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J. Biol. Chem., Vol. 277, Issue 23, 20249-20255, June 7, 2002
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From the Plant Biochemistry Laboratory and Center for
Molecular Plant Physiology, Department of Plant Biology, Royal
Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark
Received for publication, February 25, 2002, and in revised form, March 26, 2002
The formation of intermediary glucans, mature
starch, and phytoglycogen was studied using leaves of Arabidopsis
thaliana wild type and dbe mutant, which lacks
plastidic isoamylase (Zeeman, S. C., Umemoto, T., Lue, W. L.,
Au-Yeung, P., Martin, C., Smith, A. M., and Chen, J. (1998)
Plant Cell 10, 1699-1711). A new approach to the study of
starch biosynthesis was developed based on "very short pulse"
labeling of leaf starch through photosynthetic fixation of
14CO2. This allowed selective analysis of the
structure of starch formed within a 30-s period. This time frame is
shorter than the period required for the formation of a single
crystalline amylopectin lamella and consequently permits a direct
analysis of intermediary structures during granule formation. Analysis
of chain length distribution showed that the most recently formed outer
layer of the granules has a structure different from the mature starch. The outer layer is enriched in short chains that are 6-11 glucose residues long. Side chains with 6 glucose residues are the shortest abundant chains formed, and they are formed exclusively by transfer from donor chains of 12 glucose residues or longer. The labeling pattern shows that chain transfer resulting in branching is a rapid and
efficient process, and the preferential labeling of shorter chains in
the intermediary granule bound glucan is suggested to be a direct
consequence of efficient branching. Although similar, the short chain
intermediary structure is not identical to phytoglycogen, which is an
even more highly branched molecule with very few longer chains (more
than 40 glucose residues). Pulse and chase labeling profiles for the
dbe mutant showed that the final structure is more highly
branched than the intermediary structures, which implies that branching
of phytoglycogen occurs over a longer time period than branching of starch.
Starch is the major carbohydrate reserve in many plant storage
organs, including specialized roots, stems, and endosperms in which
starch may accumulate to high levels and be stored over long time
periods. The stored starch provides the energy reserve that allows
plants to survive under unfavorable conditions and serves as the carbon
source during germination. In contrast, transitory leaf starch is
accumulated during the photoperiod and remobilized during the following
night. Thereby, starch serves as a buffer for assimilates, which
enable leaves to continue the export of sugars during the night period.
This is essential for plant performance, as demonstrated by the poor
growth mutants, which are deficient in starch formation (1, 2) or
starch remobilization (3, 4).
Starch, deposited as well defined granules inside plastids, is composed
of two types of polysaccharides, amylose and amylopectin. Amylose
consists of predominantly linear Starch granules are highly organized structures, with densely (1.4 g
ml The molecular structure of starch directly affects its functionality,
and because starch is of immense agronomic importance as a major
constituent of many crops, there has been great interest in
understanding the details of the biosynthesis and structure of starch.
A large body of research has been directed at investigating starch
biosynthesis in major crops including cereal seeds and potato tubers,
as well as in important model systems such as Arabidopsis and the green algae Chlamydomonas. The major enzymes
responsible for starch biosynthesis and its regulation have been
identified (6, 11, 12), and the formation of the precise structure has
proved to be a complex process that requires the concerted action of
several isoforms of starch synthases and branching enzymes and most
likely also other modifying enzymes. Despite considerable progress in
understanding the molecular basis of starch formation, we still do not
know precisely the parameters defining the specific molecular
structures and the three-dimensional organization of amylopectin
molecules. A specific aspect that has prompted recent re-evaluation of
how the final starch structure may be achieved is the apparent
contribution of isoamylase. Mutants of maize (13), rice (14, 15),
Chlamydomonas (16), and Arabidopsis (4), which
all lack plastidic isoamylase activity, accumulate a more highly
branched polysaccharide, phytoglycogen, which is not organized in well
defined granules. This suggests a close relationship between the
packaging of the amylopectin molecules and the branching and chain
length distribution; that debranching may be essential in determining
the final amylopectin structure. Isoamylase has been proposed to remove
excess of highly branched molecules, and two models of its action have
been proposed. According to the first model, based on work with
Chlamydomonas, debranching is an integrated element in
formation of the final amylopectin structure. The model suggests the
formation of highly branched "preamylopectin" on the outer surface
of growing granules (16), and the final structure requires trimming of
excessive short chains followed by elongation of the remaining chains.
This "trimming" model would predict the structure of the outermost
layer of a starch grain to be different from the final structure. An
alternative model was proposed for the Arabidopsis system,
where data suggest that starch and phytoglycogen represent two
independent polysaccharides, which may accumulate simultaneously (4).
It has been suggested that isoamylase is required to prevent
accumulation of intermediary soluble highly branched glucans (17). This
model does not directly predict a specific structure of the outer
amylopectin layer but suggests that smaller phytoglycogen-like
molecules may exist as a separate short-lived glucan pool.
