Protoplasma DOI 10.1007/s00709-015-0832-3
ORIGINAL ARTICLE
Kinetics of nickel bioaccumulation and its relevance to selected cellular processes in leaves of Elodea canadensis during short-term exposure Maria G. Maleva 1 & Przemysław Malec 2 & Majeti Narasimha Vara Prasad 1,3 & Kazimierz Strzałka 2
Received: 8 February 2015 / Accepted: 7 May 2015 # Springer-Verlag Wien 2015
Abstract Elodea canadensis is an aquatic macrophyte used widely as a bioindicator for the monitoring of water quality and in the phytoremediation of metal-contaminated waters. This study considers the kinetics of nickel bioaccumulation and changes in accompanying metabolic and stress-related physiological parameters. These include photosynthetic activity, pigment content, the accumulation of thiol-containing compounds, thiobarbituric acid-reactive substance (TBARS) products, and the activity of selected antioxidant enzymes (catalase, glutathione reductase, superoxide dismutase). Elodea leaves accumulated nickel according to pseudosecond-order kinetics, and the protective responses followed a time sequence which was related to the apparent rates of nickel accumulation. The applicability of second-order kinetics to the Ni uptake by Elodea leaves during the first 8 h of exposure to the metal suggested that the passive binding of metal ions (chemisorption) was a rate-limiting step at the initial phase of Ni accumulation. This phase was accompanied by an increase in photosynthetic activity together with elevated photosynthetic pigments and protein synthesis, the enhanced activity of antioxidant enzymes, and increased thiol Handling Editor: Bhumi Nath Tripathi * Kazimierz Strzałka [email protected] 1
Department of Plant Physiology and Biochemistry, Ural Federal University named after the first President of Russia B.N. Yeltsin, Lenin av. 51, Ekaterinburg, Russia 620000
2
Department of Plant Physiology and Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, ul. Gronostajowa 7 30-387, Kraków, Poland
3
Department of Plant Sciences, University of Hyderabad, Hyderabad, Telangana 500 046, India
concentration. In contrast, there was a decrease in metabolic activity upon the accumulation of TBARS, and the decline in enzyme activity was observed in the saturation phase of Ni accumulation (8–24 h). These results show that a correlation exists between the protective response and the apparent kinetic rate of Ni uptake. Thus, the time of exposure to the toxicant is a crucial factor in the activation of specific mechanisms of Ni detoxification and stress alleviation. Keywords Elodea canadensis . Nickel . Bioaccumulation . Kinetic modeling . Phytoremediation . Oxidative stress . Hormesis . Antioxidants
Introduction The contamination of surface waters by trace elements originating from anthropogenic and geogenic processes has become a global problem. Heavy metals, being highly toxic, persistent, and non-biodegradable pollutants, are a serious threat to aquatic ecosystems (Lone et al. 2008). The release of metals from natural and industrial sources into the environment is harmful to human health (Järup 2003) and degrades freshwater quality in many parts of the world (FernándezLuqueño et al. 2013). Untreated domestic wastewater effluents and the smelting of non-ferrous metals also increase metal pollution (Neminen et al. 2007). Nickel is an essential trace element for plants, both for prokaryotic and eukaryotic microorganisms. As a cofactor of several enzymes, it is involved in diverse physiological processes such as the metabolism of urea, hydrogen cycling, and nitrogen fixation (Küpper and Kroneck 2007). In plants, overaccumulation of nickel is known to cause various toxic effects (Küpper and Kroneck 2007). In this context, nickel chelation and transport via metallothionein and metal
M.G. Maleva et al.
