Directory UMM :Data Elmu:jurnal:P:PlantScience:PlantScience_Elsevier:Vol151.Issue1.2000:

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Posttranslational regulation of phosphoenolpyruvate carboxylase

during germination of

Sorghum

seeds: influence of NaCl and

L

-malate

Mohamed Nhiri

_{, Naı¨ma Bakrim}

_{, Nadia Bakrim}

_{, Zakia El Hachimi-Messouak}

_,

Cristina Echevarria

_{, Jean Vidal}

_*

a_{Laboratoire de Biotechnologie}_,_{De´partement de Biologie}_,_Uni₆_{ersite´ A}_._Essaˆdi_,_{Faculte´ des Sciences et Techniques}_,_BP₄₁₆_,_Tanger_,_Morocco b_{Institut de Biotechnologie des Plantes}_,_baˆt_.₆₃₀_,_{UMR CNRS}₈₆₁₈_,_Uni₆_{ersite´ de Paris}_-_Sud_,_{Centre d}_’_Orsay_,₉₁₄₀₅_{Orsay Cedex}_,_France

c_{Laboratoire de Biochimie}_,_{De´partement de Biologie}_,_Uni₆_{ersite´ Med V}_._Rabat_,_Rabat_,_Morocco

d_{Laboratorio de Fisiologia Vegetal}_,_{Facultad de Biologia}_,_Uni₆_{ersidad de Se}₆_illa_,_A₆_da_._{Reina Mercedes}_,_Se₆_illa_,_Spain Received 23 June 1999; received in revised form 6 September 1999; accepted 20 September 1999

Abstract

Phosphoenolpyruvate carboxylase (EC 4.1.1.31: PEPC) was characterized in de-embryonated Sorghumseeds, focusing on the interaction between metabolites and posttranslational control of the enzyme by phosphorylation. Two PEPC polypeptides (108 and 110 kDa) were resolved by SDS/PAGE and shown to increase, in parallel with PEPC activity during seed germination. PEPC displayed very lowKmvalues for PEP (90mM) and inhibition constant (IC50) forL-malate (75mM) in desalted protein extracts

from de-embryonated dry seeds. The inhibition of PEPC by 0.16 mML-malate, pH 7.3, decreased from 70 to 30%, along with

a consistent increase in IC50(75 – 220mM) after 5 days of germination. PEPC phosphorylation was established both in vivo, after

imbibing the seeds with [32_{P]phosphate, and in vitro in reconstituted assays. A PEPC kinase (PEPCk) was partially purified from}

seed protein extracts by blue dextran agarose chromatography and shown to be independent of calcium and to phosphorylate both seed and recombinant C4PEPC fromSorghumon the enzyme’s N-terminal domain. Seed germination, PEPC accumulation

and phosphorylation were severely inhibited in the presence of NaCl in the imbibing medium, although PEPCk content was not altered. However, in vitro, NaCl had no effect on both PEPCk activity and PEPC phosphorylation. On the other hand,L-malate was a potent inhibitor of seed PEPCk activity in in vitro assays. Since NaCl also decreased the rate ofL-malate consumption in

the imbibing grain, the salt inhibition of PEPC phosphorylation was suggested to be due to the concentration-dependent blocking of PEPCk activity in vivo by this compound. Consistent with these data, germination and PEPC phosphorylation were inhibited, while PEPCk levels were not altered, when seeds were germinated in the presence of L-malate. © 2000 Published by Elsevier Science Ireland Ltd. All rights reserved.

Keywords:Sorghum; Seed phosphoenolpyruvate carboxylase; Enzyme kinetics; Protein phosphorylation; Salt stress

www.elsevier.com/locate/plantsci

1. Introduction

Phosphoenolpyruvate carboxylase (PEPC) is widely distributed in plants where it is involved in a number of physiological contexts [1]. In C4 plants, a specific isoenzyme is involved in the initial fixation of atmospheric CO2during C4 pho-tosynthesis. In illuminated leaves, this PEPC form is phosphorylated on a regulatory serine located in the N-terminus of the enzyme subunit [1 – 3]. The Abbre6iations:APS-IgG, anti-phosphorylation site antibody; BDA,

blue dextran agarose; IC50, concentration of the inhibitor causing a 50% decrease in the initial PEPC activity; Km, Michaelis – Menten constant; PEP, phosphoenolpyruvate; PEPC, phosphoenolpyruvate carboxylase; PEPCk, phosphoenolpyruvate carboxylase kinase.

* Corresponding author. Tel.:+33-1-6933-6344; fax:+ 33-1-6933-6423.

