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Plant Physiol. (1999) 120: 993-1004
Genetic and Biochemical Evidence for the Involvement of
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ABSTRACT |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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We describe a novel mutation in the
Chlamydomonas reinhardtii STA11 gene, which results in
significantly reduced granular starch deposition and major
modifications in amylopectin structure and granule shape. This defect
simultaneously leads to the accumulation of linear
malto-oligosaccharides. The sta11-1
mutation causes the absence of an 
-1,4 glucanotransferase known as
disproportionating enzyme (D-enzyme). D-enzyme activity was found to be
correlated with the amount of wild-type allele doses in gene dosage
experiments. All other enzymes involved in starch biosynthesis,
including ADP-glucose pyrophosphorylase, debranching enzymes, soluble
and granule-bound starch synthases, branching enzymes, phosphorylases,
-glucosidases (maltases), and amylases, were unaffected by the
mutation. These data indicate that the D-enzyme is required for normal
starch granule biogenesis in the monocellular alga C. reinhardtii.
Starch is one of the most abundant biological polymers present in
the earth's biosphere and remains the major supply of calories in both
human and animal diets. As is the case for animal, fungal, and
bacterial glycogen, starch is made solely of Glc residues linked in
Our strategy consisted of isolating mutants defective
in amylopectin synthesis, thereby defining the functions involved in starch granule biogenesis. From the analysis of mutants of the monocellular green alga Chlamydomonas reinhardtii (Mouille
et al., 1996 Eukaryotic algae are of particular relevance for studies dealing with
starch synthesis. Starch polysaccharides are not found in bacteria or
fungi. Because C. reinhardtii is the only starch-storing unicellular organism intensively studied by geneticists, it offers a
unique opportunity to understand the basic mechanisms of starch granule
biogenesis. Indeed, growth-arrested C. reinhardtii cells accumulate glucopolysaccharides that are very similar to cereal endosperm storage starch, so this organism serves as a highly useful
model for starch synthesis in crop plants. C. reinhardtii cells contain the same set of starch biosynthesis enzymes and, most
importantly, respond in an analogous fashion to mutations affecting
these activities (for review see Ball, 1998 D-enzymes define This study characterized a novel C. reinhardtii mutation,
sta11-1, which causes decreased levels of starch,
modification of amylopectin structure, increased amylose content,
modification of granule size and shape, and accumulation of unbranched
oligosaccharides. All enzymes previously suspected to be involved in
starch biosynthesis are unaffected by the presence of the
sta11-1 mutation; however, sta11-1 mutants lack an enzyme required for
maltotriose hydrolysis. Zymogram analysis established that the missing
maltotriose-metabolizing enzyme, as described for higher plants, is a
D-enzyme.
These data indicate that Materials
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
-1,4 positions and branched in -1,6 positions. Unlike glycogen,
starch granules consist of complex, semicrytalline structures of
unlimited size (for review, see Buléon et al., 1998
).
Amylopectin, the major polysaccharide fraction of starch, is considered
to be the only molecular fraction required to generate normal granules. The building of the polymer structure depends on the transfer by SS of
Glc in the -1,4 position from ADP-Glc to the nonreducing end of
growing chains. Introduction of the -1,6 branch proceeds through the
cleavage (by branching enzyme) of a pre-existing -1,4-linked glucan
and the transfer of the cleaved glucan in the -1,6 position. It was
previously thought that the specific features of starch structure
depended solely on the concerted action of multiple forms of SS and
branching enzyme; however, other enzymes are likely to be involved in
the process.
), maize (James et al., 1995), rice (Nakamura et al., 1996), and Arabidopsis (Zeeman et al., 1998b), it was determined that
debranching enzymes were required to trim -1,6 linkages from a
precursor (pre-amylopectin) into a mature amylopectin molecule (Ball et
al., 1996) or that they were required to prevent glycogen production by
the starch synthesis machinery (Zeeman et al., 1998b). Continued
genetic analysis is likely to identify additional enzymes needed for
starch biosynthesis that would not have been foreseen from the basic
biosynthetic steps (Mouille et al., 1996).
).
-glucanotransferases that transfer unbranched
malto-oligosaccharide groups from a donor -1,4 glucan of at least
three Glc residues (maltotriose) to a recipient oligosaccharide at the
final expense of Glc formation (Peat et al., 1956). It is thought that
this activity disproportionates short glucans into longer
oligosaccharides to facilitate their degradation through phosphorylases
or hydrolases (Boos and Schuman, 1998).
