OPCE-STR.vp Acta Bot. Croat. 69 (1), 107–121, 2010 CODEN: ABCRA 25 ISSN 0365–0588 Role of antioxidant enzyme responses and phytochelatins in tolerance strategies of Alhagi camelorum Fisch growing on copper mine MASOUD M. A. BOOJAR*, ZAHRA TAVAKOLI Department of Biology, University of Tarbiat Moalem, 49 Mofateh Avenue, Tehran, P.O. Box: 15614, Iran. This study was performed to clarify some aspects of tolerance mechanisms against excess copper (Cu) in Alhagi camelorum Fisch, a dominant wild type plant growing in a Cu-con- taminated zone and its vicinity. Total and available copper was at toxic levels for plants growing on the contaminated soil. There were no visual and conspicuous symptoms of Cu toxicity in this plant species. Most of the excess Cu in soil was transferred to and accumu- lated in plant leaves in which the storage rate in vacuoles and chloroplasts was 48% and 7% respectively. There was an insignificant decrease in chlorophyll content and a signifi- cant increase in tissues phytochelatins and antioxidant enzyme activities in plants col- lected from the contaminated zone as compared to plants of the same species growing on uncontaminated soil. We also observed significant elevation in oxidative damage bio- markers, malondialdehyde and dityrosine, when the aerial parts of Alhagi camelorum were compared with the same parts of the plant collected from an uncontaminated zone. Alhagi camelorum elevated its antioxidative enzyme activities, phytochelatins and accu- mulated the excess of Cu in leaf vacuoles in response to Cu-toxicity as tolerance strategy. Key words: Copper, metal, toxicity, antioxidant, phytochelatin, tolerance Abbreviations: CAT – catalase, DTPA – Diethylene triaminepenta acetic acid, EC – elec- tric conductivity, GPX – glutathione peroxidase, GSH – glutathione, HEPPS – (3-[4- (2-Hydroxyethyl)-1-piperazinyl] propanesulfonic acid), MDA – malondialdehyde, MSA – methanesulfonic acid, NBT – nitroblue tetrazolium, PC – phytochelatin, ROS – reactive oxygen species, SOD – superoxide dismutase, TBA – thiobarbituric acid, TFA – trifluoro acetic acid Introduction Copper is an essential micronutrient for plants and a component of several enzymes mainly participating in electron flow, catalyzing the redox reactions in mitochondria and chloroplast. In addition, this element is also a cofactor or a part of a prosthetic group of many key enzymes that play important roles in photosynthesis, respiration, CO2 assimila- tion, ATP synthesis and nitrogen metabolism (DEMIREVSKA-KEPOVA et al. 2004). The ab- ACTA BOT. CROAT. 69 (1), 2010 107 * Corresponding author, e-mail: aboojar@yahoo.com U:\ACTA BOTANICA\Acta-Botan 1-10\Boojar.vp 9. travanj 2010 13:21:57 Color profile: Disabled Composite 150 lpi at 45 degrees sorption of copper from soil by plants and its transport to the above-ground parts depend on the ability of plants to transfer this metal across the soil-root interface and to the total amount of Cu, present in the soil (AGATA and ERNEST 1998, BAKER and PROCTOR 1990). On the other hand, copper is a widespread contaminant originating from different human activ- ities including mining and smelting of copper ores. Mining activities generate a large amount of waste rocks and tailings, which get deposited on the surface. In this condition, a network of sequestration activities and immobilization functions regulate the uptake, dis- tribution and detoxification of excess metal ions in plants (CLEMENS 2001). An active detoxification mechanism acts by means of glutathione (GSH) and of pep- tides synthesized at the expense of GSH, the phytochelatins (PCs), (DE VOS et al. 1992). Glutathione (GSH) is a low-molecular weight thiol tripeptide, involved in cellular defense against the toxic action of xenobiotics, oxyradicals as well as of metal cations (MEISTER and ANDERSON 1983). It is able to modify metal toxicity by chelating metal ions in cells; it plays a key role in protecting macromolecules from damage by free radicals by trapping them in an aqueous phase (FREEDMAN et al. 1989). On the other hand, PCs are polymers of g-Glu-Cys units with the terminal glycine and capable of binding heavy metals via thiolate coordination. They are induced in response to many heavy metals including copper (MORELLI and SCARANO 2004) when the non-PC based