Original article Increased antioxidative capacity in maize calli during and after oxidative stress induced by a long lead treatment Massimo Zacchini a , Elvira Rea b , Monica Tullio b , Marina de Agazio a, * a Istituto di Biologia Agroambientale e Forestale-CNR, Via Salaria km 29.300, 00016 Monterotondo Scalo (Rome), Italy b Istituto Sperimentale per la Nutrizione delle Piante, MiPAF, Via della Navicella 2-4, 00184 Rome, Italy Received 16 May 2002; accepted 15 July 2002 Abstract Maize (Zea mays L., cv. Samodek) callus cultures were exposed for long period (22 months) to lead (0.5 mM lead chloride) and lead content, oxidative damage and antioxidative response were evaluated at different steps. Inductively coupled plasma (ICP) emission spectroscopy analysis showed that lead entered the cells and it accumulated, but its internal concentration was maintained 10-fold less than the external one. Increase of both polyamine and lipid peroxide content indicated that cells underwent a stress condition due to an oxidative attack, counteracted by an increase of antioxidative defence enzyme activities, ascorbate peroxidase (APX, EC 1.11.1.11) and glutathione reductase (GR, EC 1.6.4.2). After 10 months, from the start of the lead treatment, a stock of calli was transferred for 6 months in a lead-deprived medium and then re-exposed to lead for a further 6 months. Analysis indicated that lead-deprived calli maintained high levels of APX and GR activities, suggesting that, over the experimental time–course, cells with high APX and GR activity were selected and allowed to enrich the cultures. These cultures, after a new lead treatment, showed a lower oxidative damage compared to continuously lead-treated calli. © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Antioxidative enzymes; Callus culture; Lead; Lipid peroxides; Oxidative stress; Polyamines; Zea mays 1. Introduction Lead is a naturally occurring element whose diffusion in the environment occurs because of its extensive use in paints, petrol, explosives and linings. It is considered one of the most important metallic pollutants, responsible for a variety of environmental problems [23]. In plants, lead uptake and translocation occur [31], causing toxic effects resulting in a decrease of biomass production [10,34]. Among these nega- tive effects, inhibition of root growth due to the perturbation of microtubule organisation in the root meristem [9], inhibi- tion of germination [33], and reduction in chlorophyll con- tent and PSII efficiency [18] are reported. Generally, plants may prevent the toxic effect of heavy metals by induction of various defence mechanisms [2,15,26] such as adsorption to the cell wall, compartmenta- tion in vacuoles, enhancement of the active efflux, or induc- tion of higher levels of metal chelates like a protein complex (metallothioneins and phytochelatins), organic (citrates) and inorganic (sulphides) complexes. Heavy metals may induce oxidative stress with overpro- duction of active oxygen species (AOS) such as superox- ideradicals (O 2 · – ), hydroxyl radicals (·OH) and hydrogen peroxide (H 2 O 2 ) which react very rapidly with DNA, lipids and proteins causing cellular damage [6,21,28]. This damage is due to saturation of enzymatic and non-enzymatic detoxi- fication mechanisms that are able to scavenge oxygen radi- cals produced under normal metabolism. AOS tend to trans- form into the most stable and diffusive chemical form, i.e. hydrogen peroxide, that, upon entering the transduction chain of stress signalling [32], in turn stimulates the antioxi- dant defence system [7,19,22]. In plants, the antioxidant system is represented by various radical-scavenging systems, including low-molecular mass Abbreviations: AOS, active oxygen species; APX, ascorbate peroxidase; GR, glutathione reductase; MDA, malonyldialdehyde; GSSG, oxidised glu- tathione; SOD, superoxide dismutase; TLC, thin layer chromatography; TBA, thiobarbituric acid; TCA, trichloroacetic acid. * Corresponding author. E-mail address: deag@mlib.cnr.it (M. de Agazio) Plant Physiology and Biochemistry 41 (2003) 49–54 www.elsevier.com/locate/plaphy © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. PII: S 0 9 8 1 - 9 4 2 8 ( 0 2 ) 0 0 0 0 8 - 6