BDA: 1 ml equilibrated in medium B at a flow rate of 0.05 mlmin. After thorough washing with
medium B, bound proteins were eluted with medium B containing 500 mM NaCl. Peak frac-
tions were pooled and precipitated with 60 satu- ration of ammonium sulfate, after which proteins
sedimented by centrifugation were resuspended in 100 ml of 50 mM Tris – HCl pH 8, 30 glycerol, 1
mM dithiothreitol, and stored at − 20°C until use.
2
.
5
. In 6itro phosphorylation The reconstituted phosphorylation assay 50 ml
system contained: 50 mM Tris – HCl, pH 8, 5 mM MgCl
2
, 0.04 mM CaCl
2
, 20 glycerol, 1 mM DTT, 74 kBq [g-
32
P]ATP 37 GBqmmol, 0.25 mM
P1P5-diadenosine-5-pentaphosphate an
adenylate kinase inhibitor, 4 mM phosphocre- atine, 10 U of creatine phosphokinase compo-
nents of the ADP-scavenging system, and an aliquot of the desalted crude extract 30 mg of
protein. In assays where partially, BDA-purified seed protein kinase was tested, 0.2 U6 mg of C
4
PEPCs S8; wild type or S8D, Ser8 replaced by aspartate were added as phosphorylation and
control targets [14]. These Sorghum PEPC forms were produced as recombinant proteins in Es-
cherichia coli and subsequently purified from bac- terial extracts by immunoaffinity chromatography
[15]. In some assays, the PEPC target was incu- bated in the presence of 10 mg of APS-IgG for 10
min at 4°C prior to in vitro phosphorylation test. After 45 min incubation at 30°C, the phosphoryla-
tion reaction was halted by addition of 10 ml of SDS sample buffer 50 mM Tris – HCl, pH 8, 1
wv SDS, 10 vv 2-mercaptoethanol, 20 v v glycerol and 1 wv bromophenol blue and
heated for 2 min at 100°C. Denatured samples were analyzed by SDSPAGE 10 acrylamide
according to [11], and autoradiographed.
2
.
6
. In situ
32
P-labeling and immunoprecipitation of seed PEPC
De-embryonated seeds ten were allowed to imbibe in 200 ml of distilled water containing
74 × 10
5
Bq of [
32
P]phosphate specific radioactiv- ity 74 × 10
2
GBqmol for 48 h at room tempera- ture. The seeds were washed thoroughly 5 times
with distilled water to remove remaining labeled phosphate and proteins were extracted in 1 ml
medium A as described above. The homogenate was centrifuged at 15 000 × g for 5 min. An
aliquot of the clarified sample containing 14.4 mU of PEPC was incubated overnight, 4°C, with the
appropriate amount of protein A-purified APS- IgG 20 mg of protein. Protein A Sepharose beads
were added to the incubated sample to 6 wv and vortexed briefly. The beaded immunocom-
plexes were washed five times with washing medium 500 mM Tris – HCl pH 8, 1.5 M NaCl
and 1 vv Triton X-100. The final pellet of centrifugation was solubilized in 100 ml of SDS
sample buffer, boiled for 5 min, and protein were separated by 10 SDS-PAGE [11] for 2 h at 100 V
and room temperature. Proteins were electrob- loted overnight to a nitrocellulose membrane at 30
V, 4°C in a Bio-Rad transfert blot apparatus. The membrane was dried and exposed to Kodak film
at − 80°C, then seed PEPC was immunocharac- terized as described in Section 2.3.
2
.
7
. PEPC acti6ity assays and
L
-malate sensiti6ity test
Unless otherwise stated, the standard assay con- ditions were as described in [2,16].
2
.
8
. Determination of proteins and
L
-malate Soluble protein concentration was measured ac-
cording to [17] using the Bio-Rad dye reagent and bovine serum albumin as a standard.
L
-malate concentration was determined in aliquots of the
seed crude extracts by a spectrophotometric assay in the presence of NAD-dependend MDH and
NADH according to [18].
3. Results
Soluble proteins were extracted from de-embry- onated dry seeds or germinating seeds separated
from the seedling, and PEPC analyzed by SDS- PAGE and Western blot experiments. PEPC-spe-
cific APS-IgG cross-reacted with two polypeptides with slightly different molecular masses in the
range of 108 – 110 kDa, the larger one being barely detectable in gel protein patterns from dry seed
extracts Fig. 1. These PEPC polypeptides were also detected by using C-terminal IgGs raised
against a synthetic peptide not shown thus sug-
Fig. 1. Immunological detection of Sorghum seed phospho- enolpyruvate carboxylase PEPC. Soluble proteins from de-
salted seed extracts 30 ml aliquot containing, 5.2, 7.6, 9.6 mU of PEPC from dry seed, 3 and 5 days of germination in
distilled water, respectively were subjected to SDS-PAGE. Lane 1, dry seed; lane 2, germinated for 3 days in water; lane
3, germinated for 5 days in water.
found to be close to 90 mM Table 1 and did not vary significantly in the range of pH values pH
7 – 8 investigated not shown. The IC
50
value for the feedback inhibitor
L
-malate at suboptimal PEP concentration 90 mM and pH 7.3 was 75 mM
Table 1. During seed imbibition in distilled water for 5 days, the K
m
for PEP decreased slightly 65 mM, while the IC
50
for
L
-malate was increased to 220 mM, consistent with the observed decrease in
the inhibition by
L
-malate 160 mM, from 75 to 30 during the same imbibition period Table 1.
