carrot, lettuce, cabbage and onion. The GST-II-27 promoter is also suitable for use in a variety of tissues, including roots, leaves, stems and reproductive tissues .

Other suitable promoters include the Cauliflower Mosaic Virus 35S (CaMV 35S) gene promoter that is expressed at a high level in virtually all plant tissues (Benfey et al, 1990a and 1990b) ; the cauliflower meri 5 promoter that is expressed in the vegetative apical meristem as well as several well localised positions in the plant body, eg inner phloem, flower primordia, branching points in root and shoot (Medford, 1992; Medford et al, 1991) and the Arabidopsis thaliana LEAFY promoter that is expressed very early in flower development (Weigel et al , 1992) .

In a further aspect the present invention provides a gene construct including an inducible promoter operatively linked to a nucleotide sequence provided by the present invention, such as including a sequence shown in any of the figures, or any mutant, variant, derivative, allele or homologue thereof. As discussed, this enables control of expression of the gene. The present invention also provides plants transformed with said gene construct and methods including introduction of such a construct into a plant cell and/or induction of expression of a construct within a plant cell, by application of a suitable stimulus, an effective exogenous inducer .

The term "inducible" as applied to a promoter is well understood by those skilled in the art. In essence, expression under the control of an inducible promoter is "switched on" or increased in response to an applied stimulus. The nature of the stimulus varies between promoters. Some inducible promoters cause little or undetectable levels of expression (or no expression) in the absence of the appropriate stimulus. Other inducible promoters cause detectable constitutive expression in the absence of the stimulus. Whatever the level of expression is in the absence of the stimulus, expression from any inducible promoter is increased in the presence of the correct stimulus. The preferable situation is where the level of expression increases upon application of the relevant stimulus by an amount effective to alter a phenotypic characteristic. Thus an inducible (or "switchable" ) promoter may be used which causes a basic level of expression in the absence of the stimulus which level is too low to bring about a desired phenotype (and may in fact be zero) . Upon application of the stimulus, expression is increased (or switched on) to a level which brings about the desired phenotype .

The term "heterologous" may be used to indicate that the gene/sequence of nucleotides in question have been introduced into said cells of the plant or an ancestor thereof, using genetic engineering, i.e. by human intervention. A transgenic plant cell, i.e. transgenic for the nucleic acid in question, may be provided. The transgene may be on an extra-genomic vector or incorporated, preferably stably, into the genome. A heterologous gene may replace an endogenous equivalent gene, i.e. one which normally performs the same or a similar function, or the inserted sequence may be additional to the endogenous gene or other sequence. An advantage of introduction of a heterologous gene is the ability to place expression of a sequence under the control of a promoter of choice, in order to be able to influence expression according to preference.

Furthermore, mutants, variants and derivatives of the wild-type gene, e.g. with higher or lower activity than wild-type, may be used in place of the endogenous gene. Nucleic acid heterologous, or exogenous or foreign, to a plant cell may be non-naturally occurring in cells of that type, variety or species. Thus, nucleic acid may include a coding sequence of or derived from a particular type of plant cell or species or variety of plant, placed within the context of a plant cell of a different type or species or variety of plant. A further possibility is for a nucleic acid sequence to be placed within a cell in which it or a homologue is found naturally, but wherein the nucleic acid sequence is linked and/or adjacent to nucleic acid which does not occur naturally within the cell, or cells of that type or species or variety of plant, such as operably linked to one or more regulatory sequences, such as a promoter sequence, for control of expression. A sequence within a plant or other host cell may be identifiably heterologous, exogenous or foreign. Plants which include a plant cell according to the invention are also provided, along with any part or propagule thereof, seed, selfed or hybrid progeny and descendants. A plant according to the present invention may be one which does not breed true in one or more properties. Plant varieties may be excluded, particularly registrable plant varieties according to Plant Breeders' Rights. It is noted that a plant need not be considered a "plant variety" simply because it contains stably within its genome a transgene, introduced into a cell of the plant or an ancestor thereof.

In addition to a plant, the present invention provides any clone of such a plant, seed, selfed or hybrid progeny and descendants, and any part of any of these, such as cuttings, seed. The invention provides any plant propagule, that is any part which may be used in reproduction or propagation, sexual or asexual, including cuttings, seed and so on. Also encompassed by the invention is a plant which is a sexually or asexually propagated off-spring, clone or descendant of such a plant, or any part or propagule of said plant, offspring, clone or descendant.

The invention further provides a method of influencing or affecting a physical characteristic of a plant such as cell wall structure and/or composition, including causing or allowing expression of a heterologous nucleic acid sequence as discussed within cells of the plant.

