Cadmium-Induced Changes in Antioxidative Systems, Hydrogen Peroxide Content, and Differentiation in Scots Pine Roots^{<a href="#FN1" rid="FN1" class=" fn">1</a>}

Abstract

Cd is an important environmental pollutant with high toxicity to animals and plants. It is released into the environment by traffic, metal-working industries, mining, as a by-product of mineral fertilizers, and from other sources (Nriagu and Pacyna, 1988). The regulatory limit of Cd in agricultural soils is 100 mg kg ^{soil, but regionally this threshold is exceeded (}Salt et al., 1998). Heavy metal toxicity is also an important issue in reclamation of industrial sites. Cd accumulation causes reductions in photosynthesis, diminishes water and nutrient uptake (Sanita di Toppi and Gabbrielli, 1999), and results in visible symptoms of injury in plants such as chlorosis, growth inhibition, browning of root tips, and finally death (Kahle, 1993).

The question as to how Cd acts at the cellular level and how plants may defend themselves against this pollutants is receiving increasing attention. It has been shown that Cd induces the synthesis of phy-tochelatins (γ-glutamyl-Cys [γ-EC] peptides), which bind metals in the cytosol and sequester them in the vacuole (Rauser, 1995; Mehra and Tripathi, 2000). The precursor for phytochelatin synthesis is glutathione, whose cellular level was decreased after Cd exposure (Rauser, 1995; Zenk, 1996; Xiang and Oliver, 1998). Exposure to sublethal Cd concentrations resulted in the recovery of cellular glutathione concentrations and was accompanied by increased γ-EC synthetase and glutathione synthetase mRNA transcript levels (Xiang and Oliver, 1998).

Glutathione is the major non-protein thiol in plants and has many functions in plant metabolism. It is involved in the detoxification of heavy metals and xenobiotics and plays a role in gene activation and in the protection from oxidative stress (Lamoureux and Rusness, 1989; Bergmann and Rennenberg, 1993; Noctor and Foyer, 1998). As an antioxidant glutathione together with ascorbate and antioxidative enzymes, superoxide dismutases (SOD; EC 1.15.1.1), ascorbate peroxidases (APX; EC 1.11.1.11), and catalases (CAT; EC 1.11.1.6) controls the cellular concentrations of hydrogen peroxide (H₂O₂) and O₂ ⁽Noctor and Foyer, 1998). Recycling of ascorbate and GSH is achieved by monodehydroascorbate radical reductase (MDAR; EC 1.6.5.4.), dehydroascorbate reductase (DAR; EC 1.8.5.1), and glutathione reductase (GR; EC 1.6.4.2).

Cd treatment affects the activities of antioxidative enzymes, but contrasting results have been reported. For example, in leaves of Cd-exposed Helianthus annuus plants, the activities of ascorbate-glutathione-related defense enzymes were decreased (Gallego et al., 1996). Roots and leaves of Phaseolus vulgaris as well as suspension cultures of tobacco (Nicotiana tabacum) cells contained elevated APX activities after Cd exposure (Chaoui et al., 1997; Piqueras et al., 1999). In Phaseolus aureus seedlings, Cd induced elevated guaiacol peroxidase (POD) but decreased CAT activities (Shaw, 1995). Cd also caused lipid peroxidation, suggesting that the tissues suffered from oxidative stress (Shaw, 1995; Gallego et al., 1996; Lozano-Rodriguez et al., 1997; Chaoui et al., 1997). The involvement of antioxidants in plant responses against Cd toxicity is unclear because Cd does not belong to the group of transition metals like copper, iron, and zinc, which may induce oxidative stress via Fenton-type reactions. It is possible that the observed changes in the antioxidant systems occurred as a result of unspecific cellular degradation processes. However, another possibility is that Cd triggers common defense pathways in plants cells like other biotic or abiotic environmental stresses. A joint initial event of these pathways is an accumulation of H₂O₂, which acts as a signaling molecule. In plant-pathogen interactions, H₂O₂ induces an orchestrated sequence of reactions involving the activation of peroxidases, the stimulation of secondary metabolism, structural changes such as lignin deposition, and eventually cell death (Alvarez and Lamb, 1997).

Because it is not known whether Cd induces common plant defense pathways, we investigated the sequence of physiological reactions, including H₂O₂ production, changes in ascorbate-glutathione-related antioxidant systems, secondary metabolism (peroxidases, phenolics, and lignification), developmental changes, and cell death, occurring in roots after Cd exposure. Scots pine (Pinus sylvestris) seedlings were chosen as a model system because forest ecosystems are particularly vulnerable to heavy metal pollution. The seedlings were exposed to 5 or 50 μm Cd in hydroponics and used to study physiological defense reactions and anatomical changes in root tips.

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