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Plant Physiol, June 2002, Vol. 129, pp. 516-529
Starch Synthesis in Arabidopsis. Granule Synthesis, Composition,
and Structure1
Samuel C.
Zeeman,2 *
Axel
Tiessen,3
Emma
Pilling,
K. Lisa
Kato,
Athene M.
Donald, and
Alison M.
Smith
John Innes Centre, Colney Lane, Norwich NR4 7UH, United Kingdom
(S.C.Z., A.T., E.P., A.M.S.); and The Cavendish Laboratory,
Department of Physics, University of Cambridge, Madingley Road,
Cambridge CB3 0HE, United Kingdom (K.L.K., A.M.D.)
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ABSTRACT |
The aim of this work was to characterize starch synthesis,
composition, and granule structure in Arabidopsis leaves. First, the
potential role of starch-degrading enzymes during starch accumulation was investigated. To discover whether simultaneous synthesis and degradation of starch occurred during net accumulation, starch was
labeled by supplying 14CO2 to intact,
photosynthesizing plants. Release of this label from starch was
monitored during a chase period in air, using different light
intensities to vary the net rate of starch synthesis. No release of
label was detected unless there was net degradation of starch during
the chase. Similar experiments were performed on a mutant line
(dbe1) that accumulates the soluble polysaccharide, phytoglycogen. Label was not released from phytoglycogen during the
chase indicating that, even when in a soluble form, glucan is not
appreciably degraded during accumulation. Second, the effect on starch
composition of growth conditions and mutations causing starch
accumulation was studied. An increase in starch content correlated with
an increased amylose content of the starch and with an increase in the
ratio of granule-bound starch synthase to soluble starch synthase
activity. Third, the structural organization and morphology of
Arabidopsis starch granules was studied. The starch granules were
birefringent, indicating a radial organization of the polymers, and
x-ray scatter analyses revealed that granules contained alternating
crystalline and amorphous lamellae with a periodicity of 9 nm. Granules
from the wild type and the high-starch mutant sex1 were
flattened and discoid, whereas those of the high-starch mutant
sex4 were larger and more rounded. These larger granules contained "growth rings" with a periodicity of 200 to 300 nm. We
conclude that leaf starch is synthesized without appreciable turnover
and comprises similar polymers and contains similar levels of molecular
organization to storage starches, making Arabidopsis an excellent model
system for studying granule biosynthesis.
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INTRODUCTION |
The Arabidopsis leaf is an excellent
system in which to study starch granule biosynthesis for several
reasons. First, starch accumulates in large amounts over a short
period; up to one-half of the carbon assimilated through photosynthesis
is stored as starch during the light period. As a consequence, it is
possible to analyze the composition and structure of starch made over a period of a few hours by a defined set of enzymes. In contrast, starch
synthesis in storage organs occurs over a long developmental period,
during which there are usually considerable changes in the complement
of starch-synthesizing enzymes (Smith and Martin, 1993 ; Burton et al.,
1995) and in overall cellular conditions. Second, the rate of starch
synthesis in leaves can be controlled by altering the irradiance and
measured accurately by supplying 14CO2. Third, our knowledge
of the complete genome sequence of Arabidopsis and the availability of
transposon and T-DNA-tagged populations enables specific knockout
mutations to be obtained for all of the putative enzymes of starch
synthesis and degradation (Thorneycroft et al., 2001). Despite the
suitability of leaf starch as a model system, relatively little is
known about its synthesis, composition, and structure, compared with
starches from storage organs. To address this, we have studied three
major aspects of the synthesis of Arabidopsis starch where differences
between leaves and storage organs have been reported, or might be expected.
First, we investigated whether leaf starch is subject to turnover
during its synthesis. Turnover (the simultaneous occurrence of
synthesis and degradation) may be expected to affect both the amount
and nature of the starch. However, it is not known whether such
turnover occurs. In storage organs, where starch synthesis and starch
degradation usually occur in different developmental phases, the
enzymes of starch degradation may not be present during the phase of
starch accumulation. The only reported example of turnover in storage
organs is in transgenic potatoes (Solanum tuberosum)
in which the flux of carbon into starch was increased 6-fold by
elevating ADP-Glc pyrophosphorylase activity (Sweetlove et al., 1996).
However, little, if any, turnover was observed in the wild-type
tubers. This is consistent with earlier findings (Dixon and ap Rees,
1980). In contrast to storage organs, leaf starch is remobilized each
night and the enzymes responsible for starch degradation are present
and may be active in the chloroplast during starch synthesis in the
day. Under some conditions, starch degradation has been shown to occur
in leaves during the light (Fondy et al., 1989; Servaites et al.,
1989; Hausler et al., 1998). However, it is not clear whether
any degradation occurs simultaneously with synthesis. To study this, we
conducted 14C pulse-chase labeling experiments to
determine whether label incorporated during the pulse was released
during the subsequent chase period.
Second, we examined factors that influence amylose content in leaf
starch. Estimates of amylose content for leaf starch are scarce.
Typically, values of approximately 15% or less have been found,
whereas most storage starches contain between 20% and 30% amylose. In wild-type Arabidopsis leaves, the amylose content of
the starch is low (Zeeman et al., 1998b), but in starch-excess mutants,
increased amylose contents have been reported (Critchley et al., 2001;
Yu et al., 2001). We have established a robust method for the
measurement of amylose content of Arabidopsis starch and used this
method to investigate the conditions of amylose synthesis in wild-type
leaves and in starch-excess mutant lines.