The invention further provides a method of including expression from nucleic acid according to the invention within cells of a plant (thereby producing the encoded polypeptide) , following an earlier step of introduction of the nucleic acid into a cell of the plant or an ancestor thereof. Such a method may influence or affect a characteristic of the plant, such as cell wall structure and/or composition. This may be used in combination with any other transgene involved in another phenotypic trait or desirable property.

As noted, the principal physical characteristic which may be altered using the present invention is plant cell wall structure and/or composition. Manipulation of particular glycosyltransferase levels may be achieved by manipulating the level of gene expression, either reduction in expression of the gene product or over-expression.

In the present invention, over-expression may be achieved by introduction of the nucleotide sequence in a sense orientation. Thus, the present invention provides a method of influencing a physical characteristic of a plant, the method including causing or allowing expression of the product (polypeptide or nucleic acid transcript) encoded by heterologous nucleic acid according to the invention from that nucleic acid within cells of the plant.

Down-regulation of expression of a target gene may be achieved using anti-sense technology or "sense regulation" ("co-suppression") . In using anti-sense genes or partial gene sequences to down-regulate gene expression, a nucleotide sequence is placed under the control of a promoter in a "reverse orientation" such that transcription yields RNA which is complementary to normal mRNA transcribed from the "sense" strand of the target gene. See, for example, Rothstein et al, 1987; Smith et al , (1988) Nature 334, 724-726; Zhang et al , (1992) The Plant Cell 4, 1575-1588, English et al . , (1996) The Plant Cell 8, 179-188. Antisense technology is also reviewed in Bourque, (1995) , Plant Science 105, 125-149, and Flavell, (1994) PNAS USA 91, 3490-3496.

An alternative is to use a copy of all or part of the target gene inserted in sense, that is the same, orientation as the target gene, to achieve reduction in expression of the target gene by co-suppression. See, for example, van der Krol et al . , (1990) The Plant Cell 2, 291-299; Napoli et al . , (1990) The Plant Cell 2 , 279- 289; Zhang et al . , (1992) The Plant Cell 4, 1575-1588, and US-A-5,231,020.

The complete sequence corresponding to the coding sequence (in reverse orientation for anti-sense) need not be used. For example fragments of sufficient length may be used. It is a routine matter for the person skilled in the art to screen fragments of various sizes and from various parts of the coding sequence to optimise the level of anti-sense inhibition. It may be advantageous to include the initiating methionine ATG codon, and perhaps one or more nucleotides upstream of the initiating codon. A further possibility is to target a conserved sequence of a gene, e.g. a sequence that is characteristic of one or more genes, such as a regulatory sequence .

The sequence employed may be about 500 nucleotides or less, possibly about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, or about 100 nucleotides. It may be possible to use oligonucleotides of much shorter lengths, 14-23 nucleotides, although longer fragments, and generally even longer than about 500 nucleotides are preferable where possible, such as longer than about 600 nucleotides, than about 700 nucleotides, than about 800 nucleotides, than about 1000 nucleotides or more.

It may be preferable that there is complete sequence identity in the sequence used for down-regulation of expression of a target sequence, and the target sequence, though total complementarity or similarity of sequence is not essential. One or more nucleotides may differ in the sequence used from the target gene. Thus, a sequence employed in a down-regulation of gene expression in accordance with the present invention may be a wild- type sequence (e.g. gene) selected from those available, or a mutant, derivative, variant or allele, by way of insertion, addition, deletion or substitution of one or more nucleotides, of such a sequence. The sequence need not include an open reading frame or specify an RNA that would be translatable. It may be preferred for there to be sufficient homology for the respective anti-sense and sense RNA molecules to hybridise. There may be down regulation of gene expression even where there is about

5%, 10%, 15% or 20% or more mismatch between the sequence used and the target gene .

Generally, the transcribed nucleic acid may represent a fragment of a gene, such as including a nucleotide sequence shown in any figure herein, or the complement thereof, or may be a mutant, derivative, variant or allele thereof, in similar terms as discussed above in relation to alterations being made to a coding sequence and the homology of the altered sequence.^' The homology may be sufficient for the transcribed anti -sense RNA to hybridise with nucleic acid within cells of the plant, though irrespective of whether hybridisation takes place the desired effect is down-regulation of gene expression. Anti-sense regulation may itself be regulated by employing an inducible promoter in an appropriate construct .

Thus, the present invention also provides a method of influencing a characteristic of a plant, the method including causing or allowing anti-sense transcription from heterologous nucleic acid according to the invention within cells of the plant.

