The Plant Journal (2006) 48, 274–285
doi: 10.1111/j.1365-313X.2006.02870.x
Plastidial phosphorylase is required for normal starch
synthesis in Chlamydomonas reinhardtii
David Dauvillée1, Vincent Chochois2, Martin Steup3, Sophie Haebel4, Nora Eckermann3, Gerhard Ritte3, Jean-Philippe Ral1,
Christophe Colleoni1, Glenn Hicks5, Fabrice Wattebled1, Philippe Deschamps1, Christophe d’Hulst1, Luc Liénard1, Laurent
Cournac2, Jean-Luc Putaux6, Danielle Dupeyre6 and Steven G. Ball1,*
1
Unité de Glycobiologie Structurale et Fonctionnelle, UMR8576 CNRS/USTL, IFR 118, Université des Sciences et Technologies
de Lille, 59655 Villeneuve d’Ascq, Cedex, France,
2
UMR6191 CNRS/CEA/Aix Marseille II, DSV/DEVM//LB3M, Cadarache Bât 161, 13108 St-Paul-lez-Durance, France,
3
Plant Physiology, Institute of Biochemistry and Biology, University of Potsdam, 14476 Potsdam-Golm, Germany,
4
Center of Mass Spectrometry of Biopolymers of the University of Potsdam, Potsdam, Germany,
5
Center for Plant Cell Biology and Department of Botany and Plant Sciences, University of California, Riverside, CA 92521,USA,
and
6
Centre de Recherches sur les Macromolécules Végétales, ICMG-CNRS, BP 53, F-38041 Grenoble Cedex 9, France (affiliated with
the Joseph Fourier University of Grenoble)
Received 7 April 2006; revised 28 June 2006; accepted 3 July 2006.
*For correspondence (fax þ33 320 43 6555; e-mail [email protected]).
Summary
Among the three distinct starch phosphorylase activities detected in Chlamydomonas reinhardtii, two distinct
plastidial enzymes (PhoA and PhoB) are documented while a single extraplastidial form (PhoC) displays a
higher affinity for glycogen as in vascular plants. The two plastidial phosphorylases are shown to function as
homodimers containing two 91-kDa (PhoA) subunits and two 110-kDa (PhoB) subunits. Both lack the typical
80-amino-acid insertion found in the higher plant plastidial forms. PhoB is exquisitely sensitive to inhibition by
ADP-glucose and has a low affinity for malto-oligosaccharides. PhoA is more similar to the higher plant
plastidial phosphorylases: it is moderately sensitive to ADP-glucose inhibition and has a high affinity for
unbranched malto-oligosaccharides. Molecular analysis establishes that STA4 encodes PhoB. Chlamydomonas
reinhardtii strains carrying mutations at the STA4 locus display a significant decrease in amounts of starch
during storage that correlates with the accumulation of abnormally shaped granules containing a modified
amylopectin structure and a high amylose content. The wild-type phenotype could be rescued by reintroduction
of the cloned wild-type genomic DNA, thereby demonstrating the involvement of phosphorylase in storage
starch synthesis.
Keywords: Chlamydomonas, starch, amylopectin, (glycogen) starch phosphorylase.
Introduction
Glycogen (starch) phosphorylase catalyses the release of the
glucose-1-P (G1P) from the non-reducing ends of the outer
chains of polysaccharides composed of a-1,4-linked glucose
residues. The enzyme, which was demonstrated to be involved in glycogen breakdown in yeasts and humans (Burwinkel et al., 1998; Hwang et al., 1989) displays a generally
well-conserved structure. The latter consists mostly of
homodimers with each subunit linked to a pyridoxal phosphate co-factor involved in enzyme catalysis (for review see
Buchbinder et al., 2001).
274
In Escherichia coli two a-1,4-glucan phosphorylases playing distinctive roles in carbohydrate metabolism have been
documented (Chen and Segel, 1968a,b). Maltodextrin phosphorylase, the product of the malP gene, was proved to be
required for the assimilation of maltose in E. coli and
displays a high affinity for malto-oligosaccharides (MOS;
reviewed in Boos and Shuman, 1998). Glycogen phosphorylase, the product of the glgP gene, has recently been
demonstrated to be involved in glycogen breakdown
(Alonso-Casajús et al., 2006). Mutants lacking GlgP were
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Phosphorylase functions in starch metabolism 275
shown to overproduce glycogen with longer outer chains.
These results make a strong case for a predominant
catabolic function of phosphorylase in glycogen metabolism, a function which is apparently conserved in bacteria
and heterotrophic eukaryotes such as yeasts and humans.
In plants the function of phosphorylase in storage polysaccharide metabolism remains unknown. Two forms were
purified from a variety of plant species (Conrads et al., 1986;
Steup and Latzko, 1979). An extraplastidial form with high
and low affinity respectively for glycogen and MOS was
documented (Shimomura et al., 1982). When the activity of
this form was selectively decreased by antisense RNA
constructs in potato (Duwenig et al., 1997), the plant exhibited increased tuber sprouting and flowered early and
continuously with little if any change in carbohydrate
metabolism. However, cytosolic heteroglycans that are
suspected to be required for maltose assimilation during
starch breakdown are affected by modifications in cytosolic
phosphorylase (Fettke et al., 2005). Antisense RNA constructs directed against the plastidial phosphorylase also
failed to reveal any impact on starch metabolism (Sonnewald et al., 1995). In addition characterization of a mutation in
the gene coding the unique plastidial phosphorylase gene of
Arabidopsis failed to uncover a function for plastidial
phosphorylase in breakdown of leaf starch (Zeeman et al.,
2004). Nevertheless, the location and expression pattern of
the enzyme is suggestive of some function in starch
metabolism. That this is indeed the case is suggested by
the isolation of the sh4 (shrunken seed) mutant of maize,
which displays a strong reduction in amounts of starch and a
significant modification of the migration pattern of starch
phosphorylases (Tsai and Nelson, 1969). However, the
product of the sh4 gene is still unknown and contradictory
reports stating that this gene may or may not control the
supply of pyridoxal phosphate have appeared (Burr and
Nelson, 1973; Yu et al., 2001).
