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Plant Physiology and Biochemistry 46 (2008) 997–1006

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Plant Physiology and Biochemistry

journal homepage: www.elsevier .com/locate/plaphy

Research article

Ultrastructural changes, zinc hyperaccumulation and its relation withantioxidants in two ecotypes of Sedum alfredii Hance.

Xiao Fen Jin a,b, Xiao E. Yang a,b,*, Ejazul Islam a,b, Dan Liu a,b, Qaisar Mahmood c,Hong Li a,b, Junying Li d

a Ministry of Education Key Laboratory of Environmental Remediation and Ecosystem Health, P.R. Chinab College of Environmental and Natural Resources Science, Zhejiang University, #268 Kaixuan Road, Hangzhou, Zhejiang 310029, P.R. Chinac Department of Botany, Federal Government Postgraduate College, H-8, Islamabad, Pakistand Analysis and Test centre, Zhejiang University, Hangzhou 310029, P.R. China

a r t i c l e i n f o

Article history:Received 31 July 2007Accepted 14 June 2008Available online 28 June 2008

Keywords:AntioxidantGlutathionePhytoremediationSedum alfredii Hance.UltrastructureZinc (Zn)

Abbreviations: AsA, ascorbic acid; APX, ascorbatesulfoximine; Cant, cantharidin; CAT, catalase; DAB,dehydroascorbate; DHAR, dehydroascorbate reductanium; GPX, guaiacol peroxidase; GR, glutathione rGSSG, oxidized glutathione; HE, Zn-hyperaccumulatinmalonaldehyde; NBT, nitroblue tetrazolium; NHE,ecotype of S. alfredii; ROS, reactive oxygen species;TCA, trichloro acetic acid; TEM, transmissiontetramethylpiperidinooxy.

* Corresponding author. College of Environmental aZhejiang University, #268 Kaixuan Road, Hangzhou,Tel./fax: þ86 571 86971907.

E-mail address: xyang581@yahoo.com (X.E. Yang)

0981-9428/$ – see front matter � 2008 Elsevier Masdoi:10.1016/j.plaphy.2008.06.012

a b s t r a c t

Zn phytotoxicity and its possible detoxifying responses in two ecotypes of Sedum alfredii Hance, i.e.hyperaccumulating ecotype (HE) and non-hyperaccumulating ecotype (NHE) were investigated. HE grewbetter with high Zn concentrations of 29.11 g kg�1 DW in shoots when exposed to 500 mM Zn2þ. Toxicitysymptoms caused by Zn in root cells of both ecotypes mainly included plasmolysis, disruption of plasmamembranes and increased cell vacuolation. At high supplied Zn concentration, chloroplasts suffered fromstructural disorganization in both ecotypes. Zn-induced hydrogen peroxide (H2O2) and superoxideradical ðO2�� Þ productions in leaves were determined by a histochemical method, which revealed that Znstress may have involved NADPH oxidase, protein phosphatases and intracellular Ca2þ to activate thereactive oxygen species production. Inhibition of glutathione synthesis may have led to increased H2O2

and O2�� accumulations in leaves of HE. In response to higher Zn concentrations, ascorbic acid signifi-cantly increased in both ecotypes and levels of glutathione increased in both leaves and roots of HE andin roots of NHE without any change in the leaves of NHE. The enzymatic activities like those of super-oxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), guaiacol peroxidase (GPX, EC 1.11.1.7),ascorbate peroxidase (APX, EC 1.11.1.11), dehydroascorbate reductase (DHAR, EC 1.8.5.1), and glutathionereductase (GR, EC 1.6.4.2) in leaves of HE were all enhanced at supplied Zn concentration of 500 mM,which may account for its better growth.

� 2008 Elsevier Masson SAS. All rights reserved.

1. Introduction

Zinc is an essential plant micronutrient which is involved ina number of enzymatic reactions catalyzing nucleic acid metabo-lism. Conversely, excess of Zn is believed to inhibit many plantmetabolic functions, resulting in retarded growth and senescence.Elevated Zn contamination of soils demands effective

peroxidase; BSO, buthionine3,30-diaminobenzidine; DHA,se; DPI, diphenylene iodo-eductase; GSH, glutathione;g ecotype of S. alfredii; MDA,

non-Zn-hyperaccumulatingSOD, superoxide dismutase;electron microscopy; TMP,

nd Natural Resources Science,Zhejiang 310029, PR China.