mechanism of detoxification gets exhausted and free metal ions become available to induce PC synthesis (SCHAT et al. 2002). Exposure to excess Cu is capable of stress induction in which the role of oxidative stress and reactive oxygen species (ROS) production may be involved (STADMAN and OLIVER 1991, WALDERMAR et al. 1994). On the other hand, under Cu toxicity, excess copper is an efficient generator of ROS in Fenton-type reactions, leading to disturbance in metabolic pathways and macromolecule damages (HEGEDUS et al. 2001). ROS are partially reduced forms of atmospheric oxygen and under normal conditions their production in plant cells is tightly controlled by the scavenging system. ROS can oxi- dize biomolecules such as DNA, proteins and lipids, creating oxidative injury that results in a reduction of plant growth and development (HERNANDEZ-JIMENEZ et al. 2002, OGAWA and IWABUCHI 2001). Since the half-lives of ROS are extremely short, their stable end products of oxidative damage to cellular macromolecules can be used for oxidative stress monitoring (ORHANL et al. 2004). Dityrosine, a hallmark of oxidized proteins and malondialdehyde (MDA) a bio- marker of lipid peroxidation are closely correlated with oxidative stress level (HALLIWELL and GUTTERIDGE 1998). To control the level and effects of ROS, cells have developed various antioxidant de- fenses including antioxidant enzymes and low molecular mass radical scavengers. They re- generate the active form of antioxidants and eliminate or reduce the damage caused by ROS (ALSHER et al. 1997). Among plants growing on Cu-contaminated soils, Cu-tolerant genotypes evoke antiox- idant enzyme induction as a general response to the toxic effects of copper to protect them against metabolic disturbance and cellular damage (VAN ASSCHE and CLIJSTERS 1990). Superoxide dismutase (SOD), the first major enzyme found in all aerobes, catalyses dismutation of superoxide anion to H2O2 and molecular oxygen. Glutathione peroxidase protects the membrane lipids from oxidative damage and detoxifies the organic peroxides; 108 ACTA BOT. CROAT. 69 (1), 2010 BOOJAR M. M. A., TAVAKOLI Z. U:\ACTA BOTANICA\Acta-Botan 1-10\Boojar.vp 9. travanj 2010 13:21:57 Color profile: Disabled Composite 150 lpi at 45 degrees it can also act on organic hydroperoxides (KANTOL et al. 1998). The intracellular level of H2O2 is regulated by a wide range of enzymes, the most important being catalase and peroxidase. Catalase inactivates H2O2 to oxygen and water (RUSINA et al. 2004). In the present study, field surveys have been carried out on Alhagi camelorum plant growing in the area of a copper mine in Kerman state. The aim of this study was to investi- gate the Cu-accumulating ability of this wild type plant growing around the mine. We also evaluated the corresponding status of the antioxidant enzyme activities, the levels of phytochelatins, GHS and oxidative damage products of lipids and proteins to clarify some aspects of this plant toxicity tolerance. Materials and methods This study was carried out at Sarcheshmeh, located in Sirjan at Kerman province in Iran (Longitude: 55o, 52’, 20’’ E, Latitude: 29o, 56’, 40’’ N). A maximum temperature of +32 oC and an average annual air temperature of 15 oC were recorded. The annual rainfall was about 516 mm and there was no industry nearby. After a geobotanical survey, two zones were considered for plant and soil sampling. Zone 1 was located in center of the Cu-mine area and Zone 2 was approximately 9.7 km south of the waterlogged area of the Cu-mine and the ecological conditions were similar in both zones. The soil of zone 2 was taken as a control because it had never received sources of Cu. Zone 1 was one of the well-known copper mines where the main activity was copper extraction at %1.16 grade of copper. Tail- ings had been abandoned for 11 years at the time of sampling. The levels of Cd, Co, Zn and Pb in zone 1 were generally below the USA maximum tol- erant concentrations (KABATA-PENDIAS 1995). The ratio of total Cu in zone 1 to that in zone 2 was about 52-fold. The ratio for available Cu was about 83 fold, showing that the available level was higher than toxicity threshold levels (ICRCL 1987). Soil and plant sampling Alhagi camelorum Fisch was endemic and widespread in the study