Jun Li1,3, Goizeder Almagro1,3, Francisco José Muñoz1,3, Edurne Baroja-Fernández1, Abdellatif Bahaji1,2,
Manuel Montero1, Maite Hidalgo1, Angela Marı́a Sánchez-López1, Ignacio Ezquer1, Marı́a Teresa Sesma1
and Javier Pozueta-Romero1,*
1
Instituto de Agrobiotecnologı́a (CSIC/UPNA/Gobierno de Navarra), Mutiloako etorbidea z/g, 31192 Mutiloa, Nafarroa, Spain
Iden Biotechnology S.L., Av. Conde Oliveto 2, 3I, 31002 Pamplona, Nafarroa, Spain
3
These authors contributed equally to this work.
*Corresponding author: E-mail, [email protected]; Fax, +34-948232191.
(Received July 13, 2011; Accepted December 20, 2011)
2
ADP-glucose pyrophosphorylase (AGP) is a heterotetrameric
enzyme comprising two small and two large subunits that
catalyze the production of ADP-glucose linked to starch biosynthesis. The current paradigm on leaf starch metabolism
assumes that post-translational redox modification of AGP
in response to light is a major determinant of fine regulation
of transitory starch accumulation. According to this view,
under oxidizing conditions occurring during the night the
two AGP small subunits (APS1) are covalently linked via an
intermolecular disulfide bridge that inactivates the protein,
whereas under reducing conditions occurring during the day
NADP-thioredoxin reductase C (NTRC)-dependent reductive monomerization of APS1 activates the enzyme. In this
work we have analyzed changes in the redox status of APS1
during dark–light transition in leaves of plants cultured
under different light intensities. Furthermore, we have carried out time-course analyses of starch content in ntrc mutants, and in aps1 mutants expressing the Escherichia coli
redox-insensitive AGP (GlgC) in the chloroplast. We also
characterized aps1 plants expressing a redox-insensitive,
mutated APS1 (APS1mut) form in which the highly conserved Cys81 residue involved in the formation of the intermolecular disulfide bridge has been replaced by serine. We
found that a very moderate, NTRC-dependent APS1 monomerization process in response to light occurred only when
plants were cultured under photo-oxidative conditions. We
also found that starch accumulation rates during the light in
leaves of both ntrc mutants and GlgC-expressing aps1 mutants were similar to those of wild-type leaves. Furthermore,
the pattern of starch accumulation during illumination in
leaves of APS1mut-expressing aps1 mutants was similar to
that of APS1-expressing aps1 mutants at any light intensity.
Regular Paper
Post-Translational Redox Modification of ADP-Glucose
Pyrophosphorylase in Response to Light is Not a Major
Determinant of Fine Regulation of Transitory Starch
Accumulation in Arabidopsis Leaves
The overall data demonstrate that post-translational redox
modification of AGP in response to light is not a major determinant of fine regulation of transitory starch accumulation in Arabidopsis.
Keywords: ADP-glucose Metabolic regulation Redox
status Starch.
Abbreviations: ADPG, ADP-glucose; AGP, ADPG pyrophosphorylase; BAM1, b-amylase 1; DTT, dithiothreitol; FBP,
fructose 1,6-bisphosphate; GAE, gallic acid equivalent; GlgC,
bacterial AGP; G1P, glucose-1-phosphate; GWD, glucan,
water dikinase; NTRC, NADP-thioredoxin reductase C;
3PGA, 3-phosphoglycerate; Pi, inorganic phosphate; Trx,
thioredoxin; U, unit of enzymatic activity; WT, wild type.
Introduction
Plant starch and bacterial glycogen are branched homopolysaccharides of a-1,4-linked glucose subunits with a-1,6-linked glucose at the branched points. Synthesized by glycogen synthase
and starch synthases using ADP-glucose (ADPG) as the sugar
donor molecule, these polyglucans accumulate as predominant
storage carbohydrates in most bacteria and plants. Starch and
bacterial glycogen metabolism is highly interconnected with a
number of cellular processes, and is under the control of a
complex and intricate network involving transcriptional and
post-transcriptional regulation of enzymes wherein cell
energy, nutritional status and response to environmental conditions play crucial roles. Since the initial demonstration that
ADPG serves as the precursor molecule for both plant starch
and bacterial glycogen biosynthesis (Murata et al. 1963,
Recondo et al. 1963), it became widely considered that ADPG
Plant Cell Physiol. 53(2): 433–444 (2012) doi:10.1093/pcp/pcr193, available online at www.pcp.oxfordjournals.org
! The Author 2011. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
All rights reserved. For permissions, please email: [email protected]
Plant Cell Physiol. 53(2): 433–444 (2012) doi:10.1093/pcp/pcr193 ! The Author 2011.
433
J. Li et al.
pyrophosphorylase (AGP) is the sole enzyme catalyzing the
production of ADPG. However, recent studies carried out
employing AGP-lacking mutants have provided strong
evidence about the occurrence of important sources, other
than AGP, of ADPG linked to glycogen and starch biosynthesis
in bacteria and plants, respectively (Martin et al. 1997, Muñoz
et al. 2005, Eydallin et al. 2007, Morán-Zorzano et al. 2007,
Sambou et al. 2008, Bahaji et al. 2011, Montero et al. 2011).
Most bacterial AGPs (designated as GlgCs) are homotetramers (Ballicora et al. 2003), whereas the plant enzymes comprise two types of homologous but distinct subunits, the small
(APS) and the large (APL) subunits (Crevillén et al. 2003,
Crevillén et al. 2005). In Arabidopsis, six genes encode proteins
with homology to AGP. Two of these genes code for small
subunits (APS1 and APS2, the latter being in a process of pseudogenization) and four encode large subunits (APL1–APL4)
(Sokolov et al. 1998, Crevillén et al. 2003, Crevillén et al.