In this communication, the phosphorylase isozyme pattern of Chlamydomonas reinhardtii has been studied. Kinetic properties and the intracellular location of the distinct
isozymes were determined. For a functional analysis of the
isozymes, mutants of C. reinhardtii were isolated that are
defective for glycogen (starch) phosphorylase isozymes. Our
results demonstrate that starch phosphorylase is required
for the synthesis of normal polysaccharide granules when
the flux to starch is high.
Results
Biochemical characterization of the Chlamydomonas phosphorylases
Upon refining our zymogram procedures we were able to
distinguish one fast migrating phosphorylase band (PHOA)
with a low affinity for glycogen and two slowly migrating
Figure 1. Compartmentation study of phosphorylase activities.
Five micrograms of proteins from crude extract (1) and purified chloroplasts
(2) of the wild-type reference strain 330 were analyzed on glycogen-containing zymogram. The PhoC activity cannot be seen in the chloroplast fractions
while the other two phosphorylase activities appears to be plastidial. The
purity of the chloroplast preparation were ascertained by assaying several
cytosolic and plastidial marker enzymes as described in Experimental
procedures. The UDP-glucose pyrophosphorylase activities were 520 15
and 97 5 lmol G1P formed per microgram of chlorophyll and per hour
(lg chl)1 h)1) in the crude extract and chloroplast fractions, respectively. The
activities measured for the second cytosolic marker (phosphoenolpyruvate
carboxylase) were respectively 21 1 and 4.4 0.6 lmol oxaloacetate
lg chl)1 h)1. The plastidial marker enzyme activities in these two samples
were respectively 92 4 and 110 7 lmol GAP lg chl)1 h)1 for NADP
glyceraldehyde-3-phosphate (GAP) dehydrogenase and 79 4 and 94 4
GAP lg chl)1 h)1 for Rubisco. All these values are the average and standard
deviations from three chloroplast purification experiments.
phosphorylase bands (PHOB and PHOC). In order to localize
these activities, intact and photosynthetically active chloroplasts were prepared from Chlamydomonas as detailed in
the methods section. Results displayed in Figure 1 clearly
show that PhoA and PhoB are plastidial while PhoC is
extraplastidial. To get a better understanding of the properties of the two plastidial forms we set out to purify them by
affinity chromatography on an amylose column. Both
activities were purified to homogeneity and revealed unique
proteins estimated respectively at 91 and 106 kDa for PhoA
and PhoB (see Figure S1a). Yield and purification factors of
0.03% and 1050-fold and 0.1% and 1590-fold were obtained
for PhoA and PhoB, respectively. The two proteins were
identified by matrix-assisted laser desorption/ionization
(MALDI) MS following trypsic digestion and were shown to
contain distinctive peptides (see Figure S2). Both activities
exhibited a broad temperature optimum range between 25
and 40C (see Figure S1b). The pH optima of PhoA and PhoB
were estimated at 6.5 and 7.5 respectively, with <30% variation over the physiological stromal pH range (see Fig-
ª 2006 The Authors
Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 48, 274–285
276 David Dauvillée et al.
Table 1 Kinetic properties of PhoA and PhoB
PhoA
PhoB
Starch
Glycogen
MOS
PI
G1P
0.71
0.51
2.00
1.60
0.52
7.80
1.90
1.60
0.68
0.41
Km values have been calculated with the Lineweaver–Burk method.
Km values for starch, glycogen and malto-oligosaccharides (MOS) are
expressed in mg ml)1 and were obtained in the presence of 10 mM
propidium iodide (PI). Km values for PI and glucose-1-phosphate
(G1P) are expressed in mM and were calculated in the presence of
10 mg ml)1 glycogen.
ure S1c). We then pursued characterization of the two
plastidial phosphorylases by measuring their affinities for
glycogen, soluble potato starch, MOS, orthophosphate and
G1P. The results listed in Table 1 clearly demonstrate that
PhoA and PhoB displayed similar characteristics with the
noticeable exception of their affinities for MOS. The high
affinity of PhoA for MOS and its inability to significantly
interact with glycogen on zymogram gels suggests that this
activity is more similar to maltodextrin-type phosphorylases
than to glycogen phosphorylases of bacteria. Crude starch
did not act as a substrate under these conditions. To ensure
that PhoB was unable to degrade this substrate, even with
low efficiency, we measured the release of radioactive G1P
from a preparation of starch granules that had been prelabeled in vivo by supplying the wild-type or the mutant I97
(sta4-2) strain cultures with radioactive acetate. The purified
PhoB protein was unable to release any radioactive G1P
from labeled starch under these conditions while commercial amyloglucosidase released labeled glucose abundantly.
Both PhoA and PhoB were found to exhibit a mixed type of
inhibition when assayed in the presence of ADP-glucose
(Figure 2). However, the inhibition by ADP-glucose of PhoA
was less sensitive and dramatic than that displayed by PhoB
(apparent Ki for PhoA and PhoB at 1 mM orthophosphate
was respectively 1 and 0.2 mM ADP-glucose and maximal
inhibition achieved was respectively 70% and 95%). Finally,
we set out to measure more precisely the size of the native
enzyme through electrophoretic analysis. These experiments established the size of native PhoA and PhoB at 182
and 214 kDa. This result, together with the MALDI analysis of
the pure protein fragments, suggests that each activity
consists of a homodimer (see Figures S1 and S3).
Genetic characterization of phosphorylase mutants
For a functional in vivo analysis of the three phosphorylase
isozymes, we probed our collection of mutants for a selective defect in any of the three characterized activities. Three
strains were found to be selectively defective for PhoB.
Mutant strain I97 was selected because of its high amylose
phenotype among 9600 clones that had survived X-ray
mutagenesis from a wild-type isolate. The latter came from a
Figure 2. ADP-glucose inhibition of PhoA and PhoB activities.
The pure PhoA and PhoB enzyme preparations were used to study the
phosphorolysis of glycogen as described in Experimental procedures in the
presence of different ADP-glucose concentrations (0.02, 0.05, 0.15, 0.5, 1 and
3 mM). The results are displayed as per cent activity compared with the
activity measured without ADP-glucose for each purified enzyme. Remaining
activities for PhoA ( ) and PhoB (h) in the presence of the different ADPglucose concentrations (x-axis) are shown.
distinct source with respect to our standard 137C strain (see
Harris, 1989a for a discussion on the identity of the 137C
references). I97 was crossed with strain 37 (a segregant with
background derived from our standard wild-type reference)
and the meiotic progeny was analyzed through tetrad analysis (seven tetrads). The high amylose phenotype was
scored after separation of amylose and amylopectin through
gel permeation chromatography. The segregations fitted
those expected from a single Mendelian defect (two wildtype and two mutant progeny per tetrad) as far as the high
amylose phenotype is concerned (see Table S1). However,
both I97 and the particular wild-type isolate from which it
was derived displayed a starch excess phenotype. This excess phenotype did not co-segregate with the mutation
responsible for the modification of starch structure. Starch
excess in these crosses segregated in a complex fashion
consistent with the presence of several additional background mutations present in the original 137C subclone.
