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Plant Physiol. (1999) 119: 385-398
Structure, Properties, and Tissue Localization of Apoplastic
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ABSTRACT |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Apoplastic 
-glucosidases
occur widely in plants but their function is unknown because
appropriate substrates in the apoplast have not been identified.
Arabidopsis contains at least three -glucosidase
genes; Aglu-1 and Aglu-3 are sequenced
and Aglu-2 is known from six expressed sequence tags.
Antibodies raised to a portion of Aglu-1 expressed
in Escherichia coli recognize two proteins of 96 and 81 kD, respectively, in vegetative tissues of Arabidopsis, broccoli
(Brassica oleracea L.), and mustard (Brassica napus L.). The acidic -glucosidase activity from broccoli
flower buds was purified using concanavalin A and ion-exchange
chromatography. Two active fractions were resolved and both contained a
96-kD immunoreactive polypeptide. The N-terminal sequence from the
96-kD broccoli -glucosidase indicated that it corresponds to the
Arabidopsis Aglu-2 gene and that approximately 15 kD of
the predicted N terminus was cleaved. The 81-kD protein was more
abundant than the 96-kD protein, but it was not active with
4-methylumbelliferyl--D-glucopyranoside as the substrate
and it did not bind to concanavalin A. In situ activity staining using
5-bromo-4-chloro-3-indolyl--D-glucopyranoside revealed
that the acidic -glucosidase activity is predominantly located in
the outer cortex of broccoli stems and in vascular tissue, especially
in leaf traces.
The Apoplastic forms of We began characterizing the The polypeptides of the Apoplastic Plant Materials
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
-glucosidases (EC 3.2.1.20, -D-glucoside
glucohydrolase) are a widespread and diverse group of enzymes that
serve a variety of functions, depending on the subcellular location and organism in which they are found. Despite their origins in several unrelated gene families, they all share an unusually wide and overlapping substrate specificity, making identification from biochemical properties alone difficult (for review, see Frandsen and
Svensson, 1998). Various forms of -glucosidase can hydrolyze -1,1-; -1,2-; -1,3-; -1,4-; and -1,6-linked Glc from
glycoproteins or from the nonreducing ends of carbohydrates ranging in
size from disaccharides to starch. They can also catalyze
-glucosyltransferase reactions (Yamasaki and Suzuki, 1980
; Yamasaki
and Konno, 1985).
-glucosidase having acidic pH optima occur
widely in plants (Klis, 1971; Parr and Edelman, 1975; Yamasaki and
Konno, 1987, 1992; Beers et al., 1990), but their function remains
elusive because of the apparent lack of appropriate substrates in the
apoplast (Fry, 1995). A chloroplastic form of -glucosidase with a
neutral pH optimum was isolated from pea and is thought to function in
starch degradation, perhaps by removing rare -1,2 or -1,3
linkages that, if present, could block the action of the more abundant
amylases and phosphorylases (Beers et al., 1990; Sun et al., 1995). Two
different ER forms of -glucosidase are also known: glucosidase I and
II, which sequentially remove -1,2- and -1,3-linked Glc,
respectively, from nascent glycoproteins as part of the quality control
system that functions during glycoprotein folding (Hebert et al.,
1995).
-glucosidases of Arabidopsis
by searching the Arabidopsis EST collection for genes
homologous to Family 31 -glucosidases, which include mammalian
lysosomal -glucosidase (Hoefsloot et al., 1988) and intestinal
sucrase/isomaltase (Chantret et al., 1992) and several fungal
-glucosidases (Dohman et al., 1990; Sugimoto and Suzuki, 1996;
Nakamura et al., 1997a). The ER glucosidase II is a distant relative of
this family (Trombetta et al., 1996; Arendt and Ostergaard, 1997). ESTs
from several different Arabidopsis genes were identified, and one,
38A2T7 (accession no. T04333), was used as a probe to clone the
Aglu-1 gene (Monroe et al., 1997). The deduced amino acid
sequence of Aglu-1 contains 902 amino acids with a
predicted mass of 101 kD, including the putative signal sequence. The
amino acid sequence of Aglu-1 is 42% to 48% identical to
those of the recently reported cDNA clones from barley (Tibbot and
Skadsen, 1996), spinach (Sugimoto et al., 1997), and sugar beet
(Matsui et al., 1997). Although it is not completely sequenced, a
second Arabidopsis gene, Aglu-2, known from six ESTs, is
similar to Aglu-1 and each of the other plant -glucosidases.
-glucosidases all have deduced masses of
just over 100 kD, but the products of the four sequenced genes are all
subjected to posttranslational modification, including proteolysis and
glycosylation, probably in the secretory pathway, since all of the
genes contain putative signal sequences. In barley seeds antibodies
against the cloned -glucosidase expressed in Escherichia
coli recognize polypeptides of 81 and 95 kD (Tibbot et al., 1998).
Active forms of barley seed -glucosidase are also glycosylated (Sun
and Henson, 1990). Four active forms of -glucosidase from spinach
seeds were separated by Sugimoto et al. (1995) and found to be 78, 78, 82, and 82 kD. The smaller forms were much more active against soluble
starch than were the larger forms, but peptide sequences from three of
the forms were all found within a single deduced cDNA sequence
(Sugimoto et al., 1997). The active product of the sugar beet
-glucosidase is 91 kD (Chiba et al., 1978). As with spinach seeds,
four forms of the sugar beet enzyme, varying both in affinity for the
cell wall fraction and in substrate specificity, were separated from
cultured cells (Yamasaki and Konno, 1989). Apparently,
posttranslational modifications affect not only the size of the enzymes
but also their substrate specificity. Other acidic -glucosidases for
which sequence information is lacking are also glycosylated, including
those from soybean (Yamasaki and Konno, 1985) and banana (Konishi et
al., 1991). We conclude that most if not all acidic -glucosidases
are glycosylated and proteolytically processed, but for no form of the
enzyme is the extent of glycosylation or the specific proteolytic
cleavage sites known.