In addition, the activity ratio of PEPC pH 87.3 was shown to decrease from 2.6 to 1.4 during seed
germination Fig. 1.
Both the decrease in
L
-malate sensitivity of PEPC, as measured at suboptimal but physiologi-
cal pH 7.3, and in the activity ratio pH 87.3 have been observed already in the case of the
photosynthetic Sorghum and maize enzyme after a darklight transition, as well as for various CAM
and C
3
plant PEPCs, including that of germinating wheat and barley seeds [1,6,9]. It is now well
established that this is due to phosphorylation by a calcium-independent PEPCk of the regulatory
serine in the consensus, N-terminal domain of PEPC [1,3,9,14,19]. This process was therefore
investigated in more detail on Sorghum seeds that were allowed to imbibe for 3 days in the presence
of [
32
P]phosphate, during which the sensitivity of PEPC to
L
-malate was decreased to about 50. In a Western blot, APS-IgG revealed a PEPC band
Fig. 2B1 which was found to be radiolabeled following autoradiography of the membrane Fig.
2A1. In this case, the two PEPC bands were not resolved; this was presumably due to partial recov-
ery of the enzyme during the immunoprecipitation process, however, the experiment clearly estab-
lished that at least one PEPC subunit underwent in vivo phosphorylation during seed imbibition.
gesting that they are intact PEPC subunits. The APS-IgG-based observations documented that the
PEPC subunits from Sorghum seeds possess the consensus phosphorylation domain common to all
plant PEPCs known so far [3]. PEPC activity was shown to increase significantly, when measured
both in non-limiting pH 8, 2.5 mM PEP and limiting pH 7.3, 0.09 mM PEP, together with the
amount of both polypeptides during seed imbibi- tion Fig. 1.
The substrate PEP saturation curve for PEPC has been determined in desalted extracts from
de-embryonated dry seeds at pH 8. The curve was right rectangular hyperbola typical Michaelis –
Menten kinetics; not shown and the K
m
value was
Table 1 Kinetic parameters of Sorghum seed phosphoenolpyruvate carboxylase PEPC
a
PEPC mUseed Seed treatment
IC
50
mM K
m
PEP mM Inhibition by
L
-malate 5.2
Dry 75
0.09 0.075
9.6 0.22
0.065 Germinated
30
a
A homogeneous sample of seedlings ten showing similar growth was used for the kinetic analyses of PEPC from germinated seeds. PEPC activity was measured from an aliquot of desalted protein extract 30 mg proteins, at pH 8, 2.5 mM PEP and the
other components of the carboxylation reaction as described in Section 2. IC
50
values for
L
-malate and percent inhibition by
L
-malate 160 mM were determined at pH 7.3, 90 mM PEP. The K
m
was calculated from Lineweaver–Burk plots from substrate PEP saturation curves at pH 8. Data are means of three experiments with different batches of seeds; S.E. = 10.
Fig. 2. In vivo phosphorylation of phosphoenolpyruvate car- boxylase PEPC in imbibing seeds. SDS PAGE, electrotrans-
fer of proteins from the gel and radiochemicalimmunological detection of PEPC polypeptide are as described in Section 2.
Lane 1, proteins from the seed extract 14.4 mU of PEPC; lane 2, molecular weight markers. A Autoradiograph of the
membrane. B Immunological detection of PEPC.
Fig. 4. In vitro phosphorylation of C
4
phosphoenolpyruvate carboxylase PEPC by partially purified seed phospho-
enolpyruvate carboxylase kinase PEPCk. The seed PEPCk was purified by chromatography on blue dextran agarose.
The assay media contained the components of the phosphory- lation reaction Section 2 and exogenous, immunopurified C
4
PEPC from Sorghum: Lane 1, 0.2 U6 mg of S8D PEPC mutant; lane 2, 0.2 U6 mg of wild type recombinant PEPC;
lane 3, 0.2 U6 mg of wild type recombinant PEPC + 0.5 mM EGTA; lane 4, 0.2 U6 mg of wild type recombinant PEPC
preincubated with 10 mg of affinity-purified APS-IgG 15 min incubation at 4°C. A Coomassie blue staining of proteins.
B Corresponding autoradiography.
PEPC phosphorylation was also investigated in vitro in crude desalted protein extracts from de-
embryonated dry seeds followed by SDS-PAGE analysis and autoradiography of phosphorylated
proteins. The endogenous C3-like PEPC was shown to be radiolabeled in the absence Fig.