Because of the high values of the standard deviations of
wild-type (32 23 lg per million cells) and mutant
(33 17 lg per million cells) progeny, we were unable at
this stage to probe the significance of variations in amounts
of starch. Nevertheless, we were later able to create sets of
isogenic strains allowing us to show the real impact of this
novel mutation on starch content (see below). The high
amylose phenotype of I97 and its meiotic progeny can only
be seen during nutrient starvation. This property is reminiscent of that previously reported for the sta4-1 mutant of
Chlamydomonas (Libessart et al., 1995). Indeed, strain I73
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Phosphorylase functions in starch metabolism 277
containing sta4-1 did not display the PhoB band on zymogram gels. This mutation was shown to lead to a conditional
phenotype consisting of low starch accumulation and high
amylose content that could only be seen during nutrient
starvation (see Table S2). In this case the co-segregation
between low amounts of starch during nitrogen starvation
and the presence of the sta4-1 mutation was perfect
(23 6 lg per million cells for the wild-type population and
8 3 lg per million cells for the mutants) We therefore
performed complementation and recombination tests to
assay possible allelism between the mutants. We were unable to find wild-type recombinants upon crossing I97 with
the sta4-1 mutant strain of the opposite mating type, thereby
establishing linkage between the two mutations (n ¼ 147).
Four diploid strains were constructed for complementation
analysis as detailed in Experimental procedures. The
homozygous mutant diploids containing one copy each of
sta4-1 and sta4-2 accumulated low amounts of starch with
high amylose and modified amylopectin (Table 2). These
results establish that I97 carries a novel allele of STA4 that
we have named sta4-2. In addition to this we found a third
strain (I86) defective for PHOB. This strain, like I73, was
generated by X-ray mutagenesis of our 137C reference
strain. Because this mutant was selected from an independent X-ray mutagenesis experiment, it potentially defines a novel sta4 allele (sta4-3). This was confirmed by
standard complementation analysis. I73 and I86 displayed
identical phenotypes with respect to starch accumulation
Table 2 Complementation study performed in diploid strains
Strains
Ploidy Genotype
37
137C
I97
I73
IO3
137C · 37
I97 · 37
I73 · 37
I97 · IO3
n
n
n
n
n
2n
2n
2n
2n
þ
þ
sta4-2
sta4-1
sta4-1
þ/þ
sta4-2/þ
sta4-1/þ
sta4-2/sta4-1
Starch amount Am %
Ap kmax
24.3 3.1
26.2 4.2
49.1 2.1
11.0 3.8
13.6 2.4
55.0 3.2
59.6 2.7
49.4 2.9
30.0 3.1
557 2
557 3
579 2
582 2
581 4
556 5
558 6
555 3
586 5
22 5
24 3
43 7
38 4
39 2
29 8
25 3
29 8
47 5
Starches from 1 l of nitrogen-starved culture were purified and
assayed from four independent diploid strains for each genotype.
Two milligrams of each starch sample were then subjected to CL-2B
gel permeation chromatography. Both amylopectin and amylose
fractions were then assayed allowing the determination of the
amylose content (Am %). The wavelength at the maximum absorbance (kmax) was monitored for the amylopectin of each strain (Ap
kmax). The starch amounts (in lg per 106 cells), the percentage of
amylose in starch and the amylopectin kmax (in nm) are displayed as
average values with standard deviations. þ stands for a wild-type
copy of the STA4 gene. These values are compared with the haploid
(n) reference strains used to construct the diploids (2n). Three
independent experiments were performed to characterize the reference strains and three independent diploid strains of each genotype
were analyzed.
and amylose content. In order to make sure that the quantitative defect in starch accumulation witnessed during
nitrogen starvation in sta4 mutants was not due to a defect in
the efficiency of starch degradation we investigated the
kinetics of polysaccharide synthesis after the switch to
nutrient-depleted medium. Results displayed in Figure S4
show that the rate of starch synthesis is immediately lower
in the mutants and the lower starch contents are not due to
inefficient polysaccharide turnover when the maximum
starch content is reached.
Characterization of wild-type and mutant starch structure
We then proceeded to analyze the structure and morphology
of the mutant starch from nitrogen-supplied and nitrogenstarved cultures. Results from nitrogen-starved cultures
displayed in Figure 3(b) show a clear increase in both amylose and in the long glucan content of amylopectin as witnessed by the increase in kmax of the iodine polysaccharide
complex. Significant differences (Figure 4b) were also witnessed in the chain-length (CL) distribution of the mutant
amylopectin. These differences were analogous to those
seen in the previously characterized sta4-1 mutant (Figures 3c and 4c). Scanning electron microscope analysis enabled us to detect some gross alterations in the morphology
of starch granules extracted from nitrogen-starved cultures
(Figure 5). These alterations were found to co-segregate in
crosses with the sta4-2 mutation and could also be found in
the mutants carrying sta4-1.
Characterization of the enzymological defect in sta4 mutants
Mutations of the STA4 gene were first reported as low-starch
high-amylose defects of a conditional nature (Libessart
et al., 1995). Indeed their expression on the phenotype could
only be clearly seen in conditions of maximum starch synthesis (nitrogen starvation). At the time we were unable to
find any clear enzymatic defect. Upon refining our zymogram procedures we were able to show the consistent absence of a phosphorylase band in all sta4 mutants. This did
not always lead to a reproducible decrease in total phosphorylase activity in crude extracts because of the compensation afforded by two other forms of enzyme activity.
We then pursued our co-segregation analysis through
zymogram analysis of 13 tetrads and of an additional set of
27 wild-type and 44 mutant progeny from our random spore
analysis. An example of such an analysis is shown in Figure 6. We found no recombination between sta4-1 or sta4-2
and the loss of the PhoB zymogram band in a total of 50
progeny. We therefore conclude that sta4-1 and sta4-2 specifically lead to the disappearance of one form of phosphorylase activity in Chlamydomonas. No other enzyme of
starch metabolism were found to be defective in the mutants
(see Table S3).
