Cadmium detoxification

Plants are immobile and cannot avoid unfavorable conditions like enhanced heavy metal concentrations. However, plants have developed many detoxification mechanisms, which include active exclusion, immobilization, chelation, and sequestration (Benavides et al., 2005;

Hasan et al., 2009). Phytochelatins (PCs) are well studied chelating ligands, which can be found in higher plants, algae, yeast, some fungi, and also a nematode (Clemens, 2006;

Vatamaniuk et al., 2001). They mostly have the general structure (γGlu-Cys)n-Gly (with n = 2-11). The synthesis of PCs is one of the main detoxification strategies in plants (Cobbett and Goldsbrough, 2002; Hall, 2002). In this study, we focused on the determination of the different PC species instead of Cd-PC complexes (chapter 2.2.). We could clearly show that the different PC-species were induced at different Cd concentrations with the most pronounced increase of PC3 at 20 nM Cd. The increase was not linear to the applied Cd concentration. In principle, longer chained PCs can bind a higher number of Cd ions and one would expect them to occur at high Cd concentrations. Indeed, PC6 was only detected in plants treated with higher Cd concentrations (50-200 nM), but the most abundant PC in all Cd-stressed and the only significant one in Cu-stressed C. demersum samples was PC3. This could indicate a preferential role of PC3, because this complex may be more stable than longer chain PCs as suggested by Mehra and Winge (1988). To extend the PC molecules, the PC synthase cleaves the last Gly and adds another glutathione (GSH) molecule (Rea, 2012;

Vatamaniuk et al., 2000). Considered that under high Cd concentrations many PC3 molecules are already loaded with Cd, the possibility to build up longer chained PCs may therefore decrease. A shortage of GSH seems rather unlikely as this thiol peptide is extraordinary abundant at steady-state concentrations (0.1-10 mM; Rabenstein, 1989). Another imaginable possibility is that the differently long PCs fulfill different specific functions in the detoxification process. This hypothesis, however, is yet to be tested.

A possible role of PCs besides detoxification, the involvement in metal homeostasis or trafficking of essential metals was discussed in the past (Thumann et al., 1991). But the threshold metal concentrations (mostly Cu and Zn) needed to induce PCs are often far above the nutritional needs of a plant, or already toxic (Schat et al., 2000). Furthermore, the presence

of low amounts (3% of maximum) of PCs in plants from control treatments (Howden et al., 1995) may result from contaminations of the solutions and glassware with Cd. This can happen easily when not ultrapure conditions were applied (chemicals of ACS grade were used, the glassware was not acid washed, or not ultraclean water was used during the treatment). In our study, the highest concentration of each PC species was detected in those plants, which were treated with the highest or second highest concentration of metal (200 nM Cu, 100 or 200 nM Cd; chapter 2.2.). Compared to those amounts, the PC concentrations in the plants treated with 0.2-5 nM were very low, mostly less than 1% of the maximal values.

This clearly indicates that PCs are synthesized as a stress response and are not involved in normal physiological processes like metal homeostasis. Instead, this task is most likely fulfilled by gene encoded proteins, the metallothioneins (MT). Those proteins belong to a diverse family, they are of low molecular weight (<10 000 Da) with a high number of conserved cysteines and exist in animals, plants and some bacteria (Cobbett and Goldsbrough, 2002). In plants, there are four types of MTs (type 1 to type 4), the classification depending on the arrangement of the Cys residues (Freisinger, 2011). MTs are involved in storage, transport and release of zinc and copper (Grennan 2011; Moulis, 2010), but also other divalent ions (e.g. Cd) can be bound. In some studies, an enhanced expression of MTs due to toxic heavy metal concentrations was observed (see Cobbett and Goldsbrough, 2002), indicating a detoxifying role. But more research is necessary to determine all processes in which MTs are involved.

A different strategy to counteract moderate Cd toxicity in the tissues is the enhanced uptake of other ions, especially those which Cd can replace. Sunflower grown in Cd-treated soil accumulated more Zn than plants from the control treatment. On the other hand, additional contamination with Zn increased both Zn and Cd accumulation in leaves, but reduced Cd accumulation in roots (Rivelli et al., 2012). The balance of micronutrients in the plants is tightly regulated and the effects of Cd treatment on Zn concentrations depend strongly on the plant species, the applied heavy metal concentrations and the experimental duration. Therefore, both synergistic and antagonistic effects were observed (Bunluesin et al., 2006; Küpper and Kochian, 2010; Moustakas et al., 2011; Turner, 1973). In this study, there was no difference in Zn accumulation on the whole plant level after six weeks of treatment (chapter 2.1.). The concentration of Zn in the leaf of the plant treated with 20 nM Cd for three weeks was the highest in all measured samples as determined by µ-XRF. Treatment with 2 nM and with 20 nM Cd for six weeks led to approximately half of the Zn concentration compared to 20 nM Cd for three weeks. It is well imaginable that this increased Zn uptake at

moderately toxic Cd concentrations is a way of defense. Although there is competition for binding sites, at least the human ZnT exporters seem to be selective for Zn and do not export Cd out of cells (Hoch et al., 2012). Furthermore, Zn transport is mediated not only via transporters of the CDF (cation diffusion facilitator) and ZIP (iron-regulated transporter (IRT)-like protein) family transporters, but also by ATPases, ligand- (Weiss and Sensi, 2000) or voltage-gated cation channels (Gyulkhandanyan et al., 2006), which may be affected differently by Cd. Küpper and Kochian (2010) found a Cd-induced increase in cellular expression for ZNT1 in young plants of the Cd/Zn-hyperaccumulator N. caerulescens, which may be part of plant acclimation to Cd toxicity as a downregulation was found in mature plants.

Sequestration, i.e. the transport into and storage of Cd (and other excess metal ions) in organs and tissue where it interferes least with the metabolic processes, is a common detoxification mechanism in hyperaccumulator plants (Clemens, 2001, 2006; Küpper et al., 1999, 2004). More sensitive measuring methods reveal that this phenomenon also exists in non-hyperaccumulators (Lombi et al., 2011). In our study, Cd distribution changed with increasing applied Cd concentration (chapter 2.2.). While at non-toxic Cd concentrations (0.2 and 2 nM), Cd was homogenously distributed over the whole leaf area, at moderately toxic Cd concentrations (20 nM) the sequestration of Cd was observed. In these samples, most Cd was found in the epidermis and an enhanced Zn concentration was observed, suggesting a combination of detoxification mechanisms. However, after longer exposure (6 weeks) to Cd, the plants treated with moderate concentrations (20 nM) also showed symptoms of toxicity, as apparent from the reduction in photosynthetic performance (chapter 2.1. and 2.2.), the again reduced Zn content in the leaf, and the distribution pattern of Zn and Cd (chapter 2.2.).

The goals of this thesis were achieved. Threshold concentrations and causal relationships of Cd toxicity and detoxification in the model macrophyte C. demersum were succesfully determined. It was shown that the onset effects of Cd occurred at a significantly lower level than shown in previous studies.