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J. Biol. Chem., Vol. 277, Issue 13, 10834-10841, March 29, 2002
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From the Departments of
Received for publication, December 5, 2001, and in revised form, January 18, 2002
Reductions in activity of SSIII, the major
isoform of starch synthase responsible for amylopectin synthesis in the
potato tuber, result in fissuring of the starch granules. To discover the causes of the fissuring, and thus to shed light on factors that
influence starch granule morphology in general, SSIII antisense lines
were compared with lines with reductions in the major granule-bound isoform of starch synthase (GBSS) and lines with reductions in activity
of both SSIII and GBSS (SSIII/GBSS antisense lines). This revealed that
fissuring resulted from the activity of GBSS in the SSIII antisense
background. Control (untransformed) lines and GBSS and SSIII/GBSS
antisense lines had unfissured granules. Starch analyses showed that
granules from SSIII antisense tubers had a greater number of
long glucan chains than did granules from the other lines, in the form
of larger amylose molecules and a unique fraction of very long
amylopectin chains. These are likely to result from increased flux
through GBSS in SSIII antisense tubers, in response to the elevated
content of ADP-glucose in these tubers. It is proposed that the
long glucan chains disrupt organization of the semi-crystalline parts
of the matrix, setting up stresses in the matrix that lead to fissuring.
Little is known about the processes that determine the
morphology of starch granules, but important clues have been gained from studies of mutant plants with altered granule morphology. Many
such plants carry mutations in genes encoding isoforms of starch
synthase and starch-branching enzyme responsible for the synthesis of
amylopectin, the branched The altered granule morphology of the mutants presumably results from
alterations in the structures and relative amounts of the glucan
polymers of which the granule is comprised. Most mutations affecting
isoforms of starch synthase and starch branching enzyme affect both of
these parameters. The r and rug5 mutations of pea and the amylose-extender mutation of maize affect the
proportion of short and long chains in amylopectin, the chain-length
distribution within the short-chain population, and the ratio within
the granule of amylopectin to amylose, the essentially linear glucan
that makes up 20-30% of storage starches (2, 6-9). However, it is
not known which of these changes in granule composition and polymer
structure leads to the changes in granule morphology.
Transgenic potato plants with reduced activity of isoforms of starch
synthase provide a good system for elucidation of the relationship
between granule morphology and glucan structure. Plants expressing
antisense RNA for granule-bound starch synthase I (GBSS antisense
plants), the isoform exclusively responsible for the synthesis of
amylose, have much less amylose in the tuber starch but normal granule
morphology (10). Plants expressing antisense RNA for starch synthase
III (SSIII antisense plants), the major isoform responsible for
amylopectin synthesis, have a markedly altered chain-length
distribution among the short chains of amylopectin and an increased
proportion of very long chains (11, 12). Whereas granules from
wild-type and GBSS antisense plants have a smooth, ovoid profile, those
from SSIII antisense lines are deeply internally fissured and are
frequently multilobed (13). We reasoned that the altered granule
morphology in SSIII antisense lines could be caused either by the
altered chain-length distribution of short chains of amylopectin, or by
the increased proportion of very long chains in amylopectin, or by the
interaction of amylose molecules within the granule with one or both of
these abnormal features of amylopectin.
To distinguish between these possibilities, we have generated
transgenic lines down-regulated for both SSIII and GBSS simultaneously (SSIII/GBSS antisense lines), and compared the starch of
control and SSIII, GBSS, and SSIII/GBSS antisense lines with
respect to amylopectin structure, amylose content, and granule morphology.
Materials
Potato plants (Solanum tuberosum L. cv. Desiree and
transgenic plants derived from it) were grown from shoots propagated in tissue culture (14) then transferred to soil-based compost in a
greenhouse at a minimum temperature of 12 °C with supplementary lighting in winter. Tubers for enzymes assays, native gel
electrophoresis, and starch preparations were used immediately after
harvest. Tuber tissue for analysis of starch content was frozen at
Methods
Construction of Antisense Binary Vector--
The 2.2-kb
NcoI fragment encoding the full-length cDNA of potato
GBSS (15) was subcloned, in the antisense orientation, into the
NcoI site of pRAT3. pRAT3 contains a 1.1-kb fragment of the
potato SSIII cDNA in antisense orientation between the cauliflower
mosaic virus double 35S promoter and terminator (13). The resulting
[promoter]-[SSIII antisense (1 kb)]-[GBSS antisense (2.2 kb)]-[SSIII antisense (0.1 kb)]-[terminator] chimera was excised
as a KpnI/XhoI fragment and ligated between the
KpnI/SalI sites of plant transformation vector
pBIN19 (16) to give plasmid pROT1.
Transformation of Potato--
Transformation and preparation of
Agrobacterium inoculum carrying the antisense construct, inoculation of
tuber discs of potato, and regeneration of shoots were all done as
described previously (13, 14).
Enzyme Assays and Native Gels--
Assays for starch synthases
were done according to Refs. 13 and 14. Assays for starch-branching
enzyme and ADP-glucose pyrophosphorylase were according to Refs. 17 and
18, respectively. Native gels for starch synthase and starch-degrading
enzymes were prepared according to Refs. 13 and 11, respectively.
Assay and Extraction of Starch and Analyses of Granule-bound
Proteins--
Starch contents of tuber tissue were measured according
to Ref. 17. Starch was purified from tubers according to Ref. 14. Starch used in the compositional and structural analyses described below was from tubers harvested from mature, senescing plants. Granule-bound proteins were extracted from purified starch and subjected to SDS-polyacrylamide gel electrophoresis according to Ref.