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Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 48, 274–285
278 David Dauvillée et al.
Figure 3. Separation of amylopectin and amylose on CL-2B chromatography.
The optical density (open circles) was measured for each 300 ll fraction at kmax (unbroken thin line). All samples were loaded on the same column setup as described
in Delrue et al. (1992). The wild-type 137C strain starch extracted from nitrogen-starved culture (a) displays both amylopectin and low-Mr amylose. Plots (b) and (c)
correspond to the chromatograms obtained with starch extracted from the sta4-2 (I97) and sta4-1 (I73) mutant strains, respectively. The high amylose content of
starch extracted from these strains can be easily detected.
Figure 4. Chain-length distribution of amylopectin from wild-type and mutant strains.
Distribution of chain lengths of wild-type and
mutant amylopectins were obtained after isoamylase-mediated debranching as fully described
in Fontaine et al. (1993). Percentages of chains
ranging between DP 5 and DP 50 (chains containing 5–50 glucose residues) are scaled on the
y-axis.
(a) Debranched chains of gel permeation chromatography purified amylopectin from the wildtype reference strain 137C. Plots (b) and (c)
correspond respectively to the chain length distributions obtained for the amylopectins produced by the sta4-2 (I97) and sta4-1 (I73) mutant
strains. Plots (d) and (e) correspond respectively
to the subtractive analysis from sta4-2 and sta4-1
mutants minus that of the wild-type strain.
(f) Subtractive analysis from the sta4-2 mutant
minus that of the sta4-1 mutant.
Molecular characterization of PHOA and PHOB cDNAs
A combination of RT-PCR cloning, screening cDNA banks
and analysis of the growing expressed sequence tag (EST)
resources of Chlamydomonas enabled us to determine the
full-length sequences of two out of the three biochemically
identified phosphorylases. We thus identified a 3368-bp and
a 4261-bp cDNA with open reading frames (ORFs) of 2619 bp
(GenBank DQ279079) and 3033 bp (GenBank DQ279077)
encoding, respectively, a 873 and a 1011-amino-acid protein.
Analysis of the sequences and matching with the MALDI
trypsic digest analysis establish the 873- and 1011-amino-
acid ORFs, respectively, as PhoA and PhoB (Figure S2). A 37amino-acid transit peptide sequence was unambiguously
determined thanks to the acquisition of the N-terminal sequence of the PhoA protein by quadruple time of flight
(QTOF) analysis yielding a mature 94-kDa enzyme. Sequence
analysis and determination of the putative transit peptide
cleavage sites of PhoB are compatible with the existence of a
55-amino-acid transit peptide yielding a 108-kDa mature
enzyme.
Both enzymes display the canonical features of a-1,4glucan phosphorylases. However, they clearly lack the
insertion that typifies the vascular plant plastidial enzymes.
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Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 48, 274–285
Phosphorylase functions in starch metabolism 279
Figure 5. Scanning electron micrographs of starches from wild-type and
mutant sta4-1 strains.
Starch was extracted from cells cultured in nitrogen-starved medium. The
four products of one representative tetrad arising from the cross between the
wild-type reference 37 and the I73 original mutant (sta4-1) strains were
analyzed. Electron micrographs of starch extracted from the two wild-type
segregants (a and b) and the two sta4-1 mutant strains (c and d) are displayed.
The irregular shapes observed in the two mutant progeny starch granules are
co-segregating with the sta4 mutation.
The latter consist of an approximately 80-amino-acid long
insertion of variable sequence found between the C-terminal
and N-terminal domains within a starch binding site.
Sequence comparisons among different bacterial a-1,4glucan phosphorylases confirm that substrate preferences
(malto-oligosaccharide versus polysaccharide) have been
acquired several times independently and do not define two
separate types of phosphorylase sequences during evolution (Schinzell and Nidetzky, 1999).
STA4 defines the PhoB structural gene
The sta4 mutations could lead to the disappearance of PhoB
through two distinct mechanisms. STA4 could encode a
regulatory protein whose function is required for PhoB
activity or STA4 could encode PhoB. X-ray induced mutations often lead to deletions, inversions or translocations of
sufficient magnitude to be found through Southern analysis
using probes corresponding to the gene concerned. We
used the pho5 cDNA clone corresponding to the C-terminal
half of the full PHOB cDNA sequence as the probe in
Southern blot experiments. We thus found allele-specific
modifications in Southern blot analysis performed on wildtype and mutant sta4-2 DNA extracts (Figure 7a). These allele-specific modifications co-segregated with sta4-2 in all
meiotic progeny of the five tetrads analyzed and in the parental strains (n ¼ 22). In addition to this, RNA was purified
from the same segregants (Figure 7b). No mRNA could be
amplified from sta4-2 strains, further proving that STA4
encodes PhoB and that sta4-2 leads to the absence of PHOB
Figure 6. The enzymatic defect of sta4 mutant strains.
Five micrograms of crude extract was loaded on a glycogen-containing PAGE
gel and subjected to electrophoresis in native conditions (see Experimental
procedures). One representative tetrad arising from the cross between the
wild-type reference 37 and the I97 original mutant (sta4-2) strains is displayed.
The two mutant progeny (lanes 3 and 4) lack the PhoB enzymatic activities
while the two wild-type strains harbor it.
normal mRNA and consequently of PhoB protein and enzyme activity. By contrast we were able to amplify this sequence from the mRNA preparations of sta4-1 mutants. It
remains possible, however, that the mutant phenotype results selectively from the absence of PhoB and not from the
absence of another protein encoded by a gene close to the
STA4 locus. To rule this out, we chose to complement the
mutant defects by introduction of the wild-type PHOB genomic sequence by transformation. In order to do this we
have amplified the full 8.7-kbp genomic sequence corresponding to PhoB starting with the ATG and ending with the
stop codon and have cloned this into the pSL18 plasmid
containing both psaD 5¢ and 3¢ untranslated regions flanking
the multicloning site and the AphVIII gene conferring paramomycin resistance. We were able to restore a wild-type
phenotype in both sta4 mutants (see Figure S5 and Table S3). In the background of the I97 (sta4-2) strain, we were
equally able to restore a wild-type polysaccharide structure.
