Plants for waste water treatment – Effects of heavy metals on the detoxification system of Typha latifolia

Bioresource Technology 102 (2011) 996–1004

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Plants for waste water treatment – Effects of heavy metals on the detoxification system of Typha latifolia Lyudmila Lyubenova, Peter Schröder ⇑ Helmholtz Zentrum München German Research Center for Environmental Health, Department Microbe-Plant Interactions, Ingolstädter Landstr. 1, D-85764 Neuherberg, Germany

a r t i c l e

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Article history: Received 6 May 2010 Received in revised form 16 September 2010 Accepted 16 September 2010 Available online 15 October 2010 Keywords: Arsenic Cadmium Organic pollutants Lead Typha latifolia

a b s t r a c t Upon treatment with Cd and As cattail (Typha latifolia) showed induced catalase, monodehydroascorbate reductase and ascorbate peroxidase activities in leaves but strong inhibition in rhizomes. Peroxidase activity in leaves of the same plants was inhibited whereas linear increase was detected after Cd treatment in rhizomes. Glutathione S-transferase measurements resulted in identical effects of the trace elements on the substrates CDNB, DCNB, NBC, NBoC, fluorodifen. When GST was assayed with the model substrate DCNB, a different pattern of activity was observed, with strongly increasing activities at increasing HM concentrations. Consequently, to improve the success rates, future phytoremediation plans need to preselect plant species with high antioxidative enzyme activities and an alert GST pattern capable of detoxifying an array of organic xenobiotics. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Heavy metals are ubiquitous in the environment and have long biological lifetime. Their presence in a given ecosystem can lead to accumulation in the food chain with negative effects for human health. Even very low heavy metal concentrations can provoke alterations in cellular processes and structures in plants (OrtegaVillasante et al., 2007). It is well documented that higher metal concentrations cause the generation of ROS (reactive oxygen species). Oxygen is not only essential for the energy metabolism and respiration but can also be involved in degenerative workflows (Aravind and Prasad, 2005). Here, singlet oxygen, super oxide, and hydroxyl radical (HO), hydrogen peroxide (H2O2), hydrogen radical, and hydrogen ion may play crucial roles. These compounds are generated during normal metabolism as a result of redox reaction with O2 and H2O (Mittler, 2002; Foyer and Noctor, 2003). If ROS support is not controlled oxidative chain reactions will start (Ortega-Villasante et al., 2007). Although oxidation can be withstood to a certain extent by proteins, nucleic acids, lipids (Buchanan et al., 2000), plants usually respond to this stress by increasing activities of antioxidant enzymes. The antioxidant defence system includes enzymes like superoxide dismutase, catalase, ascorbate peroxidase, glutathione peroxidase, and glutathione S-transferases.

⇑ Corresponding author. E-mail address: [email protected] (P. Schröder). 0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.09.072

The catalytic properties of superoxide dismutase (SOD) were first detected by McCord and Fridovich (1969). Since then SOD is known to catalyze the dismutation from superoxide to hydrogen peroxide and oxygen. SOD is found in all aerobic organisms, where it generates activated oxygen; it is supposed to play a central role in defence mechanisms related to oxidative stress (Mittler, 2002). Catalase (CAT) will catalyze the dismutation of hydrogen peroxide. Another enzyme class responsible for the degradation of hydrogen peroxide are the peroxidases, enzymes capable of reducing H2O2 to water. A different group of peroxidizing enzymes to eliminate H2O2 are the glutathione peroxidases (GPOX), which directly convert reduced glutathione (GSH) to oxidised glutathione (GSSG) (Noctor and Foyer, 1998). Besides the antioxidative enzymes, plants possess active biomolecules for defence like ascorbate and glutathione. They are present in millimolar concentrations in plant cells and play also important defence roles in cellular metabolism (Ortega-Villasante et al., 2007). L-ascorbic acid (vitamin C) reduces significantly hazard developed by free radicals. It binds superoxide, hydrogen peroxide or tocopherol radicals and generates monodehydroascorbic acid (MDHA) or dehydroascorbic acid (DHA). Ascorbic acid is recycled using NAD(P)H+ and GSH as electron sources within the ascorbate– glutathione cycle or Halliwell–Asada cycle. Glutathione (GSH), y-L-glutamyl-L-cysteinyl-glycine, is a tripeptide ubiquitously found in plants, with numerous functions in cytosol and chloroplasts. GSH