11.
Visualization of Growth Rings--
Approximately 0.3 g of
starch was suspended in 1 ml of water, frozen in liquid nitrogen in a
mortar, and ground thoroughly to crack granules. After thawing,
granules were recovered by centrifugation and incubated with occasional
stirring at 37 °C for 7 h in 50 mM Mes-KOH (pH 5.6)
at 0.3 mg/ml, with 4 units of Small-angle X-ray Scatter Analysis--
Analyses were performed
according to Refs. 19-21.
Analysis of Starch Composition--
The size distribution and
relative amounts of amylose and amylopectin in starch and starch
fractions were examined by low pressure gel permeation chromatography.
Samples of starch (20 mg), amylopectin (15 mg), or amylose (5.2 mg)
were suspended in 1 M NaOH (0.4 ml) and diluted with 1 ml
of dH2O and boiled for 5 min. After cooling, a further 1 ml
of dH2O was added. The resulting solution was loaded onto
two 1-m (i.d. 15 mm) Sepharose CL-2B columns connected in series,
equilibrated in 10 mM NaOH, and eluted in a descending mode
at 0.16 ml/min. Fractions of 6 ml were collected, and samples were
mixed with acidified iodine (Lugol's) solution and monitored at 595 nm.
Partial Purification of Amylose and Amylopectin--
Amylose and
amylopectin were prepared by butanol precipitation. Starch samples
(1.33 g) were suspended in 100 ml of H2O and boiled for
1 h, adjusted to pH 5.9-6.3 with 100 mM phosphate
buffer, autoclaved (100 kPa, 1 h), and heated under reflux
conditions at 100 °C for 1 h. Butanol (25 ml) was added, and
the mixture was refluxed for 1 h then allowed to cool very slowly
from boiling point to ambient temperature (over a period of 36-48 h).
The amylose precipitate was collected by centrifugation, and the
amylopectin-containing supernatant was again subjected to the refluxing
procedure described above. The combined amylopectin-containing
supernatants and the combined amylose-containing precipitates were
lyophilized. Amylopectin was also prepared by gel permeation
chromatography, as described above. Fractions from the amylopectin peak
were neutralized with 2 M HCl, desalted, and lyophilized.
Analysis of Chain-length Distribution of
Amylopectin--
Analysis of the chain-length distribution of the
shorter chains of amylopectin was achieved by using
fluorophore-assisted polyacrylamide gel electrophoresis (FAGE) of
debranched starch using an Applied Biosystems 373A DNA sequencer (11).
Analysis of the chain-length profile across the full range of chain
lengths was achieved by HPLC. For routine analysis, amylopectin
obtained either by the butanol precipitation method or by gel
permeation chromatography, was suspended in boiling water (10 mg/ml)
then autoclaved (100 kPa, 30 min). For some analyses amylopectin was instead suspended in 90% (v/v) Me2SO (10 mg/ml) and
stirred in a boiling water bath for 15 min before precipitation with
four volumes of methanol. The precipitate was redissolved in a
one-fifth volume of water. Amylopectin suspensions obtained by either
of the above methods were mixed with 1/20 volume of 220 mM
sodium acetate (pH 4.8) and 4 × 104 units of
isoamylase (Sigma Chemical Co.), incubated at 37 °C for 16 h
then autoclaved (100 kPa, 30 min). A sample of 100 µl was immediately
loaded onto the HPLC system, which consisted of guard columns and three
HPLC columns connected in series (SecurityGuard (4 × 3 mm),
Biosep-Sec-S 2000 guard column (75 × 7.8 mm), two Biosep-Sec-S
2000 columns (600 × 7.8 mm) and a Biosep-Sec-S 3000 column
(600 × 7.8 mm); all from Phenomenex, Torrance, CA). The columns
and sample injector were heated to 37 °C. The system was eluted with
water at 0.28 ml/min, and 0.56-ml fractions were collected. To a sample
(0.28 ml) of each fraction was added 1/20 volume of 220 mM
sodium acetate (pH 4.8) and 0.28 units of amyloglucosidase. The mixture
was incubated at 37 °C overnight then assayed enzymatically for
glucose. Two checks were made on the authenticity of the long chains
detected in amylopectin. First, results were compared from samples of
amylopectin prepared from the same batch of starch either by gel
permeation chromatography or by the butanol precipitation method.
Second, results were compared from samples of the same batch of
amylopectin prepared either in water or in Me2SO, as described above. Pullulan standards were from Polymer
Laboratories Ltd., Church Stretton, Shropshire, UK.
Measurement of Sugar Nucleotides, ATP and ADP--
Rapid
sampling and extraction of tuber tissue was according to Ref. 22. Assay
of sugar nucleotides, ATP and ADP, was by HPLC according to Ref.
23, the authors of which provide evidence of the reliability of the
methods of extraction and analysis for the metabolites.
