Ecotoxicology and Environmental Safety 104 (2014) 349–356
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Effect of methyl jasmonate on cadmium uptake and antioxidative capacity in Kandelia obovata seedlings under cadmium stress Jun Chen, Zhongzheng Yan n, Xiuzhen Li n State Key Laboratory of Estuarine and Coastal Research, East China Normal University, Shanghai, China
art ic l e i nf o
a b s t r a c t
Article history: Received 9 November 2013 Received in revised form 11 January 2014 Accepted 19 January 2014
This study investigated the effects of methyl jasmonate (MeJA) on chlorophyll concentration, lipid peroxidation, Cd uptake, antioxidative capacity, and type-2 metallothionein gene (KoMT2) expression in the leaves of Kandelia obovata seedlings exposed to Cd stress. Deleterious effects, including decreased chlorophyll content and increased malondialdehyde concentration, were observed in leaves of K. obovata after 9 d of 200 μmol L 1 Cd treatment. Application of MeJA (0.1 to 1 μmol L 1) increased the concentration of ascorbic acid and the activities of catalase and ascorbate peroxidase in the leaves of K. obovata, which helped alleviate the oxidative damage induced by Cd stress. The concentration of endogenous jasmonic acid in the leaves of K. obovata was decreased by Cd but was positively stimulated by exogenous MeJA. The expression of KoMT2 in the leaves was enhanced after 9 d of 200 μmol L 1 Cd treatment, while the exogenous application of MeJA significantly restored the expression of KoMT2. Exogenous MeJA also inhibited the uptake of Cd to the aboveground part (leaves) of the seedlings, which helped reduce direct damages of Cd to the photosynthetic organ of the plant. The reduced uptake of Cd might be a result of stomatal closure and decreased transpiration by exogenous MeJA. & 2014 Elsevier Inc. All rights reserved.
Keywords: Jasmonic acid Mangrove Metallothionein Heavy metals Stress
1. Introduction Mangroves are under increasing heavy metal pollution pressure from human activities because of the rapid industrialization and urbanization in coastal areas (Harbison, 1986; MacFarlane, 2002; Tam, 2006). Metals at environmental concentrations exceeding the maximum tolerable amount damage the physiological functions of plants, thereby resulting in photosynthetic output reduction and growth degeneration (Prasad, 2004). The metals Cd, Pb, and Hg are strongly phytotoxic; this phytotoxicity can be partly attributed to the generation of reactive oxygen species (ROS), such as superoxide radical (O2d ), hydroxyl (∙OH), and hydrogen peroxide (H2O2) (Prasad, 2004). High levels of ROS usually damage cellular components, such as membranes, nucleic acids, and chloroplast pigments, resulting in lipid peroxidation (Tewari et al., 2002). Plants, including mangroves, develop a series 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 (MacFarlane, 2002; Prasad, 2004). Aside from their antioxidative defense systems, mangroves, similar to other
n
Corresponding authors. Fax: þ 86 21 62546441. E-mail addresses: [email protected] (Z. Yan), [email protected] (X. Li).
0147-6513/$ - see front matter & 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ecoenv.2014.01.022
plants, relieve heavy metal stress by chelating various ligands, including metallothioneins (MTs) (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 Cd and Hg, in animals, plants, eukaryotic microorganisms, and prokaryotes (Cobbett and Goldsbrough, 2002; Huang and Wang, 2010). The black mangrove Avicennia germinans has can overexpress MT responsive genes, which constitute a coordinated detoxification response mechanism targeting nonessential metals (Gonzalez-Mendoza et al., 2007). Similar findings were also reported in mangroves species Bruguiera gymnorrhiza (Huang and Wang, 2009) and Avicennia marina (Huang and Wang, 2010) when subjected to Zn, Cu, or Pb stress. Jasmonic acid (JA) and methyl jasmonate (MeJA) are members of a family of cyclopentanone compounds synthesized from linolenic acid via the octadecanoic pathway; these compounds exhibit important functions in the signaling network of plants under various biotic and abiotic stresses (Fujita et al., 2006). Plants under heavy metal stresses are prone to adjust their endogenous phytohormone levels for adaptation (Santner and Estelle, 2009). Abiotic stresses, such as metal stress, stimulate endogenous JA in herbaceous plants, such as Arabidopsis thaliana, Phaseolus coccineus, and Oryza sativa L. (Koeduka et al., 2005; Maksymiec et al., 2005). Moreover, JA also exhibits protective effects on plants under various abiotic stresses, including heavy metals (Walia et al., 2007; Keramat et al., 2009). These protective effects mainly include
