©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Molecular Cloning and Pattern of Expression of an -

L

-Fucosidase Gene from Pea Seedlings (*)

(Received for publication, June 6, 1995)

Christopher Augur (1)(§) Virginia Stiefel (2) Alan Darvill (3) Peter Albersheim (3) Pedro Puigdomenech (2)(¶)

From the  (1)From ORSTOM, the Institute of Scientific Research for Development in Cooperation, Ciceron 609, Los Morales, 11530 Mexico D.F., Mexico, the (2)Laboratory of Molecular Genetics, CID-CSIC, 18-26 Jordi Girona, 08034 Barcelona, Spain, and the (3)Complex Carbohydrate Research Center and Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602-4712

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

alpha-L-Fucosidase is a cell wall protein purified from pea (Pisum sativum) epicotyls. The alpha-L-fucosidase hydrolyzes terminal fucosyl residues from oligosaccharides of plant cell wall xyloglucan. alpha-L-Fucosidase may be an important factor in plant growth regulation, as it inactivates fucose-containing xyloglucan oligosaccharides that inhibit growth of pea stem segments. The amino acid sequences of the NH(2)-terminal region and one internal peptide were used to design redundant oligonucleotides that were utilized as primers in a polymerase chain reaction (PCR) with cDNA, generated from pea mRNA, as the template. A specific PCR amplification product containing 357 base pairs was isolated, cloned, and sequenced. The deduced amino acid sequence included the two peptides used to design the primers for PCR plus two other peptides obtained by proteinase digestion of alpha-L-fucosidase. No sequence homology to other alpha-L-fucosidases was apparent, although the NH(2)-terminal region is strongly homologous to Kunitz-type trypsin inhibitors. cDNA and genomic copies were isolated and sequenced. In pea, the gene is present in two or three copies. Its mRNA is present in roots, leaves, and elongating shoots. The spatial pattern of expression of the alpha-L-fucosidase was determined by in situ hybridization.


INTRODUCTION

Xyloglucan (XG) (^1)is a hemicellulosic polysaccharide present in the primary cell walls of all of the higher plants that have been examined. XG forms strong noncovalent bonds to cellulose microfibrils and is believed to strengthen the cell walls by cross-linking the microfibrils(1) . XG appears to have a regulatory as well as a structural role, as there is evidence that XG oligosaccharides regulate the rate of plant cell growth(2, 3, 4) . Indeed, fucosylated subunits of XG inhibit auxin-stimulated growth in pea stem segments(4, 5, 6, 7) . These oligosaccharins (oligosaccharides with biological regulatory properties) have relatively strict structural requirements for activity, including a critical terminal fucosyl residue. Hydrolysis of the alpha-L-fucosidic bonds abolishes the growth-inhibiting activity of the XG oligosaccharides. Thus, we hypothesized that plants have an alpha-L-fucosidase that participates in the regulation of plant growth by controlling the concentration of fucosylated XG oligosaccharides. This hypothesis led to the demonstration that pea stems have a developmentally regulated alpha-L-fucosidase with the ability to cleave the fucosyl residue of XG oligosaccharides. Indeed, the alpha-L-fucosidase, which was shown to reside in the primary cell walls of pea stems, has been purified to homogeneity (5) .

cDNAs encoding alpha-L-fucosidases have previously been isolated from human (8, 9) and rat (10) livers. The human and rat liver alpha-L-fucosidases have subunit molecular masses of approximately 50,100 and 55,000 Da, respectively. Both of these alpha-L-fucosidases hydrolyze artificial substrates such as p-nitrophenyl-alpha-L-fucoside and 4-methylumbelliferyl fucoside. Both of these fucosidases have broad aglycon specificities such that they hydrolyze alpha-1,2-, alpha-1,3-, alpha-1,4-, and alpha-1,6-L-fucosidic linkages. In contrast, the alpha-L-fucosidase from pea epicotyls, which hydrolyzes the terminal alpha-1,2-fucosidic linkages of xyloglucan oligosaccharides, has a molecular mass of 20,000 Da and does not cleave p-nitrophenyl-alpha-L-fucoside. The alpha-L-fucosidase from pea epicotyls is also unable to hydrolyze fucosyl linkages of intact plant cell wall polysaccharides including XG. We now report the isolation of cDNA and genomic transcripts encoding the alpha-L-fucosidase from pea. We show that the gene is expressed in elongating tissues (leaf, root, and stem) but is absent in fully elongated stems. We also describe the tissue- and position-dependent accumulation for transcripts encoding the alpha-L-fucosidase in pea.


