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Plant Physiol. (1999) 119: 511-520
Evidence for a Slow-Turnover Form of the
Ca2+-Independent Phosphoenolpyruvate
Carboxylase Kinase in the Aleurone-Endosperm Tissue of Germinating
Barley Seeds1
Lidia Osuna,
Jean-Nöel Pierre,
María-Cruz González,
Rosario Alvarez,
Francisco J. Cejudo,
Cristina Echevarría*, and
Jean Vidal
Departamento de Biología Vegetal, Facultad de
Biología, Universidad de Sevilla, Avenida Reina Mercedes no. 6, 41012 Sevilla, Spain (L.O., R.A., C.E.); Instituto de
Bioquímica Vegetal y Fotosíntesis, Centro de
Investigaciones Científicas "Isla de la Cartuja," Avda
Américo Vespucio s/n, 41092 Sevilla, Spain (M.-C.G., F.J.C.); and Institut de Biotechnologie des Plantes, Unité Associée
Centre National de la Recherche Scientifique, D 1128, Bâtiment 630, Université de Paris-Sud, Centre d Orsay,
cedex, France (J.-N.P., J.V.)
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ABSTRACT |
Phosphoenolpyruvate
carboxylase (PEPC) activity was detected in aleurone-endosperm extracts
of barley (Hordeum vulgare) seeds during germination,
and specific anti-sorghum (Sorghum bicolor) C4 PEPC polyclonal antibodies immunodecorated constitutive
103-kD and inducible 108-kD PEPC polypeptides in western analysis. The 103- and 108-kD polypeptides were radiolabeled in situ after imbibition for up to 1.5 d in 32P-labeled inorganic phosphate. In
vitro phosphorylation by a Ca2+-independent PEPC protein
kinase (PK) in crude extracts enhanced the enzyme's velocity and
decreased its sensitivity to L-malate at suboptimal pH and
[PEP]. Isolated aleurone cell protoplasts contained both
phosphorylated PEPC and a Ca2+-independent PEPC-PK that was
partially purified by affinity chromatography on blue dextran-agarose.
This PK activity was present in dry seeds, and PEPC phosphorylation in
situ during imbibition was not affected by the cytosolic
protein-synthesis inhibitor cycloheximide, by weak acids, or by various
pharmacological reagents that had proven to be effective blockers of
the light signal transduction chain and PEPC phosphorylation in
C4 mesophyll protoplasts. These collective data support the
hypothesis that this Ca2+-independent PEPC-PK was formed
during maturation of barley seeds and that its presumed underlying
signaling elements were no longer operative during germination.
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INTRODUCTION |
Higher-plant PEPC (EC 4.1.1.31) is subject to in vivo
phosphorylation of a regulatory Ser located in the N-terminal domain of
the protein. In vitro phosphorylation by a
Ca2+-independent, low-molecular-mass (30-39 kD)
PEPC-PK modulates PEPC regulation interactively by opposing metabolite
effectors (e.g. allosteric activation by Glc-6-P and feedback
inhibition by L-malate; Andreo et al., 1987 ), decreasing
significantly the extent of malate inhibition of the leaf enzyme
(Carter et al., 1991; Chollet et al., 1996; Vidal et al., 1996; Vidal
and Chollet, 1997). These metabolites control the rate of
phosphorylation of PEPC via an indirect target-protein effect (Wang and
Chollet, 1993; Echevarría et al., 1994; Vidal and Chollet,
1997).
Several lines of evidence support the view that this protein-Ser/Thr
kinase is the physiologically relevant PEPC-PK (Li and Chollet, 1993;
Chollet et al., 1996; Vidal et al., 1996; Vidal and Chollet, 1997). The
presence and inducible nature of leaf PEPC-PK have been established
further in various C3, C4,
and CAM plant species (Chollet et al., 1996). In all cases, CHX proved to be a potent inhibitor of this up-regulation process so that apparent
changes in the turnover rate of PEPC-PK itself or another, as yet
unknown, protein factor were invoked to account for this observation
(Carter et al., 1991; Jiao et al., 1991; Chollet et al., 1996).
Consistent with this proposal are recent findings about PEPC-PK from
leaves of C3, C4, and CAM
plants that determined activity levels of the enzyme to depend on
changes in the level of the corresponding translatable mRNA (Hartwell
et al., 1996).
Using a cellular approach we previously showed in
sorghum (Sorghum bicolor) and hairy crabgrass
(Digitaria sanguinalis) that PEPC-PK is
up-regulated in C4 mesophyll cell protoplasts
following illumination in the presence of a weak base
(NH4Cl or methylamine; Pierre et al., 1992;
Giglioli-Guivarc'h et al., 1996), with a time course (1-2 h) similar
to that of the intact, illuminated sorghum (Bakrim et al., 1992) or
maize leaf (Echevarría et al., 1990). This light- and
weak-base-dependent process via a complex transduction chain is likely
to involve sequentially an increase in pHc, inositol
trisphosphate-gated Ca2+ channels of the
tonoplast, an increase in cytosolic Ca2+, a
Ca2+-dependent PK, and PEPC-PK.
Considerably less is known about the up-regulation of PEPC-PK and
PEPC phosphorylation in nongreen tissues. A sorghum root PEPC-PK
purified on BDA was shown to phosphorylate in vitro both recombinant
C4 PEPC and the root
C3-like isoform, thereby decreasing the enzyme's
malate sensitivity (Pacquit et al., 1993). PEPC from soybean root
nodules was phosphorylated in vitro and in vivo by an endogenous PK
(Schuller and Werner, 1993; Zhang et al., 1995; Zhang and Chollet,
1997). A Ca2+-independent nodule PEPC-PK
containing two active polypeptides (32-37 kD) catalyzed the
incorporation of phosphate on a Ser residue of the target enzyme and
was modulated by photosynthate transported from the shoots (Zhang and
Chollet, 1997). Regulatory seryl phosphorylation of a heterotetrameric
( 2 2) banana fruit
PEPC by a copurifying, Ca2+-independent PEPC-PK
was shown to occur in vitro (Law and Plaxton, 1997). Although
phosphorylation was also detected in vivo and found to concern
primarily the -subunit, PEPC exists mainly in the dephosphorylated
form in preclimacteric, climacteric, and postclimacteric fruit.
