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Plant Physiol. (1999) 119: 321-330 Novel, Starch-Like Polysaccharides Are Synthesized by an Unbound Form of Granule-Bound Starch Synthase in Glycogen-Accumulating Mutants of Chlamydomonas reinhardtii
Laboratoire de Chimie Biologique, Unité Mixte de Recherche du Centre National de la Recherche Scientifiquen no. 8576, Université des Sciences et Technologies de Lille, 59655 Villeneuve d'Ascq cedex, France (D.D., C.C., G.M., C.D.H., S.B.); Massachusetts Institute of Technology, Department of Biology, 77 Massachussetts Avenue, Cambridge, Massachusetts 02139 (E.S., A.S.); Commonwealth Scientific and Industrial Research Organization, Division of Plant Industry, G.P.O. Box 1600, Canberra, ACT 2601, Australia (G.M., M.M., M.S.S.); and Institut National de la Recherche Agronomique, Unité de Recherches sur les Polysaccharides, leurs Organizations et Interactions, B.P. 71627, 44316 Nantes cedex 03, France (B.B., D.J.G.)
In vascular plants, mutations leading to a defect in debranching enzyme lead to the simultaneous synthesis of glycogen-like material and normal starch. In Chlamydomonas reinhardtii comparable defects lead to the replacement of starch by phytoglycogen. Therefore, debranching was proposed to define a mandatory step for starch biosynthesis. We now report the characterization of small amounts of an insoluble, amylose-like material found in the mutant algae. This novel, starch-like material was shown to be entirely dependent on the presence of granule-bound starch synthase (GBSSI), the enzyme responsible for amylose synthesis in plants. However, enzyme activity assays, solubilization of proteins from the granule, and western blots all failed to detect GBSSI within the insoluble polysaccharide matrix. The glycogen-like polysaccharides produced in the absence of GBSSI were proved to be qualitatively and quantitatively identical to those produced in its presence. Therefore, we propose that GBSSI requires the presence of crystalline amylopectin for granule binding and that the synthesis of amylose-like material can proceed at low levels without the binding of GBSSI to the polysaccharide matrix. Our results confirm that amylopectin synthesis is completely blocked in debranching-enzyme-defective mutants of C. reinhardtii.
Mutants of maize, rice, and Chlamydomonas reinhardtii
and, more recently, Arabidopsis have been reported to
accumulate in place of or in addition to starch a novel type of WSP
known as phytoglycogen (Sumner and Somers, 1944 The results obtained in maize, rice, and C. reinhardtii led
us, together with a number of other authors, to propose a novel pathway
for storage polysaccharide synthesis in plants (Ball et al., 1996 We now report the presence of a novel type of insoluble, starch-like
material entirely constituted of amylose-like chains in the
phytoglycogen-producing mutants of C. reinhardtii. We show the dependence of this material on GBSSI activity. Moreover, in contrast to all of the results obtained to date, we show that GBSSI
does not bind to the granular polysaccharide. We believe our results
demonstrate that GBSSI requires crystalline amylopectin for binding in
vivo and that phytoglycogen synthesis can proceed in the complete
absence of insoluble polysaccharide synthesis. We therefore confirm
that amylopectin synthesis is completely blocked in the sta7
mutants of C. reinhardtii.
Material
Strains, Media, Incubation, and Growth Conditions The genotypes of all of the strains of Chlamydomonas reinhardtii used in this work are listed in Table I. Starch and WSP were always prepared from nitrogen-starved media. Media and culture conditions used in our starvation experiments were as described by Ball et al. (1990) .
Formulas for Tris-acetate-phosphate and high salt-high acetate media
and genetic techniques were according to the method of Harris (1989a,
1989b). All experiments were performed under continuous light (40 µE
m 2 s1) in the presence
of acetate at 24°C in liquid cultures that were shaken vigorously
without CO2 bubbling.