2005). Whereas APS1, APL1 and APL2 are catalytically active,
APL3 and APL4 have lost their catalytic properties during evolution (Ventriglia et al. 2008). APL1 is the main large subunit in
source tissues, whereas APL2, APL3 and APL4 are mainly present in sink tissues (Sokolov et al. 1998, Crevillén et al. 2005).
In addition to its role in ADPG production, AGP is considered to catalyze a key regulatory step in the glycogen and
starch biosynthetic pathways in bacteria and plants, respectively. Plant and bacterial AGPs are highly regulated enzymes, and
are allosterically controlled by intermediates of the major
carbon assimilatory pathways of the organism. In Escherichia
coli for instance, fructose 1,6-bisphosphate (FBP) activates the
enzyme, whereas AMP is an inhibitor (Ballicora et al. 2003).
Other secondary activators of the E. coli enzyme are phosphoenolpyruvate, pyridoxal 50 -phosphate and reduced NADs. In
photosynthetic tissues of plants, AGP is tightly regulated by
3-phosphoglycerate (3PGA) and inorganic phosphate (Pi),
which serve as enzyme activator and inhibitor, respectively
(Kleczkowski 1999, Kleczkowski 2000). This property of plant
AGP makes the production of ADPG highly sensitive to changes
in the availability of photoassimilate and the Pi status of the
chloroplast stroma, and helps to coordinate starch synthesis
with photosynthetic CO2 fixation in green tissues.
In plants, illumination affects the redox status of the photosynthetic electron transport chain in the chloroplast that, in
turn, affects the activity of numerous enzymes. Chloroplasts
possess two important redox systems that independently regulate plastid metabolism by supplying reducing equivalents to
target enzymes. One is based on thioredoxins (Trxs) and the
other pathway is based on a peculiar type of NADPH Trx reductase termed NTRC (NADP-thioredoxin reductase C).
Whereas the Trx-dependent pathway obtains reducing power
from ferredoxin reduced by the photosynthetic electron chain
(Lemaire et al. 2007, Meyer et al. 2008), NTRC uses NADPH as
the reducing power source (Pérez-Ruiz and Cejudo, 2009).
Enzymes of the Calvin–Benson cycle, ATP synthesis and
NADPH export from chloroplasts are activated by
Trx-mediated reduction of cysteine residues. Trxs also regulate
434
the redox status of some starch metabolism enzymes such as
glucan, water dikinase (GWD, also termed SEX1) (Mikkelsen
et al. 2005), SEX4 (Sokolov et al. 2006) and b-amylase 1
(BAM1) (Sparla et al. 2006, Valerio et al. 2010), whereas
NTRC regulates the redox status of AGP (Michalska et al.
2009) (Fig. 1).
Under oxidizing conditions the two AGP small subunits
(APS1) are covalently linked via an intermolecular disulfide
bridge, thus forming a stable dimer within the heterotetramer
(Fu et al. 1998). This dimer can be reactivated in vitro by incubating extracts with dithiothreitol (DTT), which is accompanied by activation of AGP activity (Hendriks et al. 2003). It is
widely assumed that APS1 dimerization occurring during the
night markedly decreases the activity of AGP and alters its
kinetic properties, making it less sensitive to activation by
3PGA and increasing its Km for ATP (Hendriks et al. 2003). In
turn, NTRC-dependent reductive monomerization of two AGP
small subunits occurring during the light activates the enzyme,
which is then sensitive to fine control through allosteric regulation by 3PGA. The result of this redox sensitivity of APS1
would be to increase the activity of the enzyme during the
light period (when starch is made) and decrease it during the
dark period (when starch is degraded).
By examining the correlation existing between APS1 redox
status, AGP activity and starch levels in leaves under light and
dark conditions, evidence has been provided that transitory
starch accumulation is finely regulated by post-translational
redox modification of APS1 in response to light, sugars and
other environmental inputs (Hendriks et al. 2003, Gibon et al.
Fig. 1 Proposed mechanisms of action of thioredoxins and NTRC in
linking the photosynthetic electron transport chain with starch metabolic and Calvin–Benson cycle enzymes. According to this metabolic
scheme, redox-regulated enzymes such as AGP, GWD, SEX4 and
BAM1 are activated during illumination.
Plant Cell Physiol. 53(2): 433–444 (2012) doi:10.1093/pcp/pcr193 ! The Author 2011.
AGP redox and fine regulation of starch metabolism
2004a, Gibon et al. 2004b, Kolbe et al. 2005, Geigenberger et al.
2005, Kolbe et al. 2006, Lunn et al. 2006, Hädrich et al. 2011a).
Redox regulation of AGP activity in response to light is currently considered as a major regulatory mechanism of transitory
starch accumulation in leaves (Michalska et al. 2009, Kötting
et al. 2010, Geigenberger 2011). However, this view is not consistent with reports showing that DTT does not activate leaf
AGP from many species (Kerr et al. 1984, Kleczkowski 1999,
Chen and Qi 2007), and with a recent work showing the
occurrence of fine, light-dependent starch accumulation in
AGP-lacking aps1 Arabidopsis mutants (Bahaji et al. 2011).
Furthermore, this interpretation conflicts with works showing
that (i) most of APS1 remains oxidized during illumination in
Arabidopsis leaves (Hendriks et al. 2003, Gibon et al. 2004a,
Kolbe et al. 2005, Lunn et al. 2006, Hädrich et al. 2011a, Li
et al. 2011); and (ii) the amount of reduced APS1 accumulated
in illuminated Arabidopsis leaves is sometimes comparable
with that accumulated in the dark (cf. fig. 9A of Gibon et al.
2004a, cf. fig. 6B of Li et al. 2011).
aps1 is a T-DNA null mutant of the Arabidopsis AGP small
subunit-encoding gene that possesses <0.05% of the wild-type
(WT) AGP activity (Ventriglia et al. 2008, Bahaji et al. 2011).
