Research article An Arabidopsis mutant disrupted in ASN2 encoding asparagine synthetase 2 exhibits low salt stress tolerance Houda Maaroufi-Dguimi a , Mohamed Debouba a , Laure Gaufichon b , Gilles Clément c , Houda Gouia a , Afef Hajjaji a , Akira Suzuki b, * a Unité de recherche: Nutrition et Métabolisme Azotés et Protéines de Stress (99 UR /09-20), Campus Universitaire, Faculté des Sciences de Tunis, Département de Biologie, Université Tunis EL MANAR, Tunis 1060, Tunisia b Adaptation des Plantes à leur Environnement, IJPB, UMR1318 AgroParisTech, INRA Route de Saint-Cyr, 78026 Versailles cedex, France c Plateforme de Chimie du Végétal, IJPB, UMR1318 AgroParis Tech, Institut National de la Recherche Agronomique, Route de St-Cyr, 78026 Versailles cedex, France article info Article history: Received 28 January 2011 Accepted 15 March 2011 Available online 23 March 2011 Keywords: Ammonium assimilation ASN Arabidopsis thaliana Glutamine synthetase Salt abstract Salt tolerance of Arabidopsis knockout mutant with T-DNA insertion in ASN2 gene encoding asparagine synthetase (AS, EC 6.3.5.4) (asn2-1) was investigated. Wild-type Arabidopsis Col0 and asn2-1 mutant were grown for one month by hydroponic culture and subjected to 100 mM NaCl stress for a short time from 6 to 24 h. The salt treatment decreased chlorophyll and soluble protein contents, and increased ammonium level in the asn2-1 leaves. The salinity induced ASN1 mRNA level in the wild-type and asn2-1 leaves. By contrast, the salt treatment inhibited the transcript and protein levels of chloroplastic glutamine synthetase 2 (GS2, EC 6.3.1.2) in the wild-type and asn2-1 leaves. Increase in asparagine and proline contents in response to the salt treatment provides evidence for the role of asparagine as a prevailing stress responding aminoacid. Glutamate dehydrogenase (NADH-GDH, EC 1.4.1.2) exhibited a slight increase in the a-subunit and b-subunit in the wild-type line and the asn2-1 line, respectively under the salinity, whereas its in vitro aminating activity in the wild-type leaves was not affected. The results indicate that the asn2-1 mutant was impaired in nitrogen assimilation and translocation under salt treatment. Ó 2011 Elsevier Masson SAS. All rights reserved. 1. Introduction Salinity has become major constraint for plant growth and productivity of crop plants [1]. Among the consequences, salinity inhibits enzymes of nitrogen assimilation especially glutamine synthetase (GS) and glutamate synthase (GOGAT) [2]. As well, salinity reduces photosynthetic capacity, resulting in a decrease of carbon assimilation and an enhanced protein degradation, which leads to a rapid and excessive accumulation of ammonium and changes in amino acid metabolism [3e5]. Ammonium is generally re-assimilated into amides via the GS/GOGAT cycle. Ammonium can be assimilated into asparagine by asparagine synthetase (AS, EC 6.3.5.4) in response to environmental stresses [6]. Alternatively, aminating activity of glutamate dehydrogenase (GDH) may be involved in ammonium reassimilation under stress conditions [7], while in vivo function remained to be elucidated. Three genes encoding asparagine synthetase have been identi- fied in Arabidopsis thaliana (ASN1 , ASN2 and ASN3). Asparagine plays a key role in nitrogen storage and transport of nitrogen from source to sink organs because of its high nitrogen to carbon ratio and its stability. Asparagine is a predominant amino acid transported in phloem of A. thaliana [6]. Instability of asparagine synthetase and rapid turnover of asparagine by asparaginase [8] make it difficult to carry out biochemical approach by deter- mining asparagine synthetase activity. By using molecular approaches, Wong et al. [9] reported an importance of ASN2 func- tion in the stress-induced ammonium reassimilation. However, few data are available in distinguishing ASN functions in response to salt stress. Therefore, in the present study, we aimed at investigating a potential role of ASN2 in the regulation of ammonium metabolism using ASN2 knockout mutant under NaCl stress. 2. Results 2.1. Chlorophyll, soluble protein and ammonium contents In order to determine whether salt stress results in a phenotype of A. thaliana wild-type and asn2-1 lines, we first determined chlorophyll contents. In the wild-type and the asn2-1 lines, a salt * Corresponding author. E-mail address: suzuki@versailles.inra.fr (A. Suzuki). Contents lists available at ScienceDirect Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy 0981-9428/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.plaphy.2011.03.010 Plant Physiology and Biochemistry 49 (2011) 623e628