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Plant Science 151 2000 29 – 37 Posttranslational regulation of phosphoenolpyruvate carboxylase during germination of Sorghum seeds: influence of NaCl and L -malate Mohamed Nhiri a , Naı¨ma Bakrim a , Nadia Bakrim a , Zakia El Hachimi-Messouak c , Cristina Echevarria d , Jean Vidal b, a Laboratoire de Biotechnologie, De´partement de Biologie, Uni6ersite´ A. Essaˆdi, Faculte´ des Sciences et Techniques, BP 416 , Tanger, Morocco b Institut de Biotechnologie des Plantes, baˆt. 630 , UMR CNRS 8618 , Uni6ersite´ de Paris-Sud, Centre d ’ Orsay, 91405 Orsay Cedex, France c Laboratoire de Biochimie, De´partement de Biologie, Uni6ersite´ Med V. Rabat, Rabat, Morocco d Laboratorio de Fisiologia Vegetal, Facultad de Biologia, Uni6ersidad de Se6illa, A6da. Reina Mercedes, Se6illa, 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 Sorghum seeds, focusing on the interaction between metabolites and posttranslational control of the enzyme by phosphorylation. Two PEPC polypeptides 108 and 110 kDa were resolved by SDSPAGE and shown to increase, in parallel with PEPC activity during seed germination. PEPC displayed very low K m values for PEP 90 mM and inhibition constant IC 50 for L -malate 75 mM in desalted protein extracts from de-embryonated dry seeds. The inhibition of PEPC by 0.16 mM L -malate, pH 7.3, decreased from 70 to 30, along with a consistent increase in IC 50 75 – 220 mM 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 C 4 PEPC from Sorghum on 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 of L -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.comlocateplantsci

1. Introduction

Phosphoenolpyruvate carboxylase PEPC is widely distributed in plants where it is involved in a number of physiological contexts [1]. In C 4 plants, a specific isoenzyme is involved in the initial fixation of atmospheric CO 2 during C 4 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; IC 50 , concentration of the inhibitor causing a 50 decrease in the initial PEPC activity; K m , 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.vidalibp.u-psud.fr J. Vidal 0168-945200 - see front matter © 2000 Published by Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 9 4 5 2 9 9 0 0 1 9 1 - 0 Ca 2 + -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 C 3 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 Ca 2 + -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 C 4 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