In a previous study we showed that PEPC undergoes regulatory
phosphorylation in aleurone-endosperm tissue during germination of
wheat seeds (Osuna et al., 1996). Here we report on PEPC and the
requisite PEPC-PK in germinating barley (Hordeum vulgare) seeds. PEPC was highly phosphorylated by a
Ca2+-independent Ser/Thr PEPC-PK similar to that
found in other plant systems studied previously (Chollet et al., 1996);
however, the PK was already present in the dry seed and its activity
did not require protein synthesis during imbibition.
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MATERIALS AND METHODS |
Materials
Barley (Hordeum vulgare cv Beka) seeds were sterilized
in 2% (v/v) NaOCl for 20 to 30 min and washed sequentially with
sterile water, 10 mM HCl, and sterile water.
Whole or de-embryonated seeds were placed on filter papers soaked with
sterile water in a glass Petri dish and allowed to imbibe for the
necessary time at room temperature and in the presence or absence of
different inhibitors, as indicated in the figure legends. APS-IgG and
polyclonal antibodies were raised against a synthetic peptide
encompassing the N-terminal regulatory domain of
C4 PEPC from sorghum (Sorghum bicolor)
and the native enzyme, respectively (Vidal et al., 1981; Pacquit et al., 1995). [ -32P]ATP (10 Ci/mmol) and
[32P]Pi (200 Ci/mol) were purchased from
Amersham, Cellulase-RS was from Yakult Honsha (Tokyo, Japan), Macerase
pectinase was from Calbiochem, goat anti-rabbit IgG horseradish
peroxidase conjugate was from Bio-Rad, and protein A-Sepharose was from
Pharmacia. All other reagents were from Sigma.
Seed Extracts
Eight de-embryonated seeds were chopped and ground thoroughly in a
chilled mortar with washed sand and 1 mL of buffer A (0.1 M
Tris-HCl, pH 8.0, 5% [v/v] glycerol, 1 mM EDTA, 10 mM MgCl2, 10 µg
mL 1 chymostatin, 10 µg
mL1 leupeptin, 1 mM PMSF, 1 µM microcystin-LR, 50 mM KF, and 14 mM 2-mercaptoethanol). The homogenate was centrifuged at
15,000g for 4 min at 4°C, and the supernatant fluid (20 µL) was used as a crude extract.
For in vitro phosphorylation of seed PEPC by endogenous PEPC-PK,
proteins were extracted from 15 de-embryonated seeds in 2 mL of buffer
A. After centrifugation at 45,000g for 10 min, proteins were
precipitated from the supernatant fraction by the addition of
(NH4)2SO4
to 60% saturation, sedimented by centrifugation at 45,000g
for 5 min, and resuspended in 400 µL of buffer A. The protein
preparation was clarified by centrifugation at 45,000g for 5 min before use.
Isolation of Protoplasts
De-embryonated seeds were cut in half longitudinally, sterilized,
and soaked for 3 d in distilled water at 30°C in the dark. Endosperm, testa, and pericarp were removed, and the aleurone layers
were incubated for 20 to 22 h at 28°C in the dark in 5 mL of
digestion medium containing 20 mM Mes, pH 5.5, 0.5 M mannitol, 4% (w/v) Cellulase, 2% (w/v) Macerase
pectinase, and 10 µg mL1 chloramphenicol,
with gentle shaking. The digestion medium was sterilized by filtration
through a 0.22-µm filter (Millipore). After digestion the seed halves
were washed thoroughly with wash medium containing 0.5 M
mannitol and 20 mM Hepes, pH 7.0. Protoplasts were pooled
and filtered through a 75-µm nylon net and pelleted by centrifugation
at 600g for 5 min. The pellet was resuspended in 5 mL of
wash medium, centrifuged, and washed again. Finally, the pellet was
resuspended in 0.25 mL of buffer A, and soluble proteins were extracted
by passing the protoplast suspension several times through the needle
of a microliter syringe (Hamilton, Reno, NV).
Partial Purification of PEPC-PK from De-Embryonated Seeds and
Aleurone Protoplasts
All procedures were carried out at 4°C. Crude extracts from
de-embryonated seeds were prepared (100 seeds per 12 mL) in buffer B
(50 mM Tris-HCl, pH 8.0, 5% [v/v] glycerol, 1 mM EDTA, 1 mM DTT, and 1 mM PMSF).
The homogenate was centrifuged at 150,000g for 15 min in an
ultracentrifuge. The supernatant fraction (44 mg of protein) was loaded
at a flow rate of 0.2 mL min1 on a small column
containing 2 mL of BDA equilibrated with buffer C (50 mM Tris-HCl, pH 8.0, 5% [v/v] glycerol, and 1 mM DTT). After the column was rinsed with 40 mL
of buffer C, bound proteins were eluted with 0.5 M NaCl in buffer C. Eluted proteins were
precipitated by
(NH4)2SO4
to 60% saturation for 30 min and then centrifuged at
15,000g for 15 min. The protein pellet was resuspended in
200 µL of buffer D (50 mM Tris-HCl, pH 7.8, 20% [v/v] glycerol, and 1 mM DTT) and then
desalted by dialysis against two changes (100 mL each) of this same
buffer for 2 h before use in phosphorylation assays. The
purification procedure was essentially the same when aleurone
protoplasts were used as the source of PEPC-PK. Extracted proteins in
buffer B (0.7 mg of protein per 250 µL) were loaded onto a BDA column
equilibrated with buffer C (500 µL of packed gel). In this case, the
high-salt-eluted proteins were desalted by gel filtration through
Sephadex G-25 (1.4 mL of deposited gel) equilibrated with buffer D. The
final volume of the preparation was 200 µL.
