Role of Ascorbate and Glutathione in Cellular Defence Against Cadmium Exposure in Arabidopsis thaliana

Abstract

Cadmium (Cd) is a trace element found naturally in soils and water. Cadmium pollution is often a severe environmental problem in industrial areas; this metal is easily taken up by plants through the root system and translocated to the aerial parts. Exposure to increased Cd concentrations can lead to visible symptoms of toxicity and cause growth inhibition, root damage or chlorosis. This study focuses on three aspects of plant defence mechanisms induced by Cd exposure: (1) detoxification processes (2) oxidative stress related mechanisms and (3) the effect of Cd at the protein level in the test plant Arabidopsis thaliana. The tripeptide glutathione (GSH) is an important metabolite in Cd detoxification processes in two ways (i) GSH is the precursor for phytochelatin (PC) synthesis that detoxifies Cd through metal complexation and vacuolar sequestration and (ii) GSH is a major antioxidant metabolite and in coordination with ascorbate (AsA), both are involved in the AsA-GSH cycle to cope with oxidative stress by scavenging reactive oxygen species (ROS). The first experimental part of this work evaluates the effects of one-week exposure of three weeks old plants two Cd concentrations (1 and 10 µM in rockwool) at the leaf level. Assessment of the different objectives brought us to the following conclusions (1) a dose-response was imposed by Cd; (2) plants actively detoxified Cd by PC complexation; (3) antioxidant metabolites were depleted and (4) Cd caused a disequilibrium in the antioxidative defence mechanism leading to an oxidative cellular environment (Chapter 3: Semane et al. 2007). Moreover, using a proteomic approach, it was clear that Cd stress affected lots of proteins involved in different biological pathways (Chapter 4). Coordination of carbon, nitrogen and sulphur metabolism providing energy and precursors eliciting plant defence were highlighted. In the second experimental part, plants were grown for three weeks in hydroponic culture to allow us to study also the effects of Cd at the root level. Plants were exposed for three days to 1 and 5 µM Cd. Asa and/or GSH deficient Arabidopsis mutants containing about 30-50 % of AsA (vtc1-1), GSH (cad2-1) and AsA-GSH (vtc1-1–cad2-1) as compared to the wild type (col0) were used. The results obtained, show differential responses between roots and leaves. Roots are in direct contact with the metal and actively defend themselves by induction of PCs, and gene expression levels related to GSH metabolism. Although GSH biosynthesis genes were induced, the GSH content decreased. The latter result emphasised both (i) the importance of GSH for the synthesis of PCs and (ii) PCs are not involved in Cd translocation to the shoot as i.e. Cd-PC complex (Chapter 5). As observed in the first experimental part, the respective redox ratio GSH/GSSG dramatically decreased leading to a higher oxidative environment. A similar decrease was observed for AsA and its relative redox ratio AsA/DHA (Chapter 5). However, in leaves of all genotypes, AsA and GSH concentrations and their respective ratios increased upon Cd exposure. Therefore, we hypothesised that H2O2 might be involved in both oxidative stress induced by Cd in the roots and as a signalling molecule leading to an acclimation of the leaves by an increment of antioxidant metabolites. Performing proteomic comparisons of the different genotypes using principal component analysis (PCA), we were able to separate the mutant cad from the other genotypes. This was mainly due to either the expression or no expression at all of several proteins in both roots and leaves. Root proteomic profiling revealed that lots of proteins related to metabolism, stress responses and protein metabolism were involved (Chapter 6). Moreover, the highly induced expression of two proteins observed only in the mutants treated with Cd was identified as isoforms of cytosolic glyceraldehyde dehydrogenase (cGAPDH) (Chapter 6). Recent studies have shown this enzyme to be one of the major targets of H2O2. Although this strongly suggests that Cd induces H2O2 production, a role for the associated couple H2O2-cGAPDH as a signalling molecule or as a probable ROS scavenger needs to be further investigated. Studying the leaf proteome of the different genotypes under Cd stress, we clearly observed that the genotype effect was prominent on the Cd effect (Chapter 7). In conclusion, taken together the results provided by this work emphasise the complex relations of Cd with both plants organs and at the different cellular levels. Undoubtedly, GSH is a key component of the plants’ defence against Cd and its relation with plant physiology in adaptation to its environment is crucial. The signalling network mediated by both antioxidants and their redox couple are extensive and need to be further explored. Additional experiments integrating transcriptome and ROS-targeted proteins should be conducted to explore further plants’ adaptation to environmental stress (Chapter 8).