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enhancing antioxidant capacity and reducing malondialdehyde (MDA) and H2O2 contents as well as protecting photosynthetic pigments (Maksymiec and Krupa, 2002; Piotrowska et al., 2009; Kováčik et al., 2011b). The stress hormone abscisic acid (ABA) and the plant steroid hormone brassinolide (2,4-epibrassinolide) also protect plants from abiotic stresses by reducing the plant uptake of Cd (Salt et al., 1995; Zhao et al., 2006) and Ni (Kanwar et al., 2012). To date, studies on the mechanism of the alleviatory effects of MeJA on plants under metal stress remain insufficient, especially for mangrove species. Available studies mainly focused on model plants (A. thaliana) and herbaceous plants (Maksymiec and Krupa, 2002; Piotrowska et al., 2009; Kováčik et al., 2011b). Abiotic stresses, such as metal stress, stimulate the production of endogenous JA in herbaceous plants, such as A. thaliana, P. coccineus, and O. sativa L. (Koeduka et al., 2005; Maksymiec et al., 2005). Studies on the effect of exogenous MeJA treatment on endogenous JA responses in metal-stressed plants are insufficient, and the mechanism by which exogenous MeJA elicits protective effects remains unknown. Accordingly, this study aims to investigate whether or not exogenous MeJA also exerts protective effects on mangrove seedlings under heavy metal stress. The effect of different MeJA concentrations on the antioxidative capacity of toxic metal-stressed mangrove seedlings was examined, and variations in the uptake of toxic metals and in the expression levels of type 2 MT responsive genes were investigated. The annual perennial mangrove species, Kandelia obovata, was selected as the plant model. Cd was selected as the model heavy metal because it is a nonessential element that is very common in wetland sediment (Tam, 2006).
2. Materials and methods 2.1. Experimental setup and sample collection Healthy propagules of K. obovata were collected from mangrove swamp in Beihai, China, and the propagules were comparable in terms of size (length of 18.5 7 1.9 cm, fresh weight of 10.8 7 2.6 g) and were free of insect damage and fungal infections. The propagules were sown on sand in plastic pots [24 cm (open top) 30 cm (height) 20 cm (flat bottom)] with half-strength Japanese garden test nutrient solution (Hori, 1966). The planted pots were placed on a bench in an artificial climate chamber with a daily temperature of 25 1C, a relative humidity of 70 percent, 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 every day until the third pair of leaves completely unfolded. The pots were then randomly divided into nine groups each in triplicate. Five groups were used to determine the effects of MeJA on the responses of the seedlings under Cd treatments: (i) control treatment; (ii) 200 mmol L 1 Cd; (iii) 200 mmol L 1 Cd þ 0.1 mmol L 1 MeJA (MeJA1); (iv) 200 mmol L 1 Cd þ 1 mmol L 1 MeJA (MeJA2); and (v) 200 mmol L 1 Cd þ10 mmol L 1 MeJA (MeJA3). For the treatment of 200 mmol L 1 Cd together with different levels of MeJA, appropriate amounts of MeJA (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 200 mmol L 1 Cd (CdCl2). For the treatment with 200 mmol L 1 Cd only, 0.5 mL ethanol was supplemented to keep the consistency of the treatment. One group with appropriate amounts of ethanol served as the control group. 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 two days to prevent depletion of nutrients and changes in the concentrations of Cd and MeJA concentrations. Samples were collected at 9 d after the treatment. The treatment solutions were also collected at the end of the experiments, and the exposure concentrations of Cd and MeJA were investigated. Results showed that the nominal and real applied Cd doses were not significantly different according to t-test; therefore, we used the nominal exposure concentrations in the analysis. Changes in CAT, APX, and POD activities and AsA content at different times after the treatments were determined by setting four treatments as follows: (i) control group; (ii) 200 mmol L 1 Cd; (iii) 1 mmol L 1 MeJA; and (iv) 200 mmol L 1 Cdþ 1 mmol L 1 MeJA. The seedlings were collected at 1, 5, and 11 d after the treatment. For sample collection, the 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 1C until further analyses. Parts of the leaf and root samples were collected and then dried in an oven at 70 1C for 48 h. 2.2. Determinations 2.2.1. Chlorophyll concentration Leaf chlorophyll concentration was determined according to the method described by Wang et al. (2009). In brief, 0.1 g of fresh leaf sample was cut from a mature leaf and then 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 1C for 2 d. The absorbance readings of the extract were recorded at 645 and 663 nm, and the concentrations (mg L 1) of chlorophyll a (chl a), chlorophyll b (chl b), and total chlorophyll (total chls) in the extract were calculated according to method described by Arnon (1949). 