The Plant Journal (2012) 70, 231–242
doi: 10.1111/j.1365-313X.2011.04860.x
Mutagenesis of cysteine 81 prevents dimerization of the
APS1 subunit of ADP-glucose pyrophosphorylase and alters
diurnal starch turnover in Arabidopsis thaliana leaves
Nadja Hädrich1, Janneke H.M. Hendriks1,†, Oliver Kötting2, Stéphanie Arrivault1, Regina Feil1, Samuel C. Zeeman2,
Yves Gibon1,‡, Waltraud X. Schulze1, Mark Stitt1 and John E. Lunn1,*
1
Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm, Germany, and
2
Department of Biology, Eidgenössische Technische Hochschule Zurich, Universitätsstrasse 2, CH-8092 Zurich, Switzerland
Received 16 July 2011; revised 14 November 2011; accepted 17 November 2011; published online 28 December 2011.
*
For correspondence (fax +49 331 567 8408; e-mail [email protected]).
†
Present address: Metanomics GmbH, Tegeler Weg 33, 10589 Berlin, Germany.
‡
Present address: INRA, Université de Bordeaux, UMR1332 Fruit Biology and Pathology, F-33883 Villenave d’Ornon, France.
SUMMARY
Many plants, including Arabidopsis thaliana, retain a substantial portion of their photosynthate in leaves in the
form of starch, which is remobilized to support metabolism and growth at night. ADP-glucose pyrophosphorylase (AGPase) catalyses the first committed step in the pathway of starch synthesis, the production of
ADP-glucose. The enzyme is redox-activated in the light and in response to sucrose accumulation, via
reversible breakage of an intermolecular cysteine bridge between the two small (APS1) subunits. The
biological function of this regulatory mechanism was investigated by complementing an aps1 null mutant
(adg1) with a series of constructs containing a full-length APS1 gene encoding either the wild-type APS1
protein or mutated forms in which one of the five cysteine residues was replaced by serine. Substitution of
Cys81 by serine prevented APS1 dimerization, whereas mutation of the other cysteines had no effect. Thus,
Cys81 is both necessary and sufficient for dimerization of APS1. Compared to control plants, the adg1/
APS1C81S lines had higher levels of ADP-glucose and maltose, and either increased rates of starch synthesis or
a starch-excess phenotype, depending on the daylength. APS1 protein levels were five- to tenfold lower in
adg1/APS1C81S lines than in control plants. These results show that redox modulation of AGPase contributes
to the diurnal regulation of starch turnover, with inappropriate regulation of the enzyme having an unexpected
impact on starch breakdown, and that Cys81 may play an important role in the regulation of AGPase turnover.
Keywords: ADP-glucose pyrophosphorylase, Arabidopsis thaliana, starch, cysteine, site-directed mutagenesis,
thioredoxin.
INTRODUCTION
The daily alternation of light and dark leads to large recurrent changes in the plant carbon budget. Plants buffer these
diurnal changes by retaining part of their photosynthate in
the leaves, often as starch, and remobilizing it at night to
support respiration and export of carbon to sink organs
(Geiger and Servaites, 1994; Smith et al., 2005; Smith and
Stitt, 2007; Stitt et al., 2007; Zeeman et al., 2007). Starch
turnover is regulated such that, over a wide range of conditions, starch reserves are almost fully remobilized but not
quite exhausted at dawn (Stitt et al., 1978; Chatterton and
Silvius, 1979, 1980, 1981; Mullen and Koller, 1988; Lorenzen
and Ewing, 1992; Gibon et al., 2002). This maximizes
investment in growth, while avoiding carbon starvation
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd
(Smith and Stitt, 2007; Stitt et al., 2007; Graf and Smith,
2011). Achieving this balance requires precise regulation of
the rates of starch synthesis and degradation. In addition,
inhibition of starch synthesis at night is required to avoid a
futile cycle of degradation and resynthesis.
The first committed step in the pathway of starch biosynthesis involves conversion of ATP and glucose-1-phosphate
(Glc1P) to adenosine-5¢-diphosphoglucose (ADPGlc) and
inorganic pyrophosphate (PPi), catalysed by ADP-glucose
pyrophosphorylase (AGPase; EC 2.7.7.27; Preiss, 1988). In
leaves, AGPase is exclusively localized to the chloroplast.
Although the reaction is freely reversible in vitro, hydrolysis
of PPi by plastidial alkaline pyrophosphatase renders it
231
232 Nadja Hädrich et al.
essentially irreversible in vivo (Weiner et al., 1987; Tiessen
et al., 2002). Higher-plant AGPase is a heterotetramer (a2b2)
consisting of two small subunits (approximately 50 kDa) and
two large subunits (approximately 51 kDa; Morell et al.,
1987; Okita et al., 1990; Ballicora et al., 2004). The small
subunit (APS, also known as ADG1 or BRITTLE2) and large
subunit (APL, also known as ADG2 or SHRUNKEN2) are
closely related, having evolved from a common ancestral
form. Both subunits influence the kinetic and regulatory
properties of the AGPase holoenzyme (Cross et al., 2004;
Ventriglia et al., 2008). The A. thaliana genome contains four
large-subunit genes (APL1, At5g19220; APL2, At1g27680;
APL3, At4g39210; APL4, At2g21590) and two small-subunit
genes (APS1, At5g48300; APS2, At1g05610). Expression of
the APS2 gene is barely detectable. Furthermore, the APS2
protein contains substitutions of several active-site residues,
and had no detectable catalytic activity when heterologously
expressed (Crevillén et al., 2003, 2005). Thus, APS1 is
thought to encode the only catalytically active small subunit.
Transcript and proteomic analyses have shown that APL1 is
the predominant large-subunit isoform in leaves, whereas
APL3 and APL4 are mostly expressed in sink tissues
(Crevillén et al., 2003, 2005).
Starch synthesis is severely compromised in AGPasedeficient mutants that have lost a predominant isoform of
either the small or large subunits, e.g. the adg1 and adg2
mutants in A. thaliana, the brittle2 and shrunken2 mutants in
maize (Zea mays), and the rb mutant in pea (Pisum sativum;
Tsai and Nelson, 1966; Dickinson and Preiss, 1969; Lin et al.,
1988a,b; Smith et al., 1989; Wang et al., 1997, 1998). This
provides strong genetic evidence that AGPase activity is
essential for starch synthesis. Flux control analyses using
heterozygotes of adg1 mutants (Lin et al., 1988a,b) have
shown that AGPase exerts considerable control over the rate
of starch synthesis in A. thaliana leaves (Neuhaus and Stitt,
1990; Sun et al., 1999; Stitt et al., 2010).
Higher-plant AGPase is regulated at several levels. The
enzyme from leaves displays sigmoidal substrate kinetics
for ATP and Glc1P, and is allosterically activated by
3-phosphoglycerate (3PGA) and inhibited by orthophosphate
(Pi) (Sowokinos, 1981; Sowokinos and Preiss, 1982; Preiss,
1988). These properties mean that formation of ADPGlc is
highly sensitive to changes in the availability of photoassimilate. They are thought to promote starch synthesis and
recycling of phosphate when the rate of photosynthesis
exceeds the rate of synthesis of other end products, such as
sucrose (MacRae and Lunn, 2006). Substrate availability and
allosteric regulation of AGPase may also contribute to
re-direction of carbon into starch when sucrose accumulates
in the leaf. In heterotrophic tissues, the kinetic properties of
AGPase, including its sensitivity to regulation by 3PGA and
Pi, may differ from those of the leaf enzyme (Ballicora et al.,
2004; Boehlein et al., 2010). Crevillén et al. (2003) showed
that the substrate affinities and allosteric properties of
Arabidopsis AGPase are largely determined by which of
the four isoforms of the large subunit is present in the
holoenzyme, thus accounting for the different properties of
the enzyme between leaves and sink tissues. Expression of
allosterically up-regulated forms of AGPase led to enhanced
rates of starch synthesis and turnover in A. thaliana (Obana
et al., 2006).