EXPERIMENTAL PROCEDURES

Isolation and Sequencing of Peptides from alpha-L-Fucosidase

The pea stem alpha-L-fucosidase was purified as described(5) . Homogeneous alpha-L-fucosidase was used to obtain its amino acid composition and the sequences of four peptides. The amino acid composition analysis was performed at the University of Georgia sequencing facility on an Applied Biosystems 420A amino acid analyzer. alpha-L-Fucosidase (approx75 pmol) was hydrolyzed prior to composition analysis in 6 N HCl at 155 °C for 75 min, as recommended by the manufacturer.

Three internal peptide fragments of the alpha-L-fucosidase and the NH(2)-terminal peptide were prepared by incubating the alpha-L-fucosidase (16 µg) at 37 °C for 3 h in 0.1 ml of 600 mM ammonium bicarbonate (pH 8.0), 5 mM CaCl(2), and 1 µg of Pseudomonas gingivalis H66 (50 kDa) cysteine proteinase (11) . The resulting digest was loaded on a C-8 reversed phase Aquapore RP 300 column (Brownlee) equilibrated with 0.1% trifluoroacetic acid. The peptides, which were separated using a binary gradient (solvent A = 0.1% trifluoroacetic acid in H(2)O, solvent B = 0.085% trifluoroacetic acid in 80% acetonitrile) at a flow rate of 0.2 ml/min, were detected by their absorbance at 220 nm. Each peptide peak was manually collected, dried, and sequenced. Amino acid sequence analysis of the alpha-L-fucosidase peptides was performed at the University of Georgia protein sequencing facility by automated Edman degradation on an Applied Biosystems 470A protein sequencer.

Isolation of the alpha-L-Fucosidase Gene

alpha-L-Fucosidase-specific degenerate oligonucleotide primers for use in PCR reactions were synthesized by New England Biolabs.

Total RNA was isolated from pea tissue as described(12) . Poly(A) RNA was prepared using oligo(dT) column chromatography as described(13) . Single-stranded cDNA was synthesized using oligo(dT) primers and a kit from Clontech. Amplification of cDNA sequences, flanked by the alpha-L-fucosidase-specific degenerate oligonucleotide primers, was carried out in two sequential PCR experiments using a DNA thermal cycler (Perkin-Elmer). The reaction mixture, in a final volume of 50 µl, contained 2 ng of cDNA, 64 pmol of each primer, 50 mM potassium chloride, 10 mM Tris-HCl (pH 8.3), 1.5 mM magnesium chloride, 0.01% (w/v) gelatin, 200 mM of each dNTP, and 2.5 units of Taq polymerase (Perkin-Elmer). The initial PCR amplification consisted of 26 cycles; each cycle was as follows: 94 °C for 1.5 min, 45 °C for 1.5 min, and 72 °C for 3 min. The products of the reaction were separated on a 2% agarose gel, and the major product (approx350 bp) was eluted and used for the second PCR which used the same conditions. The major PCR product was eluted, purified, blunt-ended with T4 DNA polymerase (New England Biolabs), and blunt-end-ligated into the SmaI site of the plasmid vector pBluescript II SK (Stratagene). The cloning procedure, including transformation of the cloning vector and selection of transformants, is described in the Stratagene pBluescript II instruction manual.

The amplified alpha-L-fucosidase cDNA (in GT11) fragment was sequenced as described (14) by the Sanger dideoxy method using Taq polymerase in the thermal cycler.

A pea cDNA library from 7-day-old etiolated pea stems was constructed according to the manufacturer's specifications (in GT11 vector, Stratagene). A pea genomic library (in EMBL3) was purchased from Clontech. Both libraries were screened with the 357-bp alpha-L-fucosidase clone by standard methods(13, 15) . A cDNA clone was isolated and sequenced. A 6.5-kb genomic clone hybridizing with the probe was partially sequenced using the dideoxy method after subcloning in M13mp18 and M13mp19(16) . Both strands of one of the subclones containing the complete alpha-L-fucosidase coding region (1.0 kb) were sequenced in their entirety.

