Antioxidant enzymes responses to cadmium in radish tissues

Abstract

To investigate the antioxidant responses of radish (Raphanus sativus L.) to cadmium (Cd) treatment, seedlings of a tolerant variety were grown in increasing concentrations of CdCl₂, ranging from 0.25–1 mM, for up to 72 h in a hydroponic system. Analysis of Cd uptake indicated that most of the Cd accumulated in the roots, but some was also translocated and accumulated in the leaves, especially at the higher concentrations of Cd used in the experiments. Roots and leaves were analysed for catalase, glutathione reductase and superoxide dismutase activities. Catalase and glutathione reductase activities increased considerably in the roots and leaves after 24 h exposure to the metal, indicating a direct correlation with Cd accumulation. The analysis of native PAGE enzyme activity staining, revealed several superoxide dismutase isoenzymes in leaves, with the two predominant isoenzymes exhibiting increases in activity in response to Cd treatment. The results suggest that in radish, the activity of antioxidant enzymes responds to Cd treatment. The main response may be via the activation of the ascorbate–glutathione cycle for the removal of hydrogen peroxide, or to ensure the availability of glutathione for the synthesis of Cd-binding proteins.

Introduction

Cadmium (Cd) is a toxic element that normally occurs in low concentrations in soils (Wagner, 1993). However, in areas that have been subjected to mining the concentration can be high, varying from 100–600 mg kg⁻¹ dry weight (Ernst and Nielssen, 2000, Lombi et al., 2000). In addition, following the application of sewage sludge to agricultural land, Cd can accumulate in the topsoil (Lombi et al., 2000). Although Cd is not an essential nutrient for plants, the metal ion is taken up rapidly by the roots (Leita et al., 1991, Hart et al., 1998) and on most occasions causes inhibition of growth (Di Toppi et al., 1999, Sneller et al., 1999). However, a number of plants (termed hyperaccumulators) that grow on metalliferous soils, are able to translocate Cd from the roots and accumulate it in high concentrations in the shoots (Chardonnens et al., 1998). It has been suggested that such plants would be of considerable value in the remediation of soils that are heavily contaminated with heavy metals (Zhu et al., 1999).

Cd may be detoxified in plants by combination with a family of sulphur rich peptides termed phytochelatins (Leopold et al., 1999, Cobbett, 2000). The peptides are structurally related to glutathione and contain a varying number (normally 2–5) of glutamate and cysteine, linked through the γ-carboxyl group of glutamate. Synthesis of these phytochelatins can be induced very rapidly in tissue culture cells and roots, and is accompanied by a fall in the concentration of glutathione, following the addition of Cd or other heavy metals (Grill et al., 1987). Glutathione synthesis may then be stimulated by induction of enzymes involved in sulphate uptake and reduction and γ-glutamylcysteine synthetase (Haag-Kerwer et al., 1999, Heiss et al., 1999). Phytochelatin synthase which carried out the conversion of glutathione to phytochelatins is activated by Cd, but is inactive in the absence of heavy metal ions (Cobbett, 2000).

Proof that phytochelatins are synthesised from glutathione has come from an investigation into enzyme deficient mutants and overexpressing plant lines. cad2 mutants of Arabidopsis lacking γ-glutamylcysteine synthetase are sensitive to Cd and are unable to synthesise glutathione, whilst the cad1 mutants lacking phytochelatin synthase are sensitive to Cd and accumulate glutathione (Howden et al., 1995, Cobbet et al., 1998). Overexpression of both γ-glutamylcysteine synthetase and glutathione synthetase in Indian mustard has been shown to increase phytochelatin synthesis and confer resistance to Cd (Yong et al., 1999, Zhu et al., 1999).

Van Assche and Clijsters (1990) have reviewed in detail the early work showing that Cd has the capacity to inhibit a range of enzyme activities in plants, in particular those of the Calvin cycle and chlorophyll biosynthesis. Research into the effects of Cd on photosynthetic CO₂ assimilation has continued (Krupa, 1999), with evidence presented that the enzymes of the Calvin cycle and RUBP carboxylase/oxygenase are particularly sensitive to Cd (Chugh and Sawhney, 1999, Di Cagno et al., 1999).

