The Plant Cell, Vol. 14, 2191 2213, September 2002, www.plantcell.org © 2002 American Society of Plant Biologists
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
Axel Tiessen, Janneke H. M. Hendriks, Mark Stitt, Anja Branscheid, Yves Gibon, Eva M. Farré,
and Peter Geigenberger1
Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Golm, Germany
Transcriptional and allosteric regulation of ADP-Glc pyrophosphorylase (AGPase) plays a major role in the regulation of
starch synthesis. Analysis of the response after detachment of growing potato tubers from the mother plant revealed
that this concept requires extension. Starch synthesis was inhibited within 24 h of tuber detachment, even though the
catalytic subunit of AGPase (AGPB) and overall AGPase activity remained high, the substrates ATP and Glc-1-P in-
creased, and the glycerate-3-phosphate/inorganic orthophosphate (the allosteric activator and inhibitor, respectively)
ratio increased. This inhibition was abolished in transformants in which a bacterial AGPase replaced the potato AGPase.
Measurements of the subcellular levels of each metabolite between Suc and starch established AGPase as the only
step whose substrates increase and mass action ratio decreases after detachment of wild-type tubers. Separation of
extracts on nonreducing SDS gels revealed that AGPB is present as a mixture of monomers and dimers in growing tu-
bers and becomes dimerized completely in detached tubers. Dimerization led to inactivation of the enzyme as a result
of a marked decrease of the substrate affinity and sensitivity to allosteric effectors. Dimerization could be reversed and
AGPase reactivated in vitro by incubating extracts with DTT. Incubation of tuber slices with DTT or high Suc levels re-
duced dimerization, increased AGPase activation, and stimulated starch synthesis in vivo. In intact tubers, the Suc con-
tent correlated strongly with AGPase activation across a range of treatments, including tuber detachment, aging of the
mother plant, heterologous overexpression of Suc phosphorylase, and antisense inhibition of endogenous AGPase ac-
tivity. Furthermore, activation of AGPase resulted in a stimulation of starch synthesis and decreased levels of glycolytic
intermediates.
INTRODUCTION
ADP-Glc pyrophosphorylase (AGPase) catalyzes the con- when activity is decreased to 50% of the wild-type level
version of Glc-1-P and ATP to ADP-Glc and inorganic pyro- (Müller-Röber et al., 1992; Geigenberger et al., 1999a).
phosphate (PPi), which is the first committed step in the Two mechanisms are known to regulate AGPase activity.
pathway of starch synthesis (Figure 1) (Preiss, 1988; Martin First, AGPase is subject to transcriptional regulation, with ex-
and Smith, 1995; Smith et al., 1997). The higher plant en- pression being increased by sugars (Salanoubat and Belliard,
zyme is a heterotetramer, consisting of two  regulatory 1989; Müller-Röber et al., 1990; Sokolov et al., 1998) and
subunits (AGPS; 51 kD) and two slightly smaller  catalytic decreased by nitrate (Scheible et al., 1997) and phosphate
subunits (AGPB; 50 kD) (Okita et al., 1990). AGPase plays a (Nielsen et al., 1998). This may allow starch accumulation to
major role in the regulation of starch synthesis. Studies with respond to changes in the carbon and nutritional status
an Arabidopsis AGPS mutant have demonstrated that AGPase (Scheible et al., 1997; Stitt and Krapp, 1999). Second, AGPase
is a key site for the control of starch synthesis in leaves is exquisitely sensitive to allosteric regulation, being acti-
(Neuhaus and Stitt, 1990). In potato tubers expressing an vated by glycerate-3-phosphate (3PGA) and inhibited by Pi
antisense AGPB construct, starch synthesis is decreased (Sowokinos, 1981; Sowokinos and Preiss, 1982; Preiss, 1988).
Increasing levels of phosphorylated intermediates typi-
cally lead to a marked increase of the 3PGA/Pi ratio. There-
fore, activation of AGPase by an increasing 3PGA/Pi ratio
1
To whom correspondence should be addressed. E-mail geigenberger
allows the rate of starch synthesis to be adjusted in re-
@mpimp-golm.mpg.de; fax [phone].
sponse to changes in the balance between photosynthesis
Article, publication date, and citation information can be found at
www.plantcell.org/cgi/doi/10.1105/tpc.003640. and Suc synthesis in leaves (Heldt et al., 1977; Herold,
2192 The Plant Cell
1980; Stitt et al., 1987) and to changes in the balance be- cator (Tjaden et al., 1998; Geigenberger et al., 2001). These
tween Suc breakdown and respiration in nonphotosyn- are required to transport the carbon substrates and ATP
thetic tissues (Stark et al., 1992; Hajirezaei et al., 1994; from the cytosol to the plastid, where AGPase is located
Geigenberger et al., 1997, 1998a, 2000; Jenner et al., 2001). (Figure 1). Another possibility is that AGPase activity may be
Several situations have been reported in which starch responding to changes in plastid 3PGA/Pi ratio caused by
synthesis changes independently of overall AGPase activity changes in the activity of the envelope triose phos-
and reciprocally to the levels of phosphorylated intermediates phate:phosphate translocator. This transporter is required
(Geigenberger et al., 1994; Geiger et al., 1998; Trethewey et to transmit changes of the 3PGA/Pi ratio from one compart-
al., 1998, 2001; Geigenberger and Stitt, 2000). This fact indi- ment to the other (Borchert et al., 1989; Schott et al., 1995)
cates that there may be important gaps in our understand- (Figure 1).
ing of the regulation of starch synthesis. One such situation Here, we show that the inhibition of starch synthesis after
formed the starting point for the experiments described tuber detachment occurs via a mechanism that depends on
here. Detachment of growing potato tubers from the mother the properties of the plant AGPase; additionally, we used
plant leads within 24 h to a threefold decrease of ADP-Glc nonaqueous fractionation to investigate the response of cy-
and a 50% inhibition of starch synthesis, even though tosolic and plastidic metabolite levels and define a unique
AGPase activity remains unaltered and the overall levels of crossover point at AGPase. We also demonstrate that de-
hexose phosphates and 3PGA increase (Geigenberger et tachment does not lead to a decrease of the plastid 3PGA/
al., 1994). Pi ratio and has no effect on AGPB protein or overall AGP-
Because only the overall metabolite levels were mea- ase activity; instead, it leads to post-translational inactiva-
sured, there are various explanations for these observa- tion of AGPase via a reversible mechanism that involves re-
tions. One is that AGPase is being inhibited by a novel dox-dependent dimerization of the ABPB subunits. Finally,
mechanism. Another is that the substrate supply for AGP- we present evidence that this novel mechanism contributes
ase may be changing as a result of regulation of the enve- to the regulation of starch synthesis in response to a range
lope hexose phosphate:phosphate transporter (Kammerer of treatments that modify the Suc level in tubers.
et al., 1998), the plastidic phosphoglucomutase (PGM)
(Tauberger et al., 2000), or the envelope adenylate translo-
RESULTS
Inhibition of Starch Synthesis in Response to Tuber
Detachment Is Abolished in Transgenic Tubers That
Express a Heterologous AGPase
We used two independent approaches to determine the
step or steps in the pathway of starch synthesis that are the
targets for the novel mechanism that inhibits starch synthe-
sis when tubers are detached from the mother plant. The
first approach asked whether specific regulatory features of
potato tuber AGPase are essential for the inhibition of starch
synthesis. To answer this question, the response was com-
pared in wild-type tubers and in transformants in which
endogenous AGPase was replaced largely by a nonplant
AGPase.
The introduced construct encodes a form of the mono-
meric Escherichia coli enzyme (glgC16) that is different in its
kinetic properties from the plant enzyme (Stark et al., 1992).
It was introduced (Lloyd et al., 1999) into the AGPase anti-
sense line AGP93, which has low activity of the higher plant
Figure 1. Pathway of Suc-to-Starch Conversion and Its Subcellular
AGPase (Müller-Röber et al., 1992). AGPase activity in wild-
Compartmentation in Potato Tubers.
type tubers was strongly dependent on 3PGA (Figure 2A).
1, Suc synthase; 2, UDP-Glc pyrophosphorylase; 3, fructokinase; 4,
AGPase activity in the antisense line AGP93 was reduced by
cytosolic phosphoglucomutase; 5, phosphoglucoisomerase; 6, plas-
90% and remained dependent on 3PGA. AGPase activity
tidic phosphoglucomutase; 7, ADP-Glc pyrophosphorylase; 8, alka-
in the double-transformed lines AF1-28 and AF1-20 was
line pyrophosphatase; 9, granule-bound starch synthase; 10, solu-
threefold higher than that in AGP93 and was independent of
ble starch synthase; 11, branching enzyme; 12, hexose phosphate
translocator; 13, triose phosphate translocator; 14, adenylate trans- 3PGA (Figure 2A) and Pi (data not shown) (Lloyd et al.,
locator. TCA, tricarboxylic acid. 1999).
Redox Regulation of Starch Synthesis 2193
To measure the rate of starch synthesis, low concentra-
tions of 14C-Glc were injected into a fine borehole in growing
tubers that were attached to the mother plant or 1 and 3
days after detaching them from the mother plant by sever-
ing the stolon (for details, see Geigenberger et al., 1994). Af-
ter 1 h, the area around the injected label was removed and
analyzed to determine how much of the injected label had
been converted to starch (Figure 2B). Metabolite levels were
measured in the same material (Figures 2C and 2D). Similar
results were obtained in an independent experiment in
which 14C-Glc was supplied for 30 min to discs cut from at-
tached or detached tubers (data not shown).
In wild-type tubers, 34% of the label was incorporated
into starch in attached tubers, decreasing to 21 and 15% in
tubers that had been detached for 1 and 3 days, respec-
tively (Figure 2B). The inhibition of starch synthesis was ac-
companied by an increase of hexose phosphates (Figure
2C) and a slight increase of 3PGA (data not shown). Suc de-
creased gradually, declining by 24 and 70% after 1 and 3
days, respectively (Figure 2D). This finding confirms the re-
sults described previously (Geigenberger et al., 1994).