area. At each zone, plant samples were collected at a determined time of a single growing season and accord- ing to the actual landform of the copper-mine and the distribution of vegetation before flowering period. Care was taken to collect plant species samples from both zones while they were in growth period. We established three random regions in each study zone. We collected at least 5–8 plants of our species from each area. Fresh tissues including roots, stems and mature leaves of collected plants were considered for three replicate analysis. Plants were cleaned in abundant deionised fresh water, rinsed with distilled water and identified by an expert botanist. Care was taken to avoid sample contamination with other sources of Cu during sampling, washing, transporting and analysis. Corresponding soil samples were also collected at the location of plant sampling from the rooting zone (maxi- mum sampling depth about 30 cm) and transferred to a polythene bags. Excess air was squeezed out, the bags were sealed, transferred to the laboratory and stored at 4 oC for a maximum of 48 h prior to analysis. These samples were then air-dried and sieved through a 2 mm plastic screen. There were 6 replicates for each soil sample. ACTA BOT. CROAT. 69 (1), 2010 109 ANTIOXYDANT ENZYME RESPONSE AND PHYTOCHELATINS U:\ACTA BOTANICA\Acta-Botan 1-10\Boojar.vp 9. travanj 2010 13:21:57 Color profile: Disabled Composite 150 lpi at 45 degrees Soil analysis Dried soil samples were digested with HCl + HNO3 + HClO4 (3:1:1, w.v.) (YUAN 1988). Total Cu and other metals were determined by atomic absorption spectrophotometer (Ana- lyst 100, Perkin Elmer, USA), using an acetylene- air flame. Diethylenetriaminepen- taacetic acid (DTPA)-extractable Cu, Cd, Co, Zn and Pb contents of 10 g soil samples (sample: DTPA, 1:2, w.v.) were determined by atomic absorption spectrophotometer (PAGE et al. 1982). The reagents and standards for AAS were ultra pure. The detection limits in mg kg–1 for total and extractable metals in soils were: 0.06 for Cd, 0.15 for Co, 0.17 for Pb, 0.08 for Cu and 0.11 for Zn. This step represents the fraction that is water soluble and most easily available to plants and easily leachable into the ground water (SIEB 1995). Soil nitrate (NO3–) was analysed colorimetrically (KEENEY and NELSON 1982). The total nitrogen was determined by the Kjeldhal digestion method (DUCHAUFOUR 1970). The pH and electro- lytic conductivity (EC) were determined in a water:soil extract 1:1 using a Beckman pH-meter and a conductivity meter model HI8633, Hanna Instruments Co., respectively. Plant biomass and Cu content analysis The washed plant samples were separated into roots and shoots, and dried in an oven at 60 oC for 48 h; then the biomass presented as dry weight was measured. For elemental anal- ysis, the dried plant tissues were ashed in a muffle furnace at 550 oC for 24 h. The ash was digested with a mixture of HNO3 and HClO4 (5:3 wet weight), heated in an oven. After cooling, the extracts were diluted and made up to 25 mL with 1 M HNO3. Copper concen- tration of the extract was determined by atomic absorption spectrophotometer. Chlorophyll determination Fresh and mature leaves (0.5 g) were extracted with 10 mL 80% acetone as described by ALAN (1994). The absorbance of extract was measured at 663 and 645 nm in the UV-Vis light spectrophotometer (model UV-9100). The chlorophyll content was calculated using the equation as follows: CT = 20.2 A645 + 8.02 A663 Chloroplast isolation Fresh and mature leaves (5 g) were homogenized for 15 s with a homogenizer in 50 mL ice-cold grinding medium containing: 0.33 M sorbitol, 1mM EDTA, 0.1% BSA, 2 mM so- dium ascorbate and 50 mM K2HPO4, pH 7.5. The homogenate was filtrated through Miracloth and centrifuged for 1 min at 1000 g at 4 oC to remove whole cells and cell debris. The intact chloroplasts were pelleted through centrifugation at 4500 g for 30 s and were gently resuspended in the same buffer without BSA and centrifuged again at the same con- ditions. This washing procedure was repeated twice and pelleted chloroplasts were isolated (RUSINA et al. 2004). Vacuole isolation Leaves were floated on an enzyme solution containing 1 mM CaCl2, 500 mM sorbitol, 0.05% (w.v.) polyvinyl pyrrolidone, 15 mM MES/Tris pH 5.5, 0.2% (w.v.) bovine serum 110 ACTA BOT. CROAT. 