ФИЗИОЛОГИЯ РАСТЕНИИ, 2011, том 58, № 3, с. 403-413
ЭКСПЕРИМЕНТАЛЬНЫЕ СТАТЬИ
УДК 581.1
Antioxidant Defense Mechanisms in Response to Cadmium Treatments in Two Safflower Cultivars
© 2011 S. H. Namdjoyan*, R. A. Khavari-Nejad*, F. Bernard**, T. Nejadsattari*, H. Shaker**
* Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran ** Department of Biology, Faculty of Sciences, Shahid Beheshti University, Tehran, Iran
Received 15.09.2010
By using two safflower (Carthamus tinctorius L.) cultivars, Arak2811 and Goldasht, the experiments were conducted in order to study (i) the genotypic variation in cadmium (Cd) tolerance, (ii) Cd concentrations in plants, and (iii) changes in the antioxidant defense systems in leaves, including antioxidant enzymes and non-enzymatic antioxidants. Plants were grown under controlled environmental conditions and subjected to Cd treatments (0, 25, 50, 75, and 100 цМ Cd) for different time periods. Cd concentrations and cultivar-depen-dent response to Cd were assessed. Of the two cultivars, Goldasht showed a greater sensitivity to Cd toxicity as judged from the severity of Cd toxicity symptoms on leaves, much stronger enhancement in the MDA level, and decreases in dry matter production. Increasing Cd supply markedly reduced the shoot and root dry weights in both cultivars, but at the higher Cd concentrations and longer exposure durations this decrease was more marked in cv. Goldasht. Plants accumulated substantial amount of Cd, especially in the roots, the highest being in the roots of cv. Arak2811 at 100 цМ Cd after 4 days. Cd-induced oxidative stress as was indicated by the increase in lipid peroxidation with the increase in metal concentration and exposure duration. Under different Cd stress levels, activities of antioxidant enzymes differed in the two cultivars. The results indicated that Cd tolerance of cv. Arak2811 was related to the retention of Cd in the roots and avoiding the toxic effect by activation of the antioxidant system.
Keywords: Carthamus tinctorius — antioxidant systems — cadmium tolerance
INTRODUCTION
Cadmium is an important wide-spread trace pollutant with high toxicity to plants, animals, and humans. Cadmium causes various phytotoxic symptoms, including chlorosis, growth inhibition, water imbalance, phosphorus and nitrogen deficiency, reduced manganese transport, and accelerated senescence [1]. It has been suggested that growth inhibition by Cd is due to a direct effect of Cd on the nucleus or interaction with hormones, and, in the areal parts of the plants, it is due to the inhibition of photosynthesis [2]. Heavy metals cause molecular damage to plants, either directly or indirectly through reactive oxygen species (ROS) formation. Although the mechanism of metal damaging action is not clearly understood, there is increasing evidence suggesting that, at least in part, metal toxicity is due to the oxidative damage [3].
The production of ROS must be carefully regulated to avoid oxidative damage. Plant cells are normally protected against this oxidative damage by a broad
Abbreviations'. APX — ascorbate peroxidase; ASA — ascorbic acid; CAT — catalase; GPX — guaiacol peroxidase; GR — glutathione reductase; GSH — glutathione; SOD — superoxide dismutase. Corresponding author. Shahram Namdjoyan. Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran. Fax. +98-213-336-0514; e-mail. shahram_biotech@yahoo.com
spectrum of radical scavenger systems, including antioxidant enzymes, like superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (GPX), ascorbate peroxidase (APX), and glutathione reductase (GR), as well as non-enzymatic antioxidants, like glutathione, carotenoids, and ascorbate [1]. SOD is a key enzyme responsible for catalyzing dismutation of highly reactive O2- to O2 and H2O2. The resulting H2O2 is further decomposed to water and oxygen either by APX of the ascorbate—glutathione cycle or by GPX and CAT localized in the cytoplasm and other cellular compartments. Ascorbic acid (ASA) is considered as the most popular and powerful ROS-detoxifying compound because of its ability to donate electrons in a number of enzymatic and non-enzymatic reactions [3]. Glu-tathione (GSH) may be the most important intracel-lular compound preventing damage induced by ROS. Manipulation of GSH biosynthesis increases resistance to oxidative stress [3].
The ability of higher plants to neutralize the toxic effects of reactive oxygen seems to be a very important determinant of their tolerance to heavy metal stresses. There are a number of plant species, which can grow in the presence of heavy metals and accumulate them. They are used in various phytoremediation and phyto-stabilization strategies for decontamination of the en-
vironment. Crop plants differ greatly in Cd uptake and transport. Differences in Cd uptake and accumulation have been shown both among plant species and between genotypes of a given species [4].
Plant mechanisms affecting the Cd uptake by the roots and its transport to the shoots can also affect the expression of Cd toxicity in plants and plant yield. Therefore, breeding of plant genotypes with high ability to repress Cd uptake by the roots and its transport to the shoots is a reasonable approach to alleviate adverse effects of Cd toxicity in crop plants.