complexes have been identified (Chen et al. 2009). When released in excess, both nickel and nickel compounds are considered typical noxious agents, hazardous to human health (Cempel and Nikel 2006). Phytoremediation with aquatic macrophytes is an innovative, cost-effective technology dedicated to the removal of metallic contamination from water or wastewater (Lone et al. 2008). In particular, such aquatic plants as Lemna sp. (Kara et al. 2003; Demirezen et al. 2007; Mkandawire and Dudel 2007), Eichhornia crassipes (Hadad et al. 2011) and Ceratophyllum demersum (Chorom et al. 2012) have been shown to effectively absorb nickel from the water environment. Diverse associations of macrophytes are usually employed for efficient pollution abatement in artificial ecosystems, i.e., constructed wetlands (CWs) (Bondareva et al. 2010; Combroux et al. 2015; Hadad et al. 2006; Kadlec and Zmarthie 2010; Khan et al. 2009; Vymazal 2009). CWs are planted with rapidly multiplying water weeds such as Elodea sp., Hydrilla sp., Potamogeton sp., Typha sp., Scirpus sp., etc. (Kadlec and Wallace 2009; Vymazal 2013). In particular, species of Elodea, widely used as a bioindicator for the biomonitoring of water quality (Mal et al. 2002; Thiébaut et al. 2010), have frequently been introduced into constructed wetland for the remediation of heavy metal pollution (Bishop and Eighmy 1989; Vymazal 2013). These plants exhibit a high capacity to bioconcentrate trace metals (Malec et al. 2009a, b; Maleva et al. 2009) and are common in water bodies at metalcontaminated sites (Murphy 2002). However, in Elodea, there is scanty of knowledge on the kinetics of toxicant accumulation in tissues or on the mechanisms that regulate tolerance to pollutants. Earlier reports indicated that Elodea canadensis is efficient in the removal of nickel upon short-term exposure (24 h) at concentrations 3.5–30 μM in hydroponic culture media (Kähkönen and Manninen 1998). It has also been demonstrated by Kähkönen and Kairesalo (1998) that nickel accumulation affected the uptake of nitrogen and phosphorus and inhibited the growth of E. canadensis with prolonged exposure. In our previous work (Maleva et al. 2009), several physiological effects of nickel accumulation under pseudo-steadystate conditions (5 days of exposure, up to 50 μM nickel in the medium) were identified. It was shown that increased nickel concentration (up to 50 μM) could induce sublethal oxidative stress in Elodea leaves. In response, plants have developed detoxification mechanisms, including the biosynthesis of non-protein and protein thiol-containing compounds, and have induced two additional polypeptides of 9.5 and 15 kDa (in 18 % SDS-PAGE). Under these conditions, Ni was effectively accumulated and sequestered in plant tissues. Recently, E. canadensis populations have been identified in the middle Ural rivers which have unusually high levels of nickel contamination (Chukina and Borisova 2010). The
prospective use of plants in the phytoextraction of the metal from such water bodies requires information both on the uptake of the toxicant and plants’ response under extreme conditions. To this end, in the present study, we analyze the kinetics of nickel bioaccumulation and the accompanying metabolic and stress-related physiological parameters under shortterm exposure (24 h) at a sublethal concentration of metal (50 μM). We show that Elodea leaves accumulate nickel according to pseudo-second-order kinetics and that protective responses follow a time sequence related to the apparent rate of nickel accumulation; the first 24 h of exposure time to the toxicant are crucial for the activation of specific mechanisms of Ni detoxification and stress protection.