.

son SAS. All rights reserved.

phytoremediation approaches. Compared with the traditionalmethods, phytoremediation is more cost effective and environ-ment friendly technique. Generally, effective phytoremediation ofcontaminated soils requires fast growing hyperaccumulators withlarger biomass. Presently, several Zn hyperaccumulators havebeen identified and study of the physiological and molecularmechanisms involved in hyperaccumulation is of paramountimportance. Sedum alfredii is a newly identified Zn/Cd hyper-accumulator with faster growth producing plenty of biomass. Theplant reproduces asexually and due to its perennial habit coversnearly 100% of the land surface [1,2]. Therefore, it is an ideal plantfor phytoremediation and to study the mechanisms involved inhyperaccumulation.

The mechanisms responsible for metal hyperaccumulation arenot fully understood yet. High Zn concentrations induce oxidativestress through an increased production of ROS, i.e. superoxideradicals ðO2�� Þ and hydrogen peroxide (H2O2) which results incellular oxidative damage and membrane lipid peroxidation inplant cells [3,4]. Interpretation of the biochemical detoxificationstrategies of plants against oxidative stress induced by metal

X.F. Jin et al. / Plant Physiology and Biochemistry 46 (2008) 997–1006998

accumulation is a key to optimize phytoremediation of heavy metalaffected biomes. Ascorbate and glutathione are two important an-tioxidant defense components in plant cells [5,6]. Oxygen freeradicals may be scavenged directly by ascorbate or may lead tothe production of H2O2, which can be detoxified by ascorbate in thepresence of ascorbate peroxidase [7]. GSH (g-glutamylcysteinylglycine) possess unique structural properties of broad redoxpotential, abundance, and wide distribution in plants due to whichit takes part in thioredoxin-related regulation of many enzymesinvolved in photosynthetic metabolism [6]. Superoxide radicals canbe converted to H2O2 by the action of superoxide dismutase (SOD,EC 1.15.1.1). Catalase (CAT, EC 1.11.1.6) and peroxidases such asascorbate peroxidase (APX, EC 1.11.1.11) and guaiacol peroxidase(GPX, EC 1.11.1.7) can prevent the accumulation of H2O2. The en-zymatic mechanism of detoxification involves dehydroascorbatereductase (DHAR, EC 1.8.5.1), glutathione reductase (GR, EC 1.6.4.2)and other enzymes.

Previous studies on S. alfredii mainly focused on growth andmetal uptake, while detoxification and transportation mechanismswere not investigated. The present study was performed to in-vestigate the phytotoxicity caused by Zn and responses of twoecotypes of S. alfredii for Zn detoxification.

2. Materials and methods

2.1. Plant source and culture

The Zn-hyperaccumulating ecotype of S. alfredii was collectedfrom an old Pb/Zn mined site in Zhejiang province of China, whileits non-hyperaccumulating ecotype was obtained from a tea gardenof Hangzhou in Zhejiang province of China. Plants with healthy anduniform shoots were selected and precultured for 4 weeks in thebasic nutrient solution containing (mmol L�1) Ca(NO3)2$4H2O 2.00,KH2PO4 0.10, MgSO4$7H2O 0.50, KCl 0.10, K2SO4 0.70; and (inmmol L�1) H3BO3 10.00, MnSO4$H2O 0.50, ZnSO4$7H2O 1.0,CuSO4$5H2O 0.20, (NH4)6Mo7O24$4H2O 0.01, Fe–EDTA 100. The pHof the nutrient solution was adjusted daily at 5.5 with 0.1 mol L�1

NaOH or HCl. Plants were grown under glasshouse conditions withnatural light, day/night temperature and humidity of 26/20 �C and70/85%, respectively. The nutrient solution was continuouslyaerated and renewed after every 3 days.