Plant genotypes can differ in their tolerance to Cd toxicity. The ability of plant genotypes to detoxify Cd can differ between plant species, and this plays a critical role in the expression of high tolerance to Cd toxicity [4, 5].
Safflower (Carthamus tinctorius L.) is a crop plant of the Asteraceae family, with the wide geographical distribution. It possesses interesting characteristics of Cd accumulation [6, 7]. Although a lot of reports regarding the medicinal and agronomical aspects of this plant are available, to our knowledge, there is no study dealing with Cd tolerance of safflower genotypes or the role of antioxidant systems in the expression of Cd toxicity in safflower. Hence, this study aimed to evaluate the effects of Cd stress on non-enzymatic (ASA, GSH) and enzymatic (SOD, APX, GPX, CAT, and GR) activities in seedlings of two safflower cultivars to compare their tolerance levels.
MATERIALS AND METHODS
Plant material and growth conditions. The safflower (Carthamus tinctorius L.) cvs. Arak2811 and Goldasht were used. The seeds were germinated in petri dishes under sterile conditions at a temperature of 25 ± 1°C for 48 h, transferred to pots containing a mixture of sand and perlite (1 : 1), and irrigated with the nutrient solution (1 g/l KNO3; 250 mg/l Ca(H2PO4)2; 250 mg/l MgSO4 x 7H2O; 2.3 mg/l H3BO3; 1.8 mg/l MnCl2 x x 4 H2O; 0.22 mg/l ZnSO4 x 7 H2O; 0.08 mg/l CuSO4 x x 5 H2O; 0.02 mg/l H2MoO2; and 6.92 mg/l Fe-EDTA). The seedlings were grown for 10 days in a growth chamber (200 ^E/(m2 s), a 12-h photoperiod, 60 ± 5% relative humidity, and 25 ± 1°C).
CdCl2 treatment was performed at the end of the tenth day. Different Cd concentrations (0, 25, 50, 75, and 100 ^M) were applied; treatment lasted for 1—4 days. Before harvesting, Cd toxicity for the leaves was assessed, and then the roots were washed in deionized water. At harvest, the roots and shoots were separated and dried at 80°C in order to determine dry weight and Cd concentration.
Estimation of cellular Cd. Dried plant samples (100 mg) were digested in HNO3/HClO4 (3 : 1) mixture at 160°C. Digested material was diluted with deionized water, and Cd concentration was deter-
mined using atomic absorption spectrophotometer ("Perkin Elmer", Germany).
Analysis of lipid peroxidation. Lipid peroxidation in leaves was determined by estimation of the MDA content following the method of Heath and Packer [8] with slight modification. Leaf fresh samples (0.5 g) were homogenized in 5 ml of 0.1% trichloroacetic acid (TCA). The homogenate was centrifuged at 10000 g for 5 min. To every 1 ml of aliquot, 4 ml of 20% TCA containing 0.5% thiobarbituric acid (TBA) was added. The mixture was heated at 95°C for 30 min and then cooled quickly on the ice bath. The resulting mixture was centrifuged at 10000 g for 15 min, and the absorbance of the supernatant was taken at 532 and 600 nm. The nonspecific absorbance at 600 nm was subtracted from the absorbance at 532 nm. The concentration of MDA was calculated by using the extinction coefficient of 155/(mM cm) and expressed as ^mol/g fr wt.
Antioxidant enzyme activity assays. Leaf fresh samples from different treatments (500 mg) were harvested and homogenized with a mortar and pestle in 100 mM cold phosphate buffer (pH 7.0) containing 0.1 mM EDTA and 1% polyvinylpolypyrrolidone (PVP) at 4°C. After homogenization in cold phosphate buffer, the homogenate was centrifuged at 15000 g at 4°C for 15 min to remove plant debris. The supernatant was used for the following assays of antioxidant enzyme activities. All enzymatic activities were measured at 25°C in a UV-vis spectrophotometer (Model UV-1601 PC, "Shimadzu", Japan).
SOD (EC 1.15.1.1) activity was measured according to the method of Beauchamp and Fridovich [9]. One unit of SOD activity was defined as the amount of enzyme required to cause a 50% inhibition of the ni-troblue tetrazolium (NBT) reduction rate at 560 nm. CAT (EC 1.11.1.6) activity was assayed in the reaction mixture containing 25 mM potassium phosphate buffer (pH 7.0) containing 0.1 mM EDTA, 10 mM H2O2, and the enzyme. A decrease in the absorbance of H2O2 within 1 min at 240 nm (E = 39.4/(mM cm)) was recorded [10]. The GPX activity (EC 1.11.1.7) was measured using the method described by Hemeda and Klein [11]. The reaction mixture contained 50 mM potassium phosphate buffer (pH 6.6) containing 0.1 mM EDTA, 1% guaiacol, and 10 ml of 0.3% H2O2, and the enzyme activity was measured by the increase in absorbance at 470 nm caused by guaiacol oxidation (E = 26.6/(mM cm)). APX (EC 1.11.1.11) activity was measured according to the
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