Leaves of this mutant accumulate WT ADPG and approximately 2% of the WT starch content (Bahaji et al. 2011). Given the
occurrence of conflicting data about the involvement of
changes in the redox status of AGP in the fine regulation of
transitory starch metabolism, in this work we have studied the
pattern of starch accumulation during illumination in ntrc
mutants, and in aps1 mutants expressing in the chloroplast
the E. coli redox-insensitive GlgC cultured under different
light intensities. Furthermore, we have characterized aps1
plants expressing a site-directed mutated form of APS1, in
which the highly conserved Cys81 residue involved in the formation of an intermolecular disulfide bridge (Fu et al. 1998,
Hädrich et al. 2011a) has been replaced by serine. The overall
data presented in this work provide strong evidence that, contrary to the currently prevailing ideas on regulatory aspects of
leaf starch metabolism, redox modification of AGP in response
to light is not a major determinant of fine regulation of transitory starch accumulation in Arabidopsis.
Results and Discussion
Neither APS1 monomerization nor fine regulation
of transitory starch accumulation are controlled
by NTRC when plants are cultured under
non-photo-oxidative stress conditions
NTRC has been suggested largely to control APS1 reduction
during the day (Michalska et al. 2009), and to play an important
role in protecting plants against oxidative stress (Serrato et al.
2004, Pérez-Ruiz et al. 2006, Pulido et al. 2010). Because light
excess may result in photo-oxidative stress, we investigated the
control by NTRC of the APS1 redox status and starch
accumulation in WT and ntrc plants cultured under three different light intensity regimes: 16 h light (90, 180 or 400 mmol
photons s1 m2)/8 h dark. We must emphasize that previous
works investigating the light-dependent APS1 redox status
were carried out at 180–250 mmol photons s1 m2 (Hendriks
et al. 2003, Kolbe et al. 2005, Kolbe et al. 2006, Michalska et al.
2009, Hädrich et al. 2011a). Consistent with Serrato et al.
(2004), ntrc mutants grown at 180 and 400 mmol photons s1 m2 were much smaller than WT plants (Fig. 2A). In
clear contrast, the size of ntrc mutants was similar to that of WT
plants when cultured at 90 mmol photons s1 m2 (Fig. 2A). It
is noteworthy that WT leaves accumulated significantly higher
levels of anthocyanins and total polyphenols at 180 and
400 mmol photons s1 m2 than at 90 mmol photons s1 m2,
whereas ntrc leaves accumulated comparable levels of anthocyanins and total polyphenols at any light intensity (Fig. 2B, C).
Because anthocyanins and other polyphenols play an important role in protecting plants against photo-oxidative damage
(Smillie and Hetherington 1999, Winkel-Shirley 2001, Shao et al.
2008), the overall data indicated that NTRC would play an important role in protecting plants against photo-oxidative stress
at 180 and 400 mmol photons s1 m2.
Non-reducing Western blot analyses revealed that most of
APS1 remained oxidized (approximately 100 kDa dimeric) at
any light intensity (Fig. 2D), which is consistent with previous
reports (Hendriks et al. 2003, Gibon et al. 2004a, Kolbe et al.
2005, Lunn et al. 2006, Hädrich et al. 2011a, Li et al. 2011).
These analyses also revealed that a very moderate NTRCdependent APS1 monomerization in response to light only
occurred when plants were cultured at 180 and 400 mmol photons s1 m2, but not at 90 mmol photons s1 m2 (Fig. 2D),
indicating that NTRC controls APS1 monomerization only
under photo-oxidative stress conditions. Furthermore,
non-reducing Western blot analyses also revealed the occurrence of low levels of monomeric APS1 in the dark in both WT
and ntrc leaves (Fig. 2D). The very moderate monomerization
of APS1 occurring during dark–light transition in plants cultured at 180 and 400 mmol photons s1 m2 was consistent
with previous reports (cf. fig. 9A of Gibon et al. 2004a, cf. fig.
6B of Li et al. 2011, Hädrich et al. 2011a). The rate of starch
accumulation in leaves of WT plants cultured at 90 mmol photons s1 m2 was approximately 45 nmol of glucose transferred
to starch min1 g FW1 (Fig. 2E). No differences in the patterns
and rates of starch accumulation could be observed between
leaves of WT plants and ntrc mutants (Fig. 2E), indicating that
APS1 monomerization in response to light is not involved in
fine regulation of transitory starch accumulation when plants
are cultured at 90 mmol photons s1 m2. The fact that, despite
the lack of APS1 monomerization during light–dark transition,
transitory starch normally accumulates in leaves of plants cultured at 90 mmol photons s1 m2 would indicate that
(i) changes in the redox status of APS1 in response to light
are not a major determinant of fine regulation of starch
accumulation in Arabidopsis leaves; and (ii) activity of the
monomeric APS1 occurring in the night is sufficient to support
Plant Cell Physiol. 53(2): 433–444 (2012) doi:10.1093/pcp/pcr193 ! The Author 2011.
