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(Received for publication, June 6, 1995) From the
Xyloglucan (XG) ( cDNAs encoding
Three internal peptide fragments of
the
Total RNA was isolated from pea tissue as
described(12) . Poly(A) The amplified A pea cDNA library from 7-day-old etiolated pea stems was
constructed according to the manufacturer's specifications (in
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
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 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
Figure 1:
Peptide
fragments of pea stem
Figure 2:
Nucleotide and deduced amino acid sequence
of the
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
Comparison of the 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 No strong
homology was found when the sequence of the
Figure 3:
Comparison of the deduced
NH
We performed a genomic Southern blot
analysis to estimate the number of
Figure 4:
Determination of the number of
A single
Figure 5:
Northern blot analysis showing
The NH
Figure 6:
Localization of
Volume 270,
Number 42,
Issue of October 20, 1995 pp. 24839-24843
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
-
L
-Fucosidase
Gene from Pea Seedlings (*)
-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.
)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) .-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.
Isolation and Sequencing of Peptides from
The pea stem
-L-Fucosidase-L-fucosidase was purified as described(5) .
Homogeneous -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.
-L-Fucosidase (
75 pmol) was hydrolyzed prior to
composition analysis in 6 N HCl at 155 °C for 75 min, as
recommended by the manufacturer.-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
HO, 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.Isolation of the
-L-Fucosidase
Gene-L-Fucosidase-specific degenerate
oligonucleotide primers for use in PCR reactions were synthesized by
New England Biolabs. 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.
-L-fucosidase
cDNA (in GT11) fragment was sequenced as described (14) by
the Sanger dideoxy method using Taq polymerase in the thermal
cycler.
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.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.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.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
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
-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
-L-fucosidase RNAs were
synthesized according to the instructions of Boehringer Mannheim. ) 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. 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.
Amino Acid Composition and Amino Acid
Sequencing
The lysine and arginine content of the
-L-fucosidase (Table 1) led to the expectation of
the presence of several tryptic peptides. However, 24-h digestion of
the -L-fucosidase with L-(1-tosylamino-2-phenyl)
ethyl chloromethyl ketone-treated trypsin failed to produce any
peptides. Therefore, a portion of the purified
-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
-L-fucosidase (3 µg) was used to determine its
NH-terminal sequence. The amino acid sequences of peptides
P1, P2, P3, P4, and of the NH terminus are illustrated in Fig. 2. The amino acid sequences of peptide P1 and the NH terminus are identical.
-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.''
-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.
PCR, Cloning, and Nucleotide Sequencing
An
oligonucleotide was synthesized in its sense orientation on the basis
of the NH-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.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.cDNA and Genomic Cloning
Several genomic clones
that hybridized to the partial sequence of the
-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
-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
-L-fucosidase coding region. There are no introns within
the coding region.-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. 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) .-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.
-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).
-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.
-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.
-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) .
-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.
-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.Localization of
We
showed above that mRNA encoding the -Fucosidase Transcripts-L-fucosidase is
present in several pea tissues, all of which have some cells that are
elongating. -L-Fucosidase mRNA was not detected in
elongated stems. To localize more precisely the accumulation of
-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
-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 -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 -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
-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 -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
-L-fucosidase functions in plant growth regulation,
although the role of the enzyme in vivo remains to be
established.
-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.
))
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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