E-mail address:jean.vidal@ibp.u-psud.fr (J. Vidal)

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Ca2+_{-independent PEPC kinase (PEPCk) is}

up-regulated via a light-dependent transduction cas-cade [3]. In seeds, a heterotrophic PEPC whose activity is increased during maturation is thought to provide carbon skeletons for the synthesis of amino and fatty acids [4 – 8], thus contributing to accumulation of protein and lipid, while its role during germination is still unclear [9]. Consistent with this view are recent results on barley seeds showing that the carbon flux through PEPC rel-ative to pyruvate kinase increases 3 – 5-fold to-gether with a 5-fold increase in PEPC activity during maturation and acidification of the starchy endosperm which is due to L-malate

ac-cumulation [8]. Two immunologically-related PEPC polypeptides (103 and 108 kDa) have been identified in a variety of seeds from C3 plants like castor oil, wheat and barley [4,6,7]. Both polypeptides have been shown to be phos-phorylated in vitro and in vivo during germina-tion of wheat seeds [6]. Recent results have documented the fact that a Ca2+_-independent

PEPCk is already present in dry barley seeds and does not necessitate the functioning of a transduction cascade for up-regulation during subsequent germination [9].

Very little is known about seed PEPC from graminaceous C4 plants. It was reported that the enzyme and corresponding mRNA are synthe-sized in imbibing Sorghum seeds [10]. In the present work, the fate and properties of Sorghum

seed PEPC have been investigated, paying partic-ular attention to the metabolite and salt effect on the phosphorylation process of the enzyme during germination.

2. Materials and methods

2.1. Plant material

Sorghum 6_ulgare _{(var. Tamaran) seeds were} sterilized in 5% (v/v) NaOCl for 15 min and thoroughly washed with sterile water. Seeds were germinated either on filter paper soaked in sterile distilled water or on sterile distilled water con-taining different concentrations (50, 100, 150, 200 mM) of NaCl, or in 10 mM Hepes – KOH, pH 7+10 mM L-malate, in Petri dishes. The

dishes were placed in darkness in a sterile cham-ber at 24°C.

2.2. Seed extracts

Dry seeds were de-embryonated, or germinated seeds separated from the seedling using a razor blade prior to extraction of soluble proteins. The material (ten seeds) was chopped and ground thor-oughly on ice in a prechilled mortar with washed sand in 1 ml of medium A containing 100 mM Tris – HCl, pH 8, 10 mM MgCl2, 1 mM EDTA, 5% (v/v) glycerol, 14 mM b-mercaptoethanol, 10

mg/ml chymostatin, 10 mg/ml leupeptin, 1 mM PMSF. The homogenate was centrifuged at 120 000×g for 5 min (Beckman ultracentrifuge TL100), and the supernatant fluid was used as a crude extract or rapidly filtered through Sephadex G-25 equilibrated with medium A without b -mer-captoethanol (desalted extract).

2.3. Immunocharacterization of seed PEPC

Proteins from desalted extracts were subjected to 10% SDS-PAGE [11] for 2 h, 10 V/cm, at room temperature in a Bio-Rad electrophoresis cell. Proteins were electroblotted overnight to a nitro-cellulose membrane at 30 V, 4°C in a Bio-Rad transfer blot apparatus. The membrane was blocked at room temperature by a 4 h incubation in TBS (20 mM Tris – HCl, 0.15 M NaCl, pH 7.9) containing 5% (w/v) powdered milk. PEPC was immuno-labeled by a 3 h incubation of the mem-brane at room temperature in 10 ml of TBS con taining 20 mg of affinity-purified N-terminal IgG (APS-IgG) [12]. Subsequent detection was by a peroxidase assay (affinity-purified goat anti-rabbit IgG horseradish conjugate from Sigma).

2.4. Purification of seed PEPCk

This was performed as described by Ref. [13]. All steps were carried out at 4°C. De-embryonated dry seeds [20] were homogenized using a mortar and pestle in 2 ml of medium A, and the brei ultracentrifuged at 120 000×g as indicated above. Ammonium sulfate was added to 60% saturation and precipitated proteins collected by centrifuga-tion in an Eppendorf centrifuge. The protein pellet was disolved in 200 ml medium B containing: 50 mM Tris – HCl pH 7.8, 20% glycerol and 1 mM DTT and dialyzed against the same medium (2×

500 ml+1 l) for 2 h. Proteins were subjected to affinity chromatography on blue-dextran-agarose

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(BDA: 1 ml) equilibrated in medium B (at a flow rate of 0.05 ml/min). 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 100ml of 50 mM Tris – HCl pH 8, 30% glycerol, 1 mM dithiothreitol, and stored at −20°C until use. 2.5. In 6_{itro phosphorylation}