435
J. Li et al.
A
ntrc
WT
90 µmol photons sec -1 m-2
B
180 µmol photons sec-1 m-2
C
5
400 µmol photons sec -1 m-2
80
Total polyphenols
(mg GAE/100 g FW)
Total anthocyanins
(mg/g FW)
70
4
3
2
1
60
50
40
30
20
10
0
90
180
0
400
Light intensity (µmol photons sec-1 m-2 )
D
kDa
Light Dark
Light Dark
kDa
100
100
(dimer)
90
180
400
Light intensity (µmol photons sec-1 m-2 )
kDa
100
Light Dark
WT
50
(monomer)
kDa
50
50
kDa
kDa
100
(dimer)
100
100
50
(monomer)
50
50
180 µmol photons sec-1 m-2
400 µmol photons sec -1 m-2
ntrc
90 µmol photons sec -1 m-2
E
Starch (µmol glucose/g FW)
70
60
50
40
30
20
10
0
0
4
8
12
Time (h)
16
20
24
Fig. 2 A very moderate, NTRC-dependent monomerization of APS1 only occurs under photo-oxidative conditions. Effect of light intensity on
(A) growth, (B) total anthocyanin content and (C) total polyphenol content in 4-week-old WT and ntrc mutants (white and gray columns,
respectively) cultured under 16 h light (90, 180 or 400 mmol photons s1 m2)/8 h dark. (D) Non-reducing Western blot analyses of APS1 in leaves
of WT and ntrc plants cultured for 4 weeks under 16 h light (90, 180 or 400 mmol photons s1 m2)/8 h dark. Leaves were harvested at the end of
the light and dark periods. (E) Time-course of starch content in leaves of 4-week-old WT plants and ntrc mutants (filled squares and filled
diamonds, respectively) cultured under a 16 h light (90 mmol photons s1 m2)/8 h dark regime. In B, C and E, the results are the mean ± SE of
three independent experiments. In D, proteins from 1 mg of FW were loaded per lane.
436
Plant Cell Physiol. 53(2): 433–444 (2012) doi:10.1093/pcp/pcr193 ! The Author 2011.
AGP redox and fine regulation of starch metabolism
Rates of transitory starch accumulation in leaves
of aps1 mutants expressing in their chloroplast
the redox-insensitive E. coli GlgC are similar to
those of WT plants
The cytosol of heterotrophic bacteria is a highly reductive
environment that has evolved not only lacking components
that catalyze formation of disulfide bonds, but also having
active systems that result in the reduction of protein disulfide
bonds (Derman et al. 1993, Hatahet et al. 2010). Due to the
presence of these pathways, the production of disulfide
bond-containing proteins is thought to be impossible in the
cytosol of E. coli (Derman et al. 1993). It is thus conceivable
that, unlike many proteins occurring in the chloroplast whose
redox status varies during light–dark transitions, E. coli GlgC will
be reduced and will hardly form disulfide bonds in its natural
environment. Consistent with this presumption, non-reducing
Western blot analyses of GlgC extracted from E. coli cells revealed that this protein is entirely reduced in the cytosol of the
bacterium (Supplementary Fig. S2). GlgC shares low sequence
similarity to plant AGPs (Anderson et al. 1989), and lacks the
conserved cysteine residue involved in the redox-dependent
formation of the intermolecular disulfide bridge of plant
APS1 (Supplementary Fig. S3). Whether modification of the
APS1 redox status is a major determinant of fine regulation of
transitory starch metabolism in Arabidopsis was further investigated by carrying out a time-course analysis of the starch
content in leaves of both WT plants and aps1 plants constitutively expressing GlgC in the plastid (Bahaji et al. 2011). The
rationale behind this experimental approach was that if
changes in APS1 redox status are important for fine regulation
of transitory starch metabolism in leaves, changes in the starch
content during light–dark transitions in aps1 leaves expressing
the redox-insensitive GlgC should be different from those
of WT leaves. Conversely, if fine regulation of starch metabolism
is not subjected to regulation of the APS1 redox status,
changes in the starch content during light–dark transitions in
GlgC-expressing aps1 leaves should be similar to those of WT
leaves. Although previous attempts to express a bacterial AGP
ectopically in plants were carried out employing glgC-16, a
mutated variant of E. coli glgC whose product shows a reduced
response to FBP and AMP (Stark et al. 1992, Sweetlove et al.
1996), in this work we characterized plants expressing the
WT glgC.
As shown in Fig. 3A, non-reducing Western blot analyses of
GlgC-expressing aps1 leaves revealed that GlgC is equally
reduced under light and dark conditions, thus confirming
that the protein is redox insensitive under environmental conditions occurring in the chloroplast. Kinetic analyses of GlgC
purified from glgC-expressing aps1 leaves confirmed that the
protein typically responds to FBP and AMP (Table 1). It is
noteworthy that no differences in the rates and patterns of
starch accumulation could be observed during illumination between WT leaves and glgC-expressing aps1 leaves cultured at
90 mmol photons s1 m2 (Fig. 3B). The overall data thus
showed that (i) FBP- and AMP-responsive GlgC can be used
to complement the almost starch-less phenotype of aps1; and
(ii) fine regulation of transitory starch accumulation in response
A
kDa
Light
Dark
50
B
70
Starch (µmol glucose/g FW)
a normal rate of starch accumulation in the subsequent
light period.
Ballicora et al. (2000) and Geigenberger et al. (2005) showed
that, in vitro, APS1 from potato tubers and pea chloroplasts
can be activated by plastidial Trxs f and m, although proteomic
analyses failed to find spinach APS1 as one of the targets of
chloroplast Trxs (Balmer et al. 2003). Whether plastidial
Trx-mediated changes in APS1 redox status are major determinants of the fine regulation of transitory starch metabolism
in Arabidopsis leaves during the light/dark transitions was
investigated by carrying out a time-course analysis of starch
content in leaves of WT plants and trxf and trxm homozygous
mutants cultured under a 16 h light (90 mmol photons s1 m2)
and 8 h dark regime. As shown in Supplementary Fig. S1, no
differences in the patterns and rates of starch accumulation
during illumination could be observed between the WT and
the different plastidial Trx mutants, indicating that, similar to
NTRC, plastidial Trxs are not major determinants of fine regulation of starch accumulation in Arabidopsis leaves.
60
50
40
30
20
10
0
0
4
8
12
16
20
24
Time (h)
Fig. 3 Fine regulation of transitory starch accumulation in response to
light does not require a redox-sensitive AGP in Arabidopsis. (A) Nonreducing Western blot analysis of GlgC in 4-week-old GlgC-expressing
aps1 plants cultured for 4 weeks under 16 h light (90 mmol photons s1 m2)/8 h dark. Leaves were harvested at the end of the
light and dark periods. (B) Time-course of starch content in
4-week-old WT and GlgC-expressing aps1 plants (filled squares and
filled diamonds, respectively) cultured under a 16 h light (90 mmol
photons s1 m2)/8 h dark regime. In A, proteins from 1 mg of FW
were loaded per lane. In B, the results are the mean ± SE of three
independent experiments.