-glucosidases are well characterized biochemically, and
some have been sequenced; however, little is known about their
physiological function, especially in vegetative tissues. In this paper
we describe the Aglu gene family from Arabidopsis and report
on the isolation of a glycosylated, acidic -glucosidase from
broccoli (Brassica oleracea L.) flower buds. N-terminal
sequence from the broccoli enzyme indicates that it is derived from a
homolog of the Arabidopsis Aglu-2 gene that has undergone
proteolysis. Cellular and tissue-level location of the activity and
functional implications of that location are also described.
MATERIALS AND METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
. Mustard (Brassica napus cv Southern Giant
Curled) seeds were soaked in water with 0.1% Triton X-100 for 40 min, 95% ethanol for 4 min, 30% (v/v) sodium hypochlorite for 10 min, then
rinsed five times with sterile water. Seeds were grown in 250-mL flasks
containing 50 mL of autoclaved deionized water for 3 d. Flasks
were shaken at 120 rpm under continuous illumination. Broccoli
(Brassica oleracea) spears were purchased at a local market
and stored at 4°C until use.
DNA Analysis
ESTs were obtained from the Arabidopsis Biological Resource Center (Ohio State University, Columbus). DNA sequences from the 5
ends of the ESTs 38A2T7 (accession no. T04333) and H8A7T7 (accession
no. W43892) were obtained by cycle sequencing (Epicentre Technologies,
Madison, WI) and analyzed using an automated sequencing system (Li-Cor,
Lincoln, NE). Amino acid sequence alignments were conducted
using the Higgins-Sharp algorithm (CLUSTAL4) in DNASIS version 3.5 (for
Macintosh hardware). Identity scores were generated using default
parameters.
Construction of pPW1700, Expression, and Antibody Production
A 700-bp BamHI fragment from the cDNA 38A2T7 corresponding to amino acids 388 to 605 of the deduced Aglu-1 protein was inserted into the BamHI site of pFLAG-ATS (Kodak). The resulting plasmid, pPW1700, was transformed into Escherichia coli BL21 cells for expression. Cells containing pPW1700 were grown overnight in expression broth (Luria-Bertani medium containing 50 µg/mL ampicillin and 0.4% [w/v] Glc) using a 37°C shaker. The cells were then diluted 100-fold in 5 mL of prewarmed expression broth and incubated in a 125-mL flask at 37°C with shaking. Upon reaching an A600 of about 0.2, isopropylthio-
-galactoside was added to 0.5 mM. At 2 h postinduction, only induced cells
harboring pPW1700 contained the expected 24-kD polypeptide judging from
Coomassie blue-stained SDS-PAGE gels. We were unable to use the
anti-FLAG antibody to purify the 24-kD protein, probably because most
of the induced protein was insoluble. We therefore solubilized cells
containing the 24-kD polypeptide in 3 M urea with
sonication and electroeluted the protein from a preparative SDS-PAGE
gel (Gerhardt et al., 1994). One milligram of the purified 24-kD
polypeptide was used to raise polyclonal antibodies in New Zealand
White rabbits (Bethyl Labs, Montgomery, TX.) The antiserum specifically
recognized the 24-kD polypeptide in immunoblots.
Purification of -Glucosidase
Protein Analysis
Proteins were separated by SDS-PAGE in a minigel apparatus (Bio-Rad). The stacking and separating gels contained 4% and 12.5% acrylamide, respectively. Gels were either stained with Coomassie blue or proteins were transferred to nitrocellulose membranes. The membranes were blocked with 3% (v/v) coldwater fish gelatin in Tris-buffered saline and probed with a 1:100 dilution of anti-Aglu-1 serum or preimmune serum. Bands were visualized using peroxidase-conjugated goat anti-rabbit IgG (Sigma) diluted 1:2000 according to the manufacturer's instructions. Prestained molecular mass standards (Sigma) were used to estimate the masses of polypeptides.-glucosidase was separated by SDS-PAGE, transferred
to a PVDF membrane, and stained according to the manufacturer's instructions (ABI, Columbia, MD). The N-terminal sequence was determined using Edman degradation by Midwest Analytical (St. Louis,
MO).
Enzyme and Protein Assays
-Glucosidase assays were conducted in a final volume of 0.5 or
1.0 mL of 50 mM Na acetate, pH 4.5, or 50 mM Mes, pH 7.0, and 0.1 mM 4-MUG (unless
otherwise noted) at 37°C. Reactions were initiated with the addition
of enzyme and were stopped with 3 mL of 0.5 M Gly, pH 10.5. Reactions stopped prior to the addition of enzyme were used as inactive
controls and each assay was duplicated. Fluorescence of
4-methylumbelliferone at 450 nm was measured after excitation at 366 nm
using a Spex spectrafluorimeter (Instruments S.A., Edison, NJ) and
quantified using 4-methyl umbelliferone standards. One unit of activity
was defined as the formation of 1 µmol product
min
1 at 37°C. For pH curves 50 mM
sodium acetate, Mes, or Hepes were used as buffers. For in situ
-glucosidase assays, intact Arabidopsis or mustard seedlings or
hand-cut broccoli sections were incubated for 3 to 12 h at 30°C
in the dark in a small volume of assay medium containing the same
buffers as described above, but with 2 mM X--gluc
(Calbiochem). The staining solution was then removed and the tissues
were cleared through sequential washes with 70%, 95%, and 100%
ethanol for 10 min each prior to photography. Malate dehydrogenase
activity was assayed by monitoring the oxaloacetate-dependent oxidation
of NADH at 340 nm in 50 mM Hepes, pH 7.5, 150 µM NADH, and 8 mM oxaloacetate, pH 6.5. Protein was measured with either Bradford reagent (Bio-Rad) or
bicinchoninic acid reagent (Pierce) using BSA as the standard.
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RESULTS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Arabidopsis Contains at Least Three -Glucosidase Genes
-glucosidases. Based on overlapping EST nucleotide sequences,
alignment of deduced amino acid sequence with known -glucosidases,
and restriction mapping, we concluded that the 11 ESTs were derived
from at least three different genes, which are illustrated in Figure
1. For each of the three genes, an EST was identified containing the peptide sequence WiDMNE, which is one of
two signature peptides for -glucosidases from Family 31 recognized by Nichols et al. (1998). The Asp residue in this signature peptide is located in the active site as determined by labeling with
conduritol B epoxide (Sugimoto et al., 1997). We propose to name
the three Arabidopsis genes Aglu-1, Aglu-2, and
Aglu-3. The complete nucleotide sequences of the
Aglu-1 gene (Monroe et al., 1997) and the Aglu-3
gene (Nakamura et al., 1997b) are known.
