Ecotoxicology and Environmental Safety 115 (2015) 159–165
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Oxidative effects, nutrients and metabolic changes in aquatic macrophyte, Elodea nuttallii, following exposure to lanthanum Jingjing Zhang, Tingting Zhang, Qianqian Lu, Sanjuan Cai, Weiyue Chu, Han Qiu, Ting Xu, Feifei Li, Qinsong Xu n School of Life Sciences, Nanjing Normal University, Nanjing 210023, China
art ic l e i nf o
a b s t r a c t
Article history: Received 23 October 2014 Received in revised form 5 February 2015 Accepted 6 February 2015
We investigated the phytoremediation potential of Elodea nuttallii to remove rare earth metals from contaminated water. The laboratory experiments were designed to assess the responses induced by lanthanum (5–20 mg L 1) in E. nuttallii over a period of 7 days. The results showed that most La (approximately 85%) was associated with the cell wall. The addition of La to the culture medium reduced the concentration of K, Ca, Cu, Mg, and Mn. However, O2 levels increased with a concomitant increase in the malondialdehyde (MDA) concentration as the La concentration increased, which indicated that the cells were under oxidative stress. Significant reductions in the levels of chlorophyll (Chl) a, b, and carotenoids (Car) were observed in a concentration-dependent manner. However, the levels of reduced glutathione (GSH), total non-protein thiols (TNP-SH) and phytochelatins (PCs) increased for all La concentrations. The results suggested that La was toxic to E. nuttallii because it induced oxidative stress and disturbed mineral uptake. However, E. nuttallii was able to combat La induced damage via an immobilization mechanism, which involved the cell wall and the activation of non-enzymatic antioxidant. & 2015 Elsevier Inc. All rights reserved.
Keywords: Lanthanum Elodea nuttallii Oxidative stress Non-enzymatic antioxidant Ionome
1. Introduction Lanthanum (La) is a member of the rare earth elements (REEs), which comprise the lanthanides or lanthanoids from lanthanum to lutetium plus yttrium and scandium. They share similar physicochemical properties, but are generally different from the common transitional metals. In recent years, due to high-tech and medical applications and the intense use of fertilizers in agriculture and forestry, large amounts of REEs have been emitted to the soil and water environment (Elbaz-Poulichet et al., 2002; Oral et al., 2010). REEs are believed to have limited toxicity and are not significantly hazardous to the environment. However, their slow accumulation in the environment could become problematic (Thomas et al., 2014). Toxicological investigations have suggested that REE bioaccumulation might have significant adverse effects on aquatic biota (Barry and Meehan, 2000; Oral et al., 2010). To date, the effect of REE accumulation on plants remains fragmentary and sometimes inconsistent. For example, La significantly promoted the reproductive growth of Arabidopsis thaliana (He and Loh, 2000) and Nicotinana tabacum (Sun et al., 2003), but in a hydroponic experiment, the addition of La decreased the n
Corresponding author. Fax: þ 86 25 85898186. E-mail address: [email protected] (Q. Xu).
http://dx.doi.org/10.1016/j.ecoenv.2015.02.013 0147-6513/& 2015 Elsevier Inc. All rights reserved.
growth of Zea mays and Vigna radiate (Diatloff et al., 2008) and inhibited primary root elongation in Triticum aestivum seedlings (Hu et al., 2002). Liu and Hasenstein (2005) reported that low La3 þ concentrations ( o1 μM) promoted Z. mays root growth while higher concentrations (4100 μM) inhibited the growth of this organ. It is likely that the La influences on the cell are complex and multiple, such as ion signaling and homeostasis effects (Liu and Hasenstein, 2005) or binding to aromatic rings, carboxyl groups and phospholipids (Abe and Takeda, 1988). These contradictory results may depend of plants species, growth stage and the concentration and type of REEs (He and Loh, 2000; Hu et al., 2002; Diatloff et al., 2008; dΆquino et al., 2009; Ippolito et al., 2010; Thomas et al., 2014). Although the availability and toxicity of exogenous REEs to plants has attracted more attention recently (Chua, 1998; Yang et al., 1999; Weltje et al., 2002; Xu et al., 2012; Fu et al., 2014), The La phytoremediation potential of Elodea nuttallii, a widespread macrophyte species, has not been investigated to any great extent. Aquatic plants are represented by a variety of algal and macrophytic species that occur in many types of habitats and they are often used in water quality monitoring and assessment (Lewis, 1995; Mohan and Hosetti, 1999). It has also been shown that aquatic macrophytes can uptake metals directly from water and accumulate them in their shoots because they are completely
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submerged and have a very thin cuticle (St-Cyr et al., 1994; Wang et al., 2010b, Regier et al., 2013). E. nuttallii, a submerged plant native to North America, is known for its fast growth rate and is easily cultured in the laboratory. It is also able to accumulate pollutants. Therefore, it has been widely used as model experimental system for bioaccumulation and ecotoxicological investigations (Barrat-Segretain, 2004; Thiébaut et al., 2010; Xing et al., 2010; Regier et al., 2013; Larras et al., 2013). This study investigated the responses of E. nuttallii to exogenous La by measuring (i) the accumulation and sub-cellular distribution of La; (ii) changes in nutrient uptake; (iii) La-induced oxidative stress through the measurement of O2 , MDA changes and chlorophyll concentrations; and (iv) variations in non-enzymatic antioxidants (reduced GSH, TNP-SH and PCs). Overall these accumulation and distribution data should improve our understanding of the main cellular and molecular targets involved in the accumulation/distribution of La and further the potential use of E. nuttallii in the phytoremediation programs of aquatic bodies polluted with REEs.
2.4. Determination of O2 generation rate The concentration of O2 generation rate was measured using the hydroxylamine chloride method (Wang and Luo, 1990). The supernatant (0.5 mL) was collected and incubated at 25 °C for 60 min in the presence of 1 mM hydroxylamine hydrochloride in 50 mM sodium phosphate buffer (pH 7.8). The reaction mixture was then incubated with 1 mL of 17 mM P-aminobenzene sulfonic acid anhydrous and 1 mL of 7 mM a-naphthylamine at 25 °C for 30 min. The absorbance was measured at 530 nm. A calibration curve was established using sodium nitrite. 2.5. Lipid peroxidation The level of lipid peroxidation in the shoots (0.5 g) was determined immediately by thiobarbituric acid reaction according to Heath and Packer (1968). The concentration of MDA was calculated using 155 mM cm 1 as extinction coefficient in terms of nmol g 1 fresh weight.
2. Materials and methods
2.6. Determination of photosynthetic pigment concentration
2.1. Plant material and growth conditions
Chlorophylls (Chl) and carotenoids (Car) were extracted with 80% acetone and absorbances at 470, 647 and 663 nm recorded on a spectrophotometer (Thermo GENESYS 10). The concentrations of Chl a, Chl b and Car were estimated according to Lichtenthaler (1987).
Cleaned, healthy, 10 cm-long vegetative shoots of E. nuttallii, collected from Lake Pipa, Nanjing, China. All the materials were cultivated in 1/10 Hoagland solution under controlled conditions (114 μmol m 2 s 1 light irradiance, 14 h photoperiod and 25 °C/ 20 °C day/night). Then lanthanum was added to the containers in the form of LaCl3⋅7H2O in appropriated quantities to give the following treatments: 5, 10, 15, 20 mg L 1 [La3 þ ]. The tested concentrations of La to perform our survey were chosen within the range of previous toxicological investigations on plants (Liu and Hasenstein, 2005; dΆquino et al., 2009; Ippolito et al., 2010; Thomas et al., 2014). Each treatment had three replicates, and all of the solutions were refreshed every 2 days during the 7-day culture period. 2.2. Tissue fraction and la analysis After 7 days of metal exposure, the Elodea plants were harvested and washed with double distilled water and 20 mM EDTA to remove metals absorbed on the surface of the plant material. The sub-cellular distribution of La within the shoots was determined according to the method described by Xiong et al. (2009). Concentrations of La in cell wall, organelles and soluble fraction were determined by inductively coupled plasma atomic emission spectrometry (ICP-AES, Leeman labs, Prodigy, USA) after digestion with HNO3 and HClO4 (10:1, v/v) at 160°C (detection limit: 0.0015 mg/L). The liquid standard La reference material (GSB 04-1774-2004) was diluted and analyzed for La concentration.
2.7. Reduced glutathione (GSH), total non-protein thiol (TNP-SH), and phytochelatins (PCs) determination To determine the concentration of reduced GSH, fresh shoots (0.5 g) were homogenized in ice-cold 5% (w/v) trichloroacetic acid and then centrifuged at 10,000g for 20 min at 4 °C. The reduced GSH concentration was determined spectrophotometrically at 412 nm according to the method used by Anderson (1985), after precipitation with 0.1 M HCl, using GSH reductase, 5,5′–dithio–bis(2-nitrobenzoic acid) (DTNB) and NADPH. The reduced GSH concentration was expressed as mg g 1 fresh weight. In order to estimate the total non-protein thiol (TNP-SH) concentration, fresh shoots were extracted in 6.67% 5-sulphosalicylic acid and centrifuged at 12,000g for 10 min at 4 °C. Supernatant was reacted with Ellman's reagent (5 mM EDTA and 0.6 mM 5, 5′ dithiobis (2-nitroenzoic acid) in 120 mM phosphate buffer, pH 7.5) and absorbance was measured spectrophotometrically after 5 min at 412 nm (Ellman, 1959). The PCs were measured by following the method of Bhargava et al. (2005). The PCs were measured using the formula: total NPSH–GSH after the total NP-SH and reduced GSH concentrations had been determined.
