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Plant Physiol. (1998) 118: 451-459
Characterization of a Granule-Bound Starch Synthase Isoform Found
in the Pericarp of Wheat1
Toshiki Nakamura*,
Patricia Vrinten,
Kazuhiro Hayakawa, and
Junichi Ikeda
Tohoku National Agriculture Experimental Station, Akahira 4, Morioka 020-01, Japan (T.N., P.V., J.I.); and Nisshin Flour Milling
Co., Ohimachi, Iruma, Saitama 356, Japan (K.H.)
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ABSTRACT |
Waxy wheat (Triticum
aestivum L.) lacks the waxy protein, which is also known as
granule-bound starch synthase I (GBSSI). The starch granules of waxy
wheat endosperm and pollen do not contain amylose and therefore stain
red-brown with iodine. However, we observed that starch from pericarp
tissue of waxy wheat stained blue-black and contained amylose.
Significantly higher starch synthase activity was detected in pericarp
starch granules than in endosperm starch granules. A granule-bound
protein that differed from GBSSI in molecular mass and isoelectric
point was detected in the pericarp starch granules but not in granules
from endosperm. This protein was designated GBSSII. The N-terminal
amino acid sequence of GBSSII, although not identical to wheat GBSSI,
showed strong homology to waxy proteins or GBSSIs of cereals and
potato, and contained the motif KTGGL, which is the putative
substrate-binding site of GBSSI of plants and of glycogen synthase of
Escherichia coli. GBSSII cross-reacted specifically with
antisera raised against potato and maize GBSSI. This study indicates
that GBSSI and GBSSII are expressed in a tissue-specific manner in
different organs, with GBSSII having an important function in amylose
synthesis in the pericarp.
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INTRODUCTION |
Starch is composed of two distinct Glc polymers, amylose and
amylopectin. Amylose consists of linear molecules of (1-4)-linked  -D-glucopyranosyl units, whereas amylopectin is made up
of highly branched molecules of -D-glucopyranosyl units
linked primarily by (1-4) bonds with branches resulting from (1-6)
linkages (Whistler and Daniel, 1984 ). The two polymers differ in their
ability to form complexes with fatty acids,
low-Mr alcohols, and iodine: amylose easily
forms complexes with these compounds, whereas amylopectin does not
(Banks and Muir, 1980).
These differences in complex formation with iodine have been useful in
determining amylose content in starch (Hollo and Szeitli, 1968), as
well as in distinguishing waxy (or glutinous) mutants from the wild
type (Hixon and Brimhall, 1968). Starch of non-waxy lines, which
contains amylose, forms blue-black complexes with iodine; starch of
waxy mutants, which lacks amylose, stains red-brown. Waxy mutants have
been identified in maize, rice, barley, sorghum (Eriksson, 1963), and
amaranth (Okuno and Sakaguchi, 1982). Recently, amylose-free
(amf) mutants of potato (Hovenkamp-Hermelink et al., 1987)
and waxy wheats (Nakamura et al., 1995) have been produced. In waxy and
amf mutants, the absence of waxy protein was coincidental with a lack of amylose in storage starch. The waxy protein was thus
shown to be GBSSI, and is considered to be the only SS involved in
amylose synthesis in storage organs (Preiss, 1991; Smith and Martin,
1993).
Starch is deposited not only in storage organs but in other tissues as
well (Badenhuizen, 1969; Greenwood, 1970), and it appears that GBSSI
plays little or no role in amylose synthesis in nonstorage tissues. In
a review of starch formation in the vegetative organs of rice, starches
were classified as either permanent (storage) or temporary (Sato,
1984). Temporary starch was further divided into assimilation starch,
located in the chloroplast; waiting starch, located in the young cells
near meristems; and transitory starch, located in the parenchyma cells.
All temporary starches in waxy mutants stained blue-black with iodine.
Amylose contents of leaf and stem tissues of waxy rice were around 25%
to 35%, comparable to levels in nonwaxy types (Igaue, 1964),
suggesting the existence of a GBSS isoform(s) other than GBSSI.