In Vitro Phosphorylation Assays
An aliquot (45 µL) of BDA-purified aleurone-endosperm PEPC-PK
from protoplasts or 20 µL of BDA PEPC-PK from de-embryonated seeds
(in buffer D) was incubated with 6 µg of recombinant,
nonphosphorylated sorghum C4 PEPC (Pacquit et
al., 1993). The assay mixture (70 µL) consisted of 50 mM
Tris-HCl, pH 8.0, 5 mM MgCl2, 50 µM CaCl2, 20% (v/v) glycerol, 1 mM DTT, 1 µM microcystin-LR, and 8 µM [-32P]ATP (3 µCi). After
the sample was incubated for 1 h at 30°C in the presence or
absence of 1 mM EGTA, the assays were made 1%
(w/v) SDS, 10% (v/v) 2mercaptoethanol, and 0.002% (w/v)
bromphenol (final concentrations) and heated for 2 min at 90°C. The
samples were then subjected to SDS-PAGE (10% [w/v] acrylamide) and
autoradiography (Echevarría et al., 1990).
For PEPC-phosphorylation assays, proteins were partially purified from
crude extracts by
(NH4)2SO4
precipitation as described above. The malate sensitivity of PEPC was
recorded in the absence or presence of 1 mM
L-malate before and after incubation of the extract in the
presence or absence of either 2.5 mM ATP or 2.5 mM ATP plus 2.5 mM EGTA for up to 1 h at
30°C.
In Situ 32P Labeling and Immunoprecipitation
Four de-embryonated seed halves were soaked in 200 µL of
distilled water and 200 µCi of [32P]Pi in the
absence (control) or presence of various inhibitors (CHX, W7, W5, or
TMB-8). After 36 h of imbibition at room temperature, about
one-half of the solution was absorbed by the seeds. The seeds were
washed to remove nonabsorbed [32P]Pi, and
proteins were extracted as described above in 400 µL of buffer E
containing 100 mM Tris-HCl, pH 7.5, 20% (v/v) glycerol, 1 mM EDTA, 10 mM MgCl2, 10 µg mL1 chymostatin, 10 µg
mL1 leupeptin, 1 mM PMSF, 14 mM 2-mercaptoethanol, 50 mM KF, and 1 mM nonradioactive ATP (the addition of ATP was to minimize
in vitro 32P phosphorylation). The homogenate was
centrifuged at 12,000g for 2 min.
One-hundred microliters of the clarified supernatant fraction (0.06 unit of PEPC, which is about 225 µg of total protein) was immediately
mixed with SDS sample buffer (1% [w/v] SDS, 20% [v/v]
glycerol, 10% [v/v] 2mercaptoethanol, and 0.002% [w/v]
bromphenol blue, final concentrations). In parallel, 140 µL of the
supernatant fluid (0.084 unit of PEPC) was incubated with the
appropriate amount of purified polyclonal C4 PEPC
IgG from sorghum (34 µg of protein) for 1 h on ice. Protein
A-Sepharose beads were added to the incubated sample to 4% (w/v) and
vortexed briefly with a 5-min interval (Osuna et al., 1996). The beaded
immunocomplexes were sedimented by centrifugation at 12,000g
for 5 min, washed once with buffer containing 0.5 M Tris-HCl, pH 8.0, 1.5 M
NaCl, and 1% (v/v) Triton X-100, and once with 0.1 M Tris-HCl alone. The pellet was resuspended in
SDS sample buffer, heated for 10 min at 90°C, and centrifuged for 5 min at 12,000g at room temperature.
Both denatured preparations were analyzed by SDS-PAGE (10% [w/v]
acrylamide). Proteins in the gels were either stained with Coomassie
blue (total proteins) or transferred to a nitrocellulose membrane
(immunoprecipitated PEPC); both samples were autoradiographed (3 d at
 80°C) or analyzed with a phosphor imager (Fujix BAS 1000, Fuji,
Tokyo, Japan), and the seed PEPC was immunocharacterized as described
below.
Seed PEPC Immunocharacterization and Electrophoresis
The samples were subjected to SDS-PAGE (8% or 10% [w/v]
acrylamide, according to the method of Laemmli, 1970) for 14 h at 85 V and room temperature in an electrophoresis cell (Bio-Rad). For
western-blot experiments, proteins were electroblotted onto a
nitrocellulose membrane (N-8017, Sigma) at 10 V (3 mA
cm2) for 75 min in a semidry transfer-blot
apparatus (Bio-Rad). Protein bands were immunochemically labeled by
overnight incubation of the membrane at room temperature in 20 mL of
TBS (20 mM Tris-HCl and 0.15 M NaCl, pH 7.5)
containing protein A-purified polyclonal C4 PEPC
IgG (28 µg of protein). Subsequent detection was with a peroxidase
assay using affinity-purified goat anti-rabbit IgG horseradish
peroxidase conjugate.