In the antisense line AGP93, 19% of the injected label was
incorporated into starch in attached tubers, decreasing to
8% in detached tubers (Figure 2B). Hexose phosphates
were high in attached tubers and remained high after de-
tachment (Figure 2C), and Suc decreased even more slowly
than in wild-type tubers (Figure 2D).
In the double transformants AF1-20 and AF1-28, 24 to
26% of the label was incorporated into starch in attached
tubers (Figure 2B). Detachment did not lead to a rapid inhi-
bition of starch synthesis in these lines (Figure 2B) but in-
stead led to a marked decline of hexose phosphates (Figure
2C) and a rapid 70 to 80% decrease of Suc during the first
day after detachment (Figure 2D). There were no substantial
changes in the specific activities of the internal hexose phos-
phate pools in the various genotypes after detachment (data
not shown), demonstrating that the different response in the
(B) to (D) Changes in the rate of starch synthesis and metabolite lev-
els after tuber detachment. Tubers from 8-week-old wild-type po-
tato plants, AGPase antisense plants (AGP93), and supertrans-
formed plants expressing glgC16 (AF1-28 and AF1-20) were
analyzed either directly (black bars) or 1 day (gray bars) or 3 days
(white bars) after detachment from the plant. To measure the rate of
Figure 2. Inhibition of Starch Synthesis in Response to Tuber
starch synthesis (B), U-14C-Glc of high specific activity ( 40 to 50
Detachment Is Abolished in Transgenic Tubers Expressing the
kBq per tuber) was injected into a fine borehole of an otherwise in-
glgC16 Gene in an AGPB Antisense Background.
tact tuber. The tubers then were incubated for 1 h, and a concentric
(A) AGPase activity in wild-type tubers (WT), the parental antisense core of material around the borehole was extracted and analyzed to
AGPB line (AGP93), and four independent transgenic AF1 lines ex- determine 14C incorporation into starch. The data are expressed as a
pressing the glgC16 gene in an antisense AGPase (AGP93) back- percentage of the total label injected. The same samples were used
ground. AGPase activity was assayed using a standard protocol to measure the levels of hexose phosphates (sum of Glc-6-P, Fru-
(Müller-Röber et al., 1992) in the absence (black bars) or the pres- 6-P, and Glc-1-P) (C) and Suc (D) by enzymatic analysis. Results are
ence of 1 mM (dark-gray bars), 2 mM (medium-gray bars), or 3 mM means SE of four tubers from different plants.
(light-gray bars) 3PGA. FW, fresh weight.
2194 The Plant Cell
double transformants is not attributable to isotopic dilution cellular concentrations. This analysis yields estimates for
of the incoming label by internal pools. These results pro- the vacuole, plastids, and cytosol. The estimated values
vide strong genetic evidence that starch synthesis is inhib- for the cytosol include the mitochondrial metabolites be-
ited after detachment by a regulatory mechanism that re- cause the cytosol and mitochondria are not separated (see
quires the presence of native potato tuber AGPase. above).
Separation of Tuber Material into Changes in Subcellular Levels of Its Substrates Reveal
Subcellular Compartments That AGPase Is Involved in the Inhibition of
Starch Synthesis
Regulated enzymes can be identified by perturbing the flux
through a metabolic pathway and measuring the resulting The immediate substrates for AGPase are the pools of Glc-
changes in metabolite levels to identify the step or steps at 1-P and ATP in the plastid (Figure 1). Suc is degraded in the
which the substrate concentration(s) changes reciprocally to cytosol via Suc synthase (SuSy), UGPase, and PGM to form
the flux through the pathway (Rolleston, 1972). To allow the Glc-6-P, which is imported into the plastid via the Glc-6-P/
unbiased identification of the step(s) at which starch synthe- Pi transporter and converted back to Glc-1-P by the plas-
sis is inhibited after the detachment of wild-type tubers, we tidic PGM (Figure 1). Glc-6-P, Fru-6-P, and Glc-1-P were
investigated changes in the subcellular levels of every me- distributed between the plastid and cytosol in attached tu-
tabolite from Suc to starch. bers (Figures 3B to 3D) (Farré et al., 2001). The vacuole con-
This was performed by nonaqueous fractionation, a tech- tained negligible hexose phosphates, except for traces of
nique developed to analyze the subcellular compartmenta- Glc-1-P (Figure 3D) (Farré et al., 2001). Detachment led to
tion of metabolites in leaves (Gerhardt and Heldt, 1984; Stitt an increase of Glc-6-P, Fru-6-P, and Glc-1-P in the cytosol
et al., 1989) and adapted recently for use with potato tubers and the plastid. The error bars are large for Glc-1-P because
(Farré et al., 2001). Tubers are frozen in liquid nitrogen to this metabolite is present at low levels, leading to analytic
quench metabolism, homogenized in liquid nitrogen, ly- errors that sum during the calculations. The estimated aver-
ophilized at low temperature, and resuspended in heptane. age plastid Glc-1-P concentration increased fourfold from
Enzymic reactions are blocked for the reminder of the frac- 17 to 64 M (Table 1).
tionation procedure because water is absent. During lyoph- ATP is imported from the cytosol via the envelope adenyl-
ilization, metabolites and proteins from a particular region ate exchanger (Figure 1). A substantial proportion of the ad-
of the cell aggregate. The suspension then is ultrasoni- enine nucleotides are located in the plastid (Figures 3E and
cated to generate particles that are partially enriched for 3F), whereas uridine and guanidine nucleotides are located
different material from subcellular compartments and that mainly in the cytosol of tubers (data not shown) (Farré et al.,
can be separated by nonaqueous density gradient centri- 2001), as is found for leaves (Dancer et al., 1990; Riens et
fugation. al., 1991). Detachment led to a 50% increase of ATP in the
Marker enzyme activities are measured to reveal how ma- plastid and a 50% decrease of ATP in the cytosol (Figure
terials from different cellular compartments distribute across 3E). ADP levels were not affected substantially after detach-
the gradient (Figure 3A). The vacuolar marker mannosidase ment (Figure 3F).
(Boller and Kende, 1979) was highly enriched in the pellet, The ATP/ADP ratio was lower in the plastid than in the
the cytosolic markers UDP-Glc pyrophosphorylase (UGP- cytosol in attached tubers (1.2 compared with 3.3; data
ase; Kleczkowsky, 1994) and pyrophosphate:Fru-6-P1 phos- calculated from Figures 3E and 3F), again resembling the
photransferase (MacDonald and Preiss, 1986) and the mito- ratio in leaves (Stitt et al., 1982). The value attributed to the
chondrial marker citrate synthase were enriched in fractions cytosol underestimates the actual cytosolic value, because
0 and 1, and the plastidic marker AGPase (Kim et al., 1989) significant amounts of adenine nucleotides also are pres-
was enriched in the lightest fractions (fractions 2 and 3) (Fig- ent in the mitochondria, and the ATP/ADP ratio in the mito-
ure 3A) (Farré et al., 2001). This effect resembles the distri- chondria is lower than that in the cytosol (Stitt et al., 1982).
bution in gradients of leaf material (Stitt et al., 1989); in that After detachment, the ATP/ADP ratio in the plastid in-
earlier study, it also was shown that the distribution of en- creased to a value (1.9) similar to that estimated for the cy-
zymes tracks the distribution of metabolites that are known tosol plus the mitochondria (2.0). The estimated plastid
to be restricted to a particular compartment. ATP concentration increased after detachment from 179 to
Metabolites are measured in each fraction, and their 279 M (Table 1).
subcellular distribution are estimated by more-dimensional The estimated plastidic concentrations of Glc-1-P and
linear regression. The estimated distribution can be com- ATP were in the range of the values determined in vitro for
pared with the overall content to estimate the metabolite the substrate affinity S0.5 Glc-1-P (40 to 140 M) and S0.5
content in each compartment (per gram of total dry weight) ATP (120 to 190 M) of AGPase (Sowokinos and Preiss,
and with empirically determined values for the volume of 1982; Ballicora et al., 1995). Detachment led to an increase
each compartment (Farré et al., 2001) to estimate the sub- of the levels of both substrates, whereas the rate of starch
Redox Regulation of Starch Synthesis 2195
synthesis decreased. These results show that the inhibition of
starch synthesis involves a mechanism that acts on AGPase.
Estimation of Mass-Action Ratios for Each
Enzyme-Catalyzed Reaction and Transport Step
between Suc and Starch Identifies AGPase as the
Unique Site Involved in the Inhibition of Suc-to-Starch
Interconversion after Tuber Detachment
The cytosolic concentrations of Suc, Fru, UDP-Glc, Glc-1-P,
Glc-6-P, Fru-6-P, ATP, ADP, UTP, UDP, PPi, Pi, and 3PGA,
as well as the plastidic concentrations of Glc-1-P, Glc-6-P,
ADP-Glc, ATP, ADP, PPi, Pi, and 3PGA, are summarized in
Table 1. The variation for some metabolites, including Glc-
1-P and PPi in the plastid and ADP in the cytosol, was high.
The variation for Glc-1-P was discussed above. The varia-
tion in PPi was caused by the low level of PPi and by the
fact that only a very small fraction of the total PPi was lo-
cated in the plastid.
These results (Table 1) were used to calculate the ratio
between the in vivo concentrations of the products and the
substrates (termed the mass-action ratio) for every step be-
tween Suc and ADP-Glc (Table 2). The theoretical equilib-
rium constant (Keq; the ratio of product and substrate con-
centrations at which the reaction is at its thermodynamic
equilibrium and net flux is zero) is listed for comparison. Keq
for the transport steps is set at unity. In attached tubers, the
mass-action ratios of the reactions catalyzed by SuSy,
UGPase, and cytosolic PGM, the transport exchanges cata-
lyzed by the Glc-6-P/Pi and ATP/ADP transporters, and the
reaction catalyzed by plastidic PGM are close to their Keq.