AGPase is also subject to post-translational redox modulation. Studies on potato (Solanum tuberosum) AGPase,
heterologously expressed in Escherichia coli, revealed that
the heterotetramer contains an intermolecular disulphide
bridge, linking the two small subunits via their Cys12
residues. The enzyme is activated when the disulfide bridge
is reduced using dithiothreitol or reduced thioredoxin (Ballicora et al., 1998, 1999, 2000; Fu et al., 1998). The reduced
(dithiol) form of the enzyme is less heat-stable than the
oxidized (disulfide) form, being rapidly inactivated at temperatures above 40C, as were mutated forms of the enzyme
in which the Cys12 residue had been substituted by alanine or
serine (Ballicora et al., 1999). The reduced APS monomers
(50 kDa) are readily separated from the oxidized dimers
(100 kDa) by non-reducing SDS–PAGE and visualized by
immunoblotting to determine the redox status of the enzyme
(Tiessen et al., 2002). In maize endosperm AGPase, the large
subunits dimerize via formation of a disulfide bridge but the
small subunits do not form dimers in this way (Lyerly
Linebarger et al., 2005), presumably due to the absence of
Cys residues in the N-terminal domain (Figure S1).
The potato tuber AGPase is redox-activated in response to
higher sugar levels, providing a mechanism to coordinate
the rate of starch synthesis in the tubers with the supply of
photoassimilate from the leaves (Tiessen et al., 2002). In
A. thaliana, pea and potato leaves, redox activation of
AGPase increases upon illumination or in response to sugar
accumulation (Hendriks et al., 2003; Gibon et al., 2004b;
Lunn et al., 2006). However, it is not known which of the
cysteine residues in the APS1 protein is responsible for lightand sucrose-induced changes in the redox status of the
enzyme in vivo. Light-dependent activation of AGPase
presumably depends on ferredoxin-dependent reduction of
thioredoxins via ferredoxin–thioredoxin reductase, as is
known to occur for enzymes involved in photosynthesis.
The dual-function NADPH-dependent thioredoxin reductase
NTRC has been implicated in sugar-induced redox activation
of AGPase (Michalska et al., 2009). It has also been proposed
that trehalose-6-phosphate is an intermediary in the sugarinduced redox activation of AGPase, both in leaves and nonphotosynthetic tissues (Kolbe et al., 2005). In support of this
proposal, the redox activation state of AGPase was found to
be correlated with the levels of both sucrose and trehalose6-phosphate in A. thaliana leaves and seedlings (Lunn et al.,
2006). However, many of the molecular details of the redox
regulation of AGPase and the role of trehalose-6-phosphate
in vivo are unresolved.
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2012), 70, 231–242
Mutation of Cys81 in Arabidopsis AGPase 233
Light and sugars lead to transcriptional activation of
AGPase in leaves (Sokolov et al., 1998; Bläsing et al., 2005;
Crevillén et al., 2005), and this is counteracted by nitrate and
phosphate (Scheible et al., 1997; Nielsen et al., 1998). This
may allow the plant to adjust its accumulation of starch in
response to sustained changes in its carbon and nutritional
status. Protein turnover may also contribute to the regulation of AGPase activitiy. Maximum AGPase activity and
AGPase protein show marked changes during diurnal
cycles, which sometimes occur independently of the
changes in transcript levels (Gibon et al., 2004a,b, 2009),
and modelling indicates that AGPase protein is rapidly
turned over (Piques et al., 2009).
Thus, AGPase is subject to multiple levels of transcriptional and post-translational regulation. In leaves, these
form part of a wider regulatory network that coordinates
end-product synthesis with the rate of CO2 assimilation, and
controls the partitioning of photoassimilates between
sucrose and starch, balancing immediate export for growth
of sink organs with the need to retain enough reserves in the
leaf to last through the night. These multiple types of
regulation provide considerable flexibility and robustness,
but make it difficult to assess the importance of any single
regulatory mechanism for the control of starch synthesis.
The aims of the present experiments were to identify which
of the five cysteine residues in the A. thaliana APS1 protein
are involved in formation of the disulphide in vivo, and to
investigate the consequences of loss of redox regulation of
AGPase activity in this species. To do this, we replaced the
native APS1 protein with various modified forms in which
individual cysteine residues were replaced by serine. Mutant
lines in which formation of the disulphide bridge was
prevented by substitution of the crucial cysteine residue
were analysed in detail to determine how loss of redox
regulation of AGPase affected the diurnal pattern of starch
synthesis and degradation.
RESULTS
Site-directed mutagenesis of cysteine residues in
A. thaliana APS1 and expression in the adg1 background
Studies of the potato tuber AGPase, heterologously
expressed in E. coli, indicated that a disulphide bridge
formed between the Cys12 residues of the two small
subunits within the heterotetrameric holoenzyme (Fu et al.,
1998). Although this suggests that Cys12 is the residue most
likely to be involved in redox regulation of the enzyme in
planta, we cannot exclude the possibility that other cysteine
residues, alone or in combination with Cys12, may also be
involved. The A. thaliana small subunit (APS1) contains five
cysteine residues (Figure S1). Cys131 is located in the
nucleotidyl transferase domain and is conserved across
plants and bacteria. Cys81 (equivalent to Cys12 in the mature
potato small subunit protein after cleavage of the 70 amino
acid plastid transit peptide), Cys406, Cys411 and Cys423 are
mostly conserved in higher plants and oxygenic photosynthetic bacteria (cyanobacteria), but are absent in facultative
anaerobic bacteria (enterobacteria).
The APS1 gene (At5g48300) was amplified by PCR from
genomic DNA extracted from A. thaliana leaves. The 3085 bp
amplicon contained the entire intergenic region upstream of
the APS1 translation initiation codon, up to and including
part of the 3¢ UTR of the adjacent upstream gene (At5g48290),
the APS1 protein coding region, and 280 bp downstream of
the translation stop codon, including the 3¢ UTR and
polyadenylation site of the pre-mRNA (Figure S2). Sitedirected mutagenesis was performed to replace individual
cysteine residues in the encoded APS1 protein with serine.
The native (APS1WT) and mutated APS1 genes (APS1C81S,
APS1C131S, APS1C406S, APS1C411S and APS1C423S) were
cloned into the pGreen binary vector and introduced into
the starch-deficient adg1 mutant, which is a recessive EMS
mutant with a single base change in the APS1 coding region
(resulting in a Gly92 fi Arg92 substitution; G92R) and has no
measurable AGPase activity in the leaf (Lin et al., 1988a;
Figure 1a). Prior to transformation, all constructs were
sequenced to confirm that no mutations other than the
desired site-directed change had been introduced. After
initial selection for resistance to phosphinothricin, the
primary transformants were screened by harvesting leaves
at the end of the day for iodine staining to detect the presence
of starch, which would indicate complementation of the adg1
mutant. Wild-type Col-0 plants and the parental adg1 mutant
were used as positive and negative controls, respectively.
For each construct, 30–35 primary transformants were identified that showed restoration of starch synthesis, indicating
the presence of a functional APS1 transgene (see Figure 1b).
Cys81 is necessary and sufficient for dimerization
of APS1 in the dark
Leaves were harvested from wild-type controls and each of
the APS1-complemented adg1 mutants 2 h into the night,
when AGPase is expected to be fully oxidized and the APS1
protein is expected to exist in the dimeric form (Tiessen
et al., 2002; Hendriks et al., 2003). Protein extracts were
analysed by immunoblotting after non-reducing SDS–PAGE
to determine the redox state of AGPase (Figure 1c). A
major immunoreactive band at approximately 100 kDa,
corresponding to the APS1 dimer, was present in wild-type
extracts, with little or no signal at 50 kDa, representing
monomeric APS1 protein. A similar result was obtained for
the complemented adg1 lines, with the exception of adg1/
APS1C81S. Darkened leaves from adg1/APS1C81S contained
only the monomeric (50 kDa) form of APS1, as expected if
the mutagenized small subunit protein is unable to dimerize.
These findings provide compelling evidence that Cys81 is
both necessary and sufficent for the APS1 protein to form a
disulphide bridge.
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2012), 70, 231–242
234 Nadja Hädrich et al.
also form a disulphide bridge with one of the large (APL)
subunits in vivo. To investigate this possibility, we
harvested leaves from wild-type Col-0 plants in the dark,
separated the proteins by non-reducing SDS–PAGE, and
identified the AGPase subunits in the 100 kDa region of the
gel by peptide mass spectrometry. In experiment 1, all 11 of
the identified AGPase peptides were derived from the small
subunit (APS1), and in experiment 2, ten of the 11 peptides
were also from APS1, with only a single peptide derived
from APL1 (Table 1). We would expect to find approximately equal numbers of APS1 and APL peptides if APS1
formed a dimer with any of the large subunit isoforms, so
the observed ratio of APS1:APL1 peptides of 21:1 is inconsistent with the presence of APS1–APL1 dimers. The single
APL1 peptide detected may be attributable to contamination of the sample, which is difficult to avoid with such a
sensitive analytical technique as peptide mass spectrometry. Therefore, we conclude that the redox regulation of
AGPase in A. thaliana leaves in vivo occurs via formation of
a disulfide bridge between the Cys81 residues of the two
small subunits.