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Arabidopsis Contains Acidic and Neutral
Antibodies to Aglu-1 Recognize an 81-kD Polypeptide in
Arabidopsis
-glucosidases, mammalian sucrase/isomaltase, and the other apoplastic plant -glucosidases. Sequences from ESTs derived from Aglu-2 strongly suggest
that it is also a member of clade 1. Aglu-3 fell into clade
2 along with the ER glucosidase II from mouse (Arendt and Ostergaard, 1997) and several sequenced eukaryotic genes, including a cDNA from
potato (Taylor et al., 1998). Partial amino acid sequence from the ER
glucosidase II purified from rat liver closely matches the mouse
glucosidase II sequence (Trombetta et al., 1996). Sequence identity
between the two clades is only 12%, omitting the poorly conserved N-
and C-terminal regions, and only 8% overall, suggesting that the two
groups are very distantly related. The presence of a perfectly
conserved Asp in the WiDMNE peptide and the considerable similarity
among both family 31 signature regions illustrated in Figure 2
suggest a common evolutionary origin for the clades.
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Figure 2.
Phylogenetic relationship among Family 31 -glucosidases and aligned signature peptides indicate that there are
two clades. Left, Phylogenetic tree indicating percent identity at the
amino acid level using about 60% of each protein. Approximately 250 amino acids from the N termini and 150 amino acids from the C termini
of each protein were omitted from this analysis. Right, Sequences of
two signature peptide regions along with consensus sequences for each
clade. Consensus amino acids are uppercase if perfectly conserved and
lowercase if conserved in more than half of the sequences. The
sequences (and accession numbers) used in the analysis from clade 1 were: Aglu-1 from Arabidopsis (AF014806),
-glucosidases from spinach (D86624), sugar beet (D89615), barley
(U22450), Mucor javanicus (D67034), Aspergillus
niger aglA (D45356), Schwanniomyces occidentalis
glucoamylase GAM1 (M60207), Schizosaccharomyces pombe
C30D11.01C (1723210), lysosomal acid -glucosidase (GAA) from human
(M34424) and mouse (P70699), and the N-terminal and C-terminal halves
of the human sucrase/isomaltase (M22616). In clade 2 the sequences (and
accession numbers) used were Aglu-3 from Arabidopsis
(AB007646), cDNAs from potato (AJ001374) and human (AJ000332), GluII
from mouse (U92793), ModA from Dictyostellium discoideum
(U72236), and the Saccharomyces cerevisiae open reading
frame YBR229c (Z36098).
-Glucosidases
-glucosidase activity using
4-nitrophenyl--D-glucopyranoside as the substrate, very
low levels of activity were observed. We therefore used 4-MUG as the
substrate because of its increased sensitivity. The broad pH profile of
activity in crude extracts (data not shown) suggested that several
forms with optimal activity at acidic and neutral pHs existed in all
tissues. Neutral forms of -glucosidase from pea, banana, and barley
have negligible activity below pH 5.0 (Beers et al., 1990; Konishi et
al., 1991; Sun et al., 1995), so we assayed various extracts at pH 4.5. Figure 3A shows that stems and "buds"
(including some stem tissue, flowers, flower buds, and young siliques)
contained more acidic activity on a total protein basis than did leaves
or seeds. To determine whether this activity was the product of any of
the Aglu genes, we raised antibodies to a fragment of an EST
derived from Aglu-1.
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Figure 3.
-Glucosidase activity and immunoblot of
extracts from Arabidopsis tissues probed with anti-Aglu-1. A,
-Glucosidase activity in Arabidopsis tissues. Assays were conducted
at pH 4.5 using 4-MUG as the substrate. Bud tissue included some stem
tissue, flowers, flower buds, and young siliques. Data represent the
means ± SD (n = 3). B, Detection
of an 81-kD protein in Arabidopsis tissues with anti-Aglu-1 serum.
Equal amounts of -glucosidase activity (8 units) from the tissues
represented in A were separated by SDS-PAGE and probed with anti-Aglu-1
serum. U, Unit.
-glucosidase activity from the same tissues
represented in Figure 3A were separated by SDS-PAGE and probed with
anti-Aglu-1 serum. Figure 3B shows that an 81-kD protein was recognized
by the antiserum in some of the extracts, however, there was no clear
relationship between the amount of the 81-kD protein and the level of
-glucosidase activity measured at pH 4.5. The bud sample contained
much more of the immunoreactive protein than did other tissues (Fig.
3B). Similar blots probed with the preimmune serum did not reveal the 81-kD protein. Because of the potential for cross-reaction of the
anti-Aglu-1 serum with products of the Aglu-2 gene, it could not be determined if the 81-kD Arabidopsis protein was the product of
Aglu-1 or Aglu-2. Regardless, the size of this
protein was similar to some of the products of the homologous spinach
and barley acidic -glucosidases (Sugimoto et al., 1995; Tibbot et al., 1998). We therefore proceeded to characterize both the 81-kD polypeptide and the major acidic -glucosidase activity from
vegetative tissues to determine whether they were related.
Purification of Acidic
Acidic
Tissue-Level Localization of Acidic
The Aglu Gene Family
Structure and Processing of the Major Acid Location of Apoplastic Received October 9, 1998;
accepted October 26, 1998.
Abbreviations:
CM, carboxy methyl.
ConA, concanavalin A.
EST, expressed sequence tag.
4-MUG, 4-methylumbelliferyl- We are grateful to Kelly Poliquin for excellent technical
assistance, Maarten Chrispeels for helpful suggestions, Mark Brodl and
Jennifer Jenkins for critically reviewing the manuscript, and the
Arabidopsis Biological Resource Center for EST clones.