2.2. Zn treatment and sample preparations

After preculturing for 4 weeks, healthy and uniform seedlingswere selected for various Zn2þ treatments. NHE plants were ex-posed to different Zn concentrations, i.e. control (1 mM Zn), 5, 50,100 and 250 mM Zn, whereas, Zn treatments for HE were control(1 mM Zn), 50, 500, 1000 and 2500 mM Zn. Levels of Zn treatmentsfor NHE were one-tenth of those given to HE due to its sensitivity

Table 1Changes in dry weight, Zn concentration and Zn content in various tissues of two ecotyp

Ecotype Zn levels (mM) Dry weight Zn concentration

Shoot (g plant�1) Root (g plant�1) Shoot (g kg�1 DW)

NHE 1 0.93a 0.077a 0.59e5 0.86ab 0.076a 0.69d

50 0.81bc 0.068b 1.75b100 0.76c 0.059c 1.65c250 0.73c 0.046d 3.21a

HE 1 0.58c 0.087ab 7.77e50 0.63bc 0.090ab 9.19d

500 0.69a 0.102a 29.11a1000 0.68ab 0.091ab 26.02b2500 0.59c 0.072b 24.18c

Each value is the mean of three individual replicates. Means followed by the same letter

towards Zn. Each treatment was applied in triplicates in a com-pletely randomized design. Zinc was applied as ZnCl2. Nutrientsolution was aerated continuously and renewed after every 2 dayswith the pH maintained at 5.5. Plants were harvested after 7 days oftreatment. At the time of harvest, roots were soaked in 20 mM Na2–EDTA for 15 min to remove excess metal ions adhering to the rootsurface.

Fresh samples of leaves were immediately frozen in liquidnitrogen and stored at�80 �C for analysis of antioxidants, H2O2 andMDA.

2.3. Elemental analysis

Zn concentrations were determined using an InductivelyCoupled Plasma Mass Spectrometer (ICP/MS, Agilent 7500a, USA)according to Jin et al. [8].

2.4. Transmission electron microscopy

Fresh root tips and leaves of both the ecotypes treated withdifferent Zn concentrations were selected for TEM studies. Trans-mission electron microscopy was carried out following our pre-viously described method [8].

2.5. MDA and H2O2 content assays

The MDA and H2O2 contents were determined by the previouslydescribed method [9].

2.6. In situ localization of H2O2 and O2��

In situ H2O2 and O2�� accumulations were determined by themethod described by Romero-Puertas et al. [10] and Jin et al. [8].

2.7. Antioxidants (glutathione and ascorbic acid) assays

The GSH concentrations were estimated fluorimetrically basedon previous method [11].

2.8. Antioxidant enzymes assays

Plant samples of a known weight (1 g) were homogenized in6 ml pre-cooled 50 mM potassium phosphate buffer (pH 7.0)containing 0.2 mM EDTA and 2% (w/v) polyvinylpyrrolidone (PVP)in an ice bath using a prechilled mortar and pestle. The homogenatewas centrifuged for 20 min at 12,000g at 4 �C and the supernatantobtained was used for enzyme analysis. An aliquot of the extractwas used to determine protein content following the previousmethod [12], using bovine serum albumin as standard.

es of S. alfredii with exposure to supplying Zn levels for 7 days

Zn content Shoot/root ratio

Root (g kg�1 DW) Shoot (mg plant�1) Root (mg plant�1)

0.94d 0.25d 0.07 d 3.44b0.96d 0.27d 0.07 d 3.75b3.00c 0.63b 0.21b 3.05b4.56a 0.52c 0.27c 1.94c3.52b 0.93a 0.16a 5.80a

1.57e 2.17d 0.14e 16.13bc2.52d 2.73c 0.22d 12.34cd3.80c 9.79a 0.39c 25.73a5.64b 9.70a 0.51b 19.30b8.29a 6.36b 0.60a 10.63d

were not significantly different at P< 0.05.

Fig. 1. Transmission electron micrograph of NHE S. alfredii root tips (left panel) and HE S. alfredii (right panel). NHE was exposed to 1 mM (control, a), 50 mM (c), 100 mM (e) and250 mM (g) Zn for 7 days, respectively, while HE was exposed to 1 mM (control, b), 500 mM (d), 1000 mM (f) and 2500 mM (h) Zn for 7 days, respectively. Bars a and c¼ 1 mm, b, d, eand f¼ 2 mm, and g and h¼ 5 mm, respectively. Labels: CW: cell wall; MT: mitochondria; PL: plasmalemma.