Generation of Plants in Which Activities of both SSIII and GBSS Are
Significantly Reduced--
To achieve a simultaneous reduction of both
GBSS and SSIII isoforms, tubers of Desiree were transformed with
plasmid pROT1, a construct that expresses a chimeric antisense RNA of
SSIII and GBSS under the control of the cauliflower mosaic virus double 35S promoter. The transgenic lines thus produced are referred to as
SSIII/GBSS antisense lines. The fragments of the GBSS and SSIII
cDNAs used in the construct had both been used previously to obtain
plants expressing antisense RNA for either SSIII (11, 13: referred to
as SSIII antisense lines) or GBSS (24: referred to as GBSS antisense
lines). In subsequent experiments, the SSIII/GBSS lines were compared
with three SSIII and three GBSS antisense lines described previously
(11, 13, 24) and with control lines that were either untransformed
Desiree or a line transformed with the vector pBIN19 alone.
Developing tubers of primary transformants were assayed for activity of
soluble and granule-bound starch synthase. Of the 35 independent
transformants examined, seven had reductions of 80% or more in
granule-bound activity relative to a control line, and five of these
also had reductions of 50% or more in soluble starch synthase
activity. The latter reductions were shown to be specific for the SSIII
isoform by native gel electrophoresis of tuber extracts followed by
staining for starch synthase activity (Fig.
1A). The intensity of the band
representing SSIII was greatly reduced in transgenic plants with low
soluble starch synthase activity, but the intensities of bands
representing SSII (11) and SSI (25) were unaffected. In two of the
plants, soluble and granule-bound starch synthase activities were
reduced to levels comparable with those in the most extreme SSIII and
GBSS antisense lines characterized previously. These lines (Rot1.1 and
Rot1.4) were propagated for further study.
Soluble starch synthase activity in the SSIII/GBSS antisense tubers was
reduced by 3.4-fold (Rot1.1) and 4.9-fold (Rot 1.4) relative to the
control line (Table I), compared with
reductions of between 2.5- and 5.5-fold in the SSIII antisense lines
(11, 13). Thus the reductions in SSIII activity in Rot1.1 and Rot1.4 are comparable with those in the SSIII antisense lines. Granule-bound starch synthase activity was reduced by 8-fold (Rot 1.1) and 14-fold (Rot 1.4) compared with a reduction of 7- to 12-fold at a comparable developmental stage in the most extreme GBSS antisense lines studied previously (lines 5 and 8 (24)). Confirmation that the extent of
reduction in GBSS was comparable in the SSIII/GBSS and the most extreme
GBSS antisense lines, was obtained by probing blots of granule-bound
proteins with an antiserum against GBSS (Fig. 1B).
To check whether activity of enzymes of starch metabolism other than
SSIII and GBSS were affected in the SSIII/GBSS antisense tubers, we
assayed activities of ADP-glucose pyrophosphorylase and
starch-branching enzyme and analyzed starch-degrading activities on
native, amylopectin-containing gels. There were no major differences between the SSIII/GBSS antisense lines and untransformed Desiree with
respect to these activities (Table I, Fig. 1C).
Granule Morphology and Anatomy--
Starch contents (per gram of
fresh weight) of mature tubers of the transgenic lines were not reduced
relative to those of the control line. In three harvests, the starch
contents of SSIII and GBSS antisense lines and the SSIII/GBSS antisense
line Rot1.4 were not statistically significantly different from the
control line. The starch content of the SSIII/GBSS antisense line Rot 1.1 was about 30% higher than that of the control line in two harvests
and not statistically significantly different from the control line in
the third harvest (data not shown).
There were striking differences in the morphology of starch granules
from mature tubers of the GBSS, SSIII, and SSIII/GBSS antisense lines.
Granules of the GBSS antisense lines were ovoid and unfissured like
those of untransformed Desiree (Fig. 2,
A and B). Granules of SSIII antisense lines were
deeply internally fissured with fissuring centered on the hilum (origin
of growth), or were multilobed or clustered structures. This was true
of lines with reductions in SSIII activity of as little as 2.5- to
3-fold, as well as of lines with greater reductions in activity (11, 13; Fig. 2C). Fissures developed as SSIII granules matured,
rather than being formed continuously from the start of granule
synthesis. Small granules from young, developing tubers were not
fissured (Fig. 2D). Thus fissures represent a fracturing of
a previously continuous matrix rather than the non-continuous
development of the matrix. Granules of the SSIII/GBSS antisense lines
were not fissured. Some appeared as clusters of small granules. Mature granules were more spherical and had a more central hilum than those of
untransformed Desiree and the GBSS antisense lines (Fig. 2E).
The internal growth rings of granules also differed between the lines.
The growth rings represent the alternation of semi-crystalline and
amorphous zones within the matrix, with a repeat distance of some
hundreds of nm. Growth rings were visualized by digesting internal
surfaces with
To investigate further the disruption of the semi-crystalline zones in
granules of SSIII antisense lines, the small-angle x-ray scattering
(SAXS) patterns of starch preparations were examined. Starch granules
from many different plants, including potato, show a characteristic
peak in the SAXS pattern at a q-spacing of about 0.06 Å
The SAXS peak for SSIII antisense starch occurred at a markedly lower
q-value and was broader than that for untransformed Desiree
starch, whereas the q-value for peaks for SSIII/GBSS
antisense and GBSS antisense starches was similar to that of
untransformed Desiree (Fig. 3). This
indicates that the periodicity of the semi-crystalline repeat is
greater and the packing is less well organized in starch from SSIII
antisense tubers than in starch from control, SSIII/GBSS, and GBSS
antisense tubers.
Granule Composition--
The amylose content of starch was much
lower in GBSS and SSSIII/GBSS antisense lines than in the control line.
Amylose was almost undetectable in starch from both the most extreme
GBSS antisense line and the SSIII/GBSS antisense lines when analyzed by
gel permeation chromatography (Fig.
