Download Biochemical Evidence for the Role of the Waxy Protein fron Pea

Plant Physiol. (1993) 103: 1355-1359
Biochemical Evidence for the Role of the Waxy Protein fron
Pea (Pisum sativum 1.) as a Granule-Bound Starch Synthase
Mirta Noemi Sivak*, Margaret Wagner, and Jack Preiss
Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824
circumvented the problem by solubilizing starch from maize
kemels with a-amylase and amyloglucosidase and found that
the dominant of the two starch synthases so released had a
molecular mass of 60 kD, as determined by ultracentrifugation on Suc density gradients (Macdonald and Preiss, 1985).
Smith (1990), however, using essentially the same approach
on pea (Pisum sativum L.) starch, obtained results that led
her to propose that "the 'waxy' protein of pea. . . is not the
major granule-bound starch synthase" and that re-examination, species by species, of the identity of the starchgranule-bound starch synthase" may be required.
Because of the need to elucidate the mechanism of amylose
synthesis and the relevance of this subject to the possible
alteration of the amylose content of starch for industrial
purposes, it was considered necessary to reinvestigate the
GBSS from pea. Here we present new evidence for the role
of the Wx protein of pea as a GBSS that contradicts the
findings presented by Smith (1990).
Proteins were solubilized from starch extracted from developing
pea (Pisum sativum 1.) embryos and chromatography of these
proteins on a Mono-Q column separated two peaks of starch
synthase activity. The major activity peak comprised more than
80% of the total activity. This fraction contained only the Waxy
protein, as shown by polyacrylamide gel electrophoresis in the
presence of sodium dodecyl sulfate followed by staining for proteins or by immunoblot. A 77-kD polypeptide associated with the
starch granules and presumed by others to be a starch synthase
could not be detected in any of the active fractions. The native
molecular weight of the solubilized starch synthase was 59,600 f
1700 as determined by sucrose density gradient. It is concluded
that in pea seeds the Waxy protein and the starch synthase bound
to the granule are the same protein.
The final product of the Waxy locus is a protein of molecular
mass 58 to 60 kD associated with the starch granule in a11
the plant species and tissues studied (for a review, see Preiss,
1991). A large body of genetic evidence indicates that the
GBSS activity, which is responsible for the synthesis of
amylose, is a function of the Wx protein. Indeed, in wx
mutants there is virtually no amylose, GBSS activity is very
low, and the Wx protein is missing (Nelson and Rines, 1962;
Nelson et al., 1978; Echt and Schwartz, 1981; Shure et al.,
1983). In an amylose-free potato mutant, activity of GBSS
was very low and the Wx protein was absent (HovenkampHermelink et al., 1987). When potato plants were transformed to produce antisense RNA so that the expression of
the Wx gene would be inhibited, the activity of GBSS was
also inhibited and the tubers contained amylose-free starch
(Visser et al., 1991).
The similarity in the deduced amino acid sequences of the
Wx proteins and the glycogen synthase from Escherichia coli,
particularly at the ADP-Glc binding site (Preiss, 1991),
strongly suggests that the Wx proteins are GBSS. However,
no direct biochemical evidence of the identity between the
GBSS and the Wx protein has been presented to date. The
Wx protein can be extracted by heating the starch with SDS
or by incubating at 37OC with 9 M urea (Frydman and Cardini,
1967; Shure et al., 1983), methods too drastic for the extraction of starch synthase activity. Macdonald and Preiss (1983)
MATERIALS AND METHODS
Materiais
Pea (Pisum sativum L.) of the round-seeded var Alaska
(Burpee, Warmister, PA) was grown in a greenhouse. The
embryos were frozen on dry ice upon harvesting and stored
at -8OOC until use. Mono-Q HR 5/5 and mo1 wt standards
were from Pharmacia. Bicinchoninicacid protein reagent was
from Pierce (Rockford, IL). [U-'4C]Glc-l-P (315.9 mCi/mmol)
was from DuPont-New England Nuclear. ADP['4C]Glc was
synthesized from ATP and [U-'4C]Glc-l-P (Preiss and Greenberg, 1972). Amylopectin was purchased from Pierce, aamylase (pig pancreas, PMSF treated, 1260 units/mg protein),
prestained mo1 wt standards, pig heart malic dehydrogenase
mitochondrion), and rabbit muscle lactic dehydrogenase
came from Sigma; amyloglucosidase (Aspergillus niger, 14
units/mg protein) were from Boehringer Mannheim; Dowex
1-X8 resin (200-400 mesh, chloride form, analytical grade)
was obtained from Bio-Rad. The amylase inhibitors acarbose
and BAY e 4609 were gifts of Drs. E. Truscheit and D.