We also observed blue-black-staining starch in the pericarp tissue of
immature waxy wheat (Triticum aestivum), although starch in
the endosperm and pollen stained red-brown, indicating that starch
synthesis, in particular that of amylose, differs between pericarp and
endosperm tissues. Wheat pericarp tissue is a good material with which
to study the mechanism of starch synthesis in nonstorage organs,
because young pericarp tissue has a higher starch content than leaf or
stem tissue, and therefore pure starch is easier to obtain. We present
the characterization of a GBSS isoform, designated GBSSII, which is
found in the starch granules of wheat pericarp.
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MATERIALS AND METHODS |
Plant Material
The common wheat (Triticum aestivum L.) cvs Morikei
CD-1479 (CD-1479) and Chinese Spring (CS) were planted in a greenhouse under 22°C (day)/15°C (night) conditions. CD-1479 is a
double-haploid cultivar of waxy wheat (Hoshino et al., 1996), and CS is
a nonwaxy wheat cultivar. CS has three waxy proteins, whereas CD-1479
lacks all waxy proteins from endosperm starch.
Immature seeds were collected from spikes at 5-d intervals until 35 DPA. The immature seeds were frozen in liquid nitrogen and stored at
 70°C until starch granules were extracted.
Preparation of Starch Granules
Pericarp and endosperm were dissected from immature seeds.
Endosperm tissue could not be observed by a light microscope at 5 DPA,
so at this stage only ovaries were removed from the seeds. Separated
pericarp and endosperm were briefly washed three times with an excess
amount of 0.25 M Tris-HCl, pH 7.5. The tissues were
homogenized with a mortar and pestle in three times their fresh weight
of SDS buffer containing 0.1 M Tris-HCl, pH 7.5, 3% (w/v)
SDS, 10% (v/v) glycerol, and 0.05% (v/v) 2-mercaptoethanol. The
homogenates were centrifuged at 15,000g for 5 min. The
supernatant was stored at 20°C until it was used as the soluble
fraction in immunoblot analysis. The pellet was resuspended in SDS
buffer and left overnight. The suspension was then filtered through a 40-µm nylon mesh and centrifuged at 15,000g for 2 min. The
pellet was washed twice with SDS buffer, three times with deionized
distilled water, and three times with cold acetone. The pellet was then vacuum dried and stored at 20°C.
Starch Staining and Microscopic Analysis
Cross-sections of immature seeds and starch granules were stained
with an iodine solution (0.74 g of resublimated iodine and 1.48 g
of KI dissolved in 400 mL of distilled water) and observed under a
light microscope. For scanning electron microscopy, starch granules
were mounted on aluminum stubs with double-stick tape and then coated
with platinum using an ion coater (Eiko Engineering Co., Ibaragi,
Japan). Examination was conducted with an electron microscope (model
S-2700, Hitachi, Tokyo, Japan) at an accelerating potential of 10 kV.
Amylose Content
Amylose content was measured by the method described by Yamamori
et al. (1992). Starch granules (20 mg) were suspended in 5 mL of 0.75 N NaOH solution with 25% (v/v) ethanol, and left at room
temperature for 12 h. The sample was made up to 50 mL with
distilled water, and amylose content was determined with an analyzer
(Tecnicon, Bran-Lubbe Co., Tokyo, Japan) using KI-I2 solution (0.005% [w/v] KI and 0.003% [w/v] I2).
Potato amylose and amylopectin (Sigma) were used as standards for
regression curves.
The  max of the iodine-starch complexes was determined
according to the method of Konishi et al. (1985).
SDS-PAGE and Two-Dimensional PAGE
Starch granule-bound proteins were separated by SDS-PAGE using
10% polyacrylamide gels (Laemmli, 1970). To extract starch granule-bound proteins the purified starch was suspended in SDS buffer
and heated in boiling water. The starch solution was cooled on ice and
centrifuged at 15,000g for 10 min, and the supernatant was
subjected to SDS-PAGE. The soluble fractions were also heated in
boiling water for 5 min and cooled on ice prior to SDS-PAGE. After
electrophoresis, proteins were visualized using a silver-staining kit
(Wako Chemicals, Osaka, Japan).
