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Ecophysiological tolerance of Lemna gibba L. exposed to cadmium
Ecotoxicology and Environmental Safety 91 (2013) 79–85
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Ecophysiological tolerance of Lemna gibba L. exposed to cadmium Kadiriye Uruc- Parlak a,n, Dilek Demirezen Yilmaz b a b
_Ibrahim C ˘ Turkey - ec- en University, Faculty of Arts and Sciences, Department of Biology, Agri, Erciyes University, Faculty of Science, Department of Biology, Kayseri, Turkey
a r t i c l e i n f o
abstract
Article history: Received 8 May 2012 Received in revised form 25 December 2012 Accepted 14 January 2013 Available online 20 February 2013
In this study, an experiment was carried out to study the process of stress adaptation in Lemna gibba L. grown under cadmium stress (0–20 mg Cd L 1). The level of photosynthetic pigments and soluble proteins decreased only upon exposure to high Cd concentrations (for pigments 5 mg Cd L 1; for soluble proteins 10 mg Cd L 1). At the same time, the level of malondialdehyde (MDA) increased with increasing Cd concentration. These results suggested an alleviation of stress that was presumably the result of antioxidants such as superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and glutathione S-transferase (GST) as well as ascorbate peroxidase (APX), which increased linearly with increasing Cd levels. In addition, the proline content in L. gibba increased with increasing cadmium levels. These findings suggest that Lemna is equipped with an efficient antioxidant mechanism against Cd induced oxidative stress which protects the plant’s photosynthetic machinery from damage.We also found that moderate Cd treatment (0.05–5 mg L 1 Cd) alleviated oxidative stress in plants, while the addition of higher amounts of Cd (10–20 mg L 1) could cause an increasing generation of ROS, which was effectively scavenged by the antioxidative system. & 2013 Elsevier Inc. All rights reserved.
Keywords: Cadmium Lemna gibba Accumulation Antioxidant Proline Protein
1. Introduction Cadmium (Cd) is a globally distributed toxic element, which enters the aquatic environment from natural (weathering of rocks) as well as anthropogenic sources, and is then transferred ¨ ¨ to the food chain (Li and Xiong, 2004a, 2004b; Schutzend ubel et al., 2002). It causes an inhibition of enzyme activities, water imbalance and alterations in membrane permeability; it also disturbs mineral nutrition (Milone et al., 2003; Sharma and Dubey, 2005; Liu et al., 2007). In plants, reactive oxygen species (ROS) are continuously produced as by-products of various metabolic pathways, which are localized in different cellular compartments (Gratao et al., 2005; Reddy et al., 2005). However, under stressful conditions, their formation may exceed antioxidant scavenging capacity, thereby inducing oxidative stress and damaging most of the biomolecules (Davies, 2003). One of the most damaging effects of ROS and their products is the peroxidation of membrane lipids and ion leakage (Sharma and Dubey, 2005). To control ROS levels and to protect cells, plants produce low molecular weight antioxidants (Gratao et al., 2005). Plants possess several other mechanisms to deal with the detoxification of heavy metals thus enabling them to tolerate
n
Corresponding author. Fax: þ90 472 215 65 54. E-mail addresses: [email protected] (K. Uruc- Parlak), [email protected] (D. Demirezen Yilmaz). 0147-6513/$ - see front matter & 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ecoenv.2013.01.009
stress (Hall, 2002; Malecka et al., 2001; Tennant, 1975). Aquatic ecosystems are more sensitive to pollutants than terrestrial ones because most uptakes are facilitated by dissolution in water, which may lead to an accumulation of heavy metals. Duckweed (Lemna sp.) which often forms dense floating plants in aquatic ecosystems such as ditches, ponds and lakes was selected for study. Duckweed can be used as an indicator species to assess ecotoxicity of waters polluted by contaminants. It was selected because information on the plants responses to cadmium is insufficient. Therefore, the present study was, designed to investigate (i) the effects of Cd on the growth and Cd accumulation of Lemna gibba; (ii) the effects of Cd on the lipid peroxidation (MDA), (iii) the activities of enzymatic antioxidants (SOD, CAT, GST, APX, and GR) and accumulation of proline in L. gibba. The results could be meaningful in evaluating the role of L. gibba’s antioxidative system as regards Cd tolerance and detoxification. 2. Materials and methods 2.1. Plant material, growth conditions and metal estimation Fresh samples of L. gibba L. (Lemnaceae) were obtained from ponds in the Soysallı National Park in Kayseri, Turkey. In this study, cadmium nitrate was used in the treatments, and samples were exposed to various concentrations of cadmium: 0, 0.05, 0.5, 5, 10, and 20 mg L 1. Plants were grown in plastic cups containing Hoagland’s solution (pH 5) and grown for 7 days in a growth chamber at 257 1 1C with 16 h light: 8 h dark photo-cycle at light intensity of
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40 mmolm 2 s 1. The solutions were changed every two days. On the 7th day, the cadmium treated plants were harvested from their containers. The plants were rinsed twice with distilled water, and subsequently, their biochemical parameters were determined. Dried samples of L. gibba were digested with 10 ml of concentrated HNO3, using a CEM microwave digestion system. After digestion, the volume of each sample was adjusted to 25 ml using double deionized water (Demirezen Yılmaz, 2007). Determination of the cadmium concentrations in all samples was carried out by inductively coupled plasma optical emission spectrometry (Varian). The samples were analyzed in triplicate.
performed to determine the significant differences between treatments. In the figures, the values are marked with an n(asterisk) for the significance level (Pr0.05) as compared to the control.
2.2. Determination of pigment concentration
The results related to the accumulation of cadmium in L. gibba are presented in Table 1. The maximum Cd accumulation was found to be 1.67 mg kg 1 DW at 0.5 mg L 1 Cd. On prolonged exposure to higher concentrations of Cd (i.e. 20 mg L 1), there was a significant decline in the Cd accumulation rate. The analysis of one-way ANOVA showed that the differences in all the treatments were significant (P o0.05) for L. gibba.