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may act as an antioxidant, reducing the availability of free radicals. On the other side it is a cofactor of DHA, converting ascorbic acid from its reduced to its oxidised form (Loewus, 1988). Glutathione is frequently involved in cell metabolism, for example as transport form of reduced sulphur from leaves to roots. Furthermore it acts as co-substrate for glutathione S-transferases during detoxification of xenobiotics. Glutathione S-transferases (GST) have been detected for the first time in plants in the reaction of conjugation between atrazine and GSH in maize (Frear and Swanson, 1970). This process of substitution leads to a cleavage of electrophilic groups from a xenobiotic and results in detoxification sensu stricto. Plant GSTs are dimeric proteins found in cytosol as well as in membranes, with subunit sizes varying between 23 and 30 kDa (Schröder, 2001). Each subunit possesses two domains for binding of glutathione and electrophilic substrates. The present study aimed at quantifying the effects of sublethal concentrations of heavy metals on a typical plant used in phytoremediation, Typha latifolia. As waste water treatment plants usually exhibit intermittent high concentrations of heavy metals, the detoxification capacity of T. latifolia treated with heavy metals during 72 h was investigated. Furthermore, the defence mechanisms in cattail were explored with respect to enzymatic steps providing efficient tolerance against organic xenobiotics. 2. Methods 2.1. Plant material Typha latifolia plants were obtained from a local provider and grown in 5 l plastic pots in a greenhouse at 14 h daylength, 10 h night, at an average humidity of 50%. 2.2. Heavy metal application After an undisturbed growing period of 72 days the plants were treated with three different heavy metals at four different concentrations for 72 h. The heavy metals applied were: cadmium (as cadmium sulphate), arsenic (as sodium arsenate) and lead (as lead chloride), in defined concentrations. Heavy metals were administered by flooding the plant containing pots with contaminated water. Control plants were grown under same conditions, flooded to the same regimes with tap water, but not treated with heavy metals. Three plant replicates were used for each treatment. 2.3. Pigment analysis Ten millilitre cold 80% acetone were added to 0.5 g freshly ground leaf material. The mixture was centrifuged 20 min by 39,250g at 4 °C. The supernatant was collected in Falcon tubes. The pellet was extracted again with 10 ml 80% acetone, stirred, and centrifuged by 39,250g at 4 °C for 20 min. The supernatant was added to the first extract in the Falcon tube. This procedure was repeated once more. One millilitre of the extracted chlorophylls was withdrawn and pigments were determined spectrophotometrically with four repetitions at the following wavelengths: 663.2, 646.8 and 470 nm, according to Lichtenthaler (1987). Chlorophyll a and b contents were determined following the formula given for 80% acetone extraction and are expressed in lg/g fresh weight.