69 (1), 2010 BOOJAR M. M. A., TAVAKOLI Z. U:\ACTA BOTANICA\Acta-Botan 1-10\Boojar.vp 9. travanj 2010 13:21:57 Color profile: Disabled Composite 150 lpi at 45 degrees albumin, 1% (w.v.) cellulose, 0.5% (w.v.) Macerozym, 0.01% (w.v.) pectolyase, and agi- tated for 30 min. Vacuoles were released into the recording chamber by hyposmotic shock treatment of protoplasts in 100 mM KCl, 5 mM MgCl2, 2mM EGTA, 1 mM dithiotheritol (DTT) and 5 mM Tris/MES, pH 7.5, adjusted to p = 300 mOsm with D-sorbitol. After set- ting of the vacuoles, the hypotonic solution was carefully replaced by standard bath solu- tion (SCHOLZ-STARKE et al. 2004). Measurement of dityrosine: 1.2 grams of fresh tissue ma- terial were homogenized with 5 mL of ice-cold 50 mM HEPES-KOH, pH 7.2, containing 10 mM EDTA, 2 mM PMSF, 0.1 mM p-chloromercuribenzoic acid, 0.1 mM DL-norleu- cine and 100 mg polyclar AT. The plant tissue homogenate was centrifuged at 5000 g for 60 min to remove debris. Purification of o,o’-dityrosine in the clear tissue homogenized supernatant fluid was accomplished by preparative HPLC. o,o’-Dityrosine was recovered by gradient elution from the C-18 column (Econosil C18, 250 mm ´ 10 mm) (ORHANL et al. 2004). The composition of eluent varied linearly from acetonitrile–water–TFA (1:99:0.02) to acetonitrile–water– TFA (20:80:0.02) over 25 min. The gradient was started 5 min after the injection. A flow rate of 4 ml/min was used. o,o’-Dityrosine was analyzed by reversed-phase HPLC with simultaneous UV-detection (280 nm) and fluorescence-detection (ex. 280 nm, em. 410 nm). A phenomenex inertsil ODS 2 (150 mm ´ 4.6 mm, 5 mm) HPLC column (Bester, Amsterdam, the Netherlands) equipped with a guard column was used for these analyses. A gradient was formed from 10 mM ammonium acetate, adjusted to pH 4.5 with acetic acid, and methanol, starting with 1% methanol and increasing to 10% over 30 min. The flow rate was 0.8 mL min–1. A stan- dard dityrosine sample was prepared according to AMADO et al. (1984). Dityrosine was quantified by assuming that its generation from the reaction of tyrosine with horseradish peroxidase in the presence of H2O2 was quantitative (using the extinction coefficient e315 = 4.5 mM–1 cm–1 at pH 7.5). Malondialdehyde analysis Proteins of tissue homogenate were precipitated with 40% trichloracetic acid (TCA), w.v. The MDA assay was based on the condensation of one molecule malondialdehyde with two molecules of thiobarbituric acid (TBA) in the presence of reduced reagent vol- umes to increase sensitivity, generating a chromogen with UV absorbance. The TBA + MDA complex was analyzed by HPLC (BIRD et al. 1983). HPLC system consisted of a Hewlett + Packard 1050 gradient pump (Avondale, PA) equipped with an automatic injec- tor, a 1050 diode-array absorption detector and a personal computer using Chem Station Software from Hewlett + Packard. Aliquots of the TBA + MDA samples were injected on a 5 mm Supelcosil LC-18 reversed phase column (30 ´ 4.6 mm). The mobile phase consisted of 15% methanol in double-distilled water degassed by filtering through a 0.5 mm filter (Millipore, Bedford, MA). The flow rate was 2 mL min–1. MDA + TBA standards were prepared using tetraethoxy- propane. The absorption spectra of standards and samples were identical, with a character- istic peak at 540 nm. Measurements were expressed in terms of malondialdehyde (MDA) normalized to the sample protein content. Protein content was determined by the method of Bradford, with standard curves prepared using BSA (BRADFORD 1976). Extraction and determination of PCs and GSH were performed according to SNELLER et al. (2000). Frozen plant tissues were homogenized with a pestle and mortar with quartz sand in 2 mL of 6.3 mM diethylene triaminepenta acetic (DTPA) with 0.1% trifluoro acetic ACTA BOT. CROAT. 69 (1), 2010 111 ANTIOXYDANT ENZYME RESPONSE AND PHYTOCHELATINS U:\ACTA BOTANICA\Acta-Botan 1-10\Boojar.vp 9. travanj 2010 13:21:57 Color profile: Disabled Composite 150 lpi at 45 degrees acid (TFA) at 4 oC. The homogenate was centrifuged at 14,000 g at 4 oC for 12 min. The clear supernatants were collected for the assay by HPLC using pre-column derivatization with a fluorescent probe, monobromobiane (mBrB). 