3B1, or the presence Fig. 3B2, of EGTA, while addition of the APS-IgG markedly inhibited
PEPC phosphorylation Fig. 3B3. This suggested that Sorghum seeds contain a Ca
2 +
-independent PEPCk phosphorylating the N-terminal domain of
the seed PEPC. This PEPCk was subsequently purified from dry seeds by affinity chromatogra-
phy on blue dextran agarose and assayed in phos- phorylation
assays in
the presence
of the
recombinant, non-phosphorylated C
4
PEPC as target [13,20]. Consistently, the partially purified
enzyme was found to be insensitive to calcium chelation Fig. 4B2,3. In addition, it did not
phosphorylate either purified, recombinant S8D C
4
PEPC target aspartate mutant; Fig. 4B1, or wild type C
4
PEPC and seed PEPC in the presence of the APS IgG Fig. 4B4, thus demonstrating
that phosphorylation was on the regulatory serine Ser 8 in the case of the C
4
PEPC of the N-termi- nal domain in both type of PEPC. These findings
also established that this protein kinase is already present in dry seeds, as found in previous work
with wheat and barley seeds [6,7,9]. Further exper- iments must be performed to clarify whether this
enzyme is similar to the 30 – 37 kDa PEPCk al- ready described in various plant systems [1].
As mentioned above, PEPC activity showed an approximate 1.8-fold increase on a per seed basis
when measured under non-limiting conditions of substrate and pH, after 5 days of imbibition
Table 1; Fig. 1. Since the effect of phosphorylat- ing the enzyme on its velocity is low at this pH
value [21], this change reflected a corresponding enhancement of PEPC protein content, which is
supported by the protein pattern of the Western blot experiment Fig. 1. Therefore, both PEPC
protein accumulation and phosphorylation took place during seed imbibition. Whether the former
is due to enhancement of transcription or changes in the stability of PEPC mRNAprotein is not
known.
The posttranslational regulation of PEPC in the physiological context of the seed was investigated
further by perturbing germination and determin- ing how PEPCk activity and PEPC phosphoryla-
Fig. 3. In vitro phosphorylation of phosphoenolpyruvate carboxylase PEPC in desalted protein extracts from de-em-
bryonated dry seeds. Seed proteins containing 14.4 mU of PEPC were incubated in reconstituted assays as described in
Section 2 and subsequently analyzed by SDS PAGE. Lane 1, complete assay; lane 2, + 0.5 mM EGTA; lane 3, + 10 mg of
APS-IgG. A Coomassie blue staining of proteins. B Corre- sponding autoradiography.
Table 2 Effect of NaCl on phosphoenolpyruvate carboxylase PEPC activity, sensitivity to
L
-malate phosphorylation state and
L
-malate content during seed germination
a
PEPC mUseed Seed treatment
inhibition by
L
-malate
L
-malate nmol10 seeds Seedling mm
75 Dry
71.5 5.2
30 9
30 Germinated
9.6 Germinated+NaCl
mM 45
13.9 20
50 9
56 31
8 18
100 150
6.9 60
39 15
64 Not determined
6.4 8
200
a
Experimental conditions were as described in the legend of Table 1. Data are means of three experiments with different batches of seeds; S.E. = 10.
tion status responded to these altered conditions. Sodium chloride was shown to severely inhibit the
germination of Sorghum seeds Table 2 in a con- centration-dependent manner and therefore was
used to assess this point. In salt-treated plants, it was seen that the increase in PEPC activity
polypeptidephosphorylation state Table 2; Fig. 5 was significantly reduced as the salt concentra-
tion was raised in the imbibing medium. More- over, in vitro assays demonstrated that NaCl had
no significant effect on both PEPC not shown and PEPCk activity Fig. 6B6,7, and PEPCk con-
tent during seed germination Fig. 7B3. Therefore it is not clear how NaCl could affect negatively
PEPC phosphorylation in vivo. In previous work on C
4
PEPC,
L
-malate was shown to inhibit PEPC phosphorylation both in reconstituted assays [21]
and in situ, in mesophyll protoplasts from Sor- ghum during induction of the transduction cascade
by light and a weak base [14]. When tested in reconstituted assays containing protein extracts
from germinated seeds, this compound was found to block in a similar manner the phosphorylation
of exogenous C
4
PEPC, even at the low concentra- tion of 1 mM Fig. 6B2 – 5. Interestingly,
L
-malate was shown to be present in dry seeds and to
undergo an approximate 8-fold decrease in con- centration during germination in distilled water
Table 2. In contrast,
L
-malate remained rela- tively high in NaCl-treated seeds Table 2. Fi-
nally, imbibing Sorghum seeds in the presence of different concentrations of
L
-malate mimicked the negative effect of salt on seed germination and
caused a marked inhibition of PEPC phosphoryla- tion in vivo in keeping with a high
L
-malate sensitivity of the enzyme; Table 3, while the
PEPCk activity was not affected Fig. 7B2. These observations are consistent with the hypothesis
that NaCl inhibits PEPCk activityPEPC phos- phorylation in vivo through the
L
-malate content of seeds.
4. Discussion