Fresh plants (0.1 g) were cut into pieces and homogenized in 96% ethanol in the presence of quartz and calcium carbonate. The homogenate was filtrated and then diluted to 25 mL with deionized water. The concentrations of chlorophylls were calculated according to the formula of Ya˘gmur et al. (2006). 2.3. Estimation of lipid peroxidation (MDA) The level of lipid peroxidation was estimated following the method of Razinger et al. (2008), with modifications. The concentration of MDA (lM) in the solution obtained was calculated by Razinger et al. (2008). 2.4. Antioxidative enzymes 2.4.1. Enzyme extraction Fresh tissue (0.2 g) was homogenized in an ice-cooled mortar with 5 mL of 100 mM potassium phosphate buffer (pH 7.0) containing 0.1 mM EDTA and 1% (w/ v) polyvinylpyrrolidone. The homogenate was centrifuged at 15.000 g for 15 min at 4 1C (Hou et al., 2007). The supernatant was used for enzyme determination. 2.4.2. Superoxide dismutase (EC 1.15.1.11) The activity of SOD (EC 1.15.1.11) was assayed by measuring the inhibition of the photochemical reduction of nitroblue tetrazolium (NBT) (Beauchamp and Fridovich, 1971). The assay mixture contained 20 mM phosphate buffer (pH 7.5), 10 mM methionine, 0.1 mM NBT, 0.1 mM EDTA, 0.005 riboflavin, 50 mgmL 1 of enzyme extract and 0.25 mL of deionized H2O in a total volume of 3 mL. Finally, riboflavin was added, and the tubes were shaken and then illuminated for 15 min. The absorbance was recorded at 560 nm and the absorbance of the non-irradiated reaction mixture served as a control. A 50% inhibition of the reaction was defined as one unit of enzyme, and the enzyme activity was expressed as ng 1 FW. 2.4.3. Catalase (EC 1.11.1.6) CAT (EC 1.11.1.6) activity was measured spectrophotometrically according to Aebi’s method following the consumption of H2O2 at 240 nm (Aebi, 1984), in potassium phosphate buffer (150 mM, pH 7) containing 15 mM H2O2 and enzyme extract (exactly 50 mg of protein) in a final volume of 1 ml. The addition of H2O2 started the reaction. 2.4.4. Glutathione reductase (EC 1.6.4.2) GR (EC 1.6.4.2) was assayed as the increase in absorbance at 340 nm when DTNB is reduced by GSH to produce 2-nitro-5-thiobenzoic acid (TNB) according to Smith et al. (1988). 2.4.5. Glutathione S-transferase (EC 2.5.1.13) Conjugation of GSH to CDNB catalyzed by GST induced an increase in absorbance at 340 nm which was used to assay for GST activity as described by Drotar et al. (1985). 2.4.6. Ascorbate peroxidase (EC 1.11. 1.11) APX activity was determined according to Nakano and Asada by the decrease in the absorbance of ascorbate at 290 nm (Nakano and Asada, 1981). 2.5. Proline analysis Plants were sampled for analysis of proline after 7 days in the treatment using the methodology described by Bates et al. (1973). Concentrations of proline in the plant tissue were expressed on a FW basis. 2.6. Statistical analysis All experiments were repeated in triplicate. Values shown in the figures represent the average values7 standard deviation (SD) for each cadmium concentration. Data were subjected to a one-way analysis of variance (ANOVA) to confirm the variability of data and validity of results and a Turkey test was
3. Results 3.1. Cadmium content
3.2. Effect of cadmium on the level of photosynthetic pigment and total soluble proteins The leaves of L. gibba started to show signs of chlorosis after exposure to Cd. Accordingly, there were significant decreases in chlorophyll a and b for the plants exposed to Cd compared with the control (Fig. 1a). In other words, a concentration-dependent response to Cd stress was observed in the photosynthetic pigment. At 5 mg L 1 cadmium, total chlorophyll increased at 0.35 mg g 1 FW, whereas at 20 mg L 1 cadmium it decreased at 0.282 mg g 1 FW as compared to the control (0.285 mg g 1 FW). Similarly, the total soluble protein concentration in L. gibba decreased with higher Cd concentration in plants as compared to the control but was found to increase in plants at 10 mg L 1 Cd (Fig. 1b). 3.3. Effect of cadmium on lipid peroxidation The effect of Cd on MDA concentration is presented in Fig. 2. Significant increases in MDA concentration in L. gibba were observed in the experiments. MDA concentration increased linearly with increased Cd levels in the solution. The analysis of a one-way ANOVA showed that the differences in all the treatments were significant (Po0.05) for L. gibba. 3.4. Effect of Cd on catalase, superoxide dismutase, glutathione reductase, glutathione S-transferase, ascorbate peroxidase A decline in catalase activity was observed in L. gibba with the increasing Cd concentration. The highest concentration of Cd (20 mg L 1) was proved to be extremely toxic, reducing CAT activity (Fig. 3). Plants exposed to Cd presented significant differences (Po0.05). SOD activity increased linearly with increasing Cd levels (Fig. 4). The maximum SOD activity was recorded at 20 mg L 1 Cd (1.261 70.0085 mg 1). At the highest Cd concentration, SOD accumulation increased. Additionally, significant differences were found in SOD activity among the treatments. Although no significant differences were found in GR and GST, activity tended to be higher in plants from the control. The results Table 1 Cadmium concentration in L. gibba upon Cd exposure. Values represent mean 7S.E. (n¼ 3). Different letters indicate significant differences at P o0.05. Cd concentration (mgL 1)
Metal uptake by L. gibba (mgkg 1 DW)
Control 0.05 0.5 5 10 20
0.104 70.001 0.783 70.007 1.673 70.001 1.515 70.005 0.664 70.003 0.172 70.001
a d f e ce b
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Chlorophyll concentrations (mgg-1FW)
0.4 0.35
81
* kla
klb
klt
*
0.3 0.25 0.12 0.15 0.1
*
*
0.05 0
Total soluble protein (mgg-1FW)
Control
10 0.05 0.5 5 Cd concentration in solution (mgL-1)
20
4 3.5 3 2.5 2 1.5 1 0.5 0 Control
0.05 5 0.5 10 Cd concentration in solution (mgL-1)
20
Fig. 1. (a) Effect of different Cd concentrations on chlorophyll a, chlorophyll b and total chlorophyll in L. gibba. Values represent mean 7 S.E. (n¼ 3). Asterisks indicate significant differences at Po 0.05. and (b) Effect of different Cd concentrations on total soluble protein concentrations in L. gibba. Values represent mean 7 S.E. (n¼3).
1
MDA (nmol g-1 FW)
0.9
*
0.8 0.7
*
*
*
*
0.6 0.5 0.4 0.3 0.2 0.1 0 Control
0.05
0.5
5
Cd concentration in solution
10
20
(mgL-1)
Fig. 2. MDA concentration in L. gibba upon Cd exposure. Values represent mean 7 S.E. (n ¼ 3). Asterisks indicate significant differences at P o 0.05.
related to the effect of Cd on GR and GST activity are presented in Tables 2 and 3. GR and GST activity in L. gibba increased linearly with increasing Cd levels in the solution. The maximum GR activity was recorded at 0.05 mg L 1 Cd (0.02870.0001 mg 1). At 20 mg L 1 Cd, the GST in L. gibba increased to 0.03170.000 mg 1. According to the results, APX activity decreased linearly with increasing Cd levels. The highest concentration of Cd (20 mg L 1) proved to be extremely toxic resulting in a decline of APX activity (Table 4). 3.5. Proline To understand the contributions of non-enzymatic antioxidants in the L. gibba response to Cd toxicity, their proline contents
were examined. The amounts of proline in L. gibba grown under different concentrations of cadmium are shown in Table 5. The proline content in control plants increased gradually in the duration of the experiment.
