Psh

Ga

b

c

a

ARRAA

KCCGPP

1

msrrpepesm[

oprp

DG

0d

Journal of Hazardous Materials 182 (2010) 848–854

Contents lists available at ScienceDirect

Journal of Hazardous Materials

journa l homepage: www.e lsev ier .com/ locate / jhazmat

hysiological and biochemical responses in the leaves of two mangrove planteedlings (Kandelia candel and Bruguiera gymnorrhiza) exposed to multipleeavy metals

uo-Yong Huanga,b,c,∗, You-Shao Wanga

Key Laboratory of Tropical Marine Environmental Dynamics, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, ChinaState Key Laboratory of Organic Geochemistry, Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, Guangzhou 510640, ChinaGraduate University of Chinese Academy of Sciences, Beijing 100049, China

r t i c l e i n f o

rticle history:eceived 9 February 2010eceived in revised form 29 June 2010ccepted 30 June 2010vailable online 6 July 2010

a b s t r a c t

The accumulation of heavy metals and their effect on photosynthetic pigments, proline, glutathione(GSH) and phytochelatins (PCs-SH) were studied in the leaves of two mangrove plants seedlings (Kandeliacandel and Bruguiera gymnorrhiza) grown for 30 days in the nutrient solution containing four differentconcentrations of Cd2+, Pb2+ and Hg2+ (T1, T2, T3 and T4). An increase in Cd, Pb and Hg content was found

eywords:arotenoidshlorophyllslutathionehytochelatins

in the leaves of both species exposed to multiple heavy metal stress, whereas higher heavy metal levels(>T1) led to a remarkable breakdown of chlorophyll in the leaves of both species. The content of proline,GSH and PCs-SH in the leaves of both species exhibited a significant increase in response to heavy metalstress, at least under most of experimental conditions. Increased contents of proline, GSH and PCs-SH inmetal-treated plants suggest that metal tolerance in both K. candel and B. gymnorrhiza might be associatedto the efficiency of these antioxidants. Moreover, proline, GSH and PCs-SH in K. candel may play more

ating

roline important role in amelior

. Introduction

Mangrove ecosystems are diverse intertidal wetlands, com-only situated in tropical, sub-tropical and temperate coastal

ystems. Similar to other wetlands, mangrove ecosystems alsoeceive a number of pollutants from their related drainage andivers and have become a massive pollution sink [1]. Among thoseollutants, heavy metals are common pollutants in urban aquaticcosystems and are one of the main anthropogenic toxic com-ounds found in polluted mangrove locales, arising from industrialffluents, industrial wastes and urban runoff [2,3]. Many studiesuggest mangroves possess a remarkable capacity to retain heavyetals and tolerate relatively high levels of heavy metal pollution

1,4–7].The effect of nonessential heavy metals (such as Hg, Cd and Pb)

n plants have been widely studied and discussed in recent years,rompted mainly by the steadily increasing pollution of the envi-onment with these metals. Cd is one of the most aggressive andersistent heavy metals that may be present in natural environ-

∗ Corresponding author at: Key Laboratory of Tropical Marine Environmentalynamics, South China Sea Institute of Oceanology, Chinese Academy of Sciences,uangzhou 510301, China. Tel.: +86 20 85290795; fax: +86 20 85290795.

E-mail address: huang gyh@sina.com (G.-Y. Huang).

304-3894/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.jhazmat.2010.06.121

the effect of heavy metal toxicity than those in B. gymnorrhiza.© 2010 Elsevier B.V. All rights reserved.

ments as a by-product of human activities. It is toxic to living cellsat very low concentrations. Plants affected by Cd showed impairedphotosynthesis, altered mineral nutrition and water imbalance[8–10]. Pb, which is also one of the main inorganic pollutants in ourenvironment, exerts a wide range of adverse effect on growth andmetabolism of plants [11,12]. In plants, Hg ions can replace metalsion in photosynthetic pigments, causing a decrease in photosynthe-sis rates [12]. The toxicity of heavy metals is generally thought tobe due to uncontrolled and excessive production of reactive oxygenspecies (ROS), which can cause peroxidation of lipids, inactivationof enzymes, and damaged DNA and other constituents of cells: allof these changes accelerate senescence in the affected plants oreven kill them [13]. In response to heavy metals, higher plants haveevolved various mechanisms to scavenge ROS [14]. In addition,heavy metals entered within the cell may be sequestered by aminoacids, organic acids, metallothioneins (MTs), glutathione (GSH) orby specific metal-binding ligands the phytochelatins (PCs) [6,7,9].Free proline is known to occur widely in higher plants and normallyaccumulates in response to environmental stresses including heavymetal exposure [15,16]. It plays important role in osmoregulation,

protection of enzymes and stabilization of the sub-cellular struc-tures [15]. Moreover, it has been shown to help to scavenge ROSand sequester metal ions [15,16]. GSH, a sulphur containing tripep-tide, plays a prominent role in the defense against ROS caused byheavy metals in plants [17]. In addition, GSH is a potential chelator

