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Received 14 Jan. 2007 Accepted 28 Apr. 2007
Lead Induced Changes in the Growth and Antioxidant Metabolism of the Lead Accumulating and Non-accumulating Ecotypes of Sedum alfredii Hance
Dan Liu1, 2, Ting-Qiang Li1, Xiao-Fen Jin1, Xiao-E Yang1*, Ejazul Islam1, 3 and Qaisar Mahmood1
（1 Key Laboratory of Environmental Remediation and Ecosystem Health, Ministry of Education, College of Natural Resources and Environment Science, Zhejiang University, Huajiachi Campus, Hangzhou 310029, China；
2 School of Tourism and Health, Zhejiang Forestry College, Lin’an 311300, China；
3 Nuclear Institute of Agriculture, Tandojam, Hyderabad, Pakistan）
* Corresponding author.
Tel (Fax): +86 (0)571 8697 1907;
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Supported by the National Natural Science Foundation of China (20477039), the Program for Changjiang Scholars and Innovative Research Team in University (IRT0536), and the State Key Basic Research and Development Plan of China from the Science and Technology Ministry of China (2002CB410804).
Abstract:
The phytotoxicity and antioxidative adaptations of Pb accumulating ecotype (AE) and non-accumulating ecotype (NAE) of Sedum alfredii Hance were investigated under different Pb treatments involving 0, 0.02 mmol/L Pb, 0.1 mmol/L Pb and 0.1 mmol/L Pb/0.1 mmol/L EDTA for 6 days. With the increasing Pb level, the Pb concentration in the shoots of AE plants enhanced accordingly, and EDTA supply helped 51% Pb translocation to shoots of AE compared with those treated with 0.1 mmol/L Pb alone. Moreover, the presence of EDTA alleviated Pb phytotoxicity through changes in plant biomass, root morphology and chlorophyll contents. Lead toxicity induced hydrogen peroxide (H2O2) accumulation and lipid peroxidation in both ecotypes of S. alfredii. The activities of superoxide dismutase (SOD)， guaiacol peroxidase (G-POD,), ascorbate peroxidase (APX), and dehydroascorbate reductase (DHAR) elevated in both leaves and roots of AE as well as in leaves of NAE with the increasing Pb levels, but SOD and G-POD declined in roots of NAE. Enhancement in glutathione reductase (GR) activity was only detected in roots of NAE while a depression in catalase (CAT) activity was recorded in leaves of NAE. A significant enhancement in GSH and AsA levels occurred in both ecotypes exposed to Pb and Pb/EDTA treatment compared with CK, however, the difference between this two treatments were insignificant. The dehydroascorbate (DHA) contents in roots of both ecotypes were 1.41 to 11.22-folds higher than those in leaves, whereas the ratios of AsA to DHA (1.38 to 6.84) in leaves altering more to the reduced AsA form were much higher than those in roots. These results suggested that antioxidative enzymes and antioxidants played an important role in counteracting Pb stress in S. alfredii.
Keywords：Antioxidant; Detoxification; EDTA; Phytoremediation; Sedum alfredii ;
Heavy metal pollution is a widespread global dilemma, and has been a major environmental concern over the past several decades. A wide range of heavy metals have been detected in different biota. Lead (Pb) is one of the most abundant, ubiquitous toxic elements posing a critical concern to human and environmental health due to being persistent contaminant, low solubility, and the classification as carcinogenic and mutagenic (Diels et al. 2002).
Conventional cleanup technologies are generally too costly to be used to restore contaminated sites, and are often harmful to the normal properties of the soil (Holden, 1989). Well established cost effective and environmental friendly phytoremediation techniques have grabbed increasing attention recently (Salt et al. 1998; Garbisu and Alkorta 2001). No reliable reports on Pb hyper-accumulating species under natural conditions are available; moreover the phyto-availability of Pb is restricted by the strong complexation of Pb within solid soil fractions. To overcome the problem and increase Pb availability to plants, chelators have been used to enhance Pb solubility artificially in soil solution (Kos and Lestan 2004). Role of EDTA in increasing plants metal uptake has been documented by a number of workers (Liphadzi and Kirkham 2006). Huang et al., (1997) reported that among five chelating agents, EDTA was the most efficient in increasing shoot Pb concentration in both pea and corn.
