Short communication Pb 2þ exposure induced microsatellite instability in Pisum sativum in a locus related with glutamine metabolism E. Rodriguez 1 , R. Azevedo 1 , H. Moreira, L. Souto, Conceição Santos * Department of Biology & CESAM, University of Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro, Portugal article info Article history: Received 10 October 2012 Accepted 10 October 2012 Available online 3 November 2012 Keywords: Mutagenicity Microsatellite instability Pb Pisum sativum Glutamine synthetase abstract Lead (Pb) is a toxic element, but its putative mutagenic effects in plant cells, using molecular markers, remain to unveil. To evaluate if Pb induces mutagenicity, Pisum sativum L. seedlings were exposed to Pb 2þ (up to 2000 mg L 1 ) for 28 days and the instability of microsatellites (or Simple Sequence Repeats, SSR) was analyzed in leaves and roots. The analysis of eight selected microsatellites (SSR1eSSR8) demon- strated that only at the highest dosage microsatellite instability (MSI) occurred, at a frequency of 4.2%. Changes were detected in one microsatellite (SSR6) that is inserted in the locus for glutamine synthetase. SSR6 products of roots exposed to the highest concentration of Pb were 3 bp larger than those of the control. Our data demonstrate that: (a) SSR technique is sensitive to detect Pb-induced mutagenicity in plants. MSI instability is Pb dose dependent and organ dependent (roots are more sensitive); (b) the Pb- sensitive SSR6 is inserted in the glutamine synthetase locus, with still unknown relation with functional changes of this enzyme; (c) Pb levels inducing MSI are much above the maximum admitted levels in some European Union countries for agricultural purpose waters. In conclusion, we propose here the potential use of SSR to evaluate Pb 2þ -induced mutagenicity, in combination with other genetic markers. Ó 2012 Elsevier Masson SAS. All rights reserved. 1. Introduction Lead (Pb) is considered a priority hazardous contaminant by EU (Annex II of the Directive 2008/105/EC). The main sources of Pb release to the environment have anthropic origin, such as activities associated with smelters and mines. Lead-induced carcinogenicity and genotoxicity are widely known in humans (leading to saturnism) [1,2]. However, its toxicity (in particular of Pb 2þ ) in plants is still less known than the phytotoxicity of other metals. As with other metals, most of the absorbed Pb 2þ remains in the root, and only a small fraction is translocated to the shoots [3]. Pb 2þ was shown to cause chromosome aberration [4] in Allium cepa, DNA degradation, by Comet assay in lupin and tobacco [5,6] and genomic instability in turnips [7]. However, it still remains unclear if, as for animals, Pb induces point mutations. With the development of PCR based techniques, the study of the mechanisms involving metal-induced genotoxicity at the molec- ular level has become easier and more accessible. Among the many molecular markers that can be used, microsatellites (also known as Simple Sequence Repeats, SSR) are among the most informative and robust, presenting high mutation rates [8]. SSR are tandem repeats of DNA sequences of 1e6 base pair (bp) long, which are highly abundant, have random distribution through eukaryote genomes and high degree of polymorphism [9]. Moreover, micro- satellite instability (MSI), manifested as repeat length poly- morphisms, has often been related to mismatch repair system (MMR) deficiency in animals (e.g., [9]) and plants [10e13], but this correlation should be regarded carefully as multifactors may be involved in both damage/repair events. In particular, while in animals it was proposed that Pb-induced mutagenicity affected the DNA repair system [14], and therefore the MMR system ability, this hypothesis remains to unveil in plants. The aim of the present work was to confirm whether (and at what doses) Pb 2þ exposure induces mutagenicity in plants (roots and leaves). For that, we used the model crop species Pisum sat- ivum. Seedlings were exposed, for 28 days, to Pb 2þ in concentra- tions ranging from 20 mg L 1 (maximum admitted level in some European Union countries for agricultural purpose waters) to 2000 mg L 1 . Putative Pb-induced mutations in the DNA were assessed by analyzing MSI. 2. Results and discussion In our experiment the real Pb concentrations found in nutrient solutions (with nominal Pb concentrations of 20, 200, 1000 and * Corresponding author. Tel.: þ351 234 370 766; fax: þ351 234 372 587. E-mail address: csantos@ua.pt (C. Santos). 1 Current address: Department of Biology, University of Copenhagen, Ole Maaloes Vej 5, Copenhagen N, DK-2200, Denmark. Contents lists available at SciVerse ScienceDirect Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy 0981-9428/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.plaphy.2012.10.006 Plant Physiology and Biochemistry 62 (2013) 19e22