4A). Concentration of amylose by butanol precipitation, followed by gel permeation chromatography of
this material, revealed that the residual amylose of the GBSS antisense
lines and the SSIII/GBSS antisense line Rot1.4 was of smaller apparent
molecular size than amylose from the control line (Fig. 4, B
and C).
When stained with iodine solution, granules from the SSIII/GBSS
antisense lines appeared red with a blue-staining central core (Fig.
2F). The same staining pattern is observed in granules from
GBSS antisense lines (Fig. 2B (10)). It suggests that
amylose is confined to the core of the granule.
Previous iodine-based measurements indicated that the amylose content
of starch from SSIII antisense lines was the same as that of control
lines (12, 26). However, gel-permeation chromatography indicated that
the molecular size of amylose in the SSIII antisense lines is greater
than in the control lines. The amylose peak from solubilized starch
eluted earlier for SSIII/GBSS samples than for control samples (Fig.
4D). Butanol-purified amylose from the SSIII antisense line
18 had a higher apparent molecular mass than that from the
control line (Fig. 4E).
Gel permeation chromatography of amylopectin (purified by butanol
precipitation of amylose) from starches of control, SSIII, and
SSIII/GBSS antisense lines revealed a single peak of material of high
molecular mass. There was little or no material of a lower molecular
mass equivalent to that of amylose (data not shown).
Amylopectin Structure--
The chain-length distribution of the
shorter chains of amylopectin was examined by fluorophore-assisted gel
electrophoresis (FAGE) after debranching with isoamylase. All analyses
were performed several times, on separately prepared samples. All of
the differences discussed below were observed routinely and reproducibly.
The chain-length distributions of shorter chains from the SSIII and
SSIII/GBSS antisense lines differed considerably from that of the
control line (Fig. 5), whereas the GBSS
antisense lines were not distinguishable from the control in this
respect (not shown, see also Ref. 27). The chain-length distributions of amylopectin from the SSIII and SSIII/GBSS antisense lines were similar: Both were enriched in chains of 6 glucose units (degree of
polymerization (dp) 6), and chains of dp 11 to 14, and depleted in
chains from about dp 17 upwards, relative to the control line.
Longer amylopectin chains were analyzed by HPLC-gel permeation
chromatography. Eluted glucans were measured as glucose after hydrolysis with amyloglucosidase. For starch from tubers of control lines, this method separated amylopectin chains into three peaks (labeled I, II, and III in Fig.
6A). The largest peak
(I) was of lowest molecular mass and contained chains in the
size range analyzed by FAGE. Peak III contained very long
chains. Estimates from pullulan standards (shown on Fig.
6A) suggest that these chains were ~160-500 glucose units
in length. The elution profile for amylopectin from GBSS antisense
lines was indistinguishable from the control (data not shown). The
elution profile for amylopectin from SSIII antisense lines differed
from that of the control in three main ways. First, peak II
was less pronounced. Second, peak III was more pronounced
than for the control line for two out of three SSIII antisense lines.
Third, a fourth peak (IV) was present, containing chains
longer than those of peak III (Fig. 6B). These
chains were approximately dp 700-1300.
To check that long chains were not an artifact generated by
crystallization (retrogradation) of the sample or contamination of the
amylopectin with amylose, we compared two methods of purifying amylopectin and two methods of solubilizing material for HPLC (see
"Experimental Procedures"). The presence of the very long chains in
amylopectin from SSIII antisense lines was unaffected by these
different treatments, hence these chains probably form part of the
native amylopectin molecule.
Amylopectin from the SSIII/GBSS antisense lines was similar to that of
the SSIII antisense lines in that peak II was generally less
pronounced than in control amylopectin. However, amylopectin from the
SSIII/GBSS antisense lines, in common with that of the control and GBSS
antisense lines, lacked the very long chains (peak IV)
present in the amylopectin of SSIII antisense lines (Fig.
6C).
ADP-glucose Content of Tubers--
We reasoned that some of the
differences in starch composition and structure between the antisense
and control lines might be indirectly attributable to differences in
the concentration of ADP-glucose in the plastids. Elevated
concentrations of ADP-glucose in the antisense lines are expected
because of the large decreases in starch synthase activity. Differences
in ADP-glucose concentrations between lines could result in differences
in granule composition, because different isoforms of starch synthase,
which make distinct contributions to amylose and amylopectin synthesis,
have different affinities for ADP-glucose (28).
ADP-glucose levels in developing tubers were up to five times higher in
SSIII antisense lines than in the control line and up to 50 times
higher in the SSIII/GBSS antisense line Rot1.1 (Table
II). These very high levels were not
accompanied by decreases in the levels of ATP and ADP, indicating that
the total adenylate pool in tubers of the SSIII/GBSS antisense line was
twice as high as in tubers of the control line. The UDP-glucose content
of the tubers was not significantly changed in the transgenic
lines.
Our results indicate that the deep fissuring and multilobed
appearance of starch granules from SSIII antisense tubers probably result from a build-up during granule maturation of long-range stresses
caused by changes in the short-range organization of the amylopectin
matrix. Starch granules from tubers of the SSIII/GBSS antisense lines
lack the abnormalities of granules from SSIII antisense tubers. This
implies strongly that the abnormalities arise because organization of
the amylopectin matrix is strongly influenced by products of GBSS.