2.3. Determination of mineral concentration 2.8. Statistical analysis The mineral concentration in the shoots of E. nuttallii was determined according to previously reported methods (Xu et al., 2012; Fu et al., 2014). For each sample, 0.5 g of shoots was digested with HNO3:HClO4 (10:1, v/v) at 160 °C. The solution samples were diluted to a final volume of 10 mL and were analyzed for mineral elements (K, Ca, Fe, Cu, Zn, Mn, and Mg) by ICP-AES. Standard solution (xccc-13A, xccc-14A, SPEX CertiPrep, USA) were used for the calibration.
All results are presented as mean values 7standard deviation (SD) of at least three independent experiments. Statistical analysis was performed by computer software SPSS17.0. A correlation analysis was performed to determine the relationships between La accumulation and studies parameters. The experimental data was subjected to a one-way analysis of variance (ANOVA) and P-valueso0.05 were considered significant.
J. Zhang et al. / Ecotoxicology and Environmental Safety 115 (2015) 159–165
3. Results 3.1. Sub-cellular distribution of La We investigated the sub-cellular distribution of La in E. nuttallii shoots (Table 1). Most of the La was found in the cell wall fraction, while a minor part was associated with the organelle and the soluble fractions. The La proportion in different sub-cellular fractions remained fairly constant for all treatments. 3.2. The main mineral elements As shown in Table 2, the concentrations of the main mineral elements, such as K, Ca, Cu, Mg and Mn in the plant exposure groups were lower than in the control groups and decreased significantly with increasing concentrations of La (rK ¼ 0.93, P o0.01; rCa ¼ –0.97, Po0.01; rCu ¼–0.91, P o0.05; rMg ¼ 0.85, P o0.05 and rMn ¼ –0.99, Po 0.01), whereas Zn levels peaked at 5 mg L 1 and represented a 37% increased with respect to the control plants. However, the Zn levels began to decline as the La concentration continued to rise. In contrast, the Fe levels decreased slightly (by 14%) when treated with 5 mg L 1 La, but then increased thereafter (r ¼0.99, P o0.01).
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Table 1 Sub-cellular distribution of lanthanum (La) in Elodea nuttallii shoots. Data are means 7S.D. (n¼ 3). Different letters in a column indicate significantly different values at Po 0.05. La concentration (mg L 1 )
0 5 10 15 20
La in subcellular fractions (mg kg 1 FW) Cell wall
Organelles
Soluble fraction
Total La content
N.D. 1640d 7 45.7 2340c 7 33.9 3180b 7 24.9 3370a 7 22.3
N.D. 171d 70.6
N.D. 144c 710.9
N.D. 1950a 757.2
225c 7 17.3
167bc 7 12.2
2730c 7 4.5
319b 7 21.2
200ab 7 9.8
3700b 7 6.1
369a 7 12.8 234a 7 18.2
3970a 7 16.8
higher than the control when the plants were grown in 15 mg L 1 La. Despite the decrease in PC concentrations at 20 mg L 1 concentrations, they still remained 6.3 times higher than the controls.
4. Discussion 3.3. O2. generation rate In E. nuttallii shoots, the O2. generation rate showed a dosedependent increase as the La concentration rose (r ¼0.98; P o0.01). The peak accumulation was observed on day 7 at 20 mg L 1 La where the O2. generation rate was up to 214% higher than in the control plants (Fig. 1a). 3.4. Malondialdehyde (MDA) concentration MDA concentration (Fig. 1b) was measured in order to evaluate lipid peroxidation caused by La. MDA increased gradually in a concentration dependent manner and was significantly positively correlated with La accumulation by the plants (P o0.01). The maximum increase was recorded at 20 mg L 1 on day 7, which was 81% higher with respect to control plants. 3.5. Photosynthetic pigment levels As shown in Fig. 1c, the photosynthetic pigments (Chl a, Chl b and Car) showed a similar response after La exposure. Significant losses in Chl a (r ¼ –0.91; Po 0.05), Chl b (r ¼–0.98; P o0.01) and Car (r ¼ –0.81; P o0.05) were recorded with increasing levels of La in the nutrition medium. The maximum decreases were 78% (Chl a), 73% (Chl b) and 24% (Car) compared to their controls when the plants were exposed to 20 mg L 1 for 7 days. 3.6. Reduced GSH, TNP-SH and PCs Compared with the control, the concentration of reduced GSH increased slightly as the La concentration increased with a maximum increase of 14% when the plants were exposed to 10 mg L 1 La for 7 days. Even though the reduced GSH concentration at higher La concentrations declined, it still remained higher than the controls (Fig. 1d). Fig. 1e shows that the TNP-SH concentration in E. nuttallii shoots increased by up to 10 mg L 1 and then declined. The concentration at 10 mg L 1 was 42% greater than the control, but, it decreased to 115% of the control when plants were treated with 20 mg L 1 La. La treatment induced a massive accumulation of PCs compared to the control (Fig. 1f). The PC levels in the shoots were 12.3 times
Metal accumulation in E. nuttallii has been investigated by Regier et al. (2013), who noticed that the amount of Hg in shoots was significantly higher than in roots, and by Wang et al. (2010b), who found that Cd and Cu were accumulated by shoots from the water rather than from the sediments. In this study, E. nuttallii accumulated large concentrations of La in the shoots in a dosedependent manner up to 7 days of exposure (Table 1). Significant accumulation of REEs has also been observed in other aquatic plants, such as Eichhornia crassipes (Chua, 1998), Sperollela polyrrhiza (Yang et al., 1999), Hydrocharis dubia (Xu et al., 2012) and Nymphoides peltata (Fu et al., 2014). All these experimental data suggested that exogenous REEs were highly available to aquatic plants and that their shoots played a primary role in the uptake of metals. Previous studies showed that aquatic plants were capable of removing metals from water through a metal binding process (biosorption) and subsequent metabolism-dependent accumulation (Veglio and Beolchini, 1997; Sivaci et al., 2004). It is well documented that La has many chemical and physical characteristics in common with calcium, such as ionic radii (La3 þ : 8.5 10 2; Ca2 þ : 9.2 10 2) (Evans, 1988). Therefore, it may be transported into plant cells through Ca channels and/or by Ca transporters after an initial sorption of REEs to the cell wall (Hu et al., 2002). The data presented here also showed that approximately 15% of the La was taken up into the cells (Table 1). When attempting to understand the physiological role of REEs in plants, one of the most important questions is whether REEs enters the cell or not. In our study, La analysis at the sub-cellular plant tissue level demonstrated that most of the La (84–86%) was bound to the cell wall fraction (Table 1). These results were in line with previous studies on Dicropteris dichotoma (Shan et al., 2003), Pronephrium simplex (Lai et al., 2006) and Nymphoides peltata (Fu et al., 2014), which reported that La was predominantly present in the cell wall (81–89%, 68% and 73–86%, respectively). These observations suggested that the cell wall is the predominant sink for REEs and therefore is believed to play a role in metal tolerance. It has been stated that plant cells can modify the metal accumulation capacity of their cell walls in order to accumulate more cations in the apoplast and thereby protect the protoplast (Krzeslowska, 2010). However, our further analysis confirmed that cellulose and pectin (79–84%) were the major cell wall components involved in the retention of La in the cell wall (data not shown).
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Table 2 The effect of lanthanum (La) supply on nutrient element concentrations in Elodea nuttallii shoots. Data are means 7S.D. (n¼ 3). Different letters in a line indicate significantly different values at Po 0.05. Element concentration (mg kg 1FW )
La concentration (mg L 1) 0
Potassium Calcium Copper Magnesium Manganese Zinc Iron
5 a
3710 7 7. 1 1070a 7 18.4 3.4a 7 0. 1 298a 7 7.1 43.7a 7 2.1 12.8b 7 0.0 71.3b 7 7.1
10 b
3430 715.8 968b 74.1 3.2a 70.2 202b 75.0 41.3a 71.8 17.6a 70.3 61.7b 78.1
The bioaccumulation of REEs in aquatic plants is often accompanied by the induction of a variety of cellular changes, some of which directly contribute to the metal tolerance capacity of plants. In the present study, multiple physiological and biochemical responses in the plant tissues were induced by La and these affected nutrient uptake (Table 2), ROS (Fig. 1a), reduced GSH (Fig. 1d), TNP-SH (Fig. 1e) and PC (Fig. 1f) concentrations in E. nuttallii, subjected to La. It has been reported that REEs can cause damage to the plasma membrane and cytoskeleton structures (Zheng et al., 2002; Liu and Hasenstein, 2005; Peng et al., 2007; Wang et al., 2010a). Therefore, they can affect the flow of nutrients into and out of the cell. Our experimental results suggested that the cellular concentrations of macro-elements, such as K, Ca, Cu, Mn and Mg, in E. nuttallii shoots subjected to La, were lower than that in the control groups, while Fe ( 45 mg L 1) and Zn (o 15 mg L 1) levels increased (Table 2). Generally, because of a similar ionic radius to Ca2 þ , La3 þ is probably able to block calcium ionic channels and disturb the uptake of nutrient ions through calcium channels (Pineros and Tester, 1997; Lewis and Spalding, 1998). Previous studies have demonstrated that La3 þ can inhibit the physiological activity of protease (Li et al., 2003), inhibit plasma membrane redox system activity (Zheng et al., 2002; dΆquino et al., 2009), and reduce the transportation of ions in plants. The oxidative stress imposed on E. nuttallii after La exposure was shown by the significant increase in MDA at higher concentrations (Fig. 1b). It was mostly attributable to enhanced ROS levels, including O2. (Fig. 1a) and H2O2 (Wang et al., 2007; Ippolito et al., 2010), and the altered membrane permeability, which led to increased ion loss (Peng et al., 2007; Zheng et al., 2002; Wang et al., 2010a). Furthermore, our results also showed an increase in K þ leakage when plants were treated with La, which was also representative of membrane disruption (Table 2). Thus, exposure to La caused oxidative stress in E. nuttallii. Our results are similar to those observed in Hydrilla verticillata (Wang et al., 2007), Lemna minor (Ippolito et al., 2010), and Hydrocharis dubia (Xu et al., 2012). In the E. nuttallii was produced change in photosynthetic apparatus after La exposure. These alterations included a decrease in Chl a, Chl b and Car (Fig.1c) concentrations. The damage to photosynthetic pigments was also expressed visually as chlorosis. A decrease in photosynthetic pigment levels is a common response by plants to a variety of abiotic stresses and has been observed in a number aquatic plants after exposure to REEs, such as Nymphoides peltata (Fu et al., 2014), Hydrilla verticillata (Wang et al., 2007), Hydrocharis dubia (Xu et al., 2012) and Lemna minor (Ippolito et al., 2010). These studies attributed the decrease in pigment concentration to the disturbance in chlorophyll biosynthesis or degradation due to lipid peroxidation (Wang et al., 2007) and damage to the ultrastructure of the chloroplast (Xu et al., 2012; Fu et al., 2014).