Hazar

ocfip[w[c

aetolificitogspe

2

2

cdGlpiNsg1lwucccmrim5lc

2

d(ctit(

G.-Y. Huang, Y.-S. Wang / Journal of

f heavy metals and serves as a precursor of PCs [18,19]. PCs areonsidered to be involved in heavy metal homeostasis and detoxi-cation in the cells of higher plants, which are small, metal-bindingeptides with the general structure of (�-Glu-Cys)n-Gly (n = 2–11)14,20]. PCs are synthesized from GSH by the action of PC synthase,hich is activated by heavy metal ions such as Cd2+, Hg2+, and Pb2+

20,21]. The role of PCs in detoxification of heavy metals was wellonfirmed by mutant analyses and inhibitor studies [20].

K. candel and B. gymnorrhiza are dominant mangrove specieslong south china coast. As for K. candel and B. gymnorrhizaxposed to heavy metals, previous studies were mainly concen-rated on distribution and accumulation of heavy metals, and effectf heavy metals on growth of both species [1,22]. To date, veryittle work has been published on their physiological and biochem-cal mechanisms under multiple heavy metal stress. However, inact, mangrove plants are growing in a complicated environmentncluding multiple heavy metals. The aim of this paper was tolarify the biochemical processes behind heavy metal tolerancen the two mangrove species. With this purpose, we investigatehe effect of multiple heavy metals (Cd, Pb and Hg) on the physi-logical and biochemical changes in the leaves of K. candel and B.ymnorrhiza. Obtained results could be important for better under-tanding of the physiological and biochemical changes of mangrovelants to heavy metal stress conditions present in polluted aquaticcosystem.

. Materials and methods

.1. Plant material and treatment

Mature propagules of K. candel and B. gymnorrhiza wereollected from Futian mangrove national nature reserve in Guang-ong province and Shankou mangrove national nature reserve inuangxi province, respectively. Undamaged propagules of simi-

ar length were planted in plastic pots (five propagules in eachot) filled with sand under greenhouse conditions. Each pot was

rrigated with 500 mL of 1/2 Hoagland’s solution (containing 10‰aCl) every 3 days. After two leaves had been developed, the

eedlings of K. candel and B. gymnorrhiza were divided into fiveroups (3 pots in each group). Four groups were irrigated with/2 Hoagland’s solution containing multiple heavy metals at four

evels, namely T1, T2, T3 and T4. The fifth group was irrigatedith 1/2 Hoagland’s solution without multiple heavy metals andsed as the control (C). T1 represented the irrigation mediumontained 0.1 mg L−1 Cd, and 1 mg L−1 Pb and 0.1 mg L−1 Hg. Thehoice of T1 concentration was based on previous studies on K.andel and B. gymnorrhiza [1,5,22]. T2, T3 and T4 contained heavyetal concentrations that were 5×, 10× and 15× higher than T1,

espectively. Heavy metals were added as the chloride salt. Exper-mental conditions and heavy metal treatments were based on a

ethod described by Zhang et al. [5]. Each pot was irrigated with00 mL of corresponding liquid 2 times a week. After 30 days, the

eaves of both species were harvested for analysis of chlorophylls,arotenoids, proline, GSH, and PCs.

.2. Determination of Cd, Pb and Hg

Plant leaves for Cd, Pb and Hg determination were oven-ried at 70 ◦C and digested in concentrated HNO3 and H2O2

2:1, v/v) at 121 ◦C for 2 h. The analysis for Cd and Pb wasarried out using graphite furnace atomic absorbed spectropho-ometry, and Hg content was determined by cold vapor flownjection hydride generation atomic absorption spectrophotome-er. Cd, Pb and Hg contents were expressed as �g g−1 dry weightDW).

dous Materials 182 (2010) 848–854 849

2.3. Determination of photosynthetic pigments

Fresh leaves (about 0.5 g) were homogenized using a pestleand mortar in 5 mL of 95% (v/v) ethanol. The homogenate wasfiltered through ashless filter paper and made up to 25 mL with95% ethanol. The filtered solution was used for chlorophyll a (Chla), chlorophyll b (Chl b) and carotenoid estimation. Absorbance at470, 665, and 649 nm was measured using a method described byLou et al. [23]. Chlorophyll and carotenoid contents were expressedas mg g−1 fresh weight (FW).