2.2.2. Cd concentration in plant tissue Cd concentration in plant tissues was determined according to the method described by Wong et al. (1993). Approximately 0.2 g to 0.3 g of oven-dried plant sample was charred on a hot plate for approximately 1 h and then ashed in a muffle furnace at 500 1C for 6 h. The ash was digested and diluted in 10 mL of 1 percent nitric acid. Cd in the digested solutions was analyzed with an atomic absorption spectrometer (PerkinElmer AAnalyst 800). The accuracy of the method was determined in terms of the recovery of spiked Cd standards (as CdCl2) in homogenized plant tissue samples at 10 mg L 1. The average recoveries and SE of Cd were 91.579.9 (n¼ 3), and the limit of detection for Cd was 0.05 mg kg 1 DW. 2.2.3. MDA concentration MDA concentration in the leaf was determined according to the method described by Wang and Jin (2005) with modifications. In a typical procedure, 0.2 g of fresh plant sample was homogenized using a mortar and pestle with 4 mL of 20 percent trichloroacetic acid (TCA) (w/v). The homogenate was centrifuged at 9000 g for 5 min. Then, 1 mL of the supernatant was mixed with an equal volume of 0.6 percent (w/v) thiobarbituric acid solution comprising 10 percent 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 5000 g for 10 min at 25 1C, and the absorbance readings of the supernatant at 450, 532, and 600 nm were recorded. The MDA concentration (CMDA) was calculated according to the following equation: C MDA ¼ 6:45ðA532 A600Þ 0:56A450:
2.2.4. AsA concentration AsA concentration was determined according to the method described by Law et al. (1983). The assay is based on the reduction of Fe3 þ to Fe2 þ by ascorbic acid and on the formation of a pink color complex between Fe2 þ and 2, 20 -bipyridyl, with a maximum absorption at 525 nm. In brief, 0.2 g of fresh leaf sample was extracted by grinding fresh leaf tissue in 5 percent TCA, and the homogenate was centrifuged at 9000 g for 10 min. Then, 0.2 mL of the supernatant was mixed with equal volumes of 150 mmol L 1 phosphate buffer (pH 7.4) and deionized water. The mixture was maintained at ambient room temperature for 30 s. Then, 400 mL of 10 percent TCA, 400 mL of 44 percent (v:v) phosphate acid, 400 mL of 4 percent 2,20 bipyridyl (w:v, in 70 percent ethanol), and 200 mL of 3 percent (w/v) FeCl3 were added, mixed, and then incubated in water bath at 37 1C for 60 min. The mixture was centrifuged at 9000 g at 4 1C for 5 min, and the absorbance readings of the supernatant at 525 nm were recorded. L-AsA was used as standard. 2.2.5. Endogenous JA concentration Endogenous JA concentration was determined using an ELISA kit (Rapidbio, USA) according to the manufacturer's instructions. About 100 mg tissue was rinsed with 1X 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), 1.46 mmol L 1 potassium dihydrogen phosphate (KH2PO4), then homogenized in 1 mL of 1 PBS and stored overnight at 20 1C. After two freeze-thaw cycles were performed to break the cell membranes, the homogenates were centrifuged for 5 min at 9000 g at 4 1C. 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. 2.2.6. Antioxidative enzyme activity 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 9000 g for 10 min at 4 1C, and the supernatant was used for the enzyme assay.
3. Results 3.1. Growth of the seedlings and chlorophyll and MDA concentrations The growth of the seedling was not affected after 9 days treatments of Cd and MeJA (Fig. 1a). The concentrations of chl a, chl b, and total chls significantly reduced after 9 d of 200 mmol L 1 Cd treatment (Fig. 1b). However, 200 mmol L 1 Cd treatment with 0.1 mmol L 1 MeJA, significantly restored the chlorophyll content reduced by Cd stress (Fig. 1a). The concentration of MDA in the
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The mean and standard deviation of the three replicates for each treatment were calculated. One-way ANOVA and post-hoc multiple comparison (Tukey's test) were conducted to determine significant differences in the detected parameters among the different levels of Cd treatments. Two-way multivariate ANOVA (MANOVA), with treatment and treatment time as the independent variables, was applied to examine the significant interactive effects and differences between K. obovata seedlings based on different variables (i.e., AsA content and APX, CAT, and POD activities in the leaves). Statistical analyses were performed in SPSS version 16.0.