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(0.1 M Tris/HCl pH 7.8 5 mM EDTA, 5 mM dithioerythritol DTE, 1% Nonidet P40, 1% insoluble polyvinylpyrrolidone PVP K90). This mixture was homogenised and extracted for 30 min prior to centrifugation at 20,000 rpm. The proteins in the resulting crude extract were precipitated by addition of ammonium sulphate in two steps – 40% and 80% of saturation. The protein solution was centrifuged after each step and the pellet finally was resuspended in 2.5 ml of 25 mM Tris/HCl buffer pH 7.8. This step was followed by desalting on Sephadex PD-10 desalting columns (Pharmacia, Freiburg, Germany). 2.5. Enzyme assays All enzyme assays in this paper were performed in a Spectra max Plus 384 photometer (Molecular Devices) using 96 well plates, at 25 °C. All assays were done at least in triplicate. All enzyme activities are expressed in lkat/mg protein. 2.5.1. CAT assay Catalase was determined using a 96 well Spectramax photometer where each well contained in 150 ll total volume: 100 ll 100 mM KH2PO4/K2HPO4 buffer pH 7.0; 40 ll 200 mM H2O2 and 10 ll protein solution. Activity measurements were performed at 240 nm using an extinction coefficient of 0.036 mM 1cm 1. The method follows Verma and Dubey (2003). 2.5.2. POX assay Peroxidase was assayed according to the method of Drotar et al. (1985) using guaiacol (2-metoxy-phenol) as a substrate. The total volume for peroxidase measurements was 200 ll, where to 190 ll of the buffer/guaiacol/H2O2 solution 10 ll of extracted protein were added. The buffer/GSSG/NADPH solution consisted of 27.3 ml Tris/HCl 50 mM pH 6.0 buffer, 600 ll 3.4 mM guaiacol, 600 ll 9 mM H2O2. The extinction coefficient of guaiacol is 26.6 mM 1cm 1 at 420 nm (Vanacker et al., 1998). 2.5.3. APOX assay Ascorbate peroxidase was assayed according to Vanacker et al. (1998). To 36 ml of 55.56 mM KH2PO4/K2HPO4 buffer pH 7.0 were added 10 ll ascorbate solution (60 mM) and 41 ll 3% H2O2 dilution. One hundred eighty microlitre of this mixture were pipetted into each well and 20 ll protein was added to initiate the reaction. The photometer was set to 25 °C and 290 nm, and APOX activity was determined using an extinction coefficient of 2.8 mM 1cm 1. 2.5.4. MDHAR assay The monodehydroascorbate reductase was assayed according to the protocol of Vanacker et al. (1998). The total volume for the monodehydroascorbate reductase measurement was 200 ll, where to 180 ll of the Hepes/KOH buffer/NADPH/ascorbate solution 20 ll of extracted protein were added. The buffer/NADPH/ ascorbate solution consisted of 30 ml Hepes/KOH 111 mM pH 7.6 buffer, 100 ll 25 lM NADPH, 1000 ll 2.5 mM ascorbate. The extinction coefficient of NADPH is 6.2 mM 1cm 1 at 340 nm (Vanacker et al., 1998). 2.5.5. GPOX assay The glutathione peroxidase was appointed with 50 ll 50 mM phosphate–EDTA buffer pH 7.0; 10 ll from each: 2.5 mM NADPH, 9 mM H2O2, 100 mM GSH, GR and 10 ll protein extract. The determination took 5 min by 340 nm (NADPH, e340 nm = 6.22).

2.4. Protein extraction The protein extraction followed a procedure by Schröder et al. (2002). In short, frozen plant material was pulverized and to max 3 g powder were added 30 ml of freshly prepared extraction buffer

2.5.6. GST assay Spectrophotometer assays were carried out following the method of Schröder et al. (2002, 2008). For determination of GST the model substrates dichloronitrobenzene (DCNB, e345nm = 8.5

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mM 1cm 1), chlorodinitrobenzene (CDNB, e340nm = 9.6 mM 1 cm 1), p-nitrobenzylchloride (pNBC, e310nm = 1.8 mM 1cm 1), nitrobenzoychloride (NBoC, e310nm = 1.9 mM 1cm 1) and the diphenylether herbicide, fluorodifen (e405nm = 3.1 mM 1cm 1) and 0.1 M Tris/HCl pH 7.5 and 0.1 M Tris/HCl pH 6.4 buffers were used. In all above mentioned assays, the concentration of reduced glutathione (GSH) as well as of the model substrates was 1 mM. 2.6. Protein content The protein content was evaluated according to the method described by Bradford (1976) using serum albumin as standard.