2.4. Estimation of proline

Proline content was determined by the method described byBates et al. [24] with some modifications. Briefly, fresh leaves werehomogenized in 10 mL of 3% (w/v) sulphosalicylic acid and thehomogenate was centrifuged at 6000 rpm for 10 min. The reactionmixture containing 2 mL of supernatant, 2 mL of glacial acetic acidand 2 mL of ninhydrin reagent was incubated at 100 ◦C for 1 h. Themixture was extracted with toluene, and proline was quantifiedspectrophotometrically at 520 nm from the organic phase.

2.5. Extraction and assay of acid-soluble thiols

Total acid-soluble thiols (TAST) were assayed according toHartley-Whitaker et al. [25]. Extraction was carried out by grindingfresh leaf samples in 5 mL of 5% (w/v) sulphosalicyclic acid solu-tion with 6.3 mM diethylenetriamine-pentaacetic acid (DEPA) at4 ◦C. After centrifugation 12,000 rpm for 10 min (4 ◦C), the super-natants were immediately assayed. The concentration of TAST wasdetermined using 5, 5′-dithiobis-nitrobenzoic acid (DTNB).

Total glutathione (TG = GSH + GSSG) and oxidized glutathione(GSSG) were extracted by homogenizing fresh leaf samples (0.4 g)with ice-cold 5 mL of 5% (w/v) m-phosphoric acid. The homogenatewas centrifuged at 12,000 rpm for 30 min and the supernatant wascollected for analysis of TG and GSSG. TG and GSSG were measuredaccording to Huang et al. [19], which was based on DTNB-GSSGreductase recycling procedure. GSSG was determined after GSH hadbeen removed by 2-vinylpyridine derivatization. GSH was deter-mined by subtracting GSSG from TG content.

The concentration of PCs was calculated as:PCs = TAST − (TG − GSSG). Because PCs in peptides with differ-ent n values were not specified, PCs content was expressed in�mol PCs-SH g−1 FW.

2.6. Statistical analysis

Statistical analysis was carried out by one-way analysis ofvariance using SPSS 13.0. Duncan was performed to determinethe significant difference among the treatments and between thespecies (P < 0.05). Data are presented as means ± standard error(SE) of three replicates (three independent replicates of everytreatment). The correlation coefficient significant at P < 0.05 andP < 0.01 was evaluated between various physiological parametersand heavy metal accumulation in the leaves of both species.

3. Results

3.1. Accumulation of Cd, Pb and Hg

The accumulation of Cd, Pb and Hg in the leaves of both speciesat various concentrations of heavy metals was presented in Fig. 1.

K. candel accumulated significantly more Cd at T2 and above thanthe control. In B. gymnorrhiza, a significant increase in Cd contentwas only observed at T4 versus the control. In addition, Cd contentwas higher in K. candel than in B. gymnorrhiza at all concentra-tions of multiple heavy metals. In both species, the content of Pb

850 G.-Y. Huang, Y.-S. Wang / Journal of Hazardous Materials 182 (2010) 848–854

F B. gys 05).

itecwaa

3

gmCwChTwCbtiss(

3

uIo

ig. 1. The accumulation of Cd (A), Pb (B) and Hg (C) in the leaves of K. candel andignificantly different values among the treatments and between the species (P < 0.

ncreased significantly under heavy metal stress as compared toheir respective controls. Hg concentration in K. candel in the pres-nce of multiple heavy metals exhibited a pattern similar to Cdontent in B. gymnorrhiza. B. gymnorrhiza exposed to T2 and aboveas found to accumulate significantly more Hg than the control. Pb

nd Hg contents were lower in K. candel than in B. gymnorrhiza atll concentrations of multiple heavy metals.

.2. Effect of heavy metals on photosynthetic pigments

The concentration of photosynthetic pigments in the two man-rove species grown under different concentrations of heavyetals is shown in Fig. 2A–D. In both species, Chl a, Chl b and

hl (a + b) presented remarkable decrease above T1 in comparisonith their respective controls except for Chl a in K. candel at T2.hl a/b ratio in both species showed no significant change undereavy metal stress versus their respective controls except at T4.he correlation analysis revealed that Chl a, Chl b and Chl (a + b)ere significantly negatively correlated with the accumulation ofd, Pb and Hg in both species except Hg in K. candel (Table 1). Whenoth species were exposed to heavy metals, significant change inhe content of carotenoids was only observed in B. gymnorrhiza at T4n comparison with the control (Fig. 2E). Carotenoids have shownignificant negative correlation with the accumulation of all thetudied heavy metals in both species except Cd and Hg in K. candelTable 1).