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2.3. Statistical analyses
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2.2.7. RT-PCR expression of type-2 MT gene Total RNA of leaf tissue was extracted using Trizol reagent (Invitrogen). Briefly, 50 mg to 100 mg of fresh leaf sample was sufficiently ground with liquid nitrogen, added with 1 mL of Trizol reagent, 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 12,000 g for 10 min at 4 1C. The supernatants were transferred to new RNase-free tubes, mixed with 0.5 mL of isopropanol, and then centrifuged at 12,000 g (4 1C) for 10 min after 30 min of incubation. The supernatants were removed, and the precipitate was washed with 1 ml of 75 percent ethanol (diluted in DEPC water) and then centrifuged at 7500 g for 5 min at 4 1C. The supernatant was removed, the tubes were air dried at room temperature, and the RNA was dissolved by RNase-free H2O. First-strand cDNA was synthesized using an AMV First-Strand cDNA Synthesis kit by Sangon Biotech, Shanghai, China. Real-time quantitative PCR was performed on an ABI StepOne Plus analyzer with ABI SybrGreen PCR master mix. The K. candel complete MT cDNA sequence submitted to GenBank (Accession No. DQ414691) was used to design primers for RT-PCR. The forward and reverse primers were 50 TCTTGCTGTGGTGGAAACTG-30 and 50 -ATCTCGGCTCCCTCAAAGT-30 , respectively. 18S rRNA was used as the reference gene to normalize the expression levels between the samples, and the forward and reverse primers were F: 50 -CCCGTTGCTGCGATGAT-30 and R: 50 -GCTGCCTTCCTTGGATGTG-30 , 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 KoMT2 mRNA expression relative to the control was determined by the standard 2 ΔΔCT method (Livak and Schmittgen, 2001; Huang and Wang, 2010).
MDA conc. (µmol g-1 fw)
CAT activity was determined according to the method described by Beer and Sizer (1952) with modifications. The reaction mixture (2.5 mL) consisted of 50 mmol L 1 phosphate buffer (pH 7.4), 0.1 mmol L 1 EDTA-Na2, 20 mmol L 1H2O2, and 0.5 mL of enzyme extract. The reaction was initiated by adding the extract. The decrease in H2O2 was monitored at 240 nm for 2 min and quantified based on its molar extinction coefficient (36 mol L 1 cm 1). One unit (U) of CAT was defined as the amount of enzyme that decomposes 1 mmol of H2O2 per minute, and the activity was expressed as U mg 1 protein 1. Guaiacol POD activity was determined according to the guaiacol method (Fielding and Hall, 1978). Approximately 100 mL of enzyme extract was mixed with 3 mL of 50 mmol L 1 phosphate buffer (pH 7.4) containing 0.2 percent guaiacol (v:v). The reaction was initiated with 1 mL of 0.3 percent H2O2, and guaiacol oxidation was determined based on an increase in the absorbance at 470 nm for 2 min. POD activity was expressed as units of enzyme activity (U), and the units were calculated based on the molar extinction coefficient of tetraguaiacol (26.6 mM 1 cm 1). One unit was defined as the amount of enzyme catalyzing the formation of 1 μmol tetraguaiacol per minute, and the activity was expressed as U mg 1 protein 1. APX activity was estimated 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 1H2O2. 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.
chlorophyll conc. mg g-1FW
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Fig. 1. (a) Exterior appearance of K. obovata seedlings, changes of (b) chlorophyll a, chlorophyll b and total chlorophylls, and (c) MDA concentrations in leaves of K. obovata seedlings after 9 days treatment of Cd (200 mmol L 1) and different concentrations of MeJA (0.1, 1, and 10 μmol L 1 for MeJA 1, 2 and 3, respectively; Values are mean and SD; data with different letters are significantly different at Pr0.05).
leaves of K. obovata significantly increased by 200 mmol L 1 Cd. However, exogenous MeJA, especially at 1 mmol L 1 concentration, alleviated this Cd stress-induced increase in MDA (Fig. 1c). 3.2. Cd accumulation in leaves and roots After 9 d of 200 mmol L 1 Cd treatment, the leaves and roots of K. obovata accumulated 248.5 and 384.0 mg g 1 DW of Cd, respectively, which are significantly higher than the Cd content
J. Chen et al. / Ecotoxicology and Environmental Safety 104 (2014) 349–356
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alone (Fig. 3g). However, MeJA treatment alone did not stimulate the concentration of AsA in the leaves at 1 and 5 d after the treatment (Fig. 3h). MANOVA, followed by Wilk's lambda test, showed that treatment and treatment time as well as their interactions significantly affected the production of AsA in the leaves of K. obovata (Table S1 of supporting information).