Starch granule-bound proteins were analyzed by two-dimensional PAGE
according to the method of Nakamura et al. (1993). Starch granules were
suspended in lysis buffer (8 M urea, 2% [v/v]
Nonidet-P40, 2% ampholine, pH 3.5-10, 5% [v/v] 2-mercaptoethanol,
and 5% [w/v] PVP), and the suspension was heated, cooled on ice, and
centrifuged at 15,000g for 10 min. The supernatant was
subjected to IEF for 14 h at 400 V. The focused gel was applied to
a 15% low-Bis acrylamide gel to conduct SDS-PAGE (Kagawa et al.,
1988).
Starch Synthase Activity
To measure starch synthase activity, 500 mg of 5-DPA immature
seeds and 500 mg of endosperm tissues from 20-DPA seeds were homogenized in a mortar and pestle with 3 mL of ice-cold buffer (50 mM Hepes-NaOH, pH 7.5, 5 mM MgCl2,
2 mM DTT, and 1 mg/mL BSA). The homogenate was centrifuged
at 15,000g for 10 min at 4°C, and the supernatant was
assayed for soluble enzyme activity. The pellet was resuspended in
buffer and filtered through a 40-µm nylon mesh. The suspension was
left at 4°C for 15 min to allow the starch granules to settle, and
then the supernatant was removed. The starch granules were washed by
two cycles of resuspension and centrifugation with buffer and one cycle
with ice-cold acetone. The starch granules were air dried at 4°C and
stored at 20°C.
Starch synthase activities of the granule-bound and soluble fractions
were assayed by the incorporation of [14C]ADP-Glc
according to the method of Singletary et al. (1997), with minor
modifications. For measurement of GBSS activity, 1 mg of starch
granules was incubated in 200 µL of reaction buffer (100 mM Bicine, pH 8.3, 25 mM KCl, 10 mM
GSH, 4.5 mM EDTA, 3.5 mM
[14C]ADP-Glc [120 dpm/nmol]) at 25°C with continuous
gentle shaking. After 1 h the reaction was terminated with
methanol (70%, v/v) containing 1% KCl (Vos-Scheperkeuter et al.,
1986) and incubated on ice for 10 min.
Soluble starch synthase activity was measured using a primer-dependent
assay with 1 mg of rabbit-liver glycogen and 50 µL of soluble extract
in 200 µL of the above reaction buffer containing 1 mM
[14C]ADP-Glc (222 dpm/nmol). The sample was incubated for
30 min at 25°C, and the reaction was stopped by adding 100 µL of
0.2 N NaOH. After adding 1 mL of ice-cold ethanol, the
reaction was incubated on ice for 10 min. In both assays the control
reaction was stopped immediately after the addition of labeled
ADP-Glc.
After incubation on ice, reactions in both assays were centrifuged at
15,000g for 10 min and supernatants were removed. Pellets were washed twice by suspension and ethanol precipitation. The pellets
were then resuspended in 500 µL of 1 N HCl, boiled for 5 min, cooled, and mixed with 7 mL of scintillation cocktail. Radioactivity was measured with a liquid-scintillation counter (Aloka
Co. Ltd., Tokyo).
Protein Sequencing
After SDS-PAGE, proteins were transferred from gels onto a PVDF
membrane by electroblotting (Hirano and Watanabe, 1990), and stained
with Coomassie brilliant blue R. The band of interest was excised and
applied to a gas-phase protein sequencer (Perkin-Elmer-ABI). Comparison
of the amino acid sequence with other sequences deposited in the
database was performed using the Basic Local Alignment Search Tool
(BLAST).