Determination of PEPC Activity and Apparent Phosphorylation State
PEPC activity was recorded spectrophotometrically at 30°C at
optimal (8.0) and suboptimal (7.3 and 7.1) pH values using the NAD-malate dehydrogenase-coupled assay (at 2.5 mM PEP)
described by Echevarría et al. (1994). Assays were initiated by
addition of an aliquot of crude seed extract. An enzyme unit was
defined as the amount of PEPC that catalyzed the carboxylation of 1 µmol PEP min1 at pH 8.0 and 30°C. In the
APS-IgG-binding assay, crude extracts (25 µL, 0.02 unit of PEPC) were
incubated with 5 µL (10 µg of protein) of affinity-purified APS-IgG
for 5 min at 0°C, and then activity was recorded at pH 7.1. The
apparent phosphorylation state of PEPC was estimated by using the
velocity test, which is the PEPC activity at pH 8.0 divided by the PEPC
activity at pH 7.1 (Echevarría et al., 1994; Osuna et al.,
1996); the APS-IgG test, which is the PEPC activity at pH 7.1 plus a
saturating amount of the APS-IgG divided by the PEPC activity at pH 7.1 (Pacquit et al., 1995; Osuna et al., 1996); and the malate sensitivity test, which is the PEPC activity at pH 7.3 in the presence or absence
of 1 mM L-malate (Bakrim et al., 1992). Protein
was assayed by a sensitive dye-binding method (Bradford, 1976) using
BSA as a standard.
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RESULTS |
Characterization of PEPC in Protein Extracts from Dry and
Germinating Barley Seeds
Barley seeds were soaked for up to 4 d, and proteins from
aleurone endosperm were extracted at different times, resolved by SDS-PAGE, and analyzed by western blotting (Fig.
1A). Polyclonal IgGs (raised against
C4 PEPC from sorghum) immunodecorated a
constitutive 103-kD polypeptide in both dry and soaked seeds, and a
newly formed 108-kD polypeptide was detected after 1 d of
imbibition (Fig. 1A, lane 1) and was present for up to 4 d (Fig.
1A, lanes 2 and 4). No bands were detected with preimmune serum, and
sorghum C4-PEPC IgG immunoprecipitated all of the
PEPC activity from the barley seed extract (data not shown). We
previously obtained similar results for wheat seed PEPC using the same
sorghum C4-PEPC IgG and preimmune serum
(González et al., 1998). During this imbibition period PEPC
activity did not change significantly when expressed on a per-seed
basis, whereas its specific activity on a total-soluble-protein basis
showed an approximately 3-fold increase (Fig. 1B); this was likely due
to the mobilization of proteins from aleurone-endosperm tissues (Osuna
et al., 1996).
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| Figure 1.
Immunocharacterization of PEPC and time course of
PEPC activity changes during germination of barley seeds. A, At the
indicated times, soaked whole seeds were de-embryonated and soluble
protein from crude extracts (34 µg) was resolved by SDS-PAGE,
transferred onto nitrocellulose, and probed with polyclonal
anti-C4 PEPC IgGs (28 µg per 15 mL of incubation medium).
Immunolabeled proteins were detected by a horseradish peroxidase assay.
B, Equivalent preparations were assayed for PEPC activity at optimal pH
(8.0) and [PEP] (2.5 mM). Results are the means of six
experiments, which did not vary by more than 10%.
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In Situ Phosphorylation of Barley Seed PEPC
The in situ phosphorylation of PEPC during imbibition was
established after the seeds were fed for 36 h with
[32P]Pi and subsequent immunoprecipitation of
the enzyme from crude extracts. Both the 103- and 108-kD polypeptides
were radiolabeled (Fig. 2II, B, lane 2).
Consistent with this preliminary observation, malate inhibition of PEPC
decreased from 80% to 20% in protein extracts partially purified by
(NH4)2SO4
precipitation, indicating that the protein becomes highly
phosphorylated following seed imbibition. However, in many instances
(Nimmo et al., 1986; Rajagopalan et al., 1994; Chollet et al., 1996),
PEPC has been reported to be highly sensitive to partial proteolytic
degradation in crude extracts despite the presence of proteinase
inhibitors. Since germinating seeds contain high levels of proteolytic
enzymes, and truncation of the N-terminal phosphorylation domain causes the enzyme's functional and regulatory properties to be modified, we
devised two distinct, more reliable procedures to estimate the apparent
phosphorylation status of PEPC in seed extracts.
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| Figure 2.
Effects of various pharmacological reagents on in
situ protein phosphorylation in germinating barley seeds. Four
de-embryonated seed halves were soaked in 200 µL of distilled water
and 200 µCi of [32P]Pi, in the absence or presence of
25 µM CHX, 0.5 mM W7, or 0.5 mM
TMB-8 for 36 h. Following these treatments soluble protein
extracts were prepared and resolved by SDS-PAGE (10% acrylamide) and
visualized by Coomassie blue staining (I, A) and autoradiography (I,
B). Alternatively, the PEPC was immunoprecipitated, electrophoresed,
and transferred onto nitrocellulose for staining (II, A) and
autoradiography (II, B). Lanes 1, Purified, recombinant sorghum
C4 PEPC (5 µg); lanes 2, control seeds; lanes 3, CHX-treated seeds; lanes 4, W7-treated seeds; and lanes 5, TMB-8
treated seeds. Numbers in parentheses represent the apparent
phosphorylation state of PEPC from aleurone endosperm of soaked
de-embryonated seed halves in the presence of the corresponding
pharmacological reagents and in the control seeds using the velocity
test (A) and relative quantification (radioactive PEPC band expressed
as a percentage of the control) of the radiolabeled 103-kD PEPC subunit
using an image analyzer (B).
