Response mechanisms of antioxidants in bryophyte (Hypnum ...

Environ Monit Assess (2009) 149:291–302 DOI 10.1007/s10661-008-0203-z

Response mechanisms of antioxidants in bryophyte (Hypnum plumaeforme) under the stress of single or combined Pb and/or Ni Shou-Qin Sun & Ming He & Tong Cao & You-Chi Zhang & Wei Han

Received: 6 November 2007 / Accepted: 14 January 2008 / Published online: 15 February 2008 # Springer Science + Business Media B.V. 2008

Abstract The short-term responses and mechanisms of antioxidants in moss Hypnum plumaeforme subjected to single or combined Pb and/or Ni stress has been revealed in this study, in order to clarify (1) the relationship between the stress intensity and antioxidant fluctuation, (2) the difference between single and combined stress, and (3) the possibility of biomonitoring by the application of antioxidant fluctuation under stress. The results showed that the stress induced dose dependent formation of reactive oxygen species (ROS) and subsequent lipid peroxidation. Total chlorophyll (Chl) content and superoxide dismutase (SOD) activity were initiated under lower stress but were inhibited under higher stress. Both single and combined stress decreased catalase (CAT) activity but increased peroxidase (POD) activity, indicating POD in the moss played an important role in resisting the oxidative stress induced by Pb and Ni. The accumulation of O2 and H2O2 in H. plumaeforme was respectively related to the low activity of

S.-Q. Sun : M. He (*) : Y.-C. Zhang : W. Han School of Agriculture and Biology, Shanghai Jiaotong University, Dongchuan Road 800, Shanghai 200240, China e-mail: [email protected] T. Cao College of Life and Environmental Science, Shanghai Normal University, Shanghai 200234, China

SOD and the decreased activity of CAT. The study indicated that Pb and Ni had synergistic effect in inducing the oxidative stress in moss H. plumaeforme, especially under the combination of high concentration of Ni (0.1, 1.0 mM) and Pb. POD and CAT activity, as well as H2O2 and MDA content, which increased or decreased regularly with a dose dependent under Pb and Ni stress, could be used as an effective indicator in moss biomonitoring, especially in the case of light pollution caused by heavy metals without the changes in the appearance of mosses. Keywords Antioxidants . Bryophytes . Physiological indicator . Response mechanisms . Stress of single or combined Pb and/or Ni

Introduction Bryophytes have high capacities to accumulate metals (Sun et al. 2007) due to their high surface-to-volume ratio and frequent absence of cuticles. Consequently, they were widely used as biosensors of environmental pollution (Samecka-Cymerman et al. 2002; Zechmeister et al. 2003) and as models for morphological and genomic alteration caused by heavy metals (Bassi et al. 1995). However, effects of pollutants on plants could be determined at several levels, from changes in biological and physiological processes (Darrall 1989) through organs and whole-plant level responses, e.g., growth inhibition, reduction of yield, and/or foliar injury (Taylor 1984), to changes in plant communities

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(Folkeson and Andersson-Bringmark 1988). As visible injuries and significant changes in growth and yield usually become apparent only after exposure to relatively high levels of pollutants, the effect of pollution on biological processes may be detected much earlier (Malhotra and Khan 1984). But, biomonitoring such as moss analysis could only give an ambiguous response to atmospheric heavy metal pollution when the concentration was too low to show the changes in the apparent morphology of moss. Therefore, metabolic changes in plants could serve as suitable indicators of pollution in the absence of visible symptoms (Roy and Hänninen 1995), and the utility of antioxidant enzymes as biomarkers was established by several investigators (Ahmad et al. 2000; Geret et al. 2002, 2003). Nickel (Ni) is an essential micronutrient for plants (Brown et al. 1987), but it becomes toxic for the majority of plant species at high concentration. The inhibition of growth, chlorosis, necrosis, and wilting were commonly observed in plants exposed to phytotoxic amounts of Ni (Pandey and Sharma 2002; Gajewska et al. 2006) due to its negative effect on the photosynthesis (Tripathy et al. 1981), mineral nutrition (Parida et al. 2003), sugar transport (Samarakoon and Rauser 1979), and water retention (Pandey and Sharma 2002). Lead (Pb), on the other hand, was considered to be a nonessential element for metabolic processes. Nevertheless, it was widely spread. Pb was able to (1) pose adverse effects on growth and metabolism of plants (Moustakas et al. 1994), (2) interfere with nutrient uptake (Singh et al. 1997; Sharma and Dubey 2005), (3) reduce seeds germination and seedling growth (Sharma and Dubey 2005), (4) disturb water balance (Sharma and Dubey 2005), (5) influence the photosystems I and II (Moustakas et al. 1994), (6) alternate permeability of cell membrane (Sharma and Dubey 2005), and (7) inhibit the activities of enzymes at cellular level by reacting with the sulphydril groups (Mishra et al. 2006). Pb was also known to cause oxidative stress resulting in the increasing production of reactive oxygen species (ROS) in plants (Reddy et al. 2005). Overproduction of ROS is a common response of plants to stress factors caused by heavy metal (Mittler 2002; Schützendübel et al. 2002), which could react with lipids, proteins, pigments, and nucleic acids, and cause lipid peroxidation, membrane damage, and inactivation of enzymes, thus affecting cell viability (Dixit et al. 2001). To maintain the balance between the generation and degradation of ROS, plants induce