Plant Cell Physiol. 53(2): 433–444 (2012) doi:10.1093/pcp/pcr193 ! The Author 2011.
437
J. Li et al.
Table 1 Kinetic parameters of AGP partially purified from aps1
leaves of plants transformed with 35S-TP-P541-glgC as explained in
the Materials and Methods (see also Supplementary Fig. 7A)
Variable
Fixed
(2 mM)
FBP
(2 mM)
AMP
(0.5 mM)
Km
(mM)
Vmax
(mU mg
protein1)
G1P
ATP
+
0.09 ± 0.02
650 ± 59
G1P
ATP
+
+
0.12 ± 0.02
195 ± 20
G1P
ATP
0.14 ± 0.03
60 ± 5.4
G1P
ATP
+
0.25 ± 0.07
42 ± 3.9
ATP
G1P
+
0.25 ± 0.03
637 ± 49
ATP
G1P
+
+
0.30 ± 0.07
190 ± 23
ATP
G1P
0.30 ± 0.08
65 ± 5.5
ATP
G1P
+
0.29 ± 0.06
45 ± 2.1
AGP was measured following the ‘two-step assay method’ described in the
Materials and Methods. In ‘step one’, the AGP assay mixture contained
50 mM HEPES (pH 7.0), 3 mM MgCl2, the GlgC preparation and the indicated
concentration of G1P, ATP, FBP and AMP. After 3 min at 37 C, reactions
were stopped by boiling the assay reaction mixture for 2 min. In ‘step two’,
ADPG was measured by HPLC on a Waters Associate’s system fitted with a
Partisil-10-SAX column. The results are the mean ± SE of three independent
experiments.
to light does not strictly require a redox-sensitive AGP in
Arabidopsis.
Patterns of transitory starch accumulation during
dark–light transition in leaves of aps1 plants
expressing a redox-insensitive form of APS1 are
similar to those of aps1 plants expressing a
WT APS1
Cys12 is the residue involved in the formation of intermolecular
disulfide bridges in potato APS1 (Fu et al. 1998). Substitution of
this residue by serine eliminates the requirement for DTT for
the activation of AGP (Fu et al. 1998). From comparison with
the potato APS1, Cys81 in the Arabidopsis enzyme is the most
likely residue involved in the redox control of AGP activity
(Supplementary Fig. S3). Whether changes in the APS1
redox status are major determinants of fine regulation of transitory starch metabolism in Arabidopsis was further investigated by comparing the starch accumulation rates and
patterns during dark–light transition of different lines of
APS1-expressing aps1 plants, and of aps1 plants expressing a
mutated APS1 form (APS1mut) in which Cys81 has been
replaced by serine. The rationale behind this experimental
approach was that, if redox modification of AGP in response
to light is a major determinant of fine regulation of transitory starch metabolism, changes in the starch content during light–dark transitions in aps1 leaves expressing the
redox-insensitive APS1mut should be different from those of
APS1-expressing aps1 leaves. Conversely, if fine regulation of
starch metabolism is not subjected to regulation of the APS1
redox status, changes in the starch content during light–dark
transitions in APS1mut-expressing aps1 leaves should be similar
to those of APS1-expressing aps1 leaves.
438
We produced eight independent lines each of APS1and APS1mut-expressing aps1 plants (APS1,1–8 and
APS1mut,1–8, respectively) that showed different AGP activities (Fig. 4A). Kinetic analyses of AGP purified from APS1- and
APS1mut-expressing aps1 plants (lines APS1,8 and APS1mut,8,
respectively) confirmed that 3PGA activates the enzyme,
whereas Pi acts as a strong inhibitor (Tables 2, 3). Western
blot analyses of APS1 under non-reducing conditions revealed
that this protein was present largely in the oxidized (approximately 100 kDa dimeric) form (Fig. 4B), in both the light- and
dark-harvested leaves of APS1-expressing aps1 plants
(Supplementary Fig. S4A). APS1 of APS1-expressing plants
became fully reduced when DTT was added to the protein
extract (Supplementary Fig. S4B). In clear contrast, the
APS1mut was present in the reduced (50 kDa monomeric)
form in both the light and dark conditions in APS1mut-expressing aps1 plants (Fig. 4B, Supplementary Fig. S4A). Thus,
site-directed mutagenesis of Cys81 (i) eliminated the intermolecular disulfide bridge and the requirement for DTT to reduce
APS1; and (ii) yielded a permanently reduced, redox-insensitive
enzyme.
As shown in Fig. 4, there was a positive correlation between
the amount of reduced APS1 (approximately 50 kDa monomeric) and starch content in APS1- and APS1mut-expressing aps1
plants, which strongly supports the view that the reduced APS1
form, but not the oxidized form, highly controls starch biosynthesis. Time-course analyses of starch content were carried out
at 90 and 180 mmol photons s1 m2 using WT plants and two
lines each from APS1- and APS1mut-expressing aps1 plants
accumulating comparable levels of monomeric APS1 (APS1,3
vs. APS1mut,6 and APS1,8 vs. APS1mut,8) (Supplementary
Fig. S5). At 90 mmol photons s1 m2, WT leaves accumulated
monomeric APS1 levels that were comparable with those of
APS1mut,8 (Supplementary Fig. S5), whose AGP activity (approximately 150 mU g FW1) is sufficient to support the rate of
45 nmol of glucose transferred to starch min1 g FW1 occurring at 90 mmol photons s1 m2 (see above). No differences in
the rate and pattern of starch accumulation could be observed
during the dark–light transition between APS1- and APS1mutexpressing aps1 plants at 90 mmol photons s1 m2 (Fig. 5A, B).
