Combined toxicity of cadmium and copper in Avicennia marina seedlings and the regulation of exogenous jasmonic acid

Ecotoxicology and Environmental Safety 113 (2015) 124–132

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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv

Combined toxicity of cadmium and copper in Avicennia marina seedlings and the regulation of exogenous jasmonic acid Zhongzheng Yan a,b,n, Xiuzhen Li a,b, Jun Chen a,b, Nora Fung-Yee Tam c,nn a

State Key Laboratory of Estuarine and Coastal Research, East China Normal University, Shanghai, China Institute of Estuarine and Coastal Research, East China Normal University, Shanghai, China c Department of Biology and Chemistry, City University of Hong Kong, Hong Kong, China b

art ic l e i nf o

a b s t r a c t

Article history: Received 23 July 2014 Received in revised form 3 November 2014 Accepted 25 November 2014

Seedlings of Avicennia marina were exposed to single and combined metal treatments of cadmium (Cd) and copper (Cu) in a factorial design, and the combined toxicity of Cu and Cd was tested. The effects of the exogenous jasmonic acid (JA) on chlorophyll concentration, lipid peroxidation, Cd and Cu uptake, antioxidative capacity, endogenous JA concentration, and type-2 metallothionein gene (AmMT2) expression in seedlings of A. marina exposed to combined metal treatments were also investigated. A binary mixture of low-dose Cd (9 mmol L
1) and high-dose Cu (900 mmol L
1) showed toxicity to the seedlings, indicated by the significant augmentation in leaf malondialdehyde (MDA) and reduction in leaf chlorophylls. The toxicity of the combined metals was significantly alleviated by the addition of exogenous JA at 1 mmol L
1, and the chlorophyll and MDA contents were found to be restored to levels comparable to those of the control. Compare to treatment with Cd and Cu only, 1 and 10 mmol L
1 JA significantly enhanced the ascorbate peroxidase activity, and 10 mmol L
1 JA significantly decreased the uptake of Cd in A. marina leaves. The relative expression of leaf AmMT2 gene was also significantly enhanced by 1 and 10 mmol L
1 JA, which helped reduce Cd toxicity in A. marina seedlings. & 2014 Elsevier Inc. All rights reserved.

Keywords: Jasmonic acid Mangrove Metallothioneins Combined metals Stress

1. Introduction Mangroves are constantly exposed to increasing pollution from human activities because of the rapid industrialization and urbanization in coastal areas (Harbison, 1986; Tam, 2006; MacFarlane et al., 2007; Wu et al., 2014). Considering the anoxic nature of mangrove sediment, heavy metals from incoming tidal waters and freshwater sources can be rapidly removed from the water body and deposited in the sediment, making the mangrove sediment a depository for metals. This phenomenon poses challenges to the establishment and growth of mangrove seedlings (Harbison, 1986; Tam, 2006). Heavy metal toxicity in plants mainly occurs through the induction of oxidative stress with the production of reactive oxygen species (ROS) such as superoxide radical (O⋅− 2 ), hydroxyl radical (OH), and hydrogen peroxide (H2O2) (Blikhina et al., 2003). ROS damages the cellular components, such as membranes, nucleic acids and chloroplast pigments, and it enhances lipid peroxidation (Molas, 2002; Prasad, 2004). Plants develop a series n Corresponding author at: State Key Laboratory of Estuarine and Coastal Research, East China Normal University, Shanghai, China. Fax: 86 21 62546441. nn Corresponding author. Fax: þ 852 3442 0522. E-mail addresses: [email protected] (Z. Yan), [email protected] (N.F-Y. Tam).

http://dx.doi.org/10.1016/j.ecoenv.2014.11.031 0147-6513/& 2014 Elsevier Inc. All rights reserved.

of mechanisms involving enzymatic antioxidants [such as superoxide dismutase (SOD), peroxidase (POD), ascorbate peroxidase (APX), and catalase (CAT)] and nonenzymatic scavengers [mainly glutathione (GSH) and ascorbic acid (AsA)] to scavenge ROS and reduce the toxic effects of heavy metals (Prasad, 2004). Apart from their antioxidative defense systems, plants also relieve heavy metal stress by synthesizing chelating ligands, mainly including metallothioneins (MTs) and phytochelatins (PCs) (Gonzalez-Mendoza et al., 2007; Huang and Wang, 2010). MTs are a family of cysteine-rich proteins that are suggested to be involved in the cellular detoxification of toxic metals, such as cadmium (Cd) and mercury (Hg), in animals, plants, eukaryotic microorganisms, and prokaryotes (Cobbett and Goldsbrough, 2002). The black mangrove, Avicennia germinans, can overexpress MT responsive genes, which constitute a coordinated detoxification response mechanism targeting nonessential metals such as Cd (Gonzalez-Mendoza et al., 2007). Similar findings were also reported in mangrove species, Bruguiera gymnorrhiza (Huang and Wang, 2009) and Avicennia marina (Huang and Wang, 2010), when subjected to zinc (Zn), copper (Cu), or lead (Pb) stress. Jasmonic acid (JA) is a ubiquitously occurring lipid-derived compound with signal functions in plant responses to abiotic and biotic stresses, as well as plant growth and development (Wasternack, 2007). Plants under heavy metal stresses are prone to