4. Discussion In the present study, L. gibba was grown in hydroponic culture in the presence of increasing Cd concentrations to evaluate its Cd uptake, growth and to identify possible defense mechanisms. The results presented in this study indicated that Cd was toxic to L. gibba. The higher accumulation of Cd in L. gibba found in
K. Uruc- Parlak, D. Demirezen Yilmaz / Ecotoxicology and Environmental Safety 91 (2013) 79–85
82
0.02
CAT (mg-1 protein)
0.018
*
0.016
*
0.014
*
*
0.012 0.01 *
0.008 0.006 0.004 0.002 0 0.05 5 0.5 10 Cd concentration in solution (mgL-1)
Control
20
Fig. 3. CAT activities in L. gibba upon Cd exposure. Values represent mean 7 S.E. (n¼ 3). Asterisks indicate significant differences at Po 0.05.
1.3 * SOD (mg-1 protein)
1.25
* *
*
*
1.2 1.15 1.1 1.05 Control
0.05
0.5 5 10 Cd concentration in solution (mgL-1)
20
Fig. 4. SOD activities in L. gibba upon Cd exposure. Values represent mean 7 S.E. (n¼ 3). Asterisks indicate significant differences at Po 0.05.
Table 2 GR activity in and L. gibba upon Cd exposure. Values represent mean 7S.E. (n¼3). Different letters indicate significant differences at Po 0.05. Cd concentration (mgL 1) Control 0.05 0.5 5 10 20
GR activity in L. gibba (unit mg 0.011 70.0003 0.028 70.0001 0.026 70.0007 0.022 70.0004 0.021 70.0003 0.018 70.0002
1
)
a e d c c b
Table 3 GST activity in and L. gibba upon Cd exposure. Values represent mean 7 S.E. (n¼ 3). Different letters indicate significant differences at Po 0.05. Cd concentration (mgL 1)
GST activity in L. gibba (unit mg 1)
Control 0.05 0.5 5 10 20
0.026 70.0003 0.027 70.0004 0.028 70.0002 0.030 70.0003 0.031 70.0000 0.038 70.0000
a a b c cd d
our study could reflect different cellular mechanisms for the bioconcentration of essential and non-essential trace metals in plants. Additionally, the availability and bioaccumulation of Cd could also be influenced by chemical speciation of the metal, organic chelators, the presence of other metals and anions, ionic strength, light intensity, temperature and oxygen level (Greger, 1999).
Table 4 APX activity in and L. gibba upon Cd exposure. Values represent mean 7S.E. (n ¼3). Different letters indicate significant differences at P o 0.05. Cd concentration (mgL 1) Control 0.05 0.5 5 10 20
APX activity in L. gibba (unit mg 0.116 70.001 0.092 70.001 0.088 70.002 0.074 70.000 0.073 70.002 0.053 70.002
1
)
a b b c c d
Table 5 Proline contents in and L. gibba upon Cd exposure. Values represent mean 7S.E. (n ¼3). Different letters indicate significant differences at P o 0.05. Cd concentration (mgL 1)
Proline in L. gibba (lmol g 1)
Control 0.05 0.5 5 10 20
0.021 7 0.0002 0.024 7 0.0005 0.042 7 0.0015 0.045 7 0.0011 0.051 7 0.0011 0.083 7 0.0025
a a b bc c d
As can be seen in Fig. 1 while amount of Cd accumulated by plant tissue in gradually decreases after the 0.5 mgL 1 Cd treatment. A similar result was reported by Karatas- et al. (2009) with L. gibba L. stressed by wastewater. According to these authors, the decreases in amounts of heavy metal (selenium) can result from their consumption for preventing oxidative stresses taking place during treatment.
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Chlorophyll contents of tissues were significantly lower when Cd levels were above 10 ppm. The rise in chlorophyll contents could be regarded as a metal specific response that may have resulted in chlorophyll upgradation and synthesis of photosynthates (Davies, 2003). The decline in total chlorophyll contents in high Cd levels as well as growth inhibition can be regarded as general responses associated with metal toxicity (Boswell et al., 2002; Vazquez et al., 1987). In other words, a decline in the level of photosynthetic pigments may be attributed to the cadmiuminduced inhibition of chlorophyll biosynthesis possibly caused by induced nutrient deficiency, such as that of iron. In the present study these responses were noticed mostly in the case of Cd, as common duckweed can tolerate different heavy metals at applied concentrations without a notable effect on its photosynthetic pigment contents or significant multiplication inhibition (Fodor, 2002; Li and Xiong, 2004a, 2004b). Overall, the destruction of photosynthetic pigments by metals could be due to impairment of the electron transport chain, replacement of mg2 þ ions associated with the tetrapyrrole ring of chlorophyll molecules, inhibition of important enzymes associated with chlorophyll biosynthesis or peroxidation processes in chloroplast membrane lipids by reactive oxygen species (Van Assche and Clijsters, 1990; Sandalio et al., 2001). For example, it has been shown previously that Zn preferentially accumulates in the chloroplast where it can directly interact with the thylakoid membranes (Van Assche and Clijsters, 1990; Szalontai et al., 1999). The increased lipid peroxidation observed in the Cd treatments used in this study supports this hypothesis. The total soluble protein concentration in L. gibba decreased with higher Cd concentration in plants as compared to controls. A similar result was reported by John et al., with Lemna polyrrhiza L. stressed by Cd and Pb (John et al., 2008). In that study, it seems that there might have been greater ROS generation due to high Cd load in plants and as a result, more oxidative stress which resulted in the decline of protein concentration caused by oxidative damage. MDA is the decomposed product of polysaturated fatty acids (PUFA) of biomembranes, and its increase shows plants under a high level of antioxidative stress (Hou et al., 2007). The main site of attack by any redox-active metal in a plant cell is usually the cell membrane. MDA concentration obtained from this study increased linearly with increased Cd levels in the solution. Similar results were found with S. polyrhiza (Upadhyay and Panda, 2010). Thus, increased MDA indicates the prevalence of oxidative stress and this may be one of the possible mechanisms by which toxicity due to Cd is manifested in plant tissues (Gupta et al., 2009). However, Yamamoto et al., demonstrated that Al cannot catalyze the peroxidation reaction by itself, but enhances the Fe (II or III) mediated non-enzymatic peroxidation of phospholipids (Yamamoto et al., 2002). Boscolo et al. (2003). suggested that the possible reason for the absence of lipid peroxidation in maize is that the Fe2 þ bound to the membrane is not exposed to the attack of H2O2. Metal toxicity is often driven by ROS generation, directly via the catalytic production of superoxide (O2 ) by the Haber–Weiss and Fenton reactions, or indirectly by other mechanisms (Yamamoto et al., 2002; Boscolo et al., 2003). ROS can damage membrane lipids, proteins, pigments, and nucleic acids, and the plant evolves an antioxidant defense system to prevent the adverse effects of oxidative stress to cells (Briat, 2002). The inadequate activities of antioxidant defense systems cause oxidative damage in plants exposed to Cd (Mittler, 2002). The antioxidative enzyme system is one of the protective mechanisms in a plant. The increased activity of antioxidative enzymes in a plant indicates the formation of ROS, and the present study indicated the generation of oxidative stress in L. gibba since all enzyme activity increased with high Cd added levels (Jung et al., 2000). In