3. Results and discussion The fact that T. latifolia frequently grows near industry areas and in wetlands contaminated with cadmium, lead, zinc, copper and nickel soils indicates that this plant might be resistant against heavy metals (Ye et al., 1997). The species has even been reported in the waste water of lead–zinc mine tailings (Lan et al., 1992). It was shown that T. latifolia plants reduced both, lead and zinc concentration of the waste water by 80% (Lan et al., 1992), and that specifically cattail rhizomes show significant uptake of Pb and Zn. Corresponding to these observations, all treatments with three heavy metals (Cd, As and Pb) in four different concentrations resulted in metal uptake, and concentrations in the rhizomes higher than in the leaves of T. latifolia. Several authors (Vassilev et al., 2004; Wu and Zhang, 2002; Hegedüs et al., 2001) refer to the same effect in cadmium treated barley. Benavides et al. (2005) and Dixit et al. (2001) found correlative effects in pea, Gallego et al. (1996) in sunflowers, Mishra et al. (2006) in Bacopa monnieri. However, Garbisu and Alkorta (2001) point out that multiple pollution may be one of the major problems for phytotechnologies. 3.1. Pigment analysis The three heavy metals caused concentration depended effects of the chlorophyll concentrations in T. latifolia leaves. After the cadmium as well as after the arsenic treatment with 10 and 50 lM chlorophyll a and b concentrations measured are significantly increased above controls. Treatment with 100 lM As or Cd led to decrease of both chlorophylls to levels below controls, whereas 250 lM As or Cd increased chlorophylls to highest levels (Table 1). After the lead treatment the same effects were noticed. In this case the lowest chlorophyll a concentration (16.29 lg/g FW) was detected after treatment with 50 lM lead.

Table 1 Pigment analysis in Typha leaves. Pigments were determined spectrophotometrically with four repetitions at the following wavelengths: 663.2, 646.8 and 470 nm, according to Lichtenthaler (1987). Pigment contents were expressed in lg/g fresh weight.

Control 10 lM Cd 50 lM Cd 100 lM Cd 250 lM Cd 10 lM As 50 lM As 100 lM As 250 lM As 10 lM Pb 50 lM Pb 250 lM Pb

Chlorophyll a

Chlorophyll b

7.31 ± 0.9 11.10 ± 5.6 21.31 ± 4.5 5.70 ± 1.1 19.53 ± 3.9 19.66 ± 1.6 21.07 ± 4.1 4.62 ± 3.2 24.41 ± 8.1 21.34 ± 6.8 16.29 ± 7.8 21.10 ± 2.0

6.47 ± 1.0 7.9 ± 3.8 13.25 ± 3.3 5.82 ± 0.6 11.30 ± 2.1 11.54 ± 6.2 11.22 ± 1.8 6.06 ± 0.9 18.50 ± 4.9 13.13 ± 4.7 9.69 ± 4.1 12.96 ± 1.8

The described decrease of the chlorophyll content after 100 lM Cd and As treatments was also observed for the carotenoid concentrations in the treated samples (data not shown). However, Hegedüs et al. (2001) observe visible symptoms and chlorophyll decrease in plants up to 60% after four days exposure to 0.3–1 mM of cadmium. The chlorophyll reduction is a marker for oxidative stress (Clijsters et al., 1999; Sanita di Toppi et al., 1998) induced by HM (Hegedüs et al., 2001). After 72 h treatment we detected significant differences between the chlorophyll contents, but rather than at highest cadmium or arsenic concentrations (250 lM) the strongest pigment degradation occurred at the levels of 100 lM. Increases instead of expected decreases in photosynthetic pigments had previously been reported for the impact of sublethal concentrations of volatile chlorocarbons in various plants (Debus and Schröder, 2000). 3.2. Enzyme activities Changes in the activity of enzymes of the antioxidative detoxification pathways were in the main focus of the present investigation. When assaying them, both, significant increases as well as decreases were observed in the treated plants, depending on the respective heavy metal and its concentration (Figs. 1 and 2). After 72 h of incubation, even the lowest concentrations of HM used caused significant alterations, in both, rhizomes and shoots. In order to classify the enzyme responses with respect to the heavy metals used, effects of single metals were investigated and are depicted as percentage of controls. 3.2.1. CAT and SOD activities Catalase and SOD are generally understood as the first line of defence in stress response towards oxidative stress and increasing reactive oxygen species in plants. Cadmium causes formation of reactive oxygen species (ROS) in chloroplasts and peroxisomes (Romero-Puertas et al., 2002). The enzyme superoxide dismutase, located in the chloroplasts, counteracts this effect and degrades O2 to H2O2 and oxygen (Mittler, 2002). Also in the present investigation SOD is enhanced, obviously to remove ROS. Catalase is responsible for the degradation of the accumulating hydrogen peroxide (Dixit et al., 2001). The increase of catalase activity in T. latifolia after exposure to heavy metals indicates the functioning of the degradation of hydrogen peroxide in response to HM. Corresponding to this hypothesis, Catalase activity in all heavy metal treated T. latifolia leaves was higher than in the controls. Already at 50 lM Cd CAT was fivefold induced. At 100 and 250 lM Cd the increase of catalase activity was four times higher than in controls (Figs. 1H and 2H). In the T. latifolia rhizomes treated with cadmium the activity of catalase was inhibited to 60% of the activities determined in controls. Only in the case of 100 lM Cd the activities equalled the control level. The treatments with arsenate influenced the catalase activity in the leaves of T. latifolia in a similar way. Already 10 lM arsenate leads to 1.5 times increase of CAT in the leaves. This trend was not persistent for the 50 lM As concentration, where the activity of catalase was reduced to 67% of controls. After treatment with 100 or 250 lM arsenate a new increase of CAT activity to 550% of controls was observed. Catalase activity in the rhizomes of the cattail plants was inhibited at 10 lM, recovered to 50% and 100% of controls at 50 and 100 lM and dropped again to 20 of controls after treatment with 250 lM. Different to cadmium, lead caused increases in CAT activity from 10 lM onwards in leaves. At concentrations of 50 lM catalase was increased 2.5 times, and at 250 lM CAT was seven times higher than controls. The rhizomes of the lead treated cattail samples