In addition the amount of starch was significantly increased,
suggesting that in this high-starch background the effect of
PhoB loss of function is indeed also to decrease the amount
of starch.
Physiological characterization of starch accumulation and
phosphorylase activities in nitrogen-supplied cultures
In Chlamydomonas phases of starch synthesis and degradation are known not to coincide with night and day and
significant starch degradation occurs at the beginning of the
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Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 48, 274–285
280 David Dauvillée et al.
(a)
Figure 8. Diurnal behavior of PhoB activity.
Chlamydomonas reinhardtii wild-type strain 137C was grown under a 12-h
light/12-h dark cycle with constant bubbling of 2% (v.v) CO2 for 4 days. Cells
were then collected every 3 h and crude extracts were prepared from each
sample. Crude extract proteins (5 lg) were analyzed on a glycogen-containing
zymogram. The pattern observed shows a net increase in PhoB enzymatic
activities during the dark period.
(b)
lights off; net starch degradation occurs, however, predominantly from midnight to midday (Ral et al., 2006;
Thyssen et al., 2001). In the presence of acetate, the synthesis also starts in the middle of the light phase and continues to the middle of the night. Starch degradation thus
occurs from midnight to midday (Ral et al., 2006). We conclude that PhoB enzyme activity is tightly correlated to the
phases of starch synthesis. However, mRNA levels corresponding to PhoB were also monitored by semiquantitative
PCR. The results show that the mRNA corresponding to
PHOB clearly peaks at the onset of starch degradation and
decreases thereafter (see Figure S6).
Discussion
Figure 7. Southern blot and RT-PCR analysis of the I97 and 37 progeny.
(a) The genomic DNAs from reference strains were digested by BamHI and
separated by electrophoresis. The hybridization patterns was obtained with a
455-bp probe corresponding to the PHO5 cDNA fragment of PHOB (see
Experimental procedures). The original sta4-2 mutant strain (I97) profile is
displayed in lane 2. The wild-type strain profile 37 used in the crosses
performed (lane 1) shows a different pattern. Lanes 3 to 4 correspond
respectively to two wild-type (lanes 3 and 5) and one mutant (lane 4) progeny.
(b) Reverse transcriptase-CR analysis of the strains studied by Southern blot.
A specific 736-bp PHOB cDNA fragment was amplified in both the wild-type 37
reference strain (lane 1) and wild-type progeny (lanes 3 and 5). No PCR
product could be obtained with RNA extracted from the original mutant (lane
2) or the mutant progeny (lane 4) of the cross involving I97 and 37.
light phase. Zymogram analysis of starch phosphorylases
was carried out both under mixotrophic and autotrophic
(high CO2) growth in the absence of nutrient starvation.
Results displayed in Figure 8 show that PhoB activity
increases from the middle of the light phase to the middle of
the night while PhoA activity is abundant throughout the
cycle. Identical patterns were produced from cultures in the
presence of acetate. Under high CO2 starch synthesis starts
in the middle of the light phase and abruptly stops after
Starch phosphorylase defines the second most abundant
protein of the cereal kernel amyloplast (Yu et al., 2001). The
plastidial form was purified from potato tubers over 60 years
ago (Green and Stumpf, 1942) and its function in starch
metabolism remains unknown to this date. We show in this
paper that, unlike vascular plants, unicellular green algae
contain two distinct forms of plastidial phosphorylases. In
the case of vascular plants, it was shown that when several
plastidial phosphorylase subunits existed, they were closely
related and readily formed a heterodimeric structure (Albrecht et al., 1998). This is not the case here. Biochemical
characterization of the two Chlamydomonas plastidial
phosphorylases show that they can be easily distinguished
by their affinities for small unbranched MOS. PhoA displays
a significantly higher affinity for MOS while PhoB is more
active with respect to branched polymers such as glycogen.
The situation observed in the Chlamydomonas plastid is
thus reminiscent of that which has been documented in
E. coli (reviewed in Boos and Shuman, 1998).
The comparative kinetic properties of the Chlamydomonas PhoA and PhoB enzymes together with the absence of
cross compensation evidenced in sta4 mutants calls for
ª 2006 The Authors
Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 48, 274–285
Phosphorylase functions in starch metabolism 281
distinct functions to be assumed for the plastidial phosphorylases. Whether these functions consist of unbranched
MOS versus polysaccharide breakdown remains unknown.
If it exists, we expect the true polysaccharide substrate of
PhoB to be water-soluble since despite all our efforts we see
no evidence that this enzyme can release G1P directly from
crude starch granules. The absence of accumulation of this
hypothetical substrate in the mutants could be due to the
presence of many other hydrolases that have the ability to
degrade such branched water-soluble material in Chlamydomonas.
A yet to be proven function of PhoB in polysaccharide
breakdown could be to release G1P from relatively large
branched water-soluble dextrins within the chloroplast. This
function is likely to become apparent only in specific
physiological conditions calling for immediate intraplastidial release of G1P and assimilation within the organelle
without prior export to the cytoplasm. Indeed the major
route of starch catabolism documented through mutant
work in Arabidopsis appears to involve maltose production
through b-amylase and its export to the cytoplasm (Niittylä
et al., 2004). Because of this, the phoB mutants of Chlamydomonas fail to display a starch excess phenotype in standard
conditions. In this respect the mutants behaved in a similar
fashion to those recently characterized in Arabidopsis
(Zeeman et al., 2004).
This paper demonstrates, however, an important function of starch phosphorylase during starch anabolism. The
initial sta4 mutants were found as mutants expressing a
conditional low-starch high-amylose phenotype expressing
itself only in conditions of high polysaccharide synthesis
(nitrogen starvation; Libessart et al., 1995). The two new
mutant alleles characterized in this work behaved in a
similar fashion. We have previously proposed that nitrogen
starvation in Chlamydomonas could be used as a model
system to understand the major aspects of storage starch
synthesis, while nitrogen-supplied cultures could be used
to understand the transitory starch type of synthesis
occurring in leaves. We were also able to prove that the
structure of the storage starch of low-starch mutants
defective either for plastidial phosphoglucomutase or for
the large subunit of ADP-glucose pyrophosphorylase (AGPase) mimics that of transitory starch, suggesting that it is
the maintenance of a high flux intensity towards starch
synthesis throughout the day which is responsible for the
major differences (Van den Koornhuyse et al., 1996). The
absence of clear phenotype of the plastidial leaf phosphorylase Arabidopsis mutants or potato leaf antisense
constructs is in line with the absence of phenotype
witnessed in nitrogen-supplied culture (transitory starch
conditions) for the Chlamydomonas mutants.