X.F. Jin et al. / Plant Physiology and Biochemistry 46 (2008) 997–1006 999

SOD (EC 1.15.1.1) was determined by the photochemical method[13]. The decrease in absorbance of H2O2 within 1 min at 240 nm(E¼ 39.4 mM�1 cm�1) was recorded [14]. The GPX activity (EC1.11.1.7) was assayed following the previously described method

[14]. APX (EC 1.11.1.11) was measured according the method ofNakano and Asada [15] by monitoring the rate of ascorbateoxidation at 290 nm (E¼ 2.8 mM�1 cm�1). DHAR (EC 1.8.5.1) wasmeasured by monitoring the increase in absorbance at 265 nm

Fig. 2. Transmission electron micrograph of NHE S. alfredii leaf sections from (left panel) and HE S. alfredii (right panel). NHE was exposed to 1 mM (control, a), 50 mM (c), 100 mM (e)and 250 mM (g) for 7 Zn days, respectively. HE was exposed to 1 mM (control, b), 500 mM (d), 1000 mM (f) and 2500 mM (h) Zn for 7 days, respectively. Bars a and b¼ 1 mm, and c, d, e,f, g and h¼ 2 mm, respectively. Labels: Ch.: chloroplast; SG: Starch grain; PG: Plastoglobule; M: mitochondrion; Gr: granum.

X.F. Jin et al. / Plant Physiology and Biochemistry 46 (2008) 997–10061000

(E¼ 14 mM�1 cm�1). GR (EC 1.6.4.2) was assayed following themethod of Foyer and Halliwell [5] by monitoring the decrease inabsorbance at 340 nm caused by NADPH oxidation(E¼ 6.2 mM�1 cm�1).

2.9. Statistical analysis

Statistical analysis was performed using the SPSS statisticalpackage (version 11.0). Values reported here were the means of

X.F. Jin et al. / Plant Physiology and Biochemistry 46 (2008) 997–1006 1001

three replicates. Data were tested at significant levels of P< 0.05using one way ANOVA. Graphical work was carried out using SigmaPlot software v.10.

3. Results and discussion

3.1. Zn induced phytotoxicity

3.1.1. Growth and Zn accumulations in both ecotypes of S. alfrediiGrowth of both the ecotypes of S. alfredii in response to exces-

sive Zn addition was differential. When exposed to Zn� 500 mM,the HE plants grew well without any obvious change. However,some toxicity symptoms like slight necrosis and browning of roottips appeared when the HE plants were exposed to higher Zn levels,i.e. 1000 mM and 2500 mM. The toxic effects caused by external Znlevels> 50 mM were more severe in NHE plants, which includedextremely stunted roots, thickened root cuticle, and cracked, brownand wilted leaves.

For NHE, root and shoot biomass production expressed as dryweight, reduced significantly (P< 0.05) under Zn treatment of�50 mM (Table 1). However, both root and shoot dry weight of HEincreased significantly (P< 0.05) when treated with 500 mM Zn(Table 1). Zn concentration on the dry weight basis and its accu-mulation in roots and shoots of both ecotypes revealed linear in-crease in response to varying Zn supply levels, whereas Znconcentration in shoots of HE after 7 days treatment with 500 mMZn reached 29.11 g kg�1 DW and 9.79 mg plant�1, respectively(Table 1). In addition, Zn distribution in the both ecotypes differed

NHE

MD

A

(n

mo

l g

-1 F

W)

0

2

4

6

1 5 50 100 250

H2O2

(µm

ol g

-1 F

W)

0

2

4

6

8

10

le

a

b

Zn conce

Fig. 3. Effects of Zn on lipid peroxidation expressed in terms of MDA (a) and H2O2 contents (bars represent standard errors from three individual replicates.

remarkably (P< 0.05). The shoot/root ratios of total Zn content inHE ranged from 10.63 to 25.73 much higher than in NHE (Table 1).