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A

POX activity (µkat/mg protein)

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treatments - rhizomes

treatments - rhizomes

Fig. 1. Antioxidative enzymes in Typha rhizomes in response to heavy metal treatment. After an undisturbed growing period of 72 days the plants were treated with three different heavy metals at four different concentrations for 72 h. The heavy metals applied were: cadmium (as cadmium sulphate), arsenic (as sodium arsenate) and lead (as lead chloride), in defined concentrations in tap water. Control plants were grown under same conditions but not treated. Data are means of three replicates ± SD.

did not show an increase of the catalase activity below 50 lM. Instead the enzyme activity of the 250 lM lead treated samples was drastically reduced to 20% of the activity of the rhizome control. In leaves, catalase activity develops a concentration depended increase, probably resulting from the formation of hydrogen peroxide. In rhizomes, the catalase activity declines above concentrations of 50 lM. Increases in catalase activity are correlated with the heavy metal concentration found in the leaves, but strikingly, the rhizomes of the same plants do not exhibit increased catalase activity. Hegedüs et al. (2001) were also unable to show Cd-induced catalase activity in barley and suppose that cadmium cannot influence

the catalase of the peroxisomes. In contrast, Mishra et al. (2006) report a decrease of the catalase activities in leaves and rhizomes. According to Cakmak and Marschner (1988) catalase is very sensitive against O2 -radicals. 3.2.2. MDHAR, DHAR and APOX activities Upon treatment with heavy metals, the ascorbate dependent enzymes MDHAR, DHAR and APOX in rhizomes were generally found to be inhibited with increasing concentrations (Fig. 3), except MDHAR after lead treatment at 100 lM, where increases of 250% above controls were found, before activity dropped to control levels at 250 lM. Increased enzyme activities around 30% above

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3.0

2.0

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POX activity (µkat/mg protein)

MDHAR activity (µkat/mg protein)

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Cd As Pb

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80 60 40 20

H 300 250 200 150 100

0

50 0

control 10µM

50µM 100µM 250µM

treatments - leaves

control 10µM

50µM 100µM 250µM

treatments - leaves

Fig. 2. Antioxidative enzymes in Typha leaves in response to heavy metal treatment. After an undisturbed growing period of 72 days the plants were treated with three different heavy metals at four different concentrations for 72 h. The heavy metals applied were: cadmium (as cadmium sulphate), arsenic (as sodium arsenate) and lead (as lead chloride), in defined concentrations in tap water. Control plants were grown under same conditions but not treated. Data are means of three replicates ± SD.