Immunoblot Analysis
Proteins from starch granules and soluble fractions were separated
by SDS-PAGE using a 10% acrylamide gel and electroblotted onto PVDF
membrane. Blots were incubated in TBST (20 mM Tris-HCl, pH
7.5, 150 mM NaCl, and 0.05% [v/v] Tween 20) solution
containing 1% (w/v) BSA for 60 min, and then incubated for 60 min with
anti-GBSSI rabbit serum diluted with TBST solution. After a 60-min
incubation with rabbit anti-IgG alkaline phosphatase conjugate
(Promega), immunoreactive bands were detected with Western Blue
stabilized substrate for alkaline phosphatase (Promega).
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RESULTS |
PSGs
The pericarp is the wall of the mature ovary and surrounds the
entire seed (Sakri and Shannon, 1975). Investigations of wheat pericarp
morphology showed that blue-black-staining starch granules appear in
this tissue at about the time of anthesis (Eckerson, 1917; Sandstedt,
1946; Jenkins et al., 1975). We observed a thick pericarp layer with an
abundance of starch at 5 DPA (Fig. 1), although endosperm tissue was not yet detectable at this stage. Starch
granules were still present in pericarp tissue at 20 DPA, at which time
starch had accumulated in the developing endosperm (Fig. 1). The
gradual decrease in pericarp starch during endosperm development has
been observed previously in both wheat (Sandstedt, 1946; Chevalier and
Lingle, 1983) and rice (Sato, 1984). This starch may be stored
temporarily in the pericarp and later converted to sugar and
transferred to the endosperm as a substrate for starch synthesis
(Chevalier and Lingle, 1983).
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| Figure 1.
Iodine staining of wheat starches. Samples were
viewed under a light microscope. A, Cross-sections of immature waxy
wheat seeds harvested at 5 DPA (a) and 20 DPA (b). B, Starch granules
isolated from waxy pericarp (a), waxy endosperm (b), nonwaxy pericarp
(c), and nonwaxy endosperm (d).
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PSGs and ESGs in waxy wheat differed in starch composition and granule
morphology. PSGs of waxy wheat stained blue-black, indicating the
presence of amylose, whereas ESGs stained red-brown (Fig. 1). ESGs
consisted of large (A-type) and small (B-type) granules (Figs. 1 and
2), as previously observed by Parker
(1985). However, PSGs were relatively uniform in size, and slightly
smaller in diameter than B-type granules. PSGs were round and flat, and their average diameter was around 3 µm (Fig. 2).
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| Figure 2.
Scanning electron micrographs of PSGs and ESGs.
Starch granules were extracted from waxy wheat pericarp (A), waxy wheat
endosperm (B), nonwaxy wheat pericarp (C), and nonwaxy wheat endosperm
(D). The ruled area below the micrographs represents 12.0 µm.
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The max of starch-iodine complexes of ESGs from waxy and
nonwaxy wheat were 538 and 595 nm, respectively. This difference was
due to the absence of amylose in starch of waxy wheat. In contrast to
ESGs, max of PSGs of both waxy and nonwaxy wheat was
around 600 nm, indicating that both starches contained amylose. In waxy
wheat the amylose content of PSGs was 18%, whereas that of ESGs was
less than 1% (Table I). However, the
amylose content of PSGs from both waxy and nonwaxy wheat was lower than
that of nonwaxy ESGs.
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Table I.
Amylose content of starch granules and maximum
absorbance of iodine-starch complexes
For measurement of the max of starch-iodine complexes,
starch granules were dissolved in 1 N NaOH and neutralized
with acetic acid. Absorbances were examined using 1 mg of starch
granules with 0.2 mL of iodine solution in a total volume of 25 mL.
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Electrophoresis of Starch Granule-Bound Proteins
Starch granules from 10-DPA whole, immature seeds of nonwaxy wheat
contained two major proteins with relative molecular masses of 61 and
59 kD (Fig. 3). The 61-kD protein was
waxy protein (Yamamori et al., 1992), and was therefore not detected in
ESGs of waxy wheat. The 59-kD protein was detected in PSGs of waxy and
nonwaxy wheat (Fig. 3). Furthermore, the 59-kD protein seemed to bind tightly to the starch granules, because it remained in or on them after
washing with SDS solution, and was only released after heat-induced swelling occurred. The same characteristics have been reported for the
waxy protein of maize (Echt and Schwartz, 1981; Iman, 1989).
