At the cellular level, cadmium (Cd) induces both damaging and repair processes in which the cellular redox status plays a crucial role. Being not redox-active, Cd is unable to generate reactive oxygen species (ROS) directly, but Cd-induced oxidative stress is a common phenomenon observed in multiple studies. The current review gives an overview on Cd-induced ROS production and anti-oxidative defense in organisms under different Cd regimes. Moreover, the Cd-induced oxidative challenge is discussed with a focus on damage and signaling as downstream responses. Gathering these data, it was clear that oxidative stress related responses are affected during Cd stress, but the apparent discrepancies observed in between the different studies points towards the necessity to increase our knowledge on the spatial and temporal ROS signature under Cd stress. This information is essential in order to reveal the exact role of Cd-induced oxidative stress in the modulation of downstream responses under a diverse array of conditions.

Accurate quantification by real-time RT-PCR relies on normalisation of the measured gene expression data. Normalisation with multiple reference genes is becoming the standard, but the best reference genes for gene expression studies within one organism may depend on the applied treatments or the organs and tissues studied. Ideally, reference genes should be evaluated in all experimental systems. A number of candidate reference genes for Arabidopsis have been proposed, which can be used as a starting point to evaluate their expression stability in individual experimental systems by available computer algorithms like geNorm and NormFinder. Using this approach, we identified the best three reference genes from a set of ten candidates -that included three traditional "housekeeping" genes -for normalisation of gene expression when roots and leaves of Arabidopsis thaliana are exposed to cadmium (Cd) and copper (Cu). The expression stabilities of AT5G15710 (F-box protein), AT2G28390 (SAND family protein), and AT5G08290 (mitosis protein YLS8) were the highest when considering the effect to the roots and shoots of Cd and Cu treatments. Even though the effect of Cd and excess Cu on the plants is very different, the same best reference genes were identified when considering Cd or Cu treatments separately. This suggests that these three genes may also be suitable when studying the gene expression after exposure of Arabidopsis thaliana to increased concentrations of other metals.

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Phytoextraction has a promising potential as an environmentally friendly clean-up method for soils contaminated with toxic metals. To improve the development of efficient phytoextraction strategies, better knowledge regarding metal uptake, translocation and detoxification in planta is a prerequisite. This review highlights our current understanding on these mechanisms, and their impact on plant growth and health. Special attention is paid to the central role of glutathione (GSH) in this process. Because of the high affinity of metals to thiols and as a precursor for phytochelatins (PCs), GSH is an essential metal chelator. Being an important antioxidant, a direct link between metal detoxification and the oxidative challenge in plants growing on contaminated soils is observed, where GSH could be a key player. In addition, as redox couple, oxidized and reduced GSH transmits specific information, in this way tuning cellular signalling pathways under environmental stress conditions. Possible improvements of phytoextraction could be achieved by using transgenic plants or plant-associated microorganisms. Joined efforts should be made to cope with the challenges faced with phytoextraction in order to successfully implement this technique in the field.

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Exposure of plants to toxic concentrations of metals leads to disruption of the cellular redox status followed by an accumulation of reactive oxygen species (ROS). ROS, like hydrogen peroxide, can act as signaling molecules in the cell and induce signaling via mitogen-activated protein kinase (MAPK) cascades. MAPK cascades are evolutionary conserved signal transduction modules, able to convert extracellular signals to appropriate cellular responses. In this review, our current understanding about MAPK signaling in plant metal stress is discussed. However, this knowledge is scarce compared to research into the role of MAPK signaling in the case of other abiotic and biotic stresses. ROS production is a common response induced by different stresses and undiscovered analogies may exist with metal stress. Therefore, further attention is given to MAPK signaling in other biotic and abiotic stresses and its interplay with other signaling pathways to create a framework in which the involvement of MAPK signaling in metal stress may be studied.

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Reactive oxygen species produced by NADPH oxidase and oxylipins derived from lipoxygenase activity can signal various stress conditions and have been implicated when plants are exposed to heavy metals. Transcriptional profiling of the 10 NADPH oxidase and 6 lipoxygenase genes was performed after exposure of Arabidopsis thaliana wildtype and NADPH oxidase mutants to 5 mM CdSO 4 or 2 mM CuSO 4 for 24 h. Under these short exposures to environmentally realistic concentrations of Cd or Cu, plants modulate signalling networks that regulate the onset of adaptive responses. Metal-specific NADPH oxidase genes were upregulated by Cd but downregulated by Cu, and metalspecific lipoxygenase gene expression was observed only after Cu exposure. Genes that are responsive to both metals were upregulated and may be responsive to general oxidative stress. For all metal-responsive genes except RBOHD, distinct responses were observed between leaves and roots, which may be due to different stress intensities and signalling mechanisms. Mutation of NADPH oxidase genes had opposing effects on gene expression after Cd or Cu exposure. Upregulation of LOX1 and LOX6 in the roots after exposure to Cd depended on NADPH oxidase gene expression, whereas LOX3 and LOX6 expression was induced more strongly in NADPH oxidase mutants after Cu exposure. Furthermore, NADPH oxidases regulated their own expression level and that of other members of the gene family when exposed to Cd or Cu. The results suggest interplay between reactive oxygen species and oxylipin signalling under Cd or Cu stress, and are useful as a basis for genetic studies to unravel metal-specific signalling mechanisms.