Evidence that Cd causes the production of reactive oxygen species (ROS) (Foyer et al., 1997) in plants, came from observation that new isoenzymes of peroxidase were detectable in both the root and leaves of Phaseolus vulgaris (Van Assche and Clijsters, 1990). Further evidence of Cd induced oxidative stress came from the detection of lipid peroxidation and chlorophyll breakdown (Somashekaraiah et al., 1992, Gallego et al., 1996a, Chaoui et al., 1997, Dalurzo et al., 1997). Due to the multiplicity of processes that produce ROS during metabolism, including photosynthesis and photorespiration, fatty acid oxidation, response to pathogen attack and senescence, plants have developed a series of detoxification reactions. Peroxide can be metabolised directly by peroxidases, particularly in the cell wall, and by catalase (CAT) in the peroxisome (Azevedo et al., 1998, Polidoros and Scandalios, 1999). In the chloroplast, superoxide is converted by superoxide dismutase (SOD) to peroxide which is then detoxified to water and oxygen via the glutathione/ascorbate cycle, which involves the operation of ascorbate peroxidase, monohydroascorbate reductase/dehydroascorbate reductase and glutathione reductase (GR) (Foyer et al., 1997, Noctor and Foyer, 1998). Three distinct types of SOD activity have been detected in plants, which can be classified according to their metal cofactor, Mn, Fe and Cu/Zn. Mn–SOD is located in the mitochondria and peroxisomes, Fe–SOD (which is not found in all plants) is associated with the chloroplasts and the abundant Cu/Zn SODs are located in the cytosol, chloroplasts and peroxisomes (Bowler et al., 1992, Del Rio et al., 1998). The occurrence of SOD activity in such a wide range of metabolic compartments suggests that enzymes of the glutathione/ascorbate cycle, as well as peroxidases, may also play an important role outside the chloroplast. The synthesis of oxalate oxidase, that is present in the cytoplasm and apoplast of germinating wheat embryos and liberates peroxide, is stimulated by the presence of Cd (Berna and Bernier, 1999).

Given the above cited mechanisms utilised by plants to detoxify ROS, it is clearly important to establish whether exposing plants to Cd causes a detrimental or stimulatory effect on the enzymes involved in this detoxification process. Somewhat surprisingly, the limited number of experiments that have been carried out on the subject have frequently produced contradictory results. CAT activity has been shown to decline in Phaseolus vulgaris (Somashekariah et al., 1992), Phaseolus aureus (Shaw, 1995), Pisum sativum (Dalurzo et al., 1997), Lemna minor (Mohan and Hossetti, 1997) and Amaranthus lividus (Bhattacharjee, 1998), following the application of Cd to the growth medium. However in Phaseolus vulgaris, although decreases in CAT activity were detected in roots and leaves, there was no effect on the stem enzyme (Chaoui et al., 1997), and in Agropyron repens increases in CAT activity were detected in both leaves and stems (Brej, 1998). In Helianthus annuus (Gallego et al., 1996b, Gallego et al., 1999) considerable variation in CAT activity was detected over a time period up to 22 days with similar variations being also detected in tolerant varieties of Solanum tuberosum (Stroinski and Kozlowska, 1997).

GR activity in S. tuberosum increased following the application of Cd, but this was followed by a rapid and total inactivation and then a slow recovery (Stroinski et al., 1999). In two species of Alyssum, GR activity increased at 0.02 mM Cd but decreased at 0.05 mM Cd (Schickler and Caspi, 1999). Increases in GR activity were observed in the leaves of Phaseolus vulgaris (Chaoui et al., 1997), but decreases had been noted previously in germinating seedlings of the same species (Somashekariah et al., 1992). Decreases in GR activity following the application of Cd, were detected in a series of experiments employing H. annuus (Gallego et al., 1996b, Gallego et al., 1996b, Gallego et al., 1999). Increases in total SOD activity were detected following the application of Cd in P. sativum (Dalurzo et al., 1997) and two Alyssum species (Schickler and Caspi, 1999), along with a decrease and then an increase in SOD activity in S. tuberosum (Stroinski and Kozlowska, 1997). SOD activity remained constant or decreased in H. annuus (Gallego et al., 1996a, Gallego et al., 1999) and declined in A. lividus (Bhattacharjee, 1998), following the application of Cd. As far as we are aware no attempt has been made to examine the effect of Cd on the different isoenzymes of SOD in higher plants.

In this work, we report new findings showing that the activities of all three enzymes (CAT, GR and specific isoenzymes of SOD) increased in the leaves and roots of a resistant variety of radish, following exposure to increasing concentrations of Cd. Although the concentrations of Cd used (0.25 and 1 mM) are high when compared to that found in contaminated soils, they have been used recently in testes on species of Thlaspi that are able to accumulate high concentrations of Cd (Lombi et al., 2000).