Plant Physiol. (1998) 118: 581-590
Characterization of Starch-Debranching
Enzymes in Pea
Embryos1
Zhi-Ping Zhu,
Christopher M. Hylton,
Ute Rössner2, and
Alison M. Smith*
Shanghai Institute of Plant Physiology, 300 Fonglin Road, Shanghai,
China 200032 (Z.-P.Z.); and John Innes Centre, Colney Lane, Norwich NR4
7UH, United Kingdom (C.M.H., U.R., A.M.S.)
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ABSTRACT |
Two
distinct types of debranching enzymes have been identified in
developing pea (Pisum sativum L.) embryos using native
gel analysis and tests of substrate preference on purified or partially purified activities. An isoamylase-like activity capable of hydrolyzing amylopectin and glycogen but not pullulan is present throughout development and is largely or entirely confined to the plastid. Activities capable of hydrolyzing pullulan are present both inside and
outside of the plastid, and extraplastidial activity increases relative
to the plastidial activity during development. Both types of
debranching enzyme are also present in germinating embryos. We argue
that debranching enzymes are likely to have a role in starch metabolism
in the plastid of the developing embryo and in starch degradation
during germination.
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INTRODUCTION |
The aim of this work was to characterize the starch-debranching
enzymes of pea (Pisum sativum L.) embryos. Two distinct
types of debranching enzymes occur in higher plants. Enzymes that can hydrolyze the 
1,6-linkages in the yeast glucan pullulan and in limit-dextrins of amylopectin are known as limit-dextrinases, R-enzymes, or pullulanases (Nakamura, 1996
). Enzymes that cannot attack
pullulan but can debranch amylopectin and its limit-dextrins are known
as isoamylases. Debranching enzymes are involved in the degradation of
starch in germinating or sprouting starch-storing organs (Manners,
1985), and evidence from mutant cereals and Chlamydomonas reinhardtii indicates strongly that they are also important
in starch synthesis. The maize endosperm mutant sugary1
(su1), the rice endosperm mutant sugary1
(su1), and the sta7 mutant of C. reinhardtii accumulate a highly branched, soluble,
1,4,1,6-linked glucan polymer, phytoglycogen, in addition to or
instead of starch (Mouille et al., 1996; Nakamura, 1996). All three
mutants have reduced activity of debranching enzyme (Pan and Nelson,
1984; Doehlert et al., 1993; Mouille et al., 1996; Nakamura et al., 1996b, 1997), indicating that debranching activity is required for
normal amylopectin synthesis. Ball and colleagues (1996) recently suggested that the synthesis of amylopectin and its organization to
form a starch granule involves the "trimming" by debranching enzyme
of highly branched, phytoglycogen-like material synthesized by starch
synthase and starch-branching enzyme at the surface of the starch
granule.
The precise way in which debranching enzymes are involved in
amylopectin synthesis is difficult to assess because of the lack of
information about the occurrence and nature of these enzymes in
starch-synthesizing organs. First, it is not clear whether a lack of
isoamylase, limit-dextrinase, or both types of enzyme is responsible
for the phenotypes of the su1 and sta7 mutants. The su1 mutants are reported to have reduced activity of
limit-dextrinase, but this enzyme is not encoded at the su1
locus in either maize or rice (James et al., 1995; Nakamura et al.,
1996a). The su1 mutation of maize lies in a gene encoding a
putative isoamylase (James et al., 1995), but the effect of the
su1 mutation on isoamylase activity has not been reported.
The genes at the su1 locus of rice and the sta7
locus of C. reinhardtii have not been identified.
Second, relatively little is known about these enzymes in higher
plants. Limit-dextrinase activity occurs in leaves, embryos, and
endosperms of several species (Nakamura, 1996). There is evidence that
the enzyme in developing rice endosperm is plastidial (Nakamura et al.,
1996a), and both plastidial and extraplastidial activities have been
found in leaves (Okita et al., 1979; Li et al., 1992; Ghiena et al.,
1993), but the location of the activity and the number of different
gene products involved in other organs are not known. Isoamylase
activity has been reported only from maize endosperm, in which it is
plastidial (Doehlert and Knutson, 1991; Yu et al., 1998), and potato
tuber, in which its intracellular location is not known (Ishizaki et
al., 1983). This apparently limited distribution probably reflects the
lack of research on other organs.
To provide more information about the occurrence and possible role of
debranching enzymes in starch synthesis, we studied these enzymes in
developing pea embryos. These are among the best characterized
starch-synthesizing organs, but, to our knowledge, the occurrence of
debranching enzymes has not previously been investigated.
Limit-dextrinase is known to be present in mature seeds of legumes such
as pea (Yellowlees, 1980), broad bean (Gordon et al., 1975), and mung
bean (Morinaga et al., 1997). In this paper we describe the
identification, characterization, and localization of isoforms of
debranching enzyme present during starch synthesis in the developing
pea embryo. These isoforms are then compared with those of the
germinating embryo in which starch is being degraded.