Z. Yan et al. / Ecotoxicology and Environmental Safety 113 (2015) 124–132

adjust their endogenous JA levels for adaptation, which helps in relieving metal toxicity in plants (Koeduka et al., 2005; Maksymiec et al., 2005). Application of exogenous JA at certain levels also caused relieving effects on plants under stresses of heavy metals. These relieving effects caused by JA were reported mainly in the enhancement of antioxidant capacity and the reduction of malondialdehyde (MDA) and hydrogen peroxide (H2O2) contents (Piotrowska et al., 2009; Keramat et al., 2009; Kováčik et al., 2011), JA also shows alleviatory effect on photosynthetic pigments and activity of photosystem II that impaired by toxic metals (Maksymiec and Krupa, 2002; Maksymiec et al., 2007; Piotrowska et al., 2009). Other phytohormones, such as abscisic acid (ABA) (Salt et al., 1995; Zhao et al., 2006), 2,4-epibrassinolide (brassinolide) (Kanwar et al. 2012), and indole acetic acid (IAA) (Gangwar and Singh, 2011), were also reported to protect plants by reducing oxidative injuries from heavy metal stress. Moreover, ABA and brassinolide protect plants from heavy metal stress by reducing the uptake of toxic metals (Cd or Nickel (Ni)) to the aboveground part of the plant (Salt et al., 1995; Zhao et al., 2006; Kanwar et al., 2012). The single effects of heavy metals on metal accumulations, growth, and physiology of mangrove seedlings have been intensively studied (MacFarlane et al., 2007; Huang and Wang, 2009, 2010; Yan and Tam, 2013a, 2013b). However, in natural conditions, sediments are normally polluted by a mixture of substances. Heavy metals simultaneously exert their toxicity, and the toxicity of combined metals, as well as the mechanisms involved in the interaction, is still not fully understood (Shuhaimi-Othman and Pascoe, 2007). In general, a mixture of heavy metals can produce three types of interactions: synergistic, antagonistic, and non-interactive, and the types varied with the metal combination ratio and tolerance of the different plant parts (Sharma et al., 1999; An et al., 2004; Wani et al., 2007; Metwali et al., 2013). Previous studies have been conducted to investigate the combined effects of metal compounds on plant species. It has been previously reported that in A. marina (gray mangrove), Pb and Zn in combination resulted in an increased accumulation of both metals in leaf tissue and increased toxicity than individual metals alone (MacFarlane and Burchett, 2002). The single and combined toxicity of Cu, Cd and Pb was investigated in Cucumis sativus, and the combined treatment of Cu and Cd showed both antagonistic and additive effect in the growth of shoot and root, respectively (An et al., 2004). The combination of Cd with copper (24 and 1338 mg kg
1) showed a greater synergistic toxic effect on dry biomass and N contents of plant tissues of greengram than that observed for single application of Cd and Cu (Wani et al., 2007). To date, studies on the mechanism of the alleviatory effects of JA on plants under combined metal stress are insufficient, and the mechanisms by which exogenous JA elicits protective effects are still unknown, especially for mangrove species. Cu and Cd are the two major widespread heavy metal pollutants in mangrove wetlands (Fung and Lo, 1997; Tam and Wong, 2000; Ong Che, 1999). Cu is an essential micronutrient and a component of several enzymes participating mainly in electron flow and catalyzing redox reactions However, Cu exhibits toxicity at high concentrations, whereas Cd has no known biological and therapeutic function regardless of concentration, and is a highly toxic metal to aquatic organisms such as Lactuca sativa (Dias et al., 2013). Accordingly, the present study aims to investigate the effects of exogenous JA in mangrove seedlings, A. marina, subjected to stress caused by the combined metals.