.3. Effect of heavy metals on proline

The level of proline content in K. candel increased significantlynder heavy metal stress versus the control except at T1 (Fig. 3).

n B. gymnorrhiza, a significant increase of proline content was notbserved until T3 (Fig. 3). In addition, the content of proline was

mnorrhiza exposed to multiple heavy metals for 30 days. Different letters indicate

significantly higher in K. candel than in B. gymnorrhiza at all con-centrations of multiple heavy metals. In both species, proline hasshown significant positive correlation with the accumulation of Cd,Pb and Hg (Table 1).

3.4. Effect of heavy metals on acid-soluble thiols

The level of TAST in both species showed similar pattern withinitial increase and subsequent decrease (Fig. 4A). Both speciesexposed to multiple heavy metals produced significantly higherTAST content than their respective controls (Fig. 4A). In K. candel,GSH content significantly increased under heavy metal stress ascompared to the control except at T1 (Fig. 4B). Heavy metals signif-icantly enhanced the content of GSH in the leaves of B. gymnorrhizaat all heavy metal stress levels versus the control (Fig. 4B). In K.candel, PCs-SH showed significant increase in response to multipleheavy metals when compared to the control except at T2 (Fig. 4C).In B. gymnorrhiza, the content of PCs-SH peaked at T2 and was stillsignificantly higher than the control at T3 and T4 (Fig. 4C). In K.candel, the data analysis clearly showed that the accumulation ofCd, Pb and Hg is significantly positively correlated with TAST, GSHand PCs-SH except Hg where significant positive correlation withPCs-SH was not observed (Table 1). However, in B. gymnorrhiza,TAST, GSH and PCs-SH contents showed non-significant positivecorrelation with the accumulation of Cd, Pb and Hg (Table 1).

4. Discussion

Increased levels of heavy metals in growth solution generallyresulted in higher heavy metal contents in the leaves of K. can-del and B. gymnorrhiza, the results are in agreement with earlierreports on the accumulation of heavy metals in the leaves of man-

G.-Y. Huang, Y.-S. Wang / Journal of Hazardous Materials 182 (2010) 848–854 851

Fig. 2. The content of photosynthetic pigments (Chl a, Chl b, Chl (a + b), Chl a/b and Carotenoids) in the leaves of K. candel and B. gymnorrhiza exposed to multiple heavymetals for 30 days. Different letters indicate significantly different values among the treatments and between the species (P < 0.05).

Table 1Correlation coefficient (r) between various physiological parameters and heavy metal accumulation in the leaves of both species.

K. candel B. gymnorrhiza

Cd Pb Hg Cd Pb Hg

Chl a −0.776** −0.521* −0.488 −0.534* −0.719** −0.788**

Chl b −0.798** −0.659** −0.496 −0.548* −0.855** −0.912**

Chl (a + b) −0.805** −0.589* −0.503 −0.563* −0.806** −0.872**

Chl a/b 0.463 0.644** 0.342 0.260 0.540* 0.538*

Carotenoids −0.465 −0.529* −0.406 −0.620* −0.533* −0.531*

Proline 0.664** 0.781** 0.753** 0.646** 0.794** 0.856**

TAST 0.713** 0.681** 0.597* 0.078 0.296 0.380GSH 0.806** 0.530* 0.547* 0.050 0.319 0.404PCs-SH 0.523* 0.602* 0.492 0.086 0.263 0.340

* Pearson correlation is significant at the 0.05 level (2-tailed).** Pearson correlation is significant at the 0.01 level (2-tailed).

852 G.-Y. Huang, Y.-S. Wang / Journal of Hazar

FhDb

gaMtarfeeAtta

Fr

ig. 3. Proline in the leaves of K. candel and B. gymnorrhiza exposed to multipleeavy metals for 30 days. Vertical bars indicate the mean of three replication ± SE.ifferent letters indicate significantly different values among the treatments andetween the species (P < 0.05).

rove plants [1,2,22]. However, the accumulation amount of Cd, Pbnd Hg was low in the leaves of K. candel and B. gymnorrhiza (Fig. 1).angrove plants appear to possess a mechanism which limits the

ranslocation of metals to the aerial parts, and most heavy metalsre accumulated in the roots [1,2,22]. Heavy metal retention in theoots can be a strategy for protecting the more sensitive aerial partsrom the deleterious effect induced by heavy metal stress. This alsoxplained why growth of the mangrove plants were not inhibited

ven treated with very strong concentrations of heavy metals [26].lthough increasing concentrations of heavy metals in growth solu-

ion caused a significant increase in the content of Cd, Pb and Hg inhe leaves of both species, the accumulation patterns of heavy met-ls in the leaves of both species were different for each metal exam-

ig. 4. TAST (A), GSH (B) and PCs-SH (C) in the leaves of K. candel and B. gymnorrhiza expeplication ± SE. Different letters indicate significantly different values among the treatm

dous Materials 182 (2010) 848–854

ined (Fig. 1). In addition, the content of Cd was higher in K. candelthan in B. gymnorrhiza in each treatment, whereas the accumula-tion of Pb and Hg was lower in K. candel than in B. gymnorrhiza ineach treatment. To date, it is difficult to explain the different accu-mulation patterns among heavy metals and between the species.