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Using sorghum recombinant C4 PEPC
(Echevarría et al., 1994) and wheat seed
C3 PEPC (Osuna et al., 1996), we showed
previously that the determination of the activity ratio at pH 8.0/7.1
was a sensitive and accurate way to estimate the degree of the
enzyme's apparent phosphorylation state in rapidly prepared protein
extracts from the corresponding organ. Indeed, phosphorylation induced a large increase in PEPC activity when measured at suboptimal pH and
[PEP] values (7.1 and 2.5 mM, respectively), whereas
there was little change at optimal pH (8.0). For the purified sorghum C4 enzyme extensively phosphorylated in vitro in
the presence of the catalytic subunit of mammalian cAMP-dependent PK,
the phosphorylation-dependent stimulation of activity was found to be
in the range of 5.5- to 7-fold at pH 7.1 and approximately 1.35-fold at
pH 8.0 (Echevarría et al., 1994). Dry and imbibed barley seeds
were collected and the soluble protein from aleurone-endosperm was
extracted. The activity ratio of PEPC decreased from 6.5 in dry seeds
to 2.8 after 1 d of imbibition (Table
I). A similar trend was also observed (from 6.3 to 2.2) after 1 d of imbibition of de-embryonated seeds (Table I). For sorghum C4 PEPC, this result
suggests that the constitutive 103-kD PEPC subunit is mainly in a
nonphosphorylated state in the dry seed and undergoes phosphorylation
during imbibition, along with the newly formed 108-kD subunit.
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Table I.
Time course of changes in the apparent
phosphorylation state of barley seed PEPC during germination estimated
by using the malate sensitivity, velocity, and APS-IgG tests on whole
seeds and on de-embryonated seed halves
At the indicated time after imbibition of whole or de-embryonated half
seeds, proteins from aleurone endospem were rapidly extracted and PEPC
activity was determined at different pH values and a [PEP] of 2.5 (or
0.5) mM. Values are the average of two independent
experiments, which did not vary by more than 15%.
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A second procedure was based on the use of APS-IgG, which in previous
work with sorghum (Pacquit et al., 1995) and wheat (Osuna et al., 1996)
was shown to mimic the effect of phosphorylation on the regulatory Ser.
As a reference, upon rapid binding of a saturating amount of the
antibody to the nonphosphorylated enzyme, C4 PEPC
activity at pH 7.1 increased approximately 7- to 9-fold, whereas the
fully phosphorylated species, which binds the antibody equally well
(Pacquit et al., 1995), responded relatively weakly to this treatment
(1.5-fold increase). In addition, because of the rapidity of the assay
(the APS-IgG effect is almost instantaneous) and the protection of PEPC
by the bound antibodies against possible proteolytic degradation of the
corresponding N-terminal domain, this test ensures that the increase in
the measured PEPC activity at pH 7.1 is due to an N-terminally intact
form of the enzyme.
In practice, a saturating amount of affinity-purified APS-IgG (or
preimmune serum in the controls) was added to rapidly prepared protein
extracts from dry or soaking seeds. PEPC activity was measured
spectrophotometrically in parallel cuvettes at pH 7.1, and the
experiment was completed within minutes. The results depicted in Table
I confirm those of the malate sensitivity and velocity tests and are
also consistent with extensive phosphorylation of PEPC during
germination of barley seeds. The fact that PEPC phosphorylation showed
a similar trend in the aleurone endosperm from both soaked whole or
de-embryonated seeds (Table I) shows that GAs produced in the embryo
were not necessary to trigger this regulatory mechanism.
Characterization of Barley Seed PEPC-PK and a Pharmacological Study
of the Signal Transduction Chain Leading to the Phosphorylation of PEPC
In a second set of experiments we addressed the question of
whether the upstream signaling components of the cascade leading to the
phosphorylation of PEPC, as identified in C4
mesophyll protoplasts (Pierre et al., 1992; Giglioli-Guivarc'h et al.,
1996), are also key elements in seeds. Protoplasts were prepared from aleurone cell layers dissected from seeds after 3 d of imbibition. In crude protein extracts from these protoplasts, the 103- and 108-kD
PEPC subunits were immunodetected by western blotting (Fig. 3I, lanes 1 and 2). Based on the velocity
and APS-IgG tests, aleurone-protoplast PEPC was found to be highly
phosphorylated (data not shown). Extracted proteins were also subjected
to chromatography on BDA according to the classical procedure described
by Jiao and Chollet (1989), which, in previous experiments on various
plant materials, partially purifies the
Ca2+-independent PEPC-PK (Chollet et al., 1996).
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| Figure 3.
Immunocharacterization of PEPC and identification
of PEPC-PK in protein extracts from barley aleurone protoplasts.
Protoplasts were isolated from aleurone layers dissected from seeds
after 3 d of imbibition. I, PEPC immunoblots. Lane 1, Proteins
from aleurone protoplasts (1.4 milliunits of PEPC, 6 µg of protein);
lane 2, proteins from aleurone layers (6.6 milliunits of PEPC, 24 µg
of protein); lane 3, purified, recombinant C4 PEPC from
sorghum (15 milliunits of PEPC, 54 µg of protein). Proteins were
resolved by SDS-PAGE (8% acrylamide) and then transferred onto
nitrocellulose membranes. II, In vitro phosphorylation assays performed
in the presence of exogenous, immunopurified C4 PEPC from
sorghum (0.2 unit of PEPC), BDA-purified proteins from aleurone
protoplasts (45 µL), and the other components of the reconstituted
phosphorylation reaction in the presence (+) or absence () of 1 mM EGTA. Proteins were resolved by SDS-PAGE (10%
acrylamide) and autoradiographed. A, Coomassie blue-stained gel. B,
Corresponding autoradiograph. The unmarked lane in A corresponds to the
stained protein standards.
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BDA-purified protoplast proteins were assayed in reconstituted
phosphorylation reactions in the presence of nonphosphorylated, recombinant sorghum C4 PEPC as a target and other
components of the phosphorylation reaction. The autoradiograph of
proteins subsequently resolved by SDS-PAGE shows that PEPC was
radiolabeled in vitro by a BDA-purified PK whose activity was
insensitive to the presence of the Ca2+ chelator
EGTA (Fig. 3II, B). Therefore, a homolog of the PEPC-PK identified in
other plant systems was present in aleurone cell protoplasts from
germinating barley seeds.