The mass-action ratios of fructokinase, AGPase, and inor-
ganic pyrophosphatase are displaced from their Keq (Table
2). These results resemble those reported previously for cy-
tosol and plastids in leaves (Stitt et al., 1982, 1989) and for
phloem sap (Geigenberger et al., 1993). Thus, our results
provide evidence for the reliability of the fractionation tech-
nique and subsequent calculations.
The inhibition of starch synthesis after detachment was
accompanied by a 40-fold decrease of the mass-action ra-
tio for AGPase (Table 2). This was the result of an increase in
the concentration of the substrates Glc-1-P and ATP and a
decrease in the concentration of the products ADP-Glc and
PPi in the plastid (Table 1) (Geigenberger et al., 1994). The
mass-action ratios for all of the other steps were unaltered
Figure 3. Subcellular Analysis of Metabolite Levels in Attached and
1-Day-Detached Wild-Type Tubers.
(A) Marker enzyme distribution in a typical nonaqueous gradient of
lyophilized wild-type tuber tissues. The values represent the enzyme
activity found in each fraction as a percentage of the total applied to Metabolites in the fractions of the nonaqueous gradients were ex-
the gradient. The marker enzyme distribution in the fractions of each tracted with trichloroacetic acid before analysis of Glc-6-P (B), Fru-
gradient was used to calculate the distribution of metabolites in the 6-P (C), Glc-1-P (D), ATP (E), ADP (F), 3PGA (G), Pi (H), and Suc (I).
subcellular compartments. CS, citrate synthase; NAF, nonaqueous The subcellular compartmentation of the metabolites was calculated
fractionation; PFP, pyrophosphate:Fru-6-P1 phosphotransferase. by more-dimensional regression analysis (best-fit method). The val-
(B) to (I) Compartmentation of metabolites in potato tuber attached ues are standardized to tissue dry weight and give the mean of three
to the plant (gray bars) or after 1 day of detachment (black bars). separate gradients (means SE, n 3).
2196 The Plant Cell
estimated concentration in the plastid decreased from 974
Table 1. Estimated Cytosolic and Plastidic Concentrations of
to 502 M (Table 1).
Metabolites Involved in the Pathway of Suc to Starch
To explain why there is a 30 to 50% inhibition of starch
Metabolite Attached Tubers Detached Tubers
synthesis after tuber detachment (Figure 2B) (Geigenberger
et al., 1994) even though there is an increase in the sub-
Cytosol
Suc 82.4 22.9 47.9 4 strates and an increase in the 3PGA/Pi ratio, it is necessary
Fru 1.37 0.72 1.79 0.38
to postulate either (1) a very large decrease in expression or
UDP-Glc 571 17 627 54
(2) a novel regulatory mechanism acting on AGPase.
Glc-1-P 42.7 13 115 79
Glc-6-P 510 18 1212 177
Fru-6-P 147 12 299 85
Changes in AGPase Expression Are Not Reflected
ATP 292 9 151 39
Rapidly at the Protein Level and Do Not Explain the
ADP 87.3 7 77 47
Inhibition of Starch Synthesis in Wild-Type Tubers
UTP 238 8 121 8
after 1 Day of Detachment
UDP 52.4 3 37.1 4
PPi 12.0 0.7 6.9 2.9
Pi 2400 1120 2200 620 Although the levels of the AGPB and AGPS1 transcripts de-
3PGA 296 43 324 72
creased markedly in the first 24 h after detachment (Figure 4A),
Plastid
this did not lead to a marked change of protein or AGPase ac-
Glc-6-P 379 108 822 155
tivity (Figures 4B and 4C). AGPase protein was determined
Glc-1-P 17.4 6.7 64 22
by preparing extracts from attached and detached tubers in
ADP-Glc 21.5 2 11.2 1
the presence of DTT before SDS-PAGE and protein gel blot
ATP 179 7 279 31
analysis with an antibody raised against the maize AGPB
ADP 149 14 148 27
(brittle-2) protein (Giroux and Hannah, 1994) (Figure 4B). A
PPi 2.36 1.43 0.68 0.68
single band at 50 kD was present at a similar intensity in
Pi 870 350 500 260
extracts from attached and 1-day-detached tubers.
3PGA 198 33 244 95
AGPase activity was measured using a standard assay in
The cytosolic and plastidic metabolite levels, obtained after non-
which DTT was included in the extraction and assay buffer
aqueous fractionation, were divided by the fractional volumes of the
and activity was determined in the presence of saturating
cytosolic (0.606 mL/g dry wt) and plastidic compartments (0.747 mL/g
substrate levels. AGPase activity was unaltered at 1 day af-
dry wt) to derive the respective cytosolic and plastidic concentrations
ter detachment and started to decrease at 3 days after de-
(Farré et al., 2001). Data are means SE (n 3) and are expressed in
M, except for Suc and Fruc, which are expressed in mM. tachment (Figure 4C and data not shown) (Geigenberger et
al., 1994). These results show that the inhibition of starch
synthesis during the first day after tuber detachment is not
the result of changes in AGPase expression.
or even increased slightly after tuber detachment (Table 2).
This finding provides strong biochemical evidence that AGP-
ase is the only site at which significant regulation occurs af- Tuber Detachment Leads to Reversible Oxidation and
Dimerization of AGPase
ter detachment.
In a series of experiments that were performed to produce
heterotetrameric higher plant AGPase in E. coli, Preiss and
co-workers (Iglesias et al., 1993; Ballicora et al., 1995; Fu et
Inhibition of Starch Synthesis in Response to Tuber
al., 1998) found that to obtain high activity it was necessary
Detachment Does Not Involve Changes in the Plastidic
to incubate the overexpressed enzyme with DTT to reduce
Concentrations of 3PGA and Pi
an intermolecular disulfide bond that forms in E. coli at the
We next investigated the possibility that a decrease of the Cys-12 position in the heterologously expressed small sub-
plastidic 3PGA/Pi ratio is responsible for the inhibition of units. This Cys is located in a QTCL motif and corresponds
AGPase activity after detachment. The overall level of 3PGA to Cys-82 of the potato full-length mRNA for AGPB. Break-
increased slightly after detachment (342 and 394 nmol/g dry age of the disulfide bond can be monitored by subjecting
weight in attached and 1-day-detached tubers; data not AGPase to SDS-PAGE in nonreducing conditions and moni-
shown) (Geigenberger et al., 1994). This increase included a toring the disappearance of the dimerized 100-kD band and
slight nonsignificant increase of 3PGA in the plastid (Figure the appearance of the 50-kD monomer (Iglesias et al., 1993;
3G, Table 1). In attached tubers, more than half of the Pi Ballicora et al., 1995; Fu et al., 1998). The intramolecular
was located in the vacuole, 33% was located in the cytosol, disulfide bond also increases the heat stability of the het-
and 14% was located in the plastid. Detachment led to a erotetrameric plant enzyme (Ballicora et al., 1999).
decrease of Pi in the plastid and the cytosol (Figure 3H). The To determine whether analogous changes occur in planta,
Redox Regulation of Starch Synthesis 2197
Table 2. Estimated in Vivo Mass-Action Ratios of Enzyme and Transport Reactions Involved in Suc-to-Starch Conversion
Molar Mass-action Ratio
Reaction Formula Attached Tubers Detached Tubers Theoretical Equilibrium Constant (Keq)
SuSy [Fru] × [UDP-Glc 0.181 0.632 0.15 to 0.56a
-----------------------------------------------]
-
[Suc] × [UDP]
(cytosol)
UGPase [Glc-1-P] × [UTP] 1.48 3.21 3.2b
------------------------------------------------
-
[UDP-Glc] × [PPi]
(cytosol)
Fructokinase [Fru-6-P] × [ADP 0.032 0.085 851c
------------------------------------------------]
-
[Fru] × [ATP]
(cytosol)
Phosphoglucoisomerase [Glc6P] 3.47 4.05 2c
--------------------
-
(cytosol) [Fru6P]
PGM [Glc6P] 11.9 10.5 19c
--------------------
-
(cytosol) [Glc1P]
[Glc6P]pl × [Pi]cyt 2.04
Glc-6-P/Pi 2.97 1d
-----------------------------------------------
[Pi]pl × [Glc6P]cyt
translocator
[3PGA]pl × [Pi]cyt 1.85
3PGA/Pi 3.31 1d
----------------------------------------------
[Pi]pl × [3PGA]cyt
translocator
[ATP]pl × [ADP]cyt 0.359
Adenylate 0.961 1d
-------------------------------------------------
translocator [ADP]pl × [ATP]cyt
PGM [Glc1P] 0.046 0.078 0.053c
--------------------
-
[Glc6P]
(plastid)
AGPase [ADP-Glc] × [PPi 1.6 10 2 4 10 4 1e
-----------------------------------------------]
-
[Glc1P] × [ATP]
(plastid)
Inorganic pyrophosphatase [Pi] × [Pi] 0.323 0.373 1000b
--------------------------
[PPi]
(plastid)
The data from Table 1 were used to calculate the in vivo molar mass-action ratios (ratio of product to substrate concentration) for each reaction.