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a diverse array of enzymes, e.g., superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), and low molecular weight antioxidants such as ascorbic acid (AsA), glutathione (GSH), non-protein thiol (NPT), and cysteine (Cys) to scavenge different types of ROS, thereby protecting cells against potential tissue dysfunction (Singh et al. 2006). SOD is a key enzyme in protecting cells against oxidative stress, which could transform superoxide radicals (O2 ) into H2O2, a less destructive oxygen species, hence decreasing the risk of hydroxyl radical (·OH-) formation (Foyer et al. 1994). CAT, POD, and APX are involved in the destruction of H2O2 (Weckx and Clijsters 1996). Cys, NPT, AsA, and GSH could directly interact with and detoxify oxygen free radicals, and thus contribute significantly to non-enzymatic ROS scavenging (Garnczarska 2005). Although bryophytes were widely used in environmental monitoring in recent years, little was known about the subsequent mechanisms of oxidative stress and molecular damage in bryophytes under heavy metal pollution. In this study, the short-term responses and mechanisms of antioxidants in the moss Hypnum plumaeforme, subjected to the single or combined Pb and/or Ni stress, has been revealed in order to clarify (1) the relationship between the stress intensity and antioxidant fluctuation, (2) the differences between single and combined stress, and (3) the possibility of biomonitoring by the application of antioxidant fluctuation under the stress of heavy metals. This investigation could highlight a better understanding of the biological mechanisms adopted by the moss in response to physiologically toxic concentration of heavy metals, and could also be the first step in determining physiological parameters as toxicity bioindicators in moss biomonitoring.

Materials and methods Bryophytes and experiments Moss samples (H. plumaeforme) were collected from Tianmu Mountain, a state natural reservation zone in Zhejiang, China. The moss samples were washed with tap water and then with distilled water to remove all the dusts and other vegetation. After dried with filter paper, the samples were transferred to sterile petri


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plates supplemented with 200 ml solutions with different concentrations of single and/or combined nickel nitrate (Ni) and lead nitrate (Pb), respectively. The plates were then transferred to growth chamber at 25°C with a photoperiod of 12 h light and 12 h dark. After 48 h, the samples were harvested for various biochemical analysis. The stress treatments of single or combined Ni and/or Pb were carved into six regimes as shown in Table 1. The six regimes (T1–T6) were: T1, single Pbx; T2, single Nix; T3, Ni 0.001 mM + Pbx; T4, Ni 0.01 mM + Pbx; T5, Ni 0.1 mM + Pbx; T6, Ni 1.0 mM + Pbx, (subscript ‘x’ represented the concentration of Pb or Ni, it had five levels and ranged from 0 to 10 mM for Pb and from 0 to 1.0 mM for Ni). Each regime had five addition levels of Pb or Ni concentration (L0, L1, L2, L3, and L4). The five addition levels were: 0, 0.01, 0.1, 1.0 mM for Pb and 0, 0.001, 0.01, 0.1, 1.0 mM for Ni (Table 1). L0 was also the control. Accumulation of Pb and Ni in moss The moss samples were wet digested with HNO3– HClO4–H2O2 after oven-dried at 40°C, and then the concentration of Pb and Ni was analyzed by flame absorption spectrophotometer (FAAS Hitachi Z-5000 Japan).

Determination of total chlorophyll and lipid peroxidation Total chlorophyll was estimated spectrometrically as suggested by Arnon (1949), Lipid peroxidation was measured as the amount of MDA determined by thiobarbituric acid (TBA) reaction as described by Dionisio-Sese and Tobita (1998). H2O2 and O2 generation Hydrogen peroxide (H2O2) content was colorimetrically measured as described by Mukherjee and Choudhuri (1983), O2 production was estimated by Choudhury and Panda (2005) by monitoring the nitrate formation from hydroxyl amine. Determination of enzyme activity Extraction and assay of enzymes were prepared by homogenizing the plant materials in pre-chilled mortar and pestle under ice-cold condition in extraction buffer containing 0.1 M phosphate buffer (pH 7.0), 0.1 mM EDTA, and 1% polyvinyl pyrrolidone (PVP). The homogenates were centrifuged at 4°C for 15 min at 10,000 rpm in a cooling centrifuge and the supernatant was used for the assay of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD).

Table 1 Six treatment regimes of mosses under the stress of single or combined Ni and/or Pb with five addition levels of Pb or Ni concentrations Treatment regimes

T1 T2 T3 T4 T5 T6

Heavy metal

Ni Pb Ni Pb Ni Pb Ni Pb Ni Pb Ni Pb

Addition levels of Pb or Ni concentration (mM) L0

L1

L2

L3

L4

0 0 0 0 0 0 0 0 0 0 0 0

0 0.01 0.001 0 0.001 0.01 0.01 0.01 0.1 0.01 1.0 0.01

0 0.1 0.01 0 0.001 0.1 0.01 0.1 0.1 0.1 1.0 0.1

0 1.0 0.1 0 0.001 1.0 0.01 1.0 0.1 1.0 1.0 1.0

0 10 1.0 0 0.001 10 0.01 10 0.1 10 1.0 10

T1–T6 Six treatment regimes, T1 single Pbx, T2 single Nix, T3 Ni 0.001 mM + Pbx, T4 Ni 0.01 mM + Pbx, T5 Ni 0.1 mM + Pbx, T6 Ni 1.0 mM + Pbx, (subscript x represented the concentration of Pb or Ni; it had five levels and ranged from 0 to 10 mM for Pb and from 0 to 1 mM for Ni), L0–L4 the five addition levels of Pb or Ni concentration, i.e., 0, 0.001, 0.01, 0.1, 1.0 mM orderly for Ni and 0, 0.01, 0.1, 1.0, 10 mM orderly for Pb. L0, called also as the control in the text

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SOD activity was assayed following the procedure described by De Azevedo Neto et al. (2006). CAT and POD activity was determined according to Monnet et al. (2006), and De Azevedo Neto et al. (2006), respectively. Statistical analysis The values are the means of four experiments, the significant difference test was employed for comparison of the biochemical changes at p