15 c
3240 7 11.0 788c 7 4.1 3.2a 7 0.2 188b 7 0.6 37.0b 7 1.2 16.1a 7 3.4 79.5a 7 4.0
20 e
3030 7 12.0 684d 7 1.1 3.0b 7 0.0 184c 7 7.1 34.3c 7 0.3 13.4c 7 0.1 81.9a 7 1.8
3110d 7 4.6 659e 7 6.9 3.0b 7 0. 3 168d 7 4.1 31.4c 7 4.9 12.2b 7 0.8 83.7a 7 4.6
To mitigate the oxidative damage initiated by ROS, plants have evolved different detoxification mechanisms, including cellular/ sub-cellular compartmentalization into cell wall or the vacuole (Lombi et al., 2002), and complexation with phytochelatins (PCs) (Mishra et al., 2006; Srivastava et al., 2006; Seth et al., 2008; Son et al., 2012). It has also been shown that non-protein thiols and GSH are involved in the response mechanisms to trace metals in plants (Mishra et al., 2006; Seth et al., 2008). In this study, total NP-SH concentration increased in E. nuttallii following La exposure (Fig. 1e). This could be due to: (1) the newly formed PCs and unidentified thiols (Gupta et al., 1998); (2) stimulation of enzymes involved in the sulfate reduction pathway, such as APS reductase and serine acetyltransferase (Noctor and Foyer, 1998); and (3) its involvement in the metal-induced oxidative stress response (Seth et al., 2008). The plant increased TNP-SH synthesis during La exposure, which confirmed its ability to tolerate cellular metal increases (Seth et al., 2008). Moreover, our results confirmed the studies by Seth et al. (2008) and Mishra et al. (2006), which showed that there was an increase in reduced GSH concentration in Cd stressed plants, compared to control plants. The increased level of reduced GSH during La stress may possibly be due to induced transcription of the GSH biosynthesis genes (Xiang and Oliver, 1998) and PCs biosynthesis enzymes (Inouhe, 2005). PCs, a family of cysteine-rich peptides with the general structure (γ-GluCys)n-Gly (n ¼2–11) (Grill et al., 1985), synthesized from reduced glutathione (GSH), have been shown to play a crucial role in detoxification and homeostasis of metals in the cells of plants (Gupta et al., 1998; Srivastava et al., 2006; Mishra et al., 2006; Seth et al., 2008; Son et al., 2012). Our results showed that La significantly increased PC levels (Fig. 1f) and to our knowledge, this is the first report of phytochelatin synthesis in plant cells in response to REE exposure. The increase in PCs levels in E. nuttallii could be due to activation of PC genes (Heiss et al., 2003), which play an important role in metal detoxification. These results agree with the results for Bacopa monnieri (Mishra et al., 2006), Oenothera odorata (Son et al., 2012) and Brassica juncea (Seth et al., 2008) on exposure to Cd and Hydrilla verticillata exposed to Cu (Srivastava et al., 2006). Thus, this study has demonstrated that reduced GSH, TNP-SH and PC synthesis combat and detoxifies oxidative stress caused by La in E. nuttallii plants.
5. Conclusions Lanthanum toxicity was assessed in E. nuttallii in order to investigate its influence on mineral uptake, MDA, photosynthetic pigments and various non-enzymatic antioxidants levels. As shown by previous studies, La could enter the cytoplasm by altering the structure of the cell wall and the plasma membrane and initiate a series of degenerative processes. La toxicity effects on E. nuttallii were due to disturbances in the uptake of mineral
J. Zhang et al. / Ecotoxicology and Environmental Safety 115 (2015) 159–165
a
b
c
d
e
f
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Fig. 1. (a) The effect of lanthanum (La) on the O2. generation rate in E. nuttallii shoots. Each value is the means 7 S.D. (n ¼3). Different letters over the bars indicate significant differences at Po 0.05. (b) MDA concentration in Elodea nuttallii shoots in response to different levels of lanthanum (La). Data are the means 7S.D. (n¼3), Different letters over the bars indicate significantly differences at Po 0.05. (c) Pigment concentration in Elodea nuttallii shoots after exposure to different levels of lanthanum (La). The results are given as the means 7 S.D. (n¼ 3). The a, b, c, d letters denote significant differences between treatments for each parameter. (d) The effect of lanthanum (La) on reduced GSH in Elodea nuttallii shoots. Each value is the means 7 S.D. (n ¼3). Different letters over the bars indicate significant differences at P o0.05. (e) The effect of lanthanum (La) on TNP-SH in Elodea nuttallii shoots. Each value is the means 7 S.D. (n¼ 3). Different letters over the bars indicate significant differences at P o0.05. (f) The effect of lanthanum (La) on PCs (c) in Elodea nuttallii shoots. Each value is the means7 S.D. (n¼ 3). Different letters over the bars indicate significant differences at P o0.05.
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nutrients and oxidative stress. The latter enhanced MDA and O2. levels and reduced the chlorophyll concentration. Our results suggest that E. nuttallii is able to tolerate La stress through the immobilization by the cell wall and the sequestration of metal ions by non-protein thiols, despite their significant effect on various physiological parameters at 15 mg L 1. The plant, in view of its fast growth and high accumulation potential, seems to be a suitable candidate for the phytoextraction of REEs in aquatic environments.
Acknowledgments This research was supported by the National Natural Science Foundation of China(No. 31170162), the Priority Academic Program Development of the Jiangsu Higher Education Institutions (PAPD), NSFC for Talents Training in Basic Science (J1103507) and by the Qin Lan Project. The lanthanum samples were analyzed by Nanjing Normal University Center for Analysis and Testing.
References Abe, S., Takeda, J., 1988. Effects of LaCl3 on surface charges, dielectrophoresis, and electrofusion of barley protoplasts. Plant Cell Physiol. 87, 389–394. Anderson, M.E., 1985. Determination of glutathione and glutathione disulfide in biological samples. Methods Enzymol. 113, 545–548. Barrat-Segretain, M.H., 2004. Growth of Elodea canadensis and Elodea nuttallii in monocultures and mixture under different light and nutrient conditions. Arch. Hydrobiol. 161, 133–144. Barry, M.J., Meehan, B.J., 2000. The acute and chronic toxicity of lanthanum to Daphnia carinata. Chemosphere 41, 1669–1674. Bhargava, P., Srivastava, A.K., Urmil, S., Rai, L.C., 2005. Phytochelatin plays a role in UV-B tolerance in N2-fixing cyanobacterium Anabaena doliolum. J. Plant Physiol. 162, 1220–1225. Chua, H., 1998. Bio-accumulation of environmental residues of rare earth elements in aquatic flora Eichhornia crassipes (Mart.) Solms in Guangdong Province of China. Sci. Total Environ. 214, 79–85. Diatloff, E., Smith, F.W., Asher, C.J., 2008. Effects of lanthanum and cerium on the growth and mineral nutrition of corn and mungbean. Ann. Bot. 101, 971–982. dΆquino, L., de Pinto, M.C., Nardi, L., Morgana, M., Tommasi, F., 2009. Effect of some light rare earth elements on seed germination, seedling growth and antioxidant metabolism in Triticum durum. Chemosphere 75, 900–905. Elbaz-Poulichet, F., Seidel, J.-L., Othoniel, C., 2002. Occurrence of an anthropogenic gadolinium anomaly in river and coastal waters of Southern France. Water Res. 36, 1102–1105. Ellman, G.L., 1959. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82, 70–77. Evans, C.H., 1988. Biochemistry of Lanthanides. Plenum Press, New York/London. Fu, Y.Y., Li, F.F., Xu, T., Cai, S.J., Chu, W.Y., Qiu, H., Sha, S., Cheng, G.Y., Xu, Q.S., 2014. Bioaccumulation, subcellular, and molecular localization and damage to physiology and ultrastructure in Nymphoides peltata (Gmel.) O. Kuntze exposed to yttrium. Environ. Sci. Pollut. Res. 21, 2935–2942. Grill, E., Winnacker, E.L., Zenk, M.H., 1985. Phytochelatins: the principal heavymetal complexing peptides of higher plants. Science 230, 674–676. Gupta, M., Tripathi, R.D., Rai, U.N., Chandra, P., 1998. Role of glutathione and phytochelatin in Hydrilla verticillata (l.f) Royle and Vallisneria Spiralis L. under mercury stress. Chemosphere 37, 785–800. He, Y., Loh, C., 2000. Cerium and Lanthanum promote flora initiation and reproductive growth of Arabidopsis thaliana. Plant Sci. 159, 117–124. Heath, R.L., Packer, L., 1968. Photoperoxidation in isolated chloroplasts I. Kinetic and stoichiometry of fatty acid peroxidation. Arch. Biochem. Biophys. 125, 189–198. Heiss, S., Wachter, A., Bogs, J., Cobbett, C., Rausch, T., 2003. Phytochelatin synthase (PCS) protein is induced in Brassica juncea leaves after prolonged Cd exposure. J. Exp. Bot. 54, 1833–1839. Hu, X., Ding, Z.H., Chen, Y.J., Wang, X.R., Dai, L.M., 2002. Bioaccumulation of lanthanum and cerium and their effects on the growth of wheat (Triticum aestivum L.) seedlings. Chemosphere 48, 621–629. Inouhe, M., 2005. Phytochelatins. Braz. J. Plant Physiol. 17, 65–78. Ippolito, M.P., Fasciano, C., d’Aquino, L., Morgana, M., Tommasi, F., 2010. Responses of antioxidant systems after exposition to rare earths and their role in chilling stress in common duckweed (Lemna minor L.): a defensive weapon or a boomerang? Arch. Environ. Contam. Toxicol. 58, 42–52. Krzeslowska, M., 2010. The cell wall in plant cell response to trace metals: polysaccharide remodeling and its role in defense strategy. Acta Physiol. Plant. 33, 35–51.
