WO1999007857A1 - Pectin degrading enzymes - Google Patents

Abstract

Disclosed are nucleic acids encoding pectate lyases from plant somatic cells (e.g. Seq. ID No. 1 from Zinnia elegans c. Envy) and their promoters. Also disclosed are variant nucleic acids (e.g. alleles, homologues, derivatives) and methods and materials for obtaining the same, e.g. based on probing or PCR. Vectors, host cells, transgenic plants and parts and progeny thereof having altered pectate lyase activity are also disclosed, as are methods and materials for obtaining them. The invention also embraces pectate lyases themselves and antibodies specific for them, plus also altered pectins and other polygalacturonate-containing substrates.

Description

PECTIN DEGRADING ENZYMES

TECHNICAL FIELD

The present invention relates broadly to enzymes having pectate lyase activity, genes encoding such enzymes, and methods and materials for isolating and utilising them.

PRIOR ART

Pectate lyases (E.C. 4.2.2.2) have previously been described as microbial extracellular enzymes that assist pathogenesis by cleaving of polygalacturonate blocks in the plant host cell wall (Davis et al . 1984, Collmer and Keen 1986) .

A pectate lyase is an enzyme capable of degrading pectin via the hydrolysis of α-1, 4-galacturonosyl residues by jS-elimination. This results in an unsaturated C-4 - C-5 bond in the galacturonosyl moiety at the non- reducing end of the polysaccharide produced at the cleavage site (Rombouts and Pilnik 1980) .

This mechanism is common to another class of pectin- degrading enzymes, pectin lyases, but pectate lyase is distinguished by its preference for a glycosidic linkage next to a free carboxyl group rather than to an esterified carboxyl group and by its pH optimum (Pilnik 1990) .

All pectate lyases show calcium dependence. Microbial pectate lyases usually exist as multiple, independently-regulated isozymes that are 27 to 80 % identical in amino acid sequence, and have subtle differences in substrate specificity (Preston et al . 1992) . cDNAs with homology to microbial pectate lyases have been reported in several plant species, principally in relation to pollen tube growth. Pectate lyase-like genes are expressed in transmitting tissue (McCormick 1991) together with other hydrolases, 3-glucanase, chitinase, proteinase inhibitor, proline-rich proteins and hydroxyproline-rich glycoproteins (Gasser and Robinson- Beers 1993) . Two pollen-expressed genes, LAT 56 and LAT 59 show homology to Erwinia pectate lyases (Wing et al 1989) and are each represented as single-copies in the tomato genome. LAT56 and LAT59 sequences are strikingly similar to the major ragweed pollen allergen (Ambal) (Rafner et al . 1991), to a cDNA clone LMP131 expressed preferentially in anthers of lily (Kim et al . 1994) and to a tomato cDNA that is predominantly expressed in pistils but also detected at low levels in roots (Budelier et al . 1990) . Further homologies were found with the protein sequence of a cedar pollen allergen Cry j I(Sone et al . 1994), pollen-specific maize genes (Turcich et al . 1993), P0149 from alfalfa (Wu et al . 1996) and a genomic clone in tobacco (Rogers et al . 1992) . Southern analysis showed that there were two genes bearing 96% homology to each other in tobacco, and possibly other less closely related sequences.

It is important to note that to date, no pectate lyase activity, rather than homology, has been demonstrated in somatic plant cells. Thus in Dircks et al (1996) it is reported that neither pollen extracts, nor baculovirus-transformed insect cells expressing LAT56 and 59, demonstrated pectate lyase activity.

Pectate lyase activity has been demonstrated by Taniguchi et al (1995) . However this was only in pollen, and not in somatic plant cells; nor was it detected in extracts, but only after purification. Authentic plant pectate lyases may be useful, inter alia , for modifying plant cell walls, either in vivo (e.g. to alter storage properties or digestibility) or in vi tro (e.g. as a treatment step during brewing) .

Thus it can be seen that novel enzymes having pectate lyase activity (particularly when derived from plants), genes encoding such enzymes, and methods and materials for isolating and utilising them could provide a contribution to the art.

DISCLOSURE OF THE INVENTION

The present inventors have used a model system based around mesophyll cells from the leaves of Zinnia elegans c . Envy (see Roberts and Haigler 1994, Stacey et al . 1995) to identify a novel gene with sequence homology to pectate lyase. The gene (hereinafter ^λ ZePel') has homology to both the pectate lyase-like plant sequences and to the microbial enzymes.

In addition pectate lyase activity has been demonstrated in populations of both (a) elongating and (b) differentiating cells following treatment with auxin suggesting a general, widely applicable, utility in the modification of plant cell wall architecture. The mRNA encoding the enzyme is up-regulated in vi tro during both cell elongation and cell differentiation in response to auxin, but in si tu hybridisation suggests that in Zinnia plants it is associated specifically with vascular bundles and shoot primordia rather than with all elongating cells.

It has also been demonstrated that the ZePel protein has pectate lyase activity by using bacterially expressed recombinant enzyme. The recombinant enzyme has been characterised for calcium dependence, Michaelis-Menten constant and pH optimum. The activity of ZePel has been demonstrated at room temperature and within the range of physiological pH. This may be contrasted with the pollen pectate lyase of Taniguchi et al (1995) which apparently had an optimum temperature and pH of 60-70°C and pH 10.

In addition, it appears from observations of the time course during which the gene is up-regulated and its localisation, that the ZePel promoter has unusual and industrially applicable properties as regards induction.

These and other aspects of the present invention will now be discussed in more detail .

Thus in a first aspect of the invention there is disclosed a nucleic acid molecule encoding a plant pectate lyase wherein the pectate lyase enzyme is obtainable from a plant somatic cell and/or a prokaryotic host transformed with said nucleic acid molecule.

Transformation of suitable hosts with the nucleic acids of the present invention is discussed in more detail below.

Nucleic acid molecules (also referred to as nucleic acids) and vectors (see below) 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. Obviously nucleic acid molecules comprising, in addition to those consisting of, the stated sequences are included. 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.

Preferably the nucleic acid molecule has the nucleotide sequence shown in Seq ID No 1, optionally excluding non-coding regions as appropriate.

The nucleotide sequence of the ZePel cDNA (Seq ID No 1) and its predicted amino acid sequence (Hereinafter Seq ID No 2) are presented in Figure 1. Without the polyA tail, the cDNA is 1440 nucleotides long and contains an open reading frame (ORF) for a protein of 401 amino acid residues, beginning with the ATG initiation codon at position 28 and ending with a TGA stop codon at position 1230. The calculated molecular mass for this protein is 44,406 D and the pi is 8.15. Analysis of the predicted amino acid sequence revealed a hydrophobic N- erminal region with characteristics of a signal peptide 'von Heijne, 1986) and a potential cleavage site between Ser20 and Ser21 that would leave a protein with a calculated molecular mass of 42,362 D and a pi of 8.15. The absence of an endoplasmic reticulum retention signal (KDΞL or

HDEL) (Denecke et al . , 1992) suggests that the processed protein is exported from the endoplasmic reticulum once it is synthesized. The predicted amino acid sequence contains a consensus sequence for N-glycosylation (Asn-X- Ser/Thr) at amino acid residue 38.

Comparison of the cDNA sequence with data in the EMBL database revealed clear homology with other fungal and bacterial pectate lyases (42.2% with BsPei, 44.3% with E. chrysantemi Pel A), pectin lyases (44.3% with E . carotova) as well as with plant allergens (52.3% with Amb a I, 53.6% with Cry j I) and putative plant pectate lyases (68.7% with tomato le9612, 52.1% with tobacco G10) . The homology was also present at the amino acid level as shown in Fig 2) . The protein sequence of the protein shows typical features of a pectate lyase.