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2. Materials and methods 2.1. Experimental setup Healthy propagules of A. marina were collected from a mangrove swamp in Beihai, China on September 2013. The propagules were comparable in size (fresh weight of 3.4270.45 g) and were free of insect damage. The propagules were sown on sand in plastic pots with half-strength Japanese garden test nutrient solution: 945 mg L
1 Ca(NO3)2, 809 mg L
1 KNO3, 493 mg L
1 MgSO47H2O, 153 mg L
1 NH4HPO4, 20 mg L
1 C10H12FeN2NaO8  3H2O, 2.86 mg L
1 H3BO3, 2.13 mg L
1 MnSO4  4H2O, 0.22 mg L
1 ZnSO4  7H2O, 0.08 mg L
1 CuSO4  5H2O, 0.02 mg L
1 Na2MoO4  2H2O (Hori, 1966). The pot we used in the experiments consists of two containers, the internal container was filled with sands and sown with A. marina seedlings, and the external container filled with treatment solution. In order to decrease the deleterious effect of waterlogging, we lifted the internal container out of the treatment solution for 6 h each day during the experiment. The planted pots were placed on a bench in an artificial climate chamber with a daily temperature of 25 °C, a relative humidity of 70%, and a light intensity of 800 mmol photons m
2 s
1 with a 16:8 light:dark photoperiod. The seedlings were irrigated with deionized water once a day until the third pair of leaves completely unfolded. According to previous studies, Cd concentration in natural mangrove wetlands in south China usually ranges from 0 (not detected) to 58 μmol kg
1 DW, whereas Cu concentration, which ranges from 288 to 1440 μmol kg
1 DW, is much higher than the concentration of Cd (Fung and Lo, 1997; Tam and Wong, 2000; Ong Che, 1999). In view of this, the present study selected two different concentrations of Cd (9 and 90 μmol L
1) and Cu (90 and 900 μ mol L
1), which are relevant to their background values, to investigate their single and combined toxicity in A. marina seedlings. Nine groups of seedlings in triplicates were used for the experiments, and the treatment were set as follows: (i) control; (ii) 1 mg L
1 Cd (Cd1); (iii) 10 mg L
1 Cd (Cd2); (iii) 10 mg L
1 Cu (Cu1); (v) 50 mg L
1 Cu (Cu2); (vi) Cd1þ Cu1; (vii) Cd1þ Cu2; (viii) Cd2þCu1; and (ix) Cd2þCu2. Appropriate amounts of cadmiu chloride (CdCl2) and copper chloride (CuCl2) were dissolved in half-strength Japanese garden test nutrient solution to make the different treatment solutions. At day 9 after the treatments, the seedlings were collected, and the MDA and endogenous JA contents in leaves were determined. In order to investigate the possible mitigation effects of exogenous JA on A. marina seedling subjected to combined metals stress (HM), we set the following six groups of treatments: (i) control; (ii) HM; (iii) HM þ0.1 μmol L
1 JA (JA1); (iv) HMþ 1 μ mol L
1 JA (JA2); (v) HMþ 10 μmol L
1 JA (JA3); and (vi) HMþ 100 μmol L
1 JA (JA4). To ensure the combined metals is toxic to the seedlings, the treatment that induce the highest MDA production (i.e. Cd1 þCu2) were chosen. For the treatment of combined metals (HM) with different levels of JA, appropriate amounts of JA (Sigma-Aldrich, USA) were dissolved in 0.5 mL of ethanol and diluted to obtain appropriate concentrations with half-strength Japanese garden test nutrient solution containing 1 mg L
1 Cd and 50 mg L
1 Cu. Appropriate volumes of ethanol were also supplemented in the treatment of control and HM to maintain the consistency of the experiment. The seedlings were irrigated with deionized water every day to compensate for the water lost by evaporation. During the experiment, the treatment solution was replaced every 2 days to prevent depletion of nutrients and changes in the concentrations of the metals and JA. Seedlings of all the above 15 treatments were collected on day 9 after the treatment. To investigate the time course changes of leaf MDA, endogenous JA, and antioxidative enzymes, the leaves of the seedlings from the following treatment groups were collected on days 1, 5, and 21 after the following treatments: (i) control; (ii)

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Cd1þ Cu2 (As HM); (iii) JA2 and (iv) HM þJA2. At each sampling time, one seedling in each pot was carefully pulled from the sediment and sufficiently washed with deionized water. The leaves and roots of the seedlings were separated, rapidly frozen in liquid nitrogen, and then stored at
80 °C until further analyses. Parts of the leaf and root samples were collected and then dried in an oven at 70 °C for 48 h for the analyses of Cd and Cu. The treatment solutions were also collected at the end of the experiments (Treatments of control, HM, HMþ JA1, HM þJA2, HM þJA3 and HM þJA4 at day 9, and treatments of control, HM, JA2 and HM þJA2 at day 21), and the exposure concentrations of Cd, Cu and JA were investigated according to the methods described below. Results showed that the nominal and real applied metal and JA doses were not significantly different according to t-test; therefore, we used the nominal exposure concentrations in the following analysis. 2.2. Determinations 2.2.1. Leaf pigment concentration Leaf chlorophyll concentration was determined according to the method described by Chen and Chen (1984). In brief, 0.1 g of fresh leaf sample was finely sliced with stainless steel scissors to increase the surface area of the tissue exposed to the extractant. The sample was placed in a 15-mL amber glass screw-cap bottle containing 10 mL of a mixed solution of acetone, ethanol, and distilled water (4.5:4.5:1) and then stored in the dark at 4 °C for 2 days. The absorbance readings of the extract were recorded at 470, 645, and 663 nm, and the concentrations (mg L
1) of chla, chlorophyll b (chlb) and total chlorophylls (total chls) in the extract were calculated using the following equations:

( ) − 1 Chlorophyll b (mg g FW ) = (22.9A645−4.68A663 ) × V × 1000/FW , Total chlorophylls (mg g −1FW )

Chlorophyll a mg g −1FW = (12.7A663−2.69A645 ) × V × 1000/FW ,

= (20.2A645 + 8.02A663 ) × V × 1000/FW , where A is the optical density, V is volume (mL) of the extracting solution, and FW is the fresh weight (g) of the leaf sample. 2.2.2. MDA MDA concentration (CMDA) in the leaf was determined according to the method described by Wang and Jin (2005) with modifications. In the standard procedure, 0.2 g of fresh plant sample was homogenized using a mortar and pestle with 4 mL of 20% trichloroacetic acid (TCA) (w/v). The homogenate was centrifuged at 9000g for 5 min. Then, 1 mL of the supernatant was mixed with an equal volume of 0.6% (w/v) thiobarbituric acid solution composed of 10% TCA. The mixture was heated in boiling water for 30 min and then transferred to an ice bath to stop the reaction. The cooled mixture was centrifuged at 5000g for 10 min at 25 °C, and the absorbance readings of the supernatant at 450, 532, and 600 nm were recorded. CMDA was calculated according to the following equation:

CMDA = 6.45 × (A532–A600) –0.56 × A450, where A450, A532, and A600 represent the absorbance of the supernatant at 450, 532, and 600 nm, respectively. 2.2.3. Cd and Cu concentrations in leaves and roots Cd concentration in plant tissues was determined according to the method described by Wong et al. (1993). Approximately 0.2– 0.3 g of oven-dried plant sample was charred on a hot plate for approximately 1 h and then incinerated in a muffle furnace at

500 °C for 6 h. The ash was digested and diluted in 20 mL of 1% nitric acid. Cd and Cu in the digested solutions were analyzed using an atomic absorption spectrometer (PerkinElmer AAnalyst 800). The detection limits for Cd and Cu were 0.039 and 11 mg kg
1 DW, respectively. The accuracy of the method was determined in terms of the recovery of spiked Cd (CdCl2) and Cu (CdCl2) standards in homogenized plant tissue samples at 20 mg L
1, and the average recoveries and standard error for Cd and Cu were 91.5% 7 9.9% and 87.0% 72.4% (n ¼3), respectively. Translocation factor (TF) was calculated to estimate the transport of the accumulated metal from root to leaf (MacFarlane et al., 2007). The calculation was based on the following formula: TF = [metal conc. in leaf]/[metal conc. in root] 2.2.4. Endogenous JA Endogenous JA concentration was determined using an enzyme-linked immunosorbent assay (ELISA) kit (Rapidbio, USA) according to the manufacturer's instructions. Approximately 100 mg tissue was rinsed once with 1  phosphate-buffered saline (PBS), containing 137 mmol L
1 sodium chloride (NaCl), 2.7 mmol L
1 potassium chloride (KCl), 8 mmol L
1 disodium hydrogen phosphate (Na2HPO4), and 1.46 mmol L
1 potassium dihydrogen phosphate (KH2PO4), then homogenized in 1 mL of 1  PBS and stored overnight at
20 °C. Two freeze-thaw cycles were performed to break the cell membranes, then the homogenates were centrifuged for 5 min at 9000g at 4 °C. The supernatant was used for the hormone assay. Standards or samples were added to appropriate microtiter plate wells with horseradish peroxidase (HRP)-conjugated JA and then incubated. Subsequently, a competitive inhibition reaction was launched between JA (in standards or samples) and HRP-conjugated JA with the precoated antibody specific for JA. The detection limit for the endogenous JA was 80 pmol L
1. 2.2.5. Activity of APX Approximately 0.3 g of fresh leaf sample was extracted in 4 mL of 50 mmol L
1 ice-cold sodium phosphate buffer (pH 7.4) combined with 1.0 mmol L
1 ethylenediaminetetraacetic acid disodium salt (EDTA-Na2). The homogenate was centrifuged at 9000g for 10 min at 4 °C, and the supernatant was used for the enzyme assay. APX activity was determined according to the method described by Nakano and Asada (1981). Approximately 100 mL of enzyme extract was mixed with 2 mL of 50 mmol L
1 potassium phosphate buffer (pH 7.4) containing 0.5 mmol L
1 AsA, 1 mmol L
1 EDTA-Na2, and 0.1 mmol L
1 H2O2. Enzyme activity was determined by monitoring the decrease in absorbance at 290 nm for 2 min. The molar absorption coefficient of AsA at 290 nm (2.8 mM
1 cm
1) was used to calculate the units (U) of the enzyme activity. One unit of APX was defined as the amount of enzyme that oxidized 1 mmol AsA per minute, and the activity was expressed as U mg
1 protein
1. 2.2.6. Relative expression of type-2 metallothionein (AmMT2) gene The total RNA of leaf tissue was extracted using Trizol reagent (Invitrogen). 50–100 mg of fresh leaf sample was sufficiently ground with liquid nitrogen. 1 mL of Trizol reagent was added, and then maintained at room temperature for 5 min. Trichloromethane (0.2 mL) was added to the extractant and mixed thoroughly. The mixture was centrifuged at 12,000g for 10 min at 4 °C. The supernatants were transferred to new RNase-free tubes, mixed with 0.5 mL of isopropanol, and then centrifuged at 12,000g for 10 min at 4 °C after 30 min of incubation. The supernatants were removed, and the precipitate was washed with 1 ml of 75% ethanol (diluted in diethylpyrocarbonate (DEPC) water) and then centrifuged at 7500g for 5 min at 4 °C. The supernatant was removed, the tubes were air dried at room temperature, and the RNA