Remarkable differences in Zn uptake and accumulation werenoted in both ecotypes of S. alfredii. Considerable amounts of Znaccumulated in shoots of HE plants, while in case of NHE, most ofZn was distributed in roots. It is generally assumed that leaf Znconcentrations above 300–600 mg kg�1 are considered toxic toplants [16]. However, HE plants still grew well at 500 mM Zn with Znconcentration of 29.11 g kg�1 DW in shoot. The above results con-firmed that HE has higher Zn requirement and an amazing ability totolerate Zn and its transport to shoot, which might be a result ofstrong selection pressure under high soil heavy metal concentra-tion in the mined areas.

3.1.2. Ultrastructural changes under Zn toxicityIn control, root cells of both ecotypes, i.e. NHE (Fig. 1a) and HE

(Fig. 1b) were had granular cytoplasm densely filled with numerouscell organelles that seemed without any obvious damage. Cellspossessed smooth, clean and continuous cell membrane and cellwall.

Ultrastructural studies revealed that various Zn concentrationscaused adverse effects on cell organelles in both ecotypes, butdamage was more pronounced in the NHE. In NHE plants, at 50 mMZn (Fig. 1c) and HE plants, at 500 mM Zn (Fig. 1d), root cells hadexiguous cytoplasm with thickened cell walls and distorted butcontinued plasma membrane with some attachments. At higher Zntreatments, obvious damaged cell structures were observed in NHEand HE (Fig. 1e–h). Increased vacuolization, distorted plasma

HE

1 50 500 1000 2500

af root

ntration (µM )

b) in leaves and roots of NHE S. alfredii (left panel) and HE S. alfredii (right panel). Error

X.F. Jin et al. / Plant Physiology and Biochemistry 46 (2008) 997–10061002

membrane, various attachments on the thickened cell walls andplasma membranes were observed. Particularly, the cells of NHEand HE exposed to 250 mM Zn (Fig. 1g) and 2500 mM Zn (Fig. 1h),respectively, were severely damaged, in which cell structure androot epidermis were ruined with some bacterial infection.

Transmission electron micrographs of the leaf cells of both theecotypes are also shown (Fig. 2). In spongy mesophyll cells of bothcontrol NHE (Fig. 2a) and HE (Fig. 2b), oblong chloroplasts withregular arrangement of thylakoid membranes of the stroma and ofthe grana and few plastoglobuli were observed.

Ultrastructural studies revealed that Zn adversely affected leafcell organelles especially chloroplasts in both ecotypes. In NHEplants exposed to 50 mM Zn or HE plants exposed to 500 mM Zn, thechloroplasts were in good shape, with regular arrangement of thethylakoid membranes and grana, but the stroma was a bit lighterthan that of the chloroplasts in the control plants (Fig. 2c and d). Inthe plants exposed to higher external Zn concentrations, distortedchloroplasts were more common (Fig. 2e–h). Generally, they wererounded in shape and irregular in outline. Different Zn treatmentsespecially 250 mM for NHE and 2500 mM for HE resulted in a vari-able degrees of internal structure disruption of chloroplasts, thegrana was less compact and having smaller number of thylakoidmembranes. In severely damaged chloroplasts of NHE plants (at

Fig. 4. Histochemical detection of H2O2 in leaves of HE (a) and NHE (b) S. alfredii H. Excised l1000 mM Zn for 7 days, respectively) were immersed in DAB solution to visualize brown spotthem in boiling ethanol to visualize the spots. Scanned spots from treated leaves quantified(Adobe Systems, USA). The results were expressed as percentage of spot area in pixels, vrepresentative individual experiments.

250 mM Zn treatment), the thylakoid membranes were dissolvedand were hardly visible (Fig. 2g). With increasing Zn levels, en-larged starch grains and numerous plastoglobules were observed inthe cells. In some chloroplasts, the breakdown of chloroplastmembrane was observed. However, no obvious changes in shape ofmitochondria in both the ecotypes were observed.

These ultrastructural investigations of root and leaf cells ofS. alfredii exposed to various Zn concentrations indicated that thestructural alternation of cells was dependent on the concentrationof applied metal. At ultrastructural level, Zn exhibited a greaterdegree of cellular damage to NHE plants. It was evident that at highZn concentrations, the toxicity symptoms in root cell were mainlyplasmolysis, disruption of plasma membranes and increased cellvacuolation. In addition, the exposure of S. alfredii to higherexternal Zn resulted in the chloroplasts structural degradation,disorganization of grana, increased number of plastoglobuli,greater size starch grains and breakdown of chloroplast membrane.