To understand starch biosynthesis it would therefore be valuable to
know specifically the structure of the outermost amylopectin layer and
to investigate the potential formation of intermediary pools of glucan
polymers. Because of the technical difficulties, there are as yet no
direct observations of the structure of the most recently formed layer
of amylopectin. We embarked on developing a method to reveal immature
amylopectin structures, which would provide novel information on
possible intermediate steps in achieving the final amylopectin
structure. Based on pulse labeling of leaf starch through
photosynthesis in 14CO2 we examined herein the
structures of starch and phytoglycogen formed within a 30-s period.
This corresponds to a time frame shorter than the period required for
the formation of a single crystalline amylopectin lamella. The labeling
pattern reveals that the outer layer of the granules has a different
structure from the final starch, showing preferential formation of
shorter chains (DP6-11). This new approach to the study of starch
biosynthesis provides novel detailed information on starch and
phytoglycogen biosynthesis.
Plant Material--
Wild type
(WT)1 and a mutant line of
Arabidopsis thaliana (L.) Heynh. ecotype Columbia
were grown in peat soil in a controlled climate chamber with mercury
halide lamps supplemented with light from incandescent lamps at a
photosynthetic flux of 120 µmol of photons s 14C Labeling--
Leaves were radiolabeled by
photosynthesis in a 14CO2-containing chamber.
For each labeling four uniform leaves (each with a lamina area of about
1-2 cm2) were excised from young vegetative plants at the
end of the photoperiod. The leaves were kept in a transparent box on a
humidified paper towel in light for about 10 min before the experiment
was initiated. The leaves were then placed on a stainless steel grid in
a 15-ml labeling chamber made of a polystyrene Petri dish. 14CO2 was evolved from 0.5 ml of a
[14C]bicarbonate solution by the addition of 10-fold
excess acid (HCl) and shaking the final solution with 1 ml of air
contained in a 5-ml syringe fitted with a injection needle. The
resulting radioactive air was injected into the labeling chamber, and
the air in the chamber was immediately agitated efficiently for 2-3 s
by an electric fan fitted beneath the grid. In total, 3.7 MBq of
14CO2 was injected resulting in a specific
activity of 0.16 GBq mmol Extraction Procedure--
The labeled leaves were each extracted
first in 3× 5 ml of 90% (v/v) ethanol at 80 °C, then in 4×
5 ml of 80% (v/v) ethanol 40 °C, and finally in 5× 5 ml of 50 mM sodium acetate-HCl (pH 5.0) at 40 °C.
The extracted insoluble leaf material was transferred to a 1.5-ml
screw-cap microtube with 300 µl of 50 mM sodium
acetate-HCl (pH 5.0), and the starch in the sample was gelatinized by
heating it for 10 min at 90 °C. The sample was then cooled to room
temperature, added to 0.7 units of Pseudomonas isoamylase (2 µl of enzyme in 3.2 M NH4SO4)
(Megazyme), incubated at 42 °C 1 h, and heated for 2 min to
95 °C to inactivate remaining enzyme. Anionic compounds were removed
by applying 270 µl of the solution to a 100-µl anion-exchange column (OH Isolation of Starch Granules--
Five 14C-labeled
leaves were homogenized for 30 s at 0 °C in 30 ml of a solution
containing 50 mM Tris-HCl, pH 7.5, 0.1% sodium dodecyl
sulfate (SDS), and 5 mM EDTA using a Polytron PT 3000 (Kinematica AG) homogenizer. Cell debris was filtered off through glass
wool and the starch granules collected by centrifugation. The starch
granules were washed three times with distilled water before being
dried at room temperature.
High Performance Anion-exchange Chromatography
(HPAEC)--
HPAEC separation with on-line pulsed amperometric
detection (PAD) of debranched neutral glucans, integration of
chromatograms, and subsequent correction for varying PAD detector
response was performed as described (18). An aliquot (100 µl) of
isoamylase-debranched starch or phytoglycogen was used for each
chromatographic analysis. Fractions were collected either by automated
collection (0.25- or 2-ml fractions) or by manual collection of
individual peaks, each representing a defined degree of polymerization
(DP, number of glucose residues in chain). The fractions were made
acidic by addition of 200 µl of 2 M HCl to 0.8 ml of each
fraction, and 3 ml scintillation liquid (Ecoscint A, National
Diagnostics) was added to the mixture. Radioactivity in the samples was
determined with a liquid scintillation counter (Wallac). For manually
collected fractions, which varied in size, the whole fraction was
counted after acidification and the addition of 10 ml of scintillation liquid.