-1,4 glucanotransferases are important
components of the amylopectin synthesis machinery in C. reinhardtii. The results are generally similar to those regarding
debranching enzyme function, because in both instances enzymes thought
to be involved in the breakdown of glucopolysaccharides are apparently involved in starch biosynthesis.
MATERIALS AND METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
Chlamydomonas reinhardtii Strains, UV Mutagenesis, Growth Conditions, Cytological Observations, and Media
The wild-type reference Chlamydomonas reinhardtii strains used in this study were 330 (mt+ arg7 cw15 nit1 nit2) and 137C (mt
nit1 nit2). According
to the genotypes of the mutants or segregants tested, diploids were
selected by complementation on minimal medium after crossing either
with strain A35 (mt+ pab2 ac14),
37 (mt pab2 ac14), NV314
(mt pab2 ac14
sta1-1), strain 37E-17
(mt pab2 ac14
sta3-1), GST
(mt
nit1 nit2 sta5-1), BAFR1
(mt+ nit1 nit2 cw15 arg7-7
sta2-29::ARG7), or 18B
(mt nit1 nit2
sta2-1). The sta11-1 strains most
commonly used for complementation were CO 214 (mt+ nit1 nit2
sta11-1), CO 29 (mt+
pab2 ac14 sta11-1), and JV45J
(mt nit1 nit2
sta11-1).
2. Irradiation was followed by overnight
incubation in high-salt medium in darkness.
1) in the
presence of acetate at 24°C in liquid cultures that were shaken
vigorously without air or CO2 bubbling.
Late-log-phase cultures were inoculated at 105
cells mL1 and harvested at 2 × 106 cells mL1.
Nitrogen-starved cultures were inoculated at
5.105 cells mL1 and
harvested after 4 d at a final density of 1 to 2 × 106 cells mL1. Genetic
techniques were as described by Harris (1989a). Standard TAP (Tris
acetate phosphate) medium was fully detailed in Harris (1989b), while
nitrogen-starved medium (TAP-N) and diploid clone selection were
described in Ball et al. (1990, 1991) and Delrue et al. (1992).
Fixation and embedding protocols were as described in Dauvillée
et al. (1999).
Structural Analysis of Polysaccharides
Wide-angle x-ray diffraction and TEM and SEM analyses were as detailed in Buléon et al. (1997). Gel permeation chromatography of delipidated starch fractions dispersed in 10 mM NaOH was
as described in Delrue et al. (1992) and Libessart et al. (1995), and
was performed on Sepharose CL2B (Pharmacia). Gel permeation chromatography-purified amylose and amylopectin were debranched with
isoamylase. 1H-NMR of gel permeation
chromatography-purified starch fractions were as described previously
(Fontaine et al., 1993). The APTS-tagged chains produced by
isoamylase-mediated debranching were separated by high-resolution slab
gel electrophoresis on a DNA sequencer (O'Shea and Morell, 1996). The
results obtained were confirmed by capillary electrophoresis analysis
of debranched amylopectins obtained from two mutant and two wild-type
meiotic offspring. Capillary electrophoresis was carried out as
previously described (O'Shea et al., 1998).
Measures of Starch Levels, Starch Purification, and Spectral Properties of the Iodine-Starch Complex
A full account of amyloglucosidase assays, starch purification on Percoll gradients, and
max, the wavelength of
the maximal absorbance of the iodine-polysaccharide complex, can be
found in Delrue et al. (1992).
Crude Extract Preparation, Enzyme Assays, Partial Purification of Enzyme Activities, and Zymograms
Soluble crude extracts were always prepared from late- log-phase cells (2 × 106 cells mL1) grown in high-salt acetate medium under
continuous light (80 µE m2
s1). All assays were conducted in conditions of
linearity with respect to time and amount of crude extract.