3.2. Oxidative stress

3.2.1. Lipid peroxidation and H2O2 accumulationMembrane destabilization is generally attributed to lipid per-

oxidation, due to an increased production of toxic oxygen free

eaves from both control and Zn-treated plants (NHE and HE were grown in 100 mM ands characteristic of DAB reaction with H2O2, and then leaves were bleached by immersingH2O2 deposits and the number of pixels was quantified with PHOTOSHOP 7.0 softwareersus total leaf area [(spot area/total leaf area) * 100]. Results are reported from five

X.F. Jin et al. / Plant Physiology and Biochemistry 46 (2008) 997–1006 1003

radicals [17]. Our results revealed that excessive Zn levels may beinvolved in lipid peroxidation and membrane damage which wasobvious from the significantly higher MDA content and hydrogenperoxide on fresh weight basis in both ecotypes treated with Zn.with increasing Zn treatments (Fig. 3).

3.2.2. In situ detection of H2O2 and O2�� in leavesThe accumulation of H2O2 or O2�� can be eliminated by AsA

(H2O2 scavenger) (Fig. 4) or TMP (O2�� scavenger) (Fig. 5), re-spectively. Treatments with DPI (oxidase inhibitor), cantharidin(protein phosphatase inhibitor) and LaCl3 (Ca2þ channel blocker)greatly reduced H2O2 and O2�� accumulation induced by Zn2þ

stress, suggesting the involvement of an NADPH oxidase, proteinphosphatase and intracellular Ca2þ in the activation of ROS pro-duction by Zn2þ (Figs. 4 and 5). Treatment with BSO (a glutathionesynthesis inhibitor) brought about 4.01-fold and 1.46-fold increasein H2O2 and O2�� accumulations in leaves of HE, respectively (Figs.4a and 5a), whereas 1.19-fold increase of O2�� accumulation inleaves of NHE was also recorded (Fig. 5b).

These histochemical detections of H2O2 and O2�� verified that Zntoxicity induced ROS accumulations in leaves of both ecotypes of S.alfredii. Zinc is required for the NADPH-oxidase regulation in plantmembranes [18], and changes in cytosolic free calcium ([Ca2þ]c) are

Fig. 5. Histochemical detection of O2�� in leaves of HE (a) and NHE (b) S. alfredii H. Excised le1000 mM Zn for 7 days, respectively) were immersed in NBT solution to visualize blue fordeposits were quantified by scanning the spots from treated leaves and the numbers of pixwere expressed as percentage of spot area in pixels, versus total leaf area [(spot area/total

apparent during the transduction of a very wide variety of abioticand biotic signals [19–22].The signal transduction pathways such asNADPH oxidation, protein dephosphorylation and changes in Ca2þ

fluxes through the plasma membrane were involved in the re-sponse of S. alfredii to alleviate Zn2þ stress (Figs. 4 and 5).Theseprocesses were confirmed to be involved in the regulation of Cd-induced ROS productions in pea leave [10]. Present study providedan evidence that glutathione synthesis inhibition led to an in-creased H2O2 and O2�� accumulation in leaves of HE under Zn stress,which suggested that GSH may play an important role in de-toxification in HE.

3.3. Levels of some antioxidants like glutathione and ascorbic acid

The majority of ROS-scavenging pathways of plants includesuperoxide dismutase (SOD), found in almost all cellular compart-ments, the water cycle in chloroplasts, the ascorbate–glutathionecycle in chloroplasts, cytosol, mitochondria, apoplast and peroxi-somes, glutathione peroxidases, and catalase (CAT) metabolism inperoxisomes.

A wide range of the non-enzymatic antioxidants such as GSHand AsA are involved in the oxidative defense of plants. Ascorbate isknown to operate as an antioxidant either in direct chemical

aves from both control and Zn-treated plants (NHE and HE were grown in 100 mM andmazan deposits and then were bleached by immersing them in boiling ethanol. O2��

els were quantified with PHOTOSHOP 7.0 software (Adobe Systems, USA). The resultsleaf area) * 100]. Results are reported from five representative individual experiments.