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Two forms of were able to separate the major acidic and
neutral forms of -glucosidase from banana pulp using ConA-Sepharose affinity chromatography. A crude extract from Arabidopsis leaf tissue
was prepared in a neutral extraction buffer containing 1 M
NaCl, which is known to aid the release of apoplastic -glucosidase (Parr and Edelman, 1975). Figure 4A shows
that some -glucosidase activity passed through the ConA column,
whereas a fraction bound to ConA and was eluted with 15 mM
methyl Glc. The nonbinding (ConA) fraction had optimal activity at pH
7.0 to 7.5 (Fig. 4B), which is typical of neutral -glucosidases,
whereas the bound (ConA+) fraction had optimal activity at pH 4.5 to
6.0 (Fig. 4C), typical of acidic -glucosidases. As expected, the
neutral form was nearly inactive below pH 5.0.
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Figure 4.
Separation of neutral and acidic -glucosidases
from an Arabidopsis leaf extract using a ConA-Sepharose column, and the
pH profile of each activity peak. A, ConA-Sepharose column. Bound
proteins were eluted with 15 mM methyl Glc (MG).
-Glucosidase activity was measured at pH 4.5 (
) and pH 7.0 (
).
B, pH profile of pooled fractions 2 and 3 from A. C, pH profile of
pooled fractions 12 to 14 from A. For B and C, buffers were sodium
acetate (), Mes (), or Hepes (
). U, Unit.
fraction was
most active at pH 7.0, but unlike the ConA leaf fraction, which
contained negligible activity at pH 4.5 (Fig. 4B), the ConA stem+bud
fraction had low but significant activity at pH 4.5. The ConA+ stem+bud
fraction was active at both pH 4.5 and 7.0 (Fig. 5A). Both the ConA
and ConA+ fractions were then concentrated and probed with anti-Aglu-1
serum on an immunoblot. Surprisingly, nearly all of the 81-kD protein
was in the ConA fraction (Fig. 5B). The ConA+ fractions, which
contained most of the acidic activity, contained a 96-kD immunoreactive protein that was much less abundant than the 81-kD protein since it
required a greater degree of concentration to observe on immunoblots. This may explain why it was not observed in immunoblots of crude extracts.
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Figure 5.
Separation of -glucosidases from an Arabidopsis
stem+bud extract by ConA chromatography and an immunoblot of the pooled
active fractions probed with anti-Aglu-1. A, ConA-Sepharose column.
Bound proteins were eluted with 15 mM methyl Glc (MG).
-Glucosidase activity was measured at pH 4.5 () and pH 7.0 ().
U, Unit. B, Immunoblot of activity peaks separated in A. Nonbinding
activity (fractions 2 and 3) and bound activity (fractions 11-17) from
A was concentrated 24-, and 215-fold, respectively, and equal volumes
of each concentrated fraction were separated by SDS-PAGE and probed
with anti-Aglu-1.
fraction suggested that
it was either a novel neutral form of the enzyme or that the ConA
stem+bud fraction contained both the neutral form and an acidic, 81-kD
polypeptide that was much less active than the ConA+ acidic form of the
enzyme. To answer this question, a crude stem+bud extract from
Arabidopsis was subjected to gel filtration using Sephadex 150-G to
attempt to separate the neutral activity from the 81-kD polypeptide.
Figure 6 shows that all of the
-glucosidase activity at both pH 4.5 and 7.0 coeluted with the first
major peak of protein, but that most of the 81-kD polypeptide eluted
with a second major protein peak, which had no detectable -glucosidase activity. The 81-kD protein is therefore not the major
neutral -glucosidase, but because it had no apparent activity with
4-MUG, we did not characterize it further. It is possible that the
81-kD protein is not an -glucosidase but that it simply shares a
common epitope recognized by anti-Aglu-1. However, as noted above,
other acidic plant -glucosidases that were not assayed with 4-MUG
have a mass that is similar to the 81-kD Arabidopsis protein.
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Figure 6.
Separation of the Arabidopsis -glucosidase
activity from most of the 81-kD polypeptide using Sephadex G-150. A,
Sephadex G-150 column. -Glucosidase activity was assayed at pH 4.5 () and pH 7.0 (). Total protein was monitored at
A280 (solid line). U, Unit. B, Immunoblot of
fractions 5 and 10 from A. Equal volumes (10 µL) of each fraction
were separated by SDS-PAGE and probed with anti-Aglu-1.
-glucosidase activity in Arabidopsis appeared
to be associated with a relatively rare 96-kD protein enriched in bud
tissues, we continued our purification of the activity using broccoli
flower bud tissue, which was more readily available. We anticipated
that broccoli, being a crucifer, would contain -glucosidase proteins
to which our Arabidopsis Aglu-1 antiserum would bind. Indeed, crude
broccoli bud extracts also contained an 81-kD protein recognized by the
anti-Aglu-1 serum (data not shown).
-Glucosidase from Broccoli
-glucosidase activity at pH 4.5 was retained by the column and was
eluted with 15 mM methyl Glc. When probed with
anti-Aglu-1, this glycoprotein fraction contained the 96-kD
immunoreactive protein (data not shown).
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Figure 7.
Isolation of the major acidic -glucosidases
from broccoli flower buds using ConA- and CM-cellulose chromatography
and an immunoblot of pooled active fractions probed with anti-Aglu-1.
A, ConA-Sepharose column. Bound proteins were eluted with 15 mM methyl Glc (MG). -Glucosidase activity was assayed at
pH 4.5 () and pH 7.0 (). Total protein was monitored at
A280 (solid line) in A and B. B, Separation
of fractions 21 to 24 from A on a CM-cellulose column. -Glucosidase
activity was assayed at pH 4.5 (). C, Immunoblot of activity peaks
separated in B. G1 (fractions 5-6) and G2 (fractions 28-32)
from B were pooled and concentrated. Equal amounts of activity at pH
4.5 (37 units) were separated by SDS-PAGE and probed with anti-Aglu-1.
U, Unit.
G1 did not bind to the column,
whereas G2 eluted at approximately 0.5 M NaCl (Fig. 7B).