Lai, Y., Wang, Q.Q., Yang, L.M., Huang, B.L., 2006. Subcellular distribution of rare earth elements and characterization of their binding species in a newly discovered hyperaccumulator Pronephrium simplex. Talanta 70, 26–31. Larras, F., Regier, N., Planchon, S., Poté, J., Renaut, J., Cosio, C., 2013. Physiological and proteomic changes suggest an important role of cell walls in the high tolerance to metals of Elodea nuttallii. J. Hazard. Mater. 263, 575–583. Lewis, M.A., 1995. Use of freshwater plans for phytotoxicity testing: a review. Environ. Pollut. 87, 319–336. Lewis, B.D., Spalding, E.P., 1998. Nonselective block by La3 þ of Arabidopsis ion channels involved in signal transduction. J. Membr. Biol. 162, 81–90. Li, Y.H., Yan, C.L., Liu, J.C., Chen, Y.H., Hu, J., Xue, B., 2003. Effects of La3 þ on ATPase acctivities of plasma membrane vesicles isolated from Casuarina equisetifolia seedlings under acid rain stress. J. Rare Earths 21, 675–679. Lichtenthaler, H.K., 1987. Chlorophylls and carotenoids: pigments of photosynthetic membranes. Methods Enzymol. 148, 350–382. Liu, M., Hasenstein, K.H., 2005. La3 þ uptake and its effect on the cytoskeleton in root protoplasts of Zea mays L. Planta 220, 658–666. Lombi, E., Zhao, F.J., Fuhrmann, M., Ma, L.Q., Mcgrath, S.P., 2002. Arsenic distribution and speciation in the fronds of the hyperaccumulator Pteris vittata. New Phytol. 156, 195–203. Mishra, S., Srivastava, S., Tripathia, R.D., Govindarajan, R., Kuriakose, S.V., Prasad, M. N.V., 2006. Phytochelatin synthesis and response of antioxidants during cadmium stress in Bacopa monnieri L. Plant Physiol. Biochem. 44, 25–37. Mohan, B.S., Hosetti, B.B., 1999. Aquatic plants for toxicity assessment. Environ. Res. A 81, 259–274. Noctor, G., Foyer, C., 1998. Ascorbate and glutathione: keeping active oxygen under control. Annu. Rev. Plant Physiol. Plant Mol. Biol. 49, 249–279. Oral, R., Bustamante, P., Warnau, M., D’Ambra, A., Guida, M., Pagano, G., 2010. Cytogenetic and developmental toxicity of cerium and lanthanum to sea urchin embryos. Chemosphere 81, 194–198. Peng, L., Zhang, W.Y., Xi, L., Yi, L., 2007. Structural basis for the biological effects of Pr (III) ions: alteration of cell membrane permeability. Biol. Trace Elem. Res. 120, 141–147. Pineros, M., Tester, M., 1997. Calcium channels in higher plant cells: Selectivity, regulation and pharmacology. J. Exp. Bot. 48, 551–577. Regier, N., Larras, F., Bravi, A.G., Ungureanu, V.-G., Amouroux, D., Cosio, C., 2013. Mercury bioaccumulation in the aquatic plant Elodea nuttallii in the field and in microcosm: accumulation in shoots from the water might involve copper transporters. Chemosphere 90, 595–602. Seth, C.S., Chaturvedi, P.K., Misra, V., 2008. The role of phytochelatins and antioxidants in tolerance to Cd accumulation in Brassica juncea L. Ecotoxicol. Environ. Saf. 71, 76–85. Shan, X.Q., Wang, H.O., Zhang, S.Z., Zhou, H.F., Zheng, Y., Yu, H., Wen, B., 2003. Accumulation and uptake of light rare earth elements in hyperaccumulator Dicropteris dichotoma. Plant Sci. 165, 1343–1353. Sivaci, E.R., Sivaci, A., Sökmen, M., 2004. Biosorption of cadmium by Myriophyllum spicatum L. and Myriophyllum triphyllum orchard. Chemosphere 56, 1043–1048. Son, K.-H., Kim, D.-Y., Koo, N., Kim, K.-R., Kim, J.-G., Owens, G., 2012. Detoxification through phytochelatin synthesis in Oenothera odorata exposed to Cd solutions. Environ. Exp. Bot. 75, 9–15. Srivastava, S., Mishra, S., Mishra, S., Dwivedi, S., Gupta, D.K., 2006. Copper-induced oxidative stress and responses of antioxidants and phytochelatins in Hydrilla verticillata (L.f.) Royle. Aquat. Toxicol. 80, 405–415. St-Cyr, L., Campbell, P.G.C., Guertin, K., 1994. Evaluation of the role of submerged plant beds in the metal budget of a fluvial lake. Hydrobiologia 291, 141–156. Sun, Y., Liu, D.L., Yu, Z.Q., Zhang, Q., Bai, J., Sun, D., 2003. An apoplastic mechanism for short-term effects of rare earth elements at lower concentrations. Plant Cell Environ. 26, 887–896. Thiébaut, G., Gross, Y., Gierlinski, P., Boiche, A., 2010. Accumulation of metals in Elodea canadensis and Elodea nuttallii: implications for plant-macroinvertebrate interactions. Sci. Total Environ. 408, 5499–5505. Thomas, P.J., Carpenter, D., Boutin, C., Allison, J.E., 2014. Rare earth elements (REEs): Effects on germination and growth of selected crop and native plant species. Chemosphere 96, 57–66. Veglio, F., Beolchini, F., 1997. Removal of metals by biosorption: a review. Hydrometallurgy 44, 301–316. Wang, L.H., Zhou, Q., Huang, X.H., 2010a. Effects of heavy metal terbium on contents of cytosolic nurtrient elements in horseradish cell. Ecotoxicol. Envrion. Saf. 73, 1012–1765. Wang, X., Shi, G.X., Xu, Q.S., Xu, B.J., Zhao, J., 2007. Lanthanum- and cerium-induced oxidative stress in submerged Hydrilla verticillata plants. Russ. J. Plant Physiol. 54, 693–697. Wang, A.G., Luo, G.H., 1990. Quantitative relation between the reaction of hydroxylamine and superoxide anion radicals in plants. Plant Physiol. Commun. 6, 55–57. Wang, Q., Li, Z., Cheng, S.P., Wu, Z.B., 2010b. Effects of humic acids on phytoextraction of Cu and Cd from sediment by Elodea nuttallii. Chemosphere 78, 604–608. Weltj, L., Heidenreich, H., Zhu, W.Z., Wolterbeek, H.Th, Horhammer, S., de Goeij, J.J. M., Markert, B., 2002. Lanthanide concentrations in freshwater plants and molluscs, related to those in surface water, pore water and sediment. A case study in the Netherlands. Sci. Total Environ. 286, 191–214. Xiang, C., Oliver, D.J., 1998. Glutathione metabolic genes coordinately respond to heavy metals and jasmonic acid in Arabidopsis. Plant Cell 10, 1539–1550. Xing, W., Li, D.H., Liu, G.H., 2010. Antioxidative responses of Elodea nuttallii (Planch.) H. St. Hohn to short-term iron exposure. Plant Physiol. Biochem. 48, 873–878.
J. Zhang et al. / Ecotoxicology and Environmental Safety 115 (2015) 159–165
Xiong, J., An, L., Lu, H., Zhu, C., 2009. Exogenous nitric oxide enhances cadmium tolerance of rice by increasing pectin and hemicellulose contents in root cell wall. Planta 230, 755–765. Xu, Q.S., Fu, Y.Y., Min, H.L., Cai, S.J., Sha, S., Cheng, G.Y., 2012. Laboratory assessment of uptake and toxicity of lanthanum (La) in the leaves of Hydrocharis dubia (Bl.) Backer. Environ. Sci. Pollut. Res. 19, 3950–3958.
165
Yang, X.Y., Ying, D.Q., Sun, H., Wang, X.R., Dai, L.M., Chen, Y.J., Cao, M., 1999. Distribution and bioavailability of rare earth elements in aquatic microcosm. Chemosphere 39, 2442–2450. Zheng, H.L., Zhang, C.G., Zhao, Z.Q., Ma, J.H., Huang, X.J., 2002. Effects of lanthanum chloride on activity of redox system in plasma membrane of rice seedling roots. J. Rare Earths 20, 156–157.