Third, we investigated starch granule size, shape, and structure in
leaves. Granules from leaves are generally reported to be very small
(Badenhuizen, 1969) and discoid, whereas those from many storage organs
are larger (typically 15-100 µm; Jane et al., 1994) and roughly
spherical or oval in shape. Granules of storage starches possess two
main levels of internal structure, created by the organization of
amylopectin molecules (French, 1984; Jenkins et al., 1993).
Alternating, concentric crystalline and amorphous lamellae with a
periodicity of 9 nm make up semicrystalline zones. These alternate with
amorphous zones with a periodicity of a few hundred nanometers.
Although there are indications that leaf starch granules contain at
least some crystalline structures (Buttrose, 1963; Waigh, 1997), it
is not known to what extent they possess the levels of organization
seen in storage starches.
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RESULTS |
Starch Turnover
Starch Synthesis Is Not Accompanied by Significant Turnover
To discover whether starch turnover occurs during periods of
starch accumulation in Arabidopsis leaves, we performed pulse-chase experiments. A pulse of
14CO2 was supplied to
photosynthesizing wild-type plants and the incorporation of label into
starch measured. The 14CO2
was then removed and the plants maintained for a chase period of 5 h in the light in air. After the chase, the label in starch was
measured again to determine whether any of the starch made during the
pulse had been degraded. We found that none of the 14C incorporated during the pulse was released
during the chase (Table I). However, the
rate of starch synthesis during the chase was high. We reasoned that
the starch labeled during the pulse might rapidly be buried by newly
synthesized starch during chase, perhaps rendering it inaccessible to
degradative enzymes. This would restrict the release of
14C during the chase if turnover occurred only on
newly synthesized starch. To reduce the rate of burial of labeled
starch during the chase, and thus increase the chances of detecting any
turnover, the experiment was repeated but with a large reduction in
light intensity at the end of the pulse to limit the rate of starch synthesis during the chase. Although the rate of starch synthesis was
reduced by this treatment, there was still no detectable release of
14C from starch during the pulse (Table I). In a
further experiment, light intensity after the pulse was lowered to a
point at which starch content during the chase showed a decline rather
than an increase. In this case, as expected, there was a significant
loss of 14C from starch during the chase (Table
I).
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Table I.
The distribution of 14C in Arabidopsis
leaves during pulse and chase experiments
Plants were supplied with 14CO2 (400-600 µL
L 1, 1.25-1.88 MBq mmol1, and 170 µmol
photons m2 s1) for either 0.5 or 1 h.
The 14CO2 was then removed and the plants
allowed to photosynthesize in air for a further 5 to 6.5 h.
Samples were harvested and killed in boiling 80% (v/v) ethanol (wild
type) or frozen in liquid N2 (dbe1). Starch
content and the distribution of label were determined as described in
"Materials and Methods." Values are the mean ± SEs of four replicate samples, each comprising the leaves
of a single plant (n.d., not determined).
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Phytoglycogen Synthesis Is Not Accompanied by Significant
Turnover
Failure to observe loss of label from starch granules during a
chase period does not necessarily imply that starch-degrading enzymes
are inactive during the light period. It is possible that once in a
semicrystalline, granular form, the glucan is no longer susceptible to
attack from most enzymes. We reasoned that a soluble  -1,4-,
-1,6-linked glucan might be more sensitive to the actions of
starch-degrading enzymes during its synthesis than starch. Therefore,
we performed similar pulse-chase experiments on the Arabidopsis mutant
dbe1, which lacks an isoform of the debranching enzyme
isoamylase (Zeeman et al., 1998b). This mutant accumulates the soluble,
highly branched glucan phytoglycogen, which does not form
semicrystalline granules but remains soluble in the stroma of the
chloroplast. It is accumulated together with small amounts of starch
during photosynthesis and degraded during the subsequent dark period.
During the pulse, starch and phytoglycogen were labeled in the ratio
5:1, reflecting the relative rates of synthesis of the two glucans in
dbe1 leaves (Zeeman et al., 1998b). No
14C was lost from either starch or phytoglycogen
during the chase (Table I).
Amylose Content
Measurement of the Amylose Content of Leaf Starch
To investigate the amylose content of leaf starch,
solubilized starch was fractionated by gel permeation chromatography
(GPC) on a column of Sepharose CL2B (Fig.
1). Starch from wild-type plants eluted
as two peaks: an initial amylopectin-containing peak, with a wavelength
of maximal absorbance when complexed with iodine
( max) of 550 nm, and a second
amylose-containing peak with a max of 585 nm.
The max value for the amylose peak is substantially lower than that reported for amylose from other species
(max usually greater than 600 nm), suggesting
either that amylose from Arabidopsis leaves is more branched than that from other species, or that the amylose peak contains branched glucans
in addition to amylose. Two approaches were taken to distinguish between these possibilities. First, fractions from the amylose peak
from wild-type starch were pooled and subjected to butanol precipitation, a treatment that precipitates linear but not branched glucans. The max of the precipitated material
was 620 nm. Second, the fractionation was repeated with starch from the
sex1 mutant of Arabidopsis, a starch-accumulating mutant in
which the starch has a high amylose content (Yu et al., 2001). The
max of the amylose peak from this mutant was
620 nm.
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Figure 1.