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other words, the L. gibba analyzed in this work contained high Cd concentrations sufficient to activate ROS production and subsequently oxidative stress. According to Sharma and Dietz, antioxidant enzyme activities in metal-stressed plants are highly variable, depending on the plant species, metal ion, concentration and exposure duration, but these processes reflect the modified redox status of the stressed cells Since the primary reduced product of oxygen is the superoxide anion radical, SOD represents the first defense against oxidative stress (Sharma and Dietz, 2009). Superoxide dismutase, the first enzyme in the detoxifying process, converts superoxide radicals to H2O2 at a very fast rate. The enhanced SOD activity observed in the present study was consistent with studies in which other plant species were treated with heavy metals (Sharma and Dubey, 2007; Prasad et al., 1999). Due to the action of SOD, H2O2 concentration can be expected to increase inside the cell; this was found in duckweed plants under heavy metal toxicity (Sharma and Dubey, 2007). An increase in SOD activity could possibly be the result of both a direct effect of heavy metal ions and an indirect effect mediated via an increase in levels of superoxide radicals (Prasad et al., 1999). The effect of heavy metal stress on SOD expression is likely to be governed by the tissue and sub-cellular sites where oxidative stress is generated as supported by higher SOD activity in roots than in leaves of metal-stressed plants (Hou et al., 2007). An increase in SOD activity may be linked to an increase in superoxide radical formation as well as to the de novo synthesis of enzyme protein, which in turn may be associated with an induction of SOD genes by superoxide-mediated signal transduction (Prasad et al., 1999). Our data showed that significant increases in CAT activity were observed between the treatments. The results obtained indicated that CAT activity decreased linearly with increasing cadmium levels. Contrary to our results, a decline in the specific activity of catalase with an increase in Cr concentration (20– 80 ppm, 0.5 mM) was reported by Shankar et al. (2005). According to Willekens et al. (1997), it is likely that excess production of ROS caused by heavy metals can inactivate CAT, probably by inactivating the enzyme-bound heme group. CAT is only present in peroxisomes, but it is indispensable for ROS detoxification during stress, when high levels of ROS are produced. Boscolo et al. (2003), also reported no change in CAT activity under Al toxicity in maize, while in other plants a decline (soybean, rice) or enhancement (tobacco, wheat) of CAT activity was found. The antioxidative capacity of cells can be estimated by measuring APX activity. In contrast to CAT, APX has a higher affinity for H2O2, allowing for the scavenging of small amounts of H2O2 in more specific locations (Radic et al., 2010; Asada, 1992). In different aquatic and terrestrial plant species this type of peroxidases (APX) has been shown to be inducible on exposure to different heavy metals (Sharma and Dubey, 2005; Gupta et al., 2009). In our experiments, Cd treatments induced APX activities in L. gibba. This means that APX activity decreased linearly with increasing Cd levels for example it was significantly decreased by the addition of 20 mg L 1 Cd compared with the control. The hyperactivity of peroxidase under Cd stress indicated its role in the constant detoxification of H2O2. These results agree with earlier studies on aquatic plants. Cellular antioxidants play a significant role in providing resistance to plants by protecting labile macromolecules against attack by free radicals produced under heavy metal stress (Devi and Prasad, 1998). The present study revealed a decline in levels of ascorbate in plants exposed to Cd suggesting they play a significant role in scavenging free radicals and hence in the protection of cells from oxidative damage. However, high levels of glutathione in Cd exposed L. gibba seem to provide wide-spread antioxidant protection to the plant body and, most importantly increase the thiol concentration
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in the cytoplasm and chloroplasts, thus protecting them against photochemical damage. A similar response to an increase in glutathione levels on exposure to heavy metals such as Cd and Cr was reported in Phragmites (Iannelli et al., 2002). Increased SOD and CAT activities in L. gibba indicate that this plant has the capacity to adapt to Cd toxicity by developing an antioxidant defense system. Improvement of stress tolerance is often related to an increase in the activity of antioxidant enzymes (Murgia et al., 2004; Lee et al., 2007). Hsu and Kao (2007) reported that early accumulation of H2O2 during heat shock signaled an increase in APX and GR activities, which in turn protected rice plants from the oxidative stress caused by Cd. Glutathione is considered to be a very important signal molecule which acts as a link between environmental stress and adaptive responses, and it is regenerated from GST by GR activity (Navari-Izzo et al., 1997). Concomitant increases in proline as a function of metal accumulation have been observed in response to heavy metals (Bassi and Sharma, 1993). In our study, Cd treatments increased the accumulation of proline, regardless of the fact that soluble protein content declined in response to Cd. Aside from acting as a metal chelator and osmolyte, proline has been reported to scavenge hydroxyl radicals and singlet oxygen, thus providing protection against ROS-induced cell damage (Alia Prasad and Saradhi, 1995). Increases in both proline and lipid peroxidation levels with increasing Cd concentration are indicative of a correlation between ROS generation (hydroxyl radicals mostly) and ROS scavenging by proline. Accumulation of proline in response to excess metals such as copper, cadmium, zinc and nickel was described in several plants (Matysik et al., 2002).
5. Conclusion Considering these results, we strongly suggest that higher cadmium levels causes oxidative stress in L. gibba cells and may cause membrane damage through production of ROS and interferes with chlorophyll metabolism. Therefore, the data shown here can be used to illustrate how L. gibba responds to its stressful environment. Among the antioxidative enzymes, SOD, CAT and APX appear to play key roles in the plant’s antioxidative defense mechanism under Cd toxicity. The ability of L. gibba to both accumulate and tolerate moderate Cd level used in this study could be partly derived from ROS detoxification through an efficient antioxidant system. Successful adaptation of the Lemna species to moderately metal-stressed environments is the basis for several authors’ proposals to use this singular species for the re-vegetation and recovery of metal-contaminated areas. Interestingly, the use of L. gibba could also be used for water stabilization in abandoned low-level Cd contaminated wetlands (natural and artificial), where no other species is able to survive.