The theoretical equilibrium constant (ratio of product/substrate concentration) at which the net flux is zero) of each reaction is shown for compar-
ison.
a
Geigenberger et al. (1993).
b
Weiner et al. (1987).
c
Stryer (1990).
d
Estimated value.
e
Kruger (1997).
growing potato tubers were extracted in degassed SDS ex- DTT before SDS-PAGE completely converted the 100-kD
traction buffer, separated immediately by nonreducing SDS- band to the 50-kD band (Figure 5B, cf. lanes 3 and 4 with
PAGE to maintain the in vivo redox status of the enzyme, lanes 1 and 2). Similar results were obtained in repeated ex-
and immunoblotted with antibodies raised against maize or periments over a period of months with separate batches of
potato AGPB. To ensure the specificity of the antibodies, tubers. These results reveal that AGPB is present as a mix-
His-tagged potato AGPB and AGPS were overexpressed in ture of dimer and monomer in growing tubers and becomes
E. coli and purified; their identities were confirmed by ma- completely dimerized when the tubers are detached and
trix-assisted laser-desorption ionization time-of-flight analy- Suc import from the mother plant is interrupted.
sis. Then, they were applied in a concentration series, sepa-
rated by SDS-PAGE, and immunoblotted. The antibody
raised against maize brittle-2 was highly specific for the po- Establishment of a New Protocol to Extract and
tato AGPB subunit (Figure 5A). Measure AGPase Activity Reveals That the Changes in
Extracts from tubers that were attached to the plant gave Redox State Are Accompanied by Changes in Activity
immunopositive signals with the AGPB-specific maize anti- and Kinetic Properties
body at 50 and 100 kD (Figure 5B, lane 1). When 1-day-
detached tubers were analyzed (Figure 5B, lane 2), the 50-kD We next investigated whether AGPase activity is altered when
band disappeared almost completely and the 100-kD band AGPB becomes dimerized in planta. The standard protocol
became more intense. Incubation of the extracts in vitro with (Sowokinos, 1981; Müller-Röber et al., 1992; Ballicora et al.,
2198 The Plant Cell
tocol for the extraction and assay of AGPase by omitting all
redox agents, presaturating all extraction and assay media
with nitrogen, and decreasing the time between tissue dis-
ruption and transfer into the assay to 1 min.
When an extract from growing tubers was prepared in this
way and subjected to nonreducing SDS-PAGE, a mixture of
bands at 50 and 100 kD (Figure 5C, lane 2) was found that
resembled a parallel sample that was extracted in SDS (Fig-
ure 5C, lane 3). Large differences in AGPase activity were
found between extracts from attached tubers and tubers
that had been detached for 1 day (Figure 6). The differences
were stable for at least 10 min in the assay conditions (data
not shown) but were lost rapidly if extract was incubated in
the absence of substrate (data not shown).
For routine determination of AGPase activity, the assay
was performed in the reverse direction (Figures 6A to 6C)
using PGM and Glc-6-P dehydrogenase to couple Glc-1-P
formation in real time to NADP reduction. Detachment led
to a decrease of AGPase activity and the affinity for ADP-
Glc when activity was assayed in the absence of allosteric
effectors (Figure 6A). This inhibition was reversed com-
pletely when 5 mM DTT was included during extraction and
assay (Figure 6A).
Detachment also led to a decrease of activity and the af-
finity for ADP-Glc when activity was assayed in the pres-
ence of 300 M 3PGA and 1200 M Pi (Figure 6B), which lie
in the range of estimated concentrations in amyloplasts (Ta-
ble 1). When assayed with limiting substrate concentrations,
AGPase from detached tubers was strongly dependent on
the presence of 3PGA and was not activated until high con-
centrations of 3PGA were present (Figure 6C). It also was
more sensitive to inhibition by Pi than AGPase extracted
Figure 4. AGPB and AGPS Expression in Attached and 1-Day-
from attached tubers (Figure 6C). Maximum activity in the
Detached Wild-Type Tubers.
presence of high 3PGA and zero Pi was similar for attached
Attached (t0) and 1-day-detached (1 d) tubers were analyzed for
and detached tubers.
steady state mRNA levels of AGPB and AGPS1 (A), AGPase protein
We also investigated whether AGPase activity was modi-
after reducing SDS-PAGE and immunoblot analysis with antiserum
fied when it was assayed in the ADP-Glc producing direc-
raised against the homologous maize brittle-2 protein (Giroux and
tion. A stopped assay was used, in which each individual
Hannah, 1994) (B), and overall activity of AGPase using a standard
sample was analyzed by HPLC to determine ADP-Glc (Fig-
protocol (Müller-Röber et al., 1992) including DTT in the extraction
ures 6D to 6F). Control experiments showed that ADP-Glc
and assay buffers (C). Representative samples are shown in (A) and
was not degraded during the incubation. Detachment led to
(B); in (C), results are means SE (n 4 extracts from separate tu-
a very marked inhibition of activity. The affinity for ATP was
bers). FW, fresh weight.
decreased when assayed in the presence of 200 M 3PGA
(Figure 3D) or 300 M 3PGA plus 1200 M Pi (Figure 6E).
AGPase from detached tubers showed a strongly reduced
1995) for the extraction and assay of AGPase gave only sensitivity to 3PGA activation at physiological concentra-
small and variable differences of activity between extracts tions (Figure 6F).
from attached and detached tubers (data not shown). The To provide a routine microplate-compatible test for
solutions used in this protocol include DTT, and only the 50- changes in redox regulation in subsequent experiments,
kD band was found when aliquots from these extracts were AGPase activity was measured in the ADP-Glc cleaving di-
subjected to nonreducing SDS-PAGE (Figure 5C, lane 1). rection in the presence of 600 M ADP-Glc in the absence
To detect the large changes of activity of redox-modu- (Vsel) or presence of 5 mM DTT (Vred). Similar results were
lated Calvin cycle enzymes that occur when chloroplasts or obtained at a range of ADP-Glc concentrations between
leaves are illuminated or darkened, it was necessary to de- 200 and 1000 M (Figure 6A and data not shown). The ratio
velop very fast assay and extraction procedures (Laing et between the activities in these two assays (Vsel/Vred) is
al., 1981; Wirtz et al., 1982). Therefore, we modified the pro- termed  activation.
Redox Regulation of Starch Synthesis 2199
Incubation of Tuber Discs with DTT to Redox Activate
AGPase Leads to a Stimulation of Starch Synthesis and
a Decrease of Phosphorylated Metabolites
To provide independent evidence that redox modulation of
AGPase can lead to large changes in the rate of starch syn-
thesis, freshly cut discs from attached and 1-day-detached
tubers were incubated for 2 h in medium containing 0, 1, 5,
or 10 mM DTT. To prevent postextraction modification of
AGPase, the discs were washed before extraction to pre-
vent carryover of DTT into the extraction mixture. To mea-
14
sure the rate of starch synthesis, 2 mM C-Glc was in-
cluded in the incubation.
Increasing concentrations of DTT led to a progressive in-
crease of AGPase activation (Figure 7A) and the proportion
of AGPB that was present as a monomer (data not shown).
There was a parallel increase of label incorporation into
starch (Figure 7B). AGPase activation (Figure 7A) and starch
synthesis (Figure 7B) were lower in discs from detached tu-
bers than in discs from attached tubers in the absence of
DTT, and this difference was reversed partly by DTT. When
the results for all of the treatments were combined, a strong
correlation was found between AGPase activation and label
incorporation into starch (Figure 7C).
The levels of hexose phosphates and label in the phos-
phorylated intermediates were measured and used to calcu-
late the specific activity of the hexose phosphate pool and
the absolute rate of starch synthesis. This calculation con-
firmed that there is a strong correlation between AGPase
activation and the rate of starch synthesis (data not shown).
The stimulation of starch synthesis was accompanied by a
marked decrease of the levels of hexose phosphates (Figure
7D) and 3PGA (data not shown). This finding shows that the
Figure 5. Analysis of the Dimerization of AGPase.
stimulation of starch synthesis by DTT is not caused by an
additional action besides redox activation of AGPase, such as
(A) Demonstration of the specificity of the antiserum raised against
a stimulation of Suc breakdown or a restriction of respiration.
the homologous maize brittle-2 protein (Giroux and Hannah, 1994)
for the potato AGPB subunit. Similar amounts of heterologously
overexpressed His-tagged AGPB (B) and AGPS1 (S) proteins were
applied in a concentration series (diluted 1 to 81 times), sepa-
rated by SDS-PAGE in the presence of 4 mM DTT, and immuno-
Incubation of Tuber Discs Leads to a Decrease in
blotted.
AGPase Activation State, Which Is Prevented by Suc
(B) Detachment leads to the dimerization of AGPase. Total protein
was extracted from wild-type tubers, separated by SDS-PAGE, and
Detachment of a tuber interrupts Suc import, leading to a
examined by immunoblot analysis using maize brittle-2 protein anti-
decrease of Suc in the vacuole and the cytosol (Figure 3I). It
body. Samples from attached tubers (t0; lanes 1 and 3) and 1-day-
appeared plausible that changes in the import or level of
detached tubers (1 d; lanes 2 and 4) were prepared under anaerobic
Suc might stimulate the post-translational inactivation of
conditions in SDS and analyzed by SDS-PAGE in the absence of
AGPase. Therefore, experiments were performed to deter-
DTT (lanes 1 and 2) or in the presence of 4 mM DTT (lanes 3 and 4).
Each lane contained protein from 3 mg fresh weight of tuber mine whether redox modulation of potato tuber AGPase
tissue.
(C) Effect of different extraction methods on the dimerization of
AGPase after nonreducing SDS-PAGE and immunoblot analysis
with maize brittle-2 protein antibody. Lane 1, AGPase extracted us-
ing a standard protocol as described by Müller-Röber et al. (1992); tion) with 100 L of argon-treated 2 sample buffer lacking DTT
lane 2, AGPase extracted using a modified protocol omitting DTT and additional supplements. The sample was boiled immediately for
(see Methods); lane 3, tissue extracted in SDS. Electrophoresis sam- 5 min. Every lane contained the proteins originating from 1.5 mg
ples of the different extracts were prepared in an argon atmosphere fresh weight of tuber tissue. Representative sample blots using tu-
by mixing 100 L of the respective extract (directly after its prepara- bers from 8-week-old wild-type plants are shown.
2200 The Plant Cell
Figure 6. Enzyme Kinetics of AGPase in Extracts from Attached or 1-Day-Detached Wild-Type Tubers Prepared Using the Modified Rapid Protocol.
(A) to (C) Reverse assay coupling Glc-1-P formation in real time to NADP reduction.
(A) ADP-Glc saturation kinetics with or without DTT.
(B) ADP-Glc saturation kinetics in the presence of 300 M 3PGA and 1200 M Pi.
(C) 3PGA activation kinetics in the presence of 400 M ADP-Glc with and without 1200 M Pi.
