Acta Oceanol. Sin., 2012, Vol. 31, No. 3, P. 98-108

DOI: 10.1007/s13131-012-0210-3

http://www.hyxb.org.cn

E-mail: hyxbe@263.net

Photosynthetic activity and antioxidative response of seagrass

Thalassia hemprichii to trace metal stress

LI Lei1,2,3,4, HUANG Xiaoping1∗, Devajit Borthakur5, NI Hui6

1 State Key Laboratory of Tropical Oceanography, South China Sea Institute of Oceanology,Chinese Academy of Sciences, Guangzhou 510301, China

2 Graduate University of Chinese Academy of Sciences, Beijing 100049, China

3 Tropical Marine Biological Research Station in Hainan, Chinese Academy of Sciences, Sanya572200, China

4 The Science and Technology Library of Guangdong Province, Guangzhou 510070, China

5 Tocklai Experimental Station, Tea Research Association, Jorhat, Assam 785008, India

6 Foreign Language Department, Heze University, Heze 274000, China

Received 24 January 2011; accepted 31 March 2012

©The Chinese Society of Oceanography and Springer-Verlag Berlin Heidelberg 2012

AbstractThis study concerned the accumulation of trace metals in tissues of seagrass (Thalassia hemprichii)exposed to various concentrations of Zn2+, Cd2+, Pb2+ and Cu2+ for 10 d, and the effect ofexcessive metals on quantum yield (ΔF/F ′

m), photosynthetic pigments and antioxidative enzymeslike superoxide dismutase (SOD), guaiacol peroxidase (POD) were also examined. Cadmium wasthe most highly accumulated metal. Meanwhile, high metals levels led to a remarkable breakdownof photosynthetic parameters. Especially, ΔF/F ′

m, chlorophyll and carotenoid were significantlylow during prolonged Cu exposure. Besides, ΔF/F ′

m was more severely depressed by Cu and Znthan Pb and Cd. However, T. hemprichii had positive response by increasing the activity of SODand POD. The results indicate that T. hemprichii is the most sensitive to Cu, and the antioxidativeprotection mechanisms of T. hemprichii are more efficiently activated to avoid damage of Zn, Cdand Pb stress. Finally, due to the high Cd-accumulation and strong Cd-tolerance capacity, T.hemprichii can be used for phytoremediation in Cd-contaminated areas.

Key words: Thalassia hemprichii, metal stress, photosynthetic pigments, quantum yield, antiox-

idant enzymes

1 Introduction

Seagrass beds, having great ecological and com-mercial values, house diverse communities in intertidalzones of tropical, subtropical and temperate coastalareas where anthropogenic activity is the most intense.However, many seagrass habitats around the world arevulnerable to degradation and many of them have beencompletely destroyed with the expansion of develop-mental activities in the coastal areas. Trace metalcontamination is an important aspect of destructionof seagrass habitat (Huang et al., 2006). Similar toother wetlands (e.g., mangrove and salt marsh ecosys-tems), seagrasses sequester trace metals from the ma-rine environment via both blades and root-rhizomes

and these concentrations can be correlated with thewater column and sediments, respectively (Tiller etal., 1989). Many studies suggest that seagrasses pos-sess a remarkable capacity to retain metals and tol-erate relatively high levels of trace metal stress, butin excessive metal contamination, seagrass may initi-ate a variety of subcellular response which may causemetabolic turbulence and other damage at cellular lev-els (Hamoutene et al., 1996; Ralph and Burchett, 1998;Prange and Dennison, 2000; Macinnis-Ng and Ralph,2002). Although micro-nutrient metals such as Cu andZn are required in a number of enzyme systems, yetexcess of them can lead to senescence, chlorosis or evendeath of plants (Ebbs and Kochian, 1997).

The toxicity of metals is generally thought to be

Foundation item: The Chinese Nature Science Foundation (CNSF) Project under contract Nos 40776086 and 41076069; ForefrontProgram of the Knowledge Innovation Project, South China Sea Institute of Oceanology, Chinese Academy of Sciences undercontract No. LYQY200706; the National 908 Special Project under contract No. GD908-02-08.