The two peaks were subsequently concentrated and equal amounts of
activity at pH 4.5 were separated by SDS-PAGE and probed with
anti-Aglu-1 serum. Both peaks contained approximately equal amounts of
the 96-kD protein (Fig. 7C), suggesting that they had similar specific
activities. Figure 8 shows the effect of
varying the concentration of 4-MUG on activity at pH 4.5. A
Lineweaver-Burk plot of the activity in G1 was linear and the
Km for 4-MUG was approximately 0.3 mM. G2, however, was clearly a mixture of
activities since the Lineweaver-Burk plot was nonlinear. Extrapolation
from the ends of the Lineweaver-Burk plot suggested that G2
contained at least two -glucosidases, one having a
Km of approximately 0.3 mM and the
other of 8.6 µM 4-MUG.
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Figure 8.
Effect of 4-MUG on the activity of two broccoli
-glucosidase isoforms separated by CM-cellulose chromatography.
G1 () and G2 () from the CM-cellulose column (Fig. 7B) were
assayed at pH 4.5. Data were normalized to the level of activity at 1.5 mM 4-MUG. Inset, Lineweaver Burk plots of the same data.
-glucosidases separated by CM-cellulose is not known.
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Figure 9.
Purified 96-kD broccoli -glucosidase stained
with Coomassie blue or probed with anti-Aglu-1 serum, and the
N-terminal sequence from the same protein aligned with other plant
-glucosidase sequences. A, SDS-PAGE and immunoblot of the 96-kD
broccoli -glucosidase. Proteins (4 µg) were separated by SDS-PAGE
and either stained with Coomassie blue (lane 1) or probed with
anti-Aglu-1 serum (lane 2). The numbers on the left represent molecular
mass standards in kilodaltons. B, N-terminal sequence from the broccoli
96-kD -glucosidase aligned with other plant -glucosidases.
Sequences (and accession numbers) were from Arabidopsis
Aglu-1 (AF014806), Arabidopsis Aglu-2
(sequence from the EST H8A7T7), spinach (D86624), sugar beet (D89615),
and barley (U22450).
-glucosidases revealed that it matched a region 121 to 145 amino acids from the
translation start site of each sequence (Fig. 9). Although Aglu-2 is not completely sequenced, an EST from
Aglu-2, H8A7T7 (accession no. W43892), appeared to span this
region. We resequenced the 5 end of this cDNA and found that the
corresponding region of Arabidopsis Aglu-2 matched the
broccoli sequence at 16 of the 20 residues (Fig. 9). If the two
ambiguous residues are both Ile, then identity between the broccoli
96-kD protein and Arabidopsis Aglu-2 increases to 90%.
Identity between the broccoli peptide and each of the other plant
-glucosidases, including Arabidopsis Aglu-1, ranged from
only 39% to 47%. The broccoli protein therefore appears to be encoded
by the homolog of the Arabidopsis Aglu-2 gene. Assuming that
the structures of the broccoli and Arabidopsis Aglu genes
are similar, our results indicate that the 96-kD protein undergoes
proteolytic processing during its synthesis, in which approximately 15 kD of the N terminus is removed. Using Aglu-1 as the model
gene, removal of 15 kD would leave at most 86 kD of polypeptide,
suggesting that the 96-kD protein from Arabidopsis (Fig. 5B) and
broccoli (Fig. 9) may contain at least 10 kD of glycan. The deduced
Aglu-1 protein contains nine potential N-glycosylation sites, only one
of which would be removed with the N-terminal 15 kD if
Aglu-1 and Aglu-2 are similarly processed. Our
prediction that the enzyme contains at least 10 kD of glycan is thus
well within the maximum potential glycan mass, assuming that the glycan is N-linked, that Aglu-2 has a similar number of
N-glycosylation sites, and that all of the sites have high Man or
complex glycans attached.
-glucosidases is highest in the central 60% of the proteins.
It is interesting that the proposed cleavage site falls at the border
of a poorly conserved region and a well-conserved region (marked by the
asterisk in Fig. 10). Percent
identities within the N-terminal 15-kD region of the known plant
-glucosidases range from only 13% to 34%, whereas that of the
central region, omitting the last 150 amino acids, range from 52% to
70%. Sequence conservation drops to between 10% and 25% within the
C-terminal 13 kD.
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Figure 10.
Alignment of the known plant -glucosidase
sequences. Sequences are from Arabidopsis Aglu-1,
spinach, sugar beet, and barley. For accession numbers, see legend for
Figure 9. Dots represent amino acids that are identical to the
Arabidopsis Aglu-1 sequence. The N terminus of the
broccoli 96-kD protein is indicated with an asterisk. The 24-kD Aglu-1
polypeptide expressed in E. coli for antibody production
is underlined.
-Glucosidase Activity in Mustard Is Apoplastic
-glucosidase activity in Arabidopsis and broccoli is located in the apoplast produced equivocal results because only low levels of activity
could be extracted from Arabidopsis stems by centrifugation, and these
experiments necessitated breaking some cells and releasing the more
active neutral form of the enzyme. Moreover, our attempts to isolate
protoplasts free of the acidic -glucosidase activity were hampered
by the presence of large amounts of acidic -glucosidase in fungal
cellulase preparations. We therefore reasoned that if the acidic
-glucosidase was indeed apoplastic, as in other plants, then it
should elute from uncut tissues into a buffer containing 1 M NaCl if the tissues were small and not covered by a thick cuticle. We chose to use 3-d-old mustard seedlings that were grown under water with shaking so as to retard the development of the cuticle. Seedlings grown in this manner appeared to be identical to
soil-grown plants. Mustard also contained 81- and 96-kD proteins recognized by Anti-Aglu-1 serum (data not shown).