(D) to (F) Forward assay using a stopped assay and determination of ADP-Glc by HPLC.
(D) ATP saturation kinetics in the presence of 200 M 3PGA.
(E) ATP saturation kinetics in the presence of 300 M 3PGA and 1200 M Pi.
(F) 3PGA activation kinetics in the presence of 100 M ATP.
Results are from determinations with an extract from three pooled tubers for each condition. Similar results were obtained with extracts from
other pools of tubers. Closed circles, attached tubers; open circles, 1-day-detached tubers. FW, fresh weight.
contributes to the regulation of starch synthesis in other freshly cut discs were incubated in the absence of Suc, their
conditions in which the supply or level of Suc changes. internal Suc content decreased by 30% during the first 90
Geiger et al. (1998) reported that the addition of Suc to tu- min and then stabilized (Figure 8A). AGPase activation de-
ber discs stimulates starch synthesis and decreases the lev- creased between 30 and 120 min (Figure 8A). Inclusion of
els of glycolytic intermediate. In our experiments, when 200 mM Suc in the medium prevented the decrease of the
Redox Regulation of Starch Synthesis 2201
internal Suc pool (Figure 8B) and the decrease of AGPase
14
activation (Figure 8C). In a parallel experiment, 2 mM C-
Glc was provided to tuber slices in the absence and pres-
ence of 200 mM Suc. The increase in AGPase activation in
the presence of 200 mM Suc was accompanied by a 50%
stimulation of label incorporation into starch and a decrease
of hexose phosphates (Table 3).
Inhibition of Starch Synthesis in Transgenic Tubers
Overexpressing Suc Phosphorylase Involves the
Inactivation of AGPase
In the second approach, heterologous expression of bacte-
rial Suc phosphorylase in the cytosol was used as a tool to
decrease Suc levels in planta (Trethewey et al., 2001). Suc
phosphorylase converts Suc and Pi to Fru and Glc-1-P. This
resembles the endogenous pathway for Suc breakdown via
SuSy and UGPase in not producing Glc but differs because
it does not require PPi or produce UDP-Glc as an intermedi-
ate. Critically, Suc phosphorylase has a far lower Km(Suc)
( 1 mM; Silverstein et al., 1967) than SuSy (40 to 200 mM;
Avigad, 1982).
Introduction of Suc phosphorylase led to a threefold to
fourfold decrease of Suc (Figure 9A). Glc did not change
(data not shown) (Trethewey et al., 2001), UDP-Glc de-
creased slightly, PPi showed a slight and nonsignificant in-
crease (data not shown), starch decreased (Figure 9B), and
hexose phosphates (Figure 9C) and 3PGA (data not shown)
(Trethewey et al., 2001) increased markedly. The inhibition
of starch synthesis in the transformants was accompanied
by a 30 to 50% decrease of overall AGPase activity (Figure
9D). There also was a marked decrease of the AGPase acti-
vation state (Figure 9E) and a marked decrease in the inten-
sity of the 50-kD immunosignal relative to the 100-kD signal
in nonreductive SDS-PAGE (Figure 9F).
Redox Inactivation of AGPase Correlates with the
Decrease in Suc and the Inhibition of Starch
Accumulation as Potato Plants Age
The experiments shown in Figures 2 to 9 used tubers from
8-week-old plants, which contain high levels of Suc. A third
mM (inverted triangles) DTT. Results are means SE; n 5 sepa-
rate incubations with discs from separate tubers.
Figure 7. Redox Inactivation of AGPase and Inhibition of Starch
(A) AGPase redox activation state (Vsel/Vred).
Synthesis Both Can Be Reversed in Vivo by External Feeding of DTT
(B) Labeling of starch after incubation of discs in 2 mM 14C-Glc (spe-
to Wild-Type Tuber Discs for 2 h.
cific activity of 18.5 kBq/ mol).
Discs were taken from attached tubers (black bars, black symbols) (C) Starch labeling in relation to AGPase activation.
or 1-day-detached tubers (gray bars, gray symbols) and were incu- (D) Hexose phosphate levels relative to AGPase activation. FW,
bated with 0 mM (circles), 1 mM (squares), 5 mM (triangles), or 10 fresh weight.
2202 The Plant Cell
approach investigated changes in wild-type tubers as the
mother plant ages. Tuber starch levels increased on a fresh
weight basis between 6 and 10 weeks but did not increase
further between 10 and 12 weeks (Figure 10A). Suc levels
decreased slightly between 6 and 8 weeks and decreased
markedly at 10 and 12 weeks (Figure 10B) (Mares and
Marschner, 1980; Merlo et al., 1993). Hexose phosphate levels
increased between 10 and 12 weeks, revealing that starch
synthesis is inhibited more strongly than Suc breakdown
(Figure 10C). As shown previously (Mares and Marschner,
1980; Merlo et al., 1993), SuSy activity decreased sharply
(Figure 10D) and overall AGPase activity decreased mark-
edly (assayed with DTT; Figure 10E) as the plant aged.
There also was a marked decrease of AGPase activation
(Figure 10F).
DISCUSSION
A Novel Mechanism Involving Reductive
Post-Translational Regulation of AGPase Inhibits
Starch Synthesis after Detachment of Tubers from
the Mother Plant
The starting point for the experiments presented here was
the observation (Geigenberger et al., 1994) that removal of
growing potato tubers from the mother plant leads within 1
day to an inhibition of starch synthesis that cannot be ex-
plained easily by allosteric regulation or by changes in the
expression of AGPase. This led us to suspect that there
might be a major gap in our understanding of the regulation
of starch synthesis.
The first step in identifying the missing regulatory mecha-
nism was to determine the step(s) at which flux was being
inhibited. Substitution of higher plant AGPase with a heter-
ologous bacterial AGPase provided genetic evidence that
the inhibitory mechanism required the presence of native
potato AGPase (Figure 2). To provide biochemical evidence
that AGPase is the unique site at which flux is regulated, we
performed a systematic investigation of the subcellular lev-
els of every metabolite in the pathway between Suc and
starch. This allowed the mass-action ratio (the product/sub-
strate ratio) to be estimated for every enzyme and transport
Figure 8. Redox Inactivation of AGPase in Wild-Type Tuber Discs Is
Prevented by External Feeding of Suc.
(A) Decrease in Suc content (gray circles) and AGPase activation tent or frozen directly in liquid nitrogen to measure AGPase activity
state (black circles) after cutting and incubating discs in buffer solu- under selective (Vsel; DTT) and reductive (Vred; DTT) assay con-
tion in the absence of sugars for up to 4 h. ditions to calculate the activation state of the enzyme (Vsel/Vred).
(B) and (C) Suc levels (B) and AGPase redox activation (C) in discs Suc levels and AGPase activation in tuber slices directly after cutting
incubated for 2 h with no sugars (gray bars) or 200 mM Suc (black (white bars) are shown for comparison.
bars). After 2 h of incubation, subsamples of the discs were either Results are means SE; n 4 replicates from separate tubers. FW,
washed three times with Mes buffer before analyzing their Suc con- fresh weight.
Redox Regulation of Starch Synthesis 2203
step between Suc in the cytosol and starch synthesis in the
Table 3. Suc Feeding Leads to Increased AGPase Redox
plastid. AGPase was the only step at which this ratio de-
Activation, Increased Starch Synthesis, and Decreased Hexose
creased (by 2 orders of magnitude) after detachment of the
Phosphate Levels in Discs from Growing Tubers
tubers (Table 2). These genetic and biochemical experi-
200 mM
ments demonstrate (1) that the regulatory mechanism acts
Parameter Control Suc
at AGPase and (2) that AGPase is the sole step involved in
the inhibition of starch synthesis after tuber detachment. Suc content ( mol/g fresh wt) 11.6 0.4 22.2 1.4
AGPase activation [Vsel/Vred] (%) 33 2 49 3
The second step was to exclude the possibility that known
Starch synthesis (% of total 14C absorbed) 40 3 59 3
mechanisms are responsible for the inhibition of AGPase ac-
Hexose phosphate level (nmol/g fresh wt) 225 20 148 11
tivity. Measurements of 3PGA and Pi levels in the plastid
showed that the 3PGA/Pi ratio increased after detachment of Tuber discs were cut from an intact growing tuber and incubated in
tubers. Although there was a marked decrease in the tran- buffer with and without 200 mM Suc for 2 h before they were either
washed three times to analyze their Suc content or frozen immedi-
script levels for AGPB and AGPS1, there were no changes of
ately in liquid nitrogen to measure hexose phosphate levels and AGP-
AGPB protein or AGPase activity. AGPase activity also changes
ase activity under selective (Vsel; DTT) and reductive (Vred; DTT)
much more slowly than transcript levels during the photo-
assay conditions. In parallel samples, 2 mM 14C-Glc kBq/ mol spe-
period in Arabidopsis leaves (Sokolov et al., 1998) and po-
cific activity of 18.5) was provided to measure label incorporation
tato tubers (Geigenberger and Stitt, 2000). Furthermore, stud-
into starch. Results are means SE (n 3 replicate measurements
ies with antisense AGP transformants have shown that large
on different discs from the same tuber).
changes in the levels of AGP transcripts are required to pro-
duce a significant change in AGPase activity (Müller-Röber et
al., 1992) and that quite large changes of AGPase activity are
required to produce a significant inhibition of starch synthesis
sis after detachment of tubers would require the production
(Müller-Röber et al., 1992; Geigenberger et al., 1999a).
of transformants in which the native AGPB is replaced by a
The last step was to identify the missing mechanism.
modified form of AGPB in which Cys-82 is modified to pre-
Since Sowokinos (1981) found that potato AGPase activity
vent the formation of the intermolecular bridge. This experi-
is stimulated by DTT, it has been included routinely in the
ment would require a mutant line in which transcription of
extraction and assay media. An important clue was pro-
the native AGPB is blocked to allow effective overexpres-
vided by in vitro experiments by Preiss and co-workers in
sion without cosuppression of the modified construct. At
which they found that when the higher plant heterotet-
present, this is not possible in potato.