GBSS is exclusively responsible for the synthesis of the amylose
component of starch, and it can also contribute to the synthesis of
amylopectin. The amylopectin of mutants of cereals lacking GBSS has a
shorter average chain length than that of equivalent wild-type cereals,
probably due to the absence of a class of long external chains present
in wild-type amylopectin (29-31). Mutants of the unicellular green
alga Chlamydomonas lacking GBSS also have altered
amylopectin structure (32, 33). Direct evidence that GBSS can elongate
amylopectin chains inside starch granules comes from experiments with
sweet potato, pea, and Chlamydomonas in which
ADP[U-14C]glucose was supplied to isolated granules. In
these conditions GBSS adds glucosyl units from ADP-glucose to long
chains in the amylopectin fraction (34-36). Below we consider three
ways in which GBSS might influence granule morphology: via changes in
the chain-length distribution of short chains of amylopectin, changes
in amylose content, and changes in the long chain component of amylopectin.
Changes in the Shorter Chains of Amylopectin--
Our data provide
no evidence that GBSS significantly influences the chain-length
distribution of the shorter chains of amylopectin in potato. The
chain-length profile up to dp 40 was similar for control and GBSS
antisense lines and for SSIII and SSIII/GBSS antisense lines.
Comparable data for other plant species similarly suggest that GBSS
plays no role in synthesis of the shorter chains of amylopectin. For
example, the chain-length profiles of wild-type and near isogenic
waxy rice were not statistically significantly different
(37). Overall, it is highly unlikely that the impact of GBSS on granule
morphology is related to any direct role of the isoform in the
synthesis of shorter chains of amylopectin.
Changes in Amylose Content--
As expected, starch from both GBSS
antisense and SSIII/GBSS antisense lines contained much less amylose
than starch from Desiree. In both sorts of lines the reduction in
amylose content was accompanied by a concentration of the remaining
amylose toward the center of the granule and a sharp reduction in
apparent molecular size of amylose. Both of these effects may result
from the failure of amylose synthesis inside the amylopectin matrix to
keep pace with the growth of matrix during granule development
(24).
The presence of amylose per se is unlikely to be responsible
for the fissuring of SSIII antisense granules. Wild-type starch, which
has an amylose content comparable with that of SSIII antisense starch,
is not fissured, neither are the storage starch granules of numerous
other species with amylose contents in the range 20-35%. However, the
interaction of amylose with some specific aspect of the SSIII antisense
background may lead to fissuring. For example, amylose may interact
with the abnormal short-chain population of SSIII antisense amylopectin
to alter the way in which these chains are organized into crystalline
lamellae. In general, shorter chains are less stable as double helices,
so the abundance of very short chains in SSIII antisense amylopectin is
likely to compromise the stability of double helices in this starch.
The introduction of long chains of amylose into the matrix may further destabilize the double helices and cause them to pull apart. This would
result in loss of lamellar structure and a build up of long-range stresses, leading to fissuring of the matrix as granule size increases. Evidence that amylose can influence the organization of short chains of
amylopectin in starches generally has been provided by SAXS analysis of
normal and low amylose lines of maize and pea (20), and this idea is
further supported by our SAXS data for potato. It has been proposed
that amylose chains may either co-crystallize with short chains of
amylopectin within crystalline lamellae or that they may be orientated
transverse to the lamellar stack within amorphous lamellae. Our SAXS
data also provide specific evidence for interference by amylose in the
organization of the short chains of SSIII antisense amylopectin. The
shift in the q-value of the peak indicates that the repeat
distance in the semi-crystalline zones in SSIII antisense granules, but
not SSIII/GBSS antisense granules, is significantly greater than the
normal 9 nm seen in starches from all species thus far examined
(19).
An alternative means by which amylose may disrupt granule organization
in SSIII antisense tubers is suggested by the apparent increase in
amylose molecular size in these relative to control tubers. Synthesis
of larger amylose molecules inside the granule matrix, whether in the
semi-crystalline or the amorphous zones, might result in disruption
of granule structure.
Changes in the Long Chain Component of Amylopectin--
The
contribution of GBSS to long chains of amylopectin in normal potatoes
is small. We detected no difference in the amount of these chains
between control lines and both GBSS and GBSS/SSIII antisense lines. The
relatively small influence of GBSS on long chains in potato contrasts
with the situation in Chlamydomonas and some cereals in
which a fraction of long chains present in normal amylopectin is
eliminated by mutations that eliminate GBSS (see above). It appears
that the extent of the contribution of GBSS to synthesis of long chains
in amylopectin varies considerably from one type of starch-synthesizing
organ to another.
Amylopectin from SSIII antisense lines contains more, and longer, long
chains than amylopectin from control and GBSS antisense lines,
including the SSIII/GBSS antisense line. This finding agrees with that
of Lloyd et al. (12) who used a different set of SSIII antisense plants from those in our study. Depending on their location within the granule, long chains might potentially act to disrupt the
normal organization of the shorter chains of amylopectin in the way we
propose above for amylose: by forming double helices with short chains
or by penetrating and disrupting amorphous lamellae.