The present invention further provides the use of the nucleotide sequence of any of the figures or a fragment, mutant, derivative, allele, variant or homologue thereof for down-regulation of gene expression, particularly down-regulation of expression of a glycosyltransferase gene such as from Arabidopsis thaliana or a homologue thereof, preferably in order to influence a physical characteristic of a plant, especially cell wall structure and/or composition. When additional copies of the target gene are inserted in sense, that is the same, orientation as the target gene, a range of phenotypes is produced which includes individuals where over-expression occurs and some where under-expression of protein from the target gene occurs. When the inserted gene is only part of the endogenous gene the number of under-expressing individuals in the transgenic population increases. The mechanism by which sense regulation occurs, particularly down-regulation, is not well-understood. However, this technique is well-reported in scientific and patent literature and is used routinely for gene control. See, for example, van der Krol et al . , (1990) The Plant Cell 2 , 291-229; Napoli et al . , (1990) The Plant Cell 2, 279-289; Zhang et al , 1992 The Plant Cell 4 , 1575-1588. Again, fragments, mutants and so on may be used in similar terms as described above for use in anti-sense regulation.

Thus, the present invention also provides a method of influencing a characteristic of a plant, the method including causing or allowing expression from nucleic acid according to the invention within cells of the plant. This may be used to suppress activity of a product which has glycosyltransferase activity. Here the activity of the product is preferably suppressed as a result of under-expression within the plant cells.

To determine modification to the cell wall, cell wall composition can be analysed using various well known techniques . Cell wall components can be isolated using the principles outlined in Brett and Waldron (1996) ('Physiology and Biochemistry of Plant Cell Walls' C. Brett and K. Waldron, Chapman and Hall 1996) .

The sugar composition of cell wall polysaccharides and glycoproteins can be analysed using techniques well known to the skilled person, such as reduction to alditols, derivatisation to alditol acetates and analysis via gas-liquid chromatography, as demonstrated in Reiter et al . The Plant Journal, 12 p335-345 and described in Blackeney et al . Carbohydrate Research 113 p291.

Aspects and embodiments of the present invention will now be illustrated with reference to the following experimental section. Further aspects and embodiments and modification thereto will be apparent to those skilled in the art .

Experimen tal

Despite much effort, no genes encoding Golgi- localised transferases have been identified in plants. Genes encoding glycosyltransferases have been identified in animals and fungi. Most of these are involved in N- and O-linked glycan modifications of proteins.

Although there are often sequence similarities between glycosyltransferases with very similar sugar transfer specificities, there are very few similarities between different transferases catalysing sugar addition with different linkages. For example the human alpha (1,2) fucosyl-transferase shows no detectable sequence similarity to the alpha (1,3) alpha (1,4) fucosyl transferase. Also, the N-acetylglucosaminyl-transferases I and II, which use different substrates, show no sequence similarity to each other. Nevertheless, all the Golgi-localised glycosyltransferases studied so far (none from plants) have a similar overall structure (Paulson and Colley 1989) : they have a type II topology with a short, cytosolic N terminus, a single transmembrane domain, and a larger lumenal C-terminal domain that has catalytic activity. Furthermore, they often have conserved cysteines in the lumenal C-terminal domain that probably form a disulphide bridge.

To identify plant Golgi-localised glycosyltransferases, we considered the possibility that plants may have proteins homologous to animal enzymes for some of the transferase reactions. As far as we know, most of the glycans of the abundant cell wall glycoproteins and the cell wall polysaccharides are completely unique to plants. However, a commonality can be observed in the N- linked glycan structure conserved between animals and plants (Driouich et al . 1994) . The N- linked glycan of fungi is very different (Herscovics and Orlean 1993), consisting of long heterogeneous mannose chains in Saccharomyces cerevisiae, and mannose chains with galactose at the termini in Schizosaccharomyces pombe (Chappell et al . 1994) . The N-glycosylated proteins of fungi with these large mannose glycans can become incorporated into, and constitute much of the cell wall. Animal and plant N-linked glycans are much smaller. They are trimmed in the Golgi by removal of up to four mannose residues, and receive N-acetyl glucosamine. Further single xylose and fucose and galactose residues can then be added. Although the linkages can differ between organisms, we reasoned that the similarity between plant and animals in glycosylation of N-linked glycans could suggest that the transferases themselves may be conserved.