Separation of amylose and amylopectin fractions of
Arabidopsis starch using Sepharose CL2B chromatography. Starch from the
wild type (black symbols) and the mutant line sex1 (white
symbols) was isolated from batches of 200 plants harvested at the end
of the photoperiod. Samples of this starch were solubilized and applied
to the column. Values are the means ± SEs
of three samples. A, Fractions were analyzed to determine the
absorbance of the glucan-iodine complex at 595 nm (circles; inset;
y axis enlarged for clarity). The absorbances were summed
and each then divided by the total to give a normalized trace. The
wavelength of maximum absorption of the glucan-iodine complex
(max; triangles) was also determined for each
sample. B, The glucan content of each fraction was determined by
treatment with amyloglucosidase and -amylase and measurement of
released Glc (inset; y axis enlarged).
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These results suggest that the amylose from Arabidopsis starch is
similar to that found in storage starches. The amylose-containing peak
in the wild type consists of both amylose with a
max of 620 nm and branched glucans with a
max similar to that of amylopectin, leading to
an overall max of 585 nm. The higher
max of the amylose-containing peak from
sex1 starch reflects the fact that most of the material in
the peak is amylose. In both samples, the max
of the glucan tail following the amylose peak fell to values
approaching that of amylopectin, further indicating the presence of
small amounts of branched glucan in these fractions.
Using GPC to examine the amylose content of starch yields useful
qualitative information. However, due to the presence of the small
amounts of branched material in the amylose-containing fractions, it
was not possible to use this method to quantify accurately the amylose
content, particularly in samples containing little amylose. Therefore,
we established a separate method for determining the amylose content
based on the different iodine-binding capacities of the two polymers
(Hovenkamp-Hermelink et al., 1988). Pure amylose and amylopectin were
prepared from a bulk preparation of starch, derived from the wild type
and starch-excess mutant lines, using Sepharose CL2B chromatography
followed by butanol precipitation.
Standard curves of the absorbance of the iodine-polymer complexes were
used to generate the following equation to calculate amylopectin to
amylose ratios from mixed samples:
The wavelengths 700 and 525 nm were used to give a wider range of
ratios than possible when using the max for
amylose and amylopectin. The calculated relationship between amylose
content and the ratio of A700 to
A525 is shown in Figure
2. Mixtures of purified amylose and
amylopectin gave the predicted A700 to
A525 ratios. We then used this method and
GPC to investigate factors influencing the amylose content of
Arabidopsis starch.
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Figure 2.
Relationship between the percentage of amylose and
the ratio of the absorbance of the glucan-iodine complex at 700 and 525 nm. Known amounts of purified amylose or purified amylopectin from
Arabidopsis were dissolved, mixed with iodine solution, and the
absorption spectra for the polymer-iodine complex established. The
theoretical relationship between the absorbance at 700 and at 525 nm
was calculated for mixtures of the two polymers (solid line).
This relationship was tested by measuring the absorbance ratios
of different mixtures of amylose and amylopectin containing 5 (squares), 10 (triangles), and 20 (circles) µg of total glucan.
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The Amylose Content of Starch Is Related to Leaf Starch
Content
To discover how the amylose content of starch related to the
pattern of starch synthesis and the starch content of the leaf, we
investigated the amylose contents of starches from leaves with different starch contents either wild-type leaves kept in the light
for extended periods, or leaves from mutant plants with lesions
affecting the pathway of starch degradation. First, we measured the
amylose content of starch from batches of wild-type plants grown in
controlled conditions. At the end of a 12-h photoperiod, the amylose
content of the starch was 6% ± 1.7% (n = 4, mean ± SE). When a batch of wild-type
plants was transferred from normal light-dark conditions to continuous
light, they accumulated starch to very high levels (Fig.
3A). The amylose content rose from 4% after 12 h (the start of the extended light period) to 13%
after 84 h, 20% after 180 h, and 25% after 220 h in
the light. GPC analysis confirmed the increase in the
low-Mr, amylose-containing fractions (Fig.
3B).
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Figure 3.
Influence of conditions of leaf starch synthesis
on the amylose content of the starch. A, Wild-type plants were
transferred from a diurnal light regime to continuous light and the
starch content measured at intervals. Four plants were harvested and
treated as one sample (white symbols). The results correspond well to
data from a similar experiment conducted previously (black symbols;
Critchley et al., 2001). B, Amylose and amylopectin were separated by
Sepharose
CL2B chromatography from starch extracted from plants after 12 (white triangles), 84 (gray triangles), and 180 (black triangles) h in
the light. The absorbance of the glucan-iodine complex at 595 nm was
determined. C, Amylose and amylopectin from starch extracted from
wild-type (white circles), sex1 (gray circles), and
sex4 (black circles) plants at the end of a normal
photoperiod, as described in B.
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Two starch degradation mutants were also used in this study. The
sex1 mutant lacks a homolog of the potato R1 protein,
involved in the phosphorylation of starch (Yu et al., 2001), whereas
the sex4 mutant is deficient in chloroplastic endoamylase
(Zeeman et al., 1998a). The starch content of leaves of sex1
and sex4 is much higher than in the wild type (5- and
3-fold, respectively; Trethewey et al., 1994; Zeeman et al., 1998a).
When harvested at the end of a normal photoperiod, the starch from
sex1 and sex4 contained 21% ± 2.5%
(n = 3) and 33% ± 7% (n = 3)
amylose, respectively (see also Fig. 3C).