250 mL of supernatant was mixed with 450 mL of 200 mM HEPPS (3-[4-(2-Hydroxyethyl)-1-piperazinyl] propane sulfonic acid) at pH 8.2, with 6.3 mM DTPA, and 10 mL of 25 mM (mBrB). Derivatization was carried out in the dark at 45 oC for 30 min. The reaction was terminated by the addition of 300 mL of 1 M methane sulfonic acid (MSA). The samples were stored in the dark at 4 oC until HPLC analysis. Blank samples were used to identify the reagent peaks. The bimane derivatives were separated using a binary gradient of mobile phase A (0.1% TFA) and B (100% acetonitrile) at room temperature (22 ± 2 oC). Fluorescence was detected at 380 nm excita- tion and 470 nm emission wavelengths. The flow rate was 0.5 mL /min. Fifty mL of the de- rivatives sample was run in a linear gradient from 12% to 25% B for 15 min, then 25% to 35% B for 14 min and next 35% to 50% B for 21 min. Before a new sample was injected, the column was cleaned (5 min, 100% B) and equilibrated (10 min, 12% B) and post-time was 5 min. Total analysis time was 70 min. Analytical data were integrated by using the HP ChemStation. Retention times of PCs and GSH in biological samples were checked with PCs and GSH standards, respectively. Individual PC subtypes were quantified by using the relationship peak vs. concentrations of GSH standard solutions. Corrections for differential derivatization efficiencies were made according to the method stated by SNELLER et al. (2000). Preparation of enzyme extracts Whole tissue (leaves, stems and/or roots) were homogenized (1:5 w.v.) separately in an ice cold mortar using 50 mM sodium phosphate buffer, pH 7.0, containing 1 M NaCl, 1% polyvinylpyrrolidone and 1 mM EDTA. After centrifugation (20,000 g, 15 min), the supernatant (crude extract of leaves) was used to determine enzyme activities, which were measured at 25 oC. Enzyme assays Catalase (EC 1.11.1.6) activity was determined by following the consumption of H2O2 (extinction co-efficient 0.0394 mM mL–1) at 240 nm for 30s (AEBY 1984). The assay mix- ture contained 100 mM potassium phosphate buffer (pH 7.0), 15 mM H2O2 and 50 mL leaf extract in a 3 mL volume. Unit was defined as mmol H2O2 decomposed per 1 min. To detect glutathione peroxidase [EC 1.11.1.9 (GSH-Px)] activity, the method of Hopkins and Tudhope, with t -butyl hydroperoxide as a substrate was used (HOPKINS and TUDHOPE 1973). The reaction mixture comprised 50 mM potassium phosphate buffer, pH 7.0, 2 mM EDTA, 0.28 mM NADPH, 0.13 mM GSH, 0.16 U GR, 0.073 mM t-butyl hydroperoxide and enzyme extract (50 mg protein). One unit of GSH-Px activity was defined as the amount of enzyme that catalyzed the oxidation of NADPH [mmol min–1 mg–1 protein]. SOD activity was determined according to MINAMI and YOSHIKAWA (1979), with 50 mM Tris-Ca-codylic sodium salt buffer, pH 8.2, containing 0.1 mM EDTA. The reaction mix- ture was composed of 1.42% Triton X-100, 0.055 mM nitroblue tetrazolium (NBT), 16 mM pyrogallol and enzyme extract (50 mg protein). The principle of this reaction is based on the measurement of the concentration of the reduced form of NBT determined at 540 nm. The 50% inhibition was established according to MCCORD and FRIDOVICH (1969). This 112 ACTA BOT. CROAT. 69 (1), 2010 BOOJAR M. M. A., TAVAKOLI Z. U:\ACTA BOTANICA\Acta-Botan 1-10\Boojar.vp 9. travanj 2010 13:21:57 Color profile: Disabled Composite 150 lpi at 45 degrees inhibition was defined as the quantity of enzyme required to inhibit the reduction of NBT by 50% per 1 min. Statistical analysis All statistical analysis was carried out by using the procedure available in the SPSS v.10 (SPSS INC., Chicago, IL) statistical package. Each experiment was run on each sample at least in three replicates from all samples collected from all parts for each zone. Student‘s t-test was applied to determine the significance of results between different samples. Results Evaluations of pH and EC level revealed that the water extracts of the soils in both zones were mild acidic and there was no problem with salinity; however the soil samples associated with plants in zone 1 had a slightly lower pH than zone 2. To better characterize nitrogen species levels, we evaluated total nitrogen and nitrate levels, and the results indi- cated that both parameters were slightly lower in zone 1 than zone 2, although they did not differ significantly. Total