Numbering the residues according to the 'mature' protein i.e. from the potential Ser-Ser cleavage site, the calcium binding site is conserved (Asp 179, 203 and 207) in addition to the putative active site residues: RMPRPCR (residues 259-264 (Fig. 1) . The BsPel has three aspartic acid residues in the putative active site cleft: Asp 184 is a ligand to the putative active site calcium ion, the others being Asp 223 and 227. PelE has three Asps also at the active site but PelC has two Asps and a Glu, and the cleft is not so pronounced (Yoder et al . 1993, Pickersgill et al . 1994, Lietzke et al . 1994, 1996) . In ZePel, the active site is more basic than in pectin lyases due to the contributions of Lys227, Arg262 and Arg264 (as in BsPel) . Of these, the Arg264 is absent in pectin lyases. The highly conserved vWiDH region among petcin and pectate lyases is also present in ZePEl . In a second aspect of the present invention there is provided a nucleic acid molecule being a mutant, variant, derivative or allele of the nucleic acid of the first aspect .

Preferred mutants, variants, derivatives and alleles are those which encode a product which is homologous to the enzyme encoded by Seq ID No 1, and wherein the product retains all or part of the pectate lyase activity of that enzyme .

Particularly desirable mutations may be those which introduce or destroy restriction sites in the sequence. Other mutations may be to remove or introduce Met codons ; signal sequences; putative glycosylation or cleavage sites etc. All of these modifications may facilitate expression of the nucleic in particular hosts or circumstances. Particularly envisaged are the variants discussed hereinafter lacking N-terminal sequence, preferably corresponding to the putative mature protein.

Methods for producing or identifying such a mutant, variant, derivative or allele (or other homolog) and assessing homology and function are discussed below.

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 (i.e. 'degeneratively equivalent') are included. Methods for achieving such changes (e.g. mutant PCR primers) are well known to those skilled in the art .

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

As is well known to those skilled in the art, altering the primary structure of a peptide by a conservative substitution may not significantly alter the activity of that peptide because the side-chain of the amino acid which is inserted into the sequence may be able to form similar bonds and contacts as the side chain of the amino acid which has been substituted out. This is so even when the substitution is in a region which is critical in determining the peptides conformation.

Also included are homologs in having a few non-conservative substitutions. As is well known to those skilled in the art, substitutions to regions of a peptide which are not critical in determining its conformation may not greatly affect its activity because they do not greatly alter the peptide' s three dimensional structure. Indeed, non-conservative substitutions even in or around the active site may confer slightly advantageous properties on the peptide: in the present context that may mean an altered substrate specificity, increased turnover number, altered pH dependence or temperature optimum etc.

A mutant, variant or derivative amino acid sequence in accordance with the present invention may include within the 401 amino acid 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, variant or derivative amino acid sequence may of course include additional amino acids at the C-terminus and/or N-ter inus. In a third aspect of the present invention there is disclosed a method of identifying and cloning a pectate lyase homolog or allele from a plant species which method employs a nucleotide sequence derived from that shown in Seq ID No 1.

It may be that the nucleotide sequence used in the identification is taken from a portion of Seq ID No 1 not being identical to known pectate lyases, such that molecules can be identified which could not have been identified prior to the work of the present inventors . Even if portions of the sequence are used which are homologous to known pectate lyases, the differences may still mean that novel pectate lyases (particularly those expressed in somatic plant cells, more particularly parenchyma) may be more readily identified.

In one embodiment of the third aspect, 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 ability to degrade pectins.

In a second embodiment, an allele or homologue in accordance with the present invention is also obtainable by means of a method which includes providing a preparation of nucleic acid, e.g. from arabidopsis, providing a nucleic acid molecule having a nucleotide sequence shown in or complementary to a nucleotide sequence shown in Seq ID No 1, preferably from within the coding sequence (i.e. coding for an amino acid sequence shown in Figure 1) , contacting nucleic acid in said preparation with said nucleic acid molecule under conditions for hybridisation of said nucleic acid molecule to any said gene or homologue in said preparation, and identifying said gene or homologue if present by its hybridisation with said nucleic acid molecule .

Probing may employ the standard Southern blotting technique. For instance DNA may be extracted from cells and digested with different restriction enzymes. Restriction fragments may then be separated by electrophoresis on an agarose gel, before denaturation and transfer to a nitrocellulose filter. Labelled probe may be hybridised to the DNA fragments on the filter and binding determined. DNA for probing may be prepared from RNA preparations from cells.

Test nucleic acid may be provided from a cell as genomic DNA, cDNA or RNA, or a mixture of any of these, preferably as a library in a suitable vector. Naturally the use of genomic DNA may allow the identification of non-transcribed regions of DNA e.g. promoter or other control elements. These are discussed in more detail later.

Binding of a probe to target nucleic acid (e.g. DNA) may be measured using any of a variety of techniques at the disposal of those skilled in the art. For instance, probes may be radioactively, fluorescently or enzymatically labelled. Other methods not employing labelling of probe include examination of restriction fragment length polymorphisms, amplification using PCR (see below), RN'ase cleavage and allele specific oligonucleotide probing.

Probes for use in these methods, optionally labelled, form one part of this aspect of the invention.

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. Suitable conditions would be achieved when a large number of hybridising fragments were obtained while the background hybridisation was low. Using these conditions nucleic acid libraries, e.g. cDNA libraries representative of expressed sequences, may be searched.

Those skilled in the art, in the light of the present disclosure, are well able to employ suitable conditions of the desired stringency for selective hybridisation, taking into account factors such as oligonucleotide length and base composition, temperature and so on.

For instance, screening may initially be carried out under conditions, which comprise a temperature of about 37°C or less, a formamide concentration of less than about 50%, and a moderate to low salt (e.g. Standard Saline Citrate (^ΛSSC') = 0.15 M sodium chloride; 0.15 M sodium citrate; pH 7) concentration. Alternatively, a temperature of about 50 °C or less and a high salt (e.g. ^VSSPE'= 0.180 mM sodium chloride; 9 mM disodium hydrogen phosphate; 9 mM sodium dihydrogen phosphate; 1 mM sodium EDTA; pH 7.4) . Preferably the screening is carried out at about 37°C, a formamide concentration of about 20%, and a salt concentration of about 5 X SSC, or a temperature of about 50 °C and a salt concentration of about 2 X SSPE. These conditions will allow the identification of sequences which have a substantial degree of homology

(similarity, identity) with the probe sequence, without requiring the perfect homology for the identification of a stable hybrid.

Suitable conditions include, e.g. for detection of sequences that are about 80-90% identical, hybridization overnight at 42°C in 0.25M Na₂HP0₄, pH 7.2 , 6.5% SDS , 10% dextran sulfate and a final wash at 55°C in 0. IX SSC, 0.1% SDS. For detection of sequences that are greater than about 90% identical, suitable conditions include hybridization overnight at 65 °C in 0.25M Na₂HP0₄, pH 7.2, 6.5% SDS, 10% dextran sulfate and a final wash at 60°C in 0.1X SSC, 0.1% SDS.

Hybridisation is generally followed by identification of successful hybridisation and isolation of nucleic acid which has hybridised, which may involve one or more steps of PCR (see below) .

In a third embodiment of this aspect, hybridisation of nucleic acid molecule to an allele or homologue may be determined or identified indirectly, e.g using a nucleic acid amplification reaction, particularly the polymerase chain reaction (PCR) . PCR requires the use of two primers to specifically amplify target nucleic acid, so preferably two nucleic acid molecules with sequences characteristic of the ZePel sequence are employed.

However, if RACE is used (see below) only one such primer may be needed.