There is a gradual accumulation of starch in sex1 and
sex4 leaves during development (Zeeman and ap Rees, 1999).
In wild-type plants, leaves of all ages contain a similar amount of
starch at the end of the day; in the mutants, the oldest leaves contain the most starch, whereas the youngest, developing leaves contain little
or no more than the wild type. To determine whether the high-amylose
starch is synthesized in all tissues in the sex mutants, we
extracted starch from different-aged leaves of wild-type and sex4 plants and determined the amylose content (Table
II). The amylose content of the starch
from the wild type was low in all leaves irrespective of age, whereas
in sex4 the amylose content increased as the leaves aged,
correlating with the increase in starch content.
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Table II.
The amylose content of leaves of different ages of
wild-type and sex4 leaves
Four plants of the wild type and four of sex4 were harvested
at the end of the day and the leaves divided into six fractions.
Fraction 1 comprised the three youngest leaves (not analyzed); fraction
2, the next three youngest leaves; and so on. Fraction 6 contained all
the remaining, oldest leaves of the plant. Starch was extracted from
each fraction and the amylose content determined using the iodine-based
method described in "Materials and Methods."
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Granule-Bound Starch Synthase (GBSS) Content of Leaf
Starch
We investigated whether the different amylose contents of the
starches described above may be attributable to different contents of
the starch synthase isoform responsible for amylose synthesis, GBSS.
The identity of the GBSS protein on SDS-polyacrylamide gels of
granule-bound proteins from leaf starch was established by matrix-assisted laser-desorption ionization (MALDI)-time of
flight mass spectroscopy. Tryptic fragments of a major protein
of 59 kD (the predicted molecular mass of the mature GBSS protein
encoded in the Arabidopsis genome, chromosome locus At1g32900) were
analyzed. Comparison of the pattern of peptides using the MASCOT search engine (Matrix Science;
http://www.matrixscience.com/cgi/index.pl?page=/search_intro.html) confirmed that this protein was GBSS (probability-based Mowse score
215, coverage of fragments 33%).
Coomassie Blue-stained gels of granule-bound proteins revealed
that the GBSS content of starch from sex1 was slightly
greater than that of the wild type, whereas that of sex4 was
slightly lower (Fig. 4A). However, to
determine the GBSS content of the leaves on a fresh weight basis,
proteins derived from the insoluble material of leaves were separated
and analyzed by immunoblotting, using an antibody raised to the pea
embryo GBSS. This antibody recognized a single, 59-kD band on the blots
and densitometry measurements of the blot revealed a linear
relationship between the intensity of the band amount of sample loaded
(Fig. 4B). Immunoblots of replicate samples of insoluble material from
wild-type, sex1, and sex4 leaves were then
performed, revealing that the GBSS content of both sex1 and
sex4 was greater than the wild type on a fresh weight basis
(Fig. 4C). Densitometry readings of this blot revealed that compared
with the wild type, sex1 and sex4 leaves had 5- and 2-fold increase in GBSS content, respectively.
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Figure 4.
GBSS content of starch and leaves of wild-type,
sex1, and sex4. A, Starch was isolated from
wild-type, sex1, and sex4 leaves at the end of
the photoperiod. Granule-bound proteins were separated by SDS-PAGE and
stained using colloidal Coomassie Blue. B, Proteins were extracted from
the insoluble fraction of sex1 leaves harvested at the end
of the photoperiod. An immunoblot was performed using an antibody
raised against the pea (Pisum sativum) embryo GBSS
and the relationship between the amount of sample loaded and
densitometry measurements plotted. C, Immunoblot of proteins extracted
from the insoluble fraction of leaves from wild-type, sex1,
and sex4 leaves harvested at the end of the photoperiod.
Each sample comprised the leaves of a single plant.
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Granule Size, Shape, and Structure
Scanning electron microscopy of starch granules from wild-type
leaves showed that they were irregularly discoid in shape and increased
significantly in size when plants were kept in continuous light for
long periods (Fig. 5). At the end of a
normal photoperiod, granules were approximately 1 to 2 µm in diameter
and 0.2 to 0.5 µm thick. After 180 h in continuous light, they
had increased to approximately 2 to 3 µm in diameter and 0.4 to 0.6 µm thick.
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Figure 5.
Scanning electron micrographs of starch granules
isolated from plants at the end of the photoperiod (A, D, and E) or
after a period of continuous light (B and C). The bar represents 2 µm.
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To look for factors that influence granule size and shape, we
examined starch from the starch-excess mutants sex1 and
sex4. Granules from sex1 were larger, but similar
in shape to the wild type (Fig. 5D). However, granules of
sex4 were strikingly different from wild-type granules in
that they were much larger in both diameter (up to 6 µm) and
thickness (1-4 µm) and more were more regular in outline (Fig. 5E).
In this respect, the sex4 granules resembled starch from
storage organs. We investigated whether the alteration in the size and
shape of granules in the sex4 mutant was correlated with
changes in the chain length distribution of amylopectin. The shorter
chains of amylopectin from Arabidopsis and pea leaf starch show a much
more pronounced polymodal distribution of lengths than those of storage
starches (Tomlinson et al., 1997; Zeeman et al., 1998a). We found that
amylopectin from sex4 had increased numbers of chains
between six and 11 Glc residues in length and fewer between 19 and 29 residues compared with wild-type amylopectin (Fig.