Phosphoglucomutase, ADP-Glc pyrophosphorylase, and phosphorylase
activities were monitored by using the standard assays described in
Ball et al. (1991) and Van den Koornhuyse et al. (1996). For SS and
branching enzymes, the assays used were those described by Fontaine et
al. (1993) and Libessart et al. (1995). GBSS I was monitored as
previously described (Delrue et al., 1992) from the starch purified
from nitrogen-supplied cultures. -Glucosidase was monitored by
measuring the Glc produced from maltose hydrolysis in sodium acetate,
pH 6.5, by the standard Glc-6-P dehydrogenase assay described in Ball
et al. (1991). The analysis was completed by zymograms as detailed in
Buléon et al. (1997) and in Mouille et al. (1996). For partial
purification, a 10% (w/v) protamine sulfate precipitation was
applied to 5 ×109 cells in 4 mL of 50 mM sodium acetate, pH 6.0, and 1 mM DTT, loaded
on a FPLC anion-exchange column (1.6 × 10 cm, 1 mL
min1, 0-0.5 M NaCl gradient; DEAE
Mono-Q, Pharmacia), and 1-mL fractions were collected. Aliquots (300 µL) corresponding to each fraction were stored at 80°C, while the
different enzyme activities were detected by our standard enzyme assays
or zymogram procedures.
).
). The equivalent of 50 to
200 µg of undenatured or denatured crude extract protein was loaded
on a 30:1 (acry:bis), 7.5% (w/v) acrylamide, 1.5-mm-thick polyacrylamide gel (native conditions) or in a similar gel containing 0.1% (w/v) SDS (denaturing conditions). Migration at 15 V
cM1 was performed for 90 min at 4°C. At the
end of the run, the denaturing gels were first subjected to
renaturation as described in Mouille et al. (1996). The renatured and
native gels were incubated overnight in the dark at room temperature in
30 mL of 3 mg mL1 maltotriose; 200 mM Tris/HCl pH 8.0, 1 mM EDTA, 42 mM MgCl2, 0.014% (w/v) NADP, 0.027%
(w/v) NAD, 0.027% (w/v) MTT, 0.015% (w/v) PMS, 1.5 mM
ATP, 1 unit mL1 hexokinase, and 0.5 unit
mL1 Glc-6-PDH. The gel was subsequently rinsed
with distilled water and photographed.
Oligosaccharide Purification
Malto-oligosaccharides were prepared from 3 L of nitrogen-starved culture, inoculated at 105 cells mL1, and harvested after 7 d of growth
under continuous light (80 µE m2
s1) on high-salt acetate medium (Harris,
1989b) with gentle shaking. Algae were ruptured by passage in a
French press cell (10,000 p.s.i.) at a density of
108 cells mL1 in water.
The crude extract was immediately frozen at 80°C. After thawing,
cell debris were discarded by centrifugation at 10,000g for
15 min at 4°C. The supernatant was boiled for 5 min to inactivate
enzymatic activities, and centrifuged at 10,000g for 15 min.
The supernatant was then lyophilized, and the lyophilized material was
resuspended in 1 mL of distilled water and loaded onto a Dowex 50:2 (1- × 6-cm) column immediately coupled to a Dowex 1:2 (1- × 6-cm) column
equilibrated with water. Four microliters of each fraction (1 mL) was
subjected to TLC (Silica Gel 60 column). Malto-oligosaccharides were
revealed by spraying with orcinol (2 g L1)
dissolved in 20% (v/v) sulfuric acid. The TLC plate was subsequently incubated at 80°C for 10 min. After pooling and neutralization with
one drop of 30% ammonium hydroxide, the pool was concentrated by
rotary evaporation and desalted by gel filtration chromatography on a
column (TSK HW-40, Merck) equilibrated in 0.5% (w/v) acetic acid. The
desalted material was concentrated by rotary evaporation and
lyophilized. The sample was kept at room temperature until it was
subjected to NMR analysis.
Gene Dosage Experiments
Diploid and triploid strains were constructed as follows. To obtain the homozygous mutant diploid, we crossed CO 216 (mt ac14 nit1 nit2
sta11-1) and CO 27 (mt+ pab2 nit1 nit2
sta11-1) and selected the diploid after 4 d of growth on minimal medium supplied with ammonium. Vegetative diploid strains heterozygous for mating type display an
mt mating type. After checking the
phenotype, cellular volume, protein content, and mating type, we
crossed the homozygous mutant either with CO 29 to obtain the
homozygous triploid mutant or with strain 37 to obtain the
sta11-1/sta11-1/+ triploid. To obtain
the homozygous wild-type diploid, we crossed CO 218 (mt ac14 nit1 nit2) and CO 42 (mt+ pab2 ac14). The colonies
were selected on minimal medium supplied with acetate using nitrate as
the nitrogen source (Ball et al., 1991).