PCR techniques for the amplification of nucleic acid are described in US Patent No. 4,683,195 and Saiki en ai . Science 239: 487-491 (1988). PCR includes steps of denaturation of template nucleic acid (if double- stranded) , annealing of primer to target, and polymerisation. The nucleic acid probed or used as template in the amplification reaction may be genomic DNA, cDNA or RNA. PCR may be used to amplify specific sequences from genomic DNA, specific RNA sequences and cDNA transcribed from mRNA. References for the general use of PCR techniques include Mullis et al , Cold Spring Harbor Symp . Quant. Biol . , 51:263, (1987), Ehrlich (ed) , PCR technology, Stockton Press, NY, 1989, Ehrlich et ai , Science, 252:1643-1650, (1991), "PCR protocols; A Guide to Methods and Applications", Eds. Innis et ai , Academic Press, New York, (1990) .

Prior to any PCR that is to be performed, the complexity of a nucleic acid sample may be reduced where appropriate by creating a cDNA library for example using RT-PCR or by using the phenol emulsion reassociation technique (Clarke et al . (1992) NAR 20, 1289-1292) on a genomic library.

A method involving use of PCR in obtaining nucleic acid according to the present invention may include providing a preparation of plant nucleic acid, e.g. from wheat, providing a pair of nucleic acid molecule primers useful in (i.e. suitable for) PCR, at least one said primer having a sequence shown in or complementary to a sequence shown in Seq ID No 1, contacting nucleic acid in said preparation with said primers under conditions for performance of PCR, performing PCR and determining the presence or absence of an amplified PCR product. The presence of an amplified PCR product may indicate identification of a gene of interest or fragment thereof.

Thus the methods of the invention may include hybridisation of one or more (e.g. two) probes or primers to target nucleic acid. Where the nucleic acid is double-stranded DNA, hybridisation will generally be preceded by denaturation to produce single-stranded DNA. The hybridisation may be as part of a PCR procedure, or as part of a probing procedure not involving PCR. An example procedure would be a combination of PCR and low stringency hybridisation. A screening procedure, chosen from the many available to those skilled in the art, is used to identify successful hybridisation events and isolated hybridised nucleic acid.

Primers and probes for use in these methods form one part of this aspect of the present invention.

An oligonucleotide for use in probing or nucleic acid amplification may have about 10 or fewer codons (e.g. 6, 7 or 8), i.e. be about 30 or fewer nucleotides in length (e.g. 18, 21 or 24) . Generally specific probes/primers are upwards of 14 nucleotides in length. For optimum specificity, primers of 16-24 nucleotides in length may be preferred. Probes may be much longer e.g. 100 's or even 1000' s of bases long. Those skilled in the art are well versed in the design of primers for use processes such as PCR.

In all cases the sequence of the mutant, variant, derivative, alllele or other homolog related to Seq ID No 1 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 of Seq ID No 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. 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 or more amino acids or codons, compared with the relevant amino acid sequence or nucleotide sequence as the case may be.

Similarly the enzyme encoded by the mutant, variant, derivative, allele or other homolog related to Seq ID No 1 has pectate lyase activity.

One possible mode of analysing this activity is by transformation to assess function on introduction into a bacterial or plant or plant cell of interest and assay using a suitable pectin substrate or analog; methodology for such transformation is described in more detail below.

Thus the nucleic acid of the present invention, 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.

Such replicable vectors form a fourth aspect of the present invention.

DNA vector is defined to include, inter alia, any plasmid, cosmid, phage or Agrobacterium binary vector in double or single stranded linear or circular form which may or may not be self transmissible or mobilizable.

Vectors may transform a prokaryotic or eukaryotic host either by integration into the cellular genome or exist extrachromosomally (e.g. autonomous replicating plasmid with an origin of replication) . Vectors may be introduced into hosts by any appropriate method e.g. conjugation, mobilisation, transformation, transfection, transduction or electoporation. Also include shuttle vectors by which is meant a DNA vehicle capable, naturally or by design, of replication in both the actinomycetes and related species and in bacteria and/or eucaryotic (e.g. higher plant, mammalian, yeast or fungal cells).

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 into cells for recombination into the genome. The ZePel promoter itself, which forms a further aspect of the present invention, is described hereinafter.

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 . For further details see, for example Sambrook et al (1989) . 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 such as kanamycin, hygromycin, phosphinotricin, chlorsulfuron, methotrexate, gentamycin, spectinomycin, imidazolinones and glyphosate .

The nucleic acid of the present invention 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

In a fifth aspect of the present invention, there is provided a gene construct, preferably a replicable vector, comprising a promoter operatively linked to a nucleotide sequence provided by the present invention, such as the ZePel gene, a homolog from another plant species, or any mutant, variant or allele thereof.

By "promoter" is meant a sequence of nucleotides from which transcription may be initiated of DNA operably linked downstream (i.e. in the 3' direction on the sense strand of double-stranded DNA) .

"Operably linked" means joined as part of the same nucleic acid molecule, suitably positioned and oriented for transcription to be initiated from the promoter. DNA operably linked to a promoter is "under transcriptional initiation regulation" of the promoter.

It should be noted that the present invention represents the first demonstration of recombinant expression of a plant pectate lyase in E. coli .

The promoter may include one or more sequence motifs or elements conferring developmental and/or tissue- specific regulatory control of expression. Other regulatory sequences may be included, for instance as identified by mutation or digest assay in an appropriate expression system or by sequence comparison with available information, e.g. using a computer to search on-line databases.

Suitable promoters for plants 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) .

In one embodiment of this aspect, the promoter is an inducible oromoter. 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 present invention also provides a host (e.g. plants) transformed with said gene constructs or vectors and methods comprising introduction of such a construct into hosts and/or induction of expression of a construct within a host, by application of a suitable stimulus, an effective exogenous inducer.

One 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 .

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. 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 (see below) .

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 32/09696, WO 94/00583, EP 331083, EP 175966, Green et al . (1987) Plant Tissue and Cell Culture, 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) Plan t Cell Rep . 7, 379-384; Zhang, e t al . (1988) Theor Appl Gene t 76, 835-840; Shimamoto, et al . (1989) Nature 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 Mol ecular 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, electroporation and direct DNA 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-486234) 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 reviewd in Vasil et al . , Cell Cul ture and Somatic Cell Genetics of Plants , Vol I, II and III, Laboratory Procedures and Their Applications , Academic Press, 1984, and Weissbach and Weissbach, Methods for Plan t 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.

Methods of use of the vectors of the present invention form a further (sixth) ascect of the invention. Thus a method of making plant cell involving introduction of a vector of the present invention 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.

In a seventh aspect of the present invention there is disclosed a host cell containing nucleic acid or a vector according to the present invention, especially a plant or a microbial cell. 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 .

Within the cell, the nucleic acid may be incorporated within the chromosome . There may be more than one heterologous nucleotide sequence per haploid genome .

Thus 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.

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 etc. 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 occuring 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.

A plant may be regenerated from one or more transformed plant cells.

Thus in an eighth aspect of the invention there is disclosed a plant which includes a plant cell according to the invention. Also embraced is 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.

In a ninth aspect, the invention further provides a method of influencing or affecting a pectin content of a plant said method comprising the step of causing or allowing expression of a heterologous nucleic acid sequence encoding a pectate lyase in a plant cell of that plant (preferably a somatic cell) .

In nature, ZePel may be used to assist in the removal and modification of an existing pectin matrix in order to allow the deposition of newly-synthesized wall polymers for a specialised function or to create an architecture that is extensible.