6). However, these differences were small
and the chain length distribution still showed the characteristic leaf-type profile.
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Figure 6.
Analysis of the chain length distribution of
amylopectin from the wild type and from sex4 using
fluorophore-assisted PAGE. Starch samples were solubilized, debranched
with isoamylase, and derivatized with the fluorophore
8-amino-1,3,6-pyrenetrisulphonic acid. Chains of different
lengths were separated by gel electrophoresis in a DNA sequencer
(PE-Applied Biosystems, Foster City, CA) and the data analyzed
using GeneScan 672 software (PE-Applied Biosystems). Peak areas
of chains between three and 47 Glc residues in length were summed and
the individual peak areas expressed as a percentage of the total. Three
replicate samples of debranched, derivatized material were prepared
from bulk starch extracted from batches of 200 wild-type (A) and
sex4 (B) plants. The values are the means ± SEs of measurements made on these samples. To
obtain a percentage molar difference plot (C), wild-type values were
subtracted from those of sex4. The SEs
were added together.
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When viewed under polarized light, large starch
granules from Arabidopsis were birefringent, giving a typical
"Maltese cross" pattern (Fig. 7).
This indicates a high degree of radial molecular orientation within the
granule and is a well-documented feature of storage starches. We used
small-angle x-ray scattering (SAXS) to determine whether, as in storage
starches, Arabidopsis amylopectin is organized within the granule into
alternating crystalline and amorphous lamellae (French, 1984; Jenkins
et al., 1993). Figure 8 shows the
scattering profile for wild-type Arabidopsis starch with a peak in
scattering intensity at a q value of 0.06, indicating a crystalline
structure with periodicity of 9 nm (Jenkins et al., 1993).
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Figure 7.
Light micrographs of starch granules viewed under
polarized light. Starch granules from potato cv Desiree tuber (A) and
from wild-type Arabidopsis plants after 180 h of continuous light
(B) were suspended in water and digital images captured using Image-Pro
Plus software (Media Cybernetics Inc., Silver Spring, MD). The
bar represents 5 µm.
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Figure 8.
SAXS profile for wild-type Arabidopsis starch.
Bulk starch was extracted from plants after a period of 84 h of
continuous light. A low-divergence, high-intensity beam of
radiation ( = 1.5 Å) was focused onto starch samples, which
were in the form of a 50% (w/w) slurry with water. Three replicate
samples were analyzed and the results are the means ± SEs.
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We investigated whether leaf starch granules consist of alternating
semicrystalline and amorphous zones (growth rings) using a technique
developed to visualize these zones in storage starches (Pilling, 2001).
Granules were cracked open by mechanical grinding of starch suspensions
frozen in liquid nitrogen and incubated with -amylase to
preferentially digest amorphous regions. No growth rings were visible
in granules from wild-type leaves, though the granules were partially
digested during the incubation (Fig. 9, A
and B). However, the treatment revealed growth rings in granules from
sex4 leaves (Fig. 9, C and D). These had a periodicity of about 0.2 to 0.3 µm, a distance almost the same as the total
thickness of wild-type granules.
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Figure 9.
Scanning electron micrographs of partially
digested starch granules from the wild type (A and B) and
sex4 (C and D). Granules were cracked by grinding in liquid
nitrogen and partially digested with -amylase to reveal internal
growth ring structures.
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DISCUSSION |
Starch Is Accumulated without Turnover
We found no evidence for turnover during starch accumulation
despite conducting experiments designed specifically to reveal such a
process. Radiolabel incorporated into starch during a pulse of
14CO2 was not subsequently
released during a chase in the light in air. A similar result was
observed in pea leaves (Kruger et al., 1983). There are several
possible explanations for this result. First, label released by
degradative enzymes (as Glc or Glc-1-P) may be reincorporated into
starch. This seems unlikely because released Glc would be transported
to the cytosol and it is doubtful that the label would reenter the
plastid for starch synthesis (Weber et al., 2000). Glc-1-P released
through the action of starch phosphorylase could be reincorporated, but
phosphorolytic activity in Arabidopsis chloroplasts is low (Lin et al.,
1988) and it is unlikely that Glc-1-P would be a major product of
degradation. Alternatively, malto-oligo-saccharides released by
turnover might be transferred to nascent amylopectin molecules by
disproportionating enzyme (D-enzyme) as suggested for
Chlamydomonas reinhardtii by Colleoni et al. (1999).
This also seems unlikely because in Arabidopsis leaves, D-enzyme does
not participate in starch synthesis in this way (Critchley et al.,
2001). Second, the radiolabeled starch may not be accessible to the
degrading enzymes due to the deposition of unlabeled starch on top of
it. Reducing the light intensity during the chase to slow the
deposition of unlabeled material did not result in detectable loss of
label from the starch. If appreciable turnover were occurring, it
should be more readily detectable using these conditions. However,
label was released from the starch when the light was reduced to the
extent that starch synthesis stopped and breakdown occurred. Third, the
starch-degrading enzymes may not be active. This seems most likely
because there is good evidence that the process of starch mobilization
in leaves is regulated (Trethewey and Smith, 2000). For example, starch degradation in leaves at night often commences only after a lag, rather
than on the light-to-dark transition (Gordon et al., 1980; Fondy and
Geiger, 1982).