Degradation with Very Short Pulse Labeling of Starch--
A procedure was developed
for in vivo pulse labeling of starch in
Arabidopsis leaves during photosynthesis in
14CO2. The procedure was optimized to allow
very short pulses (30 s or less), rapid quenching of the labeled plant
material, and incorporation of sufficient radioactivity for structural
analysis. This was achieved by using ample
14CO2 and a minimized labeling chamber volume
with efficient stirring. Direct extraction of whole leaves in hot
ethanol (80 °C, 90% v/v) secured immediate quenching. To facilitate
handling of the leaf material during extraction, samples were not
homogenized or frozen before extraction. As we observed that freezing
of the debranched starch samples occasionally influenced the HPAEC
separation, the debranched samples were kept on ice but never frozen.
For the very short pulses, the major fraction of the fixed
14C was recovered in few metabolites, presumably in the
Calvin cycle, disturbing the glucan HPAEC profiles. To completely
remove these compounds the extensive washing procedure was followed by
ion-exchange purification of each sample.
The Distributions of Radiolabel in Soluble Fractions and
Starch--
Large amounts of radioactivity were fixed in the leaves
during 15-60 s of pulse labeling (Fig.
1A). Essentially all of the radioactivity was found in ethanol-soluble, water-soluble, or starch
fractions, and only insignificant amounts remained in the insoluble
fraction (cell walls, data not shown). Radioactivity incorporated per
leaf increased with pulse time (Fig. 1A). Leaves were chosen
to be of similar size (~1 cm2), but to minimize the
disturbance of the leaves after harvest the area was not determined.
With increasing pulse time the fraction of 14C found
in starch increased from 3 (15 s) to 18% (60 s). During the following
10-min chase period (photosynthesis in nonradioactive CO2),
the fraction of label recovered in starch increased to ~50%. The low
14CO2 fixation combined with low incorporation
into the starch fraction during the 15-20-s pulses resulted in
radioactivity levels too low for structural analysis, because this
requires considerable radioactivity in even less abundant individual
unit chains. Labeling for 30 s gave sufficient radioactivity in
each unit chain to allow a detailed structural analysis. Hence, the
following labeling experiments were all performed with a 30 s
pulse period.
Determination of Label in Outer Chains--
Starch is organized in
crystalline lamellae with a 9-nm pitch; and the labeling procedure was
developed to analyze selectively the structure of the outermost layer.
Ideally, the pulse label should be located exclusively at the end
sections of glucan unit chains, which are not yet substituted with an
Initial Structure Analysis--
Having confirmed that pulse and
pulse-chase labeling represents outer and inner (mature) lamellae, we
proceeded to analyze the starch structure during its formation. The
individual unit chains of the starch were separated by HPAEC (Fig.
3A, PAD trace) and 2-ml
fractions were collected over 84 ml. The radioactivity of each fraction
was determined, and the value was normalized to the total radioactivity
in the 42 fractions. This allowed a comparison of the distribution
profile for individual samples. It should be noted that the 2-ml
fractions do not correspond to specific unit chain length, because
these do not elute equidistantly. A clear difference in distribution of
radioactivity was consistently observed when comparing pulse and
pulse-chase samples. Leaves labeled for 30 s and quenched after a
10- or 20-min chase period showed a similar and characteristic
distribution (Fig. 3, B and C). This is
reminiscent to the PAD response, which corresponds to the total starch
fraction and is similar for all leaves (only shown in Fig.
3A). There was a peak plateau in radioactivity around 19-27
min (at approximately DP11-15), a valley starting at 32-33 min (DP
18) and a second peak at 40-44 min (around DP 23). On average, the
radioactivity peaked later (longer chains) as compared with the PAD
response. This difference is due to the fact that the PAD response
primarily detects the glucose residue at the reducing end and therefore
does not reflect the size of the molecules. The labeling profiles
demonstrate that a uniform label of the chains was obtained after a
10-min chase period. Leaves quenched immediately after a 30-s pulse had
a different distribution of label. Radioactivity was present over the
entire elution profile (Fig. 3A) but with a preferential
labeling of the shorter chains (DP 6-12) and a generally lower
incorporation in the longest chains.
To evaluate the 14C distribution in individual unit chains,
fractions were collected at short intervals for a pulse-labeled sample
in which the amount of 14CO2 used during
labeling was increased (Fig. 4). The
refined fractions reveal that radioactivity elutes as peaks
corresponding exactly to the PAD-detected unit chains. A high
incorporation in short chains was observed in agreement with the
results presented in Fig. 3. The reason for the more extreme peak in
the short-chain area in Fig. 3A is that the smaller unit
chains elute close to each other and are collected in fewer
fractions than the longer chains. Collection of smaller
fractions prevents this effect (Fig. 4). The first large peak of
radioactivity corresponds exactly to DP6. Only minute amounts of
radioactivity are found at DP3, DP4, and DP5.