Characterization of adg1/APS1C81S mutants under
a 12 h light/12 h dark diurnal cycle
Figure 1. Cys81 is required for formation of the intermolecular disulfide
bridge in APS1.
(a) APS1 (At5g48300) gene constructs used for transformation of the AGPasedeficient adg1 mutant. The Cys residues (red) in wild-type APS1 (top) were
individually changed to Ser (green).
(b) Iodine staining showing the presence of starch in the APS1C81S-complemented adg1 mutant.
(c) Monomerization of APS1 protein determined by non-reducing SDS–PAGE
and immunoblotting. Leaves were harvested from wild-type and mutant
plants 2 h into the night when all the APS1 protein in wild-type plants is
dimerized.
For further physiological characterization of the transformants, plants were propagated and screened for two more
generations to obtain one adg1/APS1WT line (line 21) and
three independent lines for adg1/APS1C81S [line A (35-237),
line B (11-2) and line C (13-8)] that were homozygous for the
respective transgenes. As the activity of AGPase is known to
be dependent on gene dosage (Neuhaus and Stitt, 1990), we
checked that these lines contained a single transgenic locus
by Southern hybridization (Figure S3).
Involvement of the large subunits in formation
of the disulfide bridge in vivo
Previous in vitro studies on heterologously expressed
potato tuber AGPase indicated that the disulphide bridge is
formed between the small subunits of the enzyme, but we
cannot exclude the possibility that the APS1 protein can
Wild-type Col-0, adg1/APS1WT and three independent adg1/
APS1C81S lines, each containing a single transgenic locus,
were grown under a 12 h light/12 h dark diurnal cycle in low
light (150 lE m)2 sec)1), and sampled every 4 h to investigate APS1 transcript levels, AGPase activity, AGPase activation state and starch content.
Compared to the wild-type Col-0, APS1 transcript levels
were almost exactly twofold higher in the adg1/APS1WT
controls and adg1/APS1C81S mutants throughout the diurnal
cycle (Figure 2a). The wild-type Col-0 and adg1/APS1WT
plants showed similar maximum catalytic activities of
AGPase (measured in the presence of 3PGA) (Figure 2b). In
Table 1 AGPase subunits identified in the 100 kDa region from
Arabidopsis thaliana leaf extracts
Number of peptides
Protein identifier
Sample 1
Sample 2
At5g48300.1 (APS1)
At1g05610.1 (APS2)
At5g19220.1 (APL1)
At1g27680.1 (APL2)
At4g39210.1 (APL3)
At2g21590.1 (APL4)
10
0
1
0
0
0
11
0
0
0
0
0
Extracts from wild-type Col-0 leaves were separated by non-reducing
SDS–PAGE. The gel region adjacent to the 100 kDa marker protein
was excised, and proteins were identified by peptide mass spectrometry after tryptic digestion. The number of peptides identified for
each APS and APL isoform is shown for two independent replicates.
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2012), 70, 231–242
Mutation of Cys81 in Arabidopsis AGPase 235
Figure 2. Diurnal changes in APS1 transcripts,
AGPase activity, starch content and post-translational modification of APS1 in the wild-type
and adg1 mutants complemented with APS1WT
or APS1C81S.
Wild-type Col-0, adg1/APS1WT and adg1/
APS1C81S plants were grown under a 12 h
light/12 h dark diurnal cycle with 150 lE m)2 sec)1 irradiance.
(a) Transcripts of APS1 (At5g48300).
(b) Maximum catalytic activity of AGPase.
(c) Oligomeric state of APS1.
(d) Leaf starch content.
For (a), (b) and (d), the values are means SD
(n = 5 separate samples, each comprising three
individual rosettes). Significant differences from
wild-type Col-0 according to Student’s t test are
indicated by asterisks (*P < 0.05, **P < 0.05,
***P < 0.001).
contrast, all three of the adg1/APS1C81S lines contained
much lower AGPase activity (five- to tenfold lower than the
controls, depending on the time of day), despite having
comparable APS1 mRNA expression levels to adg1/APS1WT.
The extraction buffer used to extract the leaf tissue contained DTT, and DTT was included in the AGPase assay; the
measured AGPase activities therefore represent the fully
reduced form of the enzyme.
The APS1 protein is almost immediately oxidized in
extracts prepared from A. thaliana leaves (Hendriks et al.,
2003). Therefore, to detect differences in dimerization of the
enzyme, extracts were prepared in trichloroacetic acid (TCA).
TCA immediately denatures the AGPase holoenzyme and
prevents formation of APS1 dimers during extraction and
analysis (Hendriks et al., 2003). In wild-type Col-0 plants (and
adg1/APS1WT, data not shown) APS1 was totally dimerized
at the end of the night and became progressively reduced to
the monomeric form during the day (Figure 2c). In contrast,
only the monomeric (50 kDa) form of the APS1 protein was
observed at any time point in the adg1/APS1C81S mutants,
either during the day or at night. In agreement with the large
decrease in total AGPase activity (Figure 2b), the total
amount of APS1 protein detected by immunoblotting was
noticeably lower in adg1/APS1C81S plants than in wild-type
controls (Figure 2c).
Despite the five- to tenfold decrease in maximal AGPase
activity, the starch content of leaves from all three adg1/
APS1C81S lines was consistently higher than in wild-type
Col-0 and adg1/APS1WT, although the diurnal pattern of
starch accumulation and degradation was retained (Figure 2d). The difference was especially marked at dawn,
when the adg1/APS1C81S plants contained twice as much
starch as wild-type Col-0 or adg1/APS1WT. Sugar levels
showed a slight tendency to be lower during the light period
in adg1/APS1C81S plants than in control plants (data not
shown).
In summary, replacement of Cys81 by serine not only
prevents dimerization of APS1, but also leads to the plants
having less APS1 protein and five- to tenfold lower maximum catalytic activity of AGPase activity than wild-type
plants. Despite the lower levels of AGPase protein, constitutive activation of APS1 allows the adg1/APS1C81S plants to
maintain similar or even higher rates of starch synthesis
than control plants.
ADPGlc levels
We used LC-MS/MS to investigate whether ADPGlc levels
were altered in adg1/APS1C81S plants (Figure 3). In both
wild-type Col-0 and adg1/APS1WT plants, leaves harvested in
the light at the end of the day contained approximately
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2012), 70, 231–242
236 Nadja Hädrich et al.
1.5 nmol of ADPGlc per gram fresh weight. The level of
ADPGlc was 70–110% higher in the adg1/APS1C81S plants
(Figure 3a), indicating that constitutive activation of APS1
allows a higher level of ADPGlc to be maintained despite
the much lower maximal AGPase activity in these lines. In
leaves from both wild-type and mutant plants harvested
in the dark, the level of ADPGlc was 35–45 times lower than
in the light. Both of the adg1/APS1C81S lines showed a tendency to have higher absolute levels of ADPGlc than the
wild-type Col-0 and adg1/APS1WT plants (Figure 3b), although the differences were not statistically significant. The
much lower levels of ADPGlc in the adg1/APS1C81S lines in
the dark, compared to the same plants in the light, indicate
that AGPase activity was restricted at night, despite the
inability of the mutated enzyme to be redox-regulated. This
suggests that other mechanisms, such as substrate limitation and allosteric regulation, are operating to limit ADPGlc
synthesis at night, which would otherwise allow futile cycling of ATP and hexose phosphates. Nevertheless, the
tendency of the adg1/APS1C81S lines to have slightly higher
night-time levels of ADPGlc than the control plants suggests
that there may be a very low rate of ADPGlc synthesis in the
dark in the redox-insensitive mutants.
Characterization of diurnal starch turnover in adg1/
APS1C81S mutants under various photoperiod conditions
Starch levels in the adg1/APS1C81S lines were moderately,
but consistently, higher than in wild-type plants, when
grown under a 12 h light/12 h dark diurnal cycle (Figure 2d).