The shrunken 4 (sh4) mutants of maize define the only
case where a plastidial phosphorylase defect was correlated
with a defect in storage starch synthesis within the kernel
endosperm (Tsai and Nelson, 1969). As with the sta4
Chlamydomonas mutants reported in this work, the defect
consisted of a decrease in starch content. It must be noted,
however, that following the initial report, Burr and Nelson
(1973) further suggested that the defect might be due to a
modification of the pyridoxal phosphate pool and thereby
affect several metabolic steps including AGPase. Since this
sobering rectification, additional experiments reported by
Yu et al. (2001) suggest that decreases in the synthesis of
effectors such as pyridoxal phosphate may not be responsible for the two-thirds reduction of total phosphorylase
activity assayed in sh4. In addition a net reduction in the
amount of the 112-kDa protein was witnessed. It therefore
remains possible that sh4 is indeed mainly a plastidial
phosphorylase-defective mutant. The 112-kDa plastidial
phosphorylase is basically similar to the chloroplast leaf
enzymes. Further work is now needed to clarify the
molecular nature of sh4 in maize and its impact on starch
structure.
In vascular plants there is evidence for only one type of
phosphorylase within the plastids. The kinetic properties of
the purified enzyme brings it closer to the Chlamydomonas
PhoA or the E. coli maltodextrin phosphorylase than to the
PhoB glycogen phosphorylase type of enzyme. It is indeed
possible that the function assumed by PhoB has been lost
during evolution and taken over by other enzymes On the
other hand, the dual functions (maltodextrin versus polysaccharide phosphorylase) of starch phosphorylases might
be assumed by the single higher plant plastidial enzyme. It
has been recently demonstrated that the 78-amino-acid
insertion that typifies the plastidial phosphorylase was
subjected to targeted proteolysis in mature sweet potato
roots (Chen et al., 2002). The nicked enzyme still kept its
quaternary dimeric structure through the presence of multiple weak interactions between the two subunits. Interestingly, this yielded an enzyme which displayed a significantly
higher affinity for soluble potato starch, in effect switching
the enzyme from a maltodextrin active form to a polysaccharide active form in an irreversible fashion. Thus a single
enzyme in vascular plants might still assume the dual
functions of the bacterial or Chlamydomonas phosphorylases. The fact that Chlamydomonas has apparently maintained the dual function of starch phosphorylases on distinct
enzymes may facilitate both further genetic dissection and
the interpretation of already complex mutant phenotypes.
As to the function of PhoA, the biochemical properties and
enzyme activity expression patterns is also suggestive of
function during both phases of starch synthesis (because of
pre-amylopectin trimming) and degradation in a fashion
similar to D-enzyme (Wattebled et al., 2003). We believe this
function to consist of phosphorolytic breakdown of MOS.
The absence of phenotype recorded in Arabidopsis is simply
due to the presence and importance of plastidial b-amylases
and to the fact that since the supply of ATP is unlimited in the
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Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 48, 274–285
282 David Dauvillée et al.
leaf, phosphorolysis is not necessarily favored over hydrolysis.
Our work builds a solid case for the involvement of one
form of plastidial phosphorylase in the process of polysaccharide storage. Several possibilities can account for a
selective function of this enzyme in the biosynthesis of
storage starch.
When the flux of carbon to starch increases so does the
amount of glucan trimming through isoamylase. In such
circumstances the MOS produced would need to be efficiently recycled, probably through the combined action of a1,4-glucanotransferase and phosphorylase. The reduction or
absence of phosphorylase would thus shift the balance of
MOS degradation to hydrolysis which is less favorable
energetically. Phosphorolysis is indeed energetically more
economical than hydrolysis for breakdown of storage polysaccharide since only one out of the two ATPs required to
activate glucose for storage is lost in this process. We have
previously proposed that D-enzyme plays this role in
Chlamydomonas and have explained the low-starch phenotype of mutants lacking this activity accordingly. However,
we do not feel that such a function explains the phenotype of
the sta4 mutants. Indeed among the two plastidial phosphorylases documented in this work PhoA displays the
higher affinity towards MOS and is more likely than PhoB to
play a role in the recycling of MOS during biosynthesis.
Another possible role for PhoB would be to assist
isoamylase by recessing chains that prevent efficient crystallization of amylopectin. Storage starch is known to differ
from transitory starch by many criteria, including amylopectin chain-length distribution and amylose content. This
probably reflects a modification in the nature of the
predominant starch synthase activity under conditions of
high carbon flux to starch. It is possible that in these
circumstances the polysaccharide is less prone to crystallization, thereby making the need for an additional enzyme to
assist in the trimming of the chains together with isoamylase
more acute. The altered chain-length distribution of the
mutant amylopectin and the increased amylose content
observed in the sta4 mutant starch are consistent with such a
function. However, unlike mutants defective for isoamylase
no water-soluble polysaccharides accumulate in the defective strains. We therefore do not favor this hypothesis.
Finally the role of PhoB could be indirect through its
involvement in a multienzyme complex selectively active
during starch biosynthesis under high carbon flux. Tetlow
et al. (2004) have provided good evidence for the existence
of a complex between branching enzyme IIb (BEIIb) and
plastidial phosphorylase during storage starch synthesis in
the wheat endosperm amyloplast. Formation of this complex would depend on the phosphorylation status of its
components (Tetlow et al., 2004).
Three properties of sta4 mutants and PhoB argue in favor
of the branching enzyme–phosphorylase complex hypothe-
sis. First, sta4 mutants express their phenotype only during
storage starch synthesis. Secondly, the amylose content and
the specific amylopectin chain-length distribution are very
typical and close to those of BEII mutants. In fact the mutants
described in this work can be defined as phenocopies of
branching enzyme mutants. Thirdly, the exquisite sensitivity
of PhoB to ADP-glucose inhibition makes it unlikely that this
PhoB operates directly through its catalytic activity when the
ADP-glucose concentration is high. However, such an inhibition does not preclude an indirect function of phosphorylase as an important constituent of a branching enzyme
complex.