The accumulation of heavy metals in mangrove species gener-ally leads to variations in physiological and metabolic processes,some of which directly contribute to heavy metal tolerance capac-ity of mangrove species [2,5]. Among the variety of targets reportedfor heavy metals in plants, the photosynthetic apparatus seemsthe most sensitive. Accordingly, the level of photosynthetic pig-ments is often used to assess the impact of environmental stressesin plants, because the change of photosynthetic pigments is linkedto visual symptoms of plant illness and photosynthetic produc-tivity [27]. In our study, chlorophyll pigments decreased in bothspecies under heavy metal stress, which also suggest that chloro-phyll biosynthesis and/or degradation might be an important targetof metal-induced stress in mangrove plants [2]. The decline inchlorophyll amount can be the result of increase of chlorophyllaseactivity or inhibition of �-aminolevulinic acid dehydratase [28,29].Moreover, B. gymnorrhiza appeared to be more sensitive to heavymetals than K. candel, especially as far as T4 is concerned. Chl a andChl b in K. candel decreased by 36.04% and 51.60% at T4, respec-tively, with respect to their respective controls (Fig. 2D). However,in B. gymnorrhiza, the decline of 43.21% and 60.28% were recordedin counterparts (Fig. 2D). Heavy metals had a positive effect on theratio of Chl a to Chl b in the present study. This occurs due to a

faster hydrolysis ratio of Chl b compared with Chl a, which hasbeen pointed out also by other authors [2]. Carotenoids are essen-tial components of the photosynthetic apparatus in a wide range oforganisms, which participate in the adaptation of plastids to chang-ing environmental light conditions and prevent photo-oxidative

osed to multiple heavy metals for 30 days. Vertical bars indicate the mean of threeents and between the species (P < 0.05).

Hazardous Materials 182 (2010) 848–854 853

dPchmtccfrd

miphtcptaathheiopatbcsm

snTtmSlotsoessbGGttrpocmatmtas

Fig. 5. Radar-diagrams summarizing and correlating normalized values of physio-

G.-Y. Huang, Y.-S. Wang / Journal of

amage of the photosynthetic apparatus by detoxifying ROS [30].revious studies have reported increase [8], decrease [10] or nohange [9] in the content of carotenoids in plants in response toeavy metal stress. In our study, carotenoid content in the twoangrove species decreased significantly at the highest concen-

ration of multiple heavy metals. As a result, the lowest carotenoidontent of the two mangrove species could lead to a diminishedapacity to protect photosystems against photooxidation [31]. Inact, it has been proposed that the bleaching of carotenoids in theeaction centre of PSII destabilizes PSII structure and triggers theegradation of the D1 protein [31].

Proline has been shown to accumulate in plant tissues underetal stress, ranging from algae to angiosperms [15]. For example,

n cyanobacterium subjected to heavy metals, the concentration ofroline was increased [16]. Moreover, many metal-tolerant plantsave also been reported to possess substantially elevated consti-utive proline levels in the absence of excess metal ions whenompared with their non-tolerant relatives [15]. Increased levels ofroline have been demonstrated to correlate with enhanced metalolerance in a transgenic alga, and it was suggested to act as anntioxidant in metal-stressed cells [32]. In the present work, theccumulation of proline in the two mangrove species in responseo heavy metals suggests a similar stress response to the excess ofeavy metals especially in the leaves of K. candel where there wasigher metal-induced proline accumulation. In previous studies,xposure of the two mangrove species to multiple heavy metalsnduces the stimulated generation of free radicals that leads toxidative stress [5]. Although one of the suggested functions ofroline is to scavenge different free radicals [15], whether prolineccumulation in both species resulted in increased stress tolerancehrough scavenging different free radicals is unknown. Proline haseen suggested to act as an osmoprotectant, an antioxidant, a metalhelator, and a regulator [15,32], but there has been no clear con-ensus regarding the mechanisms by which proline reduces heavyetal stress.The concentration of PCs has been carried out indirectly by