Further experiments were performed to investigate the
influence of photoperiod and irradiance on diurnal starch
turnover. Wild-type Col-0, adg1/APS1WT and adg1/APS1C81S
mutant plants were grown under short-day (8 h light/16 h
dark) and long-day (16 h light/8 h dark) conditions with low
irradiance (150 lE m)2 sec)1, comparable to the experiment
in Figure 2), or under a 12 h light/12 h dark cycle with high
irradiance (450 lE m)2 sec)1), and samples were taken
every 4 h for determination of starch content. As previously
observed in A. thaliana (see Introduction), the wild-type
Col-0 and adg1/APS1WT plants adjusted their rates of starch
synthesis and degradation according to the day length. Only
a small amount of starch was left at the end of the night,
indicating that the rates of starch accumulation in the light
and utilization during the night are tightly coordinated. This
pattern was modified in the adg1/APS1C81S plants. Under
low-light and short-day conditions, starch was synthesized
more rapidly during the 8 h light period, leading to a 40%
higher starch content at the end of the day, and degraded
more rapidly at night (Figure 4a). As already mentioned,
under low-light and equinoctial (12 h light/12 h dark) conditions, the rates of starch synthesis and degradation
resembled wild-type controls, but more starch was left at
the end of the night (Figure 2d). This pattern was even more
marked under long-day conditions (16 h light/8 h dark;
Figure 4b), under which the mutant showed a mild starch
excess phenotype. Finally, under high-light equinoctial (12 h
light/12 h dark) conditions, starch synthesis rates were
marginally increased, and even more starch remained at the
end of the night (Figure 4c). Under all growth conditions, the
wild-type Col-0 and adg1/APS1WT lines had almost identical
patterns of diurnal starch turnover to each other, and the two
independent adg1/APS1C81S lines consistently showed similar differences in behaviour compared to the control plants.
Maltose levels in wild-type and adg1/APS1C81S plants
The results in Figures 2 and 4 show that the adg1/APS1C81S
mutants appear to have lost the ability to match their rates of
starch degradation and synthesis, except when grown under
short-day conditions with low light. Under these conditions,
the mutants were able to increase the rate of starch degradation to remobilize all of their starch during the night, even
though they had more starch than the control plants at the
Figure 3. ADP glucose content in wild-type and APS1-complemented adg1 mutants.
ADPGlc content was measured by LC-MS/MS in leaves harvested at the end of the day (ED) (a) and at the end of the night (EN) (b). The measurements were
performed using material from the experiment shown in Figure 2. Values are means SD (n = 3). Significant differences from wild-type Col-0 according to
Student’s t test are indicated by asterisks (*P < 0.01).
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2012), 70, 231–242
Mutation of Cys81 in Arabidopsis AGPase 237
the rest of the night, and maltose was present at only very
low levels in the light (Figure 5). In the two adg1/APS1C81S
lines, maltose levels were consistently 50–100% higher than
in wild-type controls in the dark. The redox-insensitive APS1
mutants also contained more maltotriose and maltotetraose
(data not shown). Unexpectedly, maltose levels were also
consistently twofold higher in these mutants in the light than
in wild-type controls (Figure 5). These data suggest that
there may be a higher rate of starch degradation in adg1/
APS1C81S plants than in the wild-type, and that starch
degradation may also occur in the light.
Response of purified AGPase to redox changes
Figure 4. Influence of photoperiod and irradiance on starch turnover in wildtype and adg1/APS1C81S mutant plants during the diurnal cycle.
Starch content was determined in leaf extracts of wild-type Col-0, adg1/
APS1WT and adg1/APS1C81S plants grown under (a) 8 h light/16 h dark or (b)
16 h light/8 h dark diurnal cycles in low light (150 lE m)2 sec)1), or (c) a 12 h
light/12 h dark diurnal cycle in high light (450 lE m)2 sec)1). The values are
means SD (n = 3 separate samples, each comprising three individual
rosettes). In (c) the samples harvested at 24 h were lost during analysis and
the values shown at this time point are the same as the 0 h samples.
Significant differences from wild-type Col-0 according to Student’s t test are
indicated by asterisks (*P < 0.05, **P < 0.01, ***P < 0.001).
start of the night (Figure 4a). However, under long-day or
high-light conditions, the adg1/APS1C81S mutants did not
degrade all of their starch during the night, suggesting either
that they had insufficient starch degrading capacity to cope
with the moderately elevated amount of starch, or that
starch degradation was somehow impaired in these
mutants.
To investigate these possibilities, we measured the levels
of maltose, which is the major product of starch degradation
in leaves (Zeeman et al., 2007a). Maltose levels were measured in plants grown under equinoctial (12 h light/12 h
dark) conditions with an irradiance of 150 lE m)2 sec)1 (in
the same samples as those shown in Figure 2). In the wildtype Col-0 and adg1/APS1WT plants, maltose levels increased in the first 4 h of the night and decreased through
As already mentioned, APS1 is rapidly oxidized to form a
dimer in A. thaliana leaf extracts (Hendriks et al., 2003),
making it difficult to investigate the effect of redox changes
on the activity of the AGPase from the adg1/APS1C81S lines in
crude extracts. To overcome this problem, we purified
AGPase from both wild-type Col-0 plants and the adg1/
APS1C81S mutants by dye affinity chromatography on a
Mimetic Orange 1 column followed by size exclusion chromatography on a Superdex 200 column (for details, see
Appendix S1). Purification of the mutated C81S enzyme was
complicated by the low concentration of the protein in the
adg1/APS1C81S mutant, and by the poor recovery of activity
from the Orange1 and Superdex200 columns during the
purification, although a substantial degree of purification
(356-fold) was achieved (Table S1). The wild-type enzyme
was purified 184-fold with a yield of 6%. The final specific
activity of the wild-type enzyme was approximately tenfold
higher than that of the mutated C81S form. This was at least
partly due to a greater degree of contamination by other
proteins in the final C81S preparation (Figure S4 and
Table S2), and possibly also to partial inactivation during
purification. Peptide analysis after tryptic digestion indicated
that APS1 was the only small subunit isoform present in the
Figure 5. Diurnal changes in maltose levels.
Wild-type Col-0 plants (red circle), adg1/APS1WT plants (pink circle), and
plants of adg1/APS1C81S line A (green circle) and adg1/APS1C81S line B (green
triangle) were grown under a 12 h light/12 h dark diurnal cycle with an
irradiance of 150 lE m)2 sec)1. Maltose levels were determined by HPAECPAD using material from the experiment shown in Figure 2. Values are
means SD (n = 3).
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2012), 70, 231–242
238 Nadja Hädrich et al.
purified enzymes, and that APL1 was by far the predominant
large subunit isoform (Table S2). The activity of the wildtype AGPase was significantly decreased by incubation with
oxidized dithiothreitol, but this treatment had no effect on
the activity of the mutated C81S form (Figure S5). The low
yield of the purified enzymes prevented further analysis of
their kinetic properties.
DISCUSSION
The A. thaliana leaf AGPase is redox-activated by light and
in response to sucrose accumulation in the leaf (Hendriks
et al., 2003; Lunn et al., 2006). This undoubtedly forms part
of the wider network of regulatory mechanisms that control
carbon partitioning between starch and sucrose, and coordinate end-product synthesis with CO2 assimilation, but its
precise physiological significance is uncertain. We have
established that redox regulation of the A. thaliana AGPase
in vivo ocurs via formation of a disulphide bridge between
the Cys81 residues of the two small subunits within the
heterotetrameric holoenzyme. This leads to APS1–APS1
dimerization and decreased maximal activity. The chloroplast transit peptide of the A. thaliana APS1 protein is
predicted to be 69 amino acids long (Hädrich et al., 2011),
thus Cys81 is predicted to become Cys12 in the mature
protein after cleavage of the transit peptide. This residue is
equivalent to Cys12 in the mature potato enzyme, which
forms a disulfide bridge in the heterologously expressed
enzyme (Fu et al., 1998; Ballicora et al., 1999, 2000). Individual replacement of the other four cysteine residues by
serine did not affect the ability of the A. thaliana APS1
protein to form a dimer when oxidized. Thus, Cys81 is both
necessary and sufficient for formation of the intermolecular
disulphide bridge between the two APS1 subunits. Interestingly, all five of the mutated forms of APS1 containing
Cys/Ser substitutions were able to complement the starchless phenotype of the adg1 mutant, and thus none of the
cysteine residues in APS1 appear to be essential for catalytic activity of the holoenzyme. This was true even for the
mutated C131S form. This lacks the only cysteine residue
found in the nucleotidyl transferase domain of the APS1
protein, which is the only cysteine residue that is conserved
in all plant and bacterial AGPases (Figure S1).