) and used as an internal standard during
these experiments.
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RESULTS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Isolation and Characterization of Low-Starch Mutants
Among 5 × 104 colonies screened by the standard iodine-staining technique after UV mutagenesis of a wild-type C. reinhardtii strain (137C), we isolated a low-starch mutant (JV45J) that accumulated 8% of the normal amount of starch under conditions of maximum synthesis (Fig. 1). The defect behaved as a standard single-recessive Mendelian trait upon crossing. In addition, all diploids generated by crossing with reference mutant strains carrying a defect in the STA1, STA2, STA3, STA4, STA5, STA6, STA7, or STA8 genes proved to be wild type for starch amount and structure. Therefore, we defined a novel genetic locus, which we named STA11. In addition to starch, the sta11-1 mutants accumulated a significant amount (2% of the amount of starch in a wild-type strain) of water-soluble, amyloglucosidase-digestible material. No growth defects could be detected upon growing the mutants for over a week in continuous light with or without acetate or under a 12-h day/12-h night cycle with or without acetate (Fig. 2).
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Characterization of the Water-Soluble, Amyloglucosidase-Digestible Material
We found no evidence for the presence of high-mass, water-soluble polysaccharides. Indeed, the amount of water-soluble amyloglucosidase-digestible material excluded from the gel permeation chromatography column was less than 1% of the total water-soluble polysaccharide found in crude extracts. The water-soluble polysaccharide thus consisted solely of-1,4-linked glucans, the
size distribution of which is displayed in Figure
3. These glucans were further purified (Table I) and subjected to a detailed
structural characterization. The size distribution before and after
purification was identical. The mix of oligosaccharides generated an
averaged 1H-NMR spectrum very similar to that of
maltotriose standards (Fig. 4). As
demonstrated by 1H-NMR analysis, branches, if
present, fell below our detection level (1%). Co-segregation between
the oligosaccharide fraction and the low-starch phenotype was found
among all sta11-1-carrying strains tested
(n = 50).
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Characterization of the Residual Mutant Starch
The residual starch structure was monitored through a variety of techniques, including wide-angle x-ray diffraction analysis (not shown), TEM and SEM (Fig. 5), separation of amylose and amylopectin through gel permeation chromatography (Fig. 6), and enzymatic debranching of the purified amylose and amylopectin (Fig. 7). The starch granules showed a significant relative increase in amylose (Table II). The distributions displayed in Figure 6, A and B, suggested a decrease in the mass of the amylose fraction. This was confirmed by mixing a low amount of labeled mutant starch with a high amount of wild-type reference polysaccharide on the same column. The x-ray diffractograms switched from the wild-type A-type lattice with high crystallinity to a mix of A- and B-type lattices with very low crystallinities. The shape of the granules was particularly affected, as illustrated in Figure 5.
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Finding the Enzymatic Defect
Gene Dosage Experiments
The Expression of the Mutant Phenotype Is Partly Conditional
It was recently reported that a 98% reduction in D-enzyme
activity did not affect starch biosynthesis or structure in potato (Takaha et al., 1998 Received March 8, 1999;
accepted May 17, 1999.
Abbreviations:
APTS, 8-amino-1,3,6-pyrenetrisulfonic acid.
D-enzyme, disproportionating enzyme.
DP, degree of polymerization.
GBSS, granule-bound starch synthase.
SEM, scanning electron microscopy.
SS, soluble starch synthase.
TEM, transmission electron microscopy.
We thank André Decq and Jocelyn Celen for their excellent
technical assistance.
Ball S
(1998)
Regulation of starch biosynthesis.
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M Goldschmidt-Clermont,
S Merchant,
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Shuman H
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Colleoni C,
Shaw E,
Mouille G,
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Morell M,
Samuel MS,
Bouchet B,
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Sinskey A
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321-330
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Fontaine T,
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Van Den Koornhuyse N,
Maddelein M-L,
Fournet B,
Ball S
(1992)
Waxy Chlamydomonas reinhardtii: monocellular algal mutants defective in amylose biosynthesis and granule-bound starch synthase accumulate a structurally modified amylopectin.