Materials and methods E. canadensis Michx. shoots were cultured under laboratory conditions (25 °C in daylight) in a 5 % Hoagland medium, pH 5.5–6.0 (Hoagland and Arnon 1950). For short-term kinetic experiments, Elodea shoots (4–6 cm) were exposed (1, 4, 8, 12, 24 h) to a sublethal Ni concentration (50 μM, nickel sulfate) in this medium. In order to remove the adsorbed metal on the surface of the leaves, the treated plant material was rinsed with 0.01 % Na–EDTA solution and then gently washed twice with distilled water. All subsequent experimental and analytical techniques used were essentially as described by Maleva et al. (2009). Briefly, after initial extraction with a buffer containing Tris–HCl (0.1 M, pH 8.0), 10 mM MgCl2, and 3 mM Na–EDTA, the plant material was fractionated to isolate the subcellular fractions, including the soluble protein fraction (SP), the membrane-bound protein fraction (MP), the nonprotein soluble fraction (NPSF) and the non-protein polymeric fraction (NPPF) according to Maleva et al. (2009). The Ni content in Elodea tissues and in both non-protein and protein fractions was determined by using an AAS Vario 6 atomic absorption spectrometer (“Analytik Jena,” Germany) according to Ermachenko and Ermachenko (1999). The content of thiol groups was estimated according to Ellman (1959) and initially calculated as micromole of –SH per milligram of protein (soluble or membrane, respectively). The non-protein thiols (NPTs) were estimated according to Nagalakshmi and Prasad (2001) and calculated as micromole of –SH per gram of dry weight (DW). The content of photosynthetic pigments (chl a, chl b, total carotenoids) in Elodea leaves was measured according to Lichtenthaler (1987) and calculated as milligrams of pigments per gram of DW. The net photosynthesis was measured as described by Prasad et al. (2001) and calculated as milligrams of CO2 per gram of DW per hour. Lipid peroxidation was determined by measuring the formation of thiobarbituric acid-reactive substances (TBARS)
Kinetics of Ni bioaccumulation and selected cellular processes
in Elodea leaves, following the colorimetric method of Uchiyama and Mihara (1978), and expressed as 1 μmol of TBARS per gram of DW. Superoxide dismutase (SOD, EC 1.15.1.1) activity was measured according to Paoletti and Macali (1990) as the inhibition of NADH oxidation by mercaptoethanol in the presence of Na–EDTA and Mn at 340 nm and expressed in enzyme units per milligram of protein (one unit of SOD activity was defined as a half-maximal inhibition). Catalase (CAT, EC 1.11.1.6) activity was measured according to Aebi (1971) and expressed as micromole of H2O2 per milligram of protein per minute. Glutathione reductase (GR, EC 1.6.4.2) activity was determined according to Foyer and Halliwell (1976). Enzyme activity was expressed as micromole of NADPH oxidized per milligram of protein per minute. The protein concentration was determined according to Shakterle and Pollack (1973), using bovine serum albumin as a standard. All experiments were repeated three times, with at least three replicates. The data presented in the figures are the mean values±standard error. The statistical significance of results was determined using the non-parametric Mann–Whitney U test, p0.99. The values of the kinetic parameters of Ni bioaccumulation were calculated from the intercept and the slope of linear fits for 12 and 24 h (Table 1). These parameters were used to simulate
M.G. Maleva et al. Table 1 Pseudo-second-order kinetic parameters for the bioaccumulation of Ni(II) by Elodea canadensis calculated on dry weight basis k kqe2 R2 Bioaccumulation qe (mg g−1) (g mg−1 min−1) (mg g−1 min−1) time (h) 24 12
1.267 1.418
7.51E−3 4.54E−3
12.05E−3 9.13E−3
0.99807 0.99441
The Ni concentration is 50 μM in the medium. See text for details
bioaccumulation kinetic curves based on an integrated form of the second-order rate equation (Ho 2004): qt ¼
t 1 t þ 2 kqe qe
As shown in Fig. 1a, the time courses of Ni bioaccumulation simulated using the pseudo-second-order kinetic model of sorption reflected the experimental data. The best agreement of the model simulation with experimental data was observed during the first 8 h of Ni uptake, irrespective of the time range of bioaccumulation data used for the parameter calculations. Compartmentalization of Ni accumulation To identify the cellular compartments responsible for Ni accumulation, the plant material was fractionated and analyzed as described earlier (Maleva et al. 2009). The accumulation of Ni in Elodea leaves correlated with the relative increase in its content in both the SP and MP fractions during 24 h of exposure as measured per 1 mg of total protein in these fractions (Fig. 2a). Most of the Ni bound in the SP and MP fractions (74 and 72 %, respectively) accumulated within the first 8 h after the initial exposure to Ni. During the first hour of exposure, almost 80 % of Ni accumulated predominantly in the non-protein fractions (NPSF, NPPF) (Fig. 2b). In particular, at this initial phase of uptake, the non-protein polymeric (NPPF) fraction absorbed ca. 20 % of Ni, whereas most of the element (ca. 60 %) was in the nonprotein soluble fraction (NPSF). After 1 h of exposure, the contribution of the protein fractions (SP and MP) to Ni binding gradually increased and, after 24 h, reached ca. 40 % of the total Ni bound in the biomass. The contribution of the membrane-bound protein fraction (MP) to Ni binding did not exceed 10 % of the total of bound Ni (Fig. 2b). Metabolic activity To assess how early the effects of nickel bioaccumulation were on metabolic activity in Elodea, the content of the main photosynthetic pigments was analyzed by spectrophotometry and the net photosynthesis rate was measured by the gas