ubtracting the amount of GSH from the amount of TAST in aumber of studies [25,33–35]. Based on those studies, the level ofAST other than GSH was taken as a measure of PCs content inhe present study. PCs in plants are heavy metal-inducible, heavy

etal-binding, cysteine-rich polypeptides [20]. The content of PCs-H in K. candel peaked at T3, whereas that in B. gymnorrhiza did atower concentration of multiple heavy metals (T2) (Fig. 4C). More-ver, PCs-SH in K. candel presented a positive correlation withhe accumulation of Cd and Pb, whereas PCs-SH in B. gymnorrhizahowed non-significant positive correlation with the accumulationf Cd, Pb and Hg. These results may be resulted from the differ-nt accumulation patterns of heavy metals in the leaves of bothpecies. The increase in the content of PCs-SH in the leaves of bothpecies may be due to induction of PCs synthase gene, which cane activated by heavy metals such as Cd, Pb, and Hg [20,21]. AsSH is a substrate for PCs synthesis, it might be hypothesis thatSH depletion would occur because of PCs synthesis in response

o heavy meals. For example, a decline of GSH level is concomi-ant with the synthesis of PCs, which has been demonstrated inesponse to heavy metals [36]. Despite the increased synthesis ofhytochelatins, GSH was constant in two phytoplankton speciesver the studied Cd range [37]. However, in the present study, GSHontent in both species was significantly increased under heavyetal stress (Fig. 4B). The increase of glutathione correlated with

n increasing concentration of PCs-SH groups suggests that glu-

athione is synthesized in response to heavy metals in the two

angrove plants. Glutathione, besides being a substrate for PC syn-hesis and strong antioxidant, can exert important detoxificationction towards excess heavy metals by metal chelation, with pos-ible subsequent GSH-driven metal translocation into the vacuole

logical and biochemical parameters measured in this study after 30 days treatmentof K. candel (A) and B. gymnorrhiza (B) with increasing concentrations of multipleheavy metals. The central point of the radar-diagram represents ‘0%’ value of eachparameter (open circles represent control values 100%).

[31]. To date, very limited information is available regarding thetransport of GSH-metal complexes. Nevertheless, PCs are relativelymore efficient metal detoxifying agent than GSH [18]. Therefore,GSH is probably the in vivo donor of heavy metals to PCs, whichrefer to potentially toxic heavy metal ions are first chelated by GSHand then transferred to PCs for eventual sequestration [11]. The lev-els of PCs-SH peaked at T2 or T3 followed by a slight decline in bothspecies (Fig. 4C). The lack of a further increase in the leaf PCs-SH inboth species is probably due to storage of part of heavy metals inthe vacuoles at relatively higher cellular heavy metal levels, or tothe activity of leaf PCs synthase had reached saturation [33,35].

5. Conclusions

The radar-diagrams summarize and correlate normalized valuesof physiological parameters measured in this study after treatmentwith increasing concentrations of multiple heavy metals (Fig. 5).

8 Hazar

TcapBtaMittmhm

A

eKvL

R

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

54 G.-Y. Huang, Y.-S. Wang / Journal of

he accumulation patterns of heavy metals in the leaves of both K.andel and B. gymnorrhiza were different for each metal examined,nd the similar impact of multiple heavy metals on photosyntheticigments, proline, GSH, PCs-SH was found between K. candel and. gymnorrhiza. The results in the present study indicate that thewo mangrove species are able to tolerate multiple heavy met-ls through increasing the contents of proline, GSH and PCs-SH.oreover, proline, GSH and PCs-SH in K. candel may play more

mportant role in ameliorating the effect of heavy metal toxicityhan those in B. gymnorrhiza. Although the metal doses used inhis study might be higher than the concentration of bioavailable

etal in mangrove ecosystems, the information presented mightelp us to understand the biochemical detoxification strategies thatangrove plants adopt against heavy metal stress.

cknowledgements

This research was supported by the project of Knowl-dge Innovation Program of Chinese Academy of Sciences (No.SCX2-SW-132, KSCX2-SW-214), the project of Knowledge Inno-ation Program of South China Sea Institute of Oceanology (No.YQ200701) and the National 908 Project (No. 908-02-04-04).

eferences

[1] N.F.Y. Tam, Y.S. Wong, Accumulation and distribution of heavy metals in asimulated mangrove system treated with sewage, Hydrobiologia 352 (1997)67–75.

[2] G.R. MacFarlane, M.D. Burchett, Photosynthetic pigments and peroxidase activ-ity as indicators of heavy metal stress in the grey mangrove, Avicennia marina(Forsk.) Vierh, Mar. Pollut. Bull. 42 (2001) 233–240.