controls, as recorded at 10 lM CdSO4 for MDHAR and DHAR or at 50 lM arsenate for DHAR were found to be insignificant. MDHAR activity was strongly inhibited in the T. latifolia rhizomes treated with arsenic. Aravind and Prasad (2005) show that 10 lM cadmium leads to drastic reduction of MDHAR activity. Whereas lead inhibited DHAR and APOX completely, arsenate and cadmium were found to have a lesser inhibitory power at higher HM concentrations (Fig. 3A–C). In the leaves, the reaction of the mentioned enzymes is totally different (Fig. 3A–C). MDHAR is dramatically increased with rising

lead and Cd concentrations, but dropping at highest values. In contrast, this enzyme activity is induced sixfold at 250 lM arsenate. Lead is inhibitory to DHAR and APOX, whereas these enzymes are transiently increased fourfold by 10 and 50 lM arsenate and doubled by Cd. Whereas DHAR returns to control levels, MDHAR and APOX increase by 650% at highest arsenate levels. Ascorbate peroxidase cleaves hydrogen peroxide under consumption of ascorbate. Despite the fact that ascorbate peroxidase activity is generally higher in leaves than in rhizomes, this enzyme is inhibited in all samples except treatment with arsenate where APOX

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800

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POX-L GPOX-L GR-L

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rhizomes POX-R GPOX-R GR-R

MDHAR-R DHAR-R APOX-R

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Fig. 3. The effect of heavy metals on the antioxidative enzymes in Typha rhizomes and leaves. (A) cadmium, (B) arsenate, (C) lead. All enzyme activities are depicted relative to untreated controls.

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300

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POX-L GPOX-L GR-L

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Fig. 3 (continued)

activity increases to 400% and 600% of controls, respectively. These results conform to the results of Hegedüs et al. (2001), who describe higher ascorbate peroxidase activity after HM treatment. 3.2.3. POX, GPOX and GR activities The influence of HM on POX, GPOX and GR is generally smaller than on the ascorbate dependent enzymes. In fact, all activities were inhibited to around 50% after lead treatment in rhizomes, and arsenate and cadmium caused small changes in GPOX and GR. Only POX was increased strongly in a concentration dependent manner after Cd and As treatments in rhizomes. Whereas APOX activity of the rhizomes was inhibited in most samples, peroxidase activity increased. We detected six times higher POX activity in rhizomes than in leaves (cf. Hegedüs et al., 2001). In rhizomes the highest POX activity was detected after treatment with 250 lM arsenic. Mocquot et al. (1996) suppose that peroxidases can be used as biomarker for sublethal metal toxicity. The high peroxidase activities can be correlated with results of Blinda et al. (1996) on barley roots. The authors presume that elevated POX activity is a result of specific gene expression of roots (Blinda et al., 1996). But according to Hegedüs et al. (2001) the cytosolic peroxidase is available in different isoforms in leaves and in roots, which is also likely in the present study and makes interpretation complicated. In the leaves, POX and GR had activities around controls after lead and arsenate incubation, as in the case of Cd. Strong and concentration dependent increases between four- and tenfold above controls were recorded exclusively for GPOX activities (Fig. 3). Glutathione peroxidase activities were inhibited in all rhizome samples, with strongest effects in As and lead; but they showed strong induction in leaves. The opposite effects had previously been detected in the case of copper treated pea (Dixit et al., 2001).

Although a careful interpretation of the given enzyme activities indicates that the chosen HM initiate different oxidative stress and toxicity reactions, it is not possible to consistently identify key enzymes within the Halliwell–Asada cascade that would perform most or least of the detoxification. Moreover, the differences in enzyme activities between rhizomes and shoots indicate that not only strong differences exist in the expression patterns, but also that some kind of signalling might be needed that is independent of the heavy metals used. 3.2.4. GST activity Under field conditions heavy metal contamination is often found together with organic contaminants. Many of these substances, in particular those with halogen substitutions, are taken up by plants and detoxified by Glutathione S-transferases. It is often referred to that the activity of these enzymes is induced by heavy metals. Hence, it was expected to find induced GST activities in metal treated Typha also in the present study. GST activity of T. latifolia treated with different heavy metals was detected for five different model substrates. Four of them were chlorobenzenes with closely related structures that would undergo a halogen substitution reaction and attachment of glutathione to the benzyl moiety, and one of them, the herbicide fluorodifen, would be cleaved by GST and the resulting trifluoromethyl-nitrophenyl-moiety would be conjugated with glutathione. Despite this difference in reaction mechanism, GST activity for fluorodifen was found to react in the same way as GST for CDNB, NBC and NBoC (Fig. 4). A large difference in the development of GST activity under heavy metal stress was found only for the conjugation of DCNB, which is generally considered a minor GST activity. As GSTs consist of a superfamily of isoforms, the differences in enzyme activities found here point to significant variations or similarities in the isoenzyme