rameric AGPase is overexpressed heterologously in E. coli,
it is inactivated partly by an intermolecular disulfide bond
that forms between Cys residues in the N-terminal regions
Redox Modulation of AGPase Allows Starch Synthesis
of the AGPB subunits (Iglesias et al., 1993; Ballicora et al.,
to Be Increased, Whereas the Levels of Phosphorylated
1995; Fu et al., 1998). Following up this clue, we investi-
Metabolites Decrease
gated the biochemical and kinetic properties of AGPase ex-
tracted from tubers in the absence of DTT using methods
The dramatic changes in the kinetic properties of AGPase
adapted to allow rapid extraction in the absence of oxygen.
produced by redox modulation explain why starch synthesis
We found that AGPB exists as a mixture of monomers
is inhibited after tuber detachment even though there are
and dimers in growing tubers and is converted almost com- changes in the levels of substrates and allosteric effectors
pletely to dimers in detached tubers. Furthermore, dimeriza- that would otherwise strongly stimulate starch synthesis. To
tion led to inactivation, as a result of a strong decrease of
provide independent biochemical evidence that redox acti-
the substrate affinity, an increased requirement for and de- vation allows the rate of starch synthesis to be increased in
creased sensitivity to activation by 3PGA, and an increased
planta in the face of diametrically opposed changes in the
sensitivity to inhibition by Pi. The change in the kinetic prop- levels of substrates and metabolite effectors, discs were
erties was especially dramatic when AGPase activity was
prepared from tubers and incubated with DTT to achieve an
assayed in the forward reaction. Dimerization and inactiva- artificial activation of AGPase. This led to a strong stimula-
tion both were reversed by incubating extracts with DTT.
tion of starch synthesis and a marked decrease in the levels
We interpret these results as evidence that the redox
of phosphorylated metabolites.
changes identified by Preiss and co-workers in heterolo-
gously overexpressed AGPase operate as a regulatory
mechanism in planta. However, biochemical analyses of the Post-Translational Redox Modulation of AGPase Also
position of the Cys bridges in AGPase holoenzyme isolated Makes an Important Contribution to the Regulation of
from plants is needed to confirm this and to exclude the Starch Synthesis in Tubers in Other Conditions
possibility that other intermolecular or intramolecular Cys
bridges also form in planta. Final genetic proof that this Other situations have been reported in which the rate of
mechanism is responsible for the inhibition of starch synthe- starch synthesis in potato tubers changes independently
2204 The Plant Cell
Figure 9. Redox Activation State and Dimerization Degree of AGPase in Transgenic Tubers Expressing a Heterologous Suc Phosphorylase in
the Cytosol.
Tubers of 8-week-old wild-type plants and three independent transgenic lines were analyzed for Suc content (A), starch (B), hexose phos-
phates (C), overall AGPase activity when DTT was included in the assay (Vred) (D), redox activation state of AGPase (Vsel/Vred) (E), and
dimerization of AGPB protein in nonreductive SDS-PAGE using the maize brittle-2 antibody (F). Each lane contains the proteins originating
from 1 mg fresh weight. Results are means SE; n 3 tubers from different plants, except for (F), which documents a representative exam-
ple. FW, fresh weight.
of overall AGPase activity and reciprocally to the changes Suc phosphorylase is overexpressed in growing tubers
in the levels of phosphorylated intermediates. When Suc (Trethewey et al., 2001), starch synthesis is inhibited even
is supplied to potato tuber slices (Geiger et al., 1998), though phosphorylated intermediates increase and AGPase
starch synthesis is stimulated even though phosphory- activity decreases only slightly. The inhibition of starch
lated intermediates, including 3PGA, decrease. When synthesis in tubers as the mother plant ages is not linked
Redox Regulation of Starch Synthesis 2205
to a general decrease of phosphorylated metabolites
Post-Translational Redox Modulation of AGPase
(Merlo et al., 1993).
Represents a Component in a Novel Regulatory
These results can be explained by post-translational re- Pathway That May Link the Rate of Starch
dox modulation of AGPase. Feeding Suc to discs led to in- Synthesis to Suc Supply
creased activation of AGPase and stimulated the rate of
starch synthesis (Figure 8, Table 3), the heterologous ex- Although the evidence from the individual experiments is
pression of Suc phosphorylase led to redox inactivation of circumstantial, together, our findings indicate that post-
AGPase and a lower tuber starch content (Figure 9), and the translational redox regulation of AGPase is part of a novel
gradual changes in the sink-source balance as the mother regulatory loop that links the rate of starch synthesis to
plant aged led to a decrease of AGPase activation. In the changes in the Suc supply. Detachment of tubers led to a
latter two cases, there also was a 30 to 40% decrease of sudden interruption of phloem import, and aging of the
overall AGPase activity, but this alone (see above) is not mother plant led to a gradual change in sink-source rela-
large enough to explain the increase in hexose phosphates tions. In both cases, the decreased rate of Suc import led to
and the decrease in the rate of starch synthesis. a decrease in the tuber Suc content (Figures 2D, 3I, and
Figure 10. Redox Activation State of AGPase in Tubers from Wild-Type Plants of Different Developmental Stages.
Tubers were harvested from 6-, 8-, 10-, and 12-week-old plants and analyzed for starch levels (A), Suc levels (B), hexose phosphate levels (C),
SuSy activity (D), AGPase activity (Vred) (E), and redox activation state of AGPase (Vsel/Vred) (F). Results are means SE; n 3. FW, fresh
weight.
2206 The Plant Cell
10B) (Merlo et al., 1993), which correlates with the redox in- Candidate Components of Signal Transduction That
activation of AGPase. In these two treatments, of course,
Lead to the Redox Modulation of AGPase Have Yet to
the import of other compounds as well as Suc was inter- Be Identified and Confirmed
rupted.
When tuber discs were incubated in the absence of Suc, The light-dependent redox modulation of Calvin cycle en-
the internal Suc level of the discs decreased and there was zymes and enzymes involved in ATP synthesis and NADPH
a decrease of AGPase activation that could be reversed by export in chloroplasts is mediated by thioredoxin f and
the addition of Suc (Figure 9, Table 3). Overexpression of thioredoxin m, respectively (Schümann and Jacquot, 2000).
Suc phosphorylase to alter the Suc level and the precise Photosynthetic electron transport leads to a reduction of
way in which Suc was metabolized in the tuber also led to ferredoxin, and reducing groups then are transferred by
redox inactivation of AGPase (Trethewey et al., 2001) (Figure ferredoxin:thioredoxin reductase to the thioredoxins, which
10). Furthermore, in an antisense AGPB transformant line in react with their targets. Oxidation is thought to occur pri-
which AGPase activity was reduced by 40 to 50%, there marily by a reversal of this process. The reaction between
was an increase of the tuber Suc level (Geigenberger et al., the thioredoxins and their individual targets is modulated
2000) and a decrease in the dimerization and an increase of by changes in pH, Mg2 , and the levels of their substrates
the activation state of AGPase (data not shown). or products, which alter the mid-redox potential of the
Figure 11 summarizes the data on tuber Suc content and Cys groups on enzymes (Scheibe, 1991; Schümann and
AGPase activation from these different experiments, which Jacquot, 2000).
were performed over a period of 2 years with many separate Ballicora et al. (2000) showed in in vitro experiments with
batches of plants grown under different conditions. By com- heterologously overexpressed AGPase that DTT can be re-
paring the two parameters, a correlation coefficient of 0.85 placed by thioredoxin f isolated from spinach. More studies
was found. These results provide very strong correlative ev- are needed to identify which thioredoxin(s) interacts with
idence that post-translational redox modulation of AGPase AGPase in planta and to determine how the flow of elec-
responds to changes in Suc availability, although it is not trons to AGPase is regulated. Suc may lead to a large in-
possible at this time to determine whether a specific influx, a crease in the redox state of the plastid or, alternatively, may
particular Suc pool, or changes in related metabolites pro- act in some way to modulate the transfer of electrons from
vide the immediate stimulus for the transduction pathway. NADPH over thioredoxin to AGPase.
Figure 11. Correlation between Tuber Suc Level and AGPase Redox Activation.
Data were taken from Figures 8B and 8C and Table 3 (feeding to tuber discs), Figures 9A and 9D (Suc phosphorylase expressing tubers), and
Figures 10B and 10F (aging of mother plant). Data from another tuber detachment experiment with wild-type plants and from transgenic tubers
with decreased expression of AGPase are included (means SE; n 4). FW, fresh weight.
Redox Regulation of Starch Synthesis 2207
Finally, our results raise the question of whether the post- rapidly under the standard methods used to extract and an-
translational regulation of AGPase contributes to the regula- alyze AGPase protein and activity. Figure 12 presents a
tion of starch synthesis in other organs. Two lines of evi- model showing how this novel mechanism combines with
dence indicate that this may be the case. First, anomalies known mechanisms to coordinate AGPase activity, forming
similar to those that prompted our investigations on tubers a network that allows starch synthesis in potato tubers to
have been reported for other tissues. Starch synthesis respond across a range of time scales to a variety of physio-
changed independently of overall AGPase activity and the logical and environmental stimuli.
levels of phosphorylated intermediates after phloem trans- Allosteric control by 3PGA and Pi operates in a time frame of
port was inhibited by detaching cotyledons of germinating seconds to stimulate starch synthesis when phosphorylated
Ricinus seedlings (Geigenberger and Stitt, 1991), when
spinach leaves were cold-girdled to decrease export (Krapp
and Stitt, 1995), when sugars were supplied to detached
spinach leaves (Krapp et al., 1991) or heterotrophic Che-
nopodium rubrum suspension cells (Hatzfeld et al., 1990),
and in leaves of transgenic tobacco plants when phloem
transport was inhibited by the phloem-specific expression
of E. coli pyrophosphatase (Geigenberger et al., 1996). In-
triguingly, these all involve manipulations that were shown
to or are likely to alter sugar export or sugar levels.