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Contents lists available at SciVerse ScienceDirect
Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Ecophysiological tolerance of Lemna gibba L. exposed to cadmium Kadiriye Uruc- Parlak a,n, Dilek Demirezen Yilmaz b a b
_Ibrahim C ˘ Turkey - ec- en University, Faculty of Arts and Sciences, Department of Biology, Agri, Erciyes University, Faculty of Science, Department of Biology, Kayseri, Turkey
a r t i c l e i n f o
abstract
Article history: Received 8 May 2012 Received in revised form 25 December 2012 Accepted 14 January 2013 Available online 20 February 2013
In this study, an experiment was carried out to study the process of stress adaptation in Lemna gibba L. grown under cadmium stress (0–20 mg Cd L 1). The level of photosynthetic pigments and soluble proteins decreased only upon exposure to high Cd concentrations (for pigments 5 mg Cd L 1; for soluble proteins 10 mg Cd L 1). At the same time, the level of malondialdehyde (MDA) increased with increasing Cd concentration. These results suggested an alleviation of stress that was presumably the result of antioxidants such as superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and glutathione S-transferase (GST) as well as ascorbate peroxidase (APX), which increased linearly with increasing Cd levels. In addition, the proline content in L. gibba increased with increasing cadmium levels. These findings suggest that Lemna is equipped with an efficient antioxidant mechanism against Cd induced oxidative stress which protects the plant’s photosynthetic machinery from damage.We also found that moderate Cd treatment (0.05–5 mg L 1 Cd) alleviated oxidative stress in plants, while the addition of higher amounts of Cd (10–20 mg L 1) could cause an increasing generation of ROS, which was effectively scavenged by the antioxidative system. & 2013 Elsevier Inc. All rights reserved.
Keywords: Cadmium Lemna gibba Accumulation Antioxidant Proline Protein
1. Introduction Cadmium (Cd) is a globally distributed toxic element, which enters the aquatic environment from natural (weathering of rocks) as well as anthropogenic sources, and is then transferred ¨ ¨ to the food chain (Li and Xiong, 2004a, 2004b; Schutzend ubel et al., 2002). It causes an inhibition of enzyme activities, water imbalance and alterations in membrane permeability; it also disturbs mineral nutrition (Milone et al., 2003; Sharma and Dubey, 2005; Liu et al., 2007). In plants, reactive oxygen species (ROS) are continuously produced as by-products of various metabolic pathways, which are localized in different cellular compartments (Gratao et al., 2005; Reddy et al., 2005). However, under stressful conditions, their formation may exceed antioxidant scavenging capacity, thereby inducing oxidative stress and damaging most of the biomolecules (Davies, 2003). One of the most damaging effects of ROS and their products is the peroxidation of membrane lipids and ion leakage (Sharma and Dubey, 2005). To control ROS levels and to protect cells, plants produce low molecular weight antioxidants (Gratao et al., 2005). Plants possess several other mechanisms to deal with the detoxification of heavy metals thus enabling them to tolerate
n
Corresponding author. Fax: þ90 472 215 65 54. E-mail addresses: [email protected] (K. Uruc- Parlak), [email protected] (D. Demirezen Yilmaz). 0147-6513/$ - see front matter & 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ecoenv.2013.01.009
stress (Hall, 2002; Malecka et al., 2001; Tennant, 1975). Aquatic ecosystems are more sensitive to pollutants than terrestrial ones because most uptakes are facilitated by dissolution in water, which may lead to an accumulation of heavy metals. Duckweed (Lemna sp.) which often forms dense floating plants in aquatic ecosystems such as ditches, ponds and lakes was selected for study. Duckweed can be used as an indicator species to assess ecotoxicity of waters polluted by contaminants. It was selected because information on the plants responses to cadmium is insufficient. Therefore, the present study was, designed to investigate (i) the effects of Cd on the growth and Cd accumulation of Lemna gibba; (ii) the effects of Cd on the lipid peroxidation (MDA), (iii) the activities of enzymatic antioxidants (SOD, CAT, GST, APX, and GR) and accumulation of proline in L. gibba. The results could be meaningful in evaluating the role of L. gibba’s antioxidative system as regards Cd tolerance and detoxification. 2. Materials and methods 2.1. Plant material, growth conditions and metal estimation Fresh samples of L. gibba L. (Lemnaceae) were obtained from ponds in the Soysallı National Park in Kayseri, Turkey. In this study, cadmium nitrate was used in the treatments, and samples were exposed to various concentrations of cadmium: 0, 0.05, 0.5, 5, 10, and 20 mg L 1. Plants were grown in plastic cups containing Hoagland’s solution (pH 5) and grown for 7 days in a growth chamber at 257 1 1C with 16 h light: 8 h dark photo-cycle at light intensity of
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40 mmolm 2 s 1. The solutions were changed every two days. On the 7th day, the cadmium treated plants were harvested from their containers. The plants were rinsed twice with distilled water, and subsequently, their biochemical parameters were determined. Dried samples of L. gibba were digested with 10 ml of concentrated HNO3, using a CEM microwave digestion system. After digestion, the volume of each sample was adjusted to 25 ml using double deionized water (Demirezen Yılmaz, 2007). Determination of the cadmium concentrations in all samples was carried out by inductively coupled plasma optical emission spectrometry (Varian). The samples were analyzed in triplicate.
performed to determine the significant differences between treatments. In the figures, the values are marked with an n(asterisk) for the significance level (Pr0.05) as compared to the control.
2.2. Determination of pigment concentration
The results related to the accumulation of cadmium in L. gibba are presented in Table 1. The maximum Cd accumulation was found to be 1.67 mg kg 1 DW at 0.5 mg L 1 Cd. On prolonged exposure to higher concentrations of Cd (i.e. 20 mg L 1), there was a significant decline in the Cd accumulation rate. The analysis of one-way ANOVA showed that the differences in all the treatments were significant (P o0.05) for L. gibba.