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was dissolved using RNase-free H2O. First-strand cDNA was synthesized using an AMV First-Strand cDNA Synthesis kit by Sangon Biotech, Shanghai, China. Real-time quantitative polymer chain reaction (RT-PCR) was performed on an ABI StepOne Plus analyzer with ABI SybrGreen PCR master mix. The A. marina type-2 metallothionein mRNA complete coding sequence submitted to GenBank (Accession No. AF333385) was used to design the primers for RT-PCR. The forward and reverse primers were 5′-GAAATTCTATGAGGGAGCAGAGTCT-3′ and 5′-ATGGATTGCAGGTGCAGTTG-3′, respectively. 18S rRNA was used as the reference gene to normalize the expression levels among the samples, and the forward and reverse primers were F: 5′-GTGGAGCGATTTGTCTGGTTA-3′ and R: 5′-CCTGTTATTGCCTCAAACTTCC-3′, respectively. The products were analyzed through a melt curve analysis to determine the specificity of PCR amplification. Each reaction was performed twice, and the fold induction in AmMT2 mRNA expression relative to the control was determined by the ΔΔ standard 2
CT method (Livak and Schmittgen, 2001).

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combined metals, as well as the different levels of exogenous JA treatments. Statistical analyses were performed in SPSS version 16.0.

3. Results 3.1. Effects of single and combined CU and Cd on leaf MDA concentration MDA is the product of membrane lipid peroxidation. MDA levels usually indicate the degree of membrane oxidative damage. In the present study, two-way ANOVA indicated that both Cu and Cd alone had no significant effect on leaf MDA content (P ¼0.627 and 0.130 for Cd and Cu, respectively), the interactive effect (CdnCu) was also not significant (P¼ 0.327). However, significant increases in leaf MDA concentration were observed under the combined metal treatments of Cd1þCu2 and Cd2þ Cu1 on day 9, compare to control (Fig. 1A).

2.3. Statistical analyses The mean and standard deviations of the three replicates for each treatment were calculated. A two-way ANOVA was applied to examine any significant effects of the Cd and Cu and their interaction (CdnCu) on leaf MDA and JA contents. Parametric one-way ANOVA and post-hoc multiple comparison (Tukey's test) were conducted to determine the significant differences in other parameters among the different treatment samples of single and

3.2. Effect of exogenous JA on photosynthetic pigments and leaf MDA content The MDA content, which increased by the combined metal treatments, was restored to nontoxic level under the treatment of exogenous JA at 1 mmol L
1 (Fig. 1B), and this effect was also observed at day 9 after the treatment (Fig. 1C). Low to moderate levels of exogenous JA, including 0.1, 1, and 10 mmol L
1, also

Fig. 1. (A) Effect of single and combined effect of Cd and Cu on MDA concentration in leaves of A. marina seedlings at day 9; Changes of (B) MDA and (D) chlorophylls concentrations in leaf of A. marina seedlings after 9 days treatment of combined Cd and Cu and different concentrations of JA, and (C) changes of MDA concentration in leaves of A. marina seedlings under different treatment of combined metals and JA at different times (values are mean and SD; for each parameter, data with different letters are significantly different at P r0.05; no letter appended if the data were not significantly different).

Z. Yan et al. / Ecotoxicology and Environmental Safety 113 (2015) 124–132

a

a

+J A4

a

H M

a

+J A3

l t ro co n

+J A4 H M

+J A3 H M

+J A2 H M

b C a N.D N.D

H M

100

+J A2

abc

bc

0 +J A1

t ro co n

abc

B

200

+J A1

c D N.D N.D

l

0

ab

H M

5

C

C

a

AB AB

300

H M

C

10

A

400

H M

B

15

Cu conc. (mg g-1 DW)

A

20

leaf root

A

500

H M

Cd conc. (µ g g -1DW)

25

H M

128

Fig. 2. The accumulation of (A) Cd and (B) Cu in leaves and roots of A. marina seedlings after 9 days treatment of combined metals and different concentrations of JA (values are mean and SD; for each plant part, data with different letters are significantly different at Pr 0.05; N.D, not detected under the detection limit of Cd:0.039 μg kg
1 DW, Cu:11 μg kg
1 DW).

alleviated the deleterious effects of combined metals on chls, and the concentrations of chlb and total chls were both restored to levels comparable with the chlorophyll concentrations of the control (Fig. 1D). 3.3. Effect of exogenous JA on Cd and CU accumulation in roots and leaves After 9 days of treatment, both Cd and Cu showed significant accumulation in the leaves and roots of A. marina under the treatment of combined Cd and Cu (Fig. 2A). Treatment of exogenous JA at 1, 10, and 100 mmol L
1 significant reduced the accumulation of Cd in roots of A. marina seedlings compared with the corresponding Cd levels in the roots without JA treatment. Similarly, the uptake of Cd in leaves of A. marina was reduced by 10 mmol L
1 JA (Fig. 2A), and the translocation factor (TF) also significantly decreased by 1 and 10 mmol L
1 JA (Table S1 of Supporting information). Differently, the concentration of Cu under different levels of JA did not exhibit significant reduction and was more or less comparable with its corresponding concentration under the combined metal treatment (Fig. 2B). Exogenous JA did not exhibit significant effect on the changes of TF of Cu compared with its corresponding TF under the combined metal treatment (Table S1 of Supporting information).