Northern Blot Analysis

Tissue was collected from 7-day-old etiolated peas. Root and leaf tissues were frozen in liquid nitrogen and kept at -70 °C until use. Stem tissue (1-cm sections collected from the apical hook region (referred to as ``elongating stem'') and 1-cm sections collected from the base of the stem (referred to as ``elongated stem'')) was collected and frozen at -70 °C until use.

RNA was extracted from 7-day-old etiolated pea tissue as described (13) . Total RNA (10 µg) was size-fractionated by electrophoresis on a formaldehyde-agarose gel and blotted on a Hybond-N filter (Amersham Corp.). Hybridization conditions with P-labeled cDNA clones (the 357-bp partial cDNA clone of the alpha-L-fucosidase and maize ribosomal cDNA as a control) as well as washing conditions were the same as described for Southern analysis.

Genomic Blot Analysis

Genomic DNA was prepared from stems of 7-day-old etiolated pea seedlings as described by Burr and Burr (17) . The DNA was digested with restriction enzymes, separated by size in an 0.8% agarose gel, and blotted onto a nylon membrane as recommended by the manufacturer (Hybond N, Amersham). Hybridization was carried out at 65 °C in 200 mM sodium phosphate (pH 7.2), 1 mM EDTA, 7% SDS, 1% bovine serum albumin, and 0.5 mg/ml of sonicated salmon sperm DNA. The DNA was labeled with P to a specific activity of 10^9 cpm/µg by random priming (Boehringer Mannheim). Final washes were carried out at 65 °C in 20 mM sodium phosphate (pH 7.2), 1% SDS, and 1 mM EDTA. The membrane was then exposed to Kodak XAR5 film with intensifying screens (Du Pont Lighting Plus) at -70 °C.

In Situ Hybridization

To obtain an alpha-L-fucosidase-specific probe, the 357-bp fragment was cloned into a pBluescript SK vector (Stratagene) and used as a linear template for synthesis of riboprobes. Sense and antisense digoxigenin-labeled alpha-L-fucosidase RNAs were synthesized according to the instructions of Boehringer Mannheim.

Pea tissue (stem, leaf, root) was collected from 7-day-old etiolated peas and fixed in 3:1 ethanol/acetic acid for 1 h at room temperature. Once the fixative was removed, the samples could be stored in 70% ethanol at 40 °C indefinitely. The fixed tissue was dehydrated through an ethanol series and embedded in paraffin. Sections (8 µm) were cut and mounted on poly-L-lysine-coated slides. Treatment of the sections prior to hybridization was performed as described previously(18) . The sections were deparaffinized by rinsing in xylene and hydrated by passing through an alcohol series. The hydrated sections were then incubated with 0.5 ml of proteinase K (1 µg ml) in 0.5 M Tris/HCl (pH 7.6) for 30 min at 37 °C. The proteinase K was removed by rinsing in phosphate-buffered saline and blocking with 2 mg/ml glycine in phosphate-buffered saline. Subsequently, the sections were refixed for 20 min at room temperature in freshly prepared 4% formaldehyde followed by rinsing two times in phosphate-buffered saline. The sections were then treated with 0.25% acetic anhydride in 100 mM Tris ethyl acetate buffer (pH 8.0, freshly made) followed by rinsing three times in H(2)O. Finally, the sections were dehydrated through an alcohol series to 100% ethanol and dried.

The fixed, deproteinated sections were hybridized by incubating at 40 °C overnight in hybridization buffer while enclosed under a coverslip. Hybridization buffer consists of 200-400 ng ml digoxigenin-labeled probe, 50% formamide, 300 mM NaCl, 10 mM Tris/HCl (pH 7.5), 1 mM EDTA, 10% dextran sulfate, and 10 mM dithiothreitol. After the hybridization, the coverslip was removed in 2 times SSC at room temperature, and the sections were washed three times for 10 min at 55 °C with 0.2 M SSC. Subsequently, an RNase A treatment (20 µg ml in 500 mM NaCl/Tris-ethanolamine (pH 8.0)) was performed at 37 °C for 30 min. The RNase-treated sections were stained overnight at room temperature with alkaline phosphatase-conjugated anti-digoxigenin antibodies according to the protocol of Boehringer Mannheim, using nitro blue tetrazolium and X-phosphate as a substrate. Color development was monitored microscopically.