Fresh plants (0.1 g) were cut into pieces and homogenized in 96% ethanol in the presence of quartz and calcium carbonate. The homogenate was filtrated and then diluted to 25 mL with deionized water. The concentrations of chlorophylls were calculated according to the formula of Ya˘gmur et al. (2006). 2.3. Estimation of lipid peroxidation (MDA) The level of lipid peroxidation was estimated following the method of Razinger et al. (2008), with modifications. The concentration of MDA (lM) in the solution obtained was calculated by Razinger et al. (2008). 2.4. Antioxidative enzymes 2.4.1. Enzyme extraction Fresh tissue (0.2 g) was homogenized in an ice-cooled mortar with 5 mL of 100 mM potassium phosphate buffer (pH 7.0) containing 0.1 mM EDTA and 1% (w/ v) polyvinylpyrrolidone. The homogenate was centrifuged at 15.000 g for 15 min at 4 1C (Hou et al., 2007). The supernatant was used for enzyme determination. 2.4.2. Superoxide dismutase (EC 1.15.1.11) The activity of SOD (EC 1.15.1.11) was assayed by measuring the inhibition of the photochemical reduction of nitroblue tetrazolium (NBT) (Beauchamp and Fridovich, 1971). The assay mixture contained 20 mM phosphate buffer (pH 7.5), 10 mM methionine, 0.1 mM NBT, 0.1 mM EDTA, 0.005 riboflavin, 50 mgmL 1 of enzyme extract and 0.25 mL of deionized H2O in a total volume of 3 mL. Finally, riboflavin was added, and the tubes were shaken and then illuminated for 15 min. The absorbance was recorded at 560 nm and the absorbance of the non-irradiated reaction mixture served as a control. A 50% inhibition of the reaction was defined as one unit of enzyme, and the enzyme activity was expressed as ng 1 FW. 2.4.3. Catalase (EC 1.11.1.6) CAT (EC 1.11.1.6) activity was measured spectrophotometrically according to Aebi’s method following the consumption of H2O2 at 240 nm (Aebi, 1984), in potassium phosphate buffer (150 mM, pH 7) containing 15 mM H2O2 and enzyme extract (exactly 50 mg of protein) in a final volume of 1 ml. The addition of H2O2 started the reaction. 2.4.4. Glutathione reductase (EC 1.6.4.2) GR (EC 1.6.4.2) was assayed as the increase in absorbance at 340 nm when DTNB is reduced by GSH to produce 2-nitro-5-thiobenzoic acid (TNB) according to Smith et al. (1988). 2.4.5. Glutathione S-transferase (EC 2.5.1.13) Conjugation of GSH to CDNB catalyzed by GST induced an increase in absorbance at 340 nm which was used to assay for GST activity as described by Drotar et al. (1985). 2.4.6. Ascorbate peroxidase (EC 1.11. 1.11) APX activity was determined according to Nakano and Asada by the decrease in the absorbance of ascorbate at 290 nm (Nakano and Asada, 1981). 2.5. Proline analysis Plants were sampled for analysis of proline after 7 days in the treatment using the methodology described by Bates et al. (1973). Concentrations of proline in the plant tissue were expressed on a FW basis. 2.6. Statistical analysis All experiments were repeated in triplicate. Values shown in the figures represent the average values7 standard deviation (SD) for each cadmium concentration. Data were subjected to a one-way analysis of variance (ANOVA) to confirm the variability of data and validity of results and a Turkey test was
3. Results 3.1. Cadmium content
3.2. Effect of cadmium on the level of photosynthetic pigment and total soluble proteins The leaves of L. gibba started to show signs of chlorosis after exposure to Cd. Accordingly, there were significant decreases in chlorophyll a and b for the plants exposed to Cd compared with the control (Fig. 1a). In other words, a concentration-dependent response to Cd stress was observed in the photosynthetic pigment. At 5 mg L 1 cadmium, total chlorophyll increased at 0.35 mg g 1 FW, whereas at 20 mg L 1 cadmium it decreased at 0.282 mg g 1 FW as compared to the control (0.285 mg g 1 FW). Similarly, the total soluble protein concentration in L. gibba decreased with higher Cd concentration in plants as compared to the control but was found to increase in plants at 10 mg L 1 Cd (Fig. 1b). 3.3. Effect of cadmium on lipid peroxidation The effect of Cd on MDA concentration is presented in Fig. 2. Significant increases in MDA concentration in L. gibba were observed in the experiments. MDA concentration increased linearly with increased Cd levels in the solution. The analysis of a one-way ANOVA showed that the differences in all the treatments were significant (Po0.05) for L. gibba. 3.4. Effect of Cd on catalase, superoxide dismutase, glutathione reductase, glutathione S-transferase, ascorbate peroxidase A decline in catalase activity was observed in L. gibba with the increasing Cd concentration. The highest concentration of Cd (20 mg L 1) was proved to be extremely toxic, reducing CAT activity (Fig. 3). Plants exposed to Cd presented significant differences (Po0.05). SOD activity increased linearly with increasing Cd levels (Fig. 4). The maximum SOD activity was recorded at 20 mg L 1 Cd (1.261 70.0085 mg 1). At the highest Cd concentration, SOD accumulation increased. Additionally, significant differences were found in SOD activity among the treatments. Although no significant differences were found in GR and GST, activity tended to be higher in plants from the control. The results Table 1 Cadmium concentration in L. gibba upon Cd exposure. Values represent mean 7S.E. (n¼ 3). Different letters indicate significant differences at P o0.05. Cd concentration (mgL 1)
Metal uptake by L. gibba (mgkg 1 DW)
Control 0.05 0.5 5 10 20
0.104 70.001 0.783 70.007 1.673 70.001 1.515 70.005 0.664 70.003 0.172 70.001
a d f e ce b
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Chlorophyll concentrations (mgg-1FW)
0.4 0.35
81
* kla
klb
klt
*
0.3 0.25 0.12 0.15 0.1
*
*
0.05 0
Total soluble protein (mgg-1FW)
Control
10 0.05 0.5 5 Cd concentration in solution (mgL-1)
20
4 3.5 3 2.5 2 1.5 1 0.5 0 Control
0.05 5 0.5 10 Cd concentration in solution (mgL-1)
20
Fig. 1. (a) Effect of different Cd concentrations on chlorophyll a, chlorophyll b and total chlorophyll in L. gibba. Values represent mean 7 S.E. (n¼ 3). Asterisks indicate significant differences at Po 0.05. and (b) Effect of different Cd concentrations on total soluble protein concentrations in L. gibba. Values represent mean 7 S.E. (n¼3).
1
MDA (nmol g-1 FW)
0.9
*
0.8 0.7
*
*
*
*
0.6 0.5 0.4 0.3 0.2 0.1 0 Control
0.05
0.5
5
Cd concentration in solution
10
20
(mgL-1)
Fig. 2. MDA concentration in L. gibba upon Cd exposure. Values represent mean 7 S.E. (n ¼ 3). Asterisks indicate significant differences at P o 0.05.
related to the effect of Cd on GR and GST activity are presented in Tables 2 and 3. GR and GST activity in L. gibba increased linearly with increasing Cd levels in the solution. The maximum GR activity was recorded at 0.05 mg L 1 Cd (0.02870.0001 mg 1). At 20 mg L 1 Cd, the GST in L. gibba increased to 0.03170.000 mg 1. According to the results, APX activity decreased linearly with increasing Cd levels. The highest concentration of Cd (20 mg L 1) proved to be extremely toxic resulting in a decline of APX activity (Table 4). 3.5. Proline To understand the contributions of non-enzymatic antioxidants in the L. gibba response to Cd toxicity, their proline contents
were examined. The amounts of proline in L. gibba grown under different concentrations of cadmium are shown in Table 5. The proline content in control plants increased gradually in the duration of the experiment.