∗Correspondent author, E-mail: xphuang@scsio.ac.cn

1

LI Lei et al. Acta Oceanol. Sin., 2012, Vol. 31, No. 3, P. 98-108 99

caused by oxidative stress which is due to uncontrolledand excessive production of reactive oxygen species(ROS), such as superoxide anion radicals (O·−

2 ), sin-glet oxygen (1O2), hydrogen peroxide (H2O2) and hy-droxyl radicals (OH·) (Salin, 1988; Apel and Hirt,2004). Excessive accumulation of ROS may be theconsequence of disruption of the balance between theirproduction and the scavenging of antioxidative sys-tem, which is composed of antioxidant enzymes suchas superoxide dismutase (SOD, EC 1.1.5.1.1), gua-iacol peroxidase (POD, EC 1.11.1.7) and catalase(CAT, EC 1.11.1.6), and non-enzymic scavengers, e.g.,carotenoids, ascorbate and glutathione (Vangronsveldand Clijsters, 1994). The imbalance causes lipid perox-idation, enzyme inactivation and DNA damage, result-ing in dramatic reduction of growth and productivityor even plant death (Stobrawa and Lorenc-Plucinska,2008).

Chlorophyll a fluorescence was reported as arapid, non-destructive way for the assessment of thephysiological effects of metal stress on photosynthesisof seagrass (Schreiber et al., 1994). Among all the vari-ables (Fm, Fo, Fv/Fm and ΔF/F ′

m), quantum yield ofphotosystem II (PS II) (ΔF/F ′

m) is the most sensitiveand rapidly responding variable in terms of monitor-ing the toxic effect of metals on plants (Ralph andBurchett, 1998). In seagrasses, the previous studieshave concentrated on seagrass morphology, taxonomyand distribution (den Hartog and Yang, 1990, Huanget al., 2006), or distribution and accumulation of met-als in seagrass tissues (Malea and Haritonidis, 1995)and effect of metals on photosynthesis of Zostera capri-corni and Halophila ovalis in Australia (Ralph andBurchett, 1998; Prange and Dennison, 2000; Macinnis-Ng and Ralph, 2002). Hamoutene et al. (1996) andFerrat et al. (2002a, b) investigated the effects of Cdand Hg on oxidative metabolism (CAT or glutathioneS-transferase activity) in Posidonia oceanica in theMediterranean area. To our knowledge, little infor-mation is found on photosynthetic activity and an-tioxidative response of tropical seagrasses under met-als stress especially for Thalassia hemprichii (Ehrenb.)Aschers. T. hemprichii is one of the most widelydistributed tropical seagrasses in the western Pacific(Mukai, 1993). Also, it is one of the two dominantseagrass species in the southeast of the Hainan Island,the South China Sea (Huang et al., 2006). Hence, itis necessary to study photosynthetic activity and an-tioxidative enzymes responses of tropical seagrass T.hemprichii treated with trace metals.

In the present study, T. hemprichii was exposedto elevated levels of Zn, Cd, Pb and Cu concentrationsand then physiological response and antioxidant de-fences of the seagrass to trace metals were determined.Especially, ΔF/F ′

m and photosynthetic pigments val-ues were used to assess the physiological changes ofphotosynthetic activity. Also the enzymatic activi-ties of SOD and POD in the leaves of T. hemprichiiwere detected to evaluate the antioxidant defences ofT. hemprichii.

2 Materials and methods

2.1 Seagrass material and trace metal aquar-

ium treatment

Plants of T. hemprichii were obtained from theXincun Bay, the Hainan Island, South China. Thewhole cores including seagrass and rhizosphere sedi-ment were collected to ensure that the samples werecomplete and undamaged. Then the intact sampleswere transferred to 1 L glass beakers and sealed withplastic bags to avoid excess water loss during trans-portation to the laboratory. Intact samples of T.hemprichii were placed in aerated glass aquaria, filledwith 15 L of seawater from the Xincun Bay. Plantsof equal size were placed in each of the 18 aquaria.Five hundred milliliters of 10% Hoagland’s solution(Hoagland and Arnon, 1950) was taken in each aquar-ium. Before metals treatment, seagrass samples wereacclimatized for one week in laboratory conditions[light intensity of 250 µmol·m−2·s−1 at the temper-ature of (25±2)℃]. For concentration-dependent ex-periments, seagrass plants were treated with concen-trations of Zn2+, Cd2+ (0, 10, 100, 300 µmol/L), Pb2+