We therefore conducted a search using the TBLASTN algorithm of the plant Expressed Sequence Tag (EST) database to determine whether there were cDNA sequences with sequence similarity to mammalian enzymes involved in glycosylation of N-linked glycans. The sequences included those of N-acetylglucosaminyl-transferases I and II, the alpha (1,2) fucosyl -transferase and the alpha (1,3) alpha (1,4) fucosyl -transferase . As of March 1997, none could be found.

Although we considered it unlikely that other glycosyltransferases could be detected because of the absence of any close similarities in function, we nevertheless decided to continue the search.

Surprisingly, highly significant scores for 3 Arabidopsis thaliana ESTs (T22705, T22094, T76358) were found when searching with the S. Pombe protein gmal2p, and the related S . cerevisiae protein mnnlOp. These proteins are, respectively, involved in transfer of galactose or mannose to mannans attached to proteins of the yeast cell wall (Chappell et al . 1994, Ballou et al . 1989) . No other glycosyltransferase families assigned on the basis of sequence alignment showed significant matches .

The relevant clones were retrieved from the Arabidopsis Biological Resource Center, Ohio, USA, and the sequence of the longer inserts determined. The sequences were used to search the database for additional homologues, which were in turn retrieved, sequenced and used to search the database again. This iterative process yielded a total of seven clones (ESTs T22705, T22094, T76358, R30521, H76181, N96444, H36383) showing strong sequence similarities to each other. At first these appeared to be derived from six different genes, and were named gtll - 6 (gma twelve-l_.ike) , but complete sequencing revealed that in fact gtl3 and gtlβ are likely to be derived from the same gene. Further sequence of gtl4 was obtained by direct sequencing of a bacterial artificial chromosome (BAC) containing the structural gene.

The longest clone gtl β (identified from EST H36383) , encoded a predicted truncated protein since there was no clear initiator methionine. To obtain a full-length clone, 5' RACE was carried out, using PCR on RNA from Arabidopsis thaliana callus cells. This procedure demonstrated that the initiating methionine of this protein is 15 amino acids N-terminal to that present in the original EST clone. Significantly, the complete sequence that we had obtained revealed that this protein has all the characteristics expected in identified Golgi glycosyltransferases (Figure 1) . Golgi-localised glycosyltransferases have a particular predicted type II topology with a short, cytosolic N terminus, a single transmembrane domain, and a larger lumenal C-terminal domain with conserved cysteines. The sequence of Figure 1 contains a single transmembrane span. Furthermore, the protein has a short N-terminal domain of 42 amino acids, conforming to the norm for Golgi-localised glycosyltransferases . Finally the larger C-terminal domain contains the predicted catalytic site by homology to the gmal2 family, and contains a potential pair of conserved cysteine residues. The protein has a predicted molecular weight of 55kDa, which is close to that of other Golgi-glycosyltransferases (Paulson and Colley 1989) .

Northern analysis revealed that the gene encoding GTL6 is expressed in leaf and callus tissue, suggesting that it is widely expressed. A TFASTA search of the plant database with these sequences further revealed that a related gene (EST C20120) is expressed in rice panicle at the ripening stage, indicating that this gene family is present in both monocotyledonous and dicotyledonous plants (Figure 2) .

This is the first complete sequence of a member of a family of related plant glycosyltransferase genes encoding sugar transferases which may be useful in manipulating synthesis of plant cell wall components.

Discussion

We have identified from Arabidopsis thaliana five members of a new family of genes encoding glycosyltransferases in plants, and present the complete sequence of one member gtl β . The complete sequence of gtl β encodes a predicted protein with all the characteristics expected of a Golgi glycosyltransferase involved in cell wall protein glycosylation or - polysaccharide synthesis. The protein has a predicted short cytoplasmic N-terminal domain, a single transmembrane span, and a lumenal catalytic C-terminal domain. The plant protein shows highly significant sequence similarity and predicted topological relationship to two yeast Golgi-localised glycosyltransferases involved in yeast cell wall synthesis.

This discovery of plant genes that share both sequence homology and predicted enzyme topology with the yeast gene family suggests that there is an unexpectedly close mechanistic parallel between cell wall synthesis in plants and yeasts, and opens up the possibility of using these genes as tools to modify plant cell wall properties .

The plant glycosyltransferase family members are closely related in sequence to each other. They show highly significant sequence similarity in their predicted catalytic domains (Figure 3) , and contain conserved cysteines thought to form a disulphide bridge found in other glycosyltransferases .