We suggest that the additional long chains in the amylopectin fraction
of SSIII antisense lines are the products of GBSS activity. The
following explanation for their occurrence is based on the fact that
the ADP-glucose level in SSIII antisense tubers is higher than that in
control tubers, presumably as a direct result of the decrease in starch
synthase activity. The ADP-glucose content of a normal tuber is
severely sub-saturating for starch synthase activity in general and
more severely limiting for GBSS activity than for the activity of
soluble, amylopectin-synthesizing isoforms. Assuming that ADP-glucose
is exclusively plastidial and that plastids represent 10% of the cell
volume, an ADP-glucose content of 2 nmol g
A relationship between ADP-glucose levels and the synthesis via GBSS of
long amylopectin chains is also suggested by previous work on mutants
of Chlamydomonas. Mutants with lesions in the pathway of
ADP-glucose synthesis, which presumably have reduced levels of
ADP-glucose, lack a long-chain fraction of amylopectin present in
normal starch (40). This long-chain fraction is also missing from
mutants with no GBSS, indicating that it is synthesized by GBSS (32).
Thus it appears that a reduction in ADP-glucose reduces the extent of
synthesis of long amylopectin chains via GBSS in this organism.
In summary, possible explanations for the disruption of organization of
the granule matrix that underlies the fissuring observed in SSIII
antisense granules include the interaction of amylose with the abnormal
short-chain population of amylopectin, the increased molecular mass of
amylose, and the presence of very long chains in the amylopectin. These
possibilities are not mutually exclusive, and none can be ruled out.
Our conclusion that GBSS products can have a radical influence on the
crystalline packing of amylopectin is consistent with two sorts of
evidence obtained from studies of Chlamydomonas. First, as
in our study, elimination of GBSS from Chlamydomonas mutants
with alterations in other isoforms of starch synthase has radical
effects on granule organization. As in higher plants, loss of GBSS
alone (through mutations at the STA2 locus) has little effect on granule morphology and crystallinity (32, 41). Loss of the
major soluble starch synthase (SSSII: encoded at the STA3 locus) shifts the crystalline diffraction pattern of the granules from
A-type to B-type (41). The crystalline type of starch granules reflects
the way in which the double helices are packed in crystalline regions.
Loss of both SSSII and GBSS (in the STA2/STA3
double mutant) results in a major shift from crystalline to soluble
glucan and a C-type crystallinity (a mixture of A and B crystalline
regions) in the remaining starch (33, 41). Second, the synthesis of amylose via GBSS in the matrix of isolated starch granules of Chlamydomonas changes the crystalline type of the starch.
Over 2 days of incubation with ADP-glucose, the amylose content of isolated granules rose from 2% to 45%, and there was a shift in the
crystalline diffraction pattern of the granules from A-type to B-type
(36).
Insufficient information is available from other higher plants to show
whether our findings for potato are more widely applicable. The granule
phenotype of many starch mutants in cereals depends upon genetic
background, and most comparative studies have not employed
near-isogenic lines (42, 43). Mutations affecting starch-synthesizing
enzymes frequently have pleiotropic effects on related enzymes (44).
There are also important differences between starch synthesis in potato
tubers and cereal endosperms that may influence the determination of
granule morphology. The representation of classes of starch synthase
isoforms in the two sorts of organ is different: Starch synthase I
makes a major contribution to activity in endosperms but not in tubers
(13, 25, 45, 46). Cereal starches generally have A rather than B type
crystallinity, thus the impact of long amylopectin chains and amylose
on organization of short chains may be different in potato and cereal
starches. Finally, ADP-glucose in cereal endosperms is synthesized
primarily in the cytosol rather than in the plastid (47, 48). The
impact of reductions in starch synthase activity upon ADP-glucose
concentrations in the plastid is thus likely to be different in potato
tubers and cereal endosperms.
We thank Sam Zeeman and Kay Denyer (John
Innes Centre, Norwich, UK), Mike Gidley, Steve Jobling, and colleagues
(Unilever Research, Sharnbrook, Bedford, UK), and Jay-Lin Jane (Iowa
State University, Ames, IA) for many valuable discussions.
*
This work was supported by a competitive Strategic Grant
from the Biotechnology and Biological Sciences Research Council (BBSRC, United Kingdom), to the John Innes Centre, by BBSRC Grant D08036, by a
BBSRC research studentship (to E. P.), by a grant from the European
Union (Framework Programme IV, project CT95-0568, to A. E.),
and by Plant Biosciences Ltd., Norwich, UK.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
**
To whom correspondence should be addressed: Tel.: 44-1603-450-622;
Fax: 44-1603-450-045; E-mail: alison.smith@bbsrc.ac.uk.
Published, JBC Papers in Press, January 18, 2002, DOI 10.1074/jbc.M111579200
The abbreviations used are:
SSI, SSII, and
SSIII, soluble starch synthases I, II, and III;
GBSS, granule-bound starch synthase;
FAGE, fluorophore-assisted
polyacrylamide gel electrophoresis;
HPLC, high performance liquid
chromatography;
SAXS, small-angle x-ray scattering;
dp, degree of
polymerization;
FWT, fresh weight.
Role of Granule-bound Starch Synthase in Determination of
Amylopectin Structure and Starch Granule Morphology in Potato*
,,,,,
,**
Metabolic Biology and
§ Cell and Developmental Biology, John Innes Centre, Colney
Lane, Norwich NR4 7UH, United Kingdom, the ¶ Department of
Physics, University of Cambridge, Cavendish Laboratory, Madingley Rd.,
Cambridge CB3 0HE, United Kingdom, and the Max-Planck Institute
of Molecular Plant Physiology, Am Mühlenberg 1, Golm D-14476,
Germany
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1,4, 1,6 glucan, which forms the
semi-crystalline matrix of the granule. For example, mutations in pea
affecting starch-branching enzyme A (at the r locus (1)) and
starch synthase II (SSII,1 at
the rug5 locus (2)) convert the normally ovoid granules of
the embryo into deeply fissured, multilobed structures and highly
twisted, contorted structures, respectively (2, 3). Mutations in maize
affecting starch-branching enzyme IIb (at the amylose-extender locus (4)) convert the normally polyhedral granules of the endosperm into irregular, elongated structures (5).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C for up to 4 months before use. For a given set of
experiments, control and transgenic plants were grown in the same
greenhouse at the same time.