Schmidt of Bayer AG (Wuppertal, Germany).
Preparation of Starch Granules
Starch was isolated from pea seeds as described previously
for maize kemels (Macdonald and Preiss, 1983) with the
following minor modifications. Embryos were crushed in a
This work was supported by a grant to M.N.S. from the Research
Excellence Funds of the state of Michigan and by the U.S. Department of Agriculture/Department of Energy/National Science Foundation Plant Science Program No. 88-37271-3964.
* Corresponding author; fax 1-517-353-9334.
Abbreviations: GBSS,granule-bound starch synthase; Wx, Waxy.
1355
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Sivak et al.
1356
blender. The starch-containing pellet obtained after the first
centrifugation of the crude extract was resuspended in a
medium containing 50 m~ Tris-acetate, pH 8.0, 2 m~ EDTA,
2.5 m~ DTT, and 30% (w/v) SUC,and filtered through four
layers of cheesecloth. The filtrate was layered on top of a SUC
cushion (2 volumes of suspended granules per volume of Suc
cushion) containing 75% (w/v) SUCin the same medium as
the 30% SUCsolution (Burr and Burr, 1976) and spun for 10
min at 12,OOOg. After remova1 of the supematant and the
protein bodies that remained at the top of the cushion, the
starch pellet was resuspended in the 30% SUCsoiution and
the procedure was repeated. The starch was finally resuspended in the 30% SUCsolution, vacuum filtered through a
glass fiber filter, and rapidly washed with 100 mL of acetone
(-20°C) by resuspending the starch in the acetone and
filtering under vacuum. The starch was then vacuum dried
at room temperature and stored at -2OOC until use.
Release and Fradionation of Granule-Bound Proteins
Starch (3 g) was ground, suspended in buffer, and partially
digested essentially as described by Macdonald and Preiss
(1983), but a-amylase was used at 70 units mL-' and amyloglucosidase, when it was used, was at 0.56 unit mL-'. After
treatment of the starch with a-amylase and amyloglucosidase, the starch suspension was centrifuged for 30 min at
25,OOOg. The precipitate was subjected to a second solubilization cycle and the two supematants were pooled and concentrated using an Amicon apparatus fitted with a PM30 membrane. The concentrate was diluted 10-fold with a medium
containing 20 m~ triethanolamine/KOH, pH 7.3, 2.5 m~
DTT, 10% (v/v) glycerol, and 2 m~ EDTA (buffer A) and
applied to a Mono-Q column (HR 5 X 5, Pharmacia fastprotein liquid chromatography system) equilibrated with
buffer A. After washing the column with 5 mL of this
medium, the enzyme was eluted with a gradient of KC1 in
buffer A as indicated in the figure; the flow rate was 0.5 mL
min-1. Fractions of 1 mL were collected, assayed for starch
synthase activity, and subjected to SDS-PAGE and immunoblot as described below.
Assay of Starch Synthase
The transfer of Glc from ADP-Glc into primer was measured in an assay (essentially the same as assay B described
by Macdonald and Preiss, 1983), which contained 140 nmol
ADP['4C]Glc of specific activity 700 cpm nmol-', 20 pmol
Bicine/KOH, pH 8.4, 0.5 M citrate, 2 pmol GSH, 1 pmol
EDTA, 1 mg rabbit liver glycogen, and enzyme in a total
volume of 0.2 mL. Reactions were incubated at 3OoC and the
incorporation of radioactivity into methanol-insoluble polysaccharide was measured as described previously (Ghosh and
Preiss, 1966). When the presence of amylases interfered with
this assay, the reaction was performed in the presence of
amylase inhibitors and the product was quantified by using
Dowex 1-X8 to absorb the unreacted ADP['4C]Glc as described by Macdonald and Preiss (1983).