and Cu2+ (0, 10, 50, 100, 150 µmol/L), maintainedin seawater in aerated glass aquaria under the abovementioned laboratory conditions for a period of 1, 2, 4(5), 7 and 10 d. Seagrasses in aquaria without treatedmetals kept with each set of experiment as control.After harvesting, epiphytes were removed from leavesby scraping and then plants were washed with doubledistilled water and used for the study of various pa-rameters. All glassware, sample containers and exper-imental chambers were cleaned with anionic detergent,then soaked in a 2 mol/L HNO3 acid-bath for 24 h,and finally rinsed with double distilled H2O.

2.2 Chlorophyll fluorescence analyses

Chlorophyll fluorescence was determined by PAMfluorometer (Walz, Germany). Steady-state chloro-

100 LI Lei et al. Acta Oceanol. Sin., 2012, Vol. 31, No. 3, P. 98-108

phyll fluorescence analysis was measured at ambienttemperature, using dark-adaptation clips (Walz, Ger-many). ΔF/F ′

m analysis was performed at a standard-ized position on the second leaf from the meristem,approximately in the middle of the adaxial surface.Fluorescence measurements were made over a discon-tinuous time-scale; and after the initial exposure, sam-ples were measured at hourly intervals for 10 h, andthen daily (10:00 am) for the following 10 d.

2.3 Determination of photosynthetic pigm-

ents

Fresh leaves were homogenized using a chilled pes-tle and mortar in 8 ml 80% acetone (Arnon, 1949)in a dark room. The homogenate was centrifugedat 4 000 r/min at 4℃ and the supernatant liquidwas used for chlorophyll a (Chl a), chlorophyll b (Chlb) and carotenoid estimation at 663, 646 and 470 nm,respectively and the results were expressed as mg/gfresh weight (FW).

2.4 Estimation of Zn, Cd, Pb and Cu

After being washed three times with double dis-tilled H2O, the harvested samples were immediatelyblotted and oven-dried at 105℃ for 20 min and thenkept at 80℃ for 72 h. The dried material was ground.The powdered samples were digested with HNO3-HClO4 solution and heated at 100–200℃ until a whitefume evolved. The contents of Zn, Cd, Pb and Cu weredetermined by a Varian Spectr AA 220 flame atomicabsorption spectrometer (AAS). Plant reference ma-terials (GBW 08517, China) were used to confirm thedetection accuracy.

2.5 Extraction and assays of enzymes

The 0.5 g fresh weight leaves were homogenizedin 50 mmol/L cold sodium phosphate buffer (pH7.8) including 0.1 mmol/L EDTA and 8.75% (w/v)polyvinylpyrrolidone (PVP, molecular weight 10 000)using a prechilled mortar and pestle in an ice bath.The homogenate was centrifuged at 10 500 r/min for30 min at 4℃ and the supernatant was used for en-zymes assays.

SOD activity was assayed by monitoring the inhi-bition of photochemical reduction of nitro-blue tetra-zolium (NBT) according to the method of Beauchampand Fridovich (1971). The 3-ml reaction mixture con-tained 50 mmol/L potassium phosphate buffer (pH7.8), 13 mmol/L methionine, 75 µmol/L NBT, 0.1mmol/L EDTA, 2 µmol/L riboflavin and 0.2 ml of en-

zyme extract. The reaction mixtures were illuminatedat an intensity of 4 500 lx for 20 min. The absorbanceof the supernatant at 560 nm was measured. One unitof SOD activity was defined as the amount of enzymerequired to cause 50% inhibition of the reduction ofNBT.

POD activity was determined as oxidation of gua-iacol by H2O2 (Upadhyaya et al., 1985). The reactionmixture was 3 ml of 200 mmol/L potassium phosphatebuffer (pH 6.0), 0.18% (v/v) H2O2, 1% guaiacol and0.5 ml of enzyme extract. The oxidation of guaiacolwas measured by the increase in absorbance at 470 nmevery 30 s [extinction coefficient 26.6 L/(mmol·cm)].One unit of POD activity was defined as units permin and mg of protein.