Contents lists available at ScienceDirect
Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Oxidative effects, nutrients and metabolic changes in aquatic macrophyte, Elodea nuttallii, following exposure to lanthanum Jingjing Zhang, Tingting Zhang, Qianqian Lu, Sanjuan Cai, Weiyue Chu, Han Qiu, Ting Xu, Feifei Li, Qinsong Xu n School of Life Sciences, Nanjing Normal University, Nanjing 210023, China
art ic l e i nf o
a b s t r a c t
Article history: Received 23 October 2014 Received in revised form 5 February 2015 Accepted 6 February 2015
We investigated the phytoremediation potential of Elodea nuttallii to remove rare earth metals from contaminated water. The laboratory experiments were designed to assess the responses induced by lanthanum (5–20 mg L 1) in E. nuttallii over a period of 7 days. The results showed that most La (approximately 85%) was associated with the cell wall. The addition of La to the culture medium reduced the concentration of K, Ca, Cu, Mg, and Mn. However, O2 levels increased with a concomitant increase in the malondialdehyde (MDA) concentration as the La concentration increased, which indicated that the cells were under oxidative stress. Significant reductions in the levels of chlorophyll (Chl) a, b, and carotenoids (Car) were observed in a concentration-dependent manner. However, the levels of reduced glutathione (GSH), total non-protein thiols (TNP-SH) and phytochelatins (PCs) increased for all La concentrations. The results suggested that La was toxic to E. nuttallii because it induced oxidative stress and disturbed mineral uptake. However, E. nuttallii was able to combat La induced damage via an immobilization mechanism, which involved the cell wall and the activation of non-enzymatic antioxidant. & 2015 Elsevier Inc. All rights reserved.
Keywords: Lanthanum Elodea nuttallii Oxidative stress Non-enzymatic antioxidant Ionome
1. Introduction Lanthanum (La) is a member of the rare earth elements (REEs), which comprise the lanthanides or lanthanoids from lanthanum to lutetium plus yttrium and scandium. They share similar physicochemical properties, but are generally different from the common transitional metals. In recent years, due to high-tech and medical applications and the intense use of fertilizers in agriculture and forestry, large amounts of REEs have been emitted to the soil and water environment (Elbaz-Poulichet et al., 2002; Oral et al., 2010). REEs are believed to have limited toxicity and are not significantly hazardous to the environment. However, their slow accumulation in the environment could become problematic (Thomas et al., 2014). Toxicological investigations have suggested that REE bioaccumulation might have significant adverse effects on aquatic biota (Barry and Meehan, 2000; Oral et al., 2010). To date, the effect of REE accumulation on plants remains fragmentary and sometimes inconsistent. For example, La significantly promoted the reproductive growth of Arabidopsis thaliana (He and Loh, 2000) and Nicotinana tabacum (Sun et al., 2003), but in a hydroponic experiment, the addition of La decreased the n
Corresponding author. Fax: þ 86 25 85898186. E-mail address: [email protected] (Q. Xu).
http://dx.doi.org/10.1016/j.ecoenv.2015.02.013 0147-6513/& 2015 Elsevier Inc. All rights reserved.
growth of Zea mays and Vigna radiate (Diatloff et al., 2008) and inhibited primary root elongation in Triticum aestivum seedlings (Hu et al., 2002). Liu and Hasenstein (2005) reported that low La3 þ concentrations ( o1 μM) promoted Z. mays root growth while higher concentrations (4100 μM) inhibited the growth of this organ. It is likely that the La influences on the cell are complex and multiple, such as ion signaling and homeostasis effects (Liu and Hasenstein, 2005) or binding to aromatic rings, carboxyl groups and phospholipids (Abe and Takeda, 1988). These contradictory results may depend of plants species, growth stage and the concentration and type of REEs (He and Loh, 2000; Hu et al., 2002; Diatloff et al., 2008; dΆquino et al., 2009; Ippolito et al., 2010; Thomas et al., 2014). Although the availability and toxicity of exogenous REEs to plants has attracted more attention recently (Chua, 1998; Yang et al., 1999; Weltje et al., 2002; Xu et al., 2012; Fu et al., 2014), The La phytoremediation potential of Elodea nuttallii, a widespread macrophyte species, has not been investigated to any great extent. Aquatic plants are represented by a variety of algal and macrophytic species that occur in many types of habitats and they are often used in water quality monitoring and assessment (Lewis, 1995; Mohan and Hosetti, 1999). It has also been shown that aquatic macrophytes can uptake metals directly from water and accumulate them in their shoots because they are completely
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submerged and have a very thin cuticle (St-Cyr et al., 1994; Wang et al., 2010b, Regier et al., 2013). E. nuttallii, a submerged plant native to North America, is known for its fast growth rate and is easily cultured in the laboratory. It is also able to accumulate pollutants. Therefore, it has been widely used as model experimental system for bioaccumulation and ecotoxicological investigations (Barrat-Segretain, 2004; Thiébaut et al., 2010; Xing et al., 2010; Regier et al., 2013; Larras et al., 2013). This study investigated the responses of E. nuttallii to exogenous La by measuring (i) the accumulation and sub-cellular distribution of La; (ii) changes in nutrient uptake; (iii) La-induced oxidative stress through the measurement of O2 , MDA changes and chlorophyll concentrations; and (iv) variations in non-enzymatic antioxidants (reduced GSH, TNP-SH and PCs). Overall these accumulation and distribution data should improve our understanding of the main cellular and molecular targets involved in the accumulation/distribution of La and further the potential use of E. nuttallii in the phytoremediation programs of aquatic bodies polluted with REEs.
2.4. Determination of O2 generation rate The concentration of O2 generation rate was measured using the hydroxylamine chloride method (Wang and Luo, 1990). The supernatant (0.5 mL) was collected and incubated at 25 °C for 60 min in the presence of 1 mM hydroxylamine hydrochloride in 50 mM sodium phosphate buffer (pH 7.8). The reaction mixture was then incubated with 1 mL of 17 mM P-aminobenzene sulfonic acid anhydrous and 1 mL of 7 mM a-naphthylamine at 25 °C for 30 min. The absorbance was measured at 530 nm. A calibration curve was established using sodium nitrite. 2.5. Lipid peroxidation The level of lipid peroxidation in the shoots (0.5 g) was determined immediately by thiobarbituric acid reaction according to Heath and Packer (1968). The concentration of MDA was calculated using 155 mM cm 1 as extinction coefficient in terms of nmol g 1 fresh weight.
2. Materials and methods
2.6. Determination of photosynthetic pigment concentration
2.1. Plant material and growth conditions
Chlorophylls (Chl) and carotenoids (Car) were extracted with 80% acetone and absorbances at 470, 647 and 663 nm recorded on a spectrophotometer (Thermo GENESYS 10). The concentrations of Chl a, Chl b and Car were estimated according to Lichtenthaler (1987).
Cleaned, healthy, 10 cm-long vegetative shoots of E. nuttallii, collected from Lake Pipa, Nanjing, China. All the materials were cultivated in 1/10 Hoagland solution under controlled conditions (114 μmol m 2 s 1 light irradiance, 14 h photoperiod and 25 °C/ 20 °C day/night). Then lanthanum was added to the containers in the form of LaCl3⋅7H2O in appropriated quantities to give the following treatments: 5, 10, 15, 20 mg L 1 [La3 þ ]. The tested concentrations of La to perform our survey were chosen within the range of previous toxicological investigations on plants (Liu and Hasenstein, 2005; dΆquino et al., 2009; Ippolito et al., 2010; Thomas et al., 2014). Each treatment had three replicates, and all of the solutions were refreshed every 2 days during the 7-day culture period. 2.2. Tissue fraction and la analysis After 7 days of metal exposure, the Elodea plants were harvested and washed with double distilled water and 20 mM EDTA to remove metals absorbed on the surface of the plant material. The sub-cellular distribution of La within the shoots was determined according to the method described by Xiong et al. (2009). Concentrations of La in cell wall, organelles and soluble fraction were determined by inductively coupled plasma atomic emission spectrometry (ICP-AES, Leeman labs, Prodigy, USA) after digestion with HNO3 and HClO4 (10:1, v/v) at 160°C (detection limit: 0.0015 mg/L). The liquid standard La reference material (GSB 04-1774-2004) was diluted and analyzed for La concentration.
2.7. Reduced glutathione (GSH), total non-protein thiol (TNP-SH), and phytochelatins (PCs) determination To determine the concentration of reduced GSH, fresh shoots (0.5 g) were homogenized in ice-cold 5% (w/v) trichloroacetic acid and then centrifuged at 10,000g for 20 min at 4 °C. The reduced GSH concentration was determined spectrophotometrically at 412 nm according to the method used by Anderson (1985), after precipitation with 0.1 M HCl, using GSH reductase, 5,5′–dithio–bis(2-nitrobenzoic acid) (DTNB) and NADPH. The reduced GSH concentration was expressed as mg g 1 fresh weight. In order to estimate the total non-protein thiol (TNP-SH) concentration, fresh shoots were extracted in 6.67% 5-sulphosalicylic acid and centrifuged at 12,000g for 10 min at 4 °C. Supernatant was reacted with Ellman's reagent (5 mM EDTA and 0.6 mM 5, 5′ dithiobis (2-nitroenzoic acid) in 120 mM phosphate buffer, pH 7.5) and absorbance was measured spectrophotometrically after 5 min at 412 nm (Ellman, 1959). The PCs were measured by following the method of Bhargava et al. (2005). The PCs were measured using the formula: total NPSH–GSH after the total NP-SH and reduced GSH concentrations had been determined.