It is possible that the control of starch degradation is exercised at
the point where the starch granule is attacked to liberate soluble
glucans. This step is most likely catalyzed by -amylase because no
other enzyme has been convincingly shown to attack intact starch
granules. However, our results with the phytoglycogen-accumulating mutant dbe1 show that even when glucan is accumulated in a
soluble form, no turnover is detectable, suggesting that other
degradative enzymes may also be tightly regulated.
Amylose Content of Leaf Starch
We confirmed the earlier observation that Arabidopsis leaf
starch has a very low amylose content when grown in a normal diurnal cycle. This contrasts with a study of leaf starch composition in
tobacco, in which an amylose content of between 15% and 20% was found (Matheson, 1996). However, tobacco differs from
Arabidopsis because, in addition to cycling in a diurnal fashion, a
background level of storage starch accumulates in leaves as they mature
(Matheson and Wheatley, 1962). When Arabidopsis plants were transferred from a diurnal cycle to continuous light, far more starch was synthesized and this starch had a higher proportion of amylose. Thus,
the balance of synthesis shifts from almost exclusively amylopectin
toward a significant proportion of amylose over time. In addition to
this increased amylose synthesis in wild-type plants, high-amylose
starch is also synthesized in the mutants sex1 and sex4, which accumulate appreciably more starch than
wild-type plants.
Although it is not clear from our current results what
determines the amylose content of leaf starch, a number of factors may
be important. The increase in amylose in sex1 and
sex4 was accompanied by an increase in the GBSS content of
the leaf, whereas in both mutants, soluble starch synthase activity is
similar to (or lower than) that of the wild type (Caspar et al., 1991;
Zeeman et al., 1998a). Therefore, it is possible that the higher ratio of GBSS to soluble starch synthase activity may cause the increased amylose in these lines. A similar change in the ratio of GBSS to
soluble starch synthase could explain why wild-type plants transferred
to continuous light accumulate starch with high amylose. Furthermore,
in the mutant lines, starch is synthesized during the day but not
completely degraded during the night. As a consequence, starch builds
up over a number of diurnal cycles (Zeeman and ap Rees, 1999). It is
plausible that GBSS trapped within the undegraded starch may remain
active and may synthesize more amylose during each light period, none
of which would be degraded during the dark. This would also lead to an
accumulation of amylose correlating with the accumulation of starch.
This hypothesis is supported by the amylose content of the starch from
sex4 leaves of different ages. The amylose content in young
leaves is only 5%, whereas in the oldest leaves, which have
experienced many diurnal cycles, the starch contains 34%
amylose. In the wild type, all of the starch is degraded each night,
including amylose, so the amylose content would not increase in this
way unless the diurnal conditions were altered.
The increase in the GBSS content in the mutants cannot
account in full for the increase in amylose content. Starch from
sex4 had the highest amylose content but the increase in the
GBSS content in this mutant is not as marked as in sex1,
starch from which has a lower amylose content. The explanation may lie
in the difference in granule morphology between the two lines. It has
been suggested that amylose is preferentially synthesized in the
amorphous zones of starch granules (Blanshard, 1987). Granules from
sex4 are large and contain alternating semicrystalline and
amorphous zones similar to storage starches (see below), whereas
wild-type and sex1 granules may be too small to contain
these amorphous zones. Thus, amylose may be more readily synthesized in
sex4 granules than in wild-type or sex1 granules.
However, other factors such as the supply of substrates are also known
to influence amylose synthesis (Van den Koornhuyse et al., 1996; Clarke
et al., 1999) and may also contribute to the observed differences.
Structure and Morphology of Leaf Starch Granules
Starch granules of wild-type plants were flat and discoid. Even
when plants were transferred to continuous light to promote further
starch synthesis, the granules increased in size but did not alter
radically in appearance. The granules from the sex1 plants,
which accumulate up to 5-fold more starch than the wild type, were also
flat and discoid. It is tempting to speculate that the shape of the
granules is defined by the spaces within the chloroplast, between
layers of thylakoid membranes. However, sex4 granules were
much larger and thicker than all the other granules, even though this
mutant only accumulates 3 times as much starch as the wild type.
The cause of the different granule morphology, and how it relates to
the enzymatic deficiency in this mutant (reduced plastidial
endoamylase), is not yet clear.
The fundamental structures and layers of organization in
starch granules of Arabidopsis leaves are similar to those found in
storage starches. The birefringence of the granules indicates radial
orientation of the constituent polymers and the amylopectin forms a
repeated crystalline structure with 9-nm periodicity. The large
granules from sex4 also have an internal growth ring structure similar to granules from storage organs. Our results demonstrate that amylopectin with a chain length distribution characteristic of leaves can form granules with striking similarities in appearance, structure, and amylose content to starches from storage organs.
We conclude that, despite the presence of starch-degrading enzymes in
chloroplasts, no degradation of starch was detected during periods of
net starch synthesis. The starch granules themselves were found to
contain varying amounts of amylose, depending on the conditions of
synthesis, and exhibited very similar levels of structural
organization to granules from non-photosynthetic tissues. We suggest
that the mechanisms underlying the synthesis of Arabidopsis starch
granules are broadly similar to those of seeds, tubers, and the
leaves of other higher plants. These findings show that the
analysis of starch biosynthesis in Arabidopsis may have valuable
implications for understanding starch in commercially important crop species. Furthermore, because the factors that determine granule size, shape, and number are not known in any species,
Arabidopsis mutants such as sex1 and sex4, in
which granule morphology and number are altered, represent useful tools
with which to investigate these questions.
| |
MATERIALS AND METHODS |
Materials
All chemicals were obtained from Sigma Chemical Co. (Poole,
Dorset, UK). Radioisotopes were supplied by Amersham Pharmacia Biotech
(Amersham, Bucks, UK).