2.6 Statistical analysis

The analysis of variance (ANOVA) was done toconfirm the variability of data and validity of results,and Duncan’s multiple range test (DMRT) was per-formed to determine whether the differences betweentreatments were significant as compared with control.Data are presented as means±standard error (SE) ofthree replicates.

3 Results

3.1 Accumulation of metals in seagrass tis-

sues

The Zn, Cd, Pb and Cu accumulations in dif-ferent parts of T. hemprichii after 10-d exposure areshown in Table 1. The accumulations of four metalsin the aboveground and underground tissues were pro-portional with the increasing concentrations of metalsin seawater. In the treatments with low concentrationof Zn and Cd (10 µmol/L), metals concentrations inseagrass tissues were not significantly higher than thatof control. However, with the increasing concentrationof metals, significant increase in accumulation was ob-served at the treatment of Cu and Pb (>10 µmol/L),Cd and Zn (>100 µmol/L) as compared with control(Table 1). In all treatments, the concentrations of fourmetals in aboveground tissues were significantly higherthan that in underground tissues, with the ratio ofaboveground/underground concentrations for Zn, Cd,Pb and Cu varying between 1.7 to 4.7, 1.8 to 5.2, 1.1to 1.7 and 1.2 to 3.8, respectively. Especially, Cd accu-mulated in aboveground tissues of T. hemprichii wasmore than 7 000 times higher than control after 10-dexposure.

LI Lei et al. Acta Oceanol. Sin., 2012, Vol. 31, No. 3, P. 98-108 101

Table 1. Accumulation of metal in different parts of T. hemprichii after 10 d of growth under different concentrations

of that metal stress

Tissues Treatments/µmol·L−1 Accumulation of metals/mg·kg−1

Zn Cd Pb Cu

Aboveground control 81.03a±1.05 0.28a±0.02 14.80a±2.11 8.98a±0.23

10 186.05a±7.05 201.87a±16.90 111.40b±5.12 119.30b±6.32

50 111.80b±4.60 350.81c±19.63

100 1 144.97b±89.03 1 182.37b±205.02 107.00b±8.60 503.42d±31.36

150 108.90b±5.60 491.63d±25.32

300 1 452.24c±110.03 1 969.93c±208.04

Underground control 40.01ad±1.30 0.21ad±0.05 12.03a±5.51 9.25a±0.86

10 53.69d±4.30 33.37d±2.30 70.20c±7.20 71.83e±4.36

50 95.11bc±8.42 136.71f±6.31

100 245.88e±89.01 229.02d±12.30 97.06bc±8.91 252.30g±8.96

150 91.20dc±9.82 328.22h±6.37

300 519.83f±46.30 1 073.66e±99.30

Notes: All the values are mean of triplicates±SE. ANOVA significant at p �0.05. Different letters indicate significantly

different values at a particular duration (DMRT, p �0.05).

3.2 Effect of metals on quantum yield

(ΔF/F ′m)

Zinc exposure resulted in an obvious change to

ΔF/F ′m. It rapidly declined in all treatments dur-

ing the initial 4-h period. After 10-h exposure, thequantum yield continued to decline only under highconcentrations of Zn treatment (Fig. 1, Zn).

Fig.1. Effective quantum yields (ΔF/F ′m) for T. hemprichii over a 10-d exposure period to Zn, Cd, Pb and

Cu. Error bars indicate the mean of triplicates±SE.

102 LI Lei et al. Acta Oceanol. Sin., 2012, Vol. 31, No. 3, P. 98-108

T. hemprichii showed a limited photosyntheticstress when exposed to Cd (10–300 µmol/L) for 10d. The ΔF/F ′

m for all three Cd treatments declinedduring the initial 4-h period, with respect to the con-trol response. However, at 10 and 100 µmol/L treat-ments, it remained relatively stable, and the strongestresponse was observed at the 300 µmol/L during the10-d period (Fig. 1, Cd).