The full-length APS1 gene, including all of the upstream
intergenic region and part of the downstream region beyond
the polyadenylation site, was used to produce constructs for
complementation of the adg1 mutant. The aim was to
include the whole promoter and all other regulatory elements, so that the level and pattern of expression of the
transgene would be as close as possible to that in wild-type
plants. Expression of the APS1 gene was measured by
quantitative RT-PCR using primers that did not distinguish
between transcripts derived from the introduced APS1
transgene and non-functional APS1 transcripts from the
endogenous mutated APS1 gene in the adg1 mutant. APS1
transcript levels in the complemented adg1 mutants were
almost exactly twofold higher than in wild-type Col-0 plants
at all points during the diurnal cycle (Figure 2a). This
doubling of APS1 transcript abundance in the complemented adg1 lines, but similar diurnal patterns of expression to wild-type Col-0, indicates that the APS1 transgenes
were subject to the same transcriptional regulation as the
endogenous APS1 gene. Despite the differences in APS1
transcript levels, the AGPase activity and starch content of
the adg1/APS1WT line were remarkably similar to those of
wild-type Col-0 plants. This suggests that either the nonfunctional APS1 transcripts derived from the endogenous
mutated APS1G92R gene in the adg1 mutant were not
translated, or that the inactive APS1G92R protein is unstable
and rapidly degraded. It is worth noting that the expression
level of the APL1 transcript, which encodes the predominant
large subunit isoform in leaves, was essentially the same in
both wild-type Col-0 and the complemented adg1 mutant
lines (data not shown).
It has previously been observed that APS1 and APL1
transcript levels are not always correlated with maximal
AGPase activity and amounts of AGPase protein in A.
thaliana leaves (Gibon et al., 2004a,b; Piques et al., 2009),
which suggests that post-transcriptional regulation can also
be important in determining the amount and activity of the
enzyme. Despite having almost identical APS1 transcript
levels to the adg1/APS1WT line, the adg1/APS1C81S lines had
noticeably lower APS1 protein levels (Figure 2c) and five- to
tenfold lower AGPase activity (Figure 2b) than both the wildtype Col-0 and adg1/APS1WT controls. This suggests that
post-transcriptional regulation of the enzyme’s abundance is
perturbed in the adg1/APS1C81S mutants.
There are several possible explanations for the low levels
of AGPase protein in the adg1/APS1C81S lines. One is that
AGPase containing the mutated C81S form of the small
subunit is inherently less stable than the wild-type enzyme.
Ballicora et al. (1999) showed that the wild-type potato
enzyme was heat-stable up to 60C when oxidized (i.e. when
APS1 is dimerized), but was heat-stable up to only 40C when
reduced (i.e. when APS1 is monomerized) or when Cys12
was replaced by alanine or serine preventing formation of
the intermolecular disulphide bridge. However, below 40C,
there were no significant differences in the stability of the
various forms of the enzyme. As our plants were grown at
18–20C and AGPase activity was measured at 25C, temperatures that are well below the 40C threshold, it seems
most unlikely that differences in the thermal stability of the
mutated enzyme account for the lower APS1 protein abundance and AGPase activity in the adg1/APS1C81S plants.
Furthermore, it is possible that the thermal stability of the
AGPase in vivo is greater than the in vitro experiments on the
isolated potato enzyme may suggest, as the very high
concentration of proteins in the chloroplast stroma may be
expected to have a stabilizing effect on the enzyme.
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2012), 70, 231–242
Mutation of Cys81 in Arabidopsis AGPase 239
It is known that AGPase is subject to rapid turnover in
leaves, and that reduction of the enzyme in the light is often
accompanied by a decrease in the enzyme’s abundance
(Gibon et al., 2004a,b; Piques et al., 2009). These observations suggest that the reduced (monomeric APS1) form of
the enzyme may be more susceptible to proteolytic turnover
than the oxidized (dimeric APS1) form, which may account
for the low abundance of the enzyme in the adg1/APS1C81S
plants (in which the APS1 cannot dimerize). It is unclear
whether the monomeric APS1 form of the enzyme simply
has a conformation that makes it more accessible to
chloroplastic proteases than the dimeric APS1 form, or
whether proteolytic turnover of the enzyme is more
precisely regulated.
The level and diurnal turnover of starch were identical
in wild-type and adg1/APS1WT plants. In contrast, adg1/
APS1C81S lines, despite their lower maximal activity of
AGPase, had increased rates of starch accumulation and/
or higher levels of starch than the control plants. This
suggests that the in vivo activity of the C81S form of
AGPase is the same as, or even higher than, the in vivo
activity of the more abundant wild-type enzyme in the
control plants, or that AGPase was not limiting for starch
synthesis under our growth conditions. The differences in
the diurnal pattern of starch turnover depended on the
photoperiod and irradiance (Figures 2d and 4). Rates of
both starch synthesis and degradation were increased
under short days and low light, whereas the plants grown
under long days or high-light conditions had increased
levels of starch at the end of the light period but
incomplete remobilization of starch at night. It is well
established that plants respond to changes in photoperiod
by adjusting their rates of starch synthesis and degradation, although very little is known about the mechanisms
involved (Smith and Stitt, 2007). Under 12 and 16 h days,
the rates of starch synthesis in the adg1/APS1C81S and
wild-type control plants were remarkably similar (Figure 2d and 4). This suggests that redox regulation of
AGPase is not required to adjust the rate of starch
synthesis to longer day lengths, or that other regulatory
mechanisms compensate for the loss of redox regulation.
The adg1/APS1C81S lines contained 75–110% more
ADPGlc than wild-type Col-0 or adg1/APS1WT plants in the
light (Figure 3a). This is consistent with the in vivo flux
catalysed by AGPase being higher in the adg1/APS1C81S
lines. The increase in ADPGlc is greater than the increase in
the net rate of starch accumulation (Figure 2d). This may
indicate that the starch synthases are already near-saturated
by the wild-type levels of ADPGlc, or that there is increased
turnover of starch in the mutants during the day. In all
genotypes, the level of ADPGlc at night was 35–45 times
lower than in the day (Figure 3b). The low night-time levels
of ADPGlc indicate that the in vivo activity of the mutated
enzyme, like that of the wild-type enzyme, is much lower at
night than during the day, despite the loss of redox
regulation. Limited substrate availability (ATP and Glc1P)
and a low 3PGA:Pi ratio in the chloroplast stroma may
therefore be important factors in restricting AGPase activity
at night in both wild-type and mutant plants.
Transitory starch degradation involves an initial cycle of
glucan phosphorylation and dephosphorylation, followed
by b-amylolysis to release maltose (Zeeman et al., 2007a).
Maltose levels increased in wild-type Col-0 plants in the dark
(Figure 5), as previously observed (Niittyla et al., 2004;
Delatte et al., 2005; Lu et al., 2005; 2006), and very similar
changes were seen in the adg1/APS1WT plants (Figure 5).
The adg1/APS1C81S lines contained higher maltose levels
than wild-type Col-0 plants or the control adg1/APS1WT line,
not only in the dark but also in the light (Figure 5). The
incomplete degradation of starch at night, and the increased
levels of maltose, indicate that the presence of the APS1C81S
form of AGPase somehow disrupts starch breakdown and/or
maltose metabolism.