Furthermore, no differences could be found in the rates and
patterns of starch accumulation between WT and APS1mut,8
plants cultured at 180 mmol photons s1 m2 (Fig. 5C) and
400 mmol photons s1 m2 (not shown). The rate of starch accumulation in leaves of APS1mut,8 plants cultured at 180 mmol
photons s1 m2 was approximately 75 nmol of glucose transferred to starch min1 g FW1, which is about 80% higher than
that observed in leaves of APS1mut,8 plants cultured at
90 mmol photons s1 m2 (Fig. 5A, C). The higher rate of
starch accumulation at 180 mmol photons s1 m2 than at
90 mmol photons s1 m2 in APS1mut,8 leaves can be ascribed
to factors such as a higher CO2 fixation rate and/or a higher
plastidial 3PGA/Pi balance and/or higher starch synthase activity occurring at 180 mmol photons s1 m2 than at 90 mmol
photons s1 m2. The overall data thus show that fine
Plant Cell Physiol. 53(2): 433–444 (2012) doi:10.1093/pcp/pcr193 ! The Author 2011.
AGP redox and fine regulation of starch metabolism
A
1200
AGP activity (mU/g FW)
1000
800
600
400
200
0
WT
apsI
1
2
3
4
5
6
7
8
1
2
3
5
6
7
8
6
7
8
6
7
8
apsI::APSImut
apsI::APSI
B
4
kDa
100
(dimer)
50
(monomer)
WT
apsI
1
2
3
4
5
6
7
8
1
2
3
5
apsI::APSImut
apsI::APSI
C
4
50
Starch (µmol glucose/g FW)
45
40
35
30
25
20
15
10
5
0
WT
apsI
1
2
3
4
5
apsI::APSI
6
7
8
1
2
3
4
5
apsI::APSImut
Fig. 4 Characterization of APS1- and APS1mut-expressing aps1 plants. (A) AGP activity in leaves of plants of eight independent lines each of
APS1- and APS1mut-expressing aps1 plants. AGP was measured following the ‘two-step assay method’ described in the Materials and Methods.
In ‘step one’ the AGP assay mixture contained 50 mM HEPES (pH 7.0), 3 mM MgCl2, 2 mM G1P, 2 mM ATP, 4 mM 3PGA and the plant crude
extract. After 3 min at 37 C, reactions were stopped by boiling the assay reaction mixture for 2 min. In ‘step two’, ADPG was measured
by HPLC on a Waters Associate’s system fitted with a Partisil-10-SAX column. (B) Non-reducing Western blot analysis of APS1 in leaves of
APS1- and APS1mut-expressing aps1 plants cultured under a 16 h light (90 mmol photons s1 m2)/8 h dark regime. (C) Starch content in leaves
of APS1- and APS1mut-expressing aps1 plants cultured under a 16 h light (90 mmol photons s1 m2)/8 h dark regime. Leaves were harvested
after 12 h of illumination. In A and C, the results are the mean ± SE of three independent experiments. In B, proteins from 1 mg of FW were
loaded per lane.
Plant Cell Physiol. 53(2): 433–444 (2012) doi:10.1093/pcp/pcr193 ! The Author 2011.
439
J. Li et al.
A
Variable
Fixed
(2 mM)
3PGA
(4 mM)
Pi
(4 mM)
Km
(mM)
Vmax
(mU mg
protein1)
G1P
ATP
+
0.09 ± 0.01
160 ± 19
G1P
ATP
0.28 ± 0.03
35 ± 2.8
G1P
ATP
+
+
0.15 ± 0.03
20 ± 1.6
ATP
G1P
+
0.10 ± 0.01
153 ± 23
ATP
G1P
0.30 ± 0.02
34 ± 3.1
ATP
G1P
+
+
0.18 ± 0.02
20 ± 2.1
Starch (µmol glucose/g FW)
Table 2 Kinetic parameters of AGP partially purified from APS1,8
leaves as explained in the Materials and Methods (see also
Supplementary Fig. S7B)
B
Starch (µmol glucose/g FW)
3PGA
(4 mM)
Pi
(1 mM)
Km
(mM)
Vmax
(mU mg1
protein)
G1P
ATP
+
0.06 ± 0.01
16.0 ± 1.3
G1P
ATP
0.27 ± 0.04
2.0 ± 0.3
G1P
ATP
+
+
0.09 ± 0.03
9.1 ± 1.2
ATP
G1P
+
0.07 ± 0.01
17.3 ± 1.1
ATP
G1P
0.28 ± 0.02
1.8 ± 0.2
ATP
G1P
+
+
0.1 ± 0.02
10.1 ± 1.1
30
20
0
4
8
Time (h)
12
16
0
4
8
Time (h)
12
16
0
4
8
Time (h)
12
16
30
25
20
15
10
0
C 140
Starch (µmol glucose/g FW)
regulation of transitory starch accumulation in response to
light can normally take place with a redox-insensitive APS1.
Additional concluding remarks
440
40
5
The results are the mean ± SE of three independent experiments.
Light regulation of chloroplastic enzymes is a process that links
photosynthetic electron transport and the activity of specific
chloroplast enzymes by means of the ferredoxin/Trx system. In
chloroplasts, the physiological interpretation of a Trx-mediated
regulation of starch metabolism in response to light appears to
be straightforward, since activation of enzymes of the Calvin–
Benson cycle, AGP and starch biosynthesis occur concurrently
during the light period. However, data presented in this work
showing that (i) the amount of reduced APS1 in the dark is
sufficient to support the observed rates of starch accumulation
during illumination; and (ii) rates and patterns of starch accumulation in leaves expressing the redox-sensitive APS1 are similar to those in aps1 leaves expressing redox-insensitive AGPs
(Figs. 3, 5) refute the current paradigm according to which fine
regulation of transitory starch metabolism is subjected to
post-translational redox modification of AGP. This conclusion
is further supported by the fact that transitory starch normally
50
0
Table 3 Kinetic parameters of AGP partially purified from
APSmut,8 leaves as explained in the Materials and Methods
Fixed
(2 mM)
60
10
AGP was measured following the ‘two-step assay method’ described in the
Materials and Methods. In ‘step one’, the AGP assay mixture contained
50 mM HEPES (pH 7.0), 3 mM MgCl2, the GlgC preparation and the indicated
concentration of G1P, ATP, 3PGA and Pi. After 3 min at 37 C, reactions were
stopped by boiling the assay reaction mixture for 2 min. In ‘step two’, ADPG was
measured by HPLC on a Waters Associate’s system fitted with a Partisil-10-SAX
column. The results are the mean ± SE of three independent experiments.
