(Received for publication, June 6, 1995)
From the
-L-Fucosidase is a cell wall protein purified from
pea (Pisum sativum) epicotyls. The
-L-fucosidase
hydrolyzes terminal fucosyl residues from oligosaccharides of plant
cell wall xyloglucan.
-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
-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
-L-fucosidase. No
sequence homology to other
-L-fucosidases was apparent,
although the NH
-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
-L-fucosidase was determined
by in situ hybridization.
Xyloglucan (XG) ()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
-L-fucosidic bonds abolishes the growth-inhibiting
activity of the XG oligosaccharides. Thus, we hypothesized that plants
have an
-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
-L-fucosidase with
the ability to cleave the fucosyl residue of XG oligosaccharides.
Indeed, the
-L-fucosidase, which was shown to reside in
the primary cell walls of pea stems, has been purified to homogeneity (5) .
cDNAs encoding -L-fucosidases have
previously been isolated from human (8, 9) and rat (10) livers. The human and rat liver
-L-fucosidases have subunit molecular masses of
approximately 50,100 and 55,000 Da, respectively. Both of these
-L-fucosidases hydrolyze artificial substrates such as p-nitrophenyl-
-L-fucoside and
4-methylumbelliferyl fucoside. Both of these fucosidases have broad
aglycon specificities such that they hydrolyze
-1,2-,
-1,3-,
-1,4-, and
-1,6-L-fucosidic linkages. In contrast,
the
-L-fucosidase from pea epicotyls, which hydrolyzes
the terminal
-1,2-fucosidic linkages of xyloglucan
oligosaccharides, has a molecular mass of 20,000 Da and does not cleave p-nitrophenyl-
-L-fucoside. The
-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
-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
-L-fucosidase in pea.
Three internal peptide fragments of
the -L-fucosidase and the NH
-terminal peptide
were prepared by incubating the
-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
, 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
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
-L-fucosidase peptides was performed at the University of
Georgia protein sequencing facility by automated Edman degradation on
an Applied Biosystems 470A protein sequencer.
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
-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 (
350 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 -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
-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
-L-fucosidase coding region (1.0
kb) were sequenced in their entirety.
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
-L-fucosidase and maize ribosomal cDNA as a control)
as well as washing conditions were the same as described for Southern
analysis.
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
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
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.
Figure 1:
Peptide
fragments of pea stem -L-fucosidase generated by cleavage
with P. gingivalis H66 cysteine proteinase. The mixture of
-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 -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
-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
-L-fucosidase. The reported genomic
sequence has been deposited in the GenBank(TM) data base under the
accession number X82595.
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
-L-fucosidase sequence (5) . The
amino acid sequence of the
-L-fucosidase fragment
included the sequences corresponding to the NH
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
-L-fucosidase.
Comparison of the -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 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
-L-fucosidase is expected, as the
-L-fucosidase is located in the extracellular matrix in
pea epicotyls(5) . As confirmed by NH
-terminal
amino acid sequencing of the
-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
-L-fucosidase(5) .
No strong
homology was found when the sequence of the -L-fucosidase
cDNA clone and the encoded protein were compared with the sequences of
human and rat liver
-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
terminus
of the pea stem
-L-fucosidase, including amino acids
1-80 (see Fig. 3), has 43 and 33% sequence identity to the
NH
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
-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
-L-fucosidase. (
)On the other hand, it may not
be coincidental that the pea stem
-L-fucosidase is not
cleaved into peptides by trypsin, whereas the
-L-fucosidase is cleaved by a protease that works
differently from trypsin.
Figure 3:
Comparison of the deduced
NH-terminal amino acid sequence of pea stem
-L-fucosidase with the NH
-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
-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
-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 -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
-L-fucosidase gene is present in
two or three copies in the pea genome. Therefore, pea
-L-fucosidases are comprised of a small gene family with
at least two genes.
Figure 4:
Determination of the number of
-L-fucosidase genes in the pea genome. Autoradiogram of
the 357-bp
-L-fucosidase probe hybridized to digests of
genomic DNA from pea. Lane A, EcoRI digest; lane B,
HindIII digest.
A single -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).
-L-Fucosidase transcripts were undetected in fully
elongated stem tissue. The observed pattern of expression is consistent
with the hypothesis that the
-L-fucosidase has a role in
growth regulation (5) .
Figure 5:
Northern blot analysis showing
-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
-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-terminal region
of
-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
-L-fucosidase, Kunitz trypsin inhibitors,
and sweet potato sporamins.
-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.
Figure 6:
Localization of
-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.