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MATERIALS AND METHODS |
Plant Material
All pea (Pisum sativum L.) plants were of the BC1/RR or
BC1/rr lines derived from JI 430 (John Innes Centre germplasm
collection) by Hedley et al. (1986) and used in our laboratory for
previous work on the characterization of starch synthesis in pea
embryos. The BC1/rr line was used in the preparation of plastids, and
the BC1/RR line was used in all other experiments. For developing embryos, pea plants were grown in a greenhouse in the conditions described by Smith (1988). Seeds were harvested on ice, the testas were removed, and the embryos were used immediately.
For imbibed and germinating embryos, seeds were soaked in running tap
water overnight (imbibition), and were then germinated between
damp paper towels in the dark at 25°C. Testas were removed from dry,
imbibed, and germinating seeds before use in experiments.
Assay of Starch-Debranching Enzyme
Embryos were extracted with a mortar and pestle in approximately 3 volumes of extraction medium (100 mM Mes, pH 6.0, 50 mL L
1 ethanediol, and 5 mM DTT). The
extract was centrifuged at 15,000g for 10 min. The
supernatant was desalted on a column of Sephadex G-25 (Pharmacia)
equilibrated with extraction medium and assayed as follows.
The assay medium for limit-dextrinase (pullulanase, EC 3.2.1.41), for
glycogen-hydrolyzing activity during partial purification of
isoamylase, and for determination of the glucan substrate specificities of purified and partially purified enzymes contained 100 mM
Mes, pH 6.0, 20 g L1 glucan substrate, and
25 to 75 µL of extract in a final volume of 0.1 mL. After incubation
at 37°C for 1.5 to 2 h (during which time activity was linear
with respect to time), reducing sugars were assayed with
dinitrosalicylic acid reagent according to the method of Bernfeld
(1951). The standard curve contained known amounts of maltose and the
same volume of extraction medium as the assays. Control assays were
stopped by the addition of dinitrosalicylic acid reagent immediately
after addition of the extract. Assays using Red Pullulan were carried
out according to the manufacturer's instructions (Megazyme
International, Bray, County Wicklow, Ireland) on extracts derived as
described above.
Assays in which maltoheptaose was the substrate were performed as
described above, except that they contained 18 mM
maltoheptaose instead of glucan. After various periods of incubation
(from 15 to 90 min), assays were stopped by boiling and assayed
spectrophotometrically for Glc according to the method of Lowry and
Passonneau (1972).
Analysis of Products of Enzymic Digestion
When glucan substrates were digested to completion, the assay
contained 100 mM Mes, pH 6.0, and 2 g
L1 glucan substrate. Incubation was for 8 h at 37°C. 
-Limit-dextrin used in these experiments was prepared
from amylopectin according to the method of Enevoldsen and Manners
(1994).
For analysis of the products of digestion of amylopectin, 0.5 mL of
potato amylopectin at 1 mg mL1 was incubated at
pH 4.0 with 5000 units of commercial isoamylase from Pseudomonas
amyloderamosa (Sigma) or at pH 5.4 with either 5 units of
-amylase from pig pancreas (Boehringer Mannheim) or partially
purified isoamylase from pea embryos (25 nmol
min1, determined by assay for
glycogen-hydrolyzing activity, as described above). After incubation at
37°C for 16 h, samples were subjected to high-performance
anion-exchange chromatography (Carbopac PA100 column with pulsed
amperometric detection, Dionex Ltd., Camberley, Surrey, UK), as
described by Tomlinson et al. (1997).
Purification of Pullulanase
All steps were carried out at 0°C to 4°C. About 400 g of
developing embryos (individual embryos of 400-600 mg fresh weight) was
homogenized in a blender in 600 mL of medium A (20 mM
Bis-Tris propane, pH 7.0, and 5 mM DTT). The homogenate was
centrifuged at 10,000g for 15 min, and the supernatant was
subjected to ammonium sulfate fractionation. Protein precipitating
between 30% and 40% saturation was collected by centrifugation,
redissolved in a small volume of medium A, and then dialyzed against
medium A. The dialyzed sample was applied at a flow rate of 0.5 mL
min1 to a column (21 cm high, 2.6 cm internal
diameter) of DEAE-Sepharose Fast Flow (Pharmacia) equilibrated with
medium A. The column was washed with medium A and then eluted at a flow
rate of 2.5 mL min1 with a 250-mL linear
gradient of 0 to 1 M NaCl in medium A.
Five-milliliter fractions were collected and assayed for pullulanase
activity. Fractions with high activity were pooled and dialyzed against
medium B (20 mM Mes, pH 6.0, and 5 mM DTT). The dialyzed sample was applied at a flow rate of 0.25 mL
min1 to a column (10 cm high, 1.5 cm internal
diameter) of cyclohexa-amylose-Sepharose (prepared from epoxy-activated
Sepharose 6B, according to the method of Vretblad [1974])
equilibrated with medium B. The column was washed with medium B, eluted
with 80 mL of 20 mM Hepes, pH 7.9, 0.5 M KCl,
and 5 mM DTT, and 2-mL fractions were collected.