The reconstituted phosphorylation assay (50 ml) system contained: 50 mM Tris – HCl, pH 8, 5 mM MgCl2, 0.04 mM CaCl2, 20% glycerol, 1 mM DTT, 74 kBq [g-32_{P]ATP (37 GBq}_/_{mmol), 0.25} mM P1P5-di(adenosine-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 U/6 mg of C4 PEPCs (S8; wild type or S8/D, 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 10mg 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 reacphosphoryla-tion was halted by addiphosphoryla-tion of 10 ml of SDS sample buffer (50 mM Tris – HCl, pH 8, 1% (w/v) SDS, 10% (v/v) 2-mercaptoethanol, 20% (v/

v) glycerol and 1% (w/v) bromophenol blue) and heated for 2 min at 100°C. Denatured samples were analyzed by SDS/PAGE (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 74×105 _{Bq of [}32_{P]phosphate (specific} radioactiv-ity 74×102 _GBq_/_{mol) 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 (20mg of protein). Protein A Sepharose beads were added to the incubated sample to 6% (w/v) 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% (v/v) 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

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sug-Fig. 1. Immunological detection of Sorghum seed phospho-enolpyruvate carboxylase (PEPC). Soluble proteins from de-salted seed extracts (30ml 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 IC50 value for the feedback inhibitorL-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 Km for PEP decreased slightly (65

mM), while the IC50 for L-malate was increased to 220 mM, consistent with the observed decrease in the % inhibition by L-malate (160mM), from 75 to

30% during the same imbibition period (Table 1). In addition, the activity ratio of PEPC (pH 8/7.3) 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 8/7.3) have been observed already in the case of the photosynthetic Sorghumand maize enzyme after a dark/light transition, as well as for various CAM and C3plant 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 Sorghumseeds 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 theKmvalue was

Table 1

Kinetic parameters ofSorghumseed phosphoenolpyruvate carboxylase (PEPC)a

PEPC (mU/seed)

Seed treatment IC50(mM) KmPEP (mM) % Inhibition by L-malate

5.2

Dry 0.075 0.09 75

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 (30mg proteins), at pH 8, 2.5 mM PEP and the other components of the carboxylation reaction as described in Section 2. IC50values for L-malate and percent inhibition by L-malate (160mM) were determined at pH 7.3, 90mM PEP. TheK_mwas calculated from Lineweaver–Burk plots from substrate

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Fig. 2. In vivo phosphorylation of phosphoenolpyruvate car-boxylase (PEPC) in imbibing seeds. SDS PAGE, electrotrans-fer of proteins from the gel and radiochemical/immunological 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 C4phosphoenolpyruvate

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 C4

PEPC from Sorghum: Lane 1, 0.2 U/6 mg of S8/D PEPC mutant; lane 2, 0.2 U/6mg of wild type recombinant PEPC; lane 3, 0.2 U/6mg of wild type recombinant PEPC+0.5 mM EGTA; lane 4, 0.2 U/6 mg of wild type recombinant PEPC preincubated with 10mg 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 Ca2+_-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 C4 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 S8/D C4 PEPC target (aspartate mutant; Fig. 4B1), or wild type C4PEPC 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 C4PEPC) 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 mRNA/protein 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, +10mg of APS-IgG. (A) Coomassie blue staining of proteins. (B) Corre-sponding autoradiography.

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Table 2

Effect of NaCl on phosphoenolpyruvate carboxylase (PEPC) activity, sensitivity toL-malate (phosphorylation state) andL-malate

content during seed germinationa

PEPC (mU/seed)

Seed treatment % inhibition byL-malate L-malate (nmol/10 seeds) Seedling (mm) 75

Dry 5.2 71.5

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/

polypeptide/phosphorylation 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 C4PEPC,L-malate was shown to inhibit PEPC phosphorylation both in reconstituted assays [21] and in situ, in mesophyll protoplasts from Sor

-ghumduring 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 C4PEPC, 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 activity/PEPC phos-phorylation in vivo through the L-malate content

of seeds.

4. Discussion

In this work, phosphoenolpyruvate carboxylase was characterized in imbibing, de-embryonated

Sorghum seeds. Two PEPC polypeptides (108 and 110 kDa) were resolved by SDS/PAGE and shown to contain the N-terminal phosphorylation do-main and to accumulate, in parallel with an in-crease in PEPC activity, during seed germination. Remarkably, theSorghumseed enzyme shows con-siderably higher affinity for the substrate PEP (Km PEP of 90 and 65 mM for the non-phospho and

Fig. 5. Effect of NaCl on the phosphoenolpyruvate carboxy-lase (PEPC) content of seeds during germination. Im-munocharacterization ofSorghumseed PEPC was carried out as described in Fig. 1. Lane 1, dry seed; lane 2, 5 days of germination in distilled water; lane 3, 5 days of germination in distilled water+100 mM NaCl.