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| Figure 3.
SDS-PAGE patterns of starch granule-bound
proteins. ESGs (E) and PSGs (P) tissues were extracted at 5-d intervals
starting at 5 DPA. Starch granule-bound proteins were released by
heating 5 mg of starch granules in 80 µL of SDS buffer at 100°C for
5 min, cooling on ice for 10 min, and centrifuging at
15,000g. Equal volumes (20 µL) of the supernatant
containing starch granule-bound proteins were loaded in each lane.
Electrophoresis was conducted at room temperature. CS10DPA*, Starch
granules extracted from 10-DPA whole, immature seeds of CS wheat.
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The two-dimensional-PAGE analysis showed that GBSSI (waxy protein) and
GBSSII differ in pI range and in molecular mass (Fig. 4). The two-dimensional PAGE pattern of
GBSSII (59 kD) seemed to be composed of three overlapping proteins,
similar to GBSSI (61 kD), which separates into three proteins encoded
by three homologous waxy genes, Wx-A1, Wx-B1, and
Wx-D1 (Nakamura et al., 1993).
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| Figure 4.
Two-dimensional-PAGE analysis of pericarp starch
granule-bound proteins. Equal amounts (10 mg) of PSGs from waxy wheat
and nonwaxy wheat were mixed in lysis buffer and heated in boiling
water for 2 min. The solution was cooled on ice for 10 min and
centrifuged at 15,000g for 10 min. The supernatant (500 µL) was applied to an isofocusing gel in a 20-cm glass capillary with
a 5-mm i.d. The focused gel was equilibrated in sample buffer (0.06 M Tris-HCl, pH 6.8, 10% [v/v] glycerol, 2.5% [w/v]
SDS, and 5% [v/v]  -mercaptoethanol) for 30 min, and then
subjected to SDS-PAGE using a 15% low-Bis acrylamide gel.
Electrophoresis was conducted at 4°C, and the gel was stained with
Coomassie brilliant blue R. The pH range for the first dimension (top)
and the molecular mass range (kD) for the second dimension (left) are
indicated.
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Three minor SGPs of approximately 80, 92, and 108 kD were found in both
pericarp and endosperm starch by SDS-PAGE (Fig. 3). The relative
amounts of these proteins, particularly of the 92-kD protein, were
lower in pericarp than in endosperm. These SGPs have been referred to
as SGP1 (108 kD), SGP2 (92 kD), and SGP3 (80 kD) (Yamamori and Endo,
1996). SGP1 and SGP3 are soluble starch synthase, and SGP2 is a
branching enzyme (Takaoka et al., 1997).
Starch Synthase Activity
Starch synthase activity of waxy PSGs was similar to that of
nonwaxy PSGs, although there was a significant difference between waxy
and nonwaxy ESGs (Table II). Both waxy
and nonwaxy PSGs had starch synthase activities 12-fold higher than
waxy ESGs. Starch synthase activities of PSGs were approximately
1.5-fold higher than that of nonwaxy ESGs. However, this does not
necessarily indicate that GBSS activity is higher in pericarp than in
endosperm tissue in nonwaxy wheat, since PSGs were extracted from 5-DPA seeds and ESGs were extracted at 20 DPA. Starch sythase activities of
the soluble fractions of waxy and nonwaxy pericarp tissue were also
similar.
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Table II.
Starch synthase activity of pericarp and endosperm
tissues
Values represent the means ± SE of three
replications, and are based on milligrams of starch granules for the
granule-bound activity and on milligrams of fresh tissue weight for
soluble activity.