Pb exerts adverse effects on morphology, growth and photosynthetic pathways of plants and inhibits enzyme activities, water balance, causes alterations in membrane permeability and disturbs mineral nutrition (Sharma et al. 2005). Biochemical responses of plants to Pb include enhancement in the activity of antioxidative enzymes and production of antioxidants scavenging system controlling free radicals that are produced upon exposure to them. Pb can induce oxidative stress with over production of reactive oxygen species (ROS) including superoxide radicals (O2 •), hydroxyl radicals (·OH) and hydrogen peroxide (H2O2) (Verma and Dubey 2003; Thomas Ruley et al. 2004; Reddy et al. 2005). Free radicals and hydrogen peroxide are reported to cause the membrane damage often related to lipid peroxidation. The dismutation of superoxide into H2O2 and O2 by superoxide dismutases (SOD, EC 1.15.1.1) has been widely recorded as a detoxification mechanism. H2O2 formed can be decomposed into H2O and O2 within the peroxisome by catalase (CAT) that does not require an additional substrate. The scavenging of H2O2 in other cell compartments depends on distinct peroxidases such as guaiacol peroxidases (G-POD) and ascorbate peroxidases (APX) that use a substrate for their activity (Cakmak and Marschner, 1992; Noctor and Foyer, 1998). Glutathione reductase (GR) together with dehydroascorbate reductase (DHAR) is involved in the breakdown of H2O2 via the ascorbate-glutathione cycle (Noctor and Foyer, 1998). Glutathione, the basic unit of phytochelatins is very mobile and can be found at high concentrations in all cell compartments, phloem and in roots (Foyer and Noctor, 2000). Glutathione is an important antioxidant and forms, with oxidized glutathione (GSSG), an important redox couple, providing the conditions to support mitochondrial oxidative phosphorylation, generation of ATP, and hence key anabolic activities (May et al., 1998), and glutathione also takes part in thioredoxin-related regulation of many enzymes of photosynthetic metabolism. Ascorbate is a major metabolite in plants, which occurs in the cytosol, chloroplasts, vacuoles, mitochondria and cell wall.
Sedum alfredii Hance is a newly discovered Zn/Cd hyper-accumulator growing in the old Pb/Zn mined areas of southeast China (118°56´E, 29°17´N), and has been reported to be a Pb accumulating species (He et al. 2002; Yang et al. 2002b; Yang et al. 2004). Earlier studies on Sedum alfredii mainly focused on the accumulation and transportation mechanism (Yang et al., 2002a; Zhou and Qiu, 2005; Yang et al., 2006), and less attention was paid to the detoxification mechanisms. In this context, a study was conducted to investigate the antioxidant metabolism of two ecotypes of Sedum alfredii under the stress of Pb, and effects of EDTA on the detoxification of Pb were also studied. Pb toxicity was assayed using parameters such as biomass, root morphology, photosynthetic pigments, antioxidant enzymes activity and membrane structure.
Results
Lead uptake
After treated with 0.02 mM Pb, there were no significant differences of Pb concentration in shoot of AE plants compared with CK (P 0.05) (Table 1). Addition of 0.1mmol/L Pb significantly accelerated the Pb concentration in shoot of AE and NAE plants resulting in 45.4- and 25.8-folds increase, compared with CK, respectively. It can be seen that combined Pb and EDTA treatment caused a significant increase in the shoot Pb concentration of AE (increased by 51%) compared with those treated with 0.1mmol/L Pb alone (P 0.05).
The Pb concentration in shoot of AE was always higher than that of NAE, but the trend was on the contrary for the root (Table 1). After treating with 0.1 mmol/L Pb, the concentration in roots of AE and NAE plants increased 186.2- and 382.7-fold compared with CK, respectively. It can be seen that with the combined EDTA and Pb treatment, the concentration of Pb in roots decreased 30.8% and 20.2% compared with the treatment having 0.1mmol/L Pb alone for the AE and NAE plants, respectively.
Plant growth parameters and photosynthetic pigments
In the hydroponics experiment, AE plants exhibited strong tolerance to Pb toxicity, with erect stem, thicker and dark green colored leaves compared with NAE plants. The dry weights of both ecotype of S. alfredii are presented in Table 2 (P 0.05). Shoot dry weights of AE were higher than those of the NAE in both CK and treated plants. After increasing application rate of Pb from 0.02 mmol/L to 0.1 mmol/L, the shoot Pb in AE decreased by 15.9%; however, while 0.1 mmol/L Pb combined with 0.1mmol/L EDTA addition caused an increase in the shoot dry weight by 5.1% compared with those treated with 0.1 mmol/L Pb alone. As for root, the same trend also could be traced i.e. EDTA addition stimulated root growth compared with Pb treatment alone.
In order to compare the root morphological characteristics under various treatments, root length, root surface area, root diameter and root volume were measured (Table 3). Under the stress of 0.1 mmol/L Pb and 0.1 mmol/L Pb/0.1 mmol/L EDTA, root length of the AE was significantly reduced by 43.4% and 21.3%, respectively, as compared with CK (P 0.05). Similar trend was traced in the NAE plants, while the root length always lowered than that of AE plant in both CK and treatments. Both root surface area and root volume decreased after treating with 0.1mmol/L Pb or 0.1 mmol/LPb/0.1 mmol/L EDTA in both ecotypes of S. alfredii (P 0.05); the parameter for AE always higher than that of NAE. As for root diameter, no significant differences were observed between CK and treatments (P 0.05) for both ecotypes.
