Plant cells contain different O-acetylserine(thiol)lyase (OASTL) enzymes involved in cysteine (Cys) biosynthesis and located in different subcellular compartments. These enzymes are made up of a complex variety of isoforms resulting in different subcellular Cys pools. To unravel the contribution of cytosolic Cys to plant metabolism, we characterized the knockout oas-a1.1 and osa-a1.2 mutants, deficient in the most abundant cytosolic OASTL isoform in Arabidopsis (Arabidopsis thaliana). Total intracellular Cys and glutathione concentrations were reduced, and the glutathione redox state was shifted in favor of its oxidized form. Interestingly, the capability of the mutants to chelate heavy metals did not differ from that of the wild type, but the mutants have an enhanced sensitivity to cadmium. With the aim of establishing the metabolic network most influenced by the cytosolic Cys pool, we used the ATH1 GeneChip for evaluation of differentially expressed genes in the oas-a1.1 mutant grown under nonstress conditions. The transcriptomic footprints of mutant plants had predicted functions associated with various physiological responses that are dependent on reactive oxygen species and suggested that the mutant was oxidatively stressed. Evidences that the mutation caused a perturbation in H2O2 homeostasis are that, in the knockout, H2O2 production was localized in shoots and roots; spontaneous cell death lesions occurred in the leaves; and lignification and guaiacol peroxidase activity were significantly increased. All these findings indicate that a deficiency of OAS-A1 in the cytosol promotes a perturbation in H2O2 homeostasis and that Cys is an important determinant of the antioxidative capacity of the cytosol in Arabidopsis.

SummaryEmploying 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.