-amylase (porcine pancreas)
mg
1 starch. Granules were then washed three times by
resuspension and centrifugation in acetone at 20 °C, and dried.
Dry starch samples were brushed onto the surface of double-sided,
carbonated sticky stills attached to SEM stubs (Agar Scientific,
Cambridge, UK), coated with gold for 3 min at 15 mA in argon using a
Polaran SEM coating unit (Polaran Equipment, Watford, UK), and viewed in a Philips XL30 Field Emission Gun SEM (Philips, Eindhoven, The Netherlands).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (63K):
[in a new window]
Fig. 1.
Isoforms of starch synthase and
starch-degrading enzymes in tubers of control and transgenic
lines. A, native polyacrylamide gel of crude, soluble
extracts of potato tuber, incubated and stained for starch synthase
activity. Each lane contains extract from the same fresh weight of
tuber. A tuber of the control line (left lane) is compared
with tubers from four different SSIII/GBSS antisense plants (primary
transformants: Rot1.15, 1.21, 1.29, and 1.30). Note that the intensity
of staining of the SSIII band differs between the transgenic lines, but
the staining of all other bands is the same as in the wild-type. The
identity of bands is deduced from their positions on the gel, in
comparison to published results for SSII and SSIII antisense lines.
B, immunoblot of an SDS-polyacrylamide gel of granule-bound
proteins. Each lane contains protein from the same weight of starch.
Starch from a control line is compared with starch from GBSS, and SSIII
antisense lines, and the two SSIII/GBSS antisense lines used in all
experiments. The blot was developed with antiserum raised against the
GBSS of pea embryos (49) at a dilution of 1/1000. C, native,
amylopectin-containing polyacrylamide gel of crude, soluble extracts of
potato tuber, incubated and stained with iodine. A tuber of the control
line is compared with tubers of GBSS and GBSS/SSIII antisense line.
Bands result from the actions of several enzymes, including starch
debranching enzymes, - and 
-amylases, and disproportionating
enzyme. There were no reproducible differences in starch-degrading
enzymes between the lines. Lack of effect on these enzymes in the SSIII
antisense lines has been documented previously (11).
Activities of enzymes of starch synthesis
View larger version (128K):
[in a new window]
Fig. 2.
Morphology and internal structure of starch
granules. A-F, light micrographs of intact, hydrated
starch granules. Granules in B and F were stained
with dilute iodine solution (1/20 Lugol solution), granules in other
panels are unstained. Bars represent 50 µm.
G-J, scanning electron micrographs of granules after
mechanical cracking and incubation with -amylase to reveal growth
rings. Bars represent 2 µm. A, control line
(Desiree). B, GBSS antisense line 8. C, SSIII
antisense line 18: starch from mature tubers. D, SSIII
antisense line 18, starch from young, developing tubers (of ~0.5 g
fresh weight). E and F, SSIII/GBSS antisense line
Rot1.1. G, control line (Desiree). H, GBSS
antisense line 8. I, SSIII/GBSS antisense line Rot1.1.
J, SSIII antisense line 18.
-amylase, which preferentially digests material in
amorphous zones. Rings were distinct and evenly spaced in granules from
the control line and from the GBSS and SSIII/GBSS antisense lines (Fig.
2, G-I). However, growth rings in granules from SSIII
antisense plants were much less distinct and regular in appearance
(Fig. 2J). This indicates some disruption of the normal
organization of the semi-crystalline zones of the granule.
1, where q is the angular distance in the
scattering pattern. This peak represents a repeat distance of 9 nm
within the semi-crystalline zones of the granule matrix. It is believed
that this repeat represents the clustering of the shorter chains of
amylopectin at regular intervals along the axis of amylopectin
molecules. Adjacent chains within clusters form double helices. The
packing of the helices in regular arrays produces alternating
crystalline and amorphous lamellae (19).
View larger version (22K):
[in a new window]
Fig. 3.
Small-angle x-ray scatter analysis of
starches. Radiation with a wavelength of 1.5 Å was focused onto
starch samples in the form of 50% (w/w) slurries in water. A
gas-filled proportional wire chamber quadrant detector was used to
collect the diffraction pattern. Experiments were carried out at the
Synchrotron Radiation Source, Daresbury Laboratory, UK. The
symbols used indicate starches from a control line
transformed with BIN19 alone (open triangles), SSIII
antisense line 18 (closed triangles), GBSS antisense line 6 (closed squares), and SSIII/GBSS antisense line Rot1.1
(open squares).
View larger version (24K):
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Fig. 4.