Partial Purification of Isoamylase
All steps were carried out at 0°C to 4°C. About 400 g of
developing embryos (individual embryos of 400-600 mg fresh weight) or
about 250 g of embryos on the 3rd d of germination was homogenized in a blender in 600 mL of medium D (50 mM Mes, pH 6.0, 10 mM calcium acetate, 5 mM DTT, and 50 mL
L1 ethanediol). The homogenate was centrifuged
at 10,000g for 15 min, and the supernatant was adjusted to
pH 5.0 by the addition of 1.2 M acetic acid with stirring.
Precipitated protein was collected by centrifugation, resuspended in 80 to 100 mL of medium D, and then stirred for 16 h. This sample was
applied at a flow rate of 0.5 mL min1 to a
Mono-Q anion-exchange column (Pharmacia) equilibrated with medium D. The column was washed with medium D and eluted with a 60-mL linear
gradient of 0 to 0.6 M NaCl in medium D. One-milliliter fractions were collected, analyzed on native gels, and assayed for the
production of reducing sugars from rabbit-liver glycogen (Sigma). Those
with isoamylase activity were pooled and dialyzed against medium E (50 mM Mops, pH 7.0, 10 mM calcium acetate, 5 mM DTT, and 50 mL L1 ethanediol).
The dialyzed sample was reapplied to a Mono-Q column equilibrated with
medium E. Elution and assay conditions were identical to those used for
the first Mono-Q column except that medium E was used throughout.
Fractions were analyzed on native gels and assayed for the production
of reducing sugars from glycogen.
Determination of pH Optima
The pH optima of purified and partially purified enzymes were
determined by assay at five pH values between 5.5 and 8.0. Buffers were
Mes (pH 5.5-6.6), Mops (pH 6.6-7.2), and Hepes (pH 7.2-8.0).
Preparation of Plastids
Plastid-enriched fractions were prepared from samples of about 40 developing embryos according to the method of Denyer and Smith (1988)
for crude plastid preparations. Embryos were from a line lacking one
isoform of starch-branching enzyme (BC1/rr; Bhattacharyya et al., 1990)
but otherwise isogenic to the line used in other experiments (BC1/RR).
The low starch content of the rr line facilitates the
preparation of plastids. No differences were observed between
developing embryos of the RR and rr lines in the
pattern or intensity of bands on amylopectin- or Red
Pullulan-containing gels. Marker enzymes for plastids (ADP-Glc
pyrophosphorylase, EC 2.7.7.27) and cytosol (alcohol dehydrogenase, EC
1.1.1.1) were assayed according to the method of Denyer and Smith
(1988).
Gel Electrophoresis
SDS-PAGE was performed according to the method of Laemmli (1970)
on 6% or 7.5% acrylamide gels 1.0 or 0.75 mm thick. Native gel
electrophoresis was carried out in the same way except that SDS was
omitted from all solutions. The separating gel contained potato
amylopectin (Sigma) at 2 g L1. After
electrophoresis, native gels were rinsed in medium C (100 mM Mes, pH 6.0, 5 mM DTT, and 50 mL
L1 ethanediol), incubated in this medium for 2 to 4 h at 37°C, and then stained with a solution of 13 mM iodine and 40 mM KI. To inhibit amylase
activity in gels, 0.1 mg mL1 aplanin (BAY
e4609, Bayer AG, Wuppertal, Germany) and 0.1 mg mL1 acarbose were included in the separating
gel and in the incubation medium. The incubation medium also contained
5 mM EDTA. Aplanin and acarbose were kind gifts from Bayer
AG.
Proteins from identical amylopectin gels were transferred by
electroblotting at 36 V for 2 h onto native gels containing Red Pullulan at 10 g L1 according to the
method of Wegrzyn and MacRae (1995). Gels were then incubated in medium
C for 6 to 16 h at 37°C until the bands were visible.
Protein Assay
Protein was assayed with the Bio-Rad Protein Assay Dye Reagent
with a standard curve of BSA.
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RESULTS |
Identification and Localization of Debranching Enzymes
We used native polyacrylamide gels containing amylopectin to
detect debranching enzymes of both types in a nonquantitative manner.
Incubation of gels at an appropriate pH after electrophoresis followed
by staining with iodine solution revealed debranching activities as
blue bands against a brown background. To distinguish debranching
enzymes capable of hydrolyzing pullulan, proteins were transferred by
electroblotting from amylopectin-containing gels onto gels containing
Red Pullulan, on which pullulan-hydrolyzing debranching enzymes appear
as clear bands. We also measured the capacity of embryo extracts to
release reducing sugars (pullulan is not a substrate for other
starch-hydrolyzing enzymes) and dye from Red Pullulan. Assay by release
of reducing sugars is subject to interference by enzymes capable of
hydrolyzing the products of the action of debranching enzyme on
pullulan (maltotriose and maltotetraose). Isoamylase cannot be measured
in crude extracts because its substrates are also substrates for
amylases and/or limit-dextrinase. Because we identified debranching
enzymes according to their ability to use pullulan as a substrate, we
use the term "pullulanase" for isoforms that can use this substrate
and "isoamylase" for isoforms that cannot.