The characterised yeast enzymes are involved in transfer of galactose or mannose to a backbone polymer of mannose residues on glycoproteins of the cell wall. Disruption of the genes encoding these yeast enzymes results in altered cell wall properties (Chappell et al . 1994, Mondesert and Reed 1996) . Because the two yeast proteins recognise different nucleotide sugar substrates, it is not possible to predict which sugar (s) are likely to be used by these plant enzymes. However, the high similarity in sequence, together with the overall predicted size and topology of the plant proteins, indicates that the plant proteins are also involved in glycosylation. Disruption or alteration of the expression of these plant genes may be used to alter properties of plant cell walls, by modifying the glycosylation state of glycoproteins or specific polysaccharides. The consequence of such modifications may be to alter the mechanical or rheological properties of the cell walls and their products, or to modify the development of the plant .

By selecting plants that have increased or decreased glycosylation activities and thus modified cell wall polysaccharides or proteins, these genes will allow an alteration to plant cell wall properties to be engineered or selected. Plants with increased activities may be made by generating transgenic plants that express the glycosyltransferases under the control of regulated, tissue-specific or constitutive promoters. Reduced activities may result from selection of plants that have had the gene disrupted, or generation of antisense construct transgenic plants .

At present, it is technically difficult to screen varieties biochemically for their different cell wall constituents and properties. These genes may also be used as probes during plant breeding to screen for varieties of plants that express the glycosyltransferases at differing levels, and consequently selection of varieties with altered cell wall properties. Because of the wide range of uses of cell wall- containing products, both an increase and decrease in glycosylation patterns yield useful alterations in cell walls, for example in decreased processing time or costs by altering the rheological and textural properties of the plant products from fruits, vegetables, legumes and cereals. This is important for a wide range of foods and drinks, including fruit juices, conserves, sauces, and bakery products.

Consumption of plant cell walls has considerable medical significance, constituting much dietary fibre required for healthy digestive system and nutrient absorption. Consumption of dietary fibre is also correlated with bowel cancer incidence. An alteration to plant cell wall components may be used to modify the dietary fibre content of foods. These considerations also apply to animal feeds in agriculture. Preparation of useful fibres from wood involves extreme chemical and other treatments to remove the non- cellulosic constituents. The properties of woods from different trees vary due to the different cell wall constituents, including the Golgi-synthesised hemicelluloses . Alteration of the synthesis of these constituents may yield wood of different properties, useful for timber applications and the pulp and paper industry.

There are several related glycosyltransferase genes in Arabidopsis thaliana . These may have roles at different developmental stages, or be involved in different glycosylation processes as a result of their specificity in sugar addition and sugar polymer substrate. Modification of the expression of different members of the family may therefore have different consequences and the effects may be usefully cumulative.

References

Ballou L. et al . (1989) J^". Biol . Chem . 264, 11857-11864 Chapell, TG et al . (1994) Mol . Biol . Cell 5, 519-528

Chapell TG and G Warren (1989) J^" Cell Biol . 109, 2693-

2702

Dean, N and JB Poster (1996) Glycobiology 6, 73-81

Driouich A et al . (1994) Plant Physiol . Biochem . 32,731- 749

Gibeaut DM and NC Carpita (1994) FASEB J. 8, 904-915 Herscovics A and P Orlean (1993) FASEB J. 7, 540-550 Moore PJ et al . (1991) J. Cell Biol . 112, 589-602 Mondesert G and SI Reed (1996) J. Cell Biol . 132, 137-151 Northcote DH and JD Pickett-Heaps (1966) Biochem J. 98,159-167 Paulson JC and KJ Colley (1989) J. Biol . Chem . 264, 17615-17618

Zablackis et al . (1995) Plant Physiol . 107, 1129-1138

Claims

CLAIMS :

- 1. An isolated nucleic acid which encodes a plant glycosyltransferase which is membrane associated in plants.

2. An isolated nucleic acid according to claim 1 wherein said plant glycosyltransferase has the catalytic activity of a glycosyltransferase with a catalytic domain comprising the amino acid sequence motif E- [FW] - hydrophobic-W-W-hydrophobic-D-x-D-A-hydrophobic- hydrophobic or an amino acid sequence motif with at least 60% homology to the amino acid sequence motif above.

3. An isolated nucleic acid according to claim 1 or claim 2 wherein said plant glycosyltransferase comprises a catalytic domain comprising the amino acid sequence motif E- [FW] -hydrophobic-W-W-hydrophobic-D-x-D-A- hydrophobic-hydrophobic or an amino acid sequence motif with at least 60% homology to the amino acid sequence motif above.

4. An isolated nucleic acid according to any one of claims 1 to 3 which encodes a glycosyltransferase in which 25% or fewer of the amino acid residues in the catalytic domain of said glycosyltransferase are the same as the equivalent amino acid residues of the yeast glycosyltransferases gmal2p and mnnlOp.

