Research article Cloning and characterization of drought-stimulated phosphatidic acid phosphatase genes from Vigna unguiculata Marcel Giovanni Costa França a , Ana Rita Matos b, * , Agne ` s D’arcy-Lameta c , Chantal Passaquet c , Christiane Lichtle ´ d , Yasmine Zuily-Fodil c , Anh Thu Pham-Thi c a Departamento de Bota ˆnica, Universidade Federal de Minas Gerais, ICB, Av. Anto ˆnio Carlos 6627, Pampulha, 31270-901 Belo Horizonte, MG, Brazil b Centro de Engenharia Biolo ´gica, Faculdade de Cieˆncias da Universidade de Lisboa, Campo Grande,1749-016 Lisbon, Portugal c Laboratoire d’Ecophysiologie Mole´culaire, UMR-IRD 137 BIOSOL, Universite ´ Paris 12, 61 Av. du Gl de Gaulle, 94010 Cre ´teil Cedex, France d ENS, Laboratoire de Dynamique des Membranes Ve ´ge´tales, Complexe Prote ´ines-Pigments, 46 rue d’Ulm, 75005 Paris, France article info Article history: Received 14 December 2007 Accepted 9 July 2008 Available online 19 July 2008 Keywords: Drought Lipid phosphate phosphatase Phosphatidic acid phosphatase Vigna unguiculata abstract Under environmental stresses, several lipolytic enzymes are known to be activated and to contribute to membrane lipid turnover and generation of second messengers. In animal cells, phosphatidic acid phosphatase (PAP, EC 3.1.3.4), which dephosphorylates phosphatidic acid generating diacylglycerol, is long known as an enzyme involved in lipid synthesis and cell signalling. However, knowledge on PAP in plants remains very limited. The aim of this work was to isolate and characterize PAP genes in the tropical legume Vigna unguiculata (cowpea), and to study their expression under different stress conditions. Two cDNAs designated as VuPAPa and VuPAPb were cloned from the leaves of cowpea. Both proteins share sequence homology to animal type 2 PAP, namely, the six transmembrane regions and the consensus sequences corresponding to the catalytic domain of the phosphatase family, like the recently described Arabidopsis LPP (Lipid Phosphate Phosphatase) proteins. The recombinant protein VuPAPa expressed in Escherichia coli cells was able to convert phosphatidic acid into diacylglycerol. Unlike VuPAPb, VuPAPa has an N-terminal transit peptide and was addressed to chloroplast in vitro. Both genes are expressed in several cowpea organs and their transcripts accumulate in leaves in response to water deficit, including progressive dehydration of whole plants and rapid desiccation of detached leaves. No changes in expression of both genes were observed after wounding or by treatment with jasmonic acid. Further- more, the in silico analysis of VuPAPa promoter allowed the identification of several putative drought- related regulatory elements. The possible physiological role of the two cloned PAPs is discussed. Ó 2008 Elsevier Masson SAS. All rights reserved. 1. Introduction Membrane lipid modifications are observed when plants are submitted to water deficit. Several lipolytic enzymes are known to be activated under such conditions and to contribute to membrane lipid catabolism and generation of second messengers. Phospha- tidic acid (PA), which has been recently identified as an important signalling molecule in plants and animals, can be generated by the action of Phospholipase D (PLD) or by the sequential actions of phospholipase C (PLC) and diacylglycerol (DAG) kinase (DGK) (for a review see [40]). PA can be further converted to DAG by Phosphatidate phosphatase (PAP, EC 3.1.3.4) or used by PA kinase (PAK) [43] to generate DAG pyrophosphate (DGPP) [30]. Since the increase in PA and DGPP levels in response to stress is known to be transient, PA kinase and PAP appear as important enzymes regulating stress signalling events [40]. Besides being involved in cell signalling, PAP also plays a pivotal role in plastid glycerolipid biosynthesis [25]. In plants two distinct PAP types were characterized (for a review see [22]). PAP activity located in the microsome-associated fractions of safflower and avocado [13,17,36] is thought to be implicated in the so-called ‘‘eukaryotic’’ pathway of lipid biosynthesis [7] while the activity associated to the inner membrane of plastid envelopes of spinach and pea [1,2] is thought to be implicated in the ‘‘prokaryotic’’ pathway of glycolipid synthesis [35]. Furukawa-Stoffer and co- workers [11] identified in microspore-derived cultures of oilseed rape several PAP isoforms, one among them being probably involved in signal transduction. In Arabidopsis there are four PAP genes. The proteins encoded by two of them, designated AtLPP1 and AtLPP2 (AGI codes: At2g01180 Abbreviations: ABA, abscisic acid; DAG, diacylglycerol; DGK, diacylglycer- olkinase; DGPP, diacylglycerol pyrophosphate; JA, jasmonic acid; LPP, lipid phos- phate phosphatase; PA, phosphatidic acid; PAK, phosphatidic acid kinase; PAP, phosphatidic acid phosphatase; D-PLD, phospholipase D; C-PLC, phospholipase C; RT–PCR, reverse-transcribed polymerase chain reaction. * Corresponding author. Fax: þ351 217 500 166. E-mail address: armatos@fc.ul.pt (A.R. Matos). Contents lists available at ScienceDirect Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy 0981-9428/$ – see front matter Ó 2008 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.plaphy.2008.07.004 Plant Physiology and Biochemistry 46 (2008) 1093–1100