The control of starch degradation in leaves is poorly
understood, so we can only speculate on possible mechanisms for the effect of the mutated AGPase on starch
turnover. We observed no significant differences between
wild-type and mutant plants in terms of the levels of gene
transcripts encoding the main enzymes of starch degradation (glucan:water dikinase, phosphoglucan:water dikinase,
phosphoglucan phosphatase, b-amylase, debranching
enzymes and disproportionating enzymes) or the MEX1
maltose transporter (data not shown). Therefore, we can
probably exclude transcript level regulation as a possible
mechanism. Some of the enzymes of starch degradation
(e.g. glucan:water dikinase and some isoforms of b-amylase)
are known to be redox-sensitive, although the significance
of this is uncertain. However, it is conceivable that thioredoxins that bind to the docking domains of the APS1C81S
form of AGPase, but are unable to complete the redox
transfer reaction, remain bound to the enzyme. The partial
sequestration of these redox carriers may perturb redox
regulation of other enzymes, including the redox-sensitive
starch-degrading enzymes. It is known that the circadian
clock has a major input into regulation of starch degradation
at night (Graf et al., 2010; Graf and Smith, 2011). Current
models for understanding the role of the clock involve a
starch-sensing mechanism of some kind that, together with
the clock, allows the plant to set the appropriate rate of
starch degradation for the starch reserves to last through the
night. Therefore, a further possibility is that some aspect of
circadian regulation or the perception of starch is perturbed
in the adg1/APS1C81S mutant, leading to lower rates of starch
degradation and/or enhanced turnover of starch in the light.
This uncertainty in explaining the high-maltose and
starch-excess phenotypes of the adg1/APS1C81S mutant
highlights how little we know about the control of
diurnal starch turnover in leaves, despite the importance
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2012), 70, 231–242
240 Nadja Hädrich et al.
of this process for optimal plant growth (Smith and Stitt,
2007).
EXPERIMENTAL PROCEDURES
Plant growth
Arabidopsis thaliana plants were grown as described by Thimm
et al. (2004) under a 12 h light/12 h dark (20C/18C) cycle and
150 lE m)2 sec)1 irradiance unless stated otherwise, and harvested
at 35–40 days after germination. Harvested material was immediately frozen in liquid nitrogen, ground to a fine powder at )70C
using a cryogenic grinding robot (Labman Automation Ltd, http://
www.labman.co.uk/) and stored at )80C until analysis.
Cloning and site-directed mutagenesis
Standard molecular biology techniques were used as described by
Sambrook and Russell (2001). The APS1 gene (At5g48300) was
amplified from genomic DNA by PCR using KOD HiFi DNA polymerase (Merck, http://www.merck-chemicals.de) using forward
primer 5¢-TAGTCATATGAATAAAGCTCTGAGG-3¢ and reverse primer 5¢-AGCGCTTTAAAACGAATAATGTTGAACTAC-3¢. The PCR
product was cloned into the pGEM-T Easy vector (Promega, http://
www.promega.com/) and sequenced on both strands to check
amplification fidelity.
Point mutations were introduced into the APS1 coding sequence
to change individual TGT(Cys) or TGC(Cys) codons into TCT(Ser) or
TCA(Ser) codons, respectively, using a QuickChange site-directed
mutagenesis kit (Stratagene, http://www.stratagene.com/) according to the manufacturer’s instructions. Primers are listed in Table
S3. Mutations were confirmed by sequencing. The mutated APS1
genes were subcloned into the pGreen plant expression vector
(Hellens et al., 2000), and introduced into the adg1 mutant via
Agrobacterium-mediated transformation using the floral-dip method
(Clough and Bent, 1998). Seeds were collected from transformed
plants, germinated on soil and selected by spraying with 0.02% w/v
phosphinothricin at 5, 7, 9, 11 and 13 days after germination.
AGPase activity
Frozen tissue powder (20 mg fresh weight) was extracted using
extraction buffer containing 1 mM DTT and AGPase assayed in the
direction of ADPGlc pyrophosphorolysis, in the presence of 1 mM
DTT and 5 mM 3PGA at 25C as described by Gibon et al. (2004b).
AGPase protein and activation state
Extraction of AGPase, separation by non-reducing SDS–PAGE and
electroblotting onto nitrocellulose membrane were performed as
described by Hendriks et al. (2003). After blocking with 5% (w/v)
non-fat milk powder in 20 mM Tris-HCl, 0.5 M NaCl, pH 7.5 (1·TBS)
for 1 h at 20C, the membrane was incubated with anti-potato AGPB
antiserum (1:10 000 dilution in 1·TBS containing 2.5 % (w/v) non-fat
milk powder; Tiessen et al., 2002) for 1 h at 20C. After washing with
three changes of 1·TBS (5 min per wash, 20 C), the membrane was
incubated with IRDye800-conjugated goat anti-rabbit IgG F(c) antibody (1:7500 dilution in 1·TBS; Biomol, http://www.biomol.de/).
After washing with three changes of 1xTBS, as above, the fluorescent infrared signal from the dye-labeled secondary antibody was
detected using an Odyssey infrared imaging system (LI-COR, http://
www.licor.com/).
Determination of metabolites
Starch was measured in the insoluble fraction after ethanolic
extraction as described by Hendriks et al. (2003). Protein was
determined by a modification of the dye-binding method (Bradford,
1976) as described by Gibon et al. (2009).
Maltose and malto-oligosaccharides were measured in perchloric
acid extracts as described by Fulton et al. (2008). Anionic and
cationic compounds were removed from the extract by sequential
passage through 2 ml columns of Dowex 50Wx4-100 and Dowex
1·8-200 (Sigma-Aldrich, http://www.sigmaaldrich.com/). Neutral
compounds were eluted using 5 ml water, lyophilized, redissolved
in 100 ll water and analysed by HPAEC-PAD using a CarboPac PA20
column (Dionex, http://www.dionex.com) (Fulton et al., 2008). Sugars were identified by co-elution with authentic standards, and
quantified using Chromeleon analysis software (Dionex).
ADPGlc was measured in chloroform:methanol extracts by anion
exchange chromatography coupled to triple quadrupole mass
spectrometry (HPAEC-MS/MS; Lunn et al., 2006).
ACKNOWLEDGEMENTS
We thank Uschi Krause and Melanie Höhne for expert technical
assistance. This research was supported by the German Ministry for
Education and Research (GABI-TILL 0313123D) and the European
Commission (FP7 Collaborative Project TiMet).
SUPPORTING INFORMATION
Additional Supporting Information may be found in the online
version of this article:
Figure S1. Structure of AGPase proteins from plants and bacteria.
Figure S2. Arabidopsis thaliana APS1 (At5g48300) gene sequence.
Figure S3. Southern hybridization analysis of the adg1/APS1C81S
and adg1/APS1WT mutants.
Figure S4. SDS–PAGE of purified wild-type and C81S AGPase.
Figure S5. Sensitivity of wild-type and C81S AGPase to oxidized
DTT.
Table S1. Purification of AGPase.
Table S2. Peptide analysis of purified AGPase.
Table S3.Oligonucleotides used for site-directed mutagenesis.
Appendix S1. Purification of AGPase.
Please note: As a service to our authors and readers, this journal
provides supporting information supplied by the authors. Such
materials are peer-reviewed and may be re-organized for online
delivery, but are not copy-edited or typeset. Technical support
issues arising from supporting information (other than missing
files) should be addressed to the authors.
REFERENCES
Ballicora, M.A., Fu, Y., Nesbitt, N.M. and Preiss, J. (1998) ADP-glucose pyrophosphorylase from potato tubers. Site-directed mutagenesis studies of
the regulatory sites. Plant Physiol. 118, 265–274.
Ballicora, M.A., Fu, Y., Frueauf, J.B. and Preiss, J. (1999) Heat stability of the
potato tuber ADP-glucose pyrophosphorylase: role of Cys residue 12 in the
small subunit. Biochem. Biophys. Res. Commun. 257, 782–786.
Ballicora, M.A., Frueauf, J.B., Fu, Y., Schürmann, P. and Preiss, J. (2000)
Activation of the potato tuber ADP-glucose pyrophosphorylase by thioredoxin. J. Biol. Chem. 275, 1315–1320.
Ballicora, M.A., Iglesias, A.A. and Preiss, J. (2004) ADP-glucose pyrophosphorylase: a regulatory enzyme for starch synthesis. Photosynth. Res. 79,
1–24.
Bent, A.F. (2000) Arabidopsis in planta transformation. Uses, mechanisms, and
prospects for transformation of other species. Plant Physiol. 124, 1540–1547.
Bläsing, O.E., Gibon, Y., Günther, M., Höhne, M., Morcuende, R., Osuna, D.,
Thimm, O., Usadel, B., Scheible, W.R. and Stitt, M. (2005) Sugars and
circadian regulation make major contributions to the global regulation of
diurnal gene expression in Arabidopsis. Plant Cell, 17, 3257–3281.
Boehlein, S., Shaw, J.R., Stewart, J.D. and Hannah, L.C. (2010) Studies of the
kinetic mechanism of maize endosperm ADP-glucose pyrophosphorylase
uncovered complex regulatory properties. Plant Physiol. 152, 1056–1064.