3.4. Effect of exogenous JA on leaf APX activities APX activity in the leaves of A. marina was depressed by the treatment of combined Cd and Cu (Cd1 þCu2, represented as HM in Fig. 3A) on day 9, however, HM treatments with JA2 (1 m mol L
1) and JA3 (10 mmol L
1) significantly enhanced APX activity compared with the corresponding APX activity in the control and combined metal treatment. The effect of single JA (JA2, 1 m mol L
1) and combined JA and HM (HM þ JA2) on the antioxidative enzyme activity were also investigated on days 1, 5, and 21 after the treatment (Fig. 3B). APX activity on days 1 and 9 showed similar changing patterns, which was depressed by the combined metal treatment (HM), and restored by exogenous JA treatment (HM þ JA2) (Fig. 3B). Changes of APX activity on day 5 also showed a similar pattern although the changes was not statistically significant. However, no significant change in APX activity was observed on day 21 after the treatment (Fig. 3B). 3.5. Changes of endogenous JA Two-way ANOVA indicated that both Cu and Cd alone had a significant stimulatory effect on content of endogenous JA (P ¼0.012 and 0.002 for Cd and Cu, respectively), however, the interactive effect of Cd and Cu on leaf JA content at day 9 was not

Fig. 3. Changes of (A) APX activity in leaf of A. marina seedlings after 9 days treatment of combined metals and different concentrations of JA, and (B) leaf APX activity under different treatment of combined metals and JA at different times (values are mean and SD; for each parameter, data with different letters are significantly different at Pr 0.05; NS, not significant).

Z. Yan et al. / Ecotoxicology and Environmental Safety 113 (2015) 124–132

129

Fig. 4. (A) Single and combined effect of Cd and Cu on leaf JA concentrations in A. marina seedlings at day 9; (B) changes of leaf JA concentration under treatment of combined metals and different concentrations of exogenous JA at day 9; (C) changes of leaf JA concentration under different treatment of combined metals and exogenous JA at different times (values are mean and SD; for each parameter, data with different letters are significantly different at P r 0.05; NS, not significant).

significant (F-value ¼1.016, P ¼0.425). JA concentrations in the leaves of A. marina under single and combined treatments of Cd and Cu showed different changing patterns after 9 days of treatment (Fig. 4). On day 9, significant increases of leaf JA concentrations were observed under treatments of Cu2, Cd1 þ Cu1, Cd1þ Cu2, Cd2 þCu1, and Cd2 þCu2 (Fig. 4A). Leaf JA concentrations under the combined treatments of exogenous JA and combined metals (HM þJA2) showed a constant increasing pattern with the increase of exogenous JA concentration, and the significant increases were observed under HM þJA4 treatment (Fig. 4B). The treatment of HMþ JA2 at days 5 and 9 both showed stimulatory effects on endogenous JA comparing to the control (Fig. 4C). Treatments with JA2 (1 mmol L
1) alone showed no significant effect on endogenous JA on days 1, 5 and 21 comparing to the other treatments (Fig. 4C). 3.6. Effect of exogenous JA on the relative expression of AmMT2 The expression of AmMT2 of the A. marina seedlings were significantly enhanced under the combined treatment using Cd1 and Cu2, which was approximately 3.5-fold of the corresponding AmMT2 expression in the control (Fig. 5). Compared with the treatment of combined metals alone, the expression of AmMT2 were further enhanced by the application of exogenous JA at 1 and 10 mmol L
1 concentrations, which were 8.0 and 8.4-fold, respectively, of the corresponding AmMT2 expression in the control (Fig. 5A). Low and high doses of JA (0.1 and 100 mmol L
1) did not show stimulatory effects on the relative expression of AmMT2.

Fig. 5. (A) Relative expression of AmMT2 gene in leaves of A. marina seedlings after 9 days treatment of combined metals and different concentrations of JA (values are mean and SD; data with different letters are significantly different at P r0.05); (B) PCR gel electrophoresis.

4. Discussion In the present study, the combined treatment of low level Cd (Cd1, 9 mmol L
1) and high level Cu (Cu2, 900 mmol L
1) increased the MDA content and significantly decreased the chlorophyll concentration, indicated the toxic effect of the combined metals of Cd1 and Cu2 on the cell membrane of the A. marina seedlings. As the single treatments of Cd1 and Cu2 both had no significant toxicity in A. marina seedlings (no over-production in leaf MDA as indicated in Fig. 1A), suggested that the combined toxicity of Cd1þ Cu2 can then be induced by a potentiation effect of these two metals. MacFarlane and Burchett (2002) reported that Pb and Zn showed additive toxicity in seedlings of A. marina, that Pb treatments of 400 mg g
1 DW in combination with Zn (250, 500 and 1000 mg g
1 DW) exhibit greater toxicity than Zn alone. In the combined Cd and Cu stress, Cd can replace Cu in various cytoplasmic and membrane proteins, thus increasing the amount of unbound free Cu ions which participating in oxidative stress via

