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Nickel accumulation and its effect on biomass, protein content and antioxidative enzymes in roots and leaves of watercress ( Nasturtium officinale R. Br

Nickel accumulation and its effect on biomass, protein content and antioxidative enzymes in roots and leaves of watercress ( Nasturtium officinale R. Br
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  jesc c cn Journal of Environmental Sciences 2010, 22(4) 526–532 Nickel accumulation and its e ff  ect on biomass, protein content andantioxidative enzymes in roots and leaves of watercress(  Nasturtium o   ffi  cinale  R. Br.) Fatih Duman ∗ , Fatma Ozturk  Faculty of Arts and Sciences, Department of Biology, Erciyes University, Kayseri 38039, Turkey. E-mail: fduman@erciyes.edu.tr  Received 24 June 2009; revised 07 November 2009; accepted 24 November 2009 Abstract In order to understand its response towards nickel stress, watercress (  Nasturtium o  ffi cinale  R. Br.) was exposed to nickel (1–25 mg  /  L)for 1, 3, 5 and 7 days. The accumulation and translocation of nickel were determined and the influence of nickel on biomass, proteincontent and enzymatic antioxidants was examined for both roots and leaves. It was determined that  N. o  ffi cinale  could accumulateappreciable amounts of Ni in both roots and leaves. Nickel accumulated particularly in the roots of plants. Biomass increased at lownickel concentrations but certain measurable change was not found at high concentrations. Under stress conditions the antioxidantenzymes were up-regulated compared to control. An increase in protein content and enzyme activities was observed at moderateexposure conditions followed by a decline at both roots and leaves. The maximum enzyme activities were observed at di ff  erent exposureconditions. Our results showed that  N. o  ffi cinale  had the capacity to overcome nickel-induced stress especially at moderate nickelexposure. Therefore,  N. o  ffi cinale  may be used as a phytoremediator in moderately polluted aquatic ecosystems. Key words : nickel; accumulation; antioxidative enzymes; biomass; watercress DOI : 10.1016  /  S1001-0742(09)60137-6 Introduction The contamination of water by heavy metals is one of the most serious problems in the world. Each plant specieshas di ff  erent levels of tolerance towards di ff  erent contam-inants, as do morphologically similar species growing inthe same area (Siedlecka and Krupa, 2002). In order toidentify suitable plants for pollutants removal from theaquatic environment, we require broad knowledge aboutthe physiological and biochemical features of potentiallyuseful species.Nickel is one of the toxic heavy metals present in rawwastewater due to industries such as electroplating, dyemanufacturing, porcelain enameling, and steam-electricpower plants (Padmavathy, 2008). For humans, nickel cancause serious health problems such as allergic sensitization(Wilhelm et al., 2007), dermatitis (Bocca et al., 2007), andlung and nervous system damage (Haber et al., 2000). TheNi concentration in surface water was approximately 0.01–0.002 mg  /  L (Karadede and Unlu, 2000). Although nickelis known to be essential for plants at low concentrations(Gajewska and Sklodowska, 2007; Baccouch et al., 2001),it is phytotoxic at high concentrations (Madhava Rao andSresty, 2000). The nickel concentration in plants has beenshown to range from 0.1 to 5.0  µ g  /  g of dry matter (Mishra * Corresponding author. E-mail: fduman@erciyes.edu.tr and Kar, 1974). The Ni deficiency is rare compared toits excess, which is often caused by metal mining andsmelting. It has been reported that the negative e ff  ects of Ni on plants are closely related to dose and exposure time(Kov´aˇcik et al., 2009; Poulik, 1999). The e ff  ect of nickelon plants varies according to plant species and cultivationconditions (Hao et al., 2006).Antioxidative enzymes such as catalase (CAT), ascor-bate peroxidase (APX), and superoxide dismutase (SOD)play a key role in radicals and peroxides control that areproduced under conditions of metal stress (Tanyolac et al.,2007). Kumar et al., (2007) demonstrated that moderatenickeltreatments(100 µ mol  /  L NiSO 4 ) also lead to asignif-icant increase in the concentration of H 2 O 2  and antioxidantenzyme activities in maize leaves. The molecular mecha-nism for the generation of reactive oxygen species (ROS)and the factors a ff  ecting the synthesis of ROS in Ni-treatedplants are, however, largely unknown. Metal accumulationproperties of   N. o  ffi cinale  have been studied extensively(Zurayk et al., 2001; Aslan et al., 2003; Saygideger andDogan, 2005), however, little attention has been paid tothe ensuing antioxidant responses and resultant e ff  ect of accumulation in this plant.The objectives of this study were to determine theaccumulation and translocation properties of Ni, and todetermine the e ff  ects of Ni concentrations (1, 5, 10, 25mg  /  L) on biomass, protein content, CAT, SOD and APX  jesc c cn No. 4 Nickel accumulation and its e ff  ect on biomass, protein content and antioxidative enzymes ······  527 activities of   N. o  ffi cinale . The results may be useful when  N. o  ffi cinale  is used as a phytoremediator in Ni contami-nated water. 