RESULTS AND DISCUSSION

Amino Acid Composition and Amino Acid Sequencing

The lysine and arginine content of the alpha-L-fucosidase (Table 1) led to the expectation of the presence of several tryptic peptides. However, 24-h digestion of the alpha-L-fucosidase with L-(1-tosylamino-2-phenyl) ethyl chloromethyl ketone-treated trypsin failed to produce any peptides. Therefore, a portion of the purified alpha-L-fucosidase (16 µg) was subjected to P. gingivalis H66 cysteine proteinase, which generated peptide fragments that were separated by reversed phase high performance liquid chromatography (Fig. 1). Fractions containing peptides P1 + P2, P3, and P4 (see Fig. 1) were collected separately and sequenced. P1 and P2 did not separate, but sequence analysis of the peak containing P1 and P2 yielded a major and a minor sequence that were readily distinguishable. A second undigested portion of the alpha-L-fucosidase (3 µg) was used to determine its NH(2)-terminal sequence. The amino acid sequences of peptides P1, P2, P3, P4, and of the NH(2) terminus are illustrated in Fig. 2. The amino acid sequences of peptide P1 and the NH(2) terminus are identical.




Figure 1: Peptide fragments of pea stem alpha-L-fucosidase generated by cleavage with P. gingivalis H66 cysteine proteinase. The mixture of alpha-L-fucosidase was separated into peptides P1 through P4 by high performance liquid chromatography on an Aquapore RP 300 macroporous C-8 reversed phase column. Solvent B: 0.085% trifluoroacetic acid in 80% acetonitrile. Experimental details are described under ``Experimental Procedures.''




Figure 2: Nucleotide and deduced amino acid sequence of the alpha-L-fucosidase gene. A putative TATA box (nucleotides 125-132) and three polyadenylation signals are highlighted. The stop codon is indicated by an asterisk. The deduced amino acid sequence for the alpha-L-fucosidase is depicted as a single letter code. The numbers at the right margin are amino acid residues. The site of processing of the mature protein is indicated by a vertical arrow. The underlined regions, as indicated, are the sequences of peptides P1-P4 obtained by amino acid sequencing of protease-digested alpha-L-fucosidase. The reported genomic sequence has been deposited in the GenBank(TM) data base under the accession number X82595.



PCR, Cloning, and Nucleotide Sequencing

An oligonucleotide was synthesized in its sense orientation on the basis of the NH(2)-terminal sequence. The oligonucleotide was a 17-mer with 64-fold redundancy corresponding to the first six amino acids of P1 (Fig. 2). A second oligonucleotide was synthesized corresponding to the sequence of the fourth to the ninth amino acids of peptide P4. The oligonucleotide, which was constructed in its antisense orientation, was a 17-mer with 128-fold redundancy.

The redundant oligonucleotides were used as primers for the PCR using a single-stranded cDNA template synthesized from total pea RNA by reverse transcriptase. A prominent DNA band of 0.35 kb was visualized upon ethidium bromide staining of the reaction products that had been fractionated in an agarose gel (data not shown). The band was absent in control reactions containing one or none of the oligonucleotide primers. The 0.35-kb band was eluted from the agarose gel and further amplified by PCR using the same oligonucleotide primers. The PCR-generated fragment was purified and cloned. One recombinant clone, which was shown after size fractionation in an agarose gel to contain the PCR-amplified fragment, was sequenced; its deduced amino acid sequence consisted of a reading frame coding for 119 amino acids (starting at amino acid 27, Fig. 2), representing 65% of the estimated mature alpha-L-fucosidase sequence (5) . The amino acid sequence of the alpha-L-fucosidase fragment included the sequences corresponding to the NH(2) terminus and peptide P4, which were used to generate the primers. The partial cloned fragment also contained sequences encoding peptides P2 and P3 (Fig. 2) not used in the design of the PCR primers. This indicated that the PCR-amplified product was a partial cDNA encoding the alpha-L-fucosidase.

cDNA and Genomic Cloning

Several genomic clones that hybridized to the partial sequence of the alpha-L-fucosidase cDNA were isolated from a commercial EMBL3 library (see ``Experimental Procedures''), and one was selected for sequence analysis. The DNA sequence of 1.0 kb, including the alpha-L-fucosidase gene, is shown in Fig. 2. A TATA box is found, as expected, at -66 bp relative to the initial ATG in the 5`-flanking region of the alpha-L-fucosidase coding region. There are no introns within the coding region.