No significant effect on ΔF/F ′m was observed

when exposed to Pb (10 to 150 µmol/L) (Fig. 1, Pb).Exposure to Cu, from concentration 10 to 150

µmol/L, had a lethal effect on T. hemprichii. ΔF/F ′m

demonstrated a moderate decline in all Cu concen-trations in the initial 10 h. After 10-h exposure,the 10 µmol/L Cu treatment remained relatively sta-ble during the next 9 d exposure, while the 100 and150 µmol/L treated samples showed rapid decline inΔF/F ′

m. Leaves under 100 and 150 µmol/L treat-ments prematurely senesced from the second day andso fluorescence measurement could not be performed

for those treatments (Fig. 1, Cu).

3.3 Effect on photosynthetic pigments

Photosynthetic pigment analysis of Zn treatmentshows that Chl a and b content of all three treatmentswere lower than the control in the initial 24 h. After 2-d exposure, the Chl a and b content remained stable at10 µmol/L, whilst at 100 and 300 µmol/L treatmentscontinued declining (Figs 2a and b). All Zn treatmentsshowed elevated carotenoid content and the 10 µmol/Ltreatment had the highest level, however, no signifi-cant difference was observed when compared with thecontrol (Table 2).

Chl a and b content of all three Cd treatmentsdeclined rapidly during the initial 2-d exposure. Afterthat, it remained stable for 10 and 100 µmol/L treat-ments, while at 300 µmol/L treatments it continueddeclining. After 7-d exposure, at 10 and 100 µmol/Ltreatments, it returned to the same levels as thecontrols (Figs 2c and d). Total carotenoid increased

Fig.2. The content of chlorophyll (Chl a and Chl b) in the leaves of T. hemprichii exposed to different

concentrations of four trace metals. Error bars indicate the mean of triplicates±SE.

LI Lei et al. Acta Oceanol. Sin., 2012, Vol. 31, No. 3, P. 98-108 103

Table 2. Total carotenoids concentrations (mg/g) in leaves of T. hemprichii treated with different concentrations of

metals

Exposure Zn

Period/d Control 10 µmol/L 100 µmol/L 300 µmol/L

1 0.198a±0.011 0.212a±0.045 0.226a±0.021 0.237a±0.007

2 0.194a±0.017 0.343a±0.051 0.305a±0.067 0.294a±0.009

4 0.215a±0.009 0.324a±0.045 0.264a±0.020 0.279a±0.018

7 0.200a±0.017 0.225a±0.020 0.283a±0.014 0.242a±0.008

10 0.245a±0.045 0.281a±0.016 0.285a±0.003 0.254a±0.006

Cd

1 0.195a±0.032 0.196a±0.010 0.236a±0.028 0.260a±0.046

2 0.191a±0.014 0.268b±0.016 0.285b±0.013 0.186a±0.013

4 0.168a±0.015 0.300bc±0.011 0.374c±0.014 0.181ab±0.013

7 0.162a±0.014 0.165a±0.003 0.186a±0.011 0.222a±0.044

10 0.186a±0.024 0.248a±0.011 0.306a±0.013 0.324b±0.004

Pb

Control 10 µmol/L 100 µmol/L 150 µmol/L

1 0.192a±0.030 0.197a±0.050 0.204a±0.016 0.116a±0.006

2 0.266a±0.048 0.280a±0.036 0.289a±0.028 0.345a±0.010

5 0.278a±0.039 0.270a±0.011 0.284a±0.025 0.327a±0.071

7 0.300a±0.055 0.323a±0.028 0.301a±0.005 0.350a±0.005

10 0.280a±0.036 0.336a±0.078 0.325a±0.030 0.270a±0.002

Cu

1 0.176a±0.031 0.075b±0.008 0.037b±0.009 0.029b±0.005

2 0.156a±0.022 0.068b±0.008 0.016bc±0.003 0.006c±0.004

5 0.181a±0.018 0.047b±0.011 0.001b±0.001 0.000b±0.000

7 0.197a±0.020 0.072b±0.018 0.000c±0.000 0.000c±0.000

10 0.164a±0.007 0.080b±0.016 0.000c±0.000 0.000c±0.000

Notes: All the values are mean of triplicates±SE. ANOVA significant at p �0.05. Different letters indicate significantly

different values at a particular duration (DMRT, p �0.05).

under all Cd treatments as compared with the controland significant increase was detected under the 10 and100 µmol/L treatments during the 2-4 d exposure pe-riod (Table 2).