contents of each metal; Cd, Co, Zn and Pb in the soil samples of zone 1 were significantly higher than those from zone 2 soils. The available Cu concentration for plants in zone 1 consisted of 39% of total Cu level. There were no significant differences in the available levels of the studied metals between soils of the zones except for Cu. The soils of zone 2 did not display higher concentrations of other evaluated metals, among which Co and Cd were very low (Tab. 1). Accordingly, their levels could not be toxic for plants. Measurement of Cu contents in roots, leaves and stems of Alhagi camelorum collected from the different sites showed that all tissues of our studied plant species in zone 1 con- tained significantly higher Cu levels as compared with samples collected from zone 2 soils. In addition, analysis showed significantly high Cu contents in leaf, stem and roots, in which, the level of Cu in leaves of A. camelorum rose up to around three fold of its total content in roots. The ratio of Cu in roots of this species growing on zone 1 to that in the roots of the same plants in zone 2 was approximately 7. Distribution of Cu in leaf ACTA BOT. CROAT. 69 (1), 2010 113 ANTIOXYDANT ENZYME RESPONSE AND PHYTOCHELATINS Tab. 1. Chemical characteristics of soils of the studied zones. Data were presented as mean ± SD. * – Significant difference with respect to zone 2 (P < 0.05). T – total content; E – DTPA – extractable content; EC – electric conductivity, # – electrolytic conductivity in water: soil ex- tract (1:1). Z on e Heavy metal content [mg (Kg dw)–1] pH E C # (mS cm–1) Nitrogen (g (kg dw–1)Cu Zn Co Cd Pb T E T E T E T E T E NO3 – Total 1 842.7 ±* 38.5 332.4 ±* 15.9 38.64 ±* 7.4 4.08 ± 0.36 9.6 ± 2.34 <0.11 3.05 ± 0.81 <0.17 12.8 ±* 3.61 <0.28 5.82 ± 0.44 2.37 ± 0.24 0.27 ± 0.03 1.41 ± 0.10 2 16.36 ± 3.52 4.21 ± 0.77 26.41 ± 4.9 6.24 ± 0.61 3.140 ± 1.83 <0.15 1.68 ± 0.56 <0.12 6.9 ± 1.82 <0.28 6.65 ± 0.25 1.92 ± 0.16 0.31 ± 0.02 1.68 ± 0.14 U:\ACTA BOTANICA\Acta-Botan 1-10\Boojar.vp 9. travanj 2010 13:21:57 Color profile: Disabled Composite 150 lpi at 45 degrees organelles revealed that the plant species in zone 1 had significantly higher Cu content in their vacuoles and/or chloroplasts than the same plant species in zone 2. The concentration of copper in vacuoles of Alhagi camelthorn was 29.67; this comprised 48% total leaf Cu levels and was over six times higher than the chloroplast Cu content (Tab. 2). The assay of biomass and chlorophyll contents showed that these parameters in plant species from zone 1 were insignificantly lower than in the same plant species associated with zone 2 (Tab. 3). 114 ACTA BOT. CROAT. 69 (1), 2010 BOOJAR M. M. A., TAVAKOLI Z. Tab. 2. Copper bioconcentration in tissues [g (kg dw)–1] and in organelle [(mg (g dw–1)] of leaves. Data are presented as mean ± SD; b – Statistically different with respect to roots; c – signifi- cant difference as compared with vacuoles; statistically different with respect to stems; # – The ratio of leaf organelle Cu to total leaf Cu Zone Tissue Organelle Leaf Stem Root Chloroplasts Cu/Cu% Vacuoles Cu/Cu% 1 61.54 ± 5.68 b 29.86 ± 3.24 b 17.05 ± 0.22 4.27 ± 0.71 c 7 29.67 ± 2.88 48 2 5.61 ± 1.05 b 3.28 ± 0.65 2.54 ± 0.49 0.28 ± 0.02 c 5 1.25 ± 0.18 22 Tab. 3. Chlorophyll content, and biomass of different plant tissues. Data are presented as mean ± SD. Zone Biomass mg (g FW)–1 Chlorophyll mg (g FW)–1Shoot Root 1 98.17 ± 5.64 76.41 ± 4.32 2.18 ± 0.24 2 112 ± 6.82 83.52 ± 5.16 2.71 ± 0.32 Tab. 4. Antioxidant enzyme activities and biomarkers of lipid peroxidation and protein oxidation in tissues of studied plants. Data are presented as mean ± SD. * – significant difference as com- pared with zone 2; #-significant difference as compared with roots. Tissue Z on e SOD (U/mg protein) GPX (U/mg protein) CAT (uM/min/mg) MDA (nmol/mg protein) Dityrosine (nmol/mg protein) GSH (nmol/gFW) leaf 1 28.10 ± 2.91 *# 5.41 ± 1.30 # 57.43 ± 5.32 *# 52.10 ± 4.12 4.36 ± 0.53 416 ± 26 * 2 17.28 ± 2.62 # 6.26 ± 1.12 # 33.51 ± 2.81 23.62 ± 2.30 2.71 ± 0.22 670 ± 41 stem 1 31.58 ± 3.04 *# 8.10 ± 1.63 * 66.04 ± 5.84 * 45.70 ± 3.66 3.92 ± 0.26 285 ± 22 * 2 12.77 ± 2.55 4.31 ± 0.82 40.55 ± 3.61 40.15 ± 3.85 3.21 ± 0.28 512 ± 38 root 1 38.26 ± 3.16 * 11.07 ± 1.74 * 87.12 ± 6.47 * 42.80 ± 3.30 3.10 ± 