Second, the QTCL motif is conserved in the N-terminal re-
gion of almost all sequenced AGPB genes of dicots and in
some of the sequences available for monocots. Interest-
ingly, the QTCL motif is absent in the major AGPB transcript
in cereal endosperm (Thorbjornsen et al., 1996; Hannah et
al., 2001), which presumably encodes the cytosolic enzyme.
This finding indicates that other mechanisms may be re-
quired to link starch synthesis to the Suc supply during the
late stages of cereal grain filling.
The QTCL motif is absent in E. coli AGPase (glgC) and in
cyanobacterial AGPase and also is absent in the small sub-
unit of the heterotetrameric Chlamydomonas reinhardtii AGP-
ase (STA6) (Zabawinski et al., 2001), indicating that this
post-translational regulatory mechanism was developed
during the evolution of higher plants, possibly in parallel with
the evolution of the use of Suc as a transport metabolite and
of thioredoxin function in photosynthetic eukaryotes.
This comparison also implies that it may be necessary to
reevaluate the interpretation of experiments in which the
overexpression of glgC16 in higher plants was shown to
Figure 12. AGPase Is a Key Enzyme for the Regulation of Starch
lead to a strong stimulation of starch synthesis (Stark et al.,
Synthesis in Potato Tubers.
1992). This stimulation has been taken to date as evidence
Redox modulation of AGPase provides a novel mechanism that
for the crucial role of the allosteric properties of AGPase in
combines with known mechanisms to coordinate AGPase activity in
the regulation of starch synthesis. However, it is likely that
a network that allows starch synthesis to respond across a range of
this genetic switch also interrupts the post-translational re-
time scales to a variety of physiological and environmental stimuli.
dox inactivation of AGPase. The tuber detachment experi- Allosteric control by 3PGA and Pi operates in a time frame of sec-
ments with the double-transformed AF1 lines expressing an onds to adjust the rate of starch synthesis to the balance between
AGPase but lacking the QTCL motif and lacking redox mod- Suc breakdown and respiration. Post-translational redox modulation
leads to changes in AGPase activity in a time frame of 30 to 60
ulation suggest this possibility.
min. Activation occurs in response to factors directly or indirectly re-
lated to increased Suc availability and leads to stimulation of starch
synthesis and decreased glycolytic metabolite levels. The signaling
Regulation of AGPase at the Level of Expression,
components leading to redox modulation of AGPase are unknown
Post-Translational Regulation, and Allosteric Regulation
and may involve thioredoxins as well as putative sugar sensors.
Transcriptional regulation in response to changes of Suc allows
In conclusion, our results show that the redox modulation of
more gradual changes in AGPase activity, which can require days to
AGPase provides a powerful mechanism to regulate starch
develop. ABA, abscisic acid; ABI, abscisic acid insensitive; ISI, in-
synthesis. It was overlooked until now because it is reversed sensitive for Suc induction; SNF1, Suc non-fermenting1.
2208 The Plant Cell
many). The generation and characterization of transgenic potato
intermediates increase and to inhibit it when they decrease.
plants expressing antisense ADP-Glc pyrophosphorylase (AGPase),
Existing physiological data indicate that allosteric control
a heterologous gene from a bacterial mutated AGPase enzyme
does not play a major role in coordinating the rate of starch
(glgC16 from Escherichia coli), and a bacterial Suc phosphorylase in
synthesis with the Suc supply. Instead, it adjusts the rate of
the cytosol of their tubers were described by Müller-Röber et al.
starch synthesis to the balance between Suc breakdown
(1992), Lloyd et al. (1999), and Trethewey et al. (2001), respectively.
and carbon use. Conditions in which changes in the 3PGA/
Potato plants were grown in a growth chamber (350 mol·m 2·s 1
Pi ratio correlate with starch synthesis include high temper-
irradiance at 20 C and 50% RH) under a 14-h-light/10-h-dark regime
ature, during which an increasing rate of respiration leads to
in 3-L plastic pots in soil supplemented with Hakaphos grün (100 g
a depletion of phosphorylated intermediates and an inhibi-
per 230 L of soil; BASF, Ludwigshafen, Germany) and were watered
tion of starch synthesis (Geigenberger et al., 1998a), and daily from the top with tap water, or they were grown in a greenhouse
mild water stress, during which post-transcriptional activa- during the summer (16-h-light/8-h-dark regime at 20/18 C day/night
and 60% RH) with supplementing light (62 400-W AgroSonT lamps
tion of Suc phosphate synthase stimulates the resynthesis
per 100 m2) in 20-cm-diameter plastic pots with a 2:1 mixture of soil
of Suc and leads to a decrease of phosphorylated interme-
to sand and were irrigated daily with water ( 200 mL per plant per
diates and an inhibition of starch synthesis (Geigenberger
day) containing nutrients (0.7 g/L Hakaphos rot [8% N, 12% P, 24% K,
et al., 1997, 1999b).
and 4% Mg]).
Post-translational redox modulation leads to changes of
activity in a time frame of 30 to 60 min. Activation occurs
in responses to factors related directly or indirectly to an in-
Reagents
crease in Suc availability and leads to a stimulation of starch
synthesis and a decrease in the levels of phosphorylated in- Enzymes, reagents, and kits for molecular biology were purchased
from Roche (Mannheim, Germany), MBI Fermentas (St. Leon-Rot,
termediates when Suc increases. This could have two im-
Germany), Qiagen (Hilden, Germany), and Stratagene (Heidelberg,
portant consequences.
Germany). Biochemical enzymes were purchased from Roche and
First, lower levels of phosphorylated intermediates will fa-
Sigma (München, Germany), chemicals were purchased from Roche,
vor Suc breakdown via the reversible reactions catalyzed by
Merck (Darmstadt, Germany), and Sigma, and reagents for SDS-
SuSy and UGPase (Geigenberger and Stitt, 1993), will re-
PAGE were purchased from Bio-Rad (München). Radiochemicals
lieve feedback inhibition of fructokinase by Fru-6-P (Renz
and x-ray films were purchased from Amersham-Buchler (Braun-
and Stitt, 1993), and will decrease Suc resynthesis by inhib-
schweig, Germany). If not stated otherwise, enzymes were grade II
iting Suc phosphate synthase (Geigenberger et al., 1999b).
and chemicals were of analytical purity.
Second, stimulation of starch synthesis via a mechanism
that simultaneously decreases glycolytic intermediate levels
Tuber Detachment Experiments
may channel Suc toward starch without this leading to a
stimulation of glycolysis and respiration. This is important
Tuber detachment experiments were performed as described by
because internal oxygen concentrations decrease to very
Geigenberger et al. (1994) and were started in the second half of the
low levels during tuber bulking (Geigenberger et al., 2000),
light period (6 to 8 h into the photoperiod) using 8- to 9-week-old
and it is important to increase the flow of carbon from Suc
plants. Tubers of these plants contained high Suc synthase activity,
to starch without a large increase in oxygen consumption.
which is indicative of rapidly growing tubers.
Transcriptional regulation in response to changes in Suc al-
lows more gradual changes in AGPase activity that may re-
Sampling of Potato Tuber Tissue
quire days to develop. For example, during diurnal rhythms
(Geigenberger and Stitt, 2000) and after detachment of tubers
Immediately after harvesting the tuber, a cylinder was cut perpendic-
(Figure 4), the levels of the AGPS and AGPB transcripts
ular to the stolon-apex axis. Tuber slices that were incubated subse-
change within 12 to 24 h, but AGPase activity remains high
quently with radioactive Glc were cut (8 mm diameter, thickness of 2
for several days (Geigenberger et al., 1994). Changes in tran-
mm) and preincubated in 10 mM Mes-KOH, pH 6.5, for 10 to 20 min.
scription also may be responsible for the gradual decrease of
Tuber tissues sampled for direct analysis or for analysis after non-
overall AGPase activity on tubers as the mother plants age
aqueous fractionation were sliced immediately into liquid nitrogen
(Figure 10) and the slightly lower expression of AGPase in tu- and stored at 80 C until use. Metabolic gradients have been re-
ported for potato tubers along the stolon-apex axis (Merlo et al.,
bers that express Suc phosphorylase (Figure 9).
1993) and across the tuber (Geigenberger et al., 2000). Therefore, tu-
ber slices were taken from the middle of the tuber, avoiding the outer
3 mm and the tuber skin.
METHODS
Labeling Experiments with Intact Tubers
Plant Material and Growth Conditions
Radioactive labeling experiments were performed essentially as de-
Wild-type potato plants (Solanum tuberosum cv Desiree) were ob- scribed by Geigenberger et al. (1994). To measure carbon fluxes in
tained originally from Saatzucht Fritz Lange (Bad Schwartau, Ger- whole tubers, a 1-mm borehole was made (perpendicular to the sto-
Redox Regulation of Starch Synthesis 2209
lon-apex axis) into intact or detached tubers using a hypodermic concentrations in each compartment (vacuole, 3.384 mL/g dry
needle. Radioactive Glc (U-14C-Glc; specific activity of 11.5 GBq/ weight; plastid, 0.747 mL/g dry weight; cytosol, 0.655 mL/g dry
mmol) was injected into the borehole, the end was sealed, and after weight). There were no significant changes in subcellular volumes af-
1 h, a concentric cylinder of 8 mm diameter surrounding the injection ter detachment (data not shown).
hole was cut and frozen in liquid nitrogen. The frozen tissue was For each gradient, an aliquot of the initial homogenate was taken
weighed before extraction. to determine the recovery values of enzymes applied to the gradient
and to determine the dry weight of the potato tissue. The recoveries
of enzymes were 72, 82, 89, 78, and 81% for mannosidase, AGPase,
Labeling Experiments with Tuber Slices
UDP-Glc pyrophosphorylase, pyrophosphate:Fru-6-P1 phospho-
transferase, and citrate synthase, respectively. Analysis of starch lev-
A cylinder was cut perpendicular to the stolon-apex axis to prepare
els in the different fractions revealed that starch is 100% located in
slices (8-mm diameter, thickness of 2 mm) from intact or detached
the plastids. As has been documented for leaves (Stitt et al., 1989),
tubers. Slices were washed briefly in 10 mM Mes-KOH, pH 6.5, blot-
the distribution of enzymes tracks the distribution of metabolites that
ted with tissue paper, and then incubated for 30 min or 2 h at 22 C in
are known to be restricted to a particular compartment.