This activity can be utilised, for instance, by expressing ZePel protein in response to an applied stimulus in forage crops (e.g. forage maize, grasses, alfalfa etc.) where the induction is timed just prior to harvest, or alternatively where the ZePel is vacuolar targetted and released upon physical disruption of the plant tissue at harvest (e.g. on chopping prior to ensiling) . Breakdown of pectin may allow better access of degrading microorganisms/digestive enzymes to the plant tissue, thereby increasing digestibility of the forage (leading to increased animal productivity.)

Depending on the precise application, the ZePel enzyme may have a number of advantages over pectate lyases in the prior art. For instance (as compared to bacterial enzymes) it may be possible to use it in vivo in plants without triggering a defensive response (by producing pectin degradation products which do not function as elicitors, or are less powerful elicitors than those generated by pathogens) . Conversely it may be used to prime the plants defense mechanisms in certain tissues without causing excessive 'soft rot' type damage to the plant in the process. It is certainly probable (by virtue of its novel sequence) that the enzyme will have different specificity and activity to microbial enzymes, and that this difference may make it more appropriate for use with or in plants.

Thus, preferably the nucleic acid encodes the amino acid sequence of Figure 1, or a mutant, variant, allele or derivative of the sequence, within cells of the plant (thereby producing the encoded polypeptide) , following an earlier step of introduction of the nucleic acid into a ceil of the plant or an ancestor thereof.

In the present invention, over-expression of pectate lyase activity may be achieved by introduction of the nucleotide sequence in a sense orientation. In a separate (tenth) aspect of the present invention, 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) Na ture 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. 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. Again, fragments, mutants and so on may be used in similar terms as described above for use in anti-sense regulation.

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 speaking therefore, the transcribed nucleic acid may represent a fragment of a pectate lyase gene of the present invention, such as including a nucleotide sequence shown in Seq ID No 1, or the complement thereof, or may be a mutant, derivative, variant or allele thereof, in similar terms as discussed above. 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 or 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 or affecting a pectin content 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 . Also provided is a method of influencing or affecting a pectin content of a plant, the method including causing or allowing expression from nucleic acid according to the invention within cells of the plant. Further options for down regulation of gene expression include the use of ribozymes, e.g. hammerhead ribozymes, which can catalyse the site-specific cleavage of RNA, such as mRNA (see e.g. Jaeger (1997) "The new world of ribozymes" Curr Opin Struct Biol 7:324-335, or Gibson & Shillitoe (1997) "Ribozymes : their functions and strategies form their use" Mol Biotechnol 7: 242-251.)

In each case the pectate lyase activity of the plant is preferably suppressed as a result of under-expression within the plant cells. This may be particularly useful in controlling the rate of ripening/softening in fruits from plants in which ZePel homologs have been identified, for instance using the methods of the third aspect.

In an eleventh aspect, the present invention also encompasses the expression product (generally a pectate lyase, preferably ZePel) of any of the nucleic acid sequences of the invention disclosed above, 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.

The method of preparing the expression products of the present invention may particularly involve extraction of inclusion bodies from a microbial host, using detergent (preferably w th n-dodecyl α-D- maltoside) .

The expression product is most preferably a pectate lyase which is active at physiologically relevant temperature and pH. It may have modified properties with respect to the authentic ZePel as shown in Figure 1 when expressed in nature, or as expressed in E. coli as described herein. Such modifications are discussed in relation to the second aspect of the invention. They may include alterations for ease of expression, or to change specificity (e.g. for pectins having a pattern of methyl esterification or some other physical characteristic) .

Additionally, it is known that cell autolysis is a key event in tracheid formation, and this involves disruption of the vacuole to release hydrolytic enzymes. Since ZePel has a role in tracheid formation it is likely that ZePel contains motifs or other signal seqences which target it to the vacuole or to the cell wall of the cells in which it is induced.

Polypeptides containing these motifs or signals form one part the present invention. They can be identified, for instance, by immunolocalisation of ZePel and/or mutants differing in defined regions and/or fusion proteins incorporating defined regions of the ZePel sequence .

The expression products of the eleventh aspect may be used to raise antibodies employing techniques which are standard in the art. These antibodies, and polypeptides comprising antigen-binding fragments of antibodies, form a twelth aspect of the invention, and may be used in identifying homologues from other species as discussed further below. They can be used, for instance in labelled form e.g. in immunolocalisation experiments such as those described above.

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 immunoprecipitation 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. Thus, the present invention embraces a method of identifying or isolating a polypeptide with pectate lyase activity (in accordance with embodiments disclosed herein) , comprising screening candidate polypeptides with a polypeptide comprising the antigen-binding domain of an antibody (for example whole antibody or a fragment thereof) which is able to bind a the ZePel enzyme, or fragment, variant or derivative thereof, and preferably has binding specificity for such a polypeptide. Methods employing these anti-ZePel and related antibodies are also embraced by the present invention.

In a thirteenth aspect of the invention there is disclosed a method of modifying a substrate (preferably a pectin-containing substrate) by contacting said substrate with a recombinant expression product of the eleventh aspect, optionally in purified or partly purified form. Said substrate in this context may be an extraneous one i.e. not part of the host cell from which the enzyme is derived. Recombinant ZePel (or modified derivatives thereof) , for instance, may be used in microbial fermentation. This may be useful in fruit or vegetable juice extraction or clarification; in brewing; in sugar beet processing, in (paper) pulp processing; in treating silage. The high specificity of pectate lyases can provide advantages over the use of chemical processing. They can thus be used to provide novel precursors for the food, chemical, or pharmaceutical industries. Such products, particularly those from modified ZePel, form one part of the present invention.

In each case the ZePel could be used in purified or partially purified form, or as a microbial innoculan .

In addition to the various aspects of the invention above concerned primarily with enzymes having pectate lyase activity and nucleic acid encoding them, a yet further aspect of the present invention concerns the non- coding regions of the ZePel gene.

Thus a fourteenth aspect of the invention is a nucleic acid molecule encoding the promoter of the ZePel gene .

The present inventors have noted that cells in the Zinnia system cultured in the presence of auxin alone have a higher pectate lyase activity than cells cultured in inductive medium. One possibility is that the cell division in the cultures containing auxin results in a higher cell density in a given culture volume. After eight days of culture in inductive medium a decrease of activity is detected, which may correlate with a decrease in the percentage of viable cells present after tracheary element differentiation. A second possibility is that the auxin- induced expression may be modulated by other growth factors such as cytokinin. It has also been demonstrated that ZePel expression is correlated with sites of vascular differentiation and with cells that are recent products of meristematic divisions. The localization of the pectate lyase gene expression to new primordia on the flanks of the shoot meristem reveals a transient expression of the gene, as only some primordia are labelled. Auxin induces vascular differentiation as well as being necessary for final maturation of xylem. Jacobs (1952) demonstrated that an auxin flux in a basipetal direction was implicated in xylem differentiation. Sachs (1981) proposed that the pattern of vascular system results from initial local differences in the flow of auxin through cells, which leads to the establishment of preferred channels of auxin transport. These channels become progressively improved pathways of auxin movement and drain the surrounding regions at the same time that their cells are induced to undergo differentiation as vascular elements. The pattern of gene expression, where the formation of new vascular strands are taking place, is consistent with auxin regulation of ZePel.

Auxin-inducible promoters may have utility in controlling the expression of particular products e.g. during industrial fermentation products.

Interestingly, although expression of ZePel is dramatically up-regulated by auxin, it is not a primary auxin response as defined by the appearance of the gene product within minutes of the inductive signal (Abel and Theoiogis 1996) and it is not related to the other auxin- regulated genes isolated from the Zinnia system (Ye and Varner 1993, 1994, Demura and Fukuda 1993), that appear later in the time course of determination and differentiation. The late-induced response exemplified by ZePel (i.e. abundant at 72 hours but not at 24 hours) is hereinafter termed "a late-auxin" response. The use of an auxin, or late-auxin, inducible promoter, which may be down-regulated by cytokinin (see above) may be used to induce the expression of ZePel or heterologous (to the promoter) nucleic acids in a delayed fashion.