Detailed Analysis of WT and dbe Mutant--
For a more
robust comparison of the starch structure and distribution of
radiolabel, peaks corresponding to individual unit chains were
collected separately (Fig. 5). Consistent
with the refined automatic sampling (Fig. 4), this analysis showed that the shortest chains, which contained a considerable amount of 14C after 30 s, were DP6 (Fig. 5A). After a
10-min chase period the label distribution shifted to longer chains
(Fig. 5, B and C), and the distribution was
similar to the relative corrected weight distribution of carbohydrate
(Fig. 5D). Collecting individual unit chains permits
calculation of the differential distribution between the pulse and
pulse-chase samples (Fig. 5C). This clearly shows that
during the 30-s pulse there is a relatively higher label in chains
DP6-11 and a lower label in chains above DP12, with the largest
difference at DP15-16. The very long chains (>DP40) showed little
difference in relative intensity of label.
To gain more information on a possible phytoglycogen-like fraction and
to validate that the labeling procedure will identify differences in
glucan structures formation, we applied the procedure to the
dbe mutant of Arabidopsis, which accumulates
phytoglycogen (4). The labeling profiles showed a markedly different
distribution of label in the dbe mutant, with higher density
of label in the short chains, DP6-11, and a lower radioactivity in
longer chains, especially above DP40, than in the WT plants (compare
Fig. 5, A and F). This difference became even
more pronounced after the 10-min chase period (compare Fig. 5,
B and G). These data reflect the fact that
accumulated glucans in the dbe mutant are composed of
shorter chains and a very low abundance of the longest chains as
demonstrated for the corresponding distributions of total carbohydrate (Fig. 5, I and J). The calculated relative molar
distributions of individual unit chains (Fig. 5, E and
J) clearly illustrates that the short chains (DP3-10) are
more abundant in the dbe mutant. The difference in
distribution of radiolabel between pulse and pulse-chase for the
dbe mutant (Fig. 5H) was less prominent than for
the WT plants, but we consistently observed a shift in labeling pattern
toward shorter chains during the chase period. Although higher in the
dbe mutant than in WT plants, the carbon accumulated in the
very short chains (DP2-5) was still neither abundant (Fig. 5I) nor heavily labeled (Fig. 5, F and
G).
The preferential pulse labeling of short chains could represent either
soluble or granule-bound glucans. To investigate this possibility,
pulse-labeled leaf batches were divided in halves, and starch grains
were isolated by mild centrifugation after homogenization of a
half-sample, whereas the other half-sample was analyzed as described
above (nonhomogenized leaves). The samples were each analyzed for
distribution of radiolabel in unit chains. The recovery of radiolabel
was considerably lower in the isolated starch grains, but distribution
was almost identical to the nonhomogenized samples (data not shown).
The ratio of label in chains During the last decade, considerable progress has been made in the
understanding of starch biosynthesis. In general, the biochemical mechanisms responsible for starch formation are conserved from algae to
higher plants (19). However, starch structure varies with tissue and
species, and our understanding of the formation of the precise
structures remains incomplete. Different models have been proposed to
account for the highly organized structures (17, 20), and work done
with mutants of Chlamydomonas, Arabidopsis, maize, and rice suggests that isoamylase may play a general role in
determining starch structure. These studies have clearly demonstrated the value of specific mutants, and targeted selection of further mutants will be important in promoting a comprehensive understanding of
starch formation. However, new approaches such as our very short pulse
labeling technique will be essential to the realization of this
research potential.
The current models for amylopectin biosynthesis predict that
intermediary structures are formed during starch biosynthesis. We
decided to develop a method that would specifically radiolabel the most
recently deposited starch, corresponding to only one 9-nm lamella. The
intensity should be sufficient to allow determination of radioactivity
in each unit chain after debranching. Transitory starch is formed
during the photoperiod and degraded during the following night.
Arabidopsis starch granules reach a diameter of around 1 µm (4), which corresponds to about 110 lamellae. Most likely, new
granules are initiated over the entire photoperiod and average growth
time for the individual granule would then be a fraction of the
photoperiod. Assuming a 2-h growth period for the individual granule,
it can be calculated that one 9-nm layer is formed in about 1 min. Our procedure allowed efficient labeling and quenching within
considerably shorter time frames. Within 30 s about 10% of the
incorporated label was found in the starch fraction (Fig. 1). Sample
volumes used for starch debranching and ion-exchange purification were
minimized to allow efficient loading of the sample on the HPAEC column.
Typically, 5,000-10,000 dpm were loaded on the column for the
30-s pulse-labeled samples, enabling reliable analysis even in
fractions with less than 0.5% of the applied radioactivity. As an
additional benefit, our procedure allows for PAD analysis of the starch
unit chains from just a single small leaf (see Fig. 3A). The
ion exchange purification, introduced to remove residual ionic
compounds from starch-derived glucans, also reduced the PAD background
signal sufficiently to enable manual collection of individual unit
chains (Fig. 5).