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Fig. 6. Effect ofL-malate and NaCl on the in vitro phospho-rylation of recombinant C4phosphoenolpyruvate carboxylase

(PEPC) by seed PEPC kinase (PEPCk). The phosphorylation assays contained 30mg of seed proteins and 0.2 U of purified, recombinant non-phosphorylated C4 PEPC. They were

con-ducted as described in Section 2 in the absence of L-malate (lane 1), or presence, 1 mM (lane 2), 2 mM (lane 3), 5 mM (lane 4) and 10 mM (lane 5). Lanes 6 and 7 corresponded to phosphorylation assays performed in the presence of 10 and 100 mM NaCl, respectively. Proteins were analyzed by SDS-PAGE. (A) Coomassie blue staining of proteins. (B) Corre-sponding autoradiography.

Table 3

Effect of exogenous L-malate on the phosphoenolpyruvate

carboxylase (PEPC) phosphorylation state (as estimated in vitro by the enzyme’s sensitivity to L-malate) during seed

germinationa

Treatment % Inhibition Seedling (mm) by L-malate

Dry 70

Germinated (10 mM Hepes 42 30 KOH, pH 7)

Germinated (10 mM Hepes KOH, pH 7)+L-malate (mM)

5 67 12

10 74 8

20 69 8

50 62 6

a_{The assay for PEPC activity was as defined in Section 2,}

pH 7.3, and 160 mM L-malate. Data are means of three

experiments with different batches of seeds; S.E.=10%.

phospho-PEPC, respectively) and sensitivity to-wards the inhibitor L-malate (75 and 220 mM for

the non-phospho and phospho-PEPC, respec-tively) than does the photosynthetic form of the mesophyll (Km: 1100 – 1200 mmM; IC50: 170 – 300 and 1500 mM for non-phospho and phospho-PEPC, respectively) [21 – 23]. This observation probably reflects isoenzyme adaptation to a cellu-lar environment that in Sorghum seeds should be low in PEP.

Seed PEPC is phosphorylated during germina-tion by a PEPCk that resembles its mesophyll homologue in that it is calcium-independent and displays similar chromatographic properties, e.g. affinity chromatography on blue dextran agarose. In mesophyll cells, C4PEPC is regulated by

photo-synthesis-related metabolites; phosphorylated sug-ars, like glucose-6P, act as activators that in addition counteract the negative effect ofL-malate

[1,3,24,25]. It has been demonstrated in vitro that these effectors can also alter the phosphorylation state of C4PEPC via an indirect (target) effect on PEPCk [21,26]. This has also been documented in situ with mesophyll protoplasts incubated in the presence of the metabolites [25]. In the present work it has been established that: (1) PEPC accu-mulation and phosphorylation are inhibited in seeds imbibed in NaCl-enriched medium; (2) PEPCk is already present in dry seeds and its activity in vitro is not altered by the salt, but severely inhibited by L-malate; (3) L-malate is not

readily consumed in salt-treated seeds and its con-tent varies inversely with the phosphorylation state of PEPC; (4) seed germination and in situ PEPC phosphorylation are also inhibited by exogenous

L-malate. Altogether, these findings support the

view that metabolite regulation is the major pro-cess operating on PEPC/PEPCk in germinating seeds. Along these lines, it might be stressed that while the C4PEPC becomes phosphorylated when

L-malate is accumulated in the mesophyll cell of

an illuminatedSorghum leaf, this occurs in germi-nating seeds along with the removal of the metabolite. In fact, the decline in stored L-malate

during germination could reflect its transport to, and effective use by the embryo, but does not

Fig. 7. Effect of an incubation withL-malate or NaCl on in situ phosphoenolpyruvate carboxylase kinase (PEPCk) activ-ity during seed germination. Desalted protein extracts were obtained from ten seeds that had been germinated in 10 mM Hepes – KOH, pH 7 (control), 10 mM Hepes – KOH, pH 7+10 mM L-malate, or 10 mM Hepes – KOH, pH 7+100

mM NaCl, for 5 days. Aliquots (30mg of protein) were used to estimate PEPCk activity on recombinant C4 PEPC target

(0.2 U/6mg) in typical phosphorylation medium. The radiola-beled proteins were analyzed by SDS-PAGE and autoradiog-raphy. Lane 1, assay containing proteins from control seeds; lane 2, proteins from NaCl-treated seeds; lane 3, proteins from L-malate-treated seeds. (A) Coomassie blue staining of

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necessarily indicate that the carbon flux through PEPC is similarly decreased. Indeed, PEPC would still be committed to sustain this demand after stored L-malate has been markedly, but not

ex-haustively, depleted, and thus would still require protection against the inhibitor. This is consistent with the very low IC50 of the seed enzyme for

L-malate and the observed increase in its

phospho-rylation state which confers on PEPC significant desensitization to the inhibitor, after 5 days of germination.