Comparison of the alpha-L-fucosidase genomic sequence with the determined cDNA sequence revealed minor base mismatches but exhibited an overall identity of 97%. Sequence examination of the coding region established that the cDNA previously characterized is an mRNA of this gene. The cDNA does not contain a poly(A) tail, even though the genomic sequence contains polyadenylation signals starting at positions 855, 908, and 919.

The protein predicted by the genomic sequence is identical to that predicted by the cDNA, with the exception of an additional methionine residue at the amino terminus which was missing in the cDNA. The predicted protein begins at the NH(2) terminus with a hydrophobic stretch having features typical of a signal peptide. The putative signal peptide is absent in the mature protein, presumably due to processing that accompanies passage through the endoplasmic reticulum(19, 20) . The presence of a secretion signal in the nascent alpha-L-fucosidase is expected, as the alpha-L-fucosidase is located in the extracellular matrix in pea epicotyls(5) . As confirmed by NH(2)-terminal amino acid sequencing of the alpha-L-fucosidase itself, the amino-terminal end of the mature protein begins with the glutamate residue at position 27. Therefore, the signal peptide processing site is at the Asn-Glu junction between positions 26 and 27 of the predicted protein. This would make the molecular mass of the processed protein 19,943 Da, in agreement with the estimation of 20 kDa for purified, denatured alpha-L-fucosidase(5) .

No strong homology was found when the sequence of the alpha-L-fucosidase cDNA clone and the encoded protein were compared with the sequences of human and rat liver alpha-L-fucosidases present in the GenBank(TM) nucleic acid data base (release 71.0) and the NBRF protein data base (release 31.0). However, the NH(2) terminus of the pea stem alpha-L-fucosidase, including amino acids 1-80 (see Fig. 3), has 43 and 33% sequence identity to the NH(2) terminus of two Kunitz-type trypsin inhibitors (Fig. 3, first and third sequences, respectively). The Kunitz trypsin inhibitors have a molecular mass of about 21 kDa and include four cysteines forming two disulfide bridges(21) . Two cysteine residues are present at identical positions in both the alpha-L-fucosidase and Kunitz inhibitor sequences (Fig. 3, amino acids 34 and 80). However, we could not detect trypsin inhibitor activity in the purified pea stem alpha-L-fucosidase. (^2)On the other hand, it may not be coincidental that the pea stem alpha-L-fucosidase is not cleaved into peptides by trypsin, whereas the alpha-L-fucosidase is cleaved by a protease that works differently from trypsin.


Figure 3: Comparison of the deduced NH(2)-terminal amino acid sequence of pea stem alpha-L-fucosidase with the NH(2)-terminal sequences of two trypsin inhibitors of the Kunitz family (Carolina and Psophocarpus). A, inhibitor DE-5 from the Brazilian Carolina tree (Adenanthera pavonina)(25) . B, deduced amino acid sequence from pea stem alpha-L-fucosidase. C, inhibitor from winged bean Psophocarpus tetragonolobus(26) . Arrows indicate the positions of conserved cysteine residues. The boxed amino acids represent regions of identity between the alpha-L-fucosidase sequence and each of the Kunitz inhibitor sequences (A and C).



We performed a genomic Southern blot analysis to estimate the number of alpha-L-fucosidase sequences in the pea genome. Aliquots of genomic DNA of pea were digested with one of several restriction enzymes, blotted to nylon membrane, and probed with the 357-bp partial cDNA (see ``Experimental Procedures''). The results show (Fig. 4) that the alpha-L-fucosidase gene is present in two or three copies in the pea genome. Therefore, pea alpha-L-fucosidases are comprised of a small gene family with at least two genes.


Figure 4: Determination of the number of alpha-L-fucosidase genes in the pea genome. Autoradiogram of the 357-bp alpha-L-fucosidase probe hybridized to digests of genomic DNA from pea. Lane A, EcoRI digest; lane B, HindIII digest.



A single alpha-L-fucosidase mRNA species (0.7 kb) was detected by Northern blot analysis of total RNA extracted from pea roots, leaves, and elongating stems (Fig. 5). alpha-L-Fucosidase transcripts were undetected in fully elongated stem tissue. The observed pattern of expression is consistent with the hypothesis that the alpha-L-fucosidase has a role in growth regulation (5) .