4. Discussion In the present study, L. gibba was grown in hydroponic culture in the presence of increasing Cd concentrations to evaluate its Cd uptake, growth and to identify possible defense mechanisms. The results presented in this study indicated that Cd was toxic to L. gibba. The higher accumulation of Cd in L. gibba found in
K. Uruc- Parlak, D. Demirezen Yilmaz / Ecotoxicology and Environmental Safety 91 (2013) 79–85
82
0.02
CAT (mg-1 protein)
0.018
*
0.016
*
0.014
*
*
0.012 0.01 *
0.008 0.006 0.004 0.002 0 0.05 5 0.5 10 Cd concentration in solution (mgL-1)
Control
20
Fig. 3. CAT activities in L. gibba upon Cd exposure. Values represent mean 7 S.E. (n¼ 3). Asterisks indicate significant differences at Po 0.05.
1.3 * SOD (mg-1 protein)
1.25
* *
*
*
1.2 1.15 1.1 1.05 Control
0.05
0.5 5 10 Cd concentration in solution (mgL-1)
20
Fig. 4. SOD activities in L. gibba upon Cd exposure. Values represent mean 7 S.E. (n¼ 3). Asterisks indicate significant differences at Po 0.05.
Table 2 GR activity in and L. gibba upon Cd exposure. Values represent mean 7S.E. (n¼3). Different letters indicate significant differences at Po 0.05. Cd concentration (mgL 1) Control 0.05 0.5 5 10 20
GR activity in L. gibba (unit mg 0.011 70.0003 0.028 70.0001 0.026 70.0007 0.022 70.0004 0.021 70.0003 0.018 70.0002
1
)
a e d c c b
Table 3 GST activity in and L. gibba upon Cd exposure. Values represent mean 7 S.E. (n¼ 3). Different letters indicate significant differences at Po 0.05. Cd concentration (mgL 1)
GST activity in L. gibba (unit mg 1)
Control 0.05 0.5 5 10 20
0.026 70.0003 0.027 70.0004 0.028 70.0002 0.030 70.0003 0.031 70.0000 0.038 70.0000
a a b c cd d
our study could reflect different cellular mechanisms for the bioconcentration of essential and non-essential trace metals in plants. Additionally, the availability and bioaccumulation of Cd could also be influenced by chemical speciation of the metal, organic chelators, the presence of other metals and anions, ionic strength, light intensity, temperature and oxygen level (Greger, 1999).
Table 4 APX activity in and L. gibba upon Cd exposure. Values represent mean 7S.E. (n ¼3). Different letters indicate significant differences at P o 0.05. Cd concentration (mgL 1) Control 0.05 0.5 5 10 20
APX activity in L. gibba (unit mg 0.116 70.001 0.092 70.001 0.088 70.002 0.074 70.000 0.073 70.002 0.053 70.002
1
)
a b b c c d
Table 5 Proline contents in and L. gibba upon Cd exposure. Values represent mean 7S.E. (n ¼3). Different letters indicate significant differences at P o 0.05. Cd concentration (mgL 1)
Proline in L. gibba (lmol g 1)
Control 0.05 0.5 5 10 20
0.021 7 0.0002 0.024 7 0.0005 0.042 7 0.0015 0.045 7 0.0011 0.051 7 0.0011 0.083 7 0.0025
a a b bc c d
As can be seen in Fig. 1 while amount of Cd accumulated by plant tissue in gradually decreases after the 0.5 mgL 1 Cd treatment. A similar result was reported by Karatas- et al. (2009) with L. gibba L. stressed by wastewater. According to these authors, the decreases in amounts of heavy metal (selenium) can result from their consumption for preventing oxidative stresses taking place during treatment.
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Chlorophyll contents of tissues were significantly lower when Cd levels were above 10 ppm. The rise in chlorophyll contents could be regarded as a metal specific response that may have resulted in chlorophyll upgradation and synthesis of photosynthates (Davies, 2003). The decline in total chlorophyll contents in high Cd levels as well as growth inhibition can be regarded as general responses associated with metal toxicity (Boswell et al., 2002; Vazquez et al., 1987). In other words, a decline in the level of photosynthetic pigments may be attributed to the cadmiuminduced inhibition of chlorophyll biosynthesis possibly caused by induced nutrient deficiency, such as that of iron. In the present study these responses were noticed mostly in the case of Cd, as common duckweed can tolerate different heavy metals at applied concentrations without a notable effect on its photosynthetic pigment contents or significant multiplication inhibition (Fodor, 2002; Li and Xiong, 2004a, 2004b). Overall, the destruction of photosynthetic pigments by metals could be due to impairment of the electron transport chain, replacement of mg2 þ ions associated with the tetrapyrrole ring of chlorophyll molecules, inhibition of important enzymes associated with chlorophyll biosynthesis or peroxidation processes in chloroplast membrane lipids by reactive oxygen species (Van Assche and Clijsters, 1990; Sandalio et al., 2001). For example, it has been shown previously that Zn preferentially accumulates in the chloroplast where it can directly interact with the thylakoid membranes (Van Assche and Clijsters, 1990; Szalontai et al., 1999). The increased lipid peroxidation observed in the Cd treatments used in this study supports this hypothesis. The total soluble protein concentration in L. gibba decreased with higher Cd concentration in plants as compared to controls. A similar result was reported by John et al., with Lemna polyrrhiza L. stressed by Cd and Pb (John et al., 2008). In that study, it seems that there might have been greater ROS generation due to high Cd load in plants and as a result, more oxidative stress which resulted in the decline of protein concentration caused by oxidative damage. MDA is the decomposed product of polysaturated fatty acids (PUFA) of biomembranes, and its increase shows plants under a high level of antioxidative stress (Hou et al., 2007). The main site of attack by any redox-active metal in a plant cell is usually the cell membrane. MDA concentration obtained from this study increased linearly with increased Cd levels in the solution. Similar results were found with S. polyrhiza (Upadhyay and Panda, 2010). Thus, increased MDA indicates the prevalence of oxidative stress and this may be one of the possible mechanisms by which toxicity due to Cd is manifested in plant tissues (Gupta et al., 2009). However, Yamamoto et al., demonstrated that Al cannot catalyze the peroxidation reaction by itself, but enhances the Fe (II or III) mediated non-enzymatic peroxidation of phospholipids (Yamamoto et al., 2002). Boscolo et al. (2003). suggested that the possible reason for the absence of lipid peroxidation in maize is that the Fe2 þ bound to the membrane is not exposed to the attack of H2O2. Metal toxicity is often driven by ROS generation, directly via the catalytic production of superoxide (O2 ) by the Haber–Weiss and Fenton reactions, or indirectly by other mechanisms (Yamamoto et al., 2002; Boscolo et al., 2003). ROS can damage membrane lipids, proteins, pigments, and nucleic acids, and the plant evolves an antioxidant defense system to prevent the adverse effects of oxidative stress to cells (Briat, 2002). The inadequate activities of antioxidant defense systems cause oxidative damage in plants exposed to Cd (Mittler, 2002). The antioxidative enzyme system is one of the protective mechanisms in a plant. The increased activity of antioxidative enzymes in a plant indicates the formation of ROS, and the present study indicated the generation of oxidative stress in L. gibba since all enzyme activity increased with high Cd added levels (Jung et al., 2000). In