Since protoplasts prepared from aleurone layers after 3 d of
imbibition had a phosphorylated PEPC, we used de-embryonated seeds to
investigate the possible regulatory cascade leading to the control of
PEPC-PK activity and PEPC phosphorylation in situ. De-embryonated seeds
were soaked for 24 h, during which time the phosphorylation of
PEPC occurred (Table I) in the absence or presence of one of the
following components: propionic or acetic acid (to lower pHc); TMB-8, a
tonoplast-directed Ca2+-channel blocker; W7, a
calmodulin antagonist; CHX, a cytoplasmic protein-synthesis inhibitor
(Giglioli-Guivarc'h et al., 1996); and W5, a less-active analog of W7
(Hidaka et al., 1981). However, none of these pharmacological reagents
had a significant effect on PEPC phosphorylation in situ, as judged by
the velocity test (data shown in Fig. 2II, A) and the APS-IgG test
(data not shown). Incubation with propionic or acetic acid also did not
affect PEPC phosphorylation. The values from the velocity test were
1.98 and 2.10, respectively.
A related phosphorylation experiment was carried out to ensure that the
pharmacological reagents were taken up by the seeds and to examine in
more detail the effects of these drugs in vivo. After the seeds were
soaked for 36 h in the presence of [32P]Pi
and CHX, W7, W5, or TMB-8, soluble-protein extracts and
immunoprecipitated PEPC were analyzed by SDS-PAGE. The various drugs
did not change the protein patterns qualitatively, as judged by
Coomassie blue staining of the gel, although some quantitative
alterations were seen (Fig. 2I, A). This was also true for the
radiolabeled protein patterns in the corresponding autoradiograph (Fig.
2I, B) with the exception of those with CHX (Fig. 2I, B, lane 3) and,
more markedly, those with W7 (Fig. 2I, B, lane 4). In the latter case, this calmodulin antagonist inhibited the phosphorylation of many proteins, most of those detected in the control (Fig. 2I, B, lane 2).
The radiolabel in the major, 103-kD PEPC subunit was analyzed in more
detail by PEPC immunoprecipitation and found not to be modified by
TMB-8 (Fig. 2II, B, lane 5) and to be either slightly decreased or
increased by CHX and W7, respectively (Fig. 2II, B, lanes 3 and 4).
These latter changes could be at least partially accounted for by
corresponding variations in the amount of immunoprecipitated PEPC (Fig.
2II, A, lanes 3, 4). The radiolabel in the minor, 108-kD subunit was
detectable in the control and TMB-8-treated seeds (Fig. 2II, B, lanes 2 and 5) but was essentially absent in the CHX- and W7-treated samples
(Fig. 2II, B, lanes 3 and 4). Both compounds blocked the accumulation
of the 108-kD subunit (Fig. 2II, A, lanes 3 and 4), suggesting that a
Ca2+-/calmodulin-dependent event might be
involved in the accumulation of this inducible PEPC polypeptide. W5 did
not significantly affect either the radiolabeled protein pattern or the
accumulation of the 108-kD PEPC polypeptide (data not shown).
Therefore, the drugs were taken up by the seeds in sufficient amounts
to permit specific alterations to be exerted on a variety of proteins,
the major 103-kD PEPC polypeptide underwent modest changes at best in
terms of its phosphorylation state (Fig. 2II, B).
These results and those of the related velocity test (Fig. 2II, A,
numbers in parentheses) and APS-IgG test (data not shown) suggest that
the transduction chain controlling PEPC-PK activity in germinating
barley seeds is either at variance with that in C4 mesophyll protoplasts and leaves or is only
weakly active at the developmental stage examined. Assuming that this
latter possibility is valid, then PEPC-PK activity had to be already
present in the dry seed to account for the observed in situ
phosphorylation of PEPC during early imbibition. To test this
possibility, protein extracts from dry and 24-h soaked, de-embryonated
seeds were subjected to BDA chromatography. SDS-PAGE separation of
proteins from reconstituted phosphorylation assays containing an
aliquot of this protein fraction and a nonphosphorylated, purified
C4 PEPC from sorghum showed that the
Ca2+-independent PEPC-PK was even more active in
dry seeds than in soaked samples (Fig.
4B).
View larger version (18K):
[in this window]
[in a new window]
| Figure 4.
BDA PEPC-PK activity from dry and soaked (24 h)
whole seeds. BDA PEPC-PK from aleurone endosperm was isolated
chromatographically, and in vitro phosphorylation assays were performed
in the presence of exogenous, immunopurified C4 PEPC from
sorghum (0.2 unit of PEPC), BDA-purified proteins from aleurone (20 µL), and the other components of the reconstituted phosphorylation
reaction in the presence (+) or absence () of 1 mM EGTA.
Radiolabeled proteins were resolved by SDS-PAGE (10% acrylamide) and
detected by autoradiography. A, Coomassie blue-stained gel. B,
Corresponding autoradiograph.
|
|
The addition of ATP to an aliquot of a crude
(NH4)2SO4
fraction (0%-60% saturation) obtained from de-embryonated dry
seeds led to a markedly decreased malate sensitivity of endogenous PEPC from 80% to 16%, and the Ca2+ chelator EGTA had
no significant effect on this change (Fig. 5). Consistent with previous results from
in situ radiolabeling experiments, this latter observation shows that
essentially no Ca2+-dependent PEPC-PK
activities are present in seeds. Therefore, we conclude that dry barley
seeds contain a slow turnover, Ca2+-independent
PEPC-PK that starts phosphorylating PEPC in the aleurone tissue upon
imbibition and maintains a high phosphorylation state of the target
enzyme during subsequent germination to achieve efficient protection
against feedback inhibition by L-malate.