2.3. Determination of mineral concentration 2.8. Statistical analysis The mineral concentration in the shoots of E. nuttallii was determined according to previously reported methods (Xu et al., 2012; Fu et al., 2014). For each sample, 0.5 g of shoots was digested with HNO3:HClO4 (10:1, v/v) at 160 °C. The solution samples were diluted to a final volume of 10 mL and were analyzed for mineral elements (K, Ca, Fe, Cu, Zn, Mn, and Mg) by ICP-AES. Standard solution (xccc-13A, xccc-14A, SPEX CertiPrep, USA) were used for the calibration.
All results are presented as mean values 7standard deviation (SD) of at least three independent experiments. Statistical analysis was performed by computer software SPSS17.0. A correlation analysis was performed to determine the relationships between La accumulation and studies parameters. The experimental data was subjected to a one-way analysis of variance (ANOVA) and P-valueso0.05 were considered significant.
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3. Results 3.1. Sub-cellular distribution of La We investigated the sub-cellular distribution of La in E. nuttallii shoots (Table 1). Most of the La was found in the cell wall fraction, while a minor part was associated with the organelle and the soluble fractions. The La proportion in different sub-cellular fractions remained fairly constant for all treatments. 3.2. The main mineral elements As shown in Table 2, the concentrations of the main mineral elements, such as K, Ca, Cu, Mg and Mn in the plant exposure groups were lower than in the control groups and decreased significantly with increasing concentrations of La (rK ¼ 0.93, P o0.01; rCa ¼ –0.97, Po0.01; rCu ¼–0.91, P o0.05; rMg ¼ 0.85, P o0.05 and rMn ¼ –0.99, Po 0.01), whereas Zn levels peaked at 5 mg L 1 and represented a 37% increased with respect to the control plants. However, the Zn levels began to decline as the La concentration continued to rise. In contrast, the Fe levels decreased slightly (by 14%) when treated with 5 mg L 1 La, but then increased thereafter (r ¼0.99, P o0.01).
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Table 1 Sub-cellular distribution of lanthanum (La) in Elodea nuttallii shoots. Data are means 7S.D. (n¼ 3). Different letters in a column indicate significantly different values at Po 0.05. La concentration (mg L 1 )
0 5 10 15 20
La in subcellular fractions (mg kg 1 FW) Cell wall
Organelles
Soluble fraction
Total La content
N.D. 1640d 7 45.7 2340c 7 33.9 3180b 7 24.9 3370a 7 22.3
N.D. 171d 70.6
N.D. 144c 710.9
N.D. 1950a 757.2
225c 7 17.3
167bc 7 12.2
2730c 7 4.5
319b 7 21.2
200ab 7 9.8
3700b 7 6.1
369a 7 12.8 234a 7 18.2
3970a 7 16.8
higher than the control when the plants were grown in 15 mg L 1 La. Despite the decrease in PC concentrations at 20 mg L 1 concentrations, they still remained 6.3 times higher than the controls.
4. Discussion 3.3. O2. generation rate In E. nuttallii shoots, the O2. generation rate showed a dosedependent increase as the La concentration rose (r ¼0.98; P o0.01). The peak accumulation was observed on day 7 at 20 mg L 1 La where the O2. generation rate was up to 214% higher than in the control plants (Fig. 1a). 3.4. Malondialdehyde (MDA) concentration MDA concentration (Fig. 1b) was measured in order to evaluate lipid peroxidation caused by La. MDA increased gradually in a concentration dependent manner and was significantly positively correlated with La accumulation by the plants (P o0.01). The maximum increase was recorded at 20 mg L 1 on day 7, which was 81% higher with respect to control plants. 3.5. Photosynthetic pigment levels As shown in Fig. 1c, the photosynthetic pigments (Chl a, Chl b and Car) showed a similar response after La exposure. Significant losses in Chl a (r ¼ –0.91; Po 0.05), Chl b (r ¼–0.98; P o0.01) and Car (r ¼ –0.81; P o0.05) were recorded with increasing levels of La in the nutrition medium. The maximum decreases were 78% (Chl a), 73% (Chl b) and 24% (Car) compared to their controls when the plants were exposed to 20 mg L 1 for 7 days. 3.6. Reduced GSH, TNP-SH and PCs Compared with the control, the concentration of reduced GSH increased slightly as the La concentration increased with a maximum increase of 14% when the plants were exposed to 10 mg L 1 La for 7 days. Even though the reduced GSH concentration at higher La concentrations declined, it still remained higher than the controls (Fig. 1d). Fig. 1e shows that the TNP-SH concentration in E. nuttallii shoots increased by up to 10 mg L 1 and then declined. The concentration at 10 mg L 1 was 42% greater than the control, but, it decreased to 115% of the control when plants were treated with 20 mg L 1 La. La treatment induced a massive accumulation of PCs compared to the control (Fig. 1f). The PC levels in the shoots were 12.3 times
Metal accumulation in E. nuttallii has been investigated by Regier et al. (2013), who noticed that the amount of Hg in shoots was significantly higher than in roots, and by Wang et al. (2010b), who found that Cd and Cu were accumulated by shoots from the water rather than from the sediments. In this study, E. nuttallii accumulated large concentrations of La in the shoots in a dosedependent manner up to 7 days of exposure (Table 1). Significant accumulation of REEs has also been observed in other aquatic plants, such as Eichhornia crassipes (Chua, 1998), Sperollela polyrrhiza (Yang et al., 1999), Hydrocharis dubia (Xu et al., 2012) and Nymphoides peltata (Fu et al., 2014). All these experimental data suggested that exogenous REEs were highly available to aquatic plants and that their shoots played a primary role in the uptake of metals. Previous studies showed that aquatic plants were capable of removing metals from water through a metal binding process (biosorption) and subsequent metabolism-dependent accumulation (Veglio and Beolchini, 1997; Sivaci et al., 2004). It is well documented that La has many chemical and physical characteristics in common with calcium, such as ionic radii (La3 þ : 8.5 10 2; Ca2 þ : 9.2 10 2) (Evans, 1988). Therefore, it may be transported into plant cells through Ca channels and/or by Ca transporters after an initial sorption of REEs to the cell wall (Hu et al., 2002). The data presented here also showed that approximately 15% of the La was taken up into the cells (Table 1). When attempting to understand the physiological role of REEs in plants, one of the most important questions is whether REEs enters the cell or not. In our study, La analysis at the sub-cellular plant tissue level demonstrated that most of the La (84–86%) was bound to the cell wall fraction (Table 1). These results were in line with previous studies on Dicropteris dichotoma (Shan et al., 2003), Pronephrium simplex (Lai et al., 2006) and Nymphoides peltata (Fu et al., 2014), which reported that La was predominantly present in the cell wall (81–89%, 68% and 73–86%, respectively). These observations suggested that the cell wall is the predominant sink for REEs and therefore is believed to play a role in metal tolerance. It has been stated that plant cells can modify the metal accumulation capacity of their cell walls in order to accumulate more cations in the apoplast and thereby protect the protoplast (Krzeslowska, 2010). However, our further analysis confirmed that cellulose and pectin (79–84%) were the major cell wall components involved in the retention of La in the cell wall (data not shown).
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Table 2 The effect of lanthanum (La) supply on nutrient element concentrations in Elodea nuttallii shoots. Data are means 7S.D. (n¼ 3). Different letters in a line indicate significantly different values at Po 0.05. Element concentration (mg kg 1FW )
La concentration (mg L 1) 0
Potassium Calcium Copper Magnesium Manganese Zinc Iron
5 a
3710 7 7. 1 1070a 7 18.4 3.4a 7 0. 1 298a 7 7.1 43.7a 7 2.1 12.8b 7 0.0 71.3b 7 7.1
10 b
3430 715.8 968b 74.1 3.2a 70.2 202b 75.0 41.3a 71.8 17.6a 70.3 61.7b 78.1
The bioaccumulation of REEs in aquatic plants is often accompanied by the induction of a variety of cellular changes, some of which directly contribute to the metal tolerance capacity of plants. In the present study, multiple physiological and biochemical responses in the plant tissues were induced by La and these affected nutrient uptake (Table 2), ROS (Fig. 1a), reduced GSH (Fig. 1d), TNP-SH (Fig. 1e) and PC (Fig. 1f) concentrations in E. nuttallii, subjected to La. It has been reported that REEs can cause damage to the plasma membrane and cytoskeleton structures (Zheng et al., 2002; Liu and Hasenstein, 2005; Peng et al., 2007; Wang et al., 2010a). Therefore, they can affect the flow of nutrients into and out of the cell. Our experimental results suggested that the cellular concentrations of macro-elements, such as K, Ca, Cu, Mn and Mg, in E. nuttallii shoots subjected to La, were lower than that in the control groups, while Fe ( 45 mg L 1) and Zn (o 15 mg L 1) levels increased (Table 2). Generally, because of a similar ionic radius to Ca2 þ , La3 þ is probably able to block calcium ionic channels and disturb the uptake of nutrient ions through calcium channels (Pineros and Tester, 1997; Lewis and Spalding, 1998). Previous studies have demonstrated that La3 þ can inhibit the physiological activity of protease (Li et al., 2003), inhibit plasma membrane redox system activity (Zheng et al., 2002; dΆquino et al., 2009), and reduce the transportation of ions in plants. The oxidative stress imposed on E. nuttallii after La exposure was shown by the significant increase in MDA at higher concentrations (Fig. 1b). It was mostly attributable to enhanced ROS levels, including O2. (Fig. 1a) and H2O2 (Wang et al., 2007; Ippolito et al., 2010), and the altered membrane permeability, which led to increased ion loss (Peng et al., 2007; Zheng et al., 2002; Wang et al., 2010a). Furthermore, our results also showed an increase in K þ leakage when plants were treated with La, which was also representative of membrane disruption (Table 2). Thus, exposure to La caused oxidative stress in E. nuttallii. Our results are similar to those observed in Hydrilla verticillata (Wang et al., 2007), Lemna minor (Ippolito et al., 2010), and Hydrocharis dubia (Xu et al., 2012). In the E. nuttallii was produced change in photosynthetic apparatus after La exposure. These alterations included a decrease in Chl a, Chl b and Car (Fig.1c) concentrations. The damage to photosynthetic pigments was also expressed visually as chlorosis. A decrease in photosynthetic pigment levels is a common response by plants to a variety of abiotic stresses and has been observed in a number aquatic plants after exposure to REEs, such as Nymphoides peltata (Fu et al., 2014), Hydrilla verticillata (Wang et al., 2007), Hydrocharis dubia (Xu et al., 2012) and Lemna minor (Ippolito et al., 2010). These studies attributed the decrease in pigment concentration to the disturbance in chlorophyll biosynthesis or degradation due to lipid peroxidation (Wang et al., 2007) and damage to the ultrastructure of the chloroplast (Xu et al., 2012; Fu et al., 2014).