Lead treatment from 10 to 150 µmol/L had an ob-vious effect on chlorophyll in the initial 1-d exposure.And then Chl a and b stably increased from the 2ndto 5th day, and they decreased in the last three days(Figs 2e and f). Carotenoid content was also increasedbut no significant effect was observed (Table 2).

Exposure of the experimental material from 10 to150 µmol/L of Cu had a severe effect on photosyn-thetic pigments. Chl a and b declined rapidly at 50,100 and 150 µmol/L during the initial 24 h. After 2-d period, all treatments had a stable decrease and asignificantly lower level than the control was detected(Figs 2g and h). All Cu treatments had a significantlylower content of carotenoids than the control after 24-h exposure (Table 2).

3.4 Response of antioxidant enzymes

Different concentrations of Zn and Cd treatments(from 10 to 300 µmol/L) caused an increase of the en-

dogenous SOD activity in T. hemprichii leaves during10-d period (Fig. 3, Zn and Cd). Under Zn treat-ment at 100 and 300 µmol/L, SOD activity was sig-nificantly higher than control during 1 to 10 d whileat 10 µmol/L significant increase was observed onlyafter the 7th d. Meanwhile, significant increase wasdetected under all Cd treatments from the 4th d andthe highest response was observed after 10-d exposureas compared with the control.

Under Pb and Cu treatments (from 10 to 150µmol/L), SOD activity in T. hemprichii leaves exhib-ited a different response as compared with Zn and Cd.Under Pb treatments, SOD activity increased in theinitial two days. After 2-d exposure, SOD activityrapidly decreased. However, during all exposure pe-riod, SOD activity was lower than the control and sig-nificantly lower activity was observed from 5 to10 d.Under Cu stress, significant increase of SOD activitywas observed during 2-d exposure, and then a rapiddecrease was observed during the following eight days,of which, significant decrease was observed during 7 to10 d (Fig. 3, Pb and Cu).

POD activity in T. hemprichii leaves was higher

104 LI Lei et al. Acta Oceanol. Sin., 2012, Vol. 31, No. 3, P. 98-108

Fig.3. Effect of different concentrations of Zn, Cd, Pb and Cu on activity of superoxide dismutase (SOD)

in leaves of T. hemprichii. Error bars indicate the mean of triplicates±SE. Asterisks (*) indicate that mean

values are significantly different between metals treatments and controls (p <0.05).

than the control under all treatments of Zn, Cd andPb (5 to 10 d). Significantly higher POD activitywas observed under Zn and Cd treatments after 2-dexposure. Meanwhile, all Pb treatments showed sig-nificantly elevated POD activity during 7 to 10 d ascompared with the control (Fig. 4, Zn, Cd and Pb).Besides, a very different response of POD activity un-der Cu treatments was detected. It had already beenrestrained even at 10 µmol/L during the initial hours.In the following 9 d, POD activity of all treatmentswas lower than control (Fig. 4, Cu).

4 Discussion

T. hemprichii accumulated high amount of Zn,Cd, Pb and Cu depending on the concentration andduration. The uptake of four metals was proportionalwith its concentration in the aquarium and this co-incided with earlier studies obtained by Zn, Cd, Pb

and Cu treatments (Catalado et al., 1983; Caspi etal., 2000; MacFarlane and Burchett, 2001; Liu et al.,2010). This study shows that Zn, Cd, Pb and Cuare preferentially accumulated in aboveground tissues(blades) of T. hemprichii. The previous study re-ported that Cd and Pb were preferentially accumu-lated in blades of P. oceanica (Lafabrie et al., 2008).This result allows us to collect the samples (the blades)for the metal concentration analyses without removingthe entire T. hemprichii plants in future studies.