View larger version (20K):
[in this window]
[in a new window]
| Figure 5.
ATP-dependent changes in malate sensitivity of
PEPC in protein extracts from aleurone endosperm of dry seeds.
(NH4)2SO4-precipitated proteins in
crude extracts from aleurone-endosperm tissues from 15 de-embryonated
seeds were sedimented by centrifugation at 45,000g for 5 min and then resuspended in 400 µL of buffer A (3.1 mg protein
mL1). The protein preparation was clarified by
centrifugation at 45,000g for 5 min. ATP (2.5 mM), EGTA (2.5 mM), or ATP plus EGTA were added
to aliquots and incubation was performed at 30°C for up to 60 min.
Malate sensitivity of PEPC was recorded in the absence or presence of 1 mM L-malate (expressed as the percentage of
inhibition) before and after incubation of the extract. Values are the
averages of two independent experiments, which did not vary by more
than 15%.
|
|
| |
DISCUSSION |
PEPC specific activity was shown to increase during the
germination of barley seeds, whereas activity expressed on a per-seed basis remained almost constant. Western analysis revealed that anti-sorghum C4 PEPC polyclonal antibodies
detected two main polypeptides with molecular masses of 103 and 108 kD.
These results are reminiscent of wheat and castor seed PEPC, for which
an inducible (108-kD) PEPC subunit was immunocharacterized during the
same stage of germination (Sangwan et al., 1992; Osuna et al., 1996).
Malate sensitivity and velocity tests suggested that PEPC is
phosphorylated in vivo in soaking seeds. The APS-IgG test (Pacquit et
al., 1995) confirmed these results and also ensured both the presence
of the N-terminal phosphorylation domain and the intactness of the PEPC
N terminus in seed extracts (Nimmo et al., 1986; Rajagopalan et al., 1994). The major 103-kD and the minor 108-kD PEPC polypeptides were also shown to be radiolabeled in situ. These collective data not
only established phosphorylation of PEPC as a mechanism that regulates
the activity of the cereal grain enzyme in vivo (Osuna et al., 1996)
but also validated the aleurone endosperm of barley seed as a potential
model system to undertake the study of the requisite PEPC-PK and the
presumed signal transduction pathway.
Barley aleurone cells have been the focus of recent studies of
signaling in plants (Gilroy and Jones, 1992; Gilroy, 1996; Schuurink et
al., 1996; Swanson and Jones, 1996). These cells possess a three-cell
aleurone layer, unlike wheat, and the preparation of protoplasts has
been described (Gilroy and Jones, 1992; Heimovaara-Dijkstra et al.,
1994). In this study we report the presence of a
Ca2+-independent PEPC-PK activity partially
purified on BDA using aleurone-cell protoplasts and aleurone-endosperm
tissue of dry and germinating barley seeds (Figs. 3II, B, and 4B). This
seed PK was similar to the PEPC-PK already described in various plant systems, e.g. C4 (maize, sorghum, and hairy
crabgrass), CAM (Mesembryanthemum and
Bryophyllum), and C3 (wheat and
tobacco) leaves; sorghum, hairy crabgrass, and barley mesophyll
protoplasts; and soybean root nodules (Chollet et al., 1996; Vidal and
Chollet, 1997; Zhang and Chollet, 1997). This
Ca2+-independent PEPC-PK appears to be unique in
that its activity was not modulated directly by second messengers or by
phosphorylation/dephosphorylation processes but, rather, through rapid
changes in its apparent turnover rate (Carter et al., 1991; Jiao et
al., 1991; Chollet et al., 1996; Hartwell et al., 1996; Vidal and
Chollet, 1997).
It has been proposed that a light-modulated signal transduction cascade
involving alkalinization of pHc, an increase in cytosolic [Ca2+] and the activity of a
Ca2+-dependent PK, and protein synthesis are
required for up-regulation of the
Ca2+-independent PEPC-PK in
C4 mesophyll protoplasts (Giglioli-Guivarc'h et
al., 1996). In contrast, our results show that
Ca2+-independent PEPC-PK activity is already
present in dry seeds (Fig. 4B) and that CHX and other pharmacological
reagents (TMB-8, W7, and weak acids) that block the above-mentioned
steps of the cascade in C4 mesophyll-cell
protoplasts did not significantly impair the in situ phosphorylation of
PEPC in germinating seeds (Fig. 2II and data not shown for weak acids).
Our results allow us to conclude that this
Ca2+-independent PEPC-PK activity is not
inducible by a C4-like signaling mechanism in the
aleurone endosperm of barley during germination. The fact that PEPC
phosphorylation occurs in the aleurone endosperm from both soaked whole
and de-embryonated seeds (Table I) shows that GAs produced by the
embryo were not necessary to trigger this regulatory mechanism. A
consistent working hypothesis to account for these observations is that
the activation of the putative transduction chain and the up-regulation
of PEPC-PK have already occurred during seed maturation, when PEPC
activity and malate content show a tremendous increase in barley
pericarp, testa, aleurone, and starchy endosperm tissues (Macnicol and
Jacobsen, 1992). It has also been reported that ABA induces an
intracellular pH increase, possibly because of the activation of plasma
membrane H+ pumps in barley aleurone
(Heimovaara-Dijkstra et al., 1994).