15 c
3240 7 11.0 788c 7 4.1 3.2a 7 0.2 188b 7 0.6 37.0b 7 1.2 16.1a 7 3.4 79.5a 7 4.0
20 e
3030 7 12.0 684d 7 1.1 3.0b 7 0.0 184c 7 7.1 34.3c 7 0.3 13.4c 7 0.1 81.9a 7 1.8
3110d 7 4.6 659e 7 6.9 3.0b 7 0. 3 168d 7 4.1 31.4c 7 4.9 12.2b 7 0.8 83.7a 7 4.6
To mitigate the oxidative damage initiated by ROS, plants have evolved different detoxification mechanisms, including cellular/ sub-cellular compartmentalization into cell wall or the vacuole (Lombi et al., 2002), and complexation with phytochelatins (PCs) (Mishra et al., 2006; Srivastava et al., 2006; Seth et al., 2008; Son et al., 2012). It has also been shown that non-protein thiols and GSH are involved in the response mechanisms to trace metals in plants (Mishra et al., 2006; Seth et al., 2008). In this study, total NP-SH concentration increased in E. nuttallii following La exposure (Fig. 1e). This could be due to: (1) the newly formed PCs and unidentified thiols (Gupta et al., 1998); (2) stimulation of enzymes involved in the sulfate reduction pathway, such as APS reductase and serine acetyltransferase (Noctor and Foyer, 1998); and (3) its involvement in the metal-induced oxidative stress response (Seth et al., 2008). The plant increased TNP-SH synthesis during La exposure, which confirmed its ability to tolerate cellular metal increases (Seth et al., 2008). Moreover, our results confirmed the studies by Seth et al. (2008) and Mishra et al. (2006), which showed that there was an increase in reduced GSH concentration in Cd stressed plants, compared to control plants. The increased level of reduced GSH during La stress may possibly be due to induced transcription of the GSH biosynthesis genes (Xiang and Oliver, 1998) and PCs biosynthesis enzymes (Inouhe, 2005). PCs, a family of cysteine-rich peptides with the general structure (γ-GluCys)n-Gly (n ¼2–11) (Grill et al., 1985), synthesized from reduced glutathione (GSH), have been shown to play a crucial role in detoxification and homeostasis of metals in the cells of plants (Gupta et al., 1998; Srivastava et al., 2006; Mishra et al., 2006; Seth et al., 2008; Son et al., 2012). Our results showed that La significantly increased PC levels (Fig. 1f) and to our knowledge, this is the first report of phytochelatin synthesis in plant cells in response to REE exposure. The increase in PCs levels in E. nuttallii could be due to activation of PC genes (Heiss et al., 2003), which play an important role in metal detoxification. These results agree with the results for Bacopa monnieri (Mishra et al., 2006), Oenothera odorata (Son et al., 2012) and Brassica juncea (Seth et al., 2008) on exposure to Cd and Hydrilla verticillata exposed to Cu (Srivastava et al., 2006). Thus, this study has demonstrated that reduced GSH, TNP-SH and PC synthesis combat and detoxifies oxidative stress caused by La in E. nuttallii plants.
5. Conclusions Lanthanum toxicity was assessed in E. nuttallii in order to investigate its influence on mineral uptake, MDA, photosynthetic pigments and various non-enzymatic antioxidants levels. As shown by previous studies, La could enter the cytoplasm by altering the structure of the cell wall and the plasma membrane and initiate a series of degenerative processes. La toxicity effects on E. nuttallii were due to disturbances in the uptake of mineral
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a
b
c
d
e
f
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Fig. 1. (a) The effect of lanthanum (La) on the O2. generation rate in E. nuttallii shoots. Each value is the means 7 S.D. (n ¼3). Different letters over the bars indicate significant differences at Po 0.05. (b) MDA concentration in Elodea nuttallii shoots in response to different levels of lanthanum (La). Data are the means 7S.D. (n¼3), Different letters over the bars indicate significantly differences at Po 0.05. (c) Pigment concentration in Elodea nuttallii shoots after exposure to different levels of lanthanum (La). The results are given as the means 7 S.D. (n¼ 3). The a, b, c, d letters denote significant differences between treatments for each parameter. (d) The effect of lanthanum (La) on reduced GSH in Elodea nuttallii shoots. Each value is the means 7 S.D. (n ¼3). Different letters over the bars indicate significant differences at P o0.05. (e) The effect of lanthanum (La) on TNP-SH in Elodea nuttallii shoots. Each value is the means 7 S.D. (n¼ 3). Different letters over the bars indicate significant differences at P o0.05. (f) The effect of lanthanum (La) on PCs (c) in Elodea nuttallii shoots. Each value is the means7 S.D. (n¼ 3). Different letters over the bars indicate significant differences at P o0.05.
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nutrients and oxidative stress. The latter enhanced MDA and O2. levels and reduced the chlorophyll concentration. Our results suggest that E. nuttallii is able to tolerate La stress through the immobilization by the cell wall and the sequestration of metal ions by non-protein thiols, despite their significant effect on various physiological parameters at 15 mg L 1. The plant, in view of its fast growth and high accumulation potential, seems to be a suitable candidate for the phytoextraction of REEs in aquatic environments.
Acknowledgments This research was supported by the National Natural Science Foundation of China(No. 31170162), the Priority Academic Program Development of the Jiangsu Higher Education Institutions (PAPD), NSFC for Talents Training in Basic Science (J1103507) and by the Qin Lan Project. The lanthanum samples were analyzed by Nanjing Normal University Center for Analysis and Testing.
References Abe, S., Takeda, J., 1988. Effects of LaCl3 on surface charges, dielectrophoresis, and electrofusion of barley protoplasts. Plant Cell Physiol. 87, 389–394. Anderson, M.E., 1985. Determination of glutathione and glutathione disulfide in biological samples. Methods Enzymol. 113, 545–548. Barrat-Segretain, M.H., 2004. Growth of Elodea canadensis and Elodea nuttallii in monocultures and mixture under different light and nutrient conditions. Arch. Hydrobiol. 161, 133–144. Barry, M.J., Meehan, B.J., 2000. The acute and chronic toxicity of lanthanum to Daphnia carinata. Chemosphere 41, 1669–1674. Bhargava, P., Srivastava, A.K., Urmil, S., Rai, L.C., 2005. Phytochelatin plays a role in UV-B tolerance in N2-fixing cyanobacterium Anabaena doliolum. J. Plant Physiol. 162, 1220–1225. Chua, H., 1998. Bio-accumulation of environmental residues of rare earth elements in aquatic flora Eichhornia crassipes (Mart.) Solms in Guangdong Province of China. Sci. Total Environ. 214, 79–85. Diatloff, E., Smith, F.W., Asher, C.J., 2008. Effects of lanthanum and cerium on the growth and mineral nutrition of corn and mungbean. Ann. Bot. 101, 971–982. dΆquino, L., de Pinto, M.C., Nardi, L., Morgana, M., Tommasi, F., 2009. Effect of some light rare earth elements on seed germination, seedling growth and antioxidant metabolism in Triticum durum. Chemosphere 75, 900–905. Elbaz-Poulichet, F., Seidel, J.-L., Othoniel, C., 2002. Occurrence of an anthropogenic gadolinium anomaly in river and coastal waters of Southern France. Water Res. 36, 1102–1105. Ellman, G.L., 1959. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82, 70–77. Evans, C.H., 1988. Biochemistry of Lanthanides. Plenum Press, New York/London. Fu, Y.Y., Li, F.F., Xu, T., Cai, S.J., Chu, W.Y., Qiu, H., Sha, S., Cheng, G.Y., Xu, Q.S., 2014. Bioaccumulation, subcellular, and molecular localization and damage to physiology and ultrastructure in Nymphoides peltata (Gmel.) O. Kuntze exposed to yttrium. Environ. Sci. Pollut. Res. 21, 2935–2942. Grill, E., Winnacker, E.L., Zenk, M.H., 1985. Phytochelatins: the principal heavymetal complexing peptides of higher plants. Science 230, 674–676. Gupta, M., Tripathi, R.D., Rai, U.N., Chandra, P., 1998. Role of glutathione and phytochelatin in Hydrilla verticillata (l.f) Royle and Vallisneria Spiralis L. under mercury stress. Chemosphere 37, 785–800. He, Y., Loh, C., 2000. Cerium and Lanthanum promote flora initiation and reproductive growth of Arabidopsis thaliana. Plant Sci. 159, 117–124. Heath, R.L., Packer, L., 1968. Photoperoxidation in isolated chloroplasts I. Kinetic and stoichiometry of fatty acid peroxidation. Arch. Biochem. Biophys. 125, 189–198. Heiss, S., Wachter, A., Bogs, J., Cobbett, C., Rausch, T., 2003. Phytochelatin synthase (PCS) protein is induced in Brassica juncea leaves after prolonged Cd exposure. J. Exp. Bot. 54, 1833–1839. Hu, X., Ding, Z.H., Chen, Y.J., Wang, X.R., Dai, L.M., 2002. Bioaccumulation of lanthanum and cerium and their effects on the growth of wheat (Triticum aestivum L.) seedlings. Chemosphere 48, 621–629. Inouhe, M., 2005. Phytochelatins. Braz. J. Plant Physiol. 17, 65–78. Ippolito, M.P., Fasciano, C., d’Aquino, L., Morgana, M., Tommasi, F., 2010. Responses of antioxidant systems after exposition to rare earths and their role in chilling stress in common duckweed (Lemna minor L.): a defensive weapon or a boomerang? Arch. Environ. Contam. Toxicol. 58, 42–52. Krzeslowska, M., 2010. The cell wall in plant cell response to trace metals: polysaccharide remodeling and its role in defense strategy. Acta Physiol. Plant. 33, 35–51.