Plant Physiol. Vol. 103, 1993
PACE and lmmunoblotting
Samples were analyzed on 7% polyacrylamide gels in the
presence of SDS according to Laemmli (1970). For the analysis of starch-bound proteins, these were extracted as described by Shure et al. (1983).
After electrophoresis, proteins were stained using silver or
Coomassie brilliant blue as indicated, or electroblotted onto
nitrocellulose membrane according to Bumette (1981). After
electroblotting, nitrocellulose membranes were treated with
rabbit antiserum against the maize Wx protein (Shure et al.,
1983) or with antiserum raised against the 77-kD protein
(Smith, 1990). The antigen-antibody complex was visualized
by treating the membrane with goat anti-rabbit immunoglobulin G linked to alkaline phosphatase, followed by incubation
with a substrate mix containing 5-bromo-4-chloro-3-indoyl
phosphate and nitroblue tetrazolium. Immunological specificity was tested by substituting rabbit preimmune serum.
MOIWt Determination
The mo1 wt of the partially purified enzymes was determined on 5 to 20% SUCdensity gradients according to Martin
and Ames (1961). The 3-mL gradients were 5 to 20% SUCin
50 m~ Tris-acetate, pH 8.0, 2 m~ EDTA, 2.5 m~ DTT, and
centrifugation was in a SW60 rotor, at 4OC, 125,OOOg for 18
h. 'The markers were pig heart malate dehydrogenase (mitochondrial, molecular mass 68 kD) assayed by the method of
Ochoa (1955), and rabbit muscle lactate dehydrogenase (molecular mass 140 kD) assayed according to Komberg (1955).
Other Methods
Protein was measured by the method of Smith et al. (1985)
using commercially obtained bicinchoninic acid reagent and
BSA as standard.
RESULTS AND DISCUSSION
Starch granule-bound proteins from developing pea embryos were released from the starch using a-amylase. After
two cycles of digestion-centrifugation,70 to 80%of the starch
was recovered in the precipitate. The starch synthase activity
of the starch is likely to be severely underestimated before
digestion because of the difficulty of the ADP['4C]Glc in
reaching the granule-bound enzyme. Therefore, the a-amylase digestion appeared to solubilize about 100% of the
activity (rather than the 20-30% that could be expected from
the amount of starch digested).
The supematant containing the amylase and the proteins
released from the starch were fractionated by chromatography on a Mono-Q column that resolved two peaks of starch
synthase activity (Fig. l), the major one comprising more
than 80% of the total. The activity recovered from the MonoQ column was higher than that applied to it, probably because in the presence of high amounts of amylases starch
synthase activity is underestimated to some extent even when
using the Dowex assay and amylase inhibitors. (Macdonald
and Preiss, 1983). At the concentration of a-amylase used,
amyloglucosidasedid not seem to be necessary for solubilization. Indeed, the recovery of starch synthase in the
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Waxy Protein from Pea Is a Granule-Bound Starch Synthase
KCIIM)
07
PS FT 10 II 12 13 14 15
MW
1357
13 14 15 MW
04
(-42.7
0.3
•O.Z
O.I
03
10
Poss-Through
Figure 1. Chromatography on Mono-Q of the proteins released
from starch extracted from developing pea embryos by digestion
with a-amylase. After injecting the sample and washing the column
with buffer without KCI, the column was eluted with a gradient
containing KCI as indicated in the figure and 1-mL fractions were
collected and monitored for starch synthase activity (broken line)
and A2so (continuous line). "Pass-Through" indicates the activity and
AIM of the fraction that eluted from the column during injection.
supernatant was similar and the starch synthase activity
eluted at the same positions in the gradient (not illustrated).