Fig. 2 a Kinetics of total Ni accumulation in soluble and membranebound protein fractions during 24-h exposure to externally applied nickel (50 μM in the medium): SP soluble protein fraction, MP membrane-bound protein fraction. b The relative percentage of the total Ni bound in particular fractions of Elodea canadensis leaves during shortterm exposure. NPPF non-protein polymeric fraction, NPSF non-protein soluble fraction, SP soluble protein fraction, MP membrane-bound protein fraction. The data are expressed as a percentage of the total Ni content in 1 g of DW
exchange technique. As shown in Fig. 3a, the accumulation of chl a, chl b, and the total carotenoid pool increased significantly after the beginning of exposure to Ni. The maximum accumulation of photosynthetic pigments was after about 8 h of exposure. This elevated chlorophyll content started to decline after the 8th hour and that of carotenoid after the 12th hour, which means that chlorophyll concentration decreased faster than the total carotenoid pool. The net photosynthesis rate initially increased and stayed at an elevated level until 8 h of exposure to Ni. The prolonged exposure of leaves to Ni resulted in a significant decrease in photosynthetic activity to the level of ca. 80 % of control values (Fig. 3b). Oxidative stress markers In a previous study (Maleva et al. 2009), it was shown that lengthy exposure to elevated Ni concentrations causes
Kinetics of Ni bioaccumulation and selected cellular processes
Fig. 3 Relative changes in the content of photosynthetic pigments (a) and net photosynthesis rate (b) during 24-h exposure to nickel (50 μM in the medium). The data are mean values±SE of three independent experiments and expressed as a percentage of the respective control values (the initial calculations are presented in “Materials and methods” section). Means within each graph followed by the same letters do not differ statistically according to Mann–Whitney U test, p
ORIGINAL ARTICLE
Kinetics of nickel bioaccumulation and its relevance to selected cellular processes in leaves of Elodea canadensis during short-term exposure Maria G. Maleva 1 & Przemysław Malec 2 & Majeti Narasimha Vara Prasad 1,3 & Kazimierz Strzałka 2
Received: 8 February 2015 / Accepted: 7 May 2015 # Springer-Verlag Wien 2015
Abstract Elodea canadensis is an aquatic macrophyte used widely as a bioindicator for the monitoring of water quality and in the phytoremediation of metal-contaminated waters. This study considers the kinetics of nickel bioaccumulation and changes in accompanying metabolic and stress-related physiological parameters. These include photosynthetic activity, pigment content, the accumulation of thiol-containing compounds, thiobarbituric acid-reactive substance (TBARS) products, and the activity of selected antioxidant enzymes (catalase, glutathione reductase, superoxide dismutase). Elodea leaves accumulated nickel according to pseudosecond-order kinetics, and the protective responses followed a time sequence which was related to the apparent rates of nickel accumulation. The applicability of second-order kinetics to the Ni uptake by Elodea leaves during the first 8 h of exposure to the metal suggested that the passive binding of metal ions (chemisorption) was a rate-limiting step at the initial phase of Ni accumulation. This phase was accompanied by an increase in photosynthetic activity together with elevated photosynthetic pigments and protein synthesis, the enhanced activity of antioxidant enzymes, and increased thiol Handling Editor: Bhumi Nath Tripathi * Kazimierz Strzałka [email protected] 1
Department of Plant Physiology and Biochemistry, Ural Federal University named after the first President of Russia B.N. Yeltsin, Lenin av. 51, Ekaterinburg, Russia 620000
2
Department of Plant Physiology and Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, ul. Gronostajowa 7 30-387, Kraków, Poland
3
Department of Plant Sciences, University of Hyderabad, Hyderabad, Telangana 500 046, India
concentration. In contrast, there was a decrease in metabolic activity upon the accumulation of TBARS, and the decline in enzyme activity was observed in the saturation phase of Ni accumulation (8–24 h). These results show that a correlation exists between the protective response and the apparent kinetic rate of Ni uptake. Thus, the time of exposure to the toxicant is a crucial factor in the activation of specific mechanisms of Ni detoxification and stress alleviation. Keywords Elodea canadensis . Nickel . Bioaccumulation . Kinetic modeling . Phytoremediation . Oxidative stress . Hormesis . Antioxidants