In reconstituted assays a variety of PKs (the catalytic subunits of
mammalian cAMP-dependent PKs [Terada et al., 1990], plant Ca2+-dependent PKs [Echevarría et al.,
1988; Ogawa et al., 1992], and Ca2+-independent
PEPC-PKs [Li and Chollet, 1993]) are able to specifically phosphorylate the target PEPC in its N-terminal domain (Chollet et al.,
1996; Vidal et al., 1996; Vidal and Chollet, 1997). This casts doubt
about the nature and identification of the physiological PK. Data from
the present work show that the in vivo effect of the calmodulin
antagonist W7 was a marked decrease in the radiolabeling of many
soluble seed proteins (Fig. 2I, B, lane 4) but not the phosphorylation
state of PEPC (Fig. 2I, B, and 2II, B, lane 4). In contrast, W5, a
less-active analog of W7, affected neither the radiolabeled protein
pattern nor PEPC phosphorylation (data not shown). Since W7 is known to
act on a broad spectrum of
Ca2+-/calmodulin-dependent PKs and
Ca2+-dependent, calmodulin-like PKs (Roberts and
Harmon, 1992; Abo-El-Saad and Wu, 1995), our results rule out the
possibility that members of these classes of protein-Ser/Thr kinases
are involved in the in situ phosphorylation of barley seed PEPC. This
pharmacology-based observation provides strong support for the view
that the Ca2+-independent PEPC-PK identified to
date in a variety of plant systems, including seeds (Figs. 3II, B, and
4B), is the best candidate for the physiological PEPC-PK (Li and
Chollet, 1993; Chollet et al., 1996; Hartwell et al., 1996; Vidal et
al., 1996; Vidal and Chollet, 1997; Zhang and Chollet, 1997). Our
results also demonstrate that the accumulation of the inducible, 108-kD
PEPC subunit during germination was blocked by both CHX and the
calmodulin antagonist W7 (Fig. 2II, A, lanes 3 and 4) but not by W5
(data not shown), suggesting that a
Ca2+-/calmodulin-dependent event(s) is involved
in the synthesis of this PEPC polypeptide during germination.
Ironically, a relatively high PEPC-PK activity is found in dry seeds
along with the nonphosphorylated form of its target PEPC (Table I; Fig.
4B). It has been reported that the in vitro phosphorylation of
C4 PEPC is inhibited markedly by
L-malate via a target (i.e. substrate) effect (Wang and
Chollet, 1993; Echevarría et al., 1994). Since malate levels
are high at late stages of seed maturation and in dry seeds (Macnicol
and Jacobsen, 1992; Drozdowicz and Jones, 1995), this could explain why
the PEPC-phosphorylation state is low but PEPC-PK is present and fully
active (Fig. 4B). Alternatively, a high PEPC phosphatase activity
during late stages of seed maturation could account for the observed
anomaly between PEPC-PK activity and the apparent phosphorylation state
of PEPC (Duff and Chollet., 1995; Smith et al., 1996). This could be
the case in barley seeds, because an increase in okadaic-acid-sensitive protein phosphatases is detected in aleurone cells during maturation in
relation to the ABA response (Kuo et al., 1996).
Finally, several roles have been proposed for L-malate
production via PEPC during seed maturation: the acidification of the endosperm (Mikola and Virtanen, 1980; Macnicol and Jacobsen, 1992; Heimovaara-Dijkstra et al., 1994; Drozdowicz and Jones, 1995), the
anaplerotic replenishment of citric-acid-cycle intermediates to sustain
amino acid synthesis and protein filling of seeds (González et
al., 1998; Macnicol and Raymond, 1998), and fatty acid synthesis in
fat-rich seeds (Sangwan et al., 1992). It has been firmly established that the carbon flux through PEP by the PEPC branch is 3 to 5 times
greater than that through the pyruvate kinase branch in aleurone from
maturing barley seeds (Macnicol and Raymond, 1998). Malate is
accumulated in the endosperm during maturation (Macnicol and Jacobsen,
1992), whereas its concentration markedly decreases during germination
(Drozdowicz and Jones, 1995).
Our results show that the PEPC capacity and apparent phosphorylation
state are enhanced within the first 24 h of imbibition in aleurone
endosperm (Fig. 1; Table I), indicating that the enzyme maintains
sustained activity. This PEPC activity could account for a continued
synthesis of malate to be released in the endosperm, contributing to
its acidification (Mikola and Virtanen, 1980; Heimovaara-Dijkstra et
al., 1994), or to be efficiently transported to the developing embryo
(Drozdowicz and Jones, 1995). Whenever high levels of
L-malate are produced, PEPC needs enhanced protection
against this feedback inhibitor for the carboxylation reaction to
proceed efficiently in this adverse condition (Chollet at al., 1996).
Ours results show that this was the case in the germinating barley
grain (Table I), and this has recently been shown to occur in guard
cells during stomatal opening as well (Zhang et al., 1994; Du et al.,
1997).
| |
FOOTNOTES |
1
This research was supported by grants from the
Dirección General de Investigación Científica y
Técnica (no. PB 92-0675), the Dirección General de Ensenan
za Superior e Investigacion Científica (no. B1097-1205-C02-02),
and the Acción Integrada Hispano-Francesa (no. HF96-0200), and by
the Grupo de Investigación de Fisiología Vegetal from La
Junta de Andalucía. This work was also partially supported by
the Rhone-Poulenc Agro S.A., Torre de la Reina, Sevilla, Spain.
*
Corresponding author; e-mail echeva{at}cica.es; fax
34-95-461-5780.
Received June 12, 1998;
accepted October 29, 1998.
| |
ABBREVIATIONS |
Abbreviations:
APS, antiphosphorylation site.
BDA, blue
dextran-agarose.
CHX, cycloheximide.
PEPC, PEP carboxylase.
PK, protein kinase.
pHc, cytosolic pH.
TMB-8, 8-(diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride.
W5, N-(6-aminohexyl)-1-naphthalenesulfonamide hydrochloride.
W7, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide
hydrochloride.
| |
ACKNOWLEDGMENTS |
The authors thank Marion and Phil Isle for correcting the
manuscript. The photographic work of M.J. Cubas is appreciated.
| |
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Abstract
Introduction