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Cejudo, Expression and localization of phospho-enolpyruvate carboxylase in developing and germinating wheat grains, Plant Physiol. 116 (1998) 1249 – 1258. [8] P.K. Macnicol, P. Raymond, Role of

phosphoenolpyru-vate carboxylase in malate production by the developing barley aleurone layer, Physiol. Plant 103 (1998) 132 – 138. [9] L. Osuna, J.N. Pierre, M.C. Gonzalez, R. Alvarez, J.F. Cejudo, C. Echevarria, J. Vidal, Evidence for a slow turnover form of the calcium independent phospho-enolpyruvate carboxylase kinase in the aleurone-en-dosperm of germinating barley, Plant Physiol. 119 (1999) 511 – 520.

[10] E. Khayat, E.B. Dumbroff, B.R. Glick, The synthesis of phosphoenolpyruvate carboxylase in imbibing Sorghum

seeds, Biochem. Cell. Biol. 69 (1991) 141 – 145.

[11] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of the bacteriophage T4, Na-ture 22 (1970) 680 – 685.

[12] V. Pacquit, N. Giglioli, C. Cre´tin, J.N. Pierre, J. Vidal, C. Echevarria, Regulatory phosphorylation of C4

phos-phoenolpyruvate carboxylase from Sorghum: an im-munological study using specific anti-phosphorylation site-antibodies, Photosynth. Res. 43 (1995) 283 – 288. [13] J.A. Jiao, R. Chollet, Regulatory seryl-phosphorylation

of C4 phosphoenolpyruvate carboxylase by soluble

protein kinase from maize leaves, Arch. Biochem. Bio-phys. 269 (1989) 526 – 535.

[14] M. Nhiri, N. Bakrim, V. Pacquit, Z. El Hachimi-Mes-souak, L. Osuna, J. Vidal, Calcium-dependent and -inde-pendent phosphoenolpyruvate carboxylase kinase in

Sorghumleaves: further evidence for the involvement of the calcium-independent protein kinase in the in situ regulatory phosphorylation of C4 phosphoenolpyruvate

carboxylase, Plant Cell. Physiol. 39 (1998) 241 – 246. [15] C. Cre´tin, N. Bakrim, E. Keryer, S. Santi, L. Lepiniec, J.

Vidal, P. Gadal, Production inEscherichia coli of active

Sorghumphosphoenolpyruvate carboxylase which can be phosphorylated, Plant Mol. Biol. 17 (1991b) 83 – 88. [16] J.N. Pierre, V. Pacquit, J. Vidal, P. Gadal, Regulatory

phosphorylation of phosphoenolpyruvate carboxylase in protoplasts fromSorghummesophyll cells and the role of pH and Ca2+ _{as possible components of the}

light-trans-duction pathway, Eur. J. Biochem. 210 (1992) 531 – 537. [17] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of proteins utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248 – 254.

[18] H.J. Hohorst, L-malate estimation with malate dehydro-genase and NAD, in: H.V. Bergmeyer (Ed.), Methods in Enzymatic Analysis, Verlag Chemie, Weinheim, 1970, pp. 1544 – 1548.

[19] G.A. Nimmo, H.G. Nimmo, C.A. Fewson, M.B. Wilkins, Diurnal changes in the properties of phospho-enolpyruvate carboxylase inBryophyllumleaves: a possi-ble covalent modification, FEBS Lett. 178 (1984) 199 – 203.

[20] N. Bakrim, C. Echevarria, C. Cre´tin, M. Arrio-Dupont, J.N. Pierre, J. Vidal, R. Chollet, P. Gadal, Regulatory phosphorylation of Sorghum leaf phosphoenolpyruvate carboxylase: identification of the protein-serine kinase and some elements of the signal-transduction cascade, Eur. J. Biochem. 204 (1992) 821 – 830.

[21] C. Echevarria, V. Pacquit, N. Bakrim, L. Osuna, M. Delgado, M. Arrio-Dupont, J. Vidal, The effect of pH on the covalent and metabolic control of C4

phospho-enolpyruvate carboxylase from Sorghum leaf, Arch. Biochem. Biophys. 315 (1994) 425 – 430.