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other words, the L. gibba analyzed in this work contained high Cd concentrations sufficient to activate ROS production and subsequently oxidative stress. According to Sharma and Dietz, antioxidant enzyme activities in metal-stressed plants are highly variable, depending on the plant species, metal ion, concentration and exposure duration, but these processes reflect the modified redox status of the stressed cells Since the primary reduced product of oxygen is the superoxide anion radical, SOD represents the first defense against oxidative stress (Sharma and Dietz, 2009). Superoxide dismutase, the first enzyme in the detoxifying process, converts superoxide radicals to H2O2 at a very fast rate. The enhanced SOD activity observed in the present study was consistent with studies in which other plant species were treated with heavy metals (Sharma and Dubey, 2007; Prasad et al., 1999). Due to the action of SOD, H2O2 concentration can be expected to increase inside the cell; this was found in duckweed plants under heavy metal toxicity (Sharma and Dubey, 2007). An increase in SOD activity could possibly be the result of both a direct effect of heavy metal ions and an indirect effect mediated via an increase in levels of superoxide radicals (Prasad et al., 1999). The effect of heavy metal stress on SOD expression is likely to be governed by the tissue and sub-cellular sites where oxidative stress is generated as supported by higher SOD activity in roots than in leaves of metal-stressed plants (Hou et al., 2007). An increase in SOD activity may be linked to an increase in superoxide radical formation as well as to the de novo synthesis of enzyme protein, which in turn may be associated with an induction of SOD genes by superoxide-mediated signal transduction (Prasad et al., 1999). Our data showed that significant increases in CAT activity were observed between the treatments. The results obtained indicated that CAT activity decreased linearly with increasing cadmium levels. Contrary to our results, a decline in the specific activity of catalase with an increase in Cr concentration (20– 80 ppm, 0.5 mM) was reported by Shankar et al. (2005). According to Willekens et al. (1997), it is likely that excess production of ROS caused by heavy metals can inactivate CAT, probably by inactivating the enzyme-bound heme group. CAT is only present in peroxisomes, but it is indispensable for ROS detoxification during stress, when high levels of ROS are produced. Boscolo et al. (2003), also reported no change in CAT activity under Al toxicity in maize, while in other plants a decline (soybean, rice) or enhancement (tobacco, wheat) of CAT activity was found. The antioxidative capacity of cells can be estimated by measuring APX activity. In contrast to CAT, APX has a higher affinity for H2O2, allowing for the scavenging of small amounts of H2O2 in more specific locations (Radic et al., 2010; Asada, 1992). In different aquatic and terrestrial plant species this type of peroxidases (APX) has been shown to be inducible on exposure to different heavy metals (Sharma and Dubey, 2005; Gupta et al., 2009). In our experiments, Cd treatments induced APX activities in L. gibba. This means that APX activity decreased linearly with increasing Cd levels for example it was significantly decreased by the addition of 20 mg L 1 Cd compared with the control. The hyperactivity of peroxidase under Cd stress indicated its role in the constant detoxification of H2O2. These results agree with earlier studies on aquatic plants. Cellular antioxidants play a significant role in providing resistance to plants by protecting labile macromolecules against attack by free radicals produced under heavy metal stress (Devi and Prasad, 1998). The present study revealed a decline in levels of ascorbate in plants exposed to Cd suggesting they play a significant role in scavenging free radicals and hence in the protection of cells from oxidative damage. However, high levels of glutathione in Cd exposed L. gibba seem to provide wide-spread antioxidant protection to the plant body and, most importantly increase the thiol concentration
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in the cytoplasm and chloroplasts, thus protecting them against photochemical damage. A similar response to an increase in glutathione levels on exposure to heavy metals such as Cd and Cr was reported in Phragmites (Iannelli et al., 2002). Increased SOD and CAT activities in L. gibba indicate that this plant has the capacity to adapt to Cd toxicity by developing an antioxidant defense system. Improvement of stress tolerance is often related to an increase in the activity of antioxidant enzymes (Murgia et al., 2004; Lee et al., 2007). Hsu and Kao (2007) reported that early accumulation of H2O2 during heat shock signaled an increase in APX and GR activities, which in turn protected rice plants from the oxidative stress caused by Cd. Glutathione is considered to be a very important signal molecule which acts as a link between environmental stress and adaptive responses, and it is regenerated from GST by GR activity (Navari-Izzo et al., 1997). Concomitant increases in proline as a function of metal accumulation have been observed in response to heavy metals (Bassi and Sharma, 1993). In our study, Cd treatments increased the accumulation of proline, regardless of the fact that soluble protein content declined in response to Cd. Aside from acting as a metal chelator and osmolyte, proline has been reported to scavenge hydroxyl radicals and singlet oxygen, thus providing protection against ROS-induced cell damage (Alia Prasad and Saradhi, 1995). Increases in both proline and lipid peroxidation levels with increasing Cd concentration are indicative of a correlation between ROS generation (hydroxyl radicals mostly) and ROS scavenging by proline. Accumulation of proline in response to excess metals such as copper, cadmium, zinc and nickel was described in several plants (Matysik et al., 2002).
5. Conclusion Considering these results, we strongly suggest that higher cadmium levels causes oxidative stress in L. gibba cells and may cause membrane damage through production of ROS and interferes with chlorophyll metabolism. Therefore, the data shown here can be used to illustrate how L. gibba responds to its stressful environment. Among the antioxidative enzymes, SOD, CAT and APX appear to play key roles in the plant’s antioxidative defense mechanism under Cd toxicity. The ability of L. gibba to both accumulate and tolerate moderate Cd level used in this study could be partly derived from ROS detoxification through an efficient antioxidant system. Successful adaptation of the Lemna species to moderately metal-stressed environments is the basis for several authors’ proposals to use this singular species for the re-vegetation and recovery of metal-contaminated areas. Interestingly, the use of L. gibba could also be used for water stabilization in abandoned low-level Cd contaminated wetlands (natural and artificial), where no other species is able to survive.
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