the Fenton reaction and induced the synergistic toxicity of Cd and Cu (Valko et al., 2005). The toxicity of the combined Cd and Cu in A. marina seedlings was ameliorated by the addition of exogenous JA at a certain level, as the results showed that exogenous JA at 1 mmol L
1 significantly restored the total chlorophyll concentration and MDA contents to the levels similar to those of the control, compared with the corresponding chlorophyll and MDA contents in the combined treatment of Cd1þ Cu2 alone. Previous studies found that JA affects the activity of stress enzymes, thereby causing alleviation of oxidative stress in plant cells (Piotrowska-Niczyporuk et al., 2012; Noriega et al., 2012). JA at 0.1 mmol L
1 also activates the enzymatic (CAT, APX and POD) and nonenzymatic (AsA and GSH) antioxidant systems of Wolffia arrhiza (Piotrowska et al., 2009). Previous study also found that MeJA at 0.1–1 mmol L
1 significantly increased the activities of CAT and APX in the leaves of Kandelia obovata (Chen et al., 2014). Results of the previous

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study are in agreement with the results of the present study, which indicates that exogenous JA at 1 and 10 mmol L
1 significantly restored, or even enhanced, APX activity, which helped alleviate stress caused by ROS damages on the cell membrane and photosynthetic pigments. Aside from antioxidative enzymes, the present study also showed that exogenous JA at certain concentrations had regulatory effect on the Cd uptake in leaves and roots of the seedlings (Fig. 2A). As the aboveground part, plant leaf is more sensitive to the incoming metal ions than the roots. In the present study, the translocation of Cd from the roots to the leaves of A. marina under treatments of 1–10 mmol L
1 of JA was significantly reduced compared with the translocation of Cd in A. marina treated only with combined metals, which might also help reduce the direct injuries caused by Cd to the leaves. The reduced translocation of Cd from root to leaf was more directly reflected in the changes in TF of Cd as shown in Table S1 of Supporting information. This results are in accordance with a previous finding which indicates that the application of JA at 0.1 mmol L
1 significantly inhibits Pb accumulation in W. arrhiza (Piotrowska et al., 2009). However, as the smallest spermatophyte, W. arrhiza has neither roots nor even vein in leaf, which is different from the subject plant of the present study. Other signal molecules, such as ABA, brassinolide, and nitric oxide (NO), involved in multiple plant responses to environmental stresses were also reported to reduce the uptake of toxic metals, such as Cd (Salt et al., 1995; Zhao et al., 2006; Meng et al., 2009), Ni (Kanwar et al., 2012), and Cu (Dong et al., 2013) in the shoots of hydroponically grown plants. The reduced uptake of metals under ABA treatments was suggested to be induced by the reduced transpiration rate and symplastic loading of Cd into the xylem (Salt et al., 1995). Loading of Cd into the xylem of the plant is mainly influenced by plant transpiration (Lux et al., 2011). Similar to ABA and brassinolide, JA was believed to also reduce plant transpiration by promoting stomatal closure (Suhita et al., 2004; Hossain et al., 2011), which might help in the reduced uptake of Cd in the leaves of A. marina. Previous studies on the effect of another phytohormone, indole acetic acid (IAA), on the uptake of certain nonessential heavy metals can provide pieces of circumstantial evidence to prove that JA reduces Cd uptake. IAA is a common auxin which usually exerts diametrically adverse effect on the respiratory rate of plants compared with the effects of JA, ABA, or brassinolide on plant respiratory rate. The exogenous application of IAA (10
10 and 10
4 mol L
1) significantly increases Pb accumulation in the shoots of Sedum alfredii Hance (Liu et al., 2007) and sunflower (Fässler et al., 2010). However, exogenous JA reduces Cr uptake at low levels (10 mmol L
1) in the seedlings of Pisum sativum L. under Cr treatment (Gangwar and Singh, 2011), which indicates that the respiration theory cannot fully explain the reduced uptake of Cd investigated in the present study. To verify the effects of JA on plant respiration, future works are necessary to follow the changes of respiration rate of the seedlings under the combined metal treatment and different levels JA treatment. The reduction in metal uptake by certain levels of exogenous JA in the present study was observed only for Cd (such as 10 m mol L
1 JA for leaves and 1, 10 and 100 mmol L
1 JA for roots) and not Cu. In many cases, intracellular Cd uptake displayed Michaelis– Menten kinetics, and Cu usually caused mixed inhibition (both competitively and non-competitively) on intracellular Cd uptake (Noraho and Gaur, 1995; An et al., 2004). The addition of Cu significantly decreased Cd concentration in the shoots of maize (Zea mays L.) and wheat (Triticum aestivum L.), which is possibly attributed to metal competition at common root absorption sites (Keltjens and van Beusichem, 1998). An et al. (2004) also reported that under the binary mixture of Cu and Cd treatment, the accumulation of Cu in shoots was adversely influenced by the presence of Cd in soil, and Cd uptake to the shoots was decreased by Cu