[22] Y.H. Wang, S.M.G. Duff, L. Lepiniec, C. Cre´tin, G. Sarath, S.A. Condon, J. Vidal, P. Gadal, R. Chollet, Site-directed mutagenesis of the phosphorylatable serine (Ser8) in C4phosphoenolpyruvate carboxylase fromSor -ghum. The effect of negative charge at position 8, J. Biol. Chem. 267 (1992) 16759 – 16762.

[23] S.M.G. Duff, C. Andreo, V. Pacquit, L. Lepiniec, G. Sarath, S.A. Condon, J. Vidal, P. Gadal, R. Chollet, Kinetic analysis of non-phosphorylated, in vitro phos-phorylated, and phosphorylation-site mutant (Asp8) forms of intact recombinant C4 phosphoenolpyruvate

carboxylase fromSorghum, Eur. J. Biochem. 228 (1995) 92 – 95.

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[24] Y. Gao, K.C. Woo, Regulation of phosphoenolpyruvate carboxylase inZea maysby protein phosphorylation and metabolites and their roles in photosynthesis, Aust. J. Plant Physiol. 23 (1996) 25 – 32.

[25] N. Bakrim, M. Nhiri, J.N. Pierre, J. Vidal, Metabolite control ofSorghumC4phosphoenolpyruvate carboxylase

catalytic activity and phosphorylation state, Photosynth. Res. 58 (1998) 153 – 162.

[26] Y.H. Wang, R. Chollet, Partial purification and charac-terization of phosphoenolpyruvate carboxylase protein-serine kinase from illuminated maize leaves, Arch. Biochem. Biophys. 304 (1993) 496 – 502.

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Fig. 1. Immunological detection of Sorghum seed phospho-enolpyruvate carboxylase (PEPC). Soluble proteins from de-salted seed extracts (30ml 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

m

M (Table 1) and did not

vary significantly in the range of pH values (pH

7 – 8) investigated (not shown). The IC

value for

the feedback inhibitor

-malate at suboptimal PEP

concentration (90

m

M) and pH (7.3) was 75

m

M

(Table 1). During seed imbibition in distilled water

for 5 days, the

K

for PEP decreased slightly (65

m

M), while the IC

for

-malate was increased to

220 m

M, consistent with the observed decrease in

the % inhibition by

-malate (160

m

M), from 75 to

30% during the same imbibition period (Table 1).

In addition, the activity ratio of PEPC (pH 8

/

7.3)

was shown to decrease from 2.6 to 1.4 during seed

germination (Fig. 1).

Both the decrease in

-malate sensitivity of

PEPC, as measured at suboptimal but

physiologi-cal pH (7.3), and in the activity ratio (pH 8

/

7.3)

have been observed already in the case of the

photosynthetic

Sorghum

and maize enzyme after a

dark

/

light transition, as well as for various CAM

and C

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 [

_{P]phosphate, during which the sensitivity of}

PEPC to

-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

value was

Table 1

Kinetic parameters ofSorghumseed phosphoenolpyruvate carboxylase (PEPC)a PEPC (mU/seed)

Seed treatment IC50(mM) KmPEP (mM) % Inhibition by L-malate

5.2

Dry 0.075 0.09 75

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 (30mg proteins), at pH 8, 2.5 mM PEP and the other components of the carboxylation reaction as described in Section 2. IC50values for L-malate and percent inhibition by

L-malate (160mM) were determined at pH 7.3, 90mM PEP. TheK_mwas calculated from Lineweaver–Burk plots from substrate

(2)

Fig. 4. In vitro phosphorylation of C4phosphoenolpyruvate 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 C4 PEPC from Sorghum: Lane 1, 0.2 U/6 mg of S8/D PEPC mutant; lane 2, 0.2 U/6mg of wild type recombinant PEPC; lane 3, 0.2 U/6mg of wild type recombinant PEPC+0.5 mM EGTA; lane 4, 0.2 U/6 mg of wild type recombinant PEPC preincubated with 10mg 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

_-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

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 S8

/

D

C

PEPC target (aspartate mutant; Fig. 4B1), or

wild type C

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

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 mRNA

/

protein 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

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Table 2

Effect of NaCl on phosphoenolpyruvate carboxylase (PEPC) activity, sensitivity toL-malate (phosphorylation state) andL-malate

content during seed germinationa PEPC (mU/seed)

Seed treatment % inhibition byL-malate L-malate (nmol/10 seeds) Seedling (mm) 75

Dry 5.2 71.5

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

/

polypeptide

/

phosphorylation 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

PEPC,

-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

PEPC, even at the low

concentra-tion of 1 mM (Fig. 6B2 – 5). Interestingly,

-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,

-malate remained

rela-tively high in NaCl-treated seeds (Table 2).