In addition, the fact that the ZePel promoter may be induced preferentially in particular tissues (e.g. roots and stem) and/or stages of development means it may have particular utility in targeting ZePel or heterologous (to the promoter) nucleic acids to particular tissues or expressing them at particular times.

Since ZePel is apparently expressed in living cell types early in vascular development, the promoter may be advantageous in offering the ability to specifically up- or down-regulate genes in xylogenetic tissue e.g. parenchyma cells surrounding the xylem vessels.

This may useful when trying inter alia to modify lignification, affect forage digestibility, improve roughage content, or alter paper-pulping properties. Modification of targets specifically in vascular/lignified tissues without side effects in other tissues may be useful when the expressed factor or enzyme is detrimental to some tissues. For instance the transcription factor myb308 downregulates phenolics and leads to lowered iignin content with desirable results for forages/pulp but its expression in leaves leads to reduced ability to resist oxidative stress - hence its expression would be most beneficial if restricted to parenchyma .

Genes which may be usefully operably linked to the promoter include any of those encoding enzymes which are involved in cell wall formation, modification, or degradation (e.g. during growth, differentiation, abscission etc.) . Examples of such enzymes can be found in Brett and Waldron (1996) "Physiology and biochemistry of plant cell walls" Second Edition: Chapman & Hall, London. Particular enzymes may be concerned with the modification of cellulose, pectic polysaccharides, hemicellulose, lignin, plant proteins (including glycoproteins) plus energy storage polysaccharides, lipids, proteins etc.

It should be stressed that the pectate-lyase like genes identified to date (which, other than in pollen, have not been associated with actual functional enzymes) have generally been in the flower and (rarely) in roots. What's more they have not been shown to be auxin inducible: these observations suggest that the ZePel promoter is quite distinct from those characterised to date .

The promoter region may be readily identified using a probe or primer based on Seq ID No 1, as described in relation to the third aspect. This can be used in the identification and isolation of a promoter from a genomic library containing DNA derived from a plant source (i.e. the plant used to generate the ZePel cDNA) . Techniques and conditions for such probing are well known in the art as is discussed above. Following probing, promoter activity is assessed using a test transcription system.

"Promoter activity" is used to refer to ability to initiate transcription. The level of promoter activity is quantifiable for instance by assessment of the amount of mRNA produced by transcription from the promoter or by assessment of the amount of protein product produced by translation of mRNA produced by transcription from the promoter. The amount of a specific mRNA present in an expression system may be determined for example using specific oligonucleotides which are able to hybridise with the mRNA and which are labelled or may be used in a specific amplification reaction such as the polymerase chain reaction.

Use of a reporter gene facilitates determination of promoter activity by reference to protein production. The reporter gene preferably encodes an enzyme which catalyses a reaction which produces a detectable signal, preferably a visually detectable signal, such as a coloured product. Many examples are known, including β - galactosidase and luciferase. ?-galactosidase activity may be assayed by production of blue colour on substrate, the assay being by eye or by use of a spectro-photometer to measure absorbance. Fluorescence, for example that produced as a result of luciferase activity, may be quantitated using a spectrophotometer . Radioactive assays may be used, for instance using chloramphenicol acetyltransferase, which may also be used in non- radioactive assays . The presence and/or amount of gene product resulting from expression from the reporter gene may be determined using a molecule able to bind the product, such as an antibody or fragment thereof. The binding molecule may be labelled directly or indirectly using any standard technique.

Those skilled in the art are well aware of a multitude of possible reporter genes and assay techniques which may be used to determine promoter activity. Any suitable reporter/assay may be used and it should be appreciated that no particular choice is essential to or a limitation of the present invention.

Also embraced by the present invention is a promoter which is a mutant, variant, derivative, allele or other homolog of the ZePel promoter. These can be generated or identified as described above; they will share homology with the ZePel promoter and retain promoter activity. To find minimal elements or motifs responsible for tissue and/or developmental regulation, restriction enzyme or nucleases may be used to digest a nucleic acid molecule, followed by an appropriate assay (for example using a reporter gene such as luciferase) to determine the sequence required. Nucleic acid comprising these elements or motifs forms one part of the present invention.

Preferably the promoters of the present invention have the auxin inducible and/or tissue specific and/or developmentally specific promoter activity of the authentic ZePel promoter.

In a fifteenth aspect of the invention there is provided a nucleic acid construct, preferably an expression vector, including the ZePel promoter region or fragment, mutant, allele, derivative or variant thereof able to promote transcription, operably linked to a heterologous gene, e.g. a coding sequence, which is preferably not the coding sequence with which the promoter is operably linked in nature.

The invention will now be further illustrated with reference to the following non-limiting Figures and

Examples . Other embodiments falling within the scope of the invention will occur to those skilled in the art in the light of these.

FIGURES

Figure 1 : cDNA and predicted amino acid sequence of ZePel . The amino acid sequence predicted from the cDNA sequence of ZePel is shown in boldface letters . The predicted cleavage site for the mature protein is indicated with an asterisk. The potential N-glycosylation site is underlined. The DNA bases are numbered at the right . The nucleotide and deduced amino acid sequence have been submitted to EMBL, GenBank and DDBJ as accession number Y09541. Residues possibly involved in the active site and the calcium binding are double underlined.

Figure 2 : Homology of ZePel to conserved regions of other pectate lyases or pectin-degrading enzymes. Shown is a multiple alignment of the predicted amino acid sequence of ZePel, from Serl78 to His277 (numbered according to the 'mature' protein i.e. from the potential Ser-Ser cleavage site) to the predicted amino acid sequences of the most homologous sequences in the EMBL database, including tomato style-expressed gene 9612 (le9612) , tobacco pollen-specific gene G10 (tobGlO) , short ragweed allergen (Amb a I) , pectate lyase from B . Subtilis (BsPel) , E . carotova pectate lyase (Pelc) and pectin lyase (Pnl) . The alignment was created using PILEUP program (Genetics Computer Group) and sequence homology is shown by using the PRETTYBOX program. Dark shading indicates identical residues, and pale shading indicates residues that are conservative substitutions. Gaps introduced to improve the alignment are represented by dots .

Figure 3 : The pH dependence of recombinant ZePel enzyme activity as discussed in Example 4. The ■ and • represent the average enzyme activity in duplicate assays performed on different days.

Figure 4 : Time courses of pectate lyase activity at pH 10 in cells sampled from cultures in inductive medium, in medium containing 1.0 mgl^"1 NAA, or in maintenance medium. The specific activity was expressed in mU of activity per mg of protein (see Example 5) .

Figure 5: Michaelis-Menton analysis of ZePel carried out as described in Example 4.

EXAMPLES

Experimental procedure

Plant Ma terial and Cell Cul ture

Zinnia elegans cv. Envy (Chiltern Seeds, UK.) plants were grown from seed in a peat/sand potting compost in a controlled environment room at 20 °C with a 16 h day.