-glucosidase activity at pH 4.5 was observed in the
surrounding buffer (Fig. 11A). Over a
6-h period of shaking the level of activity in the buffer increased, indicating that the enzyme was slowly diffusing out of the seedlings. At the end of 6 h the medium contained activity that was about 3-fold higher at pH 4.5 than it was at pH 7.0, suggesting that only the
acidic enzyme had eluted (Fig. 11B). Similar, untreated plants were
ground with sand in the same extraction buffer to measure the total
extractable activity. About 3-fold more activity at pH 4.5 was
extracted from the seedlings with grinding, but activity at pH 7.0 was
over 50-fold higher in the ground extract than in the elution buffer
from shaken plants (Fig. 11B). Some of the activity at pH 4.5 from the
ground plants could have been due to the neutral mustard enzyme, but if
its pH profile is similar to that of the Arabidopsis neutral enzyme
(Fig. 4B), this contribution would be low. As a marker of cell
breakage, malate dehydrogenase activity in the buffer around the shaken
plants was measured and found to be less than 0.2% of that from the
ground plants, indicating that this method of elution was extremely
gentle. These results indicate that at least one-third of the acidic
-glucosidase activity in these seedlings was located in the
apoplast. The remainder of the activity could be inside the cell
membrane, or it might not have been able to diffuse out of the apoplast
because of the presence of a cuticle on the epidermis of some seedlings
or internal barriers to apoplastic diffusion, such as the suberized
root endodermis.
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Figure 11.
Localization of acidic -glucosidase activity
in the apoplastic fraction of mustard seedlings. A, Time course of
elution of -glucosidase activity from intact mustard seedlings.
Three-day-old mustard seedlings grown under water were shaken in
extraction buffer containing 1 M NaCl for 6 h.
-Glucosidase in the medium was assayed periodically with 4-MUG at pH
4.5. B, Activity of -glucosidase (Aglu) at either pH 4.5 or 7.0 and
malate dehydrogenase (MDH) in the elution buffer from A after 6 h,
and in extracts from similar but untreated plants. Data represent the
means ± SD (n = 3). U, Unit.
-glucosidase activity from
mustard seedlings suggested that two different pools of activity were
eluted. One pool came out within the 1st min and was perhaps located at
the surface of the seedlings, whereas the other diffused out more
slowly over a period of hours (Fig. 11A). To investigate the
possibility that plants contain multiple pools of -glucosidase
activity in surface and internal tissues, we used 2 mM
X--gluc as a substrate to stain seedlings and broccoli stem
sections.
-Glucosidase
-gluc at pH 4.5 appeared blue all over, but staining
was strongest in root tips and in vascular tissues, as seen in Figure
12A. In 3-d-old mustard seedlings
staining was much stronger at pH 4.5 than at 7.0, indicating that this
activity was probably due to the apoplastic acidic -glucosidase
(Fig. 12B). To determine the spatial distribution of staining in a
larger plant, hand sections of broccoli stems were similarly stained. Cross-sections through stems 2 to 3 cm below the apex revealed strongest staining in the cortex, especially in the outer 0.5 mm of the
stem (Fig. 12D). In addition, some staining of the vascular tissue was
observed in this section. Longitudinal sections through a leaf
abscission zone showed strongest staining in the leaf trace (Fig. 12C).
Staining of the inner xylem and outer cortex, even in the abscission
zone, could also be seen. Cross-sections through and below a leaf
abscission zone revealed a similar pattern (Fig. 12, E-G). The
strongest staining was observed in leaf traces above and below the
point of separation from the vascular cylinder. Figure 12G shows a
higher magnification of the cross-section in Figure 12F, illustrating
the relative lack of staining in the younger xylem, pith, and inner
cortex. Staining of the abscission zone was also observed prior to leaf
abscission (data not shown). The fast-eluting and slow-eluting pools of
activity observed in mustard seedlings (Fig. 11A) may correspond to the
epidermal and vascular activity, respectively (Fig. 12G).
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Figure 12.
In situ activity of -glucosidase assayed with
-X-gluc at pH 4.5 for 12 h, except where noted. A,
Three-day-old Arabidopsis seedling. B, Three-day-old mustard seedlings
stained at pH 4.5 (left) or 7.0 (right) for 3 h. C, Longitudinal
section through a broccoli stem. The curved surface on the right side
is a leaf abscission zone. Unstained tissue on the left is pith. Strong
staining is seen in the leaf trace indicated by the arrow. D,
Cross-section of a broccoli stem approximately 3 cm from the apex. E,
Cross-section of a broccoli stem through a leaf abscission zone. Arrows
point to strong staining in leaf traces at or above the point where
they separate from the vascular cylinder. A leaf abscission zone is on
the right side of the image. F, Same as E but approximately 4 mm basal.
Arrows point to the same leaf traces as in E, but both leaf traces are
below the point of separation from the vascular cylinder. G, Finer
detail of F. pi, Pith; ox, older xylem; yx, young xylem; ph, phloem;
ic, inner cortex; oc, outer cortex. Note the increased staining toward
the outer cortex and in the older xylem and in the leaf traces.
Bars = 1 mm (A, B, and D); 2 mm (C, E, and F); or 0.5 mm G.
-glucosidase in broccoli
stem sections assayed with X--gluc, incubations were carried out
over 12 h. This raised the possibility that some of the activity
could be due to de novo synthesis induced by wounding. Inclusion of 10 µM cycloheximide in the assay solution did not affect
staining, so the observed -glucosidase activity was not due to de
novo synthesis. Moreover, wounding did not appear to induce activity in
broccoli stems. Adding 1 µM 1-deoxynojirimycin, an
-glucosidase inhibitor, to the assay mixture completely blocked staining with X--gluc, but it had no effect on staining with X--gluc, suggesting that the observed staining with X--gluc was
indeed due to -glucosidase activity.
-glucosidase or, if
X--gluc penetrated cell membranes, to a neutral -glucosidase inside the cell, although, as mentioned above, staining was much stronger at pH 4.5, which suggests that the acidic apoplastic enzymes
contributed most of the activity (Fig. 12B). When we tested the
activity of crude extracts from leaf tissue with both 4-MUG and
X--gluc at pH 4.5 and 7.0, relative activities with the two substrates were quite different. Activity with 4-MUG was about 2-fold
higher at pH 7.0, whereas activity with X--gluc was 12-fold higher
at pH 4.5, suggesting that either the neutral form of the enzyme is
unstable in the presence of X--gluc or its solvent, or that the
neutral form of the enzyme simply does not act on X--gluc.