Liberation of radiolabel by Both radioactivity and PAD-detected carbohydrate (Figs. 4 and 5)
demonstrate that there are few chains shorter than DP6. Occasionally, larger PAD-detected peaks that eluted earlier than DP6 were observed (see e.g. Fig. 3A). These compounds contained
little or no radioactivity and do not represent starch-derived
small-chain malto-oligosaccharides. The nature of these compounds
remains to be identified. The profile of PAD-detected peaks alone does
not allow us to draw conclusions with respect to chain formation,
because these data reflect only the final starch structure, and
potential short-chain intermediates may not accumulate to notable
levels. The pulse labeling solves this problem; DP6 and longer chains
are efficiently labeled during the 30-s pulse period, whereas very
little label is incorporated in chains shorter than DP6 (Figs. 4 and
5). Because there are hardly any DP5 chains, the DP6 chains cannot
originate from elongation of DP5 chains. DP6 chains must therefore be
formed by transfer from longer chains, catalyzed by branching enzyme.
Furthermore, the donor chains must be DP12 or longer; otherwise the
branching reaction would result in stubs of DP5 or shorter. In
vitro studies show that starch branching enzyme (SBEI) will
primarily bind to chains DP6 and longer (21), and both SBEI and SBEII
from potato will produce chains with a minimal length of DP6 (22). This finding fits well with the labeling pattern during starch biosynthesis as observed in this study, and with the preferential formation of DP6
in glycogen from Escherichia coli expressing the maize SBEI
and SBEII (23).
During the pulse period a preferential labeling of short chains was
observed. Generally, the distribution of a total population of
polymeric unit chains may be represented as molar or weight-based distributions as shown in Fig. 5, D, E,
I, and J. However, for transitory starch granules
consisting of radially oriented amylopectin chains the distribution of
surface-exposed chains must be considered. Although an even label of
all nonreducing chains in the entire starch granule would show a molar
distribution, label restricted to the nonreducing surface exposed ends
will display a pattern similar to the weight-based distribution of the
entire population of the chains if it is assumed that the surface
structure is identical to the entire starch granule (calculated as a
hypothetical concentric surface exposed by a section of a granule).
Because a starch granule grows primarily by synthesis at the surface,
it would then be expected that a very short pulse resulted in a label
distribution similar to the weight-based distribution. Any significant
deviation from this distribution would indicate either labeling of
buried chains or that lamellar maturing processes occur that are slower than the pulse. In the first case, no shift in chain length profile would be expected with a pulse-chase experiment. In the second case, a
shift can occur as an effect of molecular rearrangements. Our results
favor the second of these options. Two contrasting scenarios could
explain a time-dependent change in distribution. 1) Newly
formed chains are efficiently branched and excessive branches are then
elongated or trimmed off again, resulting in preferential pulse
labeling of short chains. 2) Chains need first to be elongated to reach
a length above average before being branched resulting in the mature
chain length. This might result in preferential pulse labeling of
longer chains. The pulse label being enriched in shorter chains suggest
that the first option is correct. It should also be noted that about
2% of the label was recovered alone in DP6 chains. As discussed above,
the DP6 chains are radiolabeled by elongation of chains (to DP12 or
longer) followed by transfer of the six terminal glucose units to form
a new branch. The final degree of branching is about 5% of all
glucosidic bonds, and after 30 s already 2% of the label is found
in only DP6 chains (each represents one new branch point formed). This
suggests that within 30 s most of the branch points of the final
starch have already been formed. Thus, branching is a highly efficient
process. It is striking to note the shift in labeling profile
from pulse- to pulse-chase-labeled samples that occurs at DP12 (Fig.
5C). This indicates a direct connection to the branching
process, because DP12 are deduced to be the shortest unit chains used
for branching. Chains shorter than DP12 originate from branching and
elongation, and on a short time frame this fraction gain total
carbohydrate and thereby radioactivity by both processes. Chains of
DP12 and longer also lose carbon, and thereby radioactivity, by
the branching process. In the short term, this may result in the
observed preferential incorporation of radiolabel in short unit chains.
The major "loss" of radioactivity is seen for DP12-19 (Fig.
5C), which may suggest that these medium length chains are
the preferred (or just the most abundant) substrates for the branching
process. Starch branching isoforms SBEI and SBEII have different
preferences to substrate structure (24) and also make different
products (22, 23). We further suggest that the true in vivo
substrates and products are also determined by accessibility and timing
of substrate formation.
The dbe mutant, which accumulates phytoglycogen, showed a
significantly shorter average chain length as compared with the WT
plants (Fig. 5, E and J). As expected from this
structural difference, the pulse label of the dbe mutant was
found preferentially in shorter chains as compared with the WT (Fig. 5,
A and F). This probably reflects a more uniform
labeling of the phytoglycogen molecule than of starch granules.
Phytoglycogen is a noncrystalline polymer and therefore potentially has
a much larger accessible area than granular starch. Therefore, more
short chains can be assumed to be accessible for elongation.