Mesophyll cells were isolated from the first true leaves of 14 -day-old seedlings as described previously (Stacey et al . , 1995). Cells were resuspended in culture medium (Fukuda and Komamine, 1980) with 1.0 mg I^"1 BAP and 1.0 mg I^"1 NAA (inductive medium) or 1 μg l^"1 BAP and 1 μg l^"1 NAA (maintenance medium) or 1.0 mg l^"1 BAP or 1.0 mg l^"1 NAA. Cultures were maintained at 27 °C in the dark, shaking at 80 rpm, with 3 ml of cell suspension per well in 6-well plates (Sterilin, UK) at a density of 10^s cells ml^"1.

cDNA Library Construction and Screening

A Zinnia cDNA library was constructed in λZAP (Stratagene, San Diego, CA) using cells cultured in inductive medium for 72 h. The library was screened in duplicate with radiolabeled single-strand cDNA probes from cells cultured in inductive medium for 24 h or 72 h. One of the phages showing differential hybridization with the cDNA probes was selected and converted to the pBluescript SK form by co-infection with R408 helper phage (Stratagene) . Hybridization and washes of filters were done at 70 °C with the Church and Gilbert (1984) hybridization solution.

RNA and DNA Gel Blot Analysis RΝA was purified from different cultured cells and organs as previously described (Shirzadegan et al . , 1991) . For RΝA gel blot analysis, 15 μg of total RΝA was electrophoresed on 1% agarose gels containing formaldehyde and blotted onto nylon membranes as described by Sambrook et al . (1989) . Equal loading of RNA was verified by ethidium bromide staining of the gel before transfer to the membrane. DNA was isolated from leaves as described previously (Rogers and Bendich, 1988) . For DNA gel blot analysis, 10 μg of DNA was digested with restriction enzymes, electrophoresed in a 0.7% agarose gel and blotted onto nylon membranes (Rogers and Bendich, 1988) . RNA and DNA gel blots were probed with the entire cDNA insert or an EcoRI/HincII released fragment, which was radiolabelled by random priming using T7 polymerase (Pharmacia) . Hybridization and filter- washing conditions were done at high stringency (Church and Gilbert, 1984) .

DNA Sequencing

DΝA sequence analysis was performed on both strands with the use of cycle-sequencing dye terminator kits (Abi Prism, Perkin Elmer), according to the manufacturer's instructions. Sequencing reactions were analyzed with an ABI 373A sequencing system. The Genetics Computer Group (GCG, Wisconsin, USA) program was used for sequence analysis and homology searching of the EMBL databases .

In Si tu mRNA Hybridization

A fragment of 0.9 kb EcoRI-HincII fragment of the ZePel gene was subcloned into Bluescript KS(+) and SK( + ) vectors to provide templates for T7 polymerase to generate sense and antisense RΝA. The probes were labelled with digoxigenin-uridine 5 ' -triphosphate (Boehringer) according to the manufacturer's instructions. The methods for tissue preparation and in situ hybridization were as described by Bradley et al (1993) .

Expression of Recombinant Enzyme in Escherichia col i Two oligonucleotides binding at the N-terminal end of the reading frame of ZePel were synthesised so as to introduce Ncol sites either over the initiating ATG (5'- AAACCATGGCAACCACAATTCTACC-3' ) or to create a Met-1 and exchange Ser21 to Ala, so as to remove the putative signal sequence (5 ' -GCTTCCATGGCACCAAGTAGAACCCC-3 ' ) . A single terminal oligonucleotide was synthesised to introduce a BamHI site following the stop codon (5'- CTCGGATCCATAATCAACAACGAGACCC-3' ) .

Two independent PCR amplifications of the ZePel cDNA ( ca lOOng) were performed using the two alternative N- terminal oligonucleotides and the single C-terminal oligonucleotide (500ng each) with AmpliTaq (Perkin-Elmer, UK) for 25 cycles of 1 min at 94 'C to denature, 1 min at 57 °C to anneal and extension for 2 min at 72 °C. The PCR product was purified from a 0.8% agarose gel which was then made blunt ended and phosphorylated using klenow fragment and polynucleotide kinase . This product was ligated upon itself using T4 ligase which was then cut with the restriction enzymes Ncol and BamHI before ligation into a modified form of pBluescript similarly digested and transformed into E. coli Sure cells (Stratagene, Cambridge UK) and plated out on L-agar containing ampicillin (50 μg/ml) , IPTG (1 mM) and X-gal (12.5 mg) . The recombinant plasmids carrying the ZePel cDNA were sequenced and shown to carry the PCR induced mutations and otherwise to retain the native sequence. The ZePel cDNAs were then liberated with Ncol and BamHI and gel-purified before ligation into pET3d (Studier et ai . 1990) previously digested with Ncol and BamHI. These constructs were then transformed into E. coli BL21 cells carrying pLysS and plated out on L-agar with ampicillin (50 μg/ml) and chloramphenicol (30 μg/ml) .

Production of Pecta te Lyase in E. coli

500 ml L-broth was inoculated with 1 ml of overnight culture until 0.5 OD 600nm was reached, and then induced with 0.4 mM IPTG until 1 OD 600nm. The periplasmic, cytoplasmic and inclusion body fractions were checked for protein expression using 12% SDS-PAGE. The IPTG-inducible protein bands were blotted to Immobilon-P (Millipore, USA) from which the N-terminal protein sequences were determined using automated Edman degradation. For enzyme production the cells were grown in 500 ml L-broth containing chloramphenicol (30 μg/ml) and ampicillin (50 μg/ml) with protein induction as carried out above. The cells were French-pressed using a 50 mM Tris HC1 pH 7.5 buffer and 0.1 M NaCl , 0.1% n-dodecyl α-D-maltoside . The culture was centrifuged at 12000 rpm at 4°C in a Sorvall SS-34 rotor. The clarified supernatant was initially used to determine pectate lyase activity using 50 mM Tris HCl pH 8.5 buffer, 0.5% polygalacturonic acid and 1 mM CaCl₂ assay conditions (Nasser et al . 1990) . The V_max, pH optimum and calcium dependence were initially established using the enriched detergent extract before confirmation following purification by ion-exchange chromatography

(MonoQ and MonoS; Pharmacia, UK) in the presence of 0.1% n-dodecyl α-D-maltoside . The enzyme assays were performed with 50 mM Na acetate within the pH ranges 5 to 7; with 50 mM Tris HCl within the pH range 7.2 to 9 and with 50 mM Na₂ C0₃ within the pH range 9 to 11.

Analysis of pectate lyase activi ty in Zinnia cells

Cells, isolated from cultures by filtering, were ground in 50 mM Tris pH7.5 and 0.1 M NaCl buffer in the presence of 0.1% n-dodecyl α-D-maltoside and centrifuged for five minutes. The supernatant was assayed for enzymatic activity using 50 mM Na₂C0₃ pH 10 and 0.5% polygalacturonic acid by measuring the unsaturated compounds released from polygalacturonic acid at 235 nm during the course of 3 min at room temperature. 5.2 absorbance units per min correspond to the formation of 1 μmol unsaturated uronide per min (Nasser et al . 1990) . A unit (U) of Pel activity corresponds to the formation of 1 μmol unsaturated uronide per min. Specific activity was expressed as mU per mg of protein.

Example 1: Isolation and Analysis of Zinnia Pectate Lyase cDNA

A cDNA library representing mRNA extracted from cells cultured for 72 h in the TE inductive medium was used to isolate cDNA clones that were induced early in the trans-differentiation process. The library (30,000 recombinant phage) was differentially screened with radiolabeled first strand cDNA using RNA samples from cells cultured for 24 h or 72 h in inductive medium. One of the isolated cDNAs , that was abundantly present under inductive conditions at 72 h but not at 24 h, was selected for further study.