Another factor that did not affect recovery of activity or the
activity profile obtained from the Mono-Q chromatography
was whether the purified starch was used 'wet/ i.e. solubilized immediately after its purification, or whether it was first
washed with acetone and dried as described above (see
'Materials and Methods').
The first activity peak (which eluted from the Mono-Q
column at 0.085 M KCI) displayed a relatively high specific
activity, i.e. 10 fanol [I4C]Glc incorporated min"1 mg"1 protein, when assayed in the presence of 0.5 M citrate. SDSPAGE of this fraction followed by staining for proteins
showed the presence of a 60-kD polypeptide (Fig. 2), which
in immunoblot (not illustrated) was recognized by the antiserum raised against the Wx protein of maize (Shure et al.,
1983). Conversely, the 77-kD polypeptide present in pea
starch (Fig. 2) could not be detected in any of the active
fractions (peak I or II) by protein staining or by immunoblot
using an antiserum raised against the latter protein (Smith,
1990). It is worth noting that the antiserum raised against the
77-kD polypeptide present in pea starch (Smith, 1990) also
recognized other polypeptides, both in pea (Fig. 2) and in
maize starch (not illustrated). For this reason, in experiments
like the one illustrated in Figure 2, the antiserum against the
77-kD protein was used for immunoblots because it recognized both the 77-kD and the Wx protein and therefore
simplified the characterization of the fractions. The anti-77kD antiserum clearly showed the presence of the 60-kD
polypeptide in the active fraction. No Wx protein could be
detected in the 'pass-through' fraction, which contained the
«-amylase used to release the starch-bound proteins.
Figure 3 shows an example of the cenrrifugation pattern
on Sue density gradient of the GBSSI and of the internal
standards, malic dehydrogenase from pig heart (mitochondrial) with a molecular mass of 67 kD (Noyes et al., 1974),
Figure 2. SDS-PACE of proteins from a pea starch sample (1 mg)
and of Mono-Q fractions (10 jiL) as shown in Figure 1. Left, Immunoblot using antiserum raised against the 77-kD protein associated
with pea starch (Smith, 1990). Right, Protein (silver) staining. PS,
Pea starch; PT, "pass through," i.e proteins that did not bind to the
column. The numbers indicate the fraction number as in Figure 1.
For the immunoblot the molecular mass standards were prestained
with apparent molecular mass as follows: Fru-6-P kinase (rabbit
muscle), 96.4 kD; pyruvate kinase (chicken muscle), 80.4 kD; ovalbumin (chicken egg), 55.7 kD; lactic dehydrogenase (rabbit muscle),
43.7 kD. For protein staining, the standards were: phosphorylase b,
97.4 kD; BSA, 66.2 kD; ovalbumin, 42.7 kD. A polypeptide of 60
kD, recognized by the antiserum, was present in fraction 12, which
contained more than 80% of the total starch synthase activity
recovered from the Mono-Q. Note that the antiserum recognized
not only the 77-kD polypeptide present in starch but also two
bands of 59 and 60 kD.
1
0.0
Fraction number
Figure 3. Ultracentrifugation pattern on a continuous Sue density
gradient (5-20% Sue) of the major starch synthase associated with
pea starch (CBSSI) and of the internal standards, malic dehydrogenase (MDH) from pig heart (mitochondrial, molecular mass 67 kD)
and lactic dehydrogenase (LDH) from rabbit muscle (molecular
mass 140 kD). Enzyme activities are expressed for LDH and MDH
as A/W min"1 ^L"1 of fraction, and for starch synthase as nmol
[14C]Clc incorporated 15 min"' /iL"1 of fraction.
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1358
Sivak et al.
and the lactic dehydrogenase from rabbit muscle with a
molecular mass of 140 kD (Lovell and Winzor, 1974). From
the migration of the GBSSI in the gradient it was calculated
that its native molecular mass is 59.6 f 1.7 kD (average of
three determinations).