[3] F.F. Caregnato, C.E. Koller, G.R. MacFarlane, J.C.F. Moreira, The glutathioneantioxidant system as a biomarker suite for the assessment of heavy metalexposure and effect in the grey mangrove, Avicennia marina (Forsk.) Vierh, Mar.Pollut. Bull. 56 (2008) 1119–1127.

[4] E.C. Peters, N.J. Gassman, J.C. Firman, R.H. Richmond, E.A. Power, Ecotoxicologyof tropical marine ecosystems, Environ. Toxicol. Chem. 16 (1997) 12–40.

[5] F.Q. Zhang, Y.S. Wang, Z.P. Lou, J.D. Dong, Effect of heavy metal stress on antiox-idative enzymes and lipid peroxidation in leaves and roots of two mangroveplant seedlings (Kandelia candel and Bruguiera gymnorrhiza), Chemosphere 67(2007) 44–50.

[6] G.Y. Huang, Y.S. Wang, Expression analysis of type 2 metallothionein gene inmangrove species (Bruguiera gymnorrhiza) under heavy metal stress, Chemo-sphere 77 (2009) 1026–1029.

[7] G.Y. Huang, Y.S. Wang, Expression and characterization analysis of type 2 metal-lothionein from grey mangrove species (Avicennia marina) in response to heavymetal stress, Aquat. Toxicol. 99 (2010) 86–92.

[8] M. Drazkiewicz, T. Baszynski, Growth parameters and photosynthetic pigmentsin leaf segments of Zea mays exposed to cadmium, as related to protectionmechanisms, J. Plant Physiol. 162 (2005) 1013–1021.

[9] S. Mishra, S. Srivastava, R.D. Tripathi, R. Govindarajan, S.V. Kuriakose, M.N.V.Prasad, Phytochelatin synthesis and response of antioxidants during cadmiumstress in Bacopa monnieri L, Plant Physiol. Biochem. 44 (2006) 25–37.

10] Y. Ekmekci, D. Tanyolac, B. Ayhan, Effects of cadmium on antioxidant enzymeand photosynthetic activities in leaves of two maize cultivars, J. Plant Physiol.165 (2008) 600–611.

11] A. Piechalak, B. Tomaszewska, D. Baralkiewicz, A. Malecka, Accumulation anddetoxification of lead ions in legumes, Phytochemistry 60 (2002) 153–162.

12] L.A. Tabaldi, R. Ruppenthal, D. Cargnelutti, V.M. Morsch, L.B. Pereira, M.R.C.

Schetinger, Effects of metal elements on acid phosphatase activity in cucumber(Cucumis sativus L.) seedlings, Environ. Exp. Bot. 59 (2007) 43–48.

13] K. Stobrawa, G. Lorenc-Plucinska, Thresholds of heavy-metal toxicity in cut-tings of European black poplar (Populus nigra L.) determined according toantioxidant status of fine roots and morphometrical disorders, Sci. Total Envi-ron. 390 (2008) 86–96.

[

dous Materials 182 (2010) 848–854

14] S. Clemens, Molecular mechanisms of plant metal tolerance and homeostasis,Planta 212 (2001) 475–486.

15] S.S. Sharma, K.J. Dietz, The significance of amino acids and amino acid derivedmolecules in plant responses and adaptation to heavy metal stress, J. Exp. Bot.57 (2006) 711–726.

16] M. Choudhary, U.K. Jetley, M.A. Khan, S. Zutshi, T. Fatma, Effect of heavymetal stress on proline, malondialdehyde, and superoxide dismutase activ-ity in the cyanobacterium Spirulina platensis-S5, Ecotoxicol. Environ. Safe. 66(2007) 204–209.

17] N. Tsuji, N. Hirayanagi, M. Okada, H. Miyasaka, K. Hirata, M.H. Zenk, K.Miyamoto, Enhancement of tolerance to heavy metals and oxidative stress inDunaliella tertiolecta by Zn-induced phytochelatin synthesis, Biochem. Biophys.Res. Commun. 293 (2002) 653–659.

18] M. Gupta, R.D. Tripathi, U.N. Rai, P. Chandra, Role of glutathione and phy-tochelatin in Hydrilla verticillata (l.f.) Royle and Vallisneria spiralis L. undermercury stress, Chemosphere 37 (1998) 785–800.

19] G.Y. Huang, Y.S. Wang, C.C. Sun, J.D. Dong, Z.X. Sun, The effect of multiple heavymetals on ascorbate, glutathione and related enzymes in two mangrove plantseedlings (Kandelia candel and Bruguiera gymnorrhiza), Oceanol. Hydrobiol. St.39 (2010) 11–25.