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N-Terminal Amino Acid Sequence
To further investigate the similarities between GBSSII and
GBSSI, the N-terminal sequence was determined from SDS-PAGE blots of
GBSSII by a gas-phase sequencer. A 35-amino acid sequence was obtained
and a homology search was conducted. The GBSSII sequence showed
homology to GBSSIs or waxy proteins of potato, maize, sorghum, barley, rice, and wheat, and to the glycogen synthase of
Escherichia coli (Fig. 5). The
homology started at the N-terminal end of the mature proteins, which is
the cleavage site of the transit peptides. The 14th to 18th residues
(Lys-Thr-Gly-Gly-Leu) constitute a conserved motif found in GBSSI and
in soluble starch synthases (Ainsworth et al., 1993; Baba et al.,
1993). The motif is thought to be the binding site for the substrate
ADP-Glc (Furukawa et al., 1990).
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| Figure 5.
Comparison of the N-terminal sequence of wheat
GBSSII with the deduced amino acid sequences of potato (Po) (van der
Leij et al., 1991), maize (Mz) (Klosgen et al., 1986), and wheat (Wh)
(Ainsworth et al., 1993) GBSSI or waxy cDNAs.
Open boxes denote residues with identity to the GBSSI sequence and
filled boxes denote the conserved motif KTGGL. The putative cleavage
site of the transit peptides is indicated by an arrowhead. X,
Undetermined residues.
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Immunoblot Analysis of GBSSII
Antibodies against potato and maize GBSSI were used in immunoblot
analyses. Wheat GBSSII and GBSSI cross-reacted with both maize and
potato GBSSI antiserum (Fig. 6). Although
GBSSII reacted strongly with potato GBSSI antibody, a sharp band was
not obtained with the maize antibody. The structure of the epitope(s)
of maize GBSSI may be similar but not identical to that of wheat
GBSSII. Alternatively, the weaker reactivity of the maize antibody may have been due to a titer difference between the potato and maize antibodies used in this experiment. The potato antiserum reacted with
GBSSI after a 1:2000 dilution, whereas the maize antiserum could only
be diluted 100-fold. The western analysis also showed that GBSSII was
not associated with endosperm starch of waxy or nonwaxy wheat (Fig. 6).
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| Figure 6.
SDS-PAGE of starch granule-bound proteins (A) and
SDS-PAGE immunoblots using anti-maize waxy protein antibody (B) and
anti-potato GBSSI antibody (C). To extract SGPs, 5 mg of ESGs from waxy
wheat (lanes 1), PSGs from waxy wheat (lanes 2), and ESGs from nonwaxy
wheat (lanes 3) were heated in 80 µL of SDS buffer. For SDS-PAGE, 20 mL of the supernatant was applied to each track of 10% polyacrylamide
gels. Gels were stained with a silver-staining kit (A) or
electroblotted onto PVDF membranes (B and C). Blotted membranes were
incubated with antiserum to maize waxy protein at a dilution of 1:100
and to potato GBSSI at a dilution of 1:2000. For secondary antibody
binding, the membranes were incubated with anti-rabbit IgG alkaline
phosphatase conjugate (1:5000 dilution). Sites of antigen localization
were visualized by a substrate solution for alkaline phosphatase
containing nitroblue tetrazolium and
5-bromo-4-chloro-3-indolyl-phosphate.
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Due to the large number of proteins in the soluble fraction, it was not
possible to determine if GBSSII was present in this fraction by
staining of polyacrylamide gels. For this reason, soluble fractions
from pericarp and endosperm tissues were subjected to immunoblot
analysis. Potato GBSSI antibody did not cross-react with blots of the
soluble fraction from pericarp tissue (Fig. 7), even when four times the amount of
soluble fraction was blotted (data not shown), nor was GBSSII detected
in the soluble fraction from endosperm of waxy wheat. These results
indicate that GBSSII is specific to the granule-bound fraction from
pericarp tissue, as is GBSSI to the granule-bound fraction from
endosperm.
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| Figure 7.