Example 2 : Expression of the Pectate L ase mRNA in Zinnia Mesophyll Cells In Vitro and in Plant Organs Mesophyll cells from the leaves of Zinnia differentiate directly into TEs when cultured in a medium containing a 1:1 ratio of auxin to cytokinin. Alternatively, when cultured in a medium containing high levels of auxin alone, the mesophyll cells elongate after five days. To examine whether the expression of the pectate lyase gene is associated with TE differentiation, cell elongation, or both, we analyzed the accumulation of the ZePel transcript in isolated mesophyll cells cultured under different conditions. RNA gel blot analysis showed that mRNA accumulates in inductive cultures (containing

1.0 mg l^"1 of both auxin and cytokinin) , as well as those cultured in the presence of 1.0 mg l^"1 auxin alone. No mRNA accumulation was found in maintenance medium (1 μg l^"1 BAP and 1 μg l^"1 NAA) or in cultures with 1.0 mg l^"1 BAP only, where neither differentiation nor elongation occurred. A time course of ZePel mRNA accumulation in the cells cultured in the TE induction medium showed a large increase in the accumulation of the corresponding transcript after only 48h in culture, at a point when the cells still look indistinguishable from their appearance in the original Zinnia leaf, and coincident with the time at which the cells become committed to TE precursor fate (Stacey et al . 1995) . It was also shown that NAA alone can induce expression of the ZePel gene at the same time or earlier than with the combination of both hormones. The time of appearance of this mRNA is thus much earlier than cell elongation which does not occur until after 120h.

We also determined the expression patterns of the pectate lyase gene in different Zinnia organs . The mRNA was highly expressed in roots and to a lesser extent in stems, but no significant expression could be seen in fully expanded leaves. We also detected low levels of transcript in flowers and seedlings.

The localization of ZePel transcripts in si tu was investigated by examining cells in sections of stem and root from three-weeks-old Zinnia plants, using the antisense probe. In stems, ZePel mRNA was largely present in the recent products of cambial divisions on both phloem and xylem sides, and conspicuously localized in the xylem parenchyma cells of young vascular bundles. In roots, the ZePel mRNA was located in the outer (most recently formed) part of the xylem and in phloem parenchyma cells. When shoot meristems were examined, the ZePel transcript was detected in young leaf primordia.

Example 3 : Southern Analysis of ZePel

To see if ZePel was part of a family of related genes in Zinnia , we hybridized Xbal, HindiII or Xhol digested genomic DNA with the ZePel cDNA under high stringency conditions (possibly requiring 75% homology or more) . The DNA gel blot showed that in each digestion only a single band hybridizes under highly stringent conditions (60°C) . Although this indicates that the ZePel gene is probably present in the Zinnia genome at only one copy per haploid genome, further (unpublished) results indicate that there is at least one closely related gene present in Zinnia .

Example 4 : Recombinant ZePel is a Pectate Lyase

The ZePel cDNA sequence was mutated to produce initiation codons within convenient Ncol sites at the start of the ORF and at the predicted mature N-terminus (Ser 20) . The two constructs were cloned into the pET3d T7 polymerase based vector (Studier et al , 1990) and their expression induced in BL21 (pLysS) cells with IPTG. No protein expression could be obtained from the construct with its native initiation codon. However, with the predicted signal sequence removed, the construct, upon induction, produced a protein of approximately 42 kDa by SDS-PAGE. At this stage no enzyme activity could be detected in protein extracts of this material. E . coli expressing the recombinant protein at 28°C and 37°C were fractionated into cytoplasmic, periplasmic and inclusion body extracts and visualized by SDS-PAGE. Surprisingly at the lower temperature almost all of the recombinant protein was found to be in inclusion bodies. At 37 °C, however, a proportion of the protein was found to be soluble (cytoplasmic protein) . This fraction was shown to contain a low level of pectate lyase activity. However, the cytoplasmic protein was also observed to migrate just in front of the inclusion body material upon SDS-PAGE. These gels were electro-blotted and the corresponding proteins bands excised and N-terminally sequenced. The inclusion body protein proved to have the expected N- terminus (APSRT) accounting for the PCR induced change of the second codon and removal of the initiating methionine by E. coli . In contrast, the cytoplasmic fraction produced the sequence ASRRN which is consistent with the loss of an 18 amino acid peptide from the anticipated N- terminus, and which correlates with the putative N- glycosylation site. This suggests that the glycosylation may be protecting the peptide from being cleaved in the plant .

The recombinant ZePel protein was found to be considerably more soluble in low concentrations of detergent than other proteins present in the inclusion body material. Extraction of the inclusion bodies with n- dodecyl α-D-maltoside produced a solution enriched for the ZePel protein that also proved to be enzymatically active. The presence of this detergent was shown, unexpectedly, to be necessary for the recovery of the activity under the conditions used by the present inventors . The enzyme activity could be eliminated by the addition of lOOμM EDTA indicating its expected calcium dependence. The enzyme was purified using conventional ion-exchange chromatography in the presence of detergent. The pH optimum of the enzyme was found to be 10, as shown in Figure 3. The V_max was 0.12 ± 0.015 μmol min^"1 and a Km was 0.9 ± 0.23 g/100 ml as shown in Figure 5. The specific activity of recombinant ZePel was found to be 250 μmol min^"1 mg^"1.

Example 5: Pectate lyase activity in Zinnia cells

In vitro cultures of Zinnia cells were assayed for pectate lyase activity by separating the cells from the culture medium and extracting them with 50 mM Tris pH7.5 and 0.1 M NaCl buffer in the presence of 0.1% n-dodecyl α-D-maltoside . Extracts from seven-day-old cells cultured in inductive media or in the presence of 1.0 mg I^"1 auxin alone were then assayed at pH 6.7. A pectate lyase activity of 34.6 mU/mg was found in the cells cultured in inductive media and of 97.4 mU/mg in the culture with auxin. A time course was done for pectate lyase activity at pH 10, where the recombinant enzyme was found to be most active. As shown in Figure 4 the activity could be detected after four days when cells were cultured in inductive media or in the presence of 1.0 mg l^"1 auxin. The specific activity in cells cultured with auxin was higher than that in the inductive medium. No activity could be detected in cells cultured in maintenance medium or in the culture medium of any of the cultures .

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Claims

1. An isolated nucleic acid encoding a plant pectate lyase, the pectate lyase being obtainable from a plant somatic cell.

2. A nucleic acid as claimed in claim 1 comprising a nucleotide sequence identical to Seq ID No 1 or degeneratively equivalent thereto.

3. A nucleic acid as claimed in claim 1 or claim 2 wherein the nucleotide sequence encodes Seq ID No 2.

. An isolated nucleic acid comprising a homologous variant of the nucleotide sequence of claim 2 or claim 3 having about 70% or more sequence identity therewith and encoding a polypeptide which has pectate lyase activity.

5. A nucleic acid as claimed in claim 4 wherein the variant is an allelic variant of Seq ID No 1.

6. A nucleic acid as claimed in claim 4 wherein the variant is a pectate lyase obtainable from a plant other than Zinnia elegans c . Envy.

7. A nucleic acid as claimed in claim 4 having a sequence which is a derivative of Seq ID No 1 by way of addition, insertion, deletion or substitution of one or more nucleotides.

8. A nucleic acid as claimed in any one of claims 4 to 7 which encodes a polypeptide having altered activity with respect to the polypeptide shown in Seq ID No 2.

9. A nucleic acid as claimed in claim 7 or claim 8 wherein the derivative encodes a functional portion of Seq ID No 2.

10. A nucleic acid as claimed in any one of claims 7 to

9 wherein the derivative lacks the N-terminal 20 amino acid sequence of Seq ID No 2.

11. A nucleic acid as claimed in any one of claims 7 to

10 wherein the derivative encodes a vacuolar targeting sequence .

12. A nucleic acid as claimed in any one of claims 7 to 11 wherein the derivative encodes a cell-wall targeting sequence .

13. A nucleic acid which is complementary to the nucleic acid of any one of claims 1 to 12.

14. A nucleic acid molecule for use as a probe or primer, said molecule having a nucleotide sequence of at least 15, 18, 21, 24 or 30 nucleotides, which sequence is shown in, or complementary to, the coding region of Seq ID No 1 or a sequence degeneratively equivalent thereto.