X.F. Jin et al. / Plant Physiology and Biochemistry 46 (2008) 997–10061004

interaction with reactive oxygen species, or during the reactioncatalyzed by APX in chloroplasts and other cell compartments [7].Under normal conditions, 90% ascorbate pool is in the reduced form[23]. In response to higher Zn concentrations, ascorbic acid (AsA)significantly increased in both ecotypes (Fig. 6a). As compared tothe control, the AsA concentrations in leaves of NHE significantly(P< 0.05) increased at high Zn concentration, i.e. 100 mM and250 mM, while AsA concentration was particularly high in roots ofNHE when treated with 50 mM Zn treatment and 100 mM and250 mM Zn treatments brought 1.54 and 1.40 folds increase, re-spectively (Fig. 6a). Glutathione is an important water-phase anti-oxidant with proposed roles in the storage and transport of reducedsulfur, the synthesis of proteins and nucleic acids and as a modu-lator of enzyme activity [6]. The glutathione level has been shownto correlate with the plant adaptation to extreme heavy metalsstress and reduced glutathione pool shows marked alterations inresponse to heavy metal stress [24–30]. GSH levels enhanced bothin leaves and roots of HE and in roots of NHE but not in the leaves ofNHE. Particularly, HE plants with 500 mM Zn exposure exhibitedconsiderably high GSH concentration in leaves (Fig. 6b). GSH andAsA contents in leaves of HE were always greater than those inroots, the case was opposite in NHE plants (Fig. 6). The previousstudy also showed that a marked increase in GSH was present inroots, shoots and leaves of HE when exposed to increasing Znconcentrations, this relationship was weaker in non-hyper-accumulating plants [30]. These results ruled out the possibilitythat both AsA and GSH play role in tolerance to Zn and that GSH

NHE

AsA

(µg

g

-1 F

W)

0

30

60

90

120

150

Zn conce

GS

H

(µg

g

-1 F

W)

0

20

40

60

80

1 5 50 100 250

lea

a

b

Fig. 6. Concentrations of reduced AsA (a) and reduced GSH (b) in leaves and roots of NHE S.Zn. Error bars represent standard errors from three individual replicates.

may be involved in Zn transport and hyperaccumulation in HE. Ourprevious studies with same ecotypes of S. alfredii also showed anincreased GSH content in leaves and roots under Cd exposuresuggested that such increased GSH content in levels may accountfor greater ability of HE to tolerate Cd toxicity [8].

3.4. Activities of antioxidant enzymes

Apart from the effects of Zn in the non-enzyme antioxidants,zinc also affected the enzymatic antioxidants. Compared with thecontrol, SOD activity in leaves of NHE increased at high Zn con-centrations and then decreased by 41.5% for 250 mM Zn treatments.SOD activity in roots of NHE also induced by Zn treatment andremarkably decreased in the 250 mM Zn treatment. SOD activity inleaves and roots of HE was stimulated at Zn levels� 500 mM andthen leveled off at higher supplied Zn (Fig. 7a). SOD converts su-peroxide radicals to H2O2, which can be removed by the H2O2-capturing enzymes such as CAT, GPX and APX in different cellularcompartments. CAT activities were decreased with increasing Znconcentrations as compared to control in both leaves and roots ofNHE as well as in roots of HE, however, CAT activity improved inleaves of HE with increasing Zn levels (Fig. 7b). GPX activity in rootsof both NHE and HE was much higher than that in leaves. GPXactivity in leaves and roots of NHE significantly (P< 0.5) increasedwith increasing Zn concentrations. For HE, GPX activity decreasedwith increasing Zn concentrations in roots and was at its maximumfor 500 mM Zn treatment in leaves (Fig. 7c). APX activity in leaves

HE

ntration (µM )

1 50 500 1000 2500

f root

alfredii (left panel) and HE S. alfredii (right panel) exposed to various concentrations of

SO

D

(u

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)

0

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CA

T

(m

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-1)

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1 5 50 100 250 1 50 500 10002500

X.F. Jin et al. / Plant Physiology and Biochemistry 46 (2008) 997–1006 1005

and roots of NHE increased at low Zn concentrations and thenleveled off at concentrations greater than 250 mM Zn, compared tothe control. APX activity in roots of HE was much higher than thatin leaves, with the maximum at 500 mM Zn in both leaves and roots(Fig. 7d).