Plants and Growth Conditions
Wild-type Arabidopsis plants (ecotype Columbia) and the mutants
sex1-1 (Caspar et al., 1991; Zeeman and ap Rees, 1999;
Yu et al., 2001), sex4-1 (Zeeman et al., 1998a; Zeeman
and ap Rees, 1999) and dbe1-1 (Zeeman et al., 1998b)
were grown in peat-based compost in a growth chamber with a
12-h-light/12-h-dark cycle. The irradiance was 170 µmol photons
m2 s1, the temperature 20°C, and the
humidity 75%, unless otherwise specified. Wild-type and
dbe1-1 plants were used after 4 to 5 of weeks growth,
whereas sex4-1 plants were used after 5 to 6 weeks of
growth and sex1-1 plants after 6 to 7 weeks. At these ages the plants were at equivalent developmental stages.
In Vivo Labeling
To label starch with 14C in vivo, photosynthesizing
plants (total shoot mass of approximately 5 g) were exposed to
14CO2 with a specific activity between 1.25 MBq mmol1 and 1.88 MBq mmol1 and a
CO2 concentration of either 400 µL
L1 (30-min pulses) or 600 µL L1 (1-h
pulses). The plants were sealed in a Perspex chamber (12.1-L volume)
and 14CO2 liberated by acidification of sodium
[14C]bicarbonate. The light intensity was the same as
that used to grow the plants, unless specified, and the heat load was
alleviated using a water trap. Considering the rate of photosynthesis
of Arabidopsis plants growing under these conditions (Zeeman and ap
Rees, 1999), less than 50% of the CO2 supplied would have
been incorporated during a 1-h pulse. As a consequence, the
CO2 concentration would have remained above 300 µg
mL1 in all of the experiments. At the end of the pulse
period, the 14CO2 was removed, the chamber
opened, and pulse samples harvested. In the pulse and chase
experiments, chase samples were left in the chamber, through which air
was pumped at a rate of 1.2 L min1. Wild-type plants were
killed in boiling 80% (v/v) aqueous ethanol, whereas
dbe1 plants were frozen in liquid N2. Starch
content was determined by hydrolyzing the starch with -amylase and
amyloglucosidase and assaying released Glc as described by Zeeman et
al. (1998a).
The 14C in starch in wild-type plants was determined as
described in Zeeman et al. (2002). Starch and phytoglycogen in
dbe1 plants were extracted by homogenizing leaves in an
ice-cold aqueous medium because phytoglycogen, although soluble in
water, is insoluble in 80% (v/v) ethanol (Zeeman et al.,
1998b). The water-insoluble material, including starch, was removed by
centrifugation and washed twice with ice-cold extraction medium. The
soluble material and the washes were pooled and adjusted to 75% (v/v)
methanol and 1% (w/v) KCl to precipitate the phytoglycogen. This
precipitate was collected by centrifugation, redissolved in water, and
stored at  20°C. The insoluble material was washed twice further
with 80% (v/v) ethanol, resuspended in water, and stored at 20°C
(Zeeman et al., 1998b). The 14C content of the
starch, and of phytoglycogen, was determined in the same way as starch
in the wild type.
Analysis of Starch Composition and Amylopectin
Structure
Starch granules were isolated from leaves as described in
Zeeman et al. (1998a). Routine separation of amylose and amylopectin using a 9-mL Sepharose CL2B column was performed as described in Denyer
et al. (1995) except that 0.35-mL fractions were collected at a rate of
one fraction per 2 min. For improved separation, a larger column (90-mL
volume, 115-cm length, and 0.78-cm2 cross-sectional area)
was used. Starch (1 mg) was dissolved in 100 µL of 0.5 M
NaOH, applied to the column, and eluted with 10 mM NaOH.
The flow rate was 0.185 mL min1 and 2.78-mL fractions
were collected every 15 min. Each fraction was divided in two and
one-half used to determine the absorbance (at 595 nm) and the
wavelength of maximal absorbance (max) of the
polymer-iodine complex by mixing with 10% (v/v) Lugol's solution (Sigma). The other half was adjusted to pH 5 by the addition of a small
volume of 0.1 M HCl, and then lyophilized. The resultant material was dissolved in water and the glucan content measured as
described above for the determination of starch content.
For the preparation of pure amylopectin and amylose fractions, 10 to 20 mg of starch was dissolved in 1 mL of 0.5 NaOH, applied to the 90-mL
Sepharose CL2B column, and eluted with 100 mM NaOH. The two
peak fractions containing amylopectin were pooled, neutralized by
the addition of a small volume of 2 M HCl, and the glucan
content of a sample determined after digestion to Glc (described
above). The six to 10 peak fractions containing amylose were pooled,
neutralized, and the amylose precipitated as follows. After boiling for
1 h in a sealed vessel, one-quarter volume of butan-1-ol was added to the sample. The mixture was boiled for 1 h and then cooled gradually. The amylose-butanol precipitate was collected by
centrifugation and the amylose redissolved by boiling in water. To
determine the absorption spectrum of the polymer-iodine complex,
samples were mixed with 10% (v/v) Lugol's solution.