Variable
70
120
100
80
60
40
20
0
Fig. 5 Time-course of starch content in illuminated leaves
of 4-week-old APS1- and APS1mut-expressing aps1 plants cultured
under a 16 h light (90 mmol photons s1 m2)/8 h dark regime
(A, B), or under a 16 h light (180 mmol photons s1 m2)/8 h dark
regime (C). Open squares, APS1,8; open triangles, APS1mut,8;
open diamonds, WT; filled squares, APS1mut,6; filled diamonds,
APS1,3. The results are the mean ± SE of three independent
experiments.
Plant Cell Physiol. 53(2): 433–444 (2012) doi:10.1093/pcp/pcr193 ! The Author 2011.
AGP redox and fine regulation of starch metabolism
accumulates in plants cultured at 90 mmol photons s1 m2
despite the lack of APS1 monomerization during the dark–
light transition (Fig. 2). Although NTRC can moderately control
the redox status of AGP in response to oxidative stress (Fig. 2,
see also Li et al. 2011), our data strongly indicate that
NTRC-mediated changes in the redox status of APS1 play a
minor role, if any, in fine regulation of transitory starch accumulation in Arabidopsis leaves. It thus appears that the currently prevailing paradigm on fine regulation of transitory
starch biosynthesis was solely based on in vitro analyses of
AGP activation by plastidial Trxs (Ballicora et al. 2000,
Geigenberger et al. 2005, Michalska et al. 2009), and on the
correlation existing between APS1 redox status and starch
levels in illuminated and non-illuminated leaves when plants
were cultured under light intensity conditions that promote
the photo-oxidative stress response (Hendriks et al. 2003,
Kolbe et al. 2005, Kolbe et al. 2006, Michalska et al. 2009,
Hädrich et al. 2011a). Under those conditions, ntrc mutants
are small (Fig. 2), and have low Chl content (Serrato et al.
2004, also confirmed in our laboratory), which would explain
why their leaves accumulate about 70% of the WT starch content (Michalska et al. 2009).
We must emphasize that, similar to AGP, starch breakdown
enzymes such as GWD, SEX4 and BAM1 are subjected to redox
regulation, being active when reduced, and inactive when oxidized (Balmer et al. 2003, Mikkelsen et al. 2005, Sokolov et al.
2006, Sparla et al. 2006) (Fig. 1). This would imply that these
enzymes are activated during illumination (when the chloroplast is reductive and accumulates starch), and inactivated
during the dark (when the chloroplast becomes oxidative and
degrades starch), which clearly conflicts with biochemical and
genetic evidence showing that GWD, SEX4 and plastidial
b-amylases are active during starch breakdown taking place
in the dark (Ritte et al. 2004, Edner et al. 2007, Fulton et al.
2008, Kötting et al. 2009). Light activation of AGP, GWD and
SEX4 would also imply that stimulation of starch biosynthesis
and degradation occurs concurrently during illumination,
which conflicts with a view that simultaneous synthesis and
breakdown of starch does not occur in the illuminated
Arabidopsis leaf (Zeeman et al. 2002).
During the latter part of the review process of this paper, a
report by Hädrich et al. (2011b) has been published showing
that mutagenesis of Cys81 prevents dimerization of APS1.
These authors found that leaves of APS1mut-expressing AGP
mutants (adg1) accumulated slightly higher levels of transitory
starch than WT leaves. Hädrich et al. (2011b) also found that
the rates of starch synthesis in WT leaves were similar to those
of APS1mut-expressing adg1 plants cultured under 12 h/12 h
and 16 h/8 h light/dark regimes, which is consistent with the
data presented in our work, but contradictory to the current
paradigm on regulation of transitory starch metabolism.
Intriguingly, although Zeeman et al. (2002) have previously
argued that simultaneous synthesis and breakdown of starch
does not occur in Arabidopsis leaves during illumination, the
slight starch-excess phenotype of content and the normal rate
of starch accumulation of APS1mut-expressing adg1 plants
were ascribed to differences in the rate of starch degradation
occurring in WT and APS1mut-expressing adg1 leaves during
illumination.
Materials and Methods
Plants, bacterial strains and plant transformation
The work was carried out using WT Arabidopsis (ecotype
Columbia), the ntrc (Michalska et al. 2009), trxf1
(SALK_049146), trxf2 (SALK_108322), trxm2 (SALK_123570),
trxm3 (SALK_061968) and the aps1::T-DNA (SALK_040155)
(Ventriglia et al. 2008, Bahaji et al. 2011) mutants, and aps1
plants transformed with either 35S-TP-P541-glgC (Bahaji et al.
2011, line 1), 35S-APS1 or 35S-APS1mut (Supplementary Fig.
S6). Plasmid constructs necessary to produce plants transformed with 35S-APS1 or 35S-APS1mut were produced using
the Gateway technology and confirmed by sequencing.
Primers used to create plasmids necessary to produce
35S-APS1- or 35S-APS1mut-expressing aps1 plants are shown
in Supplementary Table S1. All plasmid constructs were electroporated and propagated in E. coli TOP 10. Transfer of plasmid constructs to Agrobacterium tumefaciens EHA105 cells was
carried out by electroporation. Transformations of Arabidopsis
plants were conducted as described by Clough and Bent (1998).