Among all chlorophyll a fluorescence parameters,quantum yield was considered as the most sensitiveparameter for measuring metal-induced stress (Ralphand Burchett, 1998). Estimation of ΔF/F ′

m is an in-dicator to detect responses of metal stress within 1 hof exposure. In the present study, a rapid decline ofΔF/F ′

m was observed during four hours of Cu and Znexposure in all concentrations. Especially, under highconcentrations of Cu treatments, a rapid decline of

LI Lei et al. Acta Oceanol. Sin., 2012, Vol. 31, No. 3, P. 98-108 105

Fig.4. Effect of different concentrations of Zn, Cd, Pb and Cu on activity of guaiacol peroxidase (POD) in

leaves of T. hemprichii. Error bars indicate the mean of triplicates±SE. Asterisks (*) indicate that mean

values are significantly different between metals treatments and controls (p <0.05).

ΔF/F ′m was continued during 2 to 10 d exposure. This

indicates that Cu was the most toxic metal tested.Our result consists with the previous studies in Zosteracapricorni exposed to different concentrations of met-als (Macinnis-Ng and Ralph, 2002). Although Cu isan essential microelement and a constituent of plasto-cyanins and plays important roles in electron transportsystem (Prasad and Strzalka, 1999), high concentra-tion of Cu can attributed to an overall inhibition of PSII, due to the alterations in the chloroplast membraneultrastructure, resulting in the K+ and Cu2+ leakageinto the chloroplast (Ouzounidou, 1994). In this study,ΔF/F ′

m remained stable after 2-d Zn exposure, so Znhas a less phytotoxic effect on T. hemprichii than Cu.Zinc may interact with the donor side of PS II andinhibit photosynthetic CO2 fixation and O2 release inthe Hill reaction (Prasad and Strzalka, 1999). Cad-mium and lead as nonessential elements displayed onlylimited effect on ΔF/F ′

m especially in the initial 24-h

period in this study. Seagrass may actively excludeor sequester Cd and Pb into structural componentsof the leaf tissues (e.g., vacuole), therefore preventingthem from affecting the more sensitive and importantphotosynthetic metabolic processes (Ward, 1989). Inaddition, the lower solubilities of Cd and Pb may leadto greater chelation and adsorption by colloidal parti-cles in seawater.

As a visible symptom, the reduced chlorophyllcontent can be used to monitor metals induced dam-age in seagrass leaves. In the present study, the lev-els of Chl a and Chl b decreased and to a lesser de-gree carotenoids increased in a dose-dependant man-ner with increasing exposure levels of Zn, Cd and Cu.This indicates that Zn, Cd and Cu stress could sig-nificantly reduce chlorophyll contents. The previousstudies reported that excess Zn, Cd and Cu could dam-age chloroplast, induce the loss of chlorophyll, causechloroplast disorganization and reduce the number of

106 LI Lei et al. Acta Oceanol. Sin., 2012, Vol. 31, No. 3, P. 98-108

thylakoid and grana (Ebbs and Kochian, 1997). Inaddition, high concentrations of Zn, Cd and Cu couldinhibit the uptake of nutrient elements through com-petition for binding sites (Wang et al., 2009). Thesenutrient elements are normally involved in many im-portant biological processes. For example, Mg is animportant component of chlorophyll. Iron and Man-ganese play important roles in synthesis or stabil-ity of chlorophyll (Marschner, 1995). Carotenoid, anon-enzymatic antioxidant, apart from functioning asaccessory light harvesting photosynthetic pigments,plays an important role in the protection of chlorophyllpigments under metals stress by quenching photody-namic reactions and replacing peroxidation (Sinha etal., 2010). The previous studies reported increase(Drazkiewicz and Baszynski, 2005), decrease (Huangand Wang, 2010) or no change (Mishra et al., 2006b)in the content of carotenoid in plants under differentconcentrations of various metal stresses. In this study,carotenoid was significantly increased under low Cdconcentration treatment but decreased when treatedwith high concentrations of Cd and all concentrationsof Cu. The lowest carotenoid content observed un-der Cu treatment in this study may add to the stress-induced damage of Cu to seagrass leaves. Because thiscould lead to a diminished capacity to protect photo-systems in leaves against photooxidation and resultedin more oxidative stress and senescence of leaves (DiToppi et al., 2009). Lead was reported to be lessdamaging to the photosynthetic apparatus, and nosignificant inhibitory effect of Pb on photosyntheticpigments was observed after long term exposure espe-cially under low Pb treatments (<50 µmol/L) in thisstudy which was consistent with the previous study(Ahmed and Tajmir-Riahi, 1993).