Separation of amylose and amylopectin by gel
permeation chromatography on Sepharose CL2B. Either whole starch
(A and D) or starch highly enriched in amylose by
precipitation with butanol (B, C, and
E) was solubilized and subjected to chromatography on a 2-m
column of Sepharose CL2B. Fractions were mixed with iodine solution,
and the absorbance at 595 nm was recorded. The first peak is
amylopectin and the second peak is amylose. For each type of
starch or amylose the results shown are typical of several repetitions,
each on a separately prepared sample. In A and D,
the area under each curve is the same. For preparations enriched in
amylose (B, D, and E) note that the
initial, sharp peak is contaminating amylopectin. The size of the
amylose peak for these preparations is not related to the amylose
content of the original starch, and no meaningful information can be
derived from the areas under the curves. A, whole starch
from tubers of a control line (Desiree, filled diamond),
GBSS antisense line 5 (open triangle), and SSIII/GBSS
antisense line Rot1.4 (open circle). B,
amylose-enriched preparations from control starch (filled
diamond) and starch of three GBSS antisense lines: 1 (filled
triangle), 5 (open triangle), and 8 (filled
square). Line 1 retains more GBSS activity than lines 5 and 8 (24). C, amylose-enriched preparations from control starch
(filled diamond) and starch of SSIII/GBSS antisense lines
Rot1.4 (open circle) and Rot1.1 (filled circle).
The yield of butanol-precipitable material from starch of line Rot1.1
was too low to permit detection of amylose. D, whole starch
from tubers of a control line (filled diamond) and the SSIII
antisense line 18 (dash). E, amylose-enriched
preparations from control starch (filled diamond) and starch
of the SSIII antisense line 18 (dash).
View larger version (13K):
[in a new window]
Fig. 5.
Analysis of chain-length distribution of the
short chains of amylopectin by fluorophore-assisted PAGE.
Debranched, fluorophore-derivatized material was subjected to gel
electrophoresis in an Applied Biosystems DNA sequencer. Data were
analyzed with Genescan software. Areas of peaks representing chains of
between 6 and 37 glucose units were summed, the areas of individual
peaks were expressed as a fraction of this sum, and the means of these
values were calculated from replicate starch samples from each
transgenic line. For each chain length (dp: degree of polymerization),
the mean value from an antisense line was subtracted from the mean
value for the control line, to give the percentage molar difference.
Thus the zero line on the y axis represents the chain-length
distribution of the control line (Desiree), and each plotted
line represents the percentage molar difference from that control
for a transgenic line. Values are means ± S.E. of four values,
each from a separate sample of a bulk starch preparation (tubers from
several plants) for that line. Control and transgenic lines appearing
on one graph were grown in the same greenhouse at the same time.
A, starch from SSIII antisense lines 18 (square)
and 9 (circle). These data are the same as those in Fig. 3
of (11). B, starch from SSIII/GBSS antisense line
Rot1.4.
View larger version (17K):
[in a new window]
Fig. 6.
Separation of amylopectin chains by
size-exclusion HPLC. Solubilized, debranched amylopectin was
subjected to HPLC on three size-exclusion columns connected in series.
Fractions collected from the column were incubated with
amyloglucosidase then assayed for glucose. The glucose content of each
fraction is expressed as a percentage of the total glucose recovered
from the column. For each line, three to five separate samples of
amylopectin, each prepared from starch from a different plant, were
subjected to chromatography. Data points are the mean ± S.E.
(bars) of values from these analyses. Where S.E.
bars are not shown, they were smaller than the data point
symbol. Peaks referred to in the text are marked as I,
II, III, and IV. A,
amylopectin from starch of a control line (Desiree, circle).
Pullulan standards of known molecular mass (squares,
right axis) were subjected to chromatography on the same
column system to allow estimation of the masses of amylopectin chains.
B, amylopectin from three SSIII antisense lines: 9 (diamond), 18 (square), and 26 (cross). C, amylopectin from the SSIII/GBSS
antisense lines Rot1.4 (diamond) and Rot1.1
(square).
Amounts of sugar nucleotides, ATP and ADP in developing tubers
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 fresh weight
equates to a plastidial concentration of 0.02 mM. Estimates
of the Km (ADP-glucose) of soluble,
amylopectin-synthesizing starch synthases range from 0.06 to 0.6 mM (28, 38). The Km (ADP-glucose) of
GBSS from potato tubers is about 5 mM when measured on
isolated starch granules (39) and about 1.2 mM when
measured on the recombinant isoform expressed in a soluble form in
Escherichia coli (38). Thus increases in ADP-glucose levels
are expected to increase flux into starch in general (probably
explaining why the rate of starch synthesis in the transgenic tubers is
near-normal despite drastic reductions in starch synthase activity) and
to have a particularly marked effect on flux through GBSS. Given the
evidence that GBSS can synthesize long chains in amylopectin in species
other than potato, and in isolated starch granules, it seems reasonable
to suppose that the additional long chains in amylopectin from SSIII
antisense lines result from increased flux via GBSS within the granule
matrix in response to elevated ADP-glucose levels. The same explanation
may hold for the increased apparent molecular size of amylose in the
SSIII antisense lines.
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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 P. Geigenberger Regulation of sucrose to starch conversion in growing potato tubers J. Exp. Bot., January 3, 2003; 54(382): 457 - 465. [Abstract] [Full Text] [PDF]
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 A. Edwards, J.-P. Vincken, L. C. J. M. Suurs, R. G. F. Visser, S. Zeeman, A. Smith, and C. Martin Discrete Forms of Amylose Are Synthesized by Isoforms of GBSSI in Pea PLANT CELL, August 1, 2002; 14(8): 1767 - 1785. [Abstract] [Full Text] [PDF]
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