Fi-nally, imbibing

Sorghum

seeds in the presence of

different concentrations of

-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

-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 activity

/

PEPC

phos-phorylation in vivo through the

-malate content

of seeds.

4. Discussion

In this work, phosphoenolpyruvate carboxylase

was characterized in imbibing, de-embryonated

Sorghum

seeds. Two PEPC polypeptides (108 and

110 kDa) were resolved by SDS

/

PAGE and shown

to contain the N-terminal phosphorylation

do-main and to accumulate, in parallel with an

in-crease in PEPC activity, during seed germination.

Remarkably, the

Sorghum

seed enzyme shows

con-siderably higher affinity for the substrate PEP (

K

PEP of 90 and 65

m

M for the non-phospho and

(4)

Fig. 6. Effect ofL-malate and NaCl on the in vitro phospho-rylation of recombinant C4phosphoenolpyruvate carboxylase (PEPC) by seed PEPC kinase (PEPCk). The phosphorylation assays contained 30mg of seed proteins and 0.2 U of purified, recombinant non-phosphorylated C4 PEPC. They were con-ducted as described in Section 2 in the absence of L-malate (lane 1), or presence, 1 mM (lane 2), 2 mM (lane 3), 5 mM (lane 4) and 10 mM (lane 5). Lanes 6 and 7 corresponded to phosphorylation assays performed in the presence of 10 and 100 mM NaCl, respectively. Proteins were analyzed by SDS-PAGE. (A) Coomassie blue staining of proteins. (B) Corre-sponding autoradiography.

Table 3

Effect of exogenous L-malate on the phosphoenolpyruvate

carboxylase (PEPC) phosphorylation state (as estimated in vitro by the enzyme’s sensitivity to L-malate) during seed

germinationa

Treatment % Inhibition Seedling (mm)

by L-malate

Dry 70

Germinated (10 mM Hepes 42 30

KOH, pH 7)

Germinated (10 mM Hepes KOH, pH 7)+L-malate (mM)

5 67 12

10 74 8

20 69 8

50 62 6

a_{The assay for PEPC activity was as defined in Section 2,} pH 7.3, and 160 mM L-malate. Data are means of three

experiments with different batches of seeds; S.E.=10%.

phospho-PEPC, respectively) and sensitivity

to-wards the inhibitor

-malate (75 and 220

m

M for

the non-phospho and phospho-PEPC,

respec-tively) than does the photosynthetic form of the

mesophyll (

K

: 1100 – 1200

mm

M; IC

: 170 – 300

and 1500

m

M for non-phospho and

phospho-PEPC, respectively) [21 – 23]. This observation

probably reflects isoenzyme adaptation to a

cellu-lar environment that in

Sorghum

seeds should be

low in PEP.

Seed PEPC is phosphorylated during

germina-tion by a PEPCk that resembles its mesophyll

homologue in that it is calcium-independent and

displays similar chromatographic properties, e.g.

affinity chromatography on blue dextran agarose.

In mesophyll cells, C

PEPC is regulated by

photo-synthesis-related metabolites; phosphorylated

sug-ars, like glucose-6P, act as activators that in

addition counteract the negative effect of

-malate

[1,3,24,25]. It has been demonstrated in vitro that

these effectors can also alter the phosphorylation

state of C

PEPC via an indirect (target) effect on

PEPCk [21,26]. This has also been documented in

situ with mesophyll protoplasts incubated in the

presence of the metabolites [25]. In the present

work it has been established that: (1) PEPC

accu-mulation and phosphorylation are inhibited in

seeds imbibed in NaCl-enriched medium; (2)

PEPCk is already present in dry seeds and its

activity in vitro is not altered by the salt, but

severely inhibited by

-malate; (3)

-malate is not

readily consumed in salt-treated seeds and its

con-tent varies inversely with the phosphorylation state

of PEPC; (4) seed germination and in situ PEPC

phosphorylation are also inhibited by exogenous

-malate. Altogether, these findings support the

view that metabolite regulation is the major

pro-cess operating on PEPC

/

PEPCk in germinating

seeds. Along these lines, it might be stressed that

while the C

PEPC becomes phosphorylated when

-malate is accumulated in the mesophyll cell of

an illuminated

Sorghum

leaf, this occurs in

germi-nating seeds along with the removal of the

metabolite. In fact, the decline in stored

-malate

during germination could reflect its transport to,

and effective use by the embryo, but does not

mM NaCl, for 5 days. Aliquots (30mg of protein) were used to estimate PEPCk activity on recombinant C4 PEPC target (0.2 U/6mg) in typical phosphorylation medium. The radiola-beled proteins were analyzed by SDS-PAGE and autoradiog-raphy. Lane 1, assay containing proteins from control seeds; lane 2, proteins from NaCl-treated seeds; lane 3, proteins from L-malate-treated seeds. (A) Coomassie blue staining of

(5)

necessarily indicate that the carbon flux through

PEPC is similarly decreased. Indeed, PEPC would

still be committed to sustain this demand after

stored

-malate has been markedly, but not

ex-haustively, depleted, and thus would still require

protection against the inhibitor. This is consistent

with the very low IC

of the seed enzyme for

-malate and the observed increase in its

phospho-rylation state which confers on PEPC significant

desensitization to the inhibitor, after 5 days of

germination.