4 mL of incubation buffer containing 2 mM radioactive U-14C-Glc
(18.5 kBq/ mol) or U-14C-Suc (18.5 kBq/ mol) in Erlenmeyer flasks
shaken at 90 rpm to maintain aerobic conditions. The incubation Metabolite Analysis
times, buffer compositions, and labeled compounds are specified in
the figure legends. For each treatment, 10 slices from four different Tuber material was extracted either directly or after subcellular frac-
tubers were used (2 slices per replicate sample). After incubation, the tionation (see above) in trichloroacetic acid as described by Weiner
discs were washed (three times for 30 s each) in nonlabeled incuba- et al. (1987) and Jellito et al. (1992); the reliability of the method has
tion buffer, blotted briefly with paper, and frozen in liquid nitrogen. been documented by Jellito et al. (1992). Nucleotides were mea-
sured by HPLC according to Geigenberger et al. (1997) using a Par-
tisil-10-SAX ion exchange column (4.6 mm 250 mm; Whatman)
Fractionation of 14C-Labeled Potato Tissue
and detection at 254 and 230 nm. The nucleotides were identified by
comparison of the elution times of authentic standards and the 254/
Tuber material was extracted with 80% (v/v) ethanol at 80 C (1 mL/
230-nm ratio of absorbance. The metabolite amount was calculated
0.1 g fresh weight) and reextracted in two subsequent steps with
from the peak area using Datasystem 450 MT2 software (Kontron,
50% (v/v) ethanol (1 mL/0.1 g fresh weight at each step). The com-
Munich, Germany).
bined supernatants were dried under an air stream at 40 C, taken up
The levels of Glc-1-P, Glc-6-P, Fru-6-P, UDP-Glc, glycerate-
in 1 mL of water, and separated into ionic (phosphate esters, organic
3-phosphate (3PGA), Pi, Suc, and starch were measured as described
acids, and amino acids) and neutral (Suc, Glc, and Fru) components
by Geigenberger et al. (1998b). Inorganic pyrophosphate (PPi) and
by ion exchange and thin layer chromatography as described by
ADP-Glc were measured in the 96-well format by enzymatic cycling
Geigenberger et al. (1997). The residue was analyzed for label in
assays (Gibon et al., 2002), except that arsenolysis was not used in
starch as described by Merlo et al. (1993).
the case of PPi determination. Instead, aliquots of the samples were
preincubated for 30 min in the presence of glycerokinase, glycerol-
3-phosphate oxidase, and glycerol and then heated at 95 C for 20 min
Nonaqueous Fractionation of Tuber Tissue
before determination of PPi, to decrease the amount of interfering
metabolites.
Potato tuber tissue was fractionated as described by Farré et al.
(2001), except that (1) five fractions (termed pellet, 0, 1, 2, and 3)
were collected from the nonaqueous density gradients and (2) the
Analysis of Overall Enzyme Activities in Potato Tubers
mass of the dried total aliquot was determined to allow the results to
be expressed per gram dry weight. The subcellular distribution of
The activities of Suc synthase and AGPase were measured in de-
each metabolite was estimated from the distribution of marker en-
salted tuber extracts as described by Merlo et al. (1993). As detailed
zymes and the metabolite between the gradient fractions, based on
below, AGPase also was measured using a newly developed protocol.
equations given by Stitt et al. (1989). The equations were solved to a
best fit by the method of least-squares values using an algorithm
based on the solver function incorporated into Excel version 5.0 (Mi- Analysis of AGPase Activity and Kinetic Properties
crosoft, Redmond, WA). This allowed direct and rapid calculation
and evaluation on one spreadsheet using recovery values (percent- A special extraction and assay protocol was developed to reveal the
age of the value found in the total aliquot) of marker enzymes and of in planta AGPase activity and redox activation state. The extraction
metabolites to make the calculations and regressions for subcellular buffer (50 mM Hepes-KOH, pH 7.8, and 5 mM MgCl2) was degassed
compartmentation. It also allowed more efficient detection of ana- by bubbling with nitrogen. Tuber discs were prepared rapidly (see
lytic errors than earlier methods. above) and frozen in liquid nitrogen. The discs ( 100 mg fresh
By using a higher number of fractions (five) than the number of an- weight) were homogenized to a powder under liquid nitrogen, ex-
alyzed compartments (three), the equation system is overdeter- tracted with 1 mL of extraction buffer, and centrifuged for 30 s at
mined, making the results for subcellular distribution more reliable 10,000g at 4 C; 10 L of the supernatant was used for the AGPase
and robust. Even when the data for one fraction is deleted, the re- assay. The entire procedure lasted 2 min. AGPase activity was fol-
gressions can predict a reliable value (data not shown). Estimates for lowed on line and was linear for up to 30 min. The 96-well format al-
the subcellular volumes of growing potato tuber tissue derived from lowed us to perform several comparative measurements of different
planimetry (Farré et al., 2001) were used to calculate the metabolite samples at the same time.
2210 The Plant Cell
AGPase assay in the pyrophosphorolysis direction was performed (Bio-Rad). Peroxidase activity was detected on x-ray film by en-
at 30 C in a total volume of 200 L containing 50 mM Hepes-KOH, hanced chemiluminescence.
pH 7.8, 5 mM MgCl2, 10 M Glc-1,6-bisP, 0.6 mM NADP , 2.5 mM
Na-PPi, 1 unit/mL phosphoglucomutase (from rabbit muscle), 2.5
units/mL Glc-6-P dehydrogenase (from yeast), and varying concen- Heterologous Expression of Potato AGPB and AGPS1 in E. coli
trations of ADP-Glc (0 to 1 mM) with or without 5 mM DTT. The assay
The potato sequences of the presumed mature proteins of AGPB
lacking DTT was termed Vsel, and the assay containing 5 mM DTT
(complete SWISS-PROT entry P23509) and AGPS1 (starting at
was termed Vred.
amino acid 41 of SWISS-PROT entry Q00081) were cloned into the
The reaction was started by adding freshly prepared extract. Par-
vector pQE (Qiagen), causing the encoding of the N-terminal exten-
allel assays were performed lacking either Na-PPi or ADP-Glc. Blank
sion MRGSHHHHHHGSVD. The proteins were expressed in E. coli
activity was negligible for at least 40 min and was subtracted when
and purified under denaturing conditions on a nickel nitrilotriacetic
detected. To analyze the kinetic properties of AGPase, the activator
acid agarose column. The resulting preparations were 90 and
3PGA or the inhibitor Pi was included in the assay mixtures as indi-
50% pure for AGPB and AGPS1, respectively. The identities of the
cated in the figure legends. To redox activate AGPase, extract was
purified preparations were confirmed by matrix-assisted laser-desorp-
preincubated for 10 min at 4 C by adding 1 mM ADP-Glc and 5 mM
tion ionization time-of-flight analysis of peptides, generated by in-gel
DTT before transfer to the Vred assay. The ratio of Vsel to Vred of the
digestion with trypsin (Walz et al., 2002), before they were used to
phosphorolysis reaction at 600 M ADP-Glc and without Pi and
determine the specificity of the maize brittle-2 and potato AGPB an-
3PGA was termed the activation state of AGPase.
tibodies.
AGPase activity in the ADP-Glc forming direction was analyzed
Upon request, all novel materials described in this article will be
in a stopped assay. The assay contained 50 mM Hepes-KOH, pH
made available in a timely manner for noncommercial research pur-
7.8, 5 mM MgCl2, 1.5 mM Glc-1-P, and 0 to 3 mM ATP in a total vol-
poses. No restrictions or conditions will be placed on the use of any
ume of 200 L. The assay was started by the addition of 10 L of
materials described in this article that would limit their use for non-
extract (see above), incubated at 30 C for 15 min with shaking at
commercial research purposes.
900 rpm, and stopped by incubating at 95 C for 5 min. The allo-
steric activator 3PGA or the inhibitor Pi was included in the assays
as indicated in the figure legends. The stopped assays were kept at
4 C until quantification. ADP-Glc was quantified via HPLC using a ACKNOWLEDGMENTS
short (25-min) program adapted to separate AMP, ADP-Glc, ADP,
and ATP (Geigenberger et al., 1994).
We are grateful to L. Curtis Hannah (University of Florida) for the gift
of the maize brittle-2 antibody, to Lothar Willmitzer, Jens Kossmann,
and Bernd Müller-Röber for providing transgenic plants, and to Ursula
RNA Gel Blot Analysis
La Cognata for providing the potato AGPB and AGPS cDNAs. We
are grateful to Tobias Haas, Katrin Prescha, and Melanie Höhne for
Total RNA was isolated from different pools of tubers (two samples
excellent technical help and to Joost van Dongen for help in prepar-
per condition) according to Logemann et al. (1987), and RNA gel blot
ing the figures. This work was supported by the Deutsche For-
analysis was performed according to Sambrook et al. (1989). Equal
schungsgemeinschaft, Grant Ge 878/1-1 (to A.T., A.B., and P.G.),
loading and transfer were confirmed by staining with ethidium bro-
and the Max Planck Society (support to J.H.M.H., M.S., Y.G., and
mide (data not shown). Radioactive hybridization probes were pre-
E.M.F.). The article is dedicated to Hans W. Heldt on the occasion of
pared with the random priming labeling kit (Boehringer Mannheim)
his retirement.
using 32P-dCTP and following the manufacturer s instructions. EcoRI
restriction fragments of plasmid B22-1 and plasmid S25-1 (Müller-
Röber et al., 1990; La Cognata et al., 1995) were used as hybridiza-
Received April 3, 2002; accepted June 10, 2002.
tion probes for AGPB and AGPS1, respectively.
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