J Bacteriol
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3612-3620
Fontaine T,
D'Hulst C,
Maddelein M-L,
Routier F,
Marianne-Pepin T,
Decq A,
Wieruszeski JM,
Delrue B,
Van Den Koornhuyse N,
Bossu JP
(1993)
Toward an understanding of the biogenesis of the starch granule: evidence that Chlamydomonas soluble starch starch synthase II controls the synthesis of intermediate size glucans of amylopectin.
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In
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eds, The Chlamydomonas Sourcebook. A Comprehensive Guide to Biology and Laboratory Use.
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(1989b)
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Maddelein M-L,
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Figure 8.
Chain-length distributions of amylopectin from
wild-type and mutant strains. Distribution of chain lengths of
wild-type and mutant amylopectin after isoamylase-mediated debranching
were confirmed by capillary electrophoresis of APTS-labeled glucans
following a procedure previously described (O'Shea et al., 1998 ). The
relative amount of chains corresponding to each DP is strictly
equivalent to the normalized fluorescence percentage. A and B
correspond to wild-type strains 137C (A) and CO65 (B), while C and D
correspond to sta11-1-carrying strains
JV45J (C) and CO29 (D).
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Figure 9.
Comparison of the normalized masses of
APTS-labeled oligosaccharides from isoamylase debranched amylopectin.
The percentage difference of the total mass present in each individual
oligosaccharide has been obtained by subtracting the chain-length
distribution from debranched amylopectins. A, Subtractive analysis from
the wild-type reference strain 137C minus that of the mutant
sta11-1 JV45J ( 
) and from the
wild-type strain 137C minus that of the mutant
sta11-1 CO29 (
). B, Subtractive
analysis from the wild-type reference strain 137C minus that of the
wild-type strain CO65 (). C, Subtractive analysis from the wild-type
reference strain CO65 minus that of the mutant
sta11-1 JV45J () and from the
wild-type strain CO65 minus that of the mutant
sta11-1 CO29 (). D, Subtractive
analysis from the mutant reference strain JV45J minus that of the
mutant CO29 ().
-glucosidase), and all starch
hydrolases that could be detected in starch-containing zymogram gels
(Table III).
View this table:
Table III.
Enzyme activities in wild-type and mutant
sta11-1 progeny
AGPase (assayed in direction of pyrophosphorolysis in the presence of
1.5 mM 3-PGA), starch phosphorylase, and phosphoglucomutase
units are expressed in nmol Glc-1-P produced min 1
mg1 protein. SS and GBSS are expressed in nmol ADP-Glc
incorporated into polysaccharide min1 mg1
protein (SS) or mg starch (GBSSI). Branching enzyme is expressed as
nmol Glc-1-P incorporated into polysaccharide min1
mg1 protein (phosphorylase amplification assay).
Limit-dextrinase and D-enzyme are expressed in nmol maltotriose formed
from pullulan min1 mg1 protein and nmoles
Glc formed from maltotriose min1 mg1
protein, respectively. -Glucosidase activities are expressed in nmol
Glc formed from maltose min1 mg1 protein.
N/A, Not applicable.
-glucosidase or other hydrolases is negligible in face
of the amount of D-enzyme activity present in our extracts. This result
is similar to that reported for higher plant leaf extracts (Zeeman et
al., 1998a).
). The 62-kD band obeyed the rules set
years ago by Peat et al. (1956) to define D-enzymes: it could not use
Glc or maltose as sole substrates, but could disproportionate
-1,4-linked oligosaccharides of at least three Glc residues into
longer oligosaccharides.
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Figure 10.
The enzymatic defect of
sta11-1 mutant strains. Denatured crude
extracts (100 µg of protein) from two wild-type (+) and three
sta11-1 ( ) were loaded on denaturing
polyacrylamide gels. The proteins were renatured after electrophoresis
and incubated overnight. Glc production was revealed after overnight
incubation of the gel with 3 mg mL1 maltotriose. The
62-kD blue-staining band can be easily distinguished in the wild-type
cells. This band contained the D-enzyme activity.
1 mL1) or to perform
gene dosage experiments in diploid and triploid zygotes. The enzyme
activity correlated with the relative amount of wild-type alleles, as
would be expected if STA11 encoded D-enzyme (Fig.