References

[1] R. Chollet, J. Vidal, M.H. O’Leary, Phosphoenolpyru-vate carboxylase: a ubiquitous, highly regulated enzyme in plants, Annu. Rev. Plant Physiol. Plant Mol. Biol. 47 (1996) 273 – 298.

Digitaria sanguinalis, Plant Cell. 8 (1996) 573 – 586. [3] J. Vidal, R. Chollet, Regulatory phosphorylation of

phosphoenolpyruvate carboxylase, Trends Plant Sci. 2 (1997) 230 – 237.

[5] R.G. Smith, D.A. Gauthier, D.T. Dennis, D.H. Turpin, Malate- and pyruvate-dependent fatty acid synthesis in leucoplasts from developing castor endosperm, Plant Physiol. 99 (1992) 1233 – 1238.

Cejudo, Expression and localization of phospho-enolpyruvate carboxylase in developing and germinating wheat grains, Plant Physiol. 116 (1998) 1249 – 1258. [8] P.K. Macnicol, P. Raymond, Role of

[10] E. Khayat, E.B. Dumbroff, B.R. Glick, The synthesis of phosphoenolpyruvate carboxylase in imbibing Sorghum

seeds, Biochem. Cell. Biol. 69 (1991) 141 – 145.

[11] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of the bacteriophage T4, Na-ture 22 (1970) 680 – 685.

[12] V. Pacquit, N. Giglioli, C. Cre´tin, J.N. Pierre, J. Vidal, C. Echevarria, Regulatory phosphorylation of C4 phos-phoenolpyruvate carboxylase from Sorghum: an im-munological study using specific anti-phosphorylation site-antibodies, Photosynth. Res. 43 (1995) 283 – 288. [13] J.A. Jiao, R. Chollet, Regulatory seryl-phosphorylation

of C4 phosphoenolpyruvate carboxylase by soluble protein kinase from maize leaves, Arch. Biochem. Bio-phys. 269 (1989) 526 – 535.

[14] M. Nhiri, N. Bakrim, V. Pacquit, Z. El Hachimi-Mes-souak, L. Osuna, J. Vidal, Calcium-dependent and -inde-pendent phosphoenolpyruvate carboxylase kinase in

Sorghumleaves: further evidence for the involvement of the calcium-independent protein kinase in the in situ regulatory phosphorylation of C4 phosphoenolpyruvate carboxylase, Plant Cell. Physiol. 39 (1998) 241 – 246. [15] C. Cre´tin, N. Bakrim, E. Keryer, S. Santi, L. Lepiniec, J.

Vidal, P. Gadal, Production inEscherichia coli of active

Sorghumphosphoenolpyruvate carboxylase which can be phosphorylated, Plant Mol. Biol. 17 (1991b) 83 – 88. [16] J.N. Pierre, V. Pacquit, J. Vidal, P. Gadal, Regulatory

phosphorylation of phosphoenolpyruvate carboxylase in protoplasts fromSorghummesophyll cells and the role of pH and Ca2+ _{as possible components of the}

[18] H.J. Hohorst, L-malate estimation with malate dehydro-genase and NAD, in: H.V. Bergmeyer (Ed.), Methods in Enzymatic Analysis, Verlag Chemie, Weinheim, 1970, pp. 1544 – 1548.

[21] C. Echevarria, V. Pacquit, N. Bakrim, L. Osuna, M. Delgado, M. Arrio-Dupont, J. Vidal, The effect of pH on the covalent and metabolic control of C4 phospho-enolpyruvate carboxylase from Sorghum leaf, Arch. Biochem. Biophys. 315 (1994) 425 – 430.

-ghum. The effect of negative charge at position 8, J. Biol. Chem. 267 (1992) 16759 – 16762.

(6)

[25] N. Bakrim, M. Nhiri, J.N. Pierre, J. Vidal, Metabolite control ofSorghumC4phosphoenolpyruvate carboxylase

catalytic activity and phosphorylation state, Photosynth. Res. 58 (1998) 153 – 162.