5. An isolated nucleic acid according to any one of claims 1 to 4 wherein said plant glycosyltransferase is capable of catalysing one or more steps in the biosynthesis of complex non-cellulosic plant cell wall polysaccharides .

6. An isolated nucleic acid according to any one of claims 1 to 5 wherein said plant glycosyltransferase is capable of catalysing the glycosylation of plant cell wall proteins.

7. An isolated nucleic acid according to any one of claims 1 to 6 wherein said plant glycosyltransferase is capable of catalysing the transfer of saccharide moieties from nucleotide sugar precursors to oligosaccharide precursors .

8. An isolated nucleic acid according to any one of claims 1 to 7 which comprises a nucleic acid sequence which codes for an amino acid sequence as shown in any one of Figs 1, 2 and 4.

9. An isolated nucleic acid according to any one of claims 1 to 8 which has a nucleic acid sequence comprising nucleotide sequence as shown in any one of Figs 1, 2 or 4 or a homologous variant of a said nucleotide sequence which homologous variant codes for a plant glycosyltransferase in which 25% or fewer of the amino acid residues in the catalytic domain of said glycosyltransferase are the same as the equivalent residues of the yeast glycosyltransferases gmal2p and mnnlOp .

10. An isolated nucleic acid according to claim 9 which comprises a nucleic acid sequence which has 50% or more homology to a nucleotide sequence as shown in any one of Figs 1, 2 or 4.

11. An isolated nucleic acid according to any one of claims 1 to 10 which comprises a nucleic acid sequence which is a mutant, variant, derivative or allele of a nucleotide sequence which codes for an amino acid sequence as shown in any one of Figs 1, 2 and 4.

12. An isolated nucleic acid according to any one of claims 1 to 11 which comprises a nucleic acid sequence which is a mutant, variant, derivative or allele of a - nucleotide sequence as shown in any one of Figs 1 , 2 or 4.

13. An isolated nucleic acid according to claim 11 or claim 12 which is a derivative of a nucleic acid sequence as defined in claim 11 or claim 12 by way of addition, insertion, deletion or substitution of one or more nucleotides and which encodes a polypeptide having altered activity with respect to a polypeptide having an amino acid sequence as shown in any one of Figs 1, 2 or 4.

14. An isolated nucleic acid sequence which comprises a nucleic acid sequence which is complementary to a nucleic acid sequence as defined in any one of claims 8 to 13.

15. A recombinant nucleic acid molecule which comprises nucleic acid according to any one of claims 1 to 14, 73,

74 and 76.

16. A recombinant vector which comprises nucleic acid according to any one of claims 1 to 15, 73, 74 and 76.

17. A vector according to claim 16 which is capable of replicating in a suitable host.

18. A vector according to claim 16 or claim 17 wherein the nucleic acid is operably linked to a promoter or other regulatory element for transcription in a host cell.

19. A vector according to claim 18 which further comprises one or more of a terminator sequence, a polyadenylation sequence; an enhancer sequence; a marker gene .

20. A vector according to claim 18 or claim 19 wherein the promoter is an inducible promoter.

21. A vector according to any one of claims 16 to 20 which is a plant vector.

22. A vector according to claim 21 which comprises a genetic marker which confers a selectable phenotype selected from resistance to antibiotics or herbicides.

23. A method which comprises the step of introducing a vector according to any one of claims 16 to 22 into a cell.

24. A method for transforming a plant cell which comprises a method according to claim 23 and the step of causing or allowing recombination between the vector and the plant cell genome in order to introduce the nucleic acid into the genome.

25. A host cell which comprises a vector according to any one of claims 16 to 22.

26. A host cell which is transformed with a vector according to any one of claims 16 to 22.

27. A host cell according to claim 25 or claim 26 which is a plant cell .

28. A host cell according to claim 27 which is in a plant .

29. A method for producing a transgenic plant which comprises a method according to claim 24 and the step of regenerating a plant from the transformed cell.

30. A plant which comprises a cell according to any one of claims 25 to 28.

31. A plant according to claim 30 produced according to claim 29.

32. A plant which is the progeny of a plant according to claim 30 or claim 31.

33. A part or propagule of a plant^' according to any one of claims 30 to 32.

34. A method for identifying or cloning a glycosyltransferase from a plant which employs nucleic acid according to any one of claims 1 to 15, 73, 74 and 76.

35. A method according to claim 34 which comprises the step of searching a database to find a sequence which is homologous to a nucleic acid sequence which codes for an amino acid sequence as shown in any one of Figs 1, 2 or 4.