Experimental procedures
Chlamydomonas strains, growth and media
The three wild-type strains of Chlamydomonas reinhardtii used in
this study were 37 (mt þ pab2 ac14), 137C and a subclone of the
latter named 137C* (mt)nit1 nit2). I73 and I97 have been generated
by X-ray mutagenesis of the two latter wild-type references and
carry the sta4-1 and sta4-2 alleles, respectively.
The 330 wild-type strain (mt þ nit1 nit2 cw15 arg7-7) was used to
purify chloroplasts.
Standard media (Harris, 1989b) and growth conditions have been
previously described (Ball et al., 1991; Delrue et al., 1992). Experiments performed in 12-h day/12-h night cycles were done under
high CO2 level (4%) bubbling in Sueoka medium (Sueoka, 1960).
The heterozygous diploid strains were selected on minimal
medium by crossing the original mutant strains I73 and I97 with
the wild-type reference strain 37. The wild-type homozygous
diploids were obtained by crossing 37 with 137C*. The mutant
homozygous diploids were selected after crossing the I97 mutant
strain with the mutant strain IO3 (mt þ pab2 ac14 sta4-1) selected in
the random progeny of the cross involving I73 and 37. The presence
of both mating types in all diploid strains was checked by PCR as
described in Werner and Mergenhagen (1998).
Determination of starch levels, starch purification and
amylopectin structures
A full account of amyloglucosidase assays, starch purification and
kmax (maximum wavelength of the iodine polysaccharide complex)
measurements can be found in Delrue et al. (1992). Separation of
starch polysaccharides by gel permeation chromatography on a
Sepharose CL-2B column was performed as in Wattebled et al.
(2003). The chain length distributions of wild-type and mutant
amylopectins were obtained by using the technique fully described
in Fontaine et al. (1993).
Determination of phosphorylases purification and
enzymatic properties of C. reinhardtii
The PhoB and PhoA phosphorylases were purified to homogeneity
in a two-step procedure. Crude extracts prepared from 10 liters of
late-log phase cells (2 · 106 cells ml)1) grown in high-salt acetate
medium under continuous light (40 lE m)2 sec)1) were subjected to
successive ammonium sulfate precipitations at 20%, 40%, 50% and
70% saturation. The PhoB-enriched fraction (40% ammonium sulfate pellet) and PhoA-enriched fraction (70% ammonium sulfate
ª 2006 The Authors
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Phosphorylase functions in starch metabolism 283
pellet) were then subjected to affinity chromatography on the
amylose resin high flow (New England Biolabs, Beverly, MA, USA)
normally used for purification of E. coli-expressed protein fused to
the maltose-binding protein MalE. One hundred milligrams of protein corresponding to the ammonium sulfate pellets was resuspended in 50 ml of 50 mM Tris, 500 mM citrate/NaOH buffer at pH
6.5. Samples were loaded onto a 5 ml column pre-equilibrated in the
same buffer at a flow rate of 0.5 ml h)1. The gel was then washed
with 100 ml of buffer and elution was obtained by applying on the
column 40 ml of buffer containing a MOS mix (degree of polymerisation (DP) 2 to DP 20) at 1 mg ml)1 for PhoA. To obtain a pure
PhoB sample, a first elution buffer (80 ml) containing 5 mg ml)1 of
MOS was applied to the column allowing the elution of the contaminating hydrolytic activities found in the 40% ammonium sulfate
pellet. Elution of PhoB was achieved by applying the final elution
buffer containing 10 mg ml)1 MOS. The purified protein were then
concentrated and desalted on a PD10 column (Pharmacia, Milton
Keynes, UK). The homogeneity of the purified fractions obtained
was ascertained by visualizing 1 lg of purified proteins by SDSPAGE and Coomassie Brilliant Blue staining. The purified enzymes
were used to study the enzymatic properties of both PhoA and PhoB.
Phosphorylase activities were assayed in both synthesis and
degradation directions. The phosphorolysis of polysaccharides
were assayed with the following protocol. Fifty microliters of algal
crude extract or 1 lg of purified phosphorylases was incubated in
395 ll of 50 mM HEPES/NaOH, 10 mM Pi (orthophosphate) buffer pH
7 containing 10 mg ml)1 rabbit liver glycogen for 1 h at 30C. The
reaction was stopped by boiling for 5 min. Five hundred microliters
of 50 mM Tris/HCl, 120 mM MgCl2, 0.05 mM glucose-1,6-diphosphate, 0.5 mM NADP buffer were added to the boiled sample. The
production of NADPH,Hþ was monitored spectrophotometrically at
365 nm after addition of four units of phosphoglucomutase (Sigma
Chemical Co., St Louis, MO, USA) and two units of glucose-6phosphate dehydrogenase (Sigma).
The synthesis assay was performed by incubating the protein
samples (20 ll) in 180 ll of 50 mM Tris/HCl, 2.5 mM EDTA, 5 mM
glucose-1-phosphate, 2 lM [U-14C] G1P (335 mCi mmol)1) buffer at
pH 7 containing 10 mg ml)1 of polysaccharide for 30 min at 30C.
The reaction was stopped by adding 2 ml of 70% ethanol. Samples
were then applied to Whatman Glass Fiber filters (Whatman
International Ltd., Maidstone, UK) and washed four times with
5 ml of 70% ethanol. The dried filters were counted in a scintillation
counter (Beckman, Fullerton, CA, USA).
The dimeric nature of both PhoA and PhoB in native conditions
was determined by comparing the mobility of each purified protein
on native acrylamide gels containing different acrylamide concentrations (7%, 8%, 9% and 10% monomer). These mobilities were
then compared with purified proteins of known molecular weights
including plastidial potato phosphorylase (208 kDa), cytosolic
potato phosphorylase (194 kDa), E. coli alcohol dehydrogenase
(150 kDa) and sweet potato b-amylase (200 kDa).
Compartmentation studies
Intact chloroplasts were isolated following the procedure described
by Mason et al. (1991) on a discontinuous Percoll gradient. The
chloroplast fractions obtained were used to measure marker enzyme activities including UDP-glucose pyrophosphorylase and
phosphoenolpyruvate carboxylase as cytosolic markers and NADP
glyceraldehyde 3-phosphate deshydrogenase and Rubisco as
plastidial markers. The corresponding enzyme assays have been
previously described in Borchert et al. (1993). The purified chloroplast extract was used to localize the three Chlamydomonas starch
phosphorylases detected by the zymograms.