20] C. Cobbett, P.B. Goldsbrough, Phytochelatins and metallothioneins: roles inheavy metal detoxification and homeostasis, Annu. Rev. Plant Biol. 53 (2002)159–182.

21] P.A. Rea, O.K. Vatamaniuk, D.J. Rigden, Weeds, worms, and more Papain’s longlost cousin, phytochelatin synthase, Plant Physiol. 136 (2004) 2463–2474.

22] M.W. Yim, N.F.Y. Tam, Effects of wastewater-borne heavy metals on mangroveplants and soil microbial activities, Mar. Pollut. Bull. 39 (1999) 179–186.

23] L.Q. Lou, Z.G. Shen, X.D. Li, The copper tolerance mechanisms of Elsholtzia hai-chowensis, a plant from copper-enriched soils, Environ. Exp. Bot. 51 (2004)111–120.

24] L.S. Bates, R.P. Waldren, I.D. Teare, Rapid determination of free proline for waterstress studies, Plant Soil 39 (1973) 205–207.

25] J. Hartley-Whitaker, C. Woods, A.A. Meharg, Is differential phytochelatin pro-duction related to decreased arsenate influx in arsenate tolerant Holcus lanatus?New Phytol. 155 (2002) 219–225.

26] G.Z. Chen, S.Y. Mao, N.F.Y. Tam, Y.S. Wong, S.H. Li, C.Y. Lan, Effect of syntheticwastewater on young Kandelia candel plants growing under greenhouse con-ditions, Hydrobiologia 295 (1995) 263–273.

27] A. Ben Ghnaya, G. Charles, A. Hourmant, J. Ben Hamida, M. Branchard, Morpho-logical and physiological characteristics of rapeseed plants regenerated in vitrofrom thin cell layers in the presence of zinc, C. R. Biol. 330 (2007) 728–734.

28] R. Abdel-Basset, A.A. Issa, M.S. Adam, Chlorophyllase activity: effect of heavymetals and calcium, Photosynthetica 31 (1995) 421–425.

29] V.M. Morsch, M.R.C. Schetinger, A.F. Martins, J.B.T. Rocha, Effects of cadmium,lead, mercury and zinc on �-aminolevulinic acid dehydratase acticity fromradish leaves, Biol. Plantarum 45 (2002) 85–89.

30] A.J. Simkin, H. Moreau, M. Kuntz, G. Pagny, C. Lin, S. Tanksley, J. McCarthy, Aninvestigation of carotenoid biosynthesis in Coffea canephora and Coffea arabica,J. Plant Physiol. 165 (2008) 1087–1106.

31] L.S. Di Toppi, A. Castagna, E. Andreozzi, M. Careri, G. Predieri, E. Vurro, A.Ranieri, Occurrence of different inter-varietal and inter-organ defence strate-gies towards supra-optimal zinc concentrations in two cultivars of Triticumaestivum L., Environ. Exp. Bot. 66 (2009) 220–229.

32] S. Siripornadulsil, S. Traina, D.P.S. Verma, R.T. Sayre, Molecular mechanismsof proline-mediated tolerance to toxic heavy metals in transgenic microalgae,Plant Cell 14 (2002) 2837–2847.

33] W.G. Keltjens, M.L. Van Beusichem, Phytochelatins as biomarkers for heavymetal stress in maize (Zea mays L.) and wheat (Triticum aestivum L.): combinedeffects of copper and cadmium, Plant Soil 203 (1998) 119–126.

34] Q. Sun, X.R. Wang, S.M. Ding, X.F. Yuan, Effects of exogenous organic chelatorson phytochelatins production and its relationship with cadmium toxicity inwheat (Triticum aestivum L.) under cadmium stress, Chemosphere 60 (2005)22–31.

35] R.L. Sun, Q.X. Zhou, F.H. Sun, C.X. Jin, Antioxidative defense and pro-line/phytochelatin accumulation in a newly discovered Cd-hyperaccumulator,Solanum nigrum L., Environ. Exp. Bot. 60 (2007) 468–476.

36] R.D. Tripathi, U.N. Rai, M. Gupta, P. Chandra, Induction of phytochelatins in

Hydrilla verticillata (l.f.) Royle under cadmium stress, Bull. Environ. Contam.Toxicol. 56 (1996) 505–512.

37] M. Lavoie, S. Le Faucheur, C. Fortin, P.G.C. Campbell, Cadmium detoxificationstrategies in two phytoplankton species: metal binding by newly synthesizedthiolated peptides and metal sequestration in granules, Aquat. Toxicol. 92(2009) 65–75.