Purification of the Insoluble Macrogranular Fraction Pure native starch was prepared from nitrogen-starved cultures, inoculated at 106 cells mL1, and harvested after 5 d of growth
under continuous light (40 µE m2
s1) in Tris-acetate-phosphate without nitogen
medium. Algal suspensions were passed through a French press at 10,000 p.s.i. A crude starch pellet was obtained by spinning the lysate at
10,000g for 20 min. The pellet was resuspended in 300 µL
of 10 mM Tris-HCl (pH 8.0), 1 mM EDTA per 109 starting
cells, and passed twice through a Percoll gradient (1.2 mL of Percoll
for the 300 µL of crude starch pellet). The purified starch pellet
was rinsed in distilled water, centrifuged twice at 10,000g,
and kept at 4°C for immediate use or dried for subsequent analysis.
Starch amounts were measured using the amyloglucosidase assay as
described by Delrue et al. (1992).
Purification of the WSPs WSPs were prepared from 20 L of 1 week-old nitrogen-starved cultures that were inoculated at 106 cells mL1. Algae were harvested and ruptured by
passing them in a French press (10,000 p.s.i) at a density of
108 cells mL1 in the
presence of pronase (1 mg mL1). The crude
extract was immediately frozen at  80°C and thawed after a minimum
of 4 h of storage. Cell debris were discarded by spinning at
10,000g for 30 min. The supernatant was extracted three
times with 2:0.1 (v/v) chloroform:methanol and centrifuged at
3000g for 15 min. Water was added between each extraction to keep the volume of the aqueous phase constant. Emulsions were obtained
by intensive shaking. The water-methanol soluble fraction was
concentrated by rotary evaporation and redissolved in 10% DMSO to be
subsequently loaded onto a gel-filtration column (TSK-HW-50 [F],
Merck, Darmstadt, Germany) eluted by 10% DMSO as described by
Maddelein et al. (1994). The high-Mr peak
was desalted by dialysis at 4°C overnight and lyophilized. The
low-Mr peak was desalted by gel filtration
onto a column (TSK-HW-40, Merck). The peak of dextrin was lyophilized
and stored at room temperature.
Debranching Analysis Phytoglycogens (500 µg) were suspended in 10 µL of water and debranched with 1 µL of isoamylase (200 units/mL, Megazyme International, Bray, County Wicklow, Ireland) in 40 µL of 50 mM sodium acetate buffer (pH 4.0). The reaction was incubated for 2 h at 37°C and terminated by heating in a water bath at 100°C for 5 min. Completion of the reaction was ascertained by assaying the amount of reducing ends through the standard dinitrosalicyclate procedure. The latter consists of mixing 5 µL of the sample with 45 µL of water. The diluted sample was then added to 150 µL of 1% dinitrosalicyclate solution and the A540 was read. Complete debranching was obtained when maximal and constant absorbencies that compared favorably with those of amylopectin, amylose, and glycogen standards were recorded. The samples were then evaporated to dryness in a centrifugal vacuum evaporator.Separation of Labeled Oligosaccharides The debranched and undebranched samples were labeled with 8-amino-1,3,6-pyrenetrisulfonic acid and analyzed with a DNA sequencer, as fully described by O'Shea and Morell (1996).
trans Complementation Tests The segregants (IJS) obtained from the cross between strains IJ2 and S were subjected to trans complementation tests to characterize their deficiencies. Diploid clone selection was achieved by growth after 5 d of the sexual fusion products on appropriate selective media. The diploids obtained were purified three times, checked for cellular volume, and then amplified. Cell patches incubated for 7 d on solid, nitrogen-deprived medium were stained with iodine. Diploids homozygous for the sta2 mutation appear red after staining (Delrue et al., 1992).