0.21 110 ± 14 * 2 9.62 ± 1.64 3.72 ± 0.63 23.38 ± 2.31 47.32 ± 3.72 3.81 ± 0.35 367 ± 27 U:\ACTA BOTANICA\Acta-Botan 1-10\Boojar.vp 9. travanj 2010 13:21:57 Color profile: Disabled Composite 150 lpi at 45 degrees As a comparison, the antioxidant enzyme activities in tissues of Alhagi camelorum from zone 1, were significantly higher than those of zone 2. On the other hand, the increases in the enzyme activities of this species growing on zone 1 were considerable for roots, stems and then leaves, in that order, but in zone 2, the pattern of increase was observed in leaves, stems and then roots. Moreover, A. camelorum showed significant increase in SOD and CAT activities of roots as compared stems and/ or leaves in zone 1. GPX activity of the plant increased significantly only in the roots. With regard to the levels of oxidative dam- age biomarkers of lipids as MDA and proteins as dityrosine, these parameters were consid- erable in leaves, stems and roots in order in zone 1, but in zone 2 the pattern of increase was for roots, stems and leaves in that order. The studied parameters in the leaves of this plant species growing in zone 1 were significantly higher (about two fold) in comparison with the same plants collected from zone 2 (Tab. 4). In addition, the evaluated plant tissues from zone 1 revealed significantly lower concentrations of GSH than those from zone 2. By con- trast, the levels of both PC2 and PC3 in each evaluated tissues of the studied plant from zone 1 were significantly higher than the same plant tissues collected from zone 2 (Fig 1). Discussion This investigation definitely showed that the study area was significantly contaminated with Cu at toxic levels and the rate of available concentration of this metal was high (about 39%) for plant growth. The copper levels in leaves of A. camelorum were above the critical level for copper toxicity (ROBSON and REUTER 1981). The soil analysis revealed normal levels of other heavy metals (Pb, Co and Cd). The high Cu availability may be attributed to the soil pH characteristic. Studies have confirmed that a low level of pH causes an increase in Cu solubility and its release from the soil phase leading to an elevation in copper uptake by roots (WATMOUGH and DICKINSON 1995). Our results were in accordance with the find- ings reported in the Cyprus Skouriotissa Cu mine, where pH was mildly acidic and con- tained copper up to 787 mg [kg (dry weight)]–1 (JOHANSSON et al. 2005). In our zones of in- vestigation, the normal growth of our studied plant samples in metalliferous soils without any visual and conspicuous symptoms of Cu toxicity (LEWIS et al. 2001) implied that A. camelorum was tolerant to toxic levels of Cu. ACTA BOT. CROAT. 69 (1), 2010 115 ANTIOXYDANT ENZYME RESPONSE AND PHYTOCHELATINS * * * * * * 0 10 20 30 40 50 60 70 80 90 100 leaf stem root leaf stem root phytochelatin 2 n m o l/ g r. F W zone 1 zone 2 phytochelatin 1 Fig. 1. The levels of phytochelatins in tissues of studied plants. * – significant difference as com- pared with zone 2. U:\ACTA BOTANICA\Acta-Botan 1-10\Boojar.vp 9. travanj 2010 13:21:58 Color profile: Disabled Composite 150 lpi at 45 degrees This plant species was endemic around the Cu mine and had adapted to contaminated soils by developing tolerance mechanisms to this metal stress. Most of these mechanisms have been recognized previously as exclusion, accumulation of metals and internal protec- tive responses that vary among plant species and among different tissues affected by their intrinsic characteristics (FREITAS et al. 2004, NICOLAU et al. 2005, SHU et al. 2002, BRUN et al. 2001). Alhagi camelorum had the ability to accumulate copper in its tissues at levels that were lower than 1000 mg g–1 tissue, a threshold limit that is prescribed for hyperaccu- mulators (REEVES and BAKER 2000). Accordingly, our studied plant could not be considered as hyperaccumulator. With re- gard to the importance of Cu distribution, the patterns of copper bioaccumulation and parti- tioning among different parts of tolerant plants including Paspalum distichum and Cyno- don dactylon, have been reported in many studies (MARSCHNER 1995, MULLIGAN et al. 2001, PYATT 2001, STOLTZ and GREGER 2002). In these two species that were collected from a Cu mine in China, the authors