The pulse-chase analysis of phytoglycogen revealed a smaller, but
consistently observed change in distribution in favor of shorter chains
during the chase period, i.e. opposite the pattern observed
for WT plants. Thus, in the dbe mutant a more branched glucan is formed over the chase period. We suggest this to reflect that
the more open internal structures of phytoglycogen remains accessible
to branching enzymes. The radiolabeling data also show more clearly
than PAD that the glucans in the dbe mutant have very
few long chains (above DP40). This represents a marked difference from
the WT starch.
The distribution of label in the chased dbe samples is
similar but does not exactly match the total carbohydrate distribution profile, indicating the formation of a more branched structure during
the labeling experiment (Fig. 5, G and I)
compared with glucans accumulated over the entire light period (Fig. 5,
G and I). It is quite likely that the structure
varies with the environment and metabolic activity of the leaf tissue.
The dbe mutant accumulates both normal starch and
phytoglycogen (4), and it is reasonable to expect that the distribution
between these two pools may change under different conditions.
It has been suggested that soluble pre-amylopectin or phytoglycogen
(17) may be formed as starch biosynthetic intermediates. The short
chain glucans observed in the pulse-labeled WT plants could represent
such soluble glucans with a structure similar to phytoglycogen. Our
analysis suggests that this is not the case. Despite a preferential
labeling of short chains, the average chain length is considerably
higher in the pulse-labeled WT samples than in dbe samples.
Furthermore, the incorporation into the very long chains (above DP40)
is significantly higher than in phytoglycogen (Fig. 5). Calculation of
the label distribution in the pulse-labeled WT as a sum of
phytoglycogen and starch did not result in a good description of the
observed label distribution. Furthermore, pulse-labeled starch granules
that were separated from soluble material showed a distribution very
similar to the unhomogenized whole leaf data, suggesting that the pulse
label distribution observed in our "in leaf" debranched samples
represents granule-bound glucans.
At this point, we can draw the following conclusions. 1) Chains of DP6
are the shortest chains formed in considerable amounts. 2) Chains of
DP6 are made from branching with donor chains of DP12 and longer. 3)
Branching is highly efficient and occurs mainly within the time period
required for synthesis of one 9-nm layer. 4) The efficient branching
results in an intermediary glucan structure enriched in DP6-11 chains.
5) This short chain enriched intermediary structure is granule bound.
6) Although similar, the short chain intermediary structure is not
identical to phytoglycogen.
Our data do not exclude that small soluble branched glucans are formed
in the WT plants as short-lived intermediates. These would be eluted
during the extraction procedure. Preliminary analysis did not reveal
considerable incorporation of label in branched soluble glucans, but
further analysis is required to reach conclusion. We can also not
conclude whether branches are trimmed off of starch by isoamylase in
the WT plants. However, if trimming is taking place, it appears to be
occupied primarily with removing DP6-11 chains. Phytoglycogen contains
more very short chains (DP3-5) than starch (Fig. 5, E and
J). However, even in phytoglycogen the amount of carbon and
label incorporated in these chains is low.
Neither trimming nor the existence of soluble branched glucans is
strictly required to explain the observed labeling patterns. Still,
these features may be important for initiation of correct starch
structure, and the accumulation of phytoglycogen in the dbe
mutant suggests that isoamylase activity is indeed required for correct
starch biosynthesis. It is reasonable to speculate that the formation
of phytoglycogen may be a self-enhancing reaction because the formation
of an open and highly branched structure will provide even more
nonreducing ends for further elongation and branching. The large
accumulation of phytoglycogen in mutants may therefore over-emphasize
the direct contribution of isoamylase. To reach a final conclusion
about the potential contribution of trimming, the methodology will need
to be further developed to allow for analysis of radiolabeling of
malto-oligosaccharides. Trimming would predict that these would be
transiently labeled. Work is under way to perform these studies.
We also propose that branching occurs principally as soon as chains are
sufficiently long to be accepted as donors for the branching reaction,
resulting primarily in short chains (DP6-11). This proposition
is based on the observations of: 1) efficient formation of DP6 chains,
2) excess of chains DP6-11 during pulse labeling, 3) and the
deficiency in chase-labeled chains peaking around DP15-16. Thus, most
of the branch transfers appear to take place from medium length chains
forming short chains. This is a plausible model; a very efficient
branching, as observed in our experiments, would promote branching of
chains as soon as they are long enough. This will result in
preferential branching of medium length chains, which are also more
abundant than longer chains, and will simultaneously ensure
preferential transfer of short chains (DP6-11). Possibly longer chains
also serve as donor chains, but because of the organization of
crystalline layers restricting accessibility, only the outer glucose
units are likely to participate in the branching reactions. This
situation appears to be entirely changed in the dbe mutant.