Introduction The contamination of surface waters by trace elements originating from anthropogenic and geogenic processes has become a global problem. Heavy metals, being highly toxic, persistent, and non-biodegradable pollutants, are a serious threat to aquatic ecosystems (Lone et al. 2008). The release of metals from natural and industrial sources into the environment is harmful to human health (Järup 2003) and degrades freshwater quality in many parts of the world (FernándezLuqueño et al. 2013). Untreated domestic wastewater effluents and the smelting of non-ferrous metals also increase metal pollution (Neminen et al. 2007). Nickel is an essential trace element for plants, both for prokaryotic and eukaryotic microorganisms. As a cofactor of several enzymes, it is involved in diverse physiological processes such as the metabolism of urea, hydrogen cycling, and nitrogen fixation (Küpper and Kroneck 2007). In plants, overaccumulation of nickel is known to cause various toxic effects (Küpper and Kroneck 2007). In this context, nickel chelation and transport via metallothionein and metal
M.G. Maleva et al.
complexes have been identified (Chen et al. 2009). When released in excess, both nickel and nickel compounds are considered typical noxious agents, hazardous to human health (Cempel and Nikel 2006). Phytoremediation with aquatic macrophytes is an innovative, cost-effective technology dedicated to the removal of metallic contamination from water or wastewater (Lone et al. 2008). In particular, such aquatic plants as Lemna sp. (Kara et al. 2003; Demirezen et al. 2007; Mkandawire and Dudel 2007), Eichhornia crassipes (Hadad et al. 2011) and Ceratophyllum demersum (Chorom et al. 2012) have been shown to effectively absorb nickel from the water environment. Diverse associations of macrophytes are usually employed for efficient pollution abatement in artificial ecosystems, i.e., constructed wetlands (CWs) (Bondareva et al. 2010; Combroux et al. 2015; Hadad et al. 2006; Kadlec and Zmarthie 2010; Khan et al. 2009; Vymazal 2009). CWs are planted with rapidly multiplying water weeds such as Elodea sp., Hydrilla sp., Potamogeton sp., Typha sp., Scirpus sp., etc. (Kadlec and Wallace 2009; Vymazal 2013). In particular, species of Elodea, widely used as a bioindicator for the biomonitoring of water quality (Mal et al. 2002; Thiébaut et al. 2010), have frequently been introduced into constructed wetland for the remediation of heavy metal pollution (Bishop and Eighmy 1989; Vymazal 2013). These plants exhibit a high capacity to bioconcentrate trace metals (Malec et al. 2009a, b; Maleva et al. 2009) and are common in water bodies at metalcontaminated sites (Murphy 2002). However, in Elodea, there is scanty of knowledge on the kinetics of toxicant accumulation in tissues or on the mechanisms that regulate tolerance to pollutants. Earlier reports indicated that Elodea canadensis is efficient in the removal of nickel upon short-term exposure (24 h) at concentrations 3.5–30 μM in hydroponic culture media (Kähkönen and Manninen 1998). It has also been demonstrated by Kähkönen and Kairesalo (1998) that nickel accumulation affected the uptake of nitrogen and phosphorus and inhibited the growth of E. canadensis with prolonged exposure. In our previous work (Maleva et al. 2009), several physiological effects of nickel accumulation under pseudo-steadystate conditions (5 days of exposure, up to 50 μM nickel in the medium) were identified. It was shown that increased nickel concentration (up to 50 μM) could induce sublethal oxidative stress in Elodea leaves. In response, plants have developed detoxification mechanisms, including the biosynthesis of non-protein and protein thiol-containing compounds, and have induced two additional polypeptides of 9.5 and 15 kDa (in 18 % SDS-PAGE). Under these conditions, Ni was effectively accumulated and sequestered in plant tissues. Recently, E. canadensis populations have been identified in the middle Ural rivers which have unusually high levels of nickel contamination (Chukina and Borisova 2010). The
prospective use of plants in the phytoextraction of the metal from such water bodies requires information both on the uptake of the toxicant and plants’ response under extreme conditions. To this end, in the present study, we analyze the kinetics of nickel bioaccumulation and the accompanying metabolic and stress-related physiological parameters under shortterm exposure (24 h) at a sublethal concentration of metal (50 μM). We show that Elodea leaves accumulate nickel according to pseudo-second-order kinetics and that protective responses follow a time sequence related to the apparent rate of nickel accumulation; the first 24 h of exposure time to the toxicant are crucial for the activation of specific mechanisms of Ni detoxification and stress protection.