Electrophoresis of crude, soluble extracts of developing pea embryos on
native gels containing amylopectin revealed three main groups of bands
that stained blue with iodine solution (Fig. 1, groups I, II, and III; A and B show
typical results from two separately grown batches of plants). Other
bands visible on these gels were either clear or stained pinkish-brown
or reddish. Group I (usually a single band) and group II bands were
always clearly visible, but group III bands were relatively faint and
not easily distinguished in some preparations. To determine whether
these bands were likely to be pullulanases or isoamylases, proteins were transferred immediately after electrophoresis to native gels containing Red Pullulan, which revealed clear bands in positions corresponding to groups II and III, but no bands corresponding to the
slower-migrating group I (Fig. 1, panels at right). Therefore, it
seemed likely that the group II and group III bands represented pullulanases, whereas the group I band represented isoamylase. On a
fresh-weight basis, group I bands changed little in intensity during
embryo development, but group II and group III bands increased in
intensity (Fig. 1).
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| Figure 1.
Isoforms of debranching enzymes in developing pea
embryos. A and B show results from two independent batches. Crude,
soluble extracts of developing embryos of the fresh weights (in
milligrams) indicated above the lanes were loaded onto duplicate
amylopectin-containing native gels so that each lane contained material
from approximately 2.5 mg fresh weight. After electrophoresis one gel
was incubated at 37°C for 2 h at pH 6.0, and then stained with
iodine solution (left panels), and the other was electroblotted onto a
Red Pullulan-containing native gel that was then incubated at 37°C
for 16 h at pH 6.0 (right panels). The positions of three bands or
groups of bands that stained blue on amylopectin-containing gels are
labeled as I, II, and III.
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To determine whether the putative debranching enzymes were plastidial,
crude homogenates of developing embryos were separated into
plastid-enriched pellets and supernatant fractions by established techniques (Denyer and Smith, 1988). When gels of homogenate, supernatant, and plastid-enriched fractions were loaded so that each
lane contained an equal activity of the plastidial enzyme ADP-Glc
pyrophosphorylase, the putative isoamylase band (group I) and the
faster migrating of the two pullulanase bands in group II were present
at approximately equal intensities in all of the lanes. However, the
slower-migrating pullulanase band in group II and the group III bands
were visible only in supernatant and homogenate lanes and not in lanes
containing plastid-enriched fractions. Figure
2 shows typical results from young
embryos (Fig. 2A) and older embryos (Fig. 2B). The group III bands were
visible only in the latter. These results suggest that most or all of the putative isoamylase and the faster-migrating pullulanase in group
II are plastidial, whereas the slower-migrating pullulanase in group II
and the group III pullulanases are extraplastidial. The results also
indicate that the ratio of extraplastidial to plastidial pullulanase
activity may increase during development. On Red Pullulan-containing
gels the slower-migrating pullulanase in group II and the group III
pullulanases were much more prominent in homogenates of older than
younger embryos, but the faster-migrating pullulanase in group II was
not.
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| Figure 2.
Intracellular localization of isoforms of
debranching enzymes in developing pea embryos. A, Preparation from
young embryos of approximately 200 mg fresh weight. B, Preparation from
older embryos of approximately 400 mg fresh weight. Plastid-enriched
(P) and supernatant (S) fractions were separated by centrifugation from
homogenates (H) of developing embryos. Fractions were loaded onto
duplicate amylopectin-containing gels so that each lane contained the
same activity of the plastidial enzyme ADP-glucose pyrophosphorylase.
After electrophoresis, one gel was incubated and then stained with
iodine (left panels) and the other was electroblotted onto a Red
Pullulan-containing native gel (right panels) (for details, see legend
to Fig. 1). Red Pullulan-containing gels were treated with ethanol
after incubation to enhance the contrast between the bands and the
background. The positions of group I, group II, and, where visible,
group III bands are indicated.
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The intracellular location of pullulanase was further investigated by
assay of the enzyme in homogenate and plastid-enriched fractions of
developing embryos. Total pullulanase activity, measured either by
release of reducing sugars from pullulan or by release of dye from Red
Pullulan, was low in the early stages of embryo development and
increased during development (Table I and
data not shown).
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Table I.
Activity of pullulanase in developing, dry, imbibed,
and germinating embryos
Values are means ± SE of six measurements, each made
on a separate extract of a single seed. Pullulanase activity was
assayed by the reducing-sugar method.
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The low activity in the early stages of development was not
attributable to the presence of inhibitory substances in extracts of
young embryos. In two separate experiments in which old (about 500 mg
fresh weight) and young (100-200 mg fresh weight) embryos were
co-extracted, activity in the mixed extract was 88% and 89%, respectively, of that predicted from separate extractions of the two
kinds of embryo when assayed by the reducing-sugar method, and 81% and
83%, respectively, of that predicted when assayed by the Red-Pullulan
method.
Plastid-enriched fractions were prepared from homogenates of embryos of
about 200 mg fresh weight. The percentage of pullulanase activity in
the plastid-enriched fraction was greater than that of the cytosolic
marker enzyme alcohol dehydrogenase, but considerably less than that of
the plastidial marker enzyme ADP-Glc pyrophosphorylase (Table
II). We estimate from these data that
17% of the pullulanase activity is plastidial in embryos of about 200 mg fresh weight (calculation described by Denyer et al., 1996).