Although the first activity peak that eluted from the MonoQ comprised most of the starch synthase activity, the second
peak was also studied. The properties of the two isoforms
were very similar with respect to their stimulation by citrate
and primer preference. Activity in the absence of added
primer as a percentage of the activity with primer was higher
for GBSSII, suggesting that the latter may contain more
endogenous primer (a-glucans) associated with it (Sivak,
1992). The mo1 wt of the GBSSII was found to be similar to
that of GBSSI, and SDS-PAGE showed that it also contained
Wx protein plus other polypeptides, but not one of 77 kD
(not illustrated).
The fact that the antiserum raised against the 77-kD polypeptide from pea recognizes others, most significantly the
Wx protein, would make neutralization experiments using
the antiserum too ambiguous to interpret. This is especially
true for the experiments of Smith (1990), in which very large
volumes of undiluted serum (20-100 pL of serum for 100 pL
of solubilized starch proteins, equivalent to about 0.008 starch
synthase unit) were used in the neutralization experiments and high dilutions of it (1:15,000) were used for the
immunoblots.
Dry et al. (1992) compared the amino acid sequence of the
mature pea 59-kD protein (named GBSSI in that paper)
deduced from the corresponding cDNA sequence with those
of the maize Wx and potato Wx proteins and the glycogen
synthase of Escherichia coli (see also Preiss, 1991) and could
find no obvious difference that could justify the lack of starch
synthase activity of the pea 59-kD protein.
The data presented here indicate that in developing pea
embryos the Wx protein and the starch synthase bound to
the granule are the same protein. The methodology used by
Smith (1990) was somewhat different than the one used by
Macdonald and Preiss (1983), i.e. she used only a-amylase
(and not a combination of a-amylase and amyloglucosidase)
for the solubilization of starch and only one step of purification, i.e. the chromatography on Mono-Q. These changes,
however, cannot explain the different results obtained by
Smith because when we used only a-amylase for starch
solubilization and chromatographed on Mono-Q the proteins
so released, the correlation between the presence of Wx
protein and starch synthase activity was very clear and the
77-kD protein could not be detected in the active fractions.
It is worth mentioning that in our experiments only aamylase and not Wx protein could be detected in the protein
fraction that did not bind to the column. Conversely, in the
results published by Smith, most of the Wx protein was in
the “pass-through”fraction. The a-amylase does not bind to
the Mono-Q in these conditions and would make the detection of starch synthase activity difficult.
The cause for the failure of the Wx protein to bind to the
Mono-Q column at pH 8.0 (Smith, 1990), when in our hands
it did attach at pH 7.3, is unknown, but possible explanations
are (a) the KCl present in the solubilization media was
incompletely removed by inadequate dialysis, reducing the
Plant Physiol. Vol. 103, 1993
capacity of the Mono-Q column and decreasing the capacity
of the weaker-charged proteins to bind; (b) the use of Tris
buffer by Smith negatively affected the chromatography,
since Tris is not recommended for its use on Mono-Q because
of the large pH variation experienced with temperature
changes; and/or (c) the use by Smith (1990) of only a fraction
of the a-amylase activity used by Macdonald and Preiss
(1983) may have been insufficient. As discussed above, measurement of starch synthase activity present in the starch itself
can be grossly underestimated, which could lead, in tum, to
gross overestimation of the proportion of activity released by
treatment with amylases.
It is worth mentioning that the major GBSS described by
Macdonald and Preiss, with a native molecular mass of 60
kD, is associated with the Wx protein according to the criteria
described above for the pea GBSS (not illustrated). Thus, the
biochemical reexamination of starch synthase present in
starch from two species, i.e. maize and pea, strengthens the
genetic evidence supporting the role of the Wx protein as a
GBSS with a major role in the determination of amylose
content of starch.
ACKNOWLEDCMENTS
We wish to thank Dr. Alison Smith for the gift of antiserum raised
against the 77-kD protein of pea starch, and Dr. Susan Wessler for
the antiserum raised against the Wx protein of maize.
Received June 28, 1993; accepted August 31, 1993.
Copyright Clearance Center: 0032-0889/93/l03/1355/05.
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