The Chlorophyll a, b, total Chlorophyll and carotene contents of the AE plants were significantly higher (P 0.05) than that of the NAE plants in all treatments (Figure 1). After treating with Pb or combined with EDTA, there are no significant changes in chlorophyll a and b for AE plants (P 0.05), while those of NAE decreased slightly compared with CK (P 0.05) (Figure 1A, B). After treating with 0.1mM Pb, the total chlorophyll content of NAE plants decreased significantly by 8.2% compared with CK (P 0.05), while AE plants treated with 0.1mmol/L Pb/0.1mmol/L EDTA the total chlorophyll increased 4% compared with that treated with Pb alone (P 0.05) (Fig 1C). As for the carotene content, after treating with 0.1 mmol/L Pb or 0.1 mmol/L Pb/0.1 mmol/L EDTA, carotene contents decreased significantly in both ecotypes. Also it could be seen that the carotene contents of AE plants were always higher than that of NAE (P 0.05) (Fig 1D).
H2O2 concentrations and Lipid peroxidation products
Treatments with Pb alone and combined with EDTA stimulated H2O2 accumulation (1.21 to 3.61-folds increase as compared with CK) in both leaves and roots tissues of AE and NAE, and the H2O2 concentrations in the roots were extremely higher than those in leaves. Moreover, the H2O2 concentration was much higher in the roots of NAE than those of AE treated with Pb alone and its combination with EDTA, whereas it was opposite to those in leaves. Particularly, the H2O2 concentrations in the plants treated with 0.1 mmol/L Pb combined with 0.1mmol/L EDTA were much lower than those in the plants treated with 0.1mmol/L Pb alone (except for that in roots of AE) (P 0.05) (Fig 2). Lead toxicity resulted in lipid peroxidation, where malondialdehyed (MDA) content increased significantly with increased Pb levels (Fig.3). Increased H2O2 concentration and lipid peroxidation with metal exposure revealed that Pb toxicity caused generation of reactive oxygen species and oxidative stress in S. alfredii.
Antioxidative enzymes activities
Superoxide dismutase (SOD) is considered as first defense against oxidative stress, which dismutates two superoxide radicals to H2O2 and oxygen and thus maintains superoxide radicals in steady state levels. SOD activity increased significantly in the leaves and roots of AE plants as well as in the leaves of NAE under Pb and its combinations with EDTA, whereas it was noticed that SOD activity decreased in roots of NAE (P 0.05) (Fig 4A and B).
Catalase (CAT), ascorbate peroxidase (APX), and guaiacol peroxidase (G-POD) contribute for elimination of excessive H2O2 in the plant cells. The activity of G-POD was dramatically increased by Pb treatments and Pb combined with EDTA in the leaves of both ecotypes, where the G-POD activity in leaves of AE and NAE grown under 0.1 mmol/L Pb+0.1 mmol/L EDTA were 1.48-fold and 3.88-fold, respectively, higher and those of the plants exposed to 0.1 mmol/L Pb alone (Fig.4C). Moreover, there was an elevated activity of G-POD in roots of AE other than NAE under Pb treatment (Fig.4D). However, CAT activity declined about 50% with increasing Pb toxicity in the leaves of NAE (Fig.4E), whereas an enhancement in CAT activity was observed in roots of both AE and NAE as well as in the leaves of AE (Fig.4E and F). Nevertheless, the activities of APX in leaves and roots were significantly elevated in lead stressed plants of both ecotypes of S. alfredii, and the magnitude of elevation ranged from 1.67- to 77.8- folds over CK, whereas APX activity in the leaves of AE exposed to 0.1 mmol/LPb/0.1 mmol/L EDTA was recorded 58.6% higher than that in presence of 0.1 mmol/L Pb alone (P 0.05) (Fig.4 G and H).
The two enzymes i.e. glutathione reductase (GR) and dehydroascorbate reductase (DHAR) which uses glutathione as reductant for converting DHA to AsA, are closely involved in GSH and AsA changes. Statistical analysis showed a significant increase in DHAR activity in both root and leaves of NAE and AE under Pb stress, and the highest DHAR activity in both leaves and roots were noted after exposing plants to 0.1mmol/L Pb (Fig.4 K, L). GR activity slightly decreased in leaves of both AE and NAE under lead stress as compared with their CK (Fig.4I), whereas GR activity was elevated in roots of NAE and remarkably declined in roots of AE exposed to 0.02 mmol/L and 0.1 mmol/L Pb but was recovered in the presence of 0.1 mmol/LPb combined with 0.1 mmol/L EDTA (Fig.4J).