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Table II.
Intracellular localization of pullulanase activity
Activities of pullulanase, the plastidial enzyme ADP-Glc
pyrophosphorylase, and the cytosolic enzyme alcohol dehydrogenase were
measured in a homogenate of young embryos (approximately 200 mg fresh
weight), in a plastic-enriched pellet fraction, and in a supernatant
fraction derived from the homogenate by centrifugation. The activity in
the plastid-enriched fraction is expressed as a percentage of that in
the homogenate. The recovery of activity during the fractionation is
calculated by expressing the sum of the activities in the supernatant
and plastid-enriched fractions as a percentage of that in the
homogenate. The high values indicate that no losses of activity
occurred during the fractionation. Values are means ± SE of measurements made on six separate preparations.
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Purification of Pullulanase from Developing Embryos
Pullulanase was purified to near homogeneity from developing
embryos by ammonium sulfate precipitation, anion-exchange
chromatography, and affinity chromatography on
cyclohexa-amylose-Sepharose. Activity was monitored during the
purification as the production of reducing sugars from pullulan.
Purification was approximately 1500-fold, to a final specific activity
of 33 to 44 µmol min1
mg1 protein (range of values from three
separate purifications). A typical purification is shown in Table
III.
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Table III.
Purification of pullulanase from developing
embryos
Embryos (about 400 g, 400-600 mg fresh weight each) were
homogenized and centrifuged to give an initial supernatant. A fraction
precipitating at between 30% and 40% saturation with ammonium sulfate
was subjected to further chromatography on columns of DEAE-Sepharose
Fast Flow and cyclohexa-amylose (CHA)-Sepharose. Pullulanase activity
was assayed by the reducing-sugar method. Recovery of activity is the
activity remaining after a given step expressed as a percentage of that
in the initial supernatant.
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On SDS-polyacrylamide gels stained with Coomassie brilliant blue
R, the purified preparation usually appeared as two bands of
approximately 100 kD (Fig. 3, lane 2). On
gels containing amylopectin, the purified pullulanase from pea embryos
appeared as a blue-staining band that co-migrated with group II bands
from crude extracts (Fig. 3, lanes 3-5). On gels containing Red
Pullulan, the purified enzyme appeared as a clear band (Fig. 3, lane 6)
that comigrated with the slower-migrating, extraplastidial group II
pullulanase, and not with the faster-migrating, plastidial pullulanase
(Fig. 3, lanes 7-9). In some preparations, bands that comigrated with the group III bands of crude extracts were also faintly visible. The
purified enzyme had a pH optimum of 6.0, hydrolyzed pullulan very
readily, and also hydrolyzed amylopectin; it had very little activity
with glycogen (Table IV).
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| Figure 3.
Purified pullulanase from developing embryos.
Pullulanase activity was purified as described in Table III. Left
panel, SDS-polyacrylamide gel. Middle panel, Amylopectin-containing
native gel; the positions of group I and group II bands are marked.
Right panel, Red Pullulan-containing native gel. Lane 1, Molecular
markers (in kilodaltons); lanes 2, 3, and 6, purified pullulanase;
lanes 4 and 7, plastid-enriched fraction from young embryos (as in Fig.
2); lanes 5 and 8, supernatant fraction from young embryos (as in Fig.
2); and lane 9, mixture of the samples in lanes 6 and 7. Note that on
the Red Pullulan-containing gel the purified pullulanase comigrates
with the upper, extraplastidial group II band in the supernatant
fraction, and not with the plastidial group II band.
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Table IV.
Substrate preferences of pullulanase and isoamylase
Release of reducing sugar by preparations of pullulanase (as in Table
III) and isoamylase (as in Figs. 4 and 7) was measured with
amylopectin, glycogen, and pullulan as substrates. Activity is
expressed as a percentage of that in assays containing amylopectin.
Substrate concentration in all assays was 20 g L1.
For a given enzyme preparation, all assays contained the same amount of
enzyme protein. Activity was measured for 90 min, and was linear with
respect to time during this period. Values are means of duplicate
assays made on single preparations of each enzyme and are typical of
values obtained from several preparations.
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Purification of Isoamylase from Developing Embryos
The putative isoamylase was partially purified from developing
embryos by precipitation at pH 5.0 followed by anion-exchange chromatography. As reported previously for the enzyme from potato (Ishizaki et al., 1983), activity was effectively precipitated at pH
5.0, whereas the activity of other starch-hydrolyzing enzymes was not.
Activity of the putative isoamylase was monitored throughout the
purification by analysis on native gels containing amylopectin. Fractions from the final Mono-Q column gave blue-staining bands that
comigrated with the group I band of crude extracts and contained an
activity that released reducing sugars from glycogen. They gave no
bands on gels containing Red Pullulan (Fig.
4). SDS-polyacrylamide gels revealed that
each of the active fractions contained several proteins (not shown).