Phytoglycogen is less organized, and presumably our data reflect that
the glucan chains remain accessible to branching enzymes over a longer
period. Long chains may therefore remain to serve as substrates for the
branching reaction, and efficient branching (relative to elongation)
will result in increased branching with time, which would explain the considerably lower amount of very long chains in phytoglycogen compared
with starch.
We gratefully acknowledge the skilled
technical help of Lis Byrsting Møller. The seeds of the dbe
mutant were kindly provided by Dr. Samuel Zeeman, University of Berne,
Switzerland, and Prof. Alison Smith, John Innes Center, Norwich, UK. We
thank Dr. Mark Turner for critical reading of the manuscript.
*
This research was supported by The Danish National Research
Foundation, Center for Molecular Plant Physiology.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.
Published, JBC Papers in Press, March 27, 2002, DOI 10.1074/jbc.M201866200
The abbreviations used are:
WT, wild type;
HPAEC, high performance anion-exchange chromatography;
PAD, pulsed
amperometric detection;
DP, degree of polymerization;
SBE, starch
branching enzyme.
Intermediary Glucan Structures Formed during Starch Granule
Biosynthesis Are Enriched in Short Side Chains, a Dynamic Pulse
Labeling Approach*
,
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-1,4-glucans with a molecular mass
in the range of 105 to 106. Amylopectin is a
highly branched structure containing shorter chains of -1,4-glucans
and frequent -1,6 branch points (5). The molecular mass of
amylopectin is 107-108 (6). Typically
amylopectin constitutes 70-80% of the starch. However, transitory
starch appears to contain almost exclusively amylopectin (4, 7)
1) packed molecules. At the supramolecular level, large
granules are clearly ordered in layers, so-called growth rings, readily visible by light microscopy. The granules are semicrystalline, and the
amylopectin molecules account for this crystallinity. According to the
current understanding, the amylopectin glucan chains are radially
arranged and ordered in alternating amorphous and crystalline lamellae.
The amorphous lamellae are regions with more abundant -1,6 branch
points, whereas the crystalline lamellae consist of the linear
-1,4-glucan chains organized in parallel double helices congregated
into clusters, which form crystalline lattices (8). The width of the
repeated crystalline and amorphous layers is 9 nm, a feature that is
highly conserved for starches from many botanical sources (9). By
specific hydrolysis of the -1,6 branch point using isoamylase
(debranching) and chromatographic separation of the derived linear
glucan chains, it is possible to obtain a branch length distribution
profile of the amylopectin. No matter what the botanical source or
organ, these profiles are polymodal, which is a direct reflection of
the repeated lamellar structure of the starch granule. The profile may
vary with the botanical source but is highly conserved for specific
types of starch (10).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1
m2 in an 8-h photoperiod. Seeds of mutant line
dbe1-1 (4) were kindly provided by Dr. Samuel Zeeman and Professor
Alison Smith.
1 CO2 in the
chamber. During both labeling and pre-incubation, 200 µmol
s1 m2 light was provided by fluorescent
light tubes. Labeling was continued for periods of 30 s to 10 min.
The labeling was initiated by injection and terminated by opening the
chamber and immediately transferring the leaves to hot extraction
medium as described below. Other leaves were transferred to a humid
transparent box on a wet paper towel and left in light for 2-20 min
before extraction (chase by photosynthesis in nonlabeled air).
form of AG 2-X8; BioRad) and eluting the
neutral glucans with an additional two more times with 100 µl of
distilled water. All fractions containing neutral glucans (470 µl) were collected together and stored at ice until further analysis.
-Amylase--
Leaves were extracted, washed,
and then gelatinized in 270 µl of buffer as indicated above.
Radioactivity released to the buffer by heating the leaf samples was
negligible (below 0.1% of label released by the following enzyme
treatments). Outer chains were degraded by the addition of 2 µl (4 units) of barley -amylase (Megazyme) and incubation for 1 h at
40 °C; radioactivity released to the solution was determined by
counting the total radioactivity in five successive washes with 2 ml of
buffer. The radioactivity of the fifth wash was in all cases below
0.3% of the total label released by the enzyme treatments. The
remaining starch was then degraded and released from the leaf sample by
incubating it for 1 h with an additional 2 µl (2 units) of
Aspergillus niger amyloglucosidase (Megazyme) at 40 °C.
The radioactivity released was determined in three successive washes
with 2 ml of buffer, and total released activity was calculated from
these data.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (25K):
[in a new window]
Fig. 1.
Distribution of radioactivity in pulse- and
pulse-chase-labeled Arabidopsis leaves. Small
whole leaves (1-1.5 cm2) were radiolabeled and extracted
with 80% ethanol and acetate buffer. The remaining starch was released
by isoamylase and purified over an anion-exchange column to remove
residual contamination. The relative distribution of radioactivity
recovered in the ethanol, buffer, and purified starch fractions was
calculated. The leaves were labeled by photosynthesis in the presence
of 14CO2 for 15, 20, 30, or 60 s and
immediately quenched in hot ethanol (Pulse) or left to
photosynthesize in a nonradioactive atmosphere for 10 or 20 min before
quenching (Chase).