Figure 5: Northern blot analysis showing alpha-L-fucosidase mRNA distribution in various pea tissues. RNA isolated from roots, stems, and leaves of 7-day-old etiolated pea seedlings was blotted and hybridized against a radioactive 357-bp alpha-fucosidase probe (1) and against a radioactive ribosomal probe(2) . R, root; S1, elongating stem from apical hook region; L, young etiolated leaves; S2, elongated stem from basal stem region.



The NH(2)-terminal region of alpha-L-fucosidase (amino acids 1-50) has 28% sequence identity to sweet potato sporamins A and B (data not shown). Sweet potato sporamins are a group of proteins with molecular weight of 20,000 (22) that account for 60-80% of the soluble protein in mature tubers. The amino acid sequences of the sporamins are also homologous to the Kunitz-type trypsin inhibitors of leguminosae seeds(23) . Three regions (see Fig. 3, amino acids 6-7, 9-10, and 21-23) and the positions of the two cysteine residues (Fig. 3, amino acids 34 and 80) are conserved in the pea stem alpha-L-fucosidase, Kunitz trypsin inhibitors, and sweet potato sporamins. alpha-L-Fucosidase is located in the cell wall, whereas the Kunitz inhibitors reside in the lysosome. The sequence homology between these proteins, with distinct localization patterns and with apparently different functions, suggests these genes have evolved by duplication and mutation of an ancestral genetic domain.

Localization of alpha-Fucosidase Transcripts

We showed above that mRNA encoding the alpha-L-fucosidase is present in several pea tissues, all of which have some cells that are elongating. alpha-L-Fucosidase mRNA was not detected in elongated stems. To localize more precisely the accumulation of alpha-L-fucosidase mRNA, we used the 357-bp cDNA fragment as a probe for in situ hybridization with elongating tissues (young leaf, hook region of the stem, stem region below hook). The alpha-L-fucosidase mRNA is localized to the epidermal layers of the elongating stem (Fig. 6, A and B) and the vascular system of the stem and leaf (Fig. 6, A-C). The alpha-L-fucosidase transcripts accumulated in the epidermal layer of the stem section closest to the shoot apex (Fig. 6A). However, in stem sections below the apical hook, the alpha-L-fucosidase transcript is absent from the epidermal layer but still present in the vascular system (Fig. 6C). The transcript is also present in the meristematic zone of root (data not shown), where differentiation is in progress. Hybridization with a sense control probe of stem and leaf sections similar to that in Fig. 6A exhibited only background signal and no organized pattern (Fig. 6D). The pattern of transcript accumulation in the epidermal layer and its absence in the elongated stem tissue reinforce the hypothesis that alpha-L-fucosidase plays a role in the control of cell elongation by regulating the levels of bioactive fucosylated xyloglucan oligosaccharides generated within the growing cell wall (24) . The localized accumulation of alpha-L-fucosidase mRNA in the highly dividing meristematic tissue (both root and shoot) may also be interpreted as its product's having a regulatory function. Thus, considerable evidence supports the hypothesis that alpha-L-fucosidase functions in plant growth regulation, although the role of the enzyme in vivo remains to be established.


Figure 6: Localization of alpha-L-fucosidase mRNAs in 7-day-old etiolated peas. Plant material was fixed, embedded, and cut into 8-µm sections. Hybridization was performed with digoxigenin-labeled single-stranded antisense RNA (A-C) or sense RNA (D), as outlined under ``Experimental Procedures.'' Sections were photographed by bright field microscopy. A, transverse section through pea leaf and stem. Bar = 700 µm. B, longitudinal section of pea stem in the apical hook region. Bar = 300 µm. C, transverse section through a pea stem under the node closest to the apical hook. Bar = 300 µm. D, transverse section through a pea stem in the apical hook region. Bar = 300 µm. L, leaf; S, stem.




FOOTNOTES

*
This work was supported in part by United States Department of Energy (DOE) Grant DE-FG05-93ER20114 (to P. A.) and by the DOE-funded (DE-FG05-93ER20097) Center for Plant and Microbial Complex Carbohydrates. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by an European Economic Community postdoctoral fellowship within the BRIDGE program.

To whom correspondence should be addressed. Tel.: 34-3-204-06-00; Fax: 34-3-204-59-04.

(^1)
The abbreviations used are: XG, xyloglucan; bp, base pair(s); kb, kilobase(s); PCR, polymerase chain reaction.

(^2)
C. Augur, V. Stiefel, A. Darvill, P. Albersheim, and P. Puigdomenech, unpublished results.


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