Transmission Electron Microscopy Cell suspensions prepared from nitrogen-starved media were harvested first and immediately embedded at 45°C in a 3% agar gel to prevent these highly mobile cells from adopting a heterogeneous spatial distribution. Small, solidified cubes of agar containing the samples were then cut and fixed in 3% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.1) for 3 h at room temperature. A part of these samples was postfixed in 1% osmium tetroxide for 1 h. The fixed samples were then dehydrated in an ethanol series, infiltrated with propylene oxide, and embedded in Epon. Sections of 80 nm thickness were mounted on copper or gold grids previously coated with carbon. Two complementary treatments were applied on the grids as follows: (a) sections of samples fixed by glutaraldehyde and osmium tetroxide were mounted on copper grids and then stained for 30 min in 2.8% uranyl acetate (50% methanol) at 48°C; and (b) sections of samples fixed by glutaraldehyde alone were mounted on gold grids and then stained according to the modified PATAg procedure previously described by Simon et al. (1995). The samples were first treated for 30 min in a
saturated solution of 2-4-dinitrophenylhydrazine in 15% acetic acid
to block free aldehydes. After several washes in deionized water,
samples were oxidized for 30 min in 1% periodic acid aqueous solution,
then treated for 18 h with 0.2% thiosemicarbazide in 20% acetic
acid, and finally treated for 30 min with 1% silver proteinate aqueous
solution.
Zymogram Analysis Soluble crude extracts were prepared as previously described (Fontaine et al., 1993) and immediately stored at 70°C. The lysate
was cleared by centrifuging at 10,000g for 10 min at 4°C. Proteins were measured using the Bio-Rad protein assay kit. In 100 µL
of 25 mM Tris glycine, pH 8.3, 1% SDS, 5%
 -mercaptoethanol, 100 to 500 µg of protein was denaturated by
boiling in a water bath for 4 min. The denatured proteins can be stored
at 4°C without subsequent loss of enzyme activity. Starch synthase
and DBE zymograms were as previously described by Mouille et al. (1996)
and Buléon et al. (1997).
SDS-PAGE Analysis of Granule-Bound Proteins, Western Blots, and GBSS Activity Assay Purified starches (1-6 mg) were boiled for 5 min in 80 µL of 2% SDS and 5%-mercaptoethanol and then centrifuged for 20 min at
10,000g to extract proteins from the granule. Supernatants were loaded onto SDS-PAGE and stained with Coomassie brilliant blue
R250.
Mutants Defective for the STA7 Locus Synthesize up to 0.5% of the Wild-Type Amount of Starch in the Form of Insoluble, High-Amylose Types of Granular Polysaccharides Because of the low amounts of polysaccharide found in the sta7 mutants of C. reinhardtii, we scaled up our culture volumes to 20 L and measured the amounts of the WSP and high-density-insoluble material that sedimented in the bottom of Percoll gradients. As previously reported, in the mutants we found 5% ± 3% (n = 5) of the amount of polysaccharide measured by the amyloglucosidase assay in wild-type reference strains in the form of WSP. Both high and low-molecular-mass WSPs could be separated through TSK-HW-50 (Fig. 1A) gel-permeation chromatography. Because of the high sensitivity of the technique, we used fluorescence labeling coupled to PAGE on a DNA-sequencing gel to analyze both the size distribution of the small oligosaccharide fraction and that of the high-molecular-mass polysaccharide subjected to selective debranching through bacterial isoamylase. Results displayed in Figure 1, B and C, together with those previously published (Mouille et al., 1996), clearly confirm the
high-molecular-mass fraction as glycogen-like material, whereas the
oligosaccharide fraction contained both branched and linear glucans.
The high-mass glucans display an iodine interaction (Fig. 1A), a
branching ratio (Mouille et al., 1996), a chain-length distribution
(Fig. 1B), and granule morphology (Fig. 8, C-F) similar to those
reported for glycogen. The relative amounts of oligosaccharide to
polysaccharide in the water-soluble fraction were between 20% and 60%
(40% ± 20%). We believe that these variations reflect the presence
of fluctuating amounts of  -amylase in the extracts to which the
soluble polysaccharides are highly sensitive.
The Amylose-Like Polysaccharide Displays an Entirely Novel Structure The high-amylose material consisted of both a high- (30%) and a low (70%)-molecular-mass component that could be separated through TSK-HW-75 gel-permeation chromatography (Fig. 2). The high-molecular-mass fraction displayed a max of 590 nm, comparable to that
of amylopectin components found in high-amylose starches of C. reinhardtii and the su-2 mutant of maize (Fontaine et
al., 1993; Takeda and Preiss, 1993; Libessart et al., 1995). However, unlike all other wild-type or mutant amylopectins analyzed to date, we
found less than 1% branches in this material by proton NMR (Fig.