DHAR requires extensive utilization of GSH for the conversion ofDHA to reduced AsA and GR is involved in conversion of GSSG toGSH. DHAR activity in roots of both NHE and HE was much higherthan in leaves. Zn treatments showed insignificant effects on DHARactivity in roots of NHE, except for a 27% increase at high 250 mMZn, while DHAR activity increased for 50 mM Zn treatment overcontrol in leaves of NHE and then decreased at 250 mM Zn. How-ever, DHAR activity reduced in roots of HE with increasing Znconcentration with the maximum at 500 mM Zn in leaves (Fig. 7e).Zn treatment resulted in reduced GR activity in leaves of NHE ascompared to the control; while in roots of NHE, it was unaffected atZn concentrations of �100 mM and then 76.5% reduced for 250 mMZn over control (Fig. 7f). However, GR activity in leaves of HE at500 mM Zn treatment was extremely high and then leveled off withincreasing Zn concentrations, whereas it reduced in roots of HE atenhanced Zn exposure in relation to control (Fig. 7f). The role ofglutathione in the antioxidative defense system provides a ratio-nale for its use as a stress marker. However, responses of gluta-thione concentrations and redox states are not consistent amongthe large number of available publications [31–33]. In general, aninitial stress response was related to changes in the glutathioneredox state, whereas acclimation was marked by increased gluta-thione concentrations, increased related enzyme activities, and/ora more reduced redox state of glutathione. The latter was inter-preted as overcompensation leading to enhanced regeneration ofglutathione. Deterioration effects upon strong stress impactswere related to progressive degradation and oxidation of theglutathione pool. A time-course analysis, which has rarely beendone in the published literature, showed this sequence of events[32].

Above results revealed that the antioxidant enzyme activitiessuch as SOD, APX, and DHAR in both roots and leaves of NHE im-proved for lower Zn treatments and then leveled off by 250 mM Zn.However, all these enzymes, i.e. SOD, CAT, GPX, APX, DHAR and GRactivities in leaves of HE were at their peak when treated with500 mM Zn, while Zn exposure of 2500 mM dramatically reducedthese activities in both roots and leaves of HE. It was demonstratedthat best growth of HE occurred at external 500 mM Zn with highenzymatic antioxidant and non-enzymatic antioxidants like GSHactivities.

4. Conclusion

Both ecotypes of S. alfredii differed in the Zn tolerance and ac-cumulation, and HE plants exhibited much higher Zn requirementsand an extraordinary ability to tolerate and translocation of Zn tothe shoots, which may be a consequence of strong selection pres-sure under high soil heavy metal concentration in the mined areas.

Zn induced the generation of ROS including hydrogen peroxide(H2O2) and superoxide radical ðO2�� Þ in S. alfredii. The processessuch as NADPH-oxidase regulation, changes in cytosolic Ca2þ andprotein dephosphorylation may be involved in Zn2þ-induced ROSresponse of S. alfredii. Glutathione biosynthesis, enhancement ofascorbic acid and glutathione concentrations and induction of an-tioxidant enzyme activity may account for higher Zn tolerance and

Fig. 7. Response of antioxidant enzyme activities like SOD (a), CAT (b), GPX (c), APX(d), DHAR (e) and GR (f) under Zn treatments in leaves and roots of NHE S. alfredii (leftpanel) and HE S. alfredii (right panel). Error bars represent standard errors from threeindividual replicates.

X.F. Jin et al. / Plant Physiology and Biochemistry 46 (2008) 997–10061006

uptake shown by HE of S. alfredii. The plants accumulated higheramount of Zn and showed potential to be used as a phytor-emediator species in terrestrial/aquatic habitats moderatelypolluted by Zn.

Acknowledgements

The work was financially supported by a program for Changjiangscholars, Innovative Research Team in University, China(#IRT0536), and the project from National Natural Science Foun-dation of China (#20777068 and # 30630046). The authors aregrateful to Dr. Levent Ozturk, Sabanci University, Turkey for hisvaluable instructions.

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