Cadmium toxicity

3.1.1. Cadmium influences the photosynthetic apparatus

Cadmium inhibited photosynthesis at much lower concentrations than those used for most previous studies. The inhibition or malfunction of photosynthesis is a great threat for all photosynthetic organisms, as they mostly cannot cover their energy demand with other sources. The study concentrating on Cd toxicity (chapter 2.1.) focused on the determination of threshold concentrations of various kinds of inhibition. Any kind of inhibition appeared first in the plants treated with the highest Cd concentration. The threshold concentration of observable effects was consistently oberserved at 20 nM in HL plants and also mostly in LL plants (see below). This was true for example for visible symptoms, efficiency of PS II in dark adapted state and during actinic light, the production of reactive oxygen species, and the oxygen exchange. It turned out that under high light conditions the effects of Cd were more severe than under low light conditions. This can be explained regarding the differences of HL and LL grown plants, which is the ratio of LHC molecules to RC. Under LL conditions, plants have bigger antenna systems with more Chl molecules. The higher number of Chls offers more binding sites for Cd and thereby reduces toxicity. This will be explained in the next section.

3.1.2. Substitution of other metals by cadmium

One major toxicity mechanism of Cd is the replacement of other divalent metals in proteins, enzymes, and transporter binding sites (Maret and Moulis, 2013). The replacement of Zn by

Cd is quite likely as both divalent metal ions have similar coordination properties, such as their preference for tetrahedral geometry and pronounced affinity for nitrogen and sulfur ligands (NHis, SCys) (Moulis, 2010). Under certain circumstances, Cd(II) may compete for Ca(II) and Mg(II) binding sites (Maret and Moulis, 2013) and even will replace copper coordinated to cysteine sulfur in proteins (e.g. plastocyanin, 2NHisSCysSMet metal binding site;

Sujak, 2005) or Fe in iron-sulfur proteins (Xu and Imlay, 2012). However, in vivo evidence for the latter cases is missing (Moulis, 2010). As mentioned above, plants grown under LL conditions built up big antenna systems with many LHCs per RC while under HL conditions there are more RCs and comparably less LHCs (Taiz and Zeiger, 2007). The replacement of Mg²⁺ in Chl by Cd²⁺ leads to the generation of [Cd]-Chl, which is unsuitable for photosynthesis (see chapter 2.1.; review by Küpper et al., 2006). The high amount of chlorophylls in the LHCs offers a large number of binding sites for Cd under LL conditions.

However, the generation of [Cd]-Chl in antenna molecules is less severe for the plant compared to the generation in the reaction centers. [Cd]-Chls in the LHCs will not transfer the electron to neighboring Chls, but this loss of some functional Chl molecules does not completely inhibit electron transport to the respective RC and thereby the process of photosynthesis. [Cd]-Chl in the RC means the loss of the whole photosystem, as excited [Cd]-Chl relaxes very fast by heat emission and will not reduce the first electron acceptor Q_A (Küpper et al., 2006). The relatively larger antenna system in LL plants therefore acts as a buffer, binding Cd, which cannot substitute other ions in other compartments.

The determination of [Cd]-Chl is a difficult task. As mentioned above, it is unstable and has the same absorption spectrum as [Mg]-Chl. Therefore, a different approach for the verification will be performed, which already led to promising results for C. demersum plants under Cu-stress (Thomas et al., 2013b). For this proteomics approach, proteins were isolated by grinding the frozen plant material in liquid nitrogen and membrane proteins separated from the soluble ones by ultracentrifugation and solubilization. The proteins were then separated by size-exclusion chromatography with a UHPLC system coupled to electrospray mass spectrometry (ESI-MS) and inductively couple mass spectrometry (ICP-MS). This combination allows the determination of the protein’s size (via mass to charge ratio) and at the same time specific elements within the sample. The photosynthetic complexes PS II, PS I, and the LHCs are very well distinguishable (e.g. Galka et al., 2012). Under Cu-stress the respective concentration of Cu increased in those samples, while Mg decreased (Thomas et al., 2013b), suggesting the substitution of Mg in the Chls. Comparative results are expected in the Cd-treated plants.

Another indication of whether Cd directly impairs photosynthesis is to detect it within the chloroplasts under physiological conditions, that is, without disrupting the cells. So far, we were only able to determine the distribution of Cd and other elements on the tissue level, not on the organelle level (chapter 2.2.). However, this may soon be possible at the now operating PETRA (Positron-Elektron-Tandem-Ring-Anlage) storage ring (beamline P06) at DESY (Deutsches Elektron Sychrotron) in Hamburg, Germany. The size of the storage ring, the improved mirror systems to focus the beam with compound refractive lenses (CRL), computers with much higher processing capacity and the now available high-throughput x-ray fluorescence detector system MAIA developed at the Australian synchrotron, in principle allow for a resolution of 250 nm, which is sufficient to distinguish chloroplasts from other cell compartments.

Unspecific binding of Cd to the cell walls should also be distinguishable with this approach. In our experiments, the plants were not treated with Na-EDTA or CaCl₂ to remove metals that were unspecifically bound to the cell wand as was done in some other studies (e.g.