addition. The presence of Cu may increase the Michaelis–Menten constant and decreased the maximal uptake rate of Cd, thus causing the inhibition of Cd uptake in the seedlings of Lemna polyrhiza L. (Noraho and Gaur, 1995). Reduced accumulation of Cd in the roots of A. marina might arise as a result of the two cations sharing a common mode of uptake and competing for common adsorption and transport sites on the cell surface. Endogenous JA is suggested to be involved in the cellular response to metal toxicity (Maksymiec and Krupa, 2002). Increases in endogenous JA were also observed in Arabidopsis thaliana and Phaseolus coccineus treated with Cu and Cd (Maksymiec et al., 2005), pea plants treated with Cd (Koeduka et al., 2005), and mangrove seedlings, such as K. obovata, Acanthus ilicifolius, and Excoecaria agallocha treated with Pb (Yan and Tam, 2013a, 2013b). In the present study, the amount of endogenous JA increased significantly under combined Cd and Cu treatment (HM) and HM together with different levels of exogenous JA (HM þ JA2/3/4) compare to the control at day 9, which might play roles in the anti-stress physiology of A. marina seedlings under metal toxicity. The increases of leaf endogenous JA levels under HM þJA3 treatment were also observed at day 5 but showed no significant changes at days 1. This results were in accordance to previous study on Capsicum frutescens var. fasciculatum, that Cd and JA treatments did not induce any significant increase in endogenous JA content at days 1 and 7 (Yan et al., 2013). However, prompt increases in endogenous JA (7 hours to 1 day) were also found in other species such as A. thaliana subjected to Cu or Cd stress (Maksymiec et al., 2005), and candel, A. ilicifolius, and E. agallocha subjected to Pb stress (Yan and Tam, 2013a, 2013b). These discrepancies suggest that the time series response of the endogenous JA to metal stress may vary with plant species and the metals treatment. In the case of the present study, endogenous JA possibly also underwent changes in the early stages (i.e., several hours) of the stress, which might account for the significant upregulation in leaf APX activity by 10 mmol L
1 exogenous JA at day 1. All gene-encoding enzymes of JA biosynthesis are JA-inducible, and JA biosynthesis is regulated by a positive feedback (Wasternack, 2004, 2007). In the present study, endogenous JA burst under the exogenous JA treatment may be attributed to de novo biosynthesis or the intake of exogenous JA per se, which need further investigation. Plant MT genes are divided into four types, in which type-2 MT genes are mainly expressed in the leaves instead of the roots (Cobbett and Goldsbrough, 2002). In the present study, AmMT2 expression in the leaves of A. marina was significantly upregulated by the combined metal treatment, which indicates that MTs are part of the heavy metal tolerance mechanism in the leaves of A. marina, and might help detoxify the intake of heavy metals and mitigate oxidative stress. Results of the present study is in agreement with previous studies on mangrove species, such as A. germinans exposed to Cd and Cu (Gonzalez-Mendoza et al., 2007), B. gymnorrhiza (Huang and Wang, 2009), and A. marina (Huang and Wang, 2010) exposed to Zn, Cu, and Pb, and K. candel exposed to Zn, Cu, Pb, and Cd (Huang et al., 2012), at varying treatment periods (3–11 days). In the present study, exogenous JA significantly enhanced the expression of leaf AmMT2 compared with its corresponding expression under the combined metal treatments, which suggests that enhanced tolerance in A. marina seedlings against combined metal stress may also be attributed to the upregulation in AmMT2 expression. In a previous study, exogenous MeJA did not regulate (at 0.1 mmol L
1) or even downregulated (at 1 mmol L
1) the expression of leaf type-2 MT gene (KoMT2) in K. obovata seedlings under Cd stress (Chen et al., 2014), which indicates that the regulatory effect of JA on type-2 MT gene expression might not be applicable in other members of the jasmonates family, such as MeJA. Aside from MTs, plants are also

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protected from toxic metals (mainly Cd and Cu) by phytochelatins (PCs), a group of cysteine-rich peptides formed by the polymerization of GSH catalyzed by phytochelatin synthase (Grill et al., 1985; Xiang and Oliver, 1998; Cobbett and Goldsbrough, 2002). Previous studies showed that exogenous JA treatments increased the mRNA levels and the capacity for the synthesis of the GSH in Arabidopsis under Cd stress (Xiang and Oliver, 1998). Significant increases of PCs were also observed in Arabidopsis plants under the combined treatment of Cd and MeJA (Maksymiec et al., 2007). Results of the present study suggest that exogenous JA might enhance the synthesis of both PCs and MTs on the transcript level, which helps alleviate Cd toxicity.

5. Conclusions In summary, the present study revealed that certain levels of exogenous JA (1 or 10 mmol L
1) involved in the anti-stress physiological processes in seedlings of A. marina under the combined toxicity of Cd and Cu, which indicated by the reduction in leaf and root Cd accumulation, enhancement in the expression of leaf AmMT2, and the boost in the pool of APX enzymes. These results of the present study provide new knowledge in metal stress amelioration of JA in mangrove seedlings, and also provided useful insight on the management of heavy metal pollution in the seedling culture and afforestation practice. Additional works with a wide variety of metals, species, and exposure concentrations are needed for a more comprehensive understanding on the regulatory effect of JA.

Acknowledgments The present study was jointly supported by National Natural Science Foundation of China (No. 41201525) and the Research Foundation of State Key Laboratory of Estuarine and Coastal Research (SKLEC-2012RCDW02). No conflict of interest exits in the submission of this manuscript.

Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ecoenv.2014.11. 031.

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