36. A method according to claim 34 which comprises the step of searching a database to find sequences which are homologous to a nucleic acid sequence as shown in any one of Figs 1, 2 or 4.

37. A method according to claim 34 which comprises the steps of: (a) providing a preparation of nucleic acid; (b) providing a nucleic acid according to any one of claims 1 to 15, 73, 74 and 76; (c) contacting said preparation of nucleic acid as in (a) with nucleic acid as in (b) under conditions for hybridisation of said nucleic acid as in (b) to a homologue in the preparation; and (d) identifying said homologue if present by its hybridisation with said nucleic acid as in (b) .

38. A method according to claim 37 wherein the hybridisation conditions are selected to allow the

" identification of sequences having about 50% or more sequence identity with nucleic acid according to any one of claims 1 to 15, 73, 74 and 76.

39. A method according to claim 34 which comprises the use of a primer to amplify nucleic acid according to any one of claims 1 to 15, 73, 74 and 76, said primer having a sequence comprising or complementary to a nucleic acid sequence which codes for an amino acid sequence as shown in any one of Figs 1, 2 or 4.

40. A method according to claim 34 which comprises the use of a primer to amplify nucleic acid according to any one of claims 1 to 15, 73, 74 and 76, said primer having a sequence comprising or complementary to a nucleic acid sequence as shown in any one of Figs 1, 2 or 4.

41. A method according to claim 39 or claim 40 which comprises the steps of (a) providing a preparation of plant nucleic acid; (b) providing a said primer; (c) contacting nucleic acid in said preparation with said primer under conditions for performance of amplification reaction; (d) performing said amplification reaction and determining the presence or absence of an amplified product .

42. A method according to any one of claims 39 to 41 wherein said primer has a sequence comprising or complementary to a nucleic acid sequence which codes for an amino acid sequence as shown in any one of Figs 1, 2 or 4 and wherein said primer has a sequence which comprises or is complementary to a portion of said nucleic acid sequence which codes for a sequence of amino acids which is conserved in glycosyltransferases.

43. A method according to any one of claims 39 to 41 wherein said primer has a sequence comprising or - complementary to a nucleic acid sequence as shown in any one of Figs 1, 2 or 4 and wherein said primer has a sequence which comprises or is complementary to a portion of said nucleic acid sequence which codes for a sequence of amino acids which is conserved in glycosyltransferases .

44. A method according to claim 42 or claim 43 wherein said sequence of amino acids which is conserved comprises the motif E- [FW] -hydrophobic-W-W-hydrophobic-D-x-D-A- hydrophobic-hydrophobic or a motif with at least 60% homology to the amino acid sequence motif above.

45. A method according to any one of claims 39 to 41 wherein said primer has a sequence comprising or complementary to a nucleic acid sequence which codes for an amino acid sequence as shown in any one of Figs 1, 2 or 4 and wherein said primer has a sequence which comprises or is complementary to a portion of said nucleic acid sequence which codes for a sequence of amino acids which is not conserved in glycosyltransferases.

46. A method according to any one of claims 39 to 41 wherein said primer has a sequence comprising or complementary to a nucleic acid sequence as shown in any one of Figs 1, 2 or 4 and wherein said primer has a sequence which comprises or is complementary to a portion of said nucleic acid sequence which is not conserved in glycosyltransferases .

47. A nucleic acid molecule for use as a probe or primer in a method according to any one of claims 34, 37 to 46, 78 to 80, said molecule having a sequence comprising or complementary to a nucleic acid sequence which codes for an amino acid sequence as shown in any one of Figs 1, 2 or 4 .

' 48. A nucleic acid molecule for use as a probe or primer in a method according to any one of claims 34, 37 to 46, 78 to 80, said molecule having a sequence comprising or complementary to a nucleic acid sequence as shown in any one of Figs 1, 2 or 4.

49. A polypeptide comprising the activity of a plant glycosyltransferase encoded by nucleic acid according to any one of claims 1 to 15, 73, 74 and 76.

50. A recombinant polypeptide according to claim 49.

51. A recombinant polypeptide according to claim 50 fused to another polypeptide.

52. A recombinant polypeptide according to claim 51 wherein said another polypeptide is a marker polypeptide or a polypeptide which confers resistance to an antibiotic or a herbicide.

53. A method of producing a polypeptide comprising the activity of a plant glycosyltransferase which comprises the step of causing or allowing expression from a nucleic acid according to any one of claims 1 to 15, 73, 74 and 76 in a suitable host cell.