11). Knowing the cell and organelle
volumes of the strains used in this work, we were able to calculate the
physiological enzyme concentrations (Schötz et al., 1972). We
were also able to estimate the amount of malto-oligosaccharides in
wild-type (50-200 µg mL1 of chloroplast) and
mutant (0.5-1 mg mL1) strains.
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Figure 11.
Gene dosages expressed from 0% (homozygous
mutant) to 100% (homozygous wild-type); 50% corresponds to the
heterozygous diploids, while 33% and 66% correspond to a
sta11-1/sta11-1/+
and a sta11-1/+/+ triploid respectively.
Haploid, diploid, or triploid homozygous combinations gave identical
results when the activity was expressed per milligram of protein. Means
( 
) and SDs of measures from three different
diploid or triploid constructs were calculated for each gene dose.
Total phosphoglucomutase-specific activities were monitored as internal
controls and proved similar in all constructs (2.5 ± 0.4 nmol
Glc-6-P formed from Glc-1-P min1 mg1
protein).
). In
C. reinhardtii, storage starch synthesis is obtained by
using nutrient starvation conditions, leading to the arrest of cell
division and to the accumulation of starch. On the other hand,
transitory starch synthesis is obtained under conditions of active
photosynthesis and cell division. Mutant phenotypes of C. reinhardtii fall into three classes. The first class concerns mutations whose phenotypes express themselves equally in both physiological conditions. Mutations affecting the small and large subunits of ADP-Glc pyrophosphorylase, GBSS, and debranching enzyme fall within this class (Ball et al., 1991; Delrue et al., 1992; Libessart et al., 1995; Mouille et al., 1996; Dauvillée et al., 1999). The functions thereby defined are said to be mandatory for
either amylose or starch synthesis.
). These
functions are thus involved in the process of normal starch synthesis,
but cannot be defined as mandatory in C. reinhardtii. We
believe this to be the result of some level of functional redundancy due either to the presence of multiple enzyme forms belonging to same
family or to the presence of alternative pathways such as those defined
by hydrolysis or phosphorolysis. Finally, some loci, when defective,
lead to a detectable phenotype only in storage conditions. Their
expression is said to be conditional. This applies to the high-amylose
sta4 mutants, for which no enzymatic defect has yet been
reported (Libessart et al., 1995).
DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References
). However, there are several known instances in
which potato antisense RNA technology has yet to confirm phenotypes of
mutants obtained in a wide variety of starch-synthesizing species. We
believe this to be due to the presence in many cases of a sufficient amount of residual wild-type activity to carry out the steps, which
could be far from rate controlling. This study establishes a
correlation between the disappearance of D-enzyme and major alterations
in starch structure and accumulation, and thus raises the possibility
that this enzyme is a constituent of the starch biosynthetic pathway.
-amylase on longer oligosaccharides that would otherwise
accumulate in the mutants. However, the major changes in starch
biosynthesis in conditions of nitrogen starvation were unexpected in
light of what is currently known about the functions of D-enzyme, in
particular because these enzymes are usually assumed to be utilized for
catabolism of unbranched malto-oligosaccharides. The D-enzyme
deficiency co-segregated not only with a reduction in total
glucopolysaccharide accumulation, but also with abnormal starch granule
shape and size, an altered amylose/amylopectin ratio in the mutant
granules, and changes in the chain-length distribution of amylopectin
in those granules. To explain these observations we propose that
D-enzyme has a specific function in the process of starch biosynthesis.
1
This work was supported by grants from the
Ministère de l'Education Nationale, by the Centre National de la
Recherche Scientifique (Unité Mixte de Recherche du Centre
National de la Recherche Scientifique no. 8576, Director André
Verbert), by the University of Lille, by the University of Reims, and
by a grant from Biogemma (Cambridge, UK).
FOOTNOTES
*
Corresponding author; e-mail steven.ball{at}univ-lille1.fr; fax
33-3-20-43-65-55.
ABBREVIATIONS
ACKNOWLEDGMENTS
LITERATURE CITED
Top
Abstract
Introduction
Methods
Results
Discussion
References
Copyright Clearance Center: 0032-0889/99/120//12
© 1999 American Society of Plant Physiologists
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-1,4
Glucanotransferases in Amylopectin Synthesis
) and wild-type (
-anomers of the reducing end are at 5.1 and 4.5 ppm, respectively.
If present, 