3). The 13C-NMR
spectra were indistinguishable from those published for amylose
(Fontaine et al., 1993), further establishing this material as composed
of -1,4-linked Glc residues. A low 1% ± 0.5% branching level was
measured with greater precision through comparative fluorescence
labeling of the debranched polysaccharide fraction. The size
distribution of the debranched glucans displayed a pattern similar to
that of the high-mass amylose present in wild-type starches (Fig.
4A). Because of both the high mass and
the low max of this fraction, these results
could not have been produced by contamination of low amounts of
amylopectin by amylose. The size distribution, branching ratio (<1%),
and max of the low-molecular-mass fraction
were similar to those of amylose. However, upon debranching, this
fraction yielded an unusual chain-length distribution that could
distinguish it from standard amylose and amylopectin (Fig. 4B).
The Amylose-Like Polysaccharide Is Not Required for Phytoglycogen Synthesis Strain S (sta7-4::ARG7) was crossed with IJ2 (sta2-29::ARG7 sta3-1). The interesting double- or triple-mutant genotypes were detected with trans complementation tests (see ``Materials and Methods''). We selected three genotypes for each of the following classes (Table I): wild type, sta7; sta7 sta2, sta7 sta3, sta7 sta2 sta3. The genotypes were double-checked through zymogram analysis of DBE and SS. We found no differences in behavior of the SS in a mutant sta7 background. SSII disappeared, as expected, upon introduction of the sta3 mutation. For each genotype class, three measurements of WSP and granular starch levels were taken on three randomly selected recombinants (n = 9; Table II). For each class a complete WSP characterization was made, including a determination of the chain-length distribution of the novel types of phytoglycogens (Figs. 5 and 6). These were compared with the phytoglycogen produced by our reference sta7 strains (Fig. 1, A and B).
GBSSI Can Synthesize Amylose-Like Granular Material in the Absence of Polysaccharide Binding In all cases in which starch synthesis was impaired in the presence of a wild-type STA2 gene, there was a net increase of GBSSI-specific activity within the granule (Libessart et al., 1995;
Van den Koornhuyse et al., 1996). A dramatic example of such an
increase is provided in Figure 7A,
comparing the amount of granule-bound proteins present after high or
low starch synthesis occurring, respectively, under
nitrogen-starvation-induced growth arrest (storage starch) or
nitrogen-supplied growing conditions. These differences can be
explained by the fact that an equivalent amount of GBSSI protein binds
to a restricted polysaccharide amount. Because of both the low levels
of granular material (0.5% of the wild-type amount) and the
overrepresentation of long glucans, we expected this fraction to be
filled with GBSSI protein and enzyme activity. We were surprised to
find SDS-PAGE profiles generated from granule-associated proteins that
could not be distinguished from those of strains completely lacking
GBSSI (Fig. 7B). Moreover, the amount of GBSS activity monitored under
these conditions decreased to less than 1% of the wild-type activity
and was also less than those measured for strains carrying a
gene-disrupted GBSSI structural gene (Fig. 7C). These results were
confirmed by western blotting using an antibody directed against a
C-terminal peptide sequence of higher-plant starch synthases (Fig. 7D).
Western blots demonstrated the presence of a small amount of 75-kD
cross-reacting polypeptide. Because SSI displays a 75-kD mass
(Buléon et al., 1997), we suspect that this protein represents
the residual 0.5% to 1% GBSS enzyme activity in a fashion analogous
to that described for the 77-kD GBSSII in potato and pea (Edwards et
al., 1996). Mutants lacking both GBSSI and SSII behaved in an identical
fashion. Because GBSSI and SSI display identical masses in
C. reinhardtii, we were unable to discriminate
between these two proteins and saw no convincing differences between
western blots of soluble proteins extracted from wild-type and
sta7 mutants.