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Fig. 2. Accumulation of Cd in roots and leaves of K. obovata seedlings after 9 days treatment of Cd (200 mmol L 1) and different concentrations of MeJA (0.1, 1, and 10 μmol L 1 for MeJA 1, 2 and 3, respectively) (Values are mean and SD; For each plant parts, data with different letters are significantly different at Pr 0.05; ND indicates not detected).
in the control group (not detected under the detection limits of 0.05 μg g 1 DW) (Fig. 2). By contrast, Cd treatment with different MeJA concentrations significantly inhibited the translocation of Cd to the leaves. The concentration of Cd in the leaves under MeJA treatment ranged from 22.6 mg g 1 to 98.4 mg g 1 DW, which is much lower than that in roots (582.6–757.7 μg g 1 DW). However, the addition of different MeJA concentrations did not affect the root uptake of Cd Fig. 2. 3.3. Changes in antioxidants In this study, 9 d of Cd treatment did not affect APX activity in the leaves, whereas Cd treatment with different MeJA concentrations significantly increased APX activity in the leaves compared with the seedling leaves under control and Cd treatments (Fig. 3a). However, 1 mmol L 1 MeJA did not significantly stimulate APX activity in the leaves on days 1, 5, and 11 (Fig. 3b). Two-way MANOVA analyses showed that the influences of the two factors as well as their interactions significantly affected the activity of APX (Table S1 of supporting information). Cd stress stimulated CAT activity in the leaves of K. obovata from day 5 after the treatment. The stimulations were negative on day 1 and became positive on days 5 and 11 (Fig. 3d). Treatment with MeJA and 200 mmol L 1 Cd for 9 d significantly enhanced CAT activity in the leaves (Fig. 3c); similar effects were also observed on days 5 and 11. In contrast to APX, leaf CAT activity showed positive stimulation at low levels of MeJA (0.1 and 1 mmol L 1) but not at the highest level (10 mmol L 1, Fig. 3c). Guaiacol POD detoxifies a large family of peroxides, including H2O2. In this study, POD activity showed no changes at 1, 5, 9, and 11 d after 200 mmol L 1 Cd treatment (Fig. 3e and f). In general, POD activity increased with increasing exogenous MeJA concentration, and POD activity only significantly increased compared with the control group at the highest MeJA level (Fig. 3e). Exogenous MeJA at 1 mmol L 1 significantly enhanced POD activity on day 5 only (Fig. 3f). Treatment and treatment time as well as their interactions significantly affected CAT and POD activities (Table S1 of supporting information). Cd significantly reduced AsA concentration in the leaves of K. obovata at 1 d to 5 d after the treatment (Fig. 3h). However, this effect was not observed at 9 and 11 d after the treatment (Fig. 3g and h). The addition of different levels of MeJA significantly restored the concentration of AsA in the leaves compare to that under treatment of 200 mmol L 1 Cd (Fig. 3g). The addition of 1 μmol L 1 MeJA also significantly increased the concentration of AsA in the leaves compared with the leaves under Cd treatment
3.4. Changes in endogenous JA The level of endogenous JA in the leaves of K. obovata was significantly decreased by Cd at 200 mmol L 1. This reduction effect was alleviated by the addition of MeJA, and endogenous JA concentration increased with increasing exogenous MeJA concentration (Fig. 4). 3.5. mRNA expression of KoMT2 The mRNA expression of KoMT2 mRNA in the leaves of K. obovata seedlings increased by fivefold (Po0.01) under 200 mmol L 1 Cd treatment compared with that under the control treatment (Fig. 5). However, this amplification of KoMT2 gene expression was abated or even restored by the addition of exogenous MeJA at 1 and 10 μmol L 1.