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| Figure 4.
Partially purified isoamylase from developing
embryos. Activity was purified by precipitation at pH 5.0 from crude,
soluble extracts of embryos, followed by chromatography on Mono-Q
anion-exchange columns. Top panel, Elution of glycogen-hydrolyzing
activity (micromoles of reducing sugar produced per minute per
fraction) from the final Mono-Q column. NaCl concentration increased
linearly from 0.23 to 0.34 M across the fractions shown.
Middle panel, Amylopectin-containing native gel of unfractionated
extract (left lane) and fractions from the Mono-Q column shown in the
top panel. The lower of the two bands in fractions 23, 24, and 25 stained reddish-brown with iodine solution; all other bands stained
blue. Bottom panel, Red Pullulan-containing native gel of
unfractionated extract (left lane) and of fractions from the Mono-Q
column shown in the top panel.
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Further evidence about the identity of the purified activity was sought
from studies of its substrate preferences. The activity could release
reducing sugars from -limit-dextrin, but to a lesser extent than
pancreatic -amylase. In a typical experiment in which the substrate
was digested to completion, the amount of reducing sugar produced by
the putative isoamylase from -limit-dextrin was 23% of that
produced by -amylase from the same amount of substrate. The putative
isoamylase did not release Glc from maltoheptaose, a linear glucan of
seven glucosyl units from which -amylases can release Glc. Its
activity with glycogen was slightly less than that with amylopectin,
and it had little or no activity on pullulan (Table IV). When allowed
to digest amylopectin to completion, the products of the putative
isoamylase were much more similar to those of bacterial isoamylase than
to those of pancreatic -amylase. Fractionation of the products by
high-performance anion-exchange chromatography with pulsed amperometric
detection revealed that although the putative isoamylase produced more
chains of fewer than six glucosyl units than did the bacterial
isoamylase, both enzymes produced a range of longer chains with a
maximum abundance at about 12 to 15 glucosyl units. In contrast,
pancreatic -amylase produced almost exclusively Glc and chains of
six or fewer glucosyl units in length (Fig.
5). The activity of the putative
isoamylase was maximal at pH 7.0.
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| Figure 5.
Products of hydrolysis of amylopectin by partially
purified isoamylase. A, Amylopectin after digestion with bacterial
isoamylase. B, Amylopectin after digestion with partially purified
isoamylase from pea embryos (25 nmol min1
incubation1 from the final Mono-Q column, as in Fig. 4).
C, Amylopectin incubated under the same conditions as in B, but without
enzyme activity. D, Amylopectin after digestion with pancreatic
-amylase. After digestion of the amylopectin to completion, the
products were fractionated by high-performance anion-exchange
chromatography and visualized with a pulsed amperometric detector. The
system was calibrated with maltooligosaccharides of known degrees of
polymerization. Peaks with degrees of polymerization of 10 (A and B)
and 6 (D) are indicated.
|
|
Pullulanases in Mature and Germinating Embryos
When expressed per embryo, the activity of pullulanase was similar
in embryos in late development, dry embryos, and embryos during the
first 9 d of germination (Table I). The general pattern of
isoforms also differed little between these stages. Red
Pullulan-containing native gels revealed the presence of both group II
and group III bands in dry, imbibed, and germinating embryos (Fig.
6).
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| Figure 6.
Debranching enzymes in developing, dry, imbibed,
and germinating embryos. A, Amylopectin-containing native gel (prepared
as described for Fig. 1) of crude, soluble extracts of embryos. Each
lane contains material from approximately 1/100th of an embryo. Lane 1, Developing embryo of 500 mg fresh weight; lane 2, dry embryo; lane 3, embryo after imbibition; lane 4, embryo after 2 d of germination;
lane 5, embryo after 4 d of germination. Group I and group II
bands are indicated; these are partly or wholly obscured by bands of
amylase activity. B, Amylopectin-containing native gel identical to
that in A, except that -amylase inhibitors were included in the gel
and incubation medium. Group I and group II bands are indicated. The
differences between lanes in the composition of the samples causes
slight differences in the migration of the group I band; this band is
indicated on both sides of the panel and is the upper of the prominent
doublet of bands in all lanes. C, Red Pullulan-containing native gel of
samples electroblotted from a gel identical to that shown in A. Group
II and group III bands are indicated.
|
|
Isoamylase in Mature and Germinating Embryos
The presence of large amounts of slow-migrating,
amylopectin-hydrolyzing activities (probably -amylases) prevented
debranching enzymes from being resolved clearly on
amylopectin-containing gels of germinating embryos. Incorporation into
the incubation mixture of the -amylase inhibitors aplanin, acarbose,
and EDTA strongly reduced the development of these bands (Fig. 6). The inhibitors had no effect on the appearance of the group I band on gels
of developing embryos, showing that they did not inhibit isoamylase. In
gels treated with inhibitors, a blue-staining band that comigrated with
the group I band of developing embryos was visible in extracts of
mature and germinating embryos (Fig. 6).