54. A composition comprising the polypeptide of any one of claims 49 to 52.

55. An antibody or fragment thereof, or a polypeptide comprising the antigen-binding domain of an antibody capable of specifically binding the polypeptide of any one of claims 49 to 52.

56. A method for producing the antibody, fragment or polypeptide according to claim 55 which comprises the step of immunising a mammal with a polypeptide according - to any one of claims 49 to 52.

57. A method of identifying and/or isolating a glycosyltransferase which comprises the step of screening candidate polypeptides with a polypeptide comprising the antigen-binding domain of the antibody of claim 55.

58. A method for the in vi tro synthesis of a polysaccharide which comprises the use of the polypeptide according to any one of claims 49 to 52.

59. A method for altering the quality or quantity of a polysaccharide in a host cell by influencing the glycosyltransferase activity in that cell, the method comprising use of any one or more of the following: all or part of nucleic acid according to any one of claims 1 to 15, 73, 74 and 76; the polypeptide of any one of claims 49 to 52; the antibody, fragment or polypeptide of claim 55.

60. A method according to claim 59 wherein the polysaccharide is a complex non-cellulosic plant cell wall polysaccharide.

61. A method according to claim 59 or claim 60 which comprises the step of causing or allowing expression of a nucleic acid according to any one of claims 1 to 15, 73, 74 and 76 within the cell.

62. A method according to claim 59 or claim 60 which comprises altering the glycosyltransferase activity in the cell.

63. A method according to claim 62 wherein the alteration comprises reducing the glycosyltransferase activity in the cell.

- 64. A method according to claim 62 wherein the alteration comprises elevating the glycosyltransferase activity in the cell.

65. A method according to claim 62 or claim 63 which comprises the step of causing or allowing the transcription of part of the nucleic acid of any one of claims 1 to 15, 73, 74 and 76 in the cell such as to co- suppress the expression of an endogenous glycosyltransferase .

66. A method according to claim 65 which comprises the step of causing or allowing the transcription of nucleic acid of any one of claims 1 to 15, 73, 74 and 76 in the cell.

67. A method according to claim 65 which comprises the step of causing or allowing the expression of a polypeptide comprising the antigen-binding domain of the antibody of claim 55.

68. A method according to any one of claims 59 to 67 wherein the cell is a plant cell.

69. A method according to claim 68 wherein the plant cell is part of a plant .

70. A complex non-cellulosic plant cell wall polysaccharide, the quantity or quality of which has been altered in accordance with the method of claim 69.

71. A plant product derived from a plant according to any one of claims 30 to 32 or a plant cell according to claim 27, 28 or 33, said product comprising a complex non-cellulosic plant cell wall polysaccharide of claim

70 .

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72. A commodity comprising an altered cell wall storage polysaccharide according to claim 70.

73. An isolated nucleic acid sequence according to any one of claims 1 to 15 or a polypeptide according to claim 49 which is obtainable from plant material.

74. An isolated nucleic acid sequence according to any one of claims 1 to 15 or a polypeptide according to claim 49 which is obtainable from Arabidopsis thaliana.

75. A method for identifying or cloning a glycosyltransferase from a plant which utilises a nucleic acid sequence coding for a yeast glycosyltransferase.

76. An isolated nucleic acid sequence according to any one of claims 1 to 15 encoding a glycosyltransferase capable of catalysing one or more steps in the biosynthesis of cellulose-containing cell walls.

77. A method according to claim 37 wherein the preparation nucleic acid is from plant cells.

78. A method according to any one of claims 39 to 46 which comprises the use of two primers .

79. A method according to claim 78 wherein either or both of said primers have either a sequence comprising or complementary to a nucleic acid sequence which codes for an amino acid sequence as shown in any one of Figs 1, 2 or 4 or a sequence comprising or complementary to a nucleic acid sequence as shown in any one of Figs 1, 2 or 4.

80. A method according to claim 79 wherein either or both of said primers comprise or are complementary to a portion of said nucleic acid sequence which codes for a sequence of amino acids which is conserved in glycosyltransferases .

81. A method according to claim 79 wherein either or both of said primers comprise or are complementary to a portion of said nucleic acid sequence which codes for a sequence of amino acids which is not conserved in glycosyl transferases .

82. A method for producing the antibody, fragment or polypeptide according to claim 55 which comprises the step of using a polypeptide according to any one of claims 49 to 52 to screen a library of antibodies, antibody fragments or polypeptides comprising antigen- binding domains .

83. A commodity according to claim 72 which comprises a wood pulp product in the form of paper or cardboard.

84. A method according to claim 75 wherein the yeast glycosyltransferase is one or more of gmal2p or mnnlOp.