Lai, Y., Wang, Q.Q., Yang, L.M., Huang, B.L., 2006. Subcellular distribution of rare earth elements and characterization of their binding species in a newly discovered hyperaccumulator Pronephrium simplex. Talanta 70, 26–31. Larras, F., Regier, N., Planchon, S., Poté, J., Renaut, J., Cosio, C., 2013. Physiological and proteomic changes suggest an important role of cell walls in the high tolerance to metals of Elodea nuttallii. J. Hazard. Mater. 263, 575–583. Lewis, M.A., 1995. Use of freshwater plans for phytotoxicity testing: a review. Environ. Pollut. 87, 319–336. Lewis, B.D., Spalding, E.P., 1998. Nonselective block by La3 þ of Arabidopsis ion channels involved in signal transduction. J. Membr. Biol. 162, 81–90. Li, Y.H., Yan, C.L., Liu, J.C., Chen, Y.H., Hu, J., Xue, B., 2003. Effects of La3 þ on ATPase acctivities of plasma membrane vesicles isolated from Casuarina equisetifolia seedlings under acid rain stress. J. Rare Earths 21, 675–679. Lichtenthaler, H.K., 1987. Chlorophylls and carotenoids: pigments of photosynthetic membranes. Methods Enzymol. 148, 350–382. Liu, M., Hasenstein, K.H., 2005. La3 þ uptake and its effect on the cytoskeleton in root protoplasts of Zea mays L. Planta 220, 658–666. Lombi, E., Zhao, F.J., Fuhrmann, M., Ma, L.Q., Mcgrath, S.P., 2002. Arsenic distribution and speciation in the fronds of the hyperaccumulator Pteris vittata. New Phytol. 156, 195–203. Mishra, S., Srivastava, S., Tripathia, R.D., Govindarajan, R., Kuriakose, S.V., Prasad, M. N.V., 2006. Phytochelatin synthesis and response of antioxidants during cadmium stress in Bacopa monnieri L. Plant Physiol. Biochem. 44, 25–37. Mohan, B.S., Hosetti, B.B., 1999. Aquatic plants for toxicity assessment. Environ. Res. A 81, 259–274. Noctor, G., Foyer, C., 1998. Ascorbate and glutathione: keeping active oxygen under control. Annu. Rev. Plant Physiol. Plant Mol. Biol. 49, 249–279. Oral, R., Bustamante, P., Warnau, M., D’Ambra, A., Guida, M., Pagano, G., 2010. Cytogenetic and developmental toxicity of cerium and lanthanum to sea urchin embryos. Chemosphere 81, 194–198. Peng, L., Zhang, W.Y., Xi, L., Yi, L., 2007. Structural basis for the biological effects of Pr (III) ions: alteration of cell membrane permeability. Biol. Trace Elem. Res. 120, 141–147. Pineros, M., Tester, M., 1997. Calcium channels in higher plant cells: Selectivity, regulation and pharmacology. J. Exp. Bot. 48, 551–577. Regier, N., Larras, F., Bravi, A.G., Ungureanu, V.-G., Amouroux, D., Cosio, C., 2013. Mercury bioaccumulation in the aquatic plant Elodea nuttallii in the field and in microcosm: accumulation in shoots from the water might involve copper transporters. Chemosphere 90, 595–602. Seth, C.S., Chaturvedi, P.K., Misra, V., 2008. The role of phytochelatins and antioxidants in tolerance to Cd accumulation in Brassica juncea L. Ecotoxicol. Environ. Saf. 71, 76–85. Shan, X.Q., Wang, H.O., Zhang, S.Z., Zhou, H.F., Zheng, Y., Yu, H., Wen, B., 2003. Accumulation and uptake of light rare earth elements in hyperaccumulator Dicropteris dichotoma. Plant Sci. 165, 1343–1353. Sivaci, E.R., Sivaci, A., Sökmen, M., 2004. Biosorption of cadmium by Myriophyllum spicatum L. and Myriophyllum triphyllum orchard. Chemosphere 56, 1043–1048. Son, K.-H., Kim, D.-Y., Koo, N., Kim, K.-R., Kim, J.-G., Owens, G., 2012. Detoxification through phytochelatin synthesis in Oenothera odorata exposed to Cd solutions. Environ. Exp. Bot. 75, 9–15. Srivastava, S., Mishra, S., Mishra, S., Dwivedi, S., Gupta, D.K., 2006. Copper-induced oxidative stress and responses of antioxidants and phytochelatins in Hydrilla verticillata (L.f.) Royle. Aquat. Toxicol. 80, 405–415. St-Cyr, L., Campbell, P.G.C., Guertin, K., 1994. Evaluation of the role of submerged plant beds in the metal budget of a fluvial lake. Hydrobiologia 291, 141–156. Sun, Y., Liu, D.L., Yu, Z.Q., Zhang, Q., Bai, J., Sun, D., 2003. An apoplastic mechanism for short-term effects of rare earth elements at lower concentrations. Plant Cell Environ. 26, 887–896. Thiébaut, G., Gross, Y., Gierlinski, P., Boiche, A., 2010. Accumulation of metals in Elodea canadensis and Elodea nuttallii: implications for plant-macroinvertebrate interactions. Sci. Total Environ. 408, 5499–5505. Thomas, P.J., Carpenter, D., Boutin, C., Allison, J.E., 2014. Rare earth elements (REEs): Effects on germination and growth of selected crop and native plant species. Chemosphere 96, 57–66. Veglio, F., Beolchini, F., 1997. Removal of metals by biosorption: a review. Hydrometallurgy 44, 301–316. Wang, L.H., Zhou, Q., Huang, X.H., 2010a. Effects of heavy metal terbium on contents of cytosolic nurtrient elements in horseradish cell. Ecotoxicol. Envrion. Saf. 73, 1012–1765. Wang, X., Shi, G.X., Xu, Q.S., Xu, B.J., Zhao, J., 2007. Lanthanum- and cerium-induced oxidative stress in submerged Hydrilla verticillata plants. Russ. J. Plant Physiol. 54, 693–697. Wang, A.G., Luo, G.H., 1990. Quantitative relation between the reaction of hydroxylamine and superoxide anion radicals in plants. Plant Physiol. Commun. 6, 55–57. Wang, Q., Li, Z., Cheng, S.P., Wu, Z.B., 2010b. Effects of humic acids on phytoextraction of Cu and Cd from sediment by Elodea nuttallii. Chemosphere 78, 604–608. Weltj, L., Heidenreich, H., Zhu, W.Z., Wolterbeek, H.Th, Horhammer, S., de Goeij, J.J. M., Markert, B., 2002. Lanthanide concentrations in freshwater plants and molluscs, related to those in surface water, pore water and sediment. A case study in the Netherlands. Sci. Total Environ. 286, 191–214. Xiang, C., Oliver, D.J., 1998. Glutathione metabolic genes coordinately respond to heavy metals and jasmonic acid in Arabidopsis. Plant Cell 10, 1539–1550. Xing, W., Li, D.H., Liu, G.H., 2010. Antioxidative responses of Elodea nuttallii (Planch.) H. St. Hohn to short-term iron exposure. Plant Physiol. Biochem. 48, 873–878.
J. Zhang et al. / Ecotoxicology and Environmental Safety 115 (2015) 159–165
Xiong, J., An, L., Lu, H., Zhu, C., 2009. Exogenous nitric oxide enhances cadmium tolerance of rice by increasing pectin and hemicellulose contents in root cell wall. Planta 230, 755–765. Xu, Q.S., Fu, Y.Y., Min, H.L., Cai, S.J., Sha, S., Cheng, G.Y., 2012. Laboratory assessment of uptake and toxicity of lanthanum (La) in the leaves of Hydrocharis dubia (Bl.) Backer. Environ. Sci. Pollut. Res. 19, 3950–3958.
165
Yang, X.Y., Ying, D.Q., Sun, H., Wang, X.R., Dai, L.M., Chen, Y.J., Cao, M., 1999. Distribution and bioavailability of rare earth elements in aquatic microcosm. Chemosphere 39, 2442–2450. Zheng, H.L., Zhang, C.G., Zhao, Z.Q., Ma, J.H., Huang, X.J., 2002. Effects of lanthanum chloride on activity of redox system in plasma membrane of rice seedling roots. J. Rare Earths 20, 156–157.