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2012), 70, 231–242
Mutation of Cys81 in Arabidopsis AGPase 241
Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein–dye
binding. Anal. Biochem. 72, 248–254.
Chatterton, N.J. and Silvius, J.E. (1979) Photosynthate partitioning into starch
in soybean leaves, effects of photoperiod versus photosynthetic period
duration. Plant Physiol. 64, 749–753.
Chatterton, N.J. and Silvius, J.E. (1980) Photosynthate partitioning as affected
by daily photosynthetic period duration in six species. Physiol. Plant. 49,
141–144.
Chatterton, N.J. and Silvius, J.E. (1981) Photosynthate partitioning into starch
in soybean leaves: irradiance level and daily photsynthetic period duration
effects. Plant Physiol. 67, 257–260.
Crevillén, P., Ballicora, M.A., Mérida, Á., Preiss, J. and Romero, J.M. (2003)
The different large subunit isoforms of Arabidopsis thaliana ADP-glucose
pyrophosphorylase confer distinct kinetic and regulatory properties to the
heterotetrameric enzyme. J. Biol. Chem. 31, 28508–28515.
Crevillén, P., Ventriglia, T., Pinto, F., Orea, A., Mérida, Á. and Romero, J.M.
(2005) Differential pattern of expression and sugar regulation of Arabidopsis thaliana ADP-glucose pyrophosphorylase-encoding genes. J. Biol.
Chem. 280, 8143–8149.
Cross, J.M., Clancy, M., Shaw, J.R., Greene, T.W., Schmidt, R.R., Okita, T.W.
and Hannah, L.C. (2004) Both subunits of ADP-glucose pyrophosphorylase
are regulatory. Plant Physiol. 135, 137–144.
Delatte, T., Trevisan, M., Parker, M.L. and Zeeman, S.C. (2005) Arabidopsis
mutants Atisa1 and Atisa2 have identical phenotypes and lack the same
multimeric isoamylase, which influences the branch point distribution of
amylopectin during starch synthesis. Plant J. 41, 815–830.
Dickinson, D.B. and Preiss, J. (1969) Presence of ADP-glucose pyrophosphorylase in shrunken-2 and brittle-2 mutants of maize endosperm. Plant
Physiol. 44, 1058–1062.
Fu, Y., Ballicora, M.A., Leykam, J.F. and Preiss, J. (1998) Mechanism of
reductive activation of potato tuber ADP-glucose pyrophosphorylase.
J. Biol. Chem. 273, 25045–25052.
Fulton, D.C., Stettler, M., Mettler, T. et al. (2008) b-AMYLASE4, a noncatalytic
protein required for starch breakdown, acts upstream of three active
b-amylases in Arabidopsis chloroplasts. Plant Cell, 20, 1040–1058.
Geiger, D.R. and Servaites, J.C. (1994) Diurnal regulation of photosynthetic
carbon metabolism in C3 plants. Annu. Rev. Plant Physiol. Plant Mol. Biol.
45, 235–256.
Gibon, Y., Vigeolas, H., Tiessen, A., Geigenberger, P. and Stitt, M. (2002)
Sensitive and high throughput metabolite assays for inorganic pyrophosphate, ADPGlc, nucleotide phosphates, and glycolytic intermediates based
on a novel enzymic cycling system. Plant J. 30, 221–235.
Gibon, Y., Blasing, O.E., Palacios-Rojas, N., Pankovic, D., Hendriks, J.H.,
Fisahn, J., Hohne, M., Gunther, M. and Stitt, M. (2004a) Adjustment of
diurnal starch turnover to short days: depletion of sugar during the
night leads to a temporary inhibition of carbohydrate utilization, accumulation of sugars and post-translational activation of ADP-glucose
pyrophosphorylase in the following light period. Plant J. 39, 847–862.
Gibon, Y., Blaesing, O.E., Hannemann, J., Carillo, P., Höhne, M., Hendriks,
J.H., Palacios, N., Cross, J., Selbig, T. and Stitt, M. (2004b) A robot-based
platform to measure multiple enzyme activities in Arabidopsis using a set
of cycling assays: comparison of changes of enzyme activities and transcript levels during diurnal cycles and in prolonged darkness. Plant Cell, 16,
3304–3325.
Gibon, Y., Pyl, E.T., Sulpice, R., Lunn, J.E., Hohne, M., Gunther, M. and
Stitt, M. (2009) Adjustment of growth, starch turnover, protein content and
central metabolism to a decrease of the carbon supply when Arabidopsis is
grown in very short photoperiods. Plant Cell Environ. 32, 859–874.
Graf, A. and Smith, A.M. (2011) Starch and the clock: the dark side of plant
productivity. Trends Plant Sci. 16, 169–175.
Graf, A., Schlereth, A., Stitt, M. and Smith, A.M. (2010) Starch degradation in
Arabidopsis leaves is controlled by the circadian clock. Proc. Natl Acad. Sci.
USA, 107, 9458–9463.
Hädrich, N., Gibon, Y., Schudoma, C., Altmann, T., Lunn, J.E. and Stitt, M.
(2011) Use of TILLING and robotised enzyme assays to generate an allelic
series of Arabidopsis thaliana mutants with altered ADP-glucose
pyrophosphorylase activity. J. Plant Physiol. 168, 1395–1405.
Hellens, R.P., Edwards, E.A., Leyland, N.R., Bean, S. and Mullineaux, P.M.
(2000) pGreen: a versatile and flexible binary Ti vector for Agrobacteriummediated plant transformation. Plant Mol. Biol. 42, 819–832.
Hendriks, J.H.M., Kolbe, A., Gibon, Y., Stitt, M. and Geigenberger, P. (2003)
ADP-glucose pyrophosphorylase is activated by posttranslational redoxmodification in response to light and to sugars in leaves of Arabidopsis and
other plant species. Plant Physiol. 133, 838–849.
Kolbe, A., Tiessen, A., Schluepmann, H., Paul, M., Ulrich, S. and Geigenberger, P. (2005) Trehalose 6-phosphate regulates starch synthesis via
posttranslational redox activation of ADP-glucose pyrophosphorylase.
Proc. Natl Acad. Sci. USA, 102, 11118–11123.
Lin, T.P., Caspar, T., Somerville, C. and Preiss, J. (1988a) Isolation and characterization of a starchless mutant of Arabidopsis thaliana (L.) Heynh.
lacking ADPglucose pyrophosphorylase activity. Plant Physiol. 86, 1131–
1135.
Lin, T.P., Caspar, T., Somerville, C. and Preiss, J. (1988b) A starch deficient
mutant of Arabidopsis thaliana with low ADPglucose pyrophosphorylase
activity lacks one of the two subunits of the enzyme. Plant Physiol. 88,
1175–1181.
Lorenzen, J.H. and Ewing, E.E. (1992) Starch accumulation in leaves of potato
(Solanum tuberosum L.) during the first 18 days of photoperiod treatment.
Ann. Bot. 69, 481–485.
Lu, Y., Gehan, J.P. and Sharkey, T.D. (2005) Daylength and circadian effects
on starch degradation and maltose metabolism. Plant Physiol. 138, 2280–
2291.
Lu, Y., Steichen, J.M., Weise, S.E. and Sharkey, T.D. (2006) Cellular and organ
level localization of maltose in maltose-excess Arabidopsis mutants.
Planta, 224, 935–943.
Lunn, J.E., Feil, R., Hendriks, J.H.M., Gibon, Y., Morcuende, R., Osuna, D.,
Scheible, W.R., Carillo, P., Hajirezaei, M.R. and Stitt, M. (2006) Sugarinduced increases in trehalose-6-phosphate are correlated with redox
activation of ADPglucose pyrophosphorylase and higher rates of starch
synthesis in Arabidopsis thaliana. Biochem. J. 397, 139–148.
Lyerly Linebarger, C.R., Boehlein, S.K., Sewell, A.K., Shaw, J. and Hannah,
L.C. (2005) Heat stability of maize endosperm ADP-glucose pyrophosphorylase is enhanced by insertion of a cysteine in the N terminus of the
small subunit. Plant Physiol. 139, 1625–1634.