-1,6-linked side chain, i.e. at the outer chains.
Degradation with -amylase, an enzyme that cannot bypass the -1,6
branch points and thus produces remaining glucans called -limit
dextrins, confirmed that after a 30-s pulse 94% of the radiolabel is
located in the outer chains beyond the last branching points (Fig.
2). During the following 10-20-min chase
period the fraction of 14C released by -amylase
decreased to 51%, as expected for carbon remaining in -limit
dextrin of uniformly labeled starch.
View larger version (13K):
[in a new window]
Fig. 2.
Release of radiolabel from
extracted leaves by -amylase.
Arabidopsis leaves were pulse-radiolabeled for 30 s,
chased for 0-20 min, and extracted as described in Fig. 1. The
starch-derived 14C was released from the extracted leaves
first with -amylase. The leaf sample was then washed extensively,
and the remaining -limit dextrin was released by amyloglucosidase.
The measured values are indicated as radioactivity released by
-amylase as % of total radioactivity released by -amylase and
amyloglucosidase.
View larger version (23K):
[in a new window]
Fig. 3.
Distribution of radioactivity in pulse- and
pulse-chase-labeled starch. Leaves were pulse-labeled for 30 s, chased for 0 (A), 10 (B), or 20 min
(C), and extracted as described in Fig. 1. The glucans
released by isoamylase and purified over an anion-exchange column were
separated by HPAEC. Fractions (2 ml) were collected, and radioactivity
was determined in each fraction. The relative distribution of
radiolabel was calculated as % of total radiolabel in all collected
fractions. The continuous curve in panel A
represent the PAD detection of glucans in the isoamylase treated
sample.
View larger version (34K):
[in a new window]
Fig. 4.
Detailed analysis of radioactivity in smaller
glucans derived from pulse-labeled starch. Leaves were
pulse-labeled for 30 s (no chase period). Glucans released by
isoamylase were separated by HPAEC as described for Fig. 3, but smaller
fractions (0.25 ml) were collected, and radioactivity was determined in
each fraction (open connected points, shaded area). The
solid continuous curve represents the PAD detection of
glucans in the sample. The time for PAD detection has been corrected
for the small delay in fraction collection due to tubing volume.
View larger version (43K):
[in a new window]
Fig. 5.
Analysis of individual unit chains in pulse-
and pulse-chase-labeled starch. Leaves of WT plants
(A-E) and dbe1-1 mutant plants (F-J)
were pulse-labeled for 30 s and chased for 0 (A and
F) and 10 min (B and G). The glucans
released by isoamylase and purified over an anion-exchange column were
separated by HPAEC. Fractions corresponding to individual unit chains
were collected by hand, and the radioactivity of each unit chain was
determined. The relative distribution of radiolabel was calculated as
% of total radiolabel in all collected fractions. Panels C
and H gives the differences between pulse and pulse-chase
samples. Panels D and I show the weight-based
distribution of individual unit chains calculated on the basis of
integrated and corrected PAD responses. Panels E and
J show the calculated relative number of individual unit
chains, i.e. the corresponding molar distribution. The
values are represented as % of total carbohydrate (D and
I) and % of total number of unit chains (E and
J) up to DP56.
11 and chains 
12 is diagnostic for the
chase shift in distribution (Fig. 5C). This ratio was very
similar for the two types of samples, as 29 and 30% of label in chains
11 was found in isolated starch granules and extracted leaf samples,
respectively. For a comparison, 17% of label was found in chains 11
in the 10-min chased leaf sample.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amylase degradation verifies specific
pulse labeling of the outermost 9-nm lamella. The -amylase does not
bypass the -1,6 branch points and therefore only degrades the outer
chains. The release of 94% of the radiolabel (Fig. 2) after a 30-s
pulse shows that essentially all of the radiolabel represents the
outermost lamella. On the contrary, a uniform labeling of the starch
glucose residues will result in retaining a fraction of radioactivity
corresponding to the -limit dextrin. Accordingly, -amylase
liberated only 50% of the radioactivity after a 10-20-min chase
period. We conclude that a 30-s pulse will label only the outermost
lamella chains, whereas after a 10-20-min chase period, the label will
also represent internal lamellae chains. The distributions of label
after chase periods of 10 and 20 min (Fig. 3, B and
C) were very similar and reflected the distribution as
observed by PAD, showing that the chased samples represent final starch
structure. Thus, we have verified that pulse- and pulse-chase-labeled
samples represent immature and mature starch structures, respectively.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed. Tel.: 45-35283338;
Fax: 45-35283333; E-mail: thni@kvl.dk.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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