Transgenic plants were selected on kanamycin-containing
medium. Plants were grown for 4 weeks in pots at ambient
CO2 (350 p.p.m.) and 80% humidity in growth chambers at
20 C under a 16 h light (90, 180 or 400 mmol photons s1 m2)/8 h dark regime. For time-course analyses of
starch content and AGP redox status, fully expanded source
leaves were harvested after the indicated illumination period,
immediately quenched in liquid nitrogen, and stored at 80 C
for up to 2 months before use. To assay AGP activity (see
below), 1 g of the frozen powder was resuspended at 4 C in
5 ml of 50 mM HEPES (pH 7.5), 5 mM DTT and 2 mM EDTA
(extraction medium). The homogenate was subjected to centrifugation for 10 min at 10,000g and 4 C. The supernatant was
desalted by ultrafiltration on Centricon YM-10 (Amicon) and
the retained material was resuspended in extraction medium.
For non-reducing Western blots (see below), 50 mg of frozen
leaf material was extracted in cold 16% (w/v) trichloroacetic
acid in diethyl ether, mixed, and stored at 20 C for at least 2 h
as described by Hendrik et al. (2003). The pellet was collected
by centrifugation at 10,000g for 5 min at 4 C, washed three
times with ice-cold acetone, dried briefly under vacuum and
resuspended in 1 Laemmli sample buffer containing no
reductant.
Site-directed mutagenesis of APS1
APS1mut was generated by site-directed mutagenesis using
the plasmid pDONR APS1 (Supplementary Fig. S6), the oligonucleotides O3 and O4 (Supplementary Table S1) and the
QuickChange site-directed mutagenesis kit (Stratagene) to
Plant Cell Physiol. 53(2): 433–444 (2012) doi:10.1093/pcp/pcr193 ! The Author 2011.
441
J. Li et al.
replace the codon initially encoding Cys81 by a codon
encoding Ser81.
Partial purification of AGP
AGPs from APS1,8 and APS1mut,8 (APS1- and APS1mutexpressing aps1 plants, respectively) and from glgC-expressing
aps1 plants (Bahaji et al. 2011) were purified for kinetic studies.
Crude extracts were subjected to an ammonium sulfate
fractionation (30–60%) with centrifugation for 20 min at
12,000g. The ammonium sulfate pellets were resuspended in
a buffer containing 50 mM HEPES (pH 7.5), and desalted by
ultrafiltration on Centricon YM-10 (Amicon). The retained material was resuspended in 50 mM HEPES (pH 7.5) and samples
were then applied to a Q-Sepharose column equilibrated with
the same buffer. To elute the enzymes, a NaCl linear gradient (20-bed volumes, 0–0.5 M) was applied, and fractions of
2.5 ml were collected. Fractions containing AGP activity
(Supplementary Fig. S7) were pooled, concentrated using
Centricon YM-10, resuspended in 50 mM HEPES (pH 7.5) and
stored at 80 C.
Two-step assay method for AGP activity assay
Measurements of AGP activity in both crude extracts and partially purified AGP preparations were performed in the direction of ADPG synthesis in two steps: (i) AGP reaction and (ii)
measurement of ADPG produced during the reaction. Unless
otherwise indicated, the AGP assay mixture contained 50 mM
HEPES (pH 7.0), 3 mM MgCl2, the indicated amounts of
glucose-1-phosphate (G1P), ATP, 3PGA, FBP, Pi and AMP,
and the AGP-containing protein preparation. The reaction
was initiated by adding the AGP-containing protein preparation to the assay mixture. After 3 min at 37 C, reactions
were stopped by boiling the assay reaction mixture for 2 min.
ADPG was measured by HPLC on a Waters Associate’s system
fitted with a Partisil-10-SAX column as described by Muñoz
et al. (2005). To confirm further that identification and measurements of ADPG were correct, ADPG was enzymatically
hydrolysed with purified E. coli ASPP (Moreno-Bruna et al.
2001). We define 1 unit (U) of enzyme activity as the amount
of enzyme that catalyzes the production of 1 mmol of product
per min. Kinetic parameters such as Km and Vmax were evaluated by Lineweaver–Burk plots.
Analytical procedures
Starch was measured by using an amyloglucosidase-based
test kit (Boehringer Manheim). Total polyphenol content
was measured and expressed as mg of gallic acid equivalent
(GAE) g FW1 as described by Georgé et al. (2005). Total anthocyanin content was measured as described by Teow et al.
(2007).
Western blot analyses
For immunoblot analyses, protein samples were extracted and
separated by 10% SDS–PAGE under either reducing or
442
non-reducing conditions as described by Hendriks et al.
(2003), transferred to nitrocellulose filters and immunodecorated by using the antisera raised against either the small subunit (Bt2) of maize AGP or E. coli GlgC (Bahaji et al. 2011), and a
goat anti-rabbit IgG–alkaline phosphatase conjugate as secondary antibody (Sigma).
Supplementary data
Supplementary data are available at PCP online.
Funding
This research was supported by the Comisión Interministerial
de Ciencia y Tecnologı́a and Fondo Europeo de Desarrollo
Regional (Spain) [grant BIO2010-18239]; Iden Biotechnology
S.L.; the Spanish Ministry of Science and Innovation
[pre-doctoral fellowships to I.E. and A.M.S.-L.].
Acknowledgments
We thank Dr. C. Hannah (University of Florida) who kindly
provided us with the antibody raised against the small subunit
of maize AGP. We also thank Dr. F. J. Cejudo (IBVF, Sevilla), who
kindly provided us with seeds of homozygous ntrc mutants
used in this study. We thank Dr. J. M. Romero (IBVF, Sevilla)
for fruitful scientific discussions. This paper is dedicated to
Professor Takashi Akazawa, for his pioneering studies on
ADP-glucose in plants.
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