Apart from damage to chloroplast and inducingthe loss of chlorophyll, the excessive uptake of tracemetals had also been reported to interfere in cellularmetabolism through production of ROS by autooxi-dation and Fenton reaction (Schutzendubel and Polle,2002). ROS can lead to the response of plants to ox-idative stress, including enhancement or suppressionof activities of antioxidative enzymes (Apel and Hirt,2004). SOD acts as the first line of defense againstROS, and catalyses O·−

2 to H2O2. In the present study,SOD activity was higher than the control under all Znand Cd treatments during the whole experiment whileit decreased and was much lower than the control af-ter long-term exposure to Pb and Cu. This suggeststhat SOD in T. hemprichii is more tolerant to Zn and

Cd than Cu and Pb. The increased SOD activities inleaves of T. hemprichii peaked at high concentrationof Zn and Cd exposure indicated that SOD playedan important role in protecting seagrass against Znand Cd stress. Enhanced SOD activity was also re-ported in leaves of T. caerulescens under Zn and Cdstress (Wojcik et al., 2006). The decreased SOD ac-tivities in the present study after long term exposureto high concentrations of Cu and Pb consisted withthe report by Mishra et al. (2006a). The reason forthe decrease might be inactivation of SOD by H2O2

or binding of metals to active center of the enzyme(Fatima and Ahmad, 2005). POD is another princi-pal enzyme involved in the elimination of ROS, and ismainly located in cytoplasm, vacuole, membrane andcell wall. It is considered as stress marker enzymehaving broad specificity for phenolic compounds andhigher affinity for H2O2 than CAT. The previous re-searches also reported that an increase in the level ofmetals could bring with it an increase in enzyme activ-ities, which could be used as environmental biomark-ers (Pergent-Martini and Pergent, 2000). POD canbreak down H2O2 into H2O. The previous study re-ported that POD activity in Hydrilla verticillata (L.f.)Royle increased till 7 d at 25 µmol/L Cu exposure(Srivastava et al., 2006). In this study, POD activityincreased under Zn, Cd and Pb stress but decreasedunder Cu stress as compared with the control. The de-creased POD activities also suggest that T. hemprichiiis very sensitive to Cu and is very easy to be severelydamaged by Cu stress. Meanwhile, decreased POD ac-tivities of T. hemprichii under Cu stress might resultin accumulation of H2O2 which could cause disorderof metabolism including low photosynthesis efficiencyand the decreased content of protein and sugar or evendeath of plants (Cao et al., 2004). Elevated POD ac-tivity in T. hemprichii under Zn, Cd and Pb stressmight contribute to enhancing the ability to cope withmetal stress with a stiffened cell wall by producingphenoxy compounds that are polymerized to producelignin (Reddy et al., 2005).

Finally we concluded that T. hemprichii was verysensitive to Cu: the photosynthetic metabolic organcould be severely damaged and the plant might evendie. T. hemprichii had positive response by increas-ing the activity of SOD and POD. Especially, PODwas the most important ROS scavenging enzyme inprotecting seagrass from metals stress, and the antiox-idative protection mechanisms of T. hemprichii weremore efficiently activated to avoid damage of Zn, Cd

LI Lei et al. Acta Oceanol. Sin., 2012, Vol. 31, No. 3, P. 98-108 107

and Pb stress. Besides, the seagrass T. hemprichii,in view of its high accumulation potential of Cd, highefficiency of avoiding damage to the photosyntheticsystems of leaves and high activities of SOD and PODinduced by Cd, seems to be suitable candidate to de-contaminate inshore sea areas moderately contami-nated with Cd. Although metals concentrations inthis study might be higher than the bioavailable met-als in seagrass ecosystems, the information presentedmight help us understand the physiological detoxifica-tion strategies that seagrasses adapt to metals stress.Acknowledgements

The authors wish to thank Mr. Zhang Jingping,Jiang Zhijian, Peng Bo and Miss Fan Minling for sam-pling collections. We also would like to thank the edi-tors and reviewers for comments and suggestion to themanuscript.

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