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GST-Leaf GST-rhizome

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PbCl2 concentration [µM] Fig. 4. The effect of heavy metals on glutathione S-transferase activities in Typha. Relative GST activities for four of the tested substrates, namely, CDNB, NBC, NBoC and fluorodifen have been plotted together. Data are means of three replicates ± SD for each of the substrates, joined into a curve for leaf activity (dashed line, squares) and compared with rhizome activities (solid line, circles). (A) Effect of cadmium on enzyme activity, (B) effect of arsenate on enzyme activity, (C) effect of lead on enzyme activity.

pattern in rhizomes and leaves, under the influence of increasing concentrations of different heavy metals. Usually, the model xenobiotic CDNB is used as standard substrate for GST (Schröder, 2001), but we chose additionally NBC, NBoC, the herbicide, fluorodifen, and DCNB as additional substrates for our assays, hypothesizing that these compounds would help to characterize the superenzyme family of GST. To our surprise, activities of four of the tested substrates, namely, CDNB, NBC, NBoC and fluorodifen, exhibited the same tendencies in heavy metal treated plant parts, albeit at different absolute conjugation rates. Upon all treatments, the multitude of GST isoforms tested with the four mentioned substrates in rhizomes was induced slightly at low HM concentrations and strongly induced

by 250 lM HM, whereas 100 lM did not cause significant changes in GST activity. The induction at 250 lM lead was fivefold and the strongest activation measured for these substrates. Contrary to this, all leaf GST activities were strongly inhibited by Cd (50%), As (70%) and Pb (100%). A similar effect has been described in a previous investigation on direct in vivo inhibition of GST by Cd (Lyubenova et al., 2007). Results of DCNB assays are in strong contrast to these observations. Albeit DCNB is only a weak substrate for GSTs (Schröder et al., 2002), its activity was above controls in every treatment and plant part tested, and it showed induction factors of two- to tenfold above controls, again with the exception of 100 lM treatments, where activities equalled those of controls. According to Schröder et al. (2002) there is significant GST induction in Picea abies after treatment with Cd and Pb. Some isoforms of the plant GSTs were induced, but the signalling was not defined (Schröder et al., 2002). A GST induction after cadmium stress is mentioned also for pea (Dixit et al., 2001) and wheat (Mauch and Dudler, 1993). It is supposed that GST activity can be an additional answer of the cell on the oxidative stress. Aravind and Prasad (2005) refer about increasing GST activity in Ceratophyllum demersum plants at very low cadmium concentrations. This limited role of GSTs in cadmium treated plants could be developed from the peroxidation of the GST proteins or the deactivation of the GST genes (Aravind and Prasad, 2005). In the rhizomes, GST for CDNB, NBC, NBOC, and fluorodifen showed virtually the same increases or decreases upon exposure to CdSO4 and Na2HAsO4. After a transient increase to 150% of controls at low concentrations, GST activities ceased, but increased again to values twice as high as controls at 250 lM concentrations. After exposure to lead, GST activities rose steadily to fivefold above control. In the leaves, the same GST activities were strongly inhibited, and even found to be nonexistent in the lead treated plants. Of course the lack of induction or even the inhibition of distinct GST isoenzymes has consequences for the survival of the plant in any given constructed wetland under real conditions of contamination. DCNB-GST, as has already been mentioned, reacts in a different manner upon HM. After the initial transient increase at low HM concentrations, it increases to eightfold above controls in the rhizomes, and to up to fivefold control levels in the leaves, irrespective of the heavy metal used. The transient increase in leaf GST is fourfold and hence much higher than in rhizomes (Fig. 5).

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DCNB GST activity [% of controls] - leaf

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HM concentrations [µM] Fig. 5. Conjugation of dichloronitrobenzene (DCNB) by Typha glutathione S-transferase. Relative GST activities for DCNB in response to all investigated heavy metals have been plotted together. Data are means of three replicates ± SD, joined into a curve for leaf activity (dashed line, squares) and compared with rhizome activities (solid line, circles).

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