Regardless, in situ assays with X--gluc as the substrate appear to
be specific for acidic -glucosidase.
DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References
-glucosidases are a poorly understood group of enzymes,
due in part to the fact that they have no known function in the
apoplast, where they are located. In several plants multiple forms of
the enzyme were separated and were shown to have distinct structural
and catalytic properties, but no potential substrates are known to
exist in the apoplast. Because of their broad substrate specificity and
structural heterogeneity, identification of these enzymes from
biochemical properties alone is problematic. We therefore chose to
characterize the products of the Arabidopsis -glucosidase gene
family at the molecular level.
-glucosidases that have
been cloned from barley (Tibbot and Skadsen, 1996), spinach (Sugimoto
et al., 1997), and sugar beet (Matsui et al., 1997). A third
more distantly related -glucosidase gene, Aglu-3, is most
similar to the ER glucosidase II, which catalyzes the hydrolysis of
-1,3-linked Glc from glycoproteins as they fold. Arabidopsis also
contains an abundant neutral -glucosidase, which does not bind to
ConA. It is likely that this enzyme is similar to the plastid form of -glucosidase isolated from pea (Beers et al., 1990; Sun et al., 1995). The large size difference between the pea neutral form (24 kD)
and the various acidic forms (78-96 kD) suggests that they may be
encoded by different gene families.
-glucosidases, including the mammalian
lysosomal -glucosidase and secreted sucrase/isomaltase, as well as
the secreted fungal and plant -glucosidases, are derived from an
ancestral ER glucosidase II gene. All would have retained their signal
peptides but would have then diversified in terms of their substrate
specificity, location, and function.
-Glucosidases
in Crucifers
-glucosidase genes that have been sequenced
encode deduced proteins of slightly more than 100 kD. The products of
these genes, however, fall into two size classes, 78 to 82 kD and 91 to
96 kD, indicating that they all undergo proteolytic processing. They
have optimal activity at an acidic pH and some were shown to be
apoplastic glycoproteins. The most active acidic -glucosidase in
Arabidopsis and broccoli is a 96-kD glycoprotein that we believe
contains at least 10 kD of sugar. Because it binds to ConA, the 96-kD
protein probably contains some high Man glycans. We separated two
isoforms of the 96-kD enzyme but were unable to distinguish them by
size or activity, although one fraction appeared to be a mixture
containing enzymes with different affinities for the substrate 4-MUG.
Multiple isoforms of -glucosidase with different catalytic
properties were also separated from sugar beet and spinach (Yamasaki
and Konno, 1989; Sugimoto et al., 1995). Whether the 96-kD broccoli
-glucosidase isoforms represent the products of different genes or
differential posttranslational processing of a single gene product is
not known, however, there is evidence that the latter can occur (Sturm,
1991). The glycan moieties of secreted glycoproteins are rapidly
modified in the ER and Golgi by glycosidases and transglycosidases, so
that the secreted forms are initially homogenous. After
secretion, apoplastic N-acetylglucosaminidase and/or
-mannosidase slowly modify the complex glycans so that heterogeneity
in glycan structure can exist, even at a single locus (Sturm, 1991).
The two isoforms of broccoli -glucosidase that we separated by
CM-cellulose chromatography may represent the same enzyme with or
without terminal GlcNAc on one or more complex glycans, thus affecting
their affinity for the ion-exchange resin. Because the two proteins
have similar specific activities, their structural differences may not
be important to their function; however, they may differ in substrate
specificity and/or affinity for the cell wall.
-glucosidase sequences, our data suggest that
approximately 15 kD of the polypeptide was proteolytically removed from
the enzyme. Several lines of evidence suggest that this processing
occurred in vivo and not during enzyme purification. First, no other
purified acidic -glucosidase has a subunit size larger than 96 kD.
If all of them are similarly glycosylated, then proteolytic removal of
a relatively large portion of the protein is a conserved event. Second,
the cleavage site occurs at the border between the poorly conserved
N-terminal region and the well-conserved central region of the sequence
(Fig. 10), suggesting that all of the plant -glucosidases may be
similarly processed. However, two peptide sequences from the 82-kD
spinach -glucosidase III and IV begin only 29 residues from the
N-terminal Met (Sugimoto et al., 1997). These residues are well
within the N-terminal 15-kD region. This would suggest that proteolysis
of the spinach enzyme might occur at the C terminus. Judging from the
alignment shown in Figure 10, poor sequence conservation also
occurs in the C-terminal 13 kD of the cloned -glucosidases,
suggesting that both ends of the proteins may be processed. If so, then
the level of glycosylation is larger than our estimate of 10 kD.
Clearly, there is a large degree of structural heterogeneity among
these enzymes.
) and spinach (Sugimoto et al.,
1995), in which the smaller 78- to 82-kD acidic -glucosidases were
catalytically active, the 81-kD Arabidopsis protein was not active with
4-MUG as the substrate. It may, however, have activity toward an in
vivo substrate. Because the 81-kD protein did not bind to
ConA-Sepharose, it either contains all complex glycans that fail to
bind to ConA, or it may be a highly deglycosylated degradation product
from either Aglu-1 or Aglu-2. The function of
sugar moieties on glycoproteins has been well studied, and in addition
to influencing thermal stability (Kern et al., 1992), glycosylation can
also influence catalytic activity. Mutational removal of each of the
four N-linked glycosylation sites in lecithin-cholesterol acyltransferase decreased or increased the specific activity of the
enzyme, but removal of all four sites nearly abolished activity (O et
al., 1993). Further study is necessary to characterize the 81-kD
cross-reacting protein and to determine if it is an active -glucosidase.
-Glucosidase Activity in Vegetative
Tissues and Implications for Function
-glucosidases are usually assayed using
-1,4-linked carbohydrate substrates, the lack of such linkages in
the apoplastic compartment of plants has made it difficult to speculate
on the potential function of these enzymes. Numerous studies of acidic
-glucosidase were conducted on cereal grain seeds, where it was
assumed that the enzymes act on starch during germination. Indeed, in
cereal grains an apoplastic -glucosidase could have access to starch
or its hydrolytic products, but in other seeds in which cell integrity
is maintained during germination and in vegetative tissues, apoplastic
-glucosidases should not come into contact with plastidic starch
except during cell lysis.
