Plant Biotechnology Journal (2004) 2, pp. 469–476 doi: 10.1111/j.1467-7652.2004.00092.x © 2004 Blackwell Publishing Ltd 469 Blackwell Publishing, Ltd. Increased cysteine availability is essential for cadmium tolerance and accumulation in Arabidopsis thaliana José R. Domínguez-Solís 1 , M. Carmen López-Martín 1 , Francisco J. Ager 2 , M. Dolores Ynsa 2 , Luis C. Romero 1 and Cecilia Gotor 1, * 1 Instituto de Bioquímica Vegetal y Fotosíntesis, Centro de Investigaciones Científicas Isla de la Cartuja, Consejo Superior de Investigaciones Científicas and Universidad de Sevilla, Avda. Américo Vespucio s/n, 41092 Sevilla, Spain 2 Centro Nacional de Aceleradores, Avda. Thomas Edison s/n, 41092 Sevilla, Spain Summary Employing genetic transformation using an Atcys-3A cDNA construct expressing the cytosolic O -acetylserine(thiol)lyase (OASTL), we obtained two Arabidopsis lines with different capabilities for supplying cysteine under metal stress conditions. Lines 1-2 and 10-10, grown under standard conditions, showed similar levels of cysteine and glutathione (GSH) to those of the wild-type. However, in the presence of cadmium, line 10-10 showed significantly higher levels. The increased thiol content allowed line 10-10 to survive under severe heavy metal stress conditions (up to 400 μ M of cadmium in the growth medium), and resulted in an accumulation of cadmium in the leaves to a level similar to that of metal hyperaccumulator plants. Investigation of the epidermal leaf surface clearly showed that most of the cadmium had accumulated in the trichomes. Furthermore, line 10-10 was able to accumulate more cadmium in its trichomes than the wild-type, whereas line 1-2 showed a reduced capacity for cadmium accumulation. Our results suggest that an increased rate of cysteine biosynthesis is responsible for the enhanced cadmium tolerance and accumulation in trichome leaves. Thus, molecular engineering of the cysteine biosynthesis pathway, together with modification of the number of leaf trichomes, may have considerable potential in increasing heavy metal accumulation for phytoremediation purposes. Received 2 March 2004; revised 16 April 2004; accepted 27April 2004. * Correspondence (fax 34-95-4460065; e-mail gotor@ibvf.csic.es) Keywords: O -acetylserine, cadmium stress, cysteine biosynthesis, sulphur assimilation, trichome cells. Introduction Cysteine biosynthesis is the final step in the sulphate assimi- lation pathway and is catalysed by O -acetylserine(thiol)lyase (OASTL, EC 4.2.99.8). This enzyme incorporates sulphide into O -acetylserine (OAS), the latter being produced through the acetylation of serine by the action of serine acetyltransferase (SAT, EC 2.3.1.30). Both OASTL and SAT activities have been demonstrated to be localized in three cellular compartments involved in protein synthesis: the cytosol, plastid and mito- chondrion (Hell et al ., 2002). In the Arabidopsis thaliana genome, five different SAT genes ( Howarth et al ., 2003) and at least seven different OASTL genes (Jost et al ., 2000) have been identified. Some evidence has suggested that the isoforms may have specific contributions to particular metabolic pathways. Thus, certain OASTL isoforms exhibit β -cyanoalanine synthase enzyme activity and are involved in cyanide detoxification (Hatzfeld et al ., 2000; Yamaguchi et al ., 2000), whereas the cytosolic isoform ATCYS-3A seems to play a role in cadmium (Cd) tolerance (Domínguez-Solís et al ., 2001). Cysteine is the precursor molecule for the synthesis of glutathione (GSH), the predominant non-protein thiol, which plays an important role in plant stress responses. GSH has been implicated in plant responses to toxic levels of heavy metals, as it is the precursor for the synthesis of phytochelat- ins (PCs), the thiolate peptides involved in the detoxification of Cd and other heavy metals. PCs bind toxic heavy metal ions, and these complexes are transported into the vacuole by an ABC-type transporter (Cobbett, 2000). Today, the con- tamination of soils and water by toxic heavy metals presents a major environmental and human health problem. The