To provide further information about the identity of the putative
isoamylase in mature, imbibed, and germinating embryos, extracts of
imbibed embryos were subjected to the purification procedure devised
for isoamylase from developing embryos. The final preparation from
imbibed embryos was similar to that from developing embryos. Fractions
from the final Mono-Q column appeared exclusively as
slow-migrating, blue-staining bands on amylopectin-containing gels, and
contained an activity that released reducing sugars from glycogen. No
bands were visible on Red Pullulan-containing gels of these fractions
(data not shown). The substrate preferences of the preparation were
indistinguishable from those of the isoamylase from developing embryos
(Table IV), and the pH optimum for activity was also the same (7.0).
| |
DISCUSSION |
Pullulanases of Developing and Germinating Embryos
Our data show unequivocally that developing pea embryos contain
two distinct isoforms of debranching enzyme capable of hydrolyzing both
amylopectin and pullulan. At least one of these forms is plastidial,
and is present in the embryo from an early stage of development and
probably throughout the period of starch synthesis. Both native gel
analysis and subcellular fractionation experiments indicate that the
plastidial pullulanase contributes a minor fraction of the total
pullulanase activity. The remaining pullulanase activity is
extraplastidial, and appears to increase relative to the plastidial activity as development proceeds. The extraplastidial activity forms
three or more bands on native gels. Purified extraplastidial pullulanase appears as two bands on SDS-polyacrylamide gels, and it is
possible that both of these bands are pullulanases. Purified pullulan-hydrolyzing activity from mature mung beans also migrated as
two bands of approximately 100 kD on SDS-PAGE, and production of
monoclonal antisera revealed that the two bands were immunologically very closely related (Morinaga et al., 1997). However, our results do
not exclude the possibility that products of a single gene account for
all of the extraplastidial activity.
The isoforms of pullulanase present in developing embryos are probably
also responsible for the pullulanase activity in germinating embryos.
The appearance of pullulanase bands on native gels is the same in late
development, in dry seed, after imbibition, and in germination.
Measurement of pullulanase activity by the reducing-sugar method
indicates that there is little difference in activity per embryo
between these developmental stages. However, these results and those
used to quantify the proportion of pullulanase in the plastid must be
treated with some caution. Native gels cannot be regarded as
quantitative, and the assay for pullulanase is susceptible to
interference by enzymes that can hydrolyze maltotriose and
maltotetraose. Pullulan is not an endogenous substrate for plant
enzymes.
The presence of plastidial and extraplastidial isoforms of pullulanase
(limit-dextrinase, R-enzyme) has been reported in leaves of three
species, but not in other plant organs. Cell-fractionation studies
showed that approximately one-half of the activity of spinach (Okita et
al., 1979), sugar beet (Li et al., 1992), and broad bean (Ghiena et
al., 1993) leaves is chloroplastic. Studies of other organs have for
the most part identified only one activity, for example in germinating
barley (Sissons et al., 1992; MacGregor et al., 1994); developing and
germinating rice endosperm (Iwaki and Fuwa, 1981; Nakamura et al.,
1996a); maize endosperm (Doehlert and Knutson, 1991); and broad bean
seeds (Gordon et al., 1975).
In the most detailed study to date, a single form of pullulanase was
purified from developing rice endosperm and the cDNA encoding it was
isolated and sequenced. The cDNA encoded a putative transit peptide,
indicating that the isoform is plastidial. Southern-blot analysis using
the cDNA as a probe suggested that there may be only a single
pullulanase gene in the rice genome (Nakamura et al., 1996a). In maize
three forms of pullulanase were separated by hydroxyapatite
chromatography of the partially purified enzyme from developing
endosperm (Pan and Nelson, 1984). However, the number of different gene
products responsible for these activities and their intracellular
locations were not investigated.
The properties of the extraplastidial pullulanase purified from
developing pea embryos are broadly similar to those of pullulanases from other plant sources. The purified protein(s) is approximately 100 kD, a value very similar to those reported for most of the pullulan-hydrolyzing enzymes characterized so far (Nakamura, 1996). The
pea enzyme hydrolyzes pullulan more readily than amylopectin and
displays little or no detectable activity with glycogen. This order of
substrate preference is the same as that of the enzymes from, for
example, spinach chloroplasts (Okita and Preiss, 1980; Ludwig et al.,
1984), sugar beet leaves (Li et al., 1992), mature broad bean seeds
(Gordon et al., 1975), and developing rice endosperm (Nakamura et al.,
1996a). The properties of our pea enzyme are also very similar
to those of a limit-dextrinase purified by ammonium sulfate and acetone
fractionation and cyclohexa-amylose chromatography from imbibed pea
seeds (Yellowlees, 1980). It seems likely that this limit-dextrinase
and the extraplastidial pullulan-hydrolyzing activity from developing
embryos are the same enzyme.