Crude extracts and zymograms
Soluble crude extracts were always prepared from late-log phase
cells (2 · 106 cells ml)1) grown in high-salt acetate medium under
continuous light (40 lE m)2 sec)1).
Phosphorylase activities were detected on native glycogencontaining zymograms. Five micrograms of Chlamydomonas crude
extracts proteins was buffered in 60 mM Tris/H3PO4 pH 7.3 in a final
volume of 6 ll. Two microliters of loading buffer (25% saccharose,
bromophenol blue 1&) were added to the samples before electrophoresis. Proteins were subjected to separation on a native 7.5%
acrylamide gel containing 0.45% of rabbit liver glycogen (Sigma)
and buffered with 110 mM Tris/HCl pH 7.2. The stacking gel
contained 2.5% acrylamide and was buffered with 60 mM Tris/
H3BO4 pH 7.3. Electrophoresis was performed at 4C at 25 V cm)1
for 2 h 30 min. Gels were then equilibrated in 100 mM citrate/NaOH
buffer (pH 6.5) for 20 min at room temperature (20C) then incubated overnight in the same buffer containing 20 mM G1P. The
phosphorylase activities were revealed by staining the gels in an
iodine solution (0.2% I2, 2% KI).
Southern blot and RT-PCR analyses
The algal DNA extraction procedure can be found in Rochaix et al.
(1991). Standard protocols for molecular biology as described in
Sambrook et al. (1989) were used for genomic DNA restriction,
electrophoresis on agarose gel, transfer onto nylon membranes and
hybridization. Total RNA was extracted using the Rneasy Plant Mini
Kit as described by the manufacturer (Qiagen, Valencia, CA, USA).
The PHO5 cDNA probe was obtained by RT-PCR using the primers
PHO5For (5¢-GAC TAC GTT GCC GCC ATC CTG AGC) and PHO5Rev
(5¢-ACC GAA CTT GTT GCG CAC CGT CTG). The 455-bp cDNA
fragment were then used to perform the Southern analysis. This
fragment was also used to screen approximately 500 000 lysis
plaques of a Chlamydomonas kZAP II cDNA library and allowed us
to clone the complete PHOB cDNA by completing the sequence
obtained with available algal EST sequences. The Chlamydomonas
genome sequence (http://genome.jgi-psf.org/chlre2/chlre2.home.html) was then blasted with our cDNA sequence to detect the
corresponding genomic fragment (GenBank DQ279078).
The complete PHOA cDNA was obtained by following the same
procedure with the PHO3 cDNA probe (420 bp) amplified by RT-PCR
with the following primers: PHO3FOR (5¢-ACC TAA AGT CCG CAA
ACA CA) and PHO3REV (5¢-AGC GGC CCA TCA GGA ACT CAG).
Reverse transcriptase-PCR co-segregation studies were performed by using the one-step RT-PCR kit purchased from Qiagen
with the PhoBfow1 (5¢-GCA TGT TCC GCC AGA CCA) and PhoBrev1
(5¢-TGC AGG AAG CGC CAG TTG A) specific primers allowing
detection of a 736-bp cDNA fragment.
The semi-quantitative experiments performed to study the cDNA
expression of PHOB was performed with the PHO5FOR and
PHO5REV primers. Results were corrected with the intensity of the
bands observed for a 550-bp fragment of the Chlamydomonas
beta1-tubulin cDNA amplified with the primers TubFor (5¢-CAA GGG
CCT GAA GAT GTC CGC) and TubRev (5¢-GAG AGC GCC AGC GGT
AAA AAT).
Complementation of the sta4 mutant strains
The full-length STA4 genomic DNA (8.7 kbp) was amplified by PCR
using the DynazymeTM Ext polymerase (Finnzymes, Espoo, Finland)
following the supplied standard procedure for large-fragment
amplification. Flanking restriction sites were introduced into the
ª 2006 The Authors
Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 48, 274–285
284 David Dauvillée et al.
PCR product by using the EcoRI PhoBFor (5¢-GAA TTC ATG CAA CTC
GCT TCC AGG GCC CTA G) and the XbaI PhoBRev (5¢-TCT AGA CCA
AGC CGT GGC GCG GCG ATC) primers. The genomic DNA fragment was then introduced into the pSL18 plasmid (digested by
EcoRI and XbaI) already used for complementation studies in
Chlamydomonas (Dauvillée et al., 2003). It was then used to transform the two mutant strains I73 and I97 by the glass beads method
(Kindle, 1990) after autolysin treatment. The transformants were
selected on tris-acetate-phosphate medium plates containing paromomycin (10 lg ml)1). These transformants were screened by
zymogram to select the complemented strains expressing the PhoB
activity. Three complemented strains were obtained for I97 and
were named I97C1 to I97C3. The four complemented strains obtained after transformation of the I73 mutant strain were named
I73C1 to I73C4.
Scanning electron microscopy
Drops of aqueous suspensions of purified starch granules were
deposited onto copper stubs and allowed to dry. The specimens
were coated with Au/Pd and observed in secondary electron imaging mode with a JEOL6100 microscope.
Acknowledgements
This research was funded by the French Ministry of Education, the
Centre National de la Recherche Scientifique (CNRS), by the Region
Nord Pas de Calais, by the Agence Nationale de la Recherche (Projet
PhotobioH2 NT05-2 _42699) and by the Deutsche Forschungsgemeinschaft [SFB 429 TP B2 (MS) and B7 (GR)].
Supplementary Material
The following supplementary material is available for this article
online:
Figure S1. Purification of PhoA and PhoB proteins.
Figure S2. Maldi MS and sequence analysis of PhoA and PhoB
derived peptides.
Figure S3. PhoA and PhoB are dimeric in their native states.
Figure S4. Diurnal behaviour of PhoB mRNA.
Figure S5. Functional complementation of the sta4 mutants.
Figure S6. Kinetics of starch deposition of wild-type and sta4
mutant strains during nitrogen starvation.
Table S1. Phenotypic segegation in the progeny of the I97 X 37
cross.
Table S2. Phenotypic segregation in the progeny of the I73 X 37
cross.
Table S3. Functional complementation of the sta4 mutant strains.
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ª 2006 The Authors
Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 48, 274–285