1 Material and methods 1.1 Sample collection and cultivation Watercress,  Nasturtium o  ffi cinale  R. Br., is an aquat-ic perennial plant. Its leaves and stems are partiallysubmerged during growth. Cool running water must beavailableintheirhabitatyear-round.Thisplantisharvestedand consumed as a salad green. As a medicinal plant,watercress has been traditionally considered a diuretic,purgative and tonic.  N. o  ffi cinale  seedlings were collected in April, 2008from the Karasu Stream in Kayseri, Turkey. This locationhas a semi-arid and very cold Mediterranean climate. Theaverage annual mean temperature in the sampling areais 10.6°C. The maximum mean temperature is 30.5°C inJuly and August, and the minimum mean temperature is–7.6°C in January. The annual mean precipitation is 422.8kg  /  m 2 .According to the results of preliminary range determin-ing tests (data not shown), the highest nickel exposureconcentration for  N. o  ffi cinale  was selected as 25 mg Ni  /  L.Collected samples were washed using distilled water andacclimatized for three days in a climate chamber with awater temperature of 15°C, a relative humidity of 70%and photoperiod of 16 hr  /  8 hr (light  /  dark). The plants thatwere in the best condition were selected for subsequentexperiments. 1.2 Experimental design Prior to the experiment, containers were disinfected byimmersing in 1% ( V   /  V  ) NaClO for three to five minutes.Containers were then rinsed three times with distilledwater (Hou et al., 2007). Experiments were set up intriplicates, and each replicate contained approximately 4 gof plants. NiSO 4  was used in experiments. Nickel stock so-lutions were prepared using double distilled water. Plantswere treated with di ff  erent concentrations of Ni (0, 1, 5, 10and 25 mg  /  L) maintained in 10% Hoagland’s solution in400 mL conical flasks (Srivastava et al., 2006). Flasks wereplaced into the climate chamber for a period of 1, 3, 5 and 7days. At the end of the exposure experiment, plant sampleswere collected and sieved with a plastic griddle and werewashed with 0.01% Na-EDTA solution; then samples wererinsed with deionized water to removal adsorbed metalfrom surface of leaves. Plant samples were drained andblotted on paper towel for 2 min, and weighted thereafter.Root and leaf parts were subsequently separated. 1.3 Quantification of nickel An aliquot of each sample was dried at 70°C. Eachsample was then digested with 10 mL of pure HNO 3 using a CEM Mars 5 (CEM Corporation Mathews, USA)microwave digestion system. The digestion conditionswere as follows: maximum power was 1200 W, powerwas 100%, ramp was 20 min, pressure was 1.24 MPa,temperature was 200°C and hold time was 10 min. Afterdigestion, the volume of each sample was adjusted to 25mL using double deionized water. The total concentrationof each metal was determined using Inductively coupledplasma mass spectroscopy (Agilent 7500a, USA). Thestability of the device was evaluated every ten samplesby examining the internal standard. Reagent blanks werealso prepared to detect potential contamination during thedigestion and analytical procedure. Peach Leaves (NIST,SRM-1547) were used as a reference material, and all ana-lytical procedures were also performed with this referencematerial. The samples were analyzed in triplicates.To determine the Ni translocation properties of   N.o  ffi cinale , translocation factor (TF) was calculated as theratio of the metal concentration in the leaves to the metalconcentration in the roots (Stoltz and Greger, 2002). 1.4 Antioxidant enzyme extraction and assay Plant tissues, both roots and leaves (500 mg), werehomogenized in 1 mL of 100 mmol  /  L chilled potassiumphosphate bu ff  er (pH 7.0) containing 0.1mmol  /  L EDTAand 1% polyvinyl pyrrolidone (PVP,  W   /  V  ) at 4°C. Thehomogenate was centrifuged at 15,000  × g  for 15 min at4°C. The supernatant was used to measure the activitiesof SOD and APX (Srivastava et al., 2006). The proteincontent was determined according to the method of Lowryet al., (1951) using bovine serum albumin as the standardprotein. 