15. A method for identifying or cloning a pectate lyase from a plant species, which method employs a nucleic acid molecule having a nucleotide sequence as claimed in claim 14.

16. A method as claimed in claim 15 comprising the steps of: (a) providing a preparation of nucleic acid from a plant cell, (b) providing a nucleic acid molecule as claimed in claim 14,

(c) contacting nucleic acid in said preparation with said nucleic acid molecule under conditions for hybridisation of said nucleic acid molecule to any nucleic acid encoding an isoamylase in said preparation,

(d) identifying said nucleic acid encoding a pectate lyase if present by its hybridisation with said nucleic acid molecule, and optionally

(e) confirming the identity the pectate lyase encoded by the nucleic acid by expressing it and assesssing its activity.

17. A method as claimed in claim 16 wherein the hybridisation conditions are selected to allow the identification of sequences having about 70% or more sequence identity with the nucleic acid molecule.

18. A method as claimed in claim 15 comprising use of two primers to amplify a nucleic acid encoding a pectate lyase, at least one of the primers having a nucleotide sequence of at least 15 nucleotides, which sequence is shown in, or complementary to, all or part of the coding region of Seq ID No 1,

19. A method as claimed in claim 18 comprising the steps of: (a) providing a preparation of nucleic acid from a plant cell,

(b) providing a pair of nucleic acid molecule primers suitable for PCR, at least one of the primers having a nucleotide sequence of at least 15 nucleotides, which sequence is shown in, or complementary to, all or part of the coding region of Seq ID No 1,

(c) contacting nucleic acid in said preparation with said primers under conditions for performance of PCR,

(d) performing PCR and determining the presence or absence of an amplified PCR product, and optionally

(e) confirming the identity of the amplified PCR product by by expressing it and assesssing its pectate lyase activity.

20. A recombinant vector comprising the nucleic acid of any one of claims 1 to 13.

21. A vector as claimed in claim 20 which is capable of replicating in a suitable host.

22. A vector as claimed in claim 20 or claim 21 wherein the nucleic acid is operably linked to a promoter or other regulatory element for transcription in a host cell.

23. A vector as claimed in claim 22 further comprising any one or more of the following: a terminator sequence; a polyadenylation sequence; an enhancer sequence; a marker gene .

24. A vector as claimed in claim 22 or claim 23 wherein the promoter is an inducible promoter.

25. A vector as claimed in any one of claims 21 to 24 which is a plant vector.

26. A vector as claimed in claim 25 comprising a selectable genetic marker which confers a selectable phenotype selected from: resistance to antibiotics or herbicides .

27. A method comprising the step of introducing a vector as claimed in any one of claims 21 to 26 into a cell.

28. A method for transforming a plant cell, comprising a method as claimed in claim 27, and further comprising the step of causing or allowing recombination between the vector and the plant cell genome to introduce the nucleic acid into the genome.

29. A host cell comprising a vector as claimed in any one of claims 20 to 26.

30. A host cell transformed with a vector as claimed in any one of claims 20 to 26.

31. A host cell as claimed in claim 29 or claim 30 which is a plant cell.

32. A host cell as claimed in claim 31 which is in a plant .

33. A host cell as claimed in claim 32 which is in a vascular and/or lignified tissue of a plant.

34. A method for producing a transgenic plant comprising a method as claimed in claim 28 and further comprising the step of regenerating a plant from the transformed cell.

35. A plant comprising the cell of any one of claims 31 to 33.

36. A plant as claimed in claim 35 produced by the method of claim 34.

37. A plant which is the progeny of a plant as claimed in claim 35 or claim 36.

38. A plant as claimed in any one of claims 35 to 37 which is a crop plant .

39. A part or propagule of the plant of any one of claims 35 to 38.

40. A polypeptide encoded by the nucleic acid of any one of claims 1 to 12.

41. A method of producing a polypeptide comprising the step of causing or allowing the expression from a nucleic acid of any one of claims 1 to 12 in a suitable host cell .

42. A composition comprising the polypeptide of claim 40.

43. An antibody or fragment thereof, or a polypeptide comprising the antigen-binding domain of the antibody, capable of specifically binding the polypeptide of claim 40.

44. A method of producing the antibody or fragment as claimed in claim 43 comprising the step of immunising a mammal with a polypeptide according to claim 40.

45. A method of identifying and/or isolating a pectate lyase comprising the step of screening candidate polypeptides with a polypeptide comprising the antigen- binding domain of the antibody of claim 43.

46. A method of modifying a polygalacturonate-containing substrate by contacting said substrate with a polypeptide as claimed in claim 40.

47. A method as claimed in claim 46 for use in any one or more of the following processes: microbial fermentation; fruit or vegetable juice extraction or clarification; brewing; sugar beet processing, paper-pulp processing; silage treatment.

48. A method for altering the quality or quantity of a polygalacturonate-containing substrate in a host cell by influencing the pectate lyase activity in that cell, the method comprising use of any one or more of the following: all or part of the nucleic acid of any one of claims 1 to 13; the polypeptide of claim 40; the antibody or fragment or polypeptide comprising the antigen-binding site thereof of claim 43.

49. A method as claimed in any one of claims 46 to 48 wherein the polygalacturonate-containing substrate is pectin.

50. A method as claimed in any one of claims 46 to 49 comprising the step of causing or allowing expression of a nucleic acid according to any one of claims 1 to 13 within the cell.

51. A method as claimed in any one of claims 48 to 50 comprising reducing the pectate lyase activity in the cell.

52. A method as claimed in claim 51 comprising the step of causing or allowing the transcription of part of the nucleic acid of any one of claims 1 to 12 in the cell such as to co-suppress the expression of an endogenous glycosyltransferase .

53. A method as claimed in claim 51 comprising the step of causing or allowing the transcription of nucleic acid of claim 13 in the cell.

54. A method as claimed in claim 51 comprising the step of causing or allowing the expression of a polypeptide comprising the antigen-binding domain of the antibody of claim 43.

55. A method as claimed in any one of claims 48 to 54 wherein the cell is the plant cell of any one of claims 31 to 33.

56. A plant-derived polygalacturonate-containing material the quality of which has been altered in accordance with the method of any one of claims 48 or claim 55.

57. A material as claimed in claim 56 wherein the quality altered is digestibility of the material.

58. A plant or animal foodstuff comprising the material of claim 57.

59. An isolated nucleic acid comprising a nucleotide sequence encoding the promoter region of the pectate lyase gene shown in Seq ID No 1.

60. An isolated nucleic acid comprising a homologous variant of the nucleotide sequence of claim 59 having about 70% or more sequence identity therewith and encoding an auxin-inducible promoter.

61. Use of a nucleic acid as claimed in claim 59 or claim 60 to express a nucleic acid operably linked thereto in response to an auxin stimulus.

62. Use of a nucleic acid as claimed in any one of claims 59 to 61 to express a nucleic acid operably linked thereto in xylogenetic tissue.

63. A nucleic acid construct comprising either: (i) a nucleotide sequence encoding the promoter region of the pectate lyase gene shown in Seq ID No 1, or (ii) a homologous variant thereof having about 70% or more sequence identity therewith and encoding an auxin- inducible promoter, operably linked to a heterologous gene.

64. A nucleic acid construct as claimed in claim 63 wherein the gene is selected from the genes encoding any of the following: a reporter enzyme; an enzyme involved in cell wall formation, modification, or degradation; an enzyme capable of modifying cellulose, pectic polysaccharides, hemicellulose , lignin, plant proteins, plant lipids.