Sanità di Toppi et al., 2007) for several reasons: the chelator treatment will remove some, but not all metals from the cell walls, depending on the strength of the binding. The cell wall contains many compounds which are able to bind divalent and trivalent metal ions, especially sugars (Krzesłowska, 2011). The deposition of metals in the cell wall is a common detoxification process in many plants, although in hyperaccumulators the intracellular sequetration into vacuoles is much stronger (reviewed by Krzesłowska, 2011; Leitenmaier and Küpper, 2013). Cell wall lacking mutants of the green alga Chlamydomonas reinhardtii were more sensitive to hevay metal stressed than the walled wild type strain (Macfie et al., 1994). In a following study, the authors found that after metal stress and removal of the extracellular metal by EDTA treatment, the wall-less strain contained more Cd within the cell than the walled type (Macfie and Welbourn, 2000). Increased metal tolerance therefore cannot be explained solely by increased cell wall binding. Furthermore, ions transport in macrophytes can also occur via the apoplastic route (Grignon and Sentenac, 1991). The uptake of Cd in the macrophyte Elodea canadensis was shown to be not metabolically dependant and the basipetal ¹⁰⁹Cd translocation to be passive via the apoplast (Fritioff and Greger, 2007). Increased apoplastic transport of Pb in Lemna minor was suggested by Samardakiewicz and Wozny (2000). Therefore, the results obtained from the acid digests (chapter 2.1.) display intracellular and extracellular, i.e. cell wall bound metals. Solely intracellular concentrations can be obtained by X-ray based techniques like µ-XRF (chapter 2.2.).

Not only does the intracellular organization influence the toxicity, but also the environment surrounding the plant as was obvious from the lake water study (chapter 2.3.). Water hardness reflects the concentration of calcium and magnesium in the water. The more ions are present, the harder is the water. The Ca and Mg ions compete with toxic metals for the binding sites on an organism’s surface and thus can reduce their uptake into and presence in the cell and thereby their toxicity. Especially the divalent metal ions (Cd, Zn, Ni, Pb) can enter the cell through Ca transporters (e.g. Ca²⁺-ATPase, Markich and Jeffree, 1994). Negative correlation between the concentrations of dissolved Ca and metal accumulation in plants were shown decades ago (Kinkade and Erdman, 1975). Laboratory experiments confirmed the reduction of heavy metal toxicity by preventing entrance into the cell by competition with Ca for uptake (Gagnon et al., 1998). For nickel, protective effects were mediated by increasing concentrations of Mg rather than Ca: Increasing Mg concentrations led to significant higher growth rates of the green alga Pseudokirchneriella subcapitata when incubated for 72 h with Ni (Deleebeeck et al., 2009a). In the lake water study (chapter 2.3.) there was no significant difference in Cd and Ni accumulation due to water hardness (comparison of “CdNi-P” in soft and hard water). It is possible that the applied concentrations of Ca and Mg did not exceed the transporter capacity, so that the uptake of the heavy metals was still possible. Alternatively, the heavy metals could have entered the cells differently, for example via Zn-transporters (Clemens, 2006). Furthermore, the Cd treatment alone (3 nM) had no inhibitory effect and the Ni only (300 nM) treatment was slightly toxic. In combination (Cd and Ni), the inhibitory effect on C. demersum’s photosynthetic performance (measured as electron flow through the PS in actinic light) was pronounced. Effects of a combination of many heavy metals can be additive, synergistic, antagonistic, or not different from the effect of only one metal. The accumulation of Zn and Cd was synergistic in wheat and maize which were sampled from contaminated soils in northern China. Under these actual field conditions, increasing Cd and Zn contents in the soils led to increased accumulations of Cd and Zn in the crop plants (Nan et al., 2002). However, the respective effects can be different in the terrestrial compared to the aquatic environment. Mixtures of Zn and Cu, Cr and Cu, and Zn and Cr had mostly antagonistic effects in Lemna minor (Ince et al., 1999). Consistently, different effects were observed for the terrestrial plant Lepidium sativum and the freshwater plant Spirodela polyrhiza when treated with the same mixture of heavy metals (Cr, Cu, Mn, Ni, Pb, Zn). While most effects were antagonistic in the terrestrial plant, they were synergistic or additive in the aquatic one (Montvydienė and Marčiulionienė, 2004). It is important to note that effects may differ depending on test organisms or habitats.

The pH strongly influences the bioavailability and uptake of macro- and micronutrients by plants (Tyler and Olsson, 2001). Alfalfa grown on soils with different pHs accumulated different amounts of heavy metals (Cd, Cu, Ni, Zn) with the highest accumulation at the highest pH (Peralta-Videa et al., 2002). In aqueous solutions, it seems to be different. There, metal toxicity often decreases with increasing pH (Plette et al., 1999). The pH in the epilimnion water of lake Ammelshain (chapter 2.3.) was higher (pH 7.2-8.0) than in the hypolimnion (pH 6.6-7.4) and while the metal concentrations were not different in the water, plants treated with hypolimnion water accumulated more Cd and less Ni than those plants treated with epilimnion water, displaying different bioavailability or uptake of the ions.