1.5 Superoxide dismutase activity determination The SOD activity was determined by the method of Beauchamp and Fridovich (1971) by monitoring the pho-toreduction of nitroblue tetrazolium (NBT). The requiredcocktail for SOD activity estimation was prepared bymixing 27 mL sodium phosphate bu ff  er (pH 7.8), 1.5 mLmethionine (300 mg  /  mL), 1 mL NBT (14.4 mg  /  10 mL),0.75 mL Triton-X-100 and 1.5 mL 2 mmol  /  L EDTA. To 1mL of this cocktail, 10  µ L riboflavin (4.4 mg in 100 mL)and 50  µ g sample protein were added. The test tubes wereshaken and placed 30 cm below a 15-W fluorescent lamp.A tube containing sample protein kept in the dark servedas a blank, while the control tube did not have the enzymeand was kept under the light. We measured the absorbanceat 560 nm. The NBT reduction under illumination wasmeasured both in the absence and presence of the enzyme.The activity of SOD correlates with the amount of NBTreduction in light without protein minus the NBT reductionwith protein, and is expressed as units per mg protein.One unit of activity is the amount of protein requiredto inhibit 50% of the initial reduction of NBT under lightconditions. 1.6 Catalase activity determination For the measurement of the CAT activity, we performedan extraction step using a bu ff  er containing 50 mmol  /  LTris-HCl (pH 7.0), 0.1 mmol  /  L EDTA, 1 mmol  /  L PMSFand 0.3 g  /  (g fresh weight) PVP. Activity was measuredby the method of Aebi (1974). The reaction mixture  jesc c cn 528 Fatih Duman et al. Vol. 22 comprised 2.5 mL of 50 mmol  /  L sodium phosphate bu ff  er(pH 7.0), 300 µ L of 20 mmol  /  L H 2 O 2  and a suitable aliquotof enzyme. The change in absorbance was measured at240 nm (extinction coe ffi cient 40 mmol  /  (L · cm)). Enzymeactivity was expressed as units per mg protein. 1.7 Ascorbate peroxidase activity determination The activity of APX was measured by estimating therate of ascorbate oxidation (extinction coe ffi cient 2.8mmol  /  (L · cm)). The reaction mixture (3 mL) contained 50mmol  /  L phosphate bu ff  er (pH 7.0), 0.1 mmol  /  L H 2 O 2 , 0.5mmol  /  L sodium ascorbate, 0.1 mmol  /  L EDTA and a suit-able aliquot of enzyme extract. The change in absorbancewas monitored at 290 nm (Nakano and Asada, 1981) andthe enzyme activity was expressed as units per mg protein. 1.8 Statistical analysis Data were expressed as mean with standard error. Theexperiments were performed in a randomized order. TheKolmogorov-Simirnov test and Levene’s test were usedto ensure the normality assumption and the homogene-ity of variances, respectively. Heterogeneity of variancewere recognised, data were log transformed ln(  x + 1) andreevaluated. Significant di ff  erences were calculated usingStudent’s  t  -test wherever applicable. Analysis of vari-ance (ANOVA) was performed to confirm significantdi ff  erences among treatments. All pairwise mean compar-isons were made using post-hoc analyses. Duncan’s testwas used to determine the significant di ff  erence betweentreatments. We used 0.05 as the statistical significancethreshold. All statistical analyses were performed with theSPSS 15.0 software package. 2 Results 2.1 Nickel accumulation and translocation in  N. o   ffi  ci- nale The accumulation of Ni in the roots and leaves of   N.o  ffi cinale  was found to depend on both the concentrationand duration of exposure. The highest accumulation of Ni(5757.9  µ g  /  g dry weight) was found in the roots of the  N.o  ffi cinale  specimens that had been exposed to 25 mg  /  L Nifor 7 days (Fig. 1). The Ni translocation is significantlyhigher than that of control plants (Fig. 2). The highesttranslocation factor (TF) ratio was obtained when plantswere exposed to 1 mg  /  L of Ni. The TF values at all Niconcentrations tested were lower than 1. 2.2 E ff  ectofnickelonplantgrowthandproteincontent We observed that biomass increased in plants exposedto 1 mg  /  L Ni (Fig. 3). In comparison with the control asignificant decrease in biomass was observed only at 25mg  /  L Ni for 7 days. Visual changes including chlorosiswere observed on the leaves of plants exposed to 25 mg  /  LNi for 5 and 7 days. The leaf protein content was higherthan the root protein content ( P    0.05). An incrementof 155% was observed in the root protein content at 5mg  /  L by day 7 (Fig. 3). Leaves showed an increment of  Fig. 1  Ni contents in  N. o  ffi cinale  roots and leaves exposed to di ff  erentconcentrations of nickel. Values are represented as means  ±  SD (  N   =  3).Means with the same letter(s) are not significantly di ff  erent at  P    0.05according to Duncan’s test. Fig. 2  Nickel translocation factor (TF) ratios for  N. o  ffi cinale . Valuesrepresent means  ±  SD (  N   =  3). Means with the same letter(s) are notsignificantly di ff  erent at  P  0.05 according to Duncan’s test. approximately 40% at 5 mg  /  L within 3 days (Fig. 3). 