MacRae, E.A. and Lunn, J.E. (2006) Control of sucrose biosynthesis. In
Advances in Plant Research, Volume 22: Control of Primary Metabolism
in Plants (Plaxton, W.C. and McManus, M.T., eds). Oxford: Blackwell, pp.
234–257.
Michalska, J., Zauber, H., Buchanan, B.B., Cejudo, F.J. and Geigenberger, P.
(2009) NTRC links built-in thioredoxin to light and sucrose in regulating
starch synthesis in chloroplasts and amyloplasts. Proc. Natl Acad. Sci.
USA, 106, 9908–9913.
Morell, M., Bloom, M., Knowles, V. and Preiss, J. (1987) Subunit structure of
spinach leaf ADPglucose pyrophosphorylase. Plant Physiol. 85, 182–187.
Mullen, J.A. and Koller, R. (1988) Daytime and nighttime carbon balance and
assimilate export in soybean leaves at different photon flux densities. Plant
Physiol. 86, 880–884.
Neuhaus, H.E. and Stitt, M. (1990) Control analysis of photosynthate partitioning – impact of reduced activity of ADP-glucose pyrophosphorylase or
plastid phosphoglucomutase on the fluxes to starch and sucrose in
Arabidopsis thaliana (L.) Heynh. Planta, 182, 445–454.
Neuhaus, H.E., Kruckeberg, A.L., Feil, R., Gottlieb, L. and Stitt, M. (1989)
Dosage mutants of phosphoglucose isomerase in the cytosol and chloroplasts of Clarkia xantiana. II. Study of the mechanisms which regulate
photosynthate partitioning. Planta, 178, 110–122.
Nielsen, T.H., Krapp, A., Roper-Schwarz, U. and Stitt, M. (1998) The sugarmediated regulation of genes encoding the small subunit of Rubisco and
the regulatory subunit of ADP glucose pyrophosphorylase is modified by
phosphate and nitrogen. Plant Cell Environ. 21, 443–454.
Niittyla, T., Messerli, G., Trevisan, M., Chen, J., Smith, A.M. and Zeeman, S.C.
(2004) A previously unknown maltose transporter essential for starch
degradation in leaves. Science, 303, 87–89.
Obana, Y., Omoto, D., Kato, C. et al. (2006) Enhanced turnover of transitory
starch by expression of up-regulated ADP-glucose pyrophosphorylase in
Arabidopsis thaliana. Plant Sci. 170, 1–11.
Okita, T.W., Nakata, P.A., Anderson, J.M., Sowokinos, J., Morell, M. and
Preiss, J. (1990) The subunit structure of potato tuber ADPglucose pyrophosphorylase. Plant Physiol. 93, 785–790.
Piques, M., Schulze, W.X., Höhne, M., Usadel, B., Gibon, Y., Rohwer, J. and
Stitt, M. (2009) Ribosome and transcript copy numbers, polysome occupancy and enzyme dynamics in Arabidopsis. Mol. Syst. Biol. 5, 314.
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2012), 70, 231–242
242 Nadja Hädrich et al.
Preiss, J. (1988) Biosynthesis of starch and its regulation. In The Biochemistry
of Plants, Volume 14: Carbohydrates, Structure and Function (Preiss, J.,
ed.). New York: Academic Press, pp. 181–254.
Sambrook, J. and Russell, D.R. (2001) Molecular Cloning: A Laboratory
Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Scheible, W.R., Gonzalez-Fontes, A., Lauerer, M., Müller-Röber, B., Caboche, M.
and Stitt, M. (1997) Nitrate acts as a signal to induce organic acid metabolism and repress starch metabolism in tobacco. Plant Cell, 9, 783–798.
Smith, A.M. and Stitt, M. (2007) Coordination of carbon supply and plant
growth. Plant Cell Environ. 30, 1126–1149.
Smith, A.M., Bettey, M. and Bedford, I.D. (1989) Evidence that the rb locus
alters the starch content of developing pea embryos through an effect on
ADP glucose pyrophosphorylase. Plant Physiol. 89, 1279–1284.
Smith, A.M., Zeeman, S.C. and Smith, S.M. (2005) Starch degradation. Annu.
Rev. Plant Biol. 56, 73–98.
Sokolov, L.N., Dejardin, A. and Kleczkowski, L.A. (1998) Sugars and light/dark
exposure trigger differential regulation of ADP-glucose pyrophosphorylase
genes in Arabidopsis thaliana (thale cress). Biochem. J. 336, 681–687.
Sowokinos, J.R. (1981) Pyrophosphorylases in Solanum tuberosum. 2. Catalytic properties and regulation of ADP-glucose and UDP-glucose pyrophosphorylase activities in potatoes. Plant Physiol. 68, 924–929.
Sowokinos, J.R. and Preiss, J. (1982) Pyrophosphorylases in Solanum
tuberosum. 3. Purification, physical and catalytic properties of ADP-glucose pyrophosphorylase in potatoes. Plant Physiol. 69, 1459–1466.
Stitt, M., Bulpin, P.V. and ap Rees, T. (1978) Pathway of starch breakdown in
photosynthetic tissue of Pisum sativum. Biochim. Biophys. Acta, 544, 200–
214.
Stitt, M., Gibon, Y., Lunn, J.E. and Piques, M. (2007) Multilevel genomics
analysis of carbon signalling during low carbon availability: coordinating
the supply and utilisation of carbon in a fluctuating environment. Funct.
Plant Biol. 34, 526–549.
Stitt, M., Lunn, J. and Usadel, B. (2010) Primary photosynthetic metabolism –
more than the icing on the cake. Plant J. 61, 1067–1091.
Sun, J., Okita, T.W. and Edwards, G.E. (1999) Modification of carbon partitioning, photosynthetic capacity, and O2 sensitivity in Arabidopsis plants
with low ADP-glucose pyrophosphorylase activity. Plant Physiol. 119, 267–
276.
Thimm, O., Bläsing, O., Gibon, Y., Nagel, A., Meyer, S., Krüger, P., Selbig, J.,
Müller, L.A., Rhee, S.Y. and Stitt, M. (2004) MapMan, a user-driven tool to
display genomics data sets onto diagrams of metabolic pathways and
other biological processes. Plant J. 37, 914–939.
Tiessen, A., Hendriks, J.H.M., Stitt, M., Branscheid, A., Gibon, Y., Farré, E.M.
and Geigenberger, P. (2002) Starch synthesis in potato tubers is regulated
by post-translational redox modification of ADP-glucose pyrophosphorylase: a novel regulatory mechanism linking starch synthesis to the sucrose
supply. Plant Cell, 14, 2191–2213.
Tsai, C.Y. and Nelson, O.E. (1966) Starch-deficient maize mutant lacking
adenosine diphosphate glucose pyrophosphorylase activity. Science, 151,
341–343.
Ventriglia, T., Kuhn, M.L., Ruiz, M.T., Ribeiro-Pedro, M., Valverde, F., Ballicora, M.A., Preiss, J. and Romero, J.M. (2008) Two Arabidopsis ADP-glucose
pyrophosphorylase large subunits (APL1 and APL2) are catalytic. Plant
Physiol. 148, 65–76.
Wang, S.-M., Chu, B., Lue, W.-L., Yu, T.-S., Eimert, K. and Chen, J. (1997) adg21 represents a missense mutation in the ADPG pyrophosphorylase large
subunit gene of Arabidopsis thaliana. Plant J. 11, 1121–1126.
Wang, S.-M., Lue, W.-L., Yu, T.-S., Long, J.-H., Wang, C.-N., Eimert, K. and
Chen, J. (1998) Characterization of ADG1, an Arabidopsis locus encoding
for ADPG pyrophosphorylase small subunit, demonstrates that the presence of the small subunit is required for large subunit stability. Plant J. 13,
63–70.
Weiner, H., Stitt, M. and Heldt, H.W. (1987) Subcellular compartmentation of
pyrophosphate and alkaline pyrophosphatase in leaves. Biochim. Biophys.
Acta, 893, 13–21.
Zeeman, S.C., Umemoto, T., Lue, W.L., Au-Yeung, P., Martin, C., Smith, A.M.
and Chen, J. (1998) A mutant of Arabidopsis lacking a chloroplastic
isoamylase accumulates both starch and phytoglycogen. Plant Cell, 10,
1699–1711.
Zeeman, S.C., Smith, S.M. and Smith, A.M. (2007) The diurnal metabolism of
leaf starch. Biochem. J. 401, 13–28.
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2012), 70, 231–242