The analysis of the distribution of chain lengths using
fluorophore-assisted PAGE was performed exactly as described by Edwards et al. (1999).
Scanning Electron Microscopy of Starch Granules
Starch granules were viewed using a scanning electron microscope
(model XL 30 FEG; Phillips Electronics NV, Eindhoven, The Netherlands).
To visualize the internal structure of the starch granules, starch
preparations were washed with acetone, dried in air, and ground in a
liquid N2-cooled mortar to crack the granules. Cracked
granules were then treated with -amylase (5 units for 30 min in
0.5-mL reaction medium containing 100 mM MES-NaOH, pH 6.0)
to preferentially digest amorphous regions of the starch granules
(Pilling, 2001). The granules were collected by centrifugation, washed
three times in cold acetone (20°C), dried, and then viewed under
the scanning electron microscope.
Light Microscopy and X-Ray Diffraction
Light micrographs were obtained using a Microphot microscope
(Zeiss, Jena, Germany). Images were captured using Image-Pro Plus software. SAXS profiles were obtained at the Daresbury Laboratory (Daresbury, Cheshire, UK) as described by Jenkins and Donald
(1995).
Gel Electrophoresis, MALDI-Mass Spectroscopy, and
Immunoblotting
Starch granule-bound proteins were extracted by boiling starch
in SDS sample buffer (Laemmli, 1970; 100 mg starch mL1)
for 10 min. Gelatinized starch was removed by centrifugation and the
proteins in the supernatant resolved by SDS-PAGE as described by Denyer
et al. (1995). To determine GBSS content of fresh tissue, leaves (200 mg) were homogenized in ice-cold medium containing 100 mM
Tris, pH 7.2; 5 mM EDTA; and 1% (w/v) SDS. The insoluble material was removed by centrifugation and washed twice in extraction medium. The pellet was resuspended in 0.5 mL of SDS sample buffer and
boiled for 10 min. Insoluble material was removed by centrifugation and
proteins in the supernatant resolved by SDS-PAGE. GBSS was detected by
immunoblotting using a polyclonal antibody raised against the pea
(Pisum sativum) embryo GBSS (Smith, 1990)
according to the method described by Bhattacharyya et al. (1990).
MALDI-mass spectroscopy was performed using a Bruker Reflex III (Bruker
Daltonics, Coventry, UK). Protein bands were cut from the gel, digested
with trypsin, and prepared for mass spectroscopy using the optimal
conditions established by Speicher et al. (2000).
| |
ACKNOWLEDGMENTS |
We thank Jane Crawshaw for her assistance in handling the SAXS
data, and Mike Naldrett and Andrew Bottrill for performing the MALDI
mass spectroscopy.
| |
FOOTNOTES |
Received February 4, 2002; accepted February 25, 2002.
1
This work was supported by the Biotechnology and
Biological Science Research Council, UK (grant no. 208/D11090) and by
the Gatsby Charitable Foundation. The John Innes Centre is funded by a
competitive Strategic Grant from the Biotechnology and Biological Science Research Council.
2
Present address: Institute of Plant Sciences, University
of Bern, Altenbergrain 21, CH-3013 Bern, Switzerland.
3
Present address: Max Planck Institute for Molecular
Plant Physiology, Am Mühlenberg 1, 14476 Golm, Germany.
*
Corresponding author; e-mail sam.zeeman{at}ips.unibe.ch; fax
41-31-332-2059.
Article, publication date, and citation information can be found at
www.plantphysiol.org/cgi/doi/10.1104/pp.003756.
| |
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© 2002 American Society of Plant Physiologists
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I. J. Tetlow, M. K. Morell, and M. J. Emes
Recent developments in understanding the regulation of starch metabolism in higher plants
J. Exp. Bot.,
October 1, 2004;
55(406):
2131 - 2145.
[Abstract]
[Full Text]
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S. M. Smith, D. C. Fulton, T. Chia, D. Thorneycroft, A. Chapple, H. Dunstan, C. Hylton, S. C. Zeeman, and A. M. Smith
Diurnal Changes in the Transcriptome Encoding Enzymes of Starch Metabolism Provide Evidence for Both Transcriptional and Posttranscriptional Regulation of Starch Metabolism in Arabidopsis Leaves
Plant Physiology,
September 1, 2004;
136(1):
2687 - 2699.
[Abstract]
[Full Text]
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G. Ritte, A. Scharf, N. Eckermann, S. Haebel, and M. Steup
Phosphorylation of Transitory Starch Is Increased during Degradation
Plant Physiology,
August 1, 2004;
135(4):
2068 - 2077.
[Abstract]
[Full Text]
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R. G. Walters, D. G. Ibrahim, P. Horton, and N. J. Kruger
A Mutant of Arabidopsis Lacking the Triose-Phosphate/Phosphate Translocator Reveals Metabolic Regulation of Starch Breakdown in the Light
Plant Physiology,
June 1, 2004;
135(2):
891 - 906.
[Abstract]
[Full Text]
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A. M. Smith, S. C. Zeeman, D. Thorneycroft, and S. M. Smith
Starch mobilization in leaves
J. Exp. Bot.,
January 3, 2003;
54(382):
577 - 583.
[Abstract]
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TOP
ABSTRACT
INTRODUCTION