2.3 Response of antioxidant enzymes In this study, we examined the activities of certainantioxidant enzymes such as SOD, APX and CAT, whichshowed varying responses with induction at various con-centrations and exposure durations.The SOD activity in the roots of   N. o  ffi cinale  showedthe highest activity at 5 mg  /  L Ni on day 3 comparing withcontrol, while in leaves the highest activity was detected at5 mg  /  L Ni on day 5 (Fig. 4). After reaching their maximumlevels, SOD activity showed a decline.  jesc c cn No. 4 Nickel accumulation and its e ff  ect on biomass, protein content and antioxidative enzymes ······  529 Fig. 3  E ff  ects of Ni treatment on biomass, and on the protein content of the roots and leaves of   N. o  ffi cinale . Values are represented as means  ± SD (  N   =  3). Means with the same letter(s) are not significantly di ff  erentat  P  0.05 according to Duncan’s test. The CAT activity of both roots and leaves showed asimilar trend (Fig. 5). The maximum level was obtainedat 5 mg  /  L Ni on day 5. The CAT activity showed a declineat all durations in roots and leaves exposed to 10 and 25mg  /  L Ni, and the maximum decline rate was recorded as45% in leaves on day 7 at 25 mg  /  L.Ni stress resulted in increased APX activity in boththe roots and leaves of   N. o  ffi cinale  only at 1 mg  /  L Nitreatment (Fig. 6). At 1 mg  /  L Ni, the APX activity inroots and leaves reached the highest value on treatment day3. The maximum decline was determined to be 130% inleaves on day 3 at 5 mg  /  L. 3 Discussion In this study, it was shown that  N. o  ffi cinale  can growin water contaminated with Ni. Maleva et al. (2009) eval-uated the accumulation properties of Ni from an aquaticplant, Elodea  canadensis , and confirmed that Elodea planttolerated higher concentrations of Ni than the amountnormally present in the contaminated areas. Moreover,  N. o  ffi cinale  accumulated appreciable amounts of Ni in Fig. 4  Time course of changes in the activity of SOD in the roots andleaves of   N. o  ffi cinale . Values are represented as means  ±  SD (  N   =  3).Means with the same letter(s) are not significantly di ff  erent at  P    0.05according to Duncan’s test. Fig. 5  Time course of changes in activity of CAT in the roots and leavesof   N. o  ffi cinale . Values represent means  ±  SD (  N   =  3). Means with thesame letter(s) are not significantly di ff  erent at  P    0.05 according toDuncan’s test. both roots and leaves. Similarly, Kara (2005) examinedthe bioaccumulation of Cu, Zn and Ni from the wastew-ater by treated  N. o  ffi cinale  and found that high amountof Ni could be accumulated by  N. o  ffi cinale . The highbioaccumulation of Ni in plants may be due to its roleas a micronutrient (Assuncao et al., 2003). Similar to theresults obtained by the earlier studies (Kov´aˇcik et al.,2009; Madhava R and Sresty, 2000), in the present studyit was found that the uptake of Ni was concentration-dependent. A plant that contains more than 1000 mg  /  kg of Ni in its tissues is considered to be a Ni-hyperaccumulator  jesc c cn 530 Fatih Duman et al. Vol. 22 Fig. 6  Time course of changes in the activity of APX in the roots andleaves of   N. o  ffi cinale . Values represent means ± SD (  N   = 3). Means withthe same letter(s) are not significantly di ff  erent at  P    0.05 according toDuncan’s test. (Brooks et al., 1977). As a result of the present findings,  N. o  ffi cinale  may be evaluated as a hyperaccumulator forNi. Similarly, Maleva et al. (2009) found that  Elodeacanadensis  accumulated more than 1000 mg  /  kg of Ni. Theroots are thought to be important for element uptake inaquatic plants (Sharma and Gaur, 1995). Nickel can berapidly taken up by the plant root system (Ali et al., 2008),and the roots of aquatic plants accumulate a higher amountof metal than stems and leaves (Aksoy et al., 2005; Chooet al., 2006, Duman et al., 2007). As expected, the rootsof   N. o  ffi cinale  accumulated a larger amount of Ni thanleaves in a concentration and duration-dependent manner.The rate and extent of translocation