Immunoblots of granule-bound and soluble fractions
of waxy wheat pericarp and endosperm. Lane 1, Granule-bound fraction of
pericarp; lane 2, soluble fraction of pericarp; lane 3, granule-bound
fraction of endosperm; lane 4, soluble fraction of endosperm. To
prepare granule-bound fractions, 5 mg of starch granules was heated in
80 µL of SDS buffer. The soluble fractions, prepared as described in
"Materials and Methods," were also heated before loading. The
granule-bound and soluble fractions (20 µL of each) were applied to a
10% polyacrylamide gel for SDS-PAGE. Separated proteins were
electroblotted onto PVDF membrane, and blots were probed with antiserum
to potato GBSSI diluted 1:2000.
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DISCUSSION |
The basic process of starch synthesis is similar in all plant
tissues (Badenhuizen, 1969; Shannon and Garwood, 1984; Preiss, 1988)
and consists of three main events in the chloroplast and amyloplast
(Muller-Rober and Kossmann, 1994): (a) the supply of Glc-1-P into the
plastid; (b) the synthesis of ADP-Glc from Glc-1-P; and (c) the
synthesis of starch (amylose and amylopectin) from ADP-Glc. However,
starch characteristics, especially granule shape and
amylose:amylopectin ratios, differ among tissues in the same plant
(Sato, 1984; Tomlinson et al., 1997). The third step in starch
synthesis, the formation of starch from ADP-Glc, must therefore involve
different enzyme isoforms and/or processes in different tissues.
Wheat pericarp tissue accumulates starch granules that are different in
appearance (Fig. 1) and in the amylose:amylopectin ratio (Table I) from
ESGs. The differences between the two tissues were particularly obvious
in waxy wheat, since waxy PSGs contained amylose and ESGs had a
negligibly low amount (Fig. 1; Table I). The presence of amylose in
PSGs was confirmed by a high-performance size-exclusion chromatography
analysis, in which PSGs showed an amylose molecule peak that could not
be detected in ESGs of waxy wheat (data not shown).
The presence of amylose in the PSGs of waxy wheat implied the existence
of either GBSSI or a GBSSI isoform in the pericarp tissue since this
enzyme is required for amylose synthesis from ADP-Glc (for review, see
Nelson and Pan, 1995). If GBSSI produced amylose in pericarp tissue, a
61-kD protein would be present in PSGs, and no such protein was found
in PSGs from waxy or nonwaxy wheats (Fig. 3). This result is consistent
with the endosperm- and pollen-specific expression of the
waxy genes encoding GBSSI as reported in wheat
(Ainsworth et al., 1993) and other cereals (Klosgen et al., 1986;
Hirano and Sano, 1991; Baba et al., 1993). However, another
granule-bound protein, GBSSII, which had characteristics similar to
those reported for GBSSI (or waxy protein) in maize (Echt and Schwartz,
1981), rice (Sano, 1984), wheat (Yamamori et al., 1992), potato
(Vos-Scheperkeuter et al., 1986), and pea (Smith, 1990), was detected
in pericarp tissue. GBSSII was strongly bound to the starch granules,
could not be detected in the soluble fraction, and its relative
molecular mass was close to those reported for GBSSIs. Three
observations strongly suggest that this new protein is a GBSS: (a)
significant SS activity was detected in PSGs; (b) the N-terminal
sequence of GBSSII showed that this protein was not a wheat GBSSI or a
GBSSI degradation product, yet it had homology to waxy proteins and
GBSSIs (Fig. 5); and (c) GBSSII was antigenically related to GBSSI of
potato and maize (Fig. 6).
Starch syntase isoforms referred to as GBSSII have been reported in pea
(Dry et al., 1992) and potato (Edwards et al., 1995). However, these
enzymes, which are likely to be involved in amylopectin synthesis, are
present in both the soluble and granule-bound phases, and probably
become entrapped in the granules during starch formation (Martin and
Smith, 1995). Recently, GBSSII from pea and potato has been referred to
simply as starch synthase II (SSII) (Smith et al., 1997). Therefore, we
feel that the designation "GBSS" should be reserved for starch
synthase isoforms that are largely limited to and active in the
granule-bound fraction and are involved in the synthesis of amylose.