Electron Microscopy Because both the glycogen found in animal cells and the maize phytoglycogen were reported to display some level of organization, including rosette-like structures (Lavintman, 1966), we embarked on
cytochemical observations made by transmission electron microscopy using the PATAg stain. This procedure enables the selective
staining of starch granules and WSP in the wild-type and single or
double mutants (Fig. 8). The wild-type
cells prepared from nitrogen-starved media (Fig. 8, A and B) showed a
significant amount of starch granules, which were electron dense with
the PATAg staining (Fig. 8B) and electron transparent (inverted
contrast) with osmium-uranyl staining (Fig. 8A). The latter, which is
used for contrasting unsaturated lipids and for proteins and cellular
membranes, has no contrasting effect on polysaccharides. In both the
single sta7-carrying mutant reference (Mouille et al., 1996)
and in the double-mutant strains (Fig. 8, C-F), starch granules
disappeared, whereas other polysaccharide structures were selectively
revealed in the mutants through the PATAg staining procedure.
These polysaccharides displayed no detectable morphological
organization at this scale. According to both the biochemical and
staining results, these structures correspond to phytoglycogen. This
material appeared in the form of either small granular units, as in the
single sta7 mutant or in the double sta2 sta7
mutant (Fig. 8, E and F), or in the form of elongated groups of units
in the double sta3 sta7 mutants (Fig. 8, C and D). Moreover,
the size distribution of the WSP fit that which was expected for
glycogen polymers. No analogous structures were ever detected in
the wild-type algae. As usual, many lipid droplets were observed in all
cells undergoing nitrogen starvation.
In this work we report the purification and characterization of an
insoluble and dense polysaccharide that is synthesized in very low
amounts in DBE mutants of C. reinhardtii. To account for the
low
Received July 21, 1998;
accepted October 16, 1998.
Abbreviations:
DBE, debranching enzyme.
DP, degree of
polymerization.
GBSS, granule-bound starch synthase.
We would like to thank André Decq and Jocelyn Celen for their excellent technical assistance.
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D. Dauvillée, C. Colleoni, G. Mouille, M. K. Morell, C. d'Hulst, F. Wattebled, L. Liénard, D. Delvallé, J.-P. Ral, A. M. Myers, et al. Biochemical Characterization of Wild-Type and Mutant Isoamylases of Chlamydomonas reinhardtii Supports a Function of the Multimeric Enzyme Organization in Amylopectin Maturation Plant Physiology, April 1, 2001; 125(4): 1723 - 1731. [Abstract] [Full Text]
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A. E. Hall, J. L. Findell, G. E. Schaller, E. C. Sisler, and A. B. Bleecker Ethylene Perception by the ERS1 Protein in Arabidopsis Plant Physiology, August 1, 2000; 123(4): 1449 - 1458. [Abstract] [Full Text]
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P. L. Vrinten and T. Nakamura Wheat Granule-Bound Starch Synthase I and II Are Encoded by Separate Genes That Are Expressed in Different Tissues Plant Physiology, January 1, 2000; 122(1): 255 - 264. [Abstract] [Full Text] [PDF]
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C. Colleoni, D. Dauvillée, G. Mouille, A. Buléon, D. Gallant, B. |
]) of the undebranched
fractions is scaled on the left y axis and the amount of
Glc (in micrograms per milliliter [
]) measured for each fraction
is indicated on the right y axis. The x
axis shows the elution volume scale (in milliliters). B and C,
Histograms of chain-length distributions obtained through gel
electrophoresis of fluorescent glucans with or without
isoamylase-mediated enzymatic debranching. Both chain-length
distributions of debranched high-molecular-mass (B) and undebranched
low-molecular-mass (C) WSPs eluted from the TSK-HW-50 column (A) are
displayed as percentages of chains of DP between 2 and 16. The
x axis displays a DP scale, and the y
axis represents the relative frequencies of the chains expressed as
percentages. The peak seen between DPs 3 and 4 is consistent with the
position of a branched trisaccharide.
1 sta3
sta7