of metals within plantsdepended on metal and plant species (Deng et al., 2004).Figure 2 indicates that the Ni accumulated was largelyretained in the roots (TF ratio  <  1). These results suggestthat protective barriers exist to prevent the penetration of Ni from the roots into the leaves (Hozhina et al., 2001).Yang et al., (1996) stated that high nickel concentrationswould cause weak plant growth, leading to depression,metabolic disorders and chlorosis. In this study, low con-centrations of nickel caused an increase in biomass. Thisincrease may due to an increase in low molecular weightstress proteins such as antioxidant enzymes (Srivastava etal., 2006). As the concentration of nickel and the durationof exposure increase, the degradation of cell membraneand wall may occur, and as a consequence this may lead toa decline in growth rate and biomass (Kabala et al., 2008).Protein content may be considered a reliable indicator of oxidative metal stress in plants (Singh et al., 2006; Sinhaet al., 2005). In the present study, we observed an increasein protein content in the roots and leaves of   N. o  ffi cinale up to a certain concentration of Ni (5 mg  /  L). This increasemay be due to the increasing activity of some other metalsequestration mechanisms, involved in the detoxificationof high Ni doses (Rauser, 1999). However, the proteincontent decreased at high Ni concentrations (10–25 mg  /  L).This decrease could be due to the degradation of a numberof proteins. Maleva et al. (2009) studied nickel-inducedoxidative stress on  Elodea canadensis , and observed anincrease in protein content at up to 10  µ mol  /  L Ni, butat higher concentrations a decrease was recorded. Thisphenomenon is in agreement with our results.Plants are equipped with a defence system to repair thedamage created by ROS. Antioxidant enzymes such asSOD, CAT and APX play important roles in this process.It has been reported that antioxidant enzyme activity mayincrease, decrease or remain unchanged in response toheavy metal exposure (Zhang et al., 2007). In literature,it has been reported that the extent of alteration variedwith the type of metal, metal concentration, the enzymetested, plant species and the specific plant part (Shri etal., 2009; Srivastava et al., 2006; Devi and Prasad, 1998).It was known that Ni treatment induced oxidative stressin plants (Kumar et al., 2007). Sheoran et al., (1990)stated that Ni may inhibit many plant enzymes, includingthose involved in the Calvin cycle. Our results showthat in contrast to CAT, the activity of SOD and APXwas more pronounced in leaves than that in roots. Ourresults show that the di ff  erence in the antioxidative enzymeactivities of leaves and roots may explain the di ff  erenttolerance levels of roots and leaves. Moreover, our studyindicates that the accumulation of Ni in  N. o  ffi cinale  overa 7-day period increased as Ni concentration increasedin the nutrient solution. However, the activities of theantioxidant enzymes were a ff  ected to di ff  erent extents. Allenzymes exhibited similar response curves, with increaseof activities at lower concentrations of Ni but decreasedactivitiesathigherconcentrations.Similarresultshavealsobeen observed for other metals such as arsenic, copper andchromium (Mishra et al., 2006; Srivastava et al., 2006;Sinha et al., 2005). Reduced activities of SOD, CAT andAPX at higher Ni exposures could be the result of enzymemodulation by stress-related e ff  ector molecules (Takahashiet al., 1997).SOD is considered to play a key role in cellular defencemechanisms against ROS. The activity of SOD decreasesthe risk of OH radical formation which may cause severedamage to membranes, proteins and DNA (Zhang et al.,2007). When the activity of SOD increased significantlyin response to exposure to low levels of Ni (up to 5mg  /  L), it decreased at high Ni concentrations. Similarto the results observed in the present study, Bacccouchet al. (1998) recorded an initial increase followed by asubsequent decrease in SOD activity of   Zea mays  shootsexposed to 250  µ mol  /  L Ni for 7 days. In contrast to ourfindings, however, Baccouch et al. (2001) determined thatthe SOD activity of   Zea mays  roots exposed to 250  µ mol  /  LNi for 5 days was unchanged. The increase in SOD activityat low exposures indicates that  N. o  ffi cinale  is able totolerate moderate exposures to Ni and combat oxidativestresse ffi ciently.ThereductioninSODactivityatbothrootand leaves of   N. o  ffi cinale  may be due to an increased level
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