GBSSII of wheat fits all of these criteria.
Although the presence of additional GBSS isoforms has not been reported
in other cereals, several studies present evidence that such isoforms
occur. In waxy maize, starch in the pollen, embryo sac, and endosperm
lacks amylose, but starch in other tissues, including pericarp, stains
blue-black (Hixon and Brimhall, 1968; Badenhuizen, 1969). Two rice waxy
mutants that produced inactive waxy proteins in endosperm starch had
blue-black-staining starch in leaf tissue, clearly indicating that
separate waxy genes are active in these two tissues (Sano,
1985). In barley, Ono and Suzuki (1957) reported that
reddish-brown-staining starch was detected not only in endosperm and
pollen, but also in leaves of several waxy cultivars. However,
re-examination of the same waxy barley lines showed only blue-staining
starch in leaves (N. Ishikawa, personal communication). The reason for
this discrepancy is unclear but it should be noted that the waxy barley
lines used in these experiments were not complete waxy mutants, since
they contained 2% to 10% amylose and detectable levels of waxy
protein in ESGs (Ishikawa et al., 1994). Recently, a barley waxy mutant
lacking both amylose and waxy protein in the endosperm starch was
produced (Ishikawa et al., 1994), and blue-black-staining starch was
found in the young pericarp tissue (T. Nakamura, P. Vrinten, K. Hayakawa, and J. Ikeda, unpublished data). These results provide
evidence that there is more than one gene encoding GBSS isoforms in
monocot plants and that the expression of these genes is regulated in a
tissue-specific manner.
In dicot plants the situation may be somewhat more variable. For
example, low-amylose (lam) mutants of pea (Denyer et al., 1995) had a major SGP in starch from leaf (Tomlinson et al., 1998) and
pod (Denyer et al., 1997) tissues. This protein was reported to be a
GBSSI isoform (Denyer et al., 1997; Tomlinson et al., 1998), suggesting
that pea also has more than one gene encoding GBSS. In contrast, a
single GBSS may be responsible for the synthesis of amylose in potato.
An amf mutant (Vos-Scheperkeuter et al., 1987) that carries
a point deletion in the GBSS gene (van der Leij et al.,
1991) displayed red-brown-staining starch not only in the tuber but
also in the roots, leaves, and pollen (Jacobsen et al., 1989).
The level of homology between the waxy (GBSSI) and
GBSSII genes is apparently not high enough to allow
cross-hybridization. When a waxy cDNA was used as a probe in
Southern hybridizations, no extra genomic fragments with strong
homology to the waxy gene were identified (T. Nakamura, P. Vriten, K. Hayakawa, and J. Ikeda, unpublished data). Similar
experiments in other cereals have also identified only a single gene
with homology to waxy cDNA (Shure et al., 1983; Hirano and
Sano, 1991). Cloning of the gene for GBSSII will provide more detail
about the differences between GBSSI and GBSSII, and will increase our
understanding of amylose synthesis in plants.
| |
FOOTNOTES |
1
This research was supported by the Ministry of
Agriculture, Forestry, and Fisheries and the Science Technology Agency
of Japan.
*
Corresponding author; e-mail tnaka{at}tnaes.affrc.go.jp; fax
81-19-643-3514.
Received May 26, 1998;
accepted July 20, 1998.
| |
ABBREVIATIONS |
Abbreviations:
max, maximum absorbance.
DPA, days post- anthesis.
ESG, endosperm starch granule.
GBSS, granule-bound
starch synthase.
PSG, pericarp starch granule.
SGP, starch granule
protein.
| |
ACKNOWLEDGMENTS |
We thank Drs. T. Baba and K. Takaha for antisera of maize and
potato, and Dr. R. Giroux for comments on an earlier version of this
manuscript.
| |
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Abstract
Introduction