reported higher copper contents in roots of Cynodon dactylon and in the shoots of Paspalum distichum than in their other tissues. As another in- dication, Pistacia terebinthus and Cistus creticus collected from the Skouriotissa Cu mine accumulated a considerable amount of the absorbed Cu in their roots, although Bosea cypria accumulated most Cu in the leaves (JOHANSSON et al. 2005). In agreement with these documents, the studied plant, A. camelorum, showed Cu accumulation and partitioning in which Cu was transported and stored into the above ground parts, particularly into the leaves. The immobilization of excess heavy metals via their storage in cell walls (HUGHES and WILLIAMS 1988) or accumulation in vacuoles (MCCAIN and MARKLEY 1989), were sug- gested as another strategy to increase plant internal tolerance to Cu toxicity. We found that A. camelorum from zone 1 accumulated about 48% of total Cu in the leaves vacuoles that was considerable as compared with the same plant collected from zone 2. Many studies have confirmed that the main portion of metals including copper enter the vacuoles in the form of a phytochelatin complex, where they gain acid-labile sulphur and form high molecular weight complexes (SANITA DI TOPPI et al. 2002). Potentially toxic heavy metal ions are firstly chelated by GSH and then transferred to PCs for eventual se- questration. However, GSH act as a first line of defense against metal toxicity by com- plexing metals before the induced synthesis of PCs arrives at effective levels (FREEDMAN et al. 1989, SINGHAL et al. 1987). PC synthesis induced by metals is accompanied by a rapid depletion of total GSH in plants, because GSH is the direct precursor/substrate for the syn- thesis of PCs (GUPTA et al. 1998). In conformity with these documents, we observed an ele- vation in PC levels and decrease in GSH concentrations in plants of zone 1 as compared to plants of zone 2. On the other hand and in agreement with our study, SUDHAKAR et al. (2006), showed that exposure of Hydrilla verticillata (L.f.) Royle to high doses of copper led to decrease in GSH and an elevation in PC levels. In addition, PCs usually play only a part in integrated mechanisms of copper homeostasis and detoxification. Tolerance strate- gies of plants to copper exposure may include other responses such as variation in antioxidative enzyme activities. It has also been confirmed in many investigations that when copper is in excess, it can promote and stimulate generation of Fenton-type reactive oxygen species leading to in- crease in MDA and dityrosine as biomarkers of oxidative damages (WECKX and CLIJESTERS 1996, DEVI and PRASDA 1998, LOMBARDI and SEBASTIANI 2005). In response to ROS gener- 116 ACTA BOT. CROAT. 69 (1), 2010 BOOJAR M. M. A., TAVAKOLI Z. U:\ACTA BOTANICA\Acta-Botan 1-10\Boojar.vp 9. travanj 2010 13:21:58 Color profile: Disabled Composite 150 lpi at 45 degrees ation, antioxidant enzyme activities are elevated as defense system. These responses to ex- cess copper can vary among plant species and among different tissues (LOMBARDI and SEBASTINI 2005). In this study, Cu-accumulation by vacuoles caused a decrease in the level of copper outside this organelle, leading to decrease in ROS production and low induction on antioxidative enzymes in the leaves as compared with roots of this plant. Accordingly, the limited but significant increase in MDA and dityrosine in leaves as compared to the roots of A. camelorum could be the consequence of considerably low ac- tivities of antioxidative enzymes in leaves. However, the slight loss in chlorophyll content may be due to lipid peroxidation of chloroplast and thylakoid membranes and chlorophyll degradation mediated by Cu (BASZYNSKI et al. 1988, VINIT-DUNAND et al. 2002). In agree- ment with our findings, CHETTRI et al. (1998), reported a considerable decrease in total chlorophyll levels in Cladonia convoluta collected from a copper mine. They found a cop- per content in this plant species exceeding 175 mg. g–1 dry weight. Moreover, although A. camelorum leaves in zone 1 showed a significant increase in oxidative damage parameters with respect to zone 2, the parameter of biomass decreased insignificantly in this tissue of the plant in zone 1. 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