-1,3-linked Glc residues from nascent glycoproteins. Apoplastic -glucosidases can also hydrolyze -1,3-linkages, sometimes with more efficiency than -1,4-linkages (Yamasaki and Konno, 1989). Perhaps the apoplastic -glucosidases act on an apoplastic
glycoprotein containing -linked Glc. However, to our
knowledge, secreted glycoproteins containing -linked Glc have not
been reported, and incorrectly processed glycoproteins are unlikely to
escape the ER. Alternatively, apoplastic -glucosidases could act on
glycoproteins secreted by other organisms such as plant pathogens. The
location of the enzyme activity near cuticular surfaces and especially
in open leaf traces, where pathogens attempt to infect plants, makes it tempting to speculate that the enzyme plays a role in defense. Several
reports have shown that exogenous application of -glucosidase can
interfere with the development of pathogenic fungi (Xaio et al., 1994;
Hollenstein et al., 1995).
). For some fungi it was
shown that if the plant is not a host, an appressorium is not formed
and the fungus lives saprophytically (Beckerman and Ebbole, 1996).
Recognition of the host thus appears to be critical for the development
of some fungi, and probably involves signaling molecules. Working with
rice blast fungus, Xaio et al. (1994) observed that conidia that
germinated on an artificial substrate in the presence of
-glucosidase, -mannosidase, or protease failed to develop
appressoria. No inhibition was observed when conidia were germinated in
the presence of -glucosidase, -galactosidase, or chitinase. The
authors concluded that a secreted glycoprotein involved in cell
signaling was perhaps being inactivated by the enzymes before it could
induce appressorium formation. Similar results were observed by
Hollenstein et al. (1995) using Phytophthora megasperma f.
sp. glycinae conidia on soybean hypocotyls. In that study
treatment with -glucosidase caused a delay in appressorial development and subsequent infection of soybean cells.
-glucosidase activity in surface tissues and
in leaf traces below the abscission zone is consistent with a role in
defense, since these tissues are the first that pathogens would contact
upon landing on a plant. Vessel elements of leaf traces that are
exposed after leaf abscission are known to be the route for infection
by some bacterial pathogens, including the causal agent of apple canker
(Crowdy, 1952) and cherry canker (Crosse, 1956). Immediately after
abscission the water in a vessel element retreats into the stem,
carrying with it any propagules on the exposed surface. Staining of the
oldest xylem tissue in broccoli stems (Fig. 12, E-G) may be due to the
fact that the vessels of this xylem would have been exposed to the air
through the cut base of the spear, thus representing another route for
pathogen invasion. Unlike the well-studied PR proteins that are induced upon infection and constitute part of the active defense system, we
could not detect any induction of acidic -glucosidase activity by
wounding or treatment of tissues with ethylene, salicylic acid, or
jasmonic acid in preliminary experiments. If the enzyme is involved in
defense, it probably plays a passive role.
-glucosidase can be
envisaged that are also consistent with our localization data.
For example, the enzyme may play a role in suberin or cutin synthesis
since the enzyme appears to be located in places where the synthesis of
those molecules takes place. Little is known about the synthesis of
suberin or cutin, but perhaps glycosylated precursors are secreted to
the apoplast where -glucosidase could remove the Glc prior to
polymerization or deposition. Leaf traces are filled with waxy
materials after leaf abscission, so high levels of -glucosidase
activity in leaf traces is consistent with this hypothesis. The
isolation of mutants should aid in testing these hypotheses.
1
This work was supported by the U.S. Department
of Agriculture-National Research Initiative Competitive Research Grants
Program (grant nos. 94-04200 and 96-00679).
FOOTNOTES
*
Corresponding author; e-mail monroejd{at}jmu.edu; fax
1-540-568-3333.
ABBREVIATIONS
D-glucopyranoside.
X--gluc, 5-bromo-4-chloro-3-indolyl--D-glucopyranoside.
ACKNOWLEDGMENTS
LITERATURE CITED
Top
Abstract
Introduction
Methods
Results
Discussion
References
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38:
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30:
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Agric Biol Chem
49:
849-850
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Phytochemistry
26:
711-713
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28:
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Copyright Clearance Center: 0032-0889/99/119//14
© 1999 American Society of Plant Physiologists
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 N. Iglesias, J. A. Abelenda, M. Rodino, J. Sampedro, G. Revilla, and I. Zarra Apoplastic Glycosidases Active Against Xyloglucan Oligosaccharides of Arabidopsis thaliana Plant Cell Physiol., January 1, 2006; 47(1): 55 - 63. [Abstract] [Full Text] [PDF]
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 E.H. Muslin, S.E. Clark, and C.A. Henson The effect of proline insertions on the thermostability of a barley {alpha}-glucosidase Protein Eng. Des. Sel., January 1, 2002; 15(1): 29 - 33. [Abstract] [Full Text] [PDF]
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 F. de la Torre, J. Sampedro, I. Zarra, and G. Revilla AtFXG1, an Arabidopsis Gene Encoding alpha -L-Fucosidase Active against Fucosylated Xyloglucan Oligosaccharides Plant Physiology, January 1, 2002; 128(1): 247 - 255. [Abstract] [Full Text] [PDF]
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J. Sampedro, C. Sieiro, G. Revilla, T. Gonzalez-Villa, and I. Zarra Cloning and Expression Pattern of a Gene Encoding an {alpha}-Xylosidase Active against Xyloglucan Oligosaccharides from Arabidopsis Plant Physiology, June 1, 2001; 126(2): 910 - 920. [Abstract] [Full Text] [PDF]
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T. P. Frandsen, F. Lok, E. Mirgorodskaya, P. Roepstorff, and B. Svensson Purification, Enzymatic Characterization, and Nucleotide Sequence of a High-Isoelectric-Point alpha -Glucosidase from Barley Malt Plant Physiology, May 1, 2000; 123(1): 275 - 286. [Abstract] [Full Text]
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-Glucosidase in Crucifers