The Isoamylase of Developing and Germinating Embryos
In addition to pullulanases, native gel analysis shows that
developing pea embryos contain a second type of debranching enzyme that
is capable of hydrolyzing amylopectin but not pullulan. Most or all of
this activity is plastidial and is present during development. The
following characteristics of partially purified preparations of this
enzyme suggest strongly that it is an isoamylase. First, the enzyme
does not release reducing sugars from pullulan or Glc from
maltoheptaose, showing that it is not pullulanase, -amylase, -glucosidase, or disproportionating enzyme. Second, the enzyme liberates reducing sugars from amylopectin, -limit-dextrin, and glycogen, all of which are substrates for isoamylases (Nakamura, 1996;
Manners, 1997). -Limit-dextrin is not a substrate for -amylase. Third, the action of the enzyme on amylopectin produces a pattern of
Glc chains similar to that produced by bacterial isoamylase, and very
different from that produced by -amylase. The enzyme preparation
from pea produced more short chains than did bacterial isoamylase (Fig.
5). Further purification of the enzyme will be required to determine
whether this is a real difference between the two isoamylases, or if it
was caused by contaminating activities in the pea preparation.
Dry, imbibed, and germinating pea seeds contain an isoamylase activity
that is indistinguishable in behavior on native gels, substrate
specificity, and pH optimum from the isoamylase from developing
embryos.
There is only limited information on the properties of isoamylase in
the two plant organs from which it has been reported previously. A
single form of the enzyme purified from potato tuber hydrolyzed
glycogen and amylopectin at an almost equal rate (Ishizaki et al.,
1983). One of two forms of the enzyme separated by anion-exchange chromatography from developing maize kernels hydrolyzed amylopectin about four times faster than phytoglycogen, and the other form was not
characterized (Doehlert and Knutson, 1991). Activity of isoamylase in
developing maize endosperm appears to be mainly or exclusively
plastidial (Yu et al., 1998).
Possible Roles of Debranching Enzymes in Starch Metabolism in the
Pea Embryo
The most commonly described role for starch-debranching enzymes is
in the degradation of starch. Limit-dextrinases are so called because
their substrate in vivo is held to be limit-dextrins generated during
hydrolytic starch degradation (Manners, 1997). Their presence in the
acellular endosperm of germinating cereals is certainly consistent with
such a role, and we assume that debranching enzymes also participate in
starch degradation in the cells of the germinating pea embryo. It has
been proposed on the basis of electron micrographs that the plastid
membranes surrounding the large starch granules break down during the
germination of legume embryos (Bain and Mercer, 1966; Harris, 1976),
which would allow cytosolic rather than plastidial isoforms of
debranching enzyme to participate in starch degradation. The
extraplastidial pullulanase may therefore be responsible for hydrolysis
of the 1,6-linkages of starch during germination.
A role for the plastidial isoform of pullulanase and for isoamylase in
the germinating embryo remains to be discovered. A situation that may
be similar to that of debranching enzymes has been described for
isoforms of starch phosphorylase in the germinating pea embryo. An
isoform of phosphorylase revealed by immunofluorescence microscopy to
be cytosolic contributes most of the activity of phosphorylase, and a
plastidial isoform is confined to the small plastids, where it
presumably cannot participate in starch degradation (Steup, 1988; van
Berkel et al., 1991).
The role of debranching enzymes in the developing embryo is unclear. It
might be argued that the extraplastidial pullulanase is required for
starch degradation during germination, and that its presence during
development of seeds simply represents accumulation in advance of this
role. The fact that limit-dextrinases of some developing and mature
cereal endosperms are reported to be present in a "latent" form,
requiring reduction to achieve full activity (Yamada, 1981; Toguri,
1991; Sissons et al., 1993; MacGregor et al., 1994), lends support to
this view. However, the presence of plastidial isoamylase and
pullulanase from an early stage of development of the pea embryo
suggests to us that these enzymes are likely to have a role in starch
metabolism during its synthesis in the developing embryo.
The demonstration that both pullulanase and isoamylase occur in the
plastids of developing pea embryos as well as in potato tubers and
maize endosperm, and the dramatic effect of mutations affecting these
enzymes on starch synthesis in organisms as distantly related as
C. reinhardtii and cereals, are consistent with a
fundamental role for debranching enzymes in starch synthesis. The
precise roles of the two kinds of enzyme remain to be discovered.
| |
FOOTNOTES |
1
This research was supported by a competitive
strategic grant from the Biotechnology and Biological Sciences Research
Council (UK) to the John Innes Centre, by an International
Collaboration Project funded by the European Union (contract no.
CI1*/0417/00), by funding from the Chinese National Natural Foundation
of Science to Z.-P.Z., and by an Erasmus Award (European Union) to
U.R.
2
Present address: Max-Planck-Institut für
Molekulare Pflanzenphysiologie, Karl-Liebknecht Strasse 25, 14476 Golm,
Germany.
*
Corresponding author; e-mail smitha{at}bbsrc.ac.uk; fax
44-1603-456844.
Received March 20, 1998;
accepted June 26, 1998.
| |
ACKNOWLEDGMENTS |
We are grateful to Sam Zeeman, Takayuki Umemoto, Cathie Martin,
Rod Casey, Kay Denyer, and Professor David Manners for their helpful
comments during the course of this work and about the manuscript, and
to Cliff Hedley and Trevor Wang for the gift of pea seeds.
| |
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Abstract
Introduction