Materials and methods E. canadensis Michx. shoots were cultured under laboratory conditions (25 °C in daylight) in a 5 % Hoagland medium, pH 5.5–6.0 (Hoagland and Arnon 1950). For short-term kinetic experiments, Elodea shoots (4–6 cm) were exposed (1, 4, 8, 12, 24 h) to a sublethal Ni concentration (50 μM, nickel sulfate) in this medium. In order to remove the adsorbed metal on the surface of the leaves, the treated plant material was rinsed with 0.01 % Na–EDTA solution and then gently washed twice with distilled water. All subsequent experimental and analytical techniques used were essentially as described by Maleva et al. (2009). Briefly, after initial extraction with a buffer containing Tris–HCl (0.1 M, pH 8.0), 10 mM MgCl2, and 3 mM Na–EDTA, the plant material was fractionated to isolate the subcellular fractions, including the soluble protein fraction (SP), the membrane-bound protein fraction (MP), the nonprotein soluble fraction (NPSF) and the non-protein polymeric fraction (NPPF) according to Maleva et al. (2009). The Ni content in Elodea tissues and in both non-protein and protein fractions was determined by using an AAS Vario 6 atomic absorption spectrometer (“Analytik Jena,” Germany) according to Ermachenko and Ermachenko (1999). The content of thiol groups was estimated according to Ellman (1959) and initially calculated as micromole of –SH per milligram of protein (soluble or membrane, respectively). The non-protein thiols (NPTs) were estimated according to Nagalakshmi and Prasad (2001) and calculated as micromole of –SH per gram of dry weight (DW). The content of photosynthetic pigments (chl a, chl b, total carotenoids) in Elodea leaves was measured according to Lichtenthaler (1987) and calculated as milligrams of pigments per gram of DW. The net photosynthesis was measured as described by Prasad et al. (2001) and calculated as milligrams of CO2 per gram of DW per hour. Lipid peroxidation was determined by measuring the formation of thiobarbituric acid-reactive substances (TBARS)
Kinetics of Ni bioaccumulation and selected cellular processes
in Elodea leaves, following the colorimetric method of Uchiyama and Mihara (1978), and expressed as 1 μmol of TBARS per gram of DW. Superoxide dismutase (SOD, EC 1.15.1.1) activity was measured according to Paoletti and Macali (1990) as the inhibition of NADH oxidation by mercaptoethanol in the presence of Na–EDTA and Mn at 340 nm and expressed in enzyme units per milligram of protein (one unit of SOD activity was defined as a half-maximal inhibition). Catalase (CAT, EC 1.11.1.6) activity was measured according to Aebi (1971) and expressed as micromole of H2O2 per milligram of protein per minute. Glutathione reductase (GR, EC 1.6.4.2) activity was determined according to Foyer and Halliwell (1976). Enzyme activity was expressed as micromole of NADPH oxidized per milligram of protein per minute. The protein concentration was determined according to Shakterle and Pollack (1973), using bovine serum albumin as a standard. All experiments were repeated three times, with at least three replicates. The data presented in the figures are the mean values±standard error. The statistical significance of results was determined using the non-parametric Mann–Whitney U test, p0.99. The values of the kinetic parameters of Ni bioaccumulation were calculated from the intercept and the slope of linear fits for 12 and 24 h (Table 1). These parameters were used to simulate
M.G. Maleva et al. Table 1 Pseudo-second-order kinetic parameters for the bioaccumulation of Ni(II) by Elodea canadensis calculated on dry weight basis k kqe2 R2 Bioaccumulation qe (mg g−1) (g mg−1 min−1) (mg g−1 min−1) time (h) 24 12
1.267 1.418
7.51E−3 4.54E−3
12.05E−3 9.13E−3
0.99807 0.99441
The Ni concentration is 50 μM in the medium. See text for details
bioaccumulation kinetic curves based on an integrated form of the second-order rate equation (Ho 2004): qt ¼
t 1 t þ 2 kqe qe
As shown in Fig. 1a, the time courses of Ni bioaccumulation simulated using the pseudo-second-order kinetic model of sorption reflected the experimental data. The best agreement of the model simulation with experimental data was observed during the first 8 h of Ni uptake, irrespective of the time range of bioaccumulation data used for the parameter calculations. Compartmentalization of Ni accumulation To identify the cellular compartments responsible for Ni accumulation, the plant material was fractionated and analyzed as described earlier (Maleva et al. 2009). The accumulation of Ni in Elodea leaves correlated with the relative increase in its content in both the SP and MP fractions during 24 h of exposure as measured per 1 mg of total protein in these fractions (Fig. 2a). Most of the Ni bound in the SP and MP fractions (74 and 72 %, respectively) accumulated within the first 8 h after the initial exposure to Ni. During the first hour of exposure, almost 80 % of Ni accumulated predominantly in the non-protein fractions (NPSF, NPPF) (Fig. 2b). In particular, at this initial phase of uptake, the non-protein polymeric (NPPF) fraction absorbed ca. 20 % of Ni, whereas most of the element (ca. 60 %) was in the nonprotein soluble fraction (NPSF). After 1 h of exposure, the contribution of the protein fractions (SP and MP) to Ni binding gradually increased and, after 24 h, reached ca. 40 % of the total Ni bound in the biomass. The contribution of the membrane-bound protein fraction (MP) to Ni binding did not exceed 10 % of the total of bound Ni (Fig. 2b). Metabolic activity To assess how early the effects of nickel bioaccumulation were on metabolic activity in Elodea, the content of the main photosynthetic pigments was analyzed by spectrophotometry and the net photosynthesis rate was measured by the gas
Fig. 2 a Kinetics of total Ni accumulation in soluble and membranebound protein fractions during 24-h exposure to externally applied nickel (50 μM in the medium): SP soluble protein fraction, MP membrane-bound protein fraction. b The relative percentage of the total Ni bound in particular fractions of Elodea canadensis leaves during shortterm exposure. NPPF non-protein polymeric fraction, NPSF non-protein soluble fraction, SP soluble protein fraction, MP membrane-bound protein fraction. The data are expressed as a percentage of the total Ni content in 1 g of DW
exchange technique. As shown in Fig. 3a, the accumulation of chl a, chl b, and the total carotenoid pool increased significantly after the beginning of exposure to Ni. The maximum accumulation of photosynthetic pigments was after about 8 h of exposure. This elevated chlorophyll content started to decline after the 8th hour and that of carotenoid after the 12th hour, which means that chlorophyll concentration decreased faster than the total carotenoid pool. The net photosynthesis rate initially increased and stayed at an elevated level until 8 h of exposure to Ni. The prolonged exposure of leaves to Ni resulted in a significant decrease in photosynthetic activity to the level of ca. 80 % of control values (Fig. 3b). Oxidative stress markers In a previous study (Maleva et al. 2009), it was shown that lengthy exposure to elevated Ni concentrations causes
Kinetics of Ni bioaccumulation and selected cellular processes
Fig. 3 Relative changes in the content of photosynthetic pigments (a) and net photosynthesis rate (b) during 24-h exposure to nickel (50 μM in the medium). The data are mean values±SE of three independent experiments and expressed as a percentage of the respective control values (the initial calculations are presented in “Materials and methods” section). Means within each graph followed by the same letters do not differ statistically according to Mann–Whitney U test, p