4. Discussion Most plants are sensitive to low Cd concentrations, which inhibit plant growth as a consequence of alterations in photosynthetic rate (Sandalio et al., 2001; Benavides et al., 2005; RodriguezSerrano et al., 2009). In the present study, the concentrations of chl a, chl b, and total chls in the leaves of K. obovata significantly decreased after 9 d of 200 mmol L 1 Cd treatment. This result indicates that the synthesis/accumulation of the pigments was interfered by Cd. The inhibition in photosynthetic pigment accumulation in response to heavy metal stress might be a consequence of chloroplast membrane peroxidation through enhanced rates of H2O2 production and lipid peroxidation in chloroplast membranes (Piotrowska-Niczyporuk et al., 2012). Low concentrations of MeJA (normally within the levels of 0.1 to 1 mmol L 1) usually exert protective effect on chlorophylls of plant under toxic metal treatment (Maksymiec and Krupa 2002; Keramat et al., 2009; Piotrowska et al., 2009; Kovač́ ik et al., 2011b). Similar results were observed in the present study. That is, low level (0.1 mmol L 1) of MeJA significantly restored the impairment induced by Cd in leaf chlorophylls, especially chl a (under 0.1 mmol L 1 MeJAþ 200 mmol L 1 Cd treatment). These results suggested that MeJA elicited protective effects during leaf photosynthesis under Cd stress. Other studies also demonstrated similar findings in Wolffia arrhiza treated with 0.1 mmol L 1 JA and Pb (Piotrowska et al., 2009) as well as in soybean plants treated with 10 mmol L 1 MeJA and Cd (Keramat et al., 2009). Cd stress indirectly induces the overproduction of different ROS, such as H2O2, O2d , and dOH, resulting in membrane lipid peroxidation and MDA accumulation (Olmos et al., 2003). In the present study, significant increases in MDA content also indicated oxidative impairment in the leaves of K. obovata. The MDA content in the leaves treated with 200 mmol L 1 Cdþ 1 mmol L 1 MeJA was significantly lower than that in the leaves treated with 200 mmol L 1 Cd alone. Therefore, the low level of MeJA exhibited protective effects on the cell membrane lipid by alleviating lipid peroxidation in K. obovata seedlings under Cd stress. The protective effect of MeJA on leaf chlorophylls under metal stress may be attributed to the mitigated oxidative damage induced by the exogenous MeJA.
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Fig. 3. Changes of APX, CAT, POD activities and AsA concentration in leaves of K. obovata seedlings under different treatments of Cd (200 mg L 1) and MeJA (0.1, 1 and 10 μmol L 1 for MeJA 1, 2 and 3, respectively) at day 9, and (b) at different time after the treatments (Values are mean and SD; bars with different letters are significantly different at Pr 0.05; for time series changes of the parameters, statistical was made in separate groups of time, NS indicates not significant).
The translocation of Cd from roots to leaves of K. obovata under different MeJA concentrations (0.1 to 10 mmol L 1) was significantly reduced compared with that under Cd treatment alone. These results are in accordance with a previous finding that the application of JA at 0.1 mmol L 1 significantly inhibits Pb accumulation in W. arrhiza (Piotrowska et al., 2009). Other stress hormones, such as ABA and brassinolide (2,4-epibrassinolide), were also reported to reduce the uptake of toxic metals (Cd or Ni) in both xylem sap and shoot of hydroponically grown plants (Salt et al., 1995; Zhao et al., 2006; Kanwar et al., 2012). The reduced uptake of metals under ABA treatments is due to the reduction in transpiration rate and symplastic loading of Cd into the xylem (Salt et al., 1995). The xylem loading of Cd in the plant is mainly influenced by plant transpiration (Lux et al., 2011). Similar to ABA and brassinolide, MeJA also affects plant transpiration by promoting stomatal closure (Suhita et al., 2004; Hossain et al., 2011). In the
present study, the reduced uptake of Cd in the shoot of K. obovata might be a result of stomatal closure and decreased transpiration by exogenous MeJA. Besides transpiration, metal uptake in the plant can also be influenced by other factors such as accumulation/exudation of organic compounds, such as phenolic compounds. It was reported that the strong accumulation of phenolic compounds in aerial parts of Chamomile plants significantly repressed the uptake of Ni and Cd in shoot (Kovač́ ik et al., 2011a). Significant increases of phenolic compounds in plants to exogenous MeJA have been reported in many studies (Kim et al., 2007; Li et al., 2007). There also remains a possibility that MeJA treatments increased the phenolic compounds in leaves of K. obovata, which thereby repressed the uptake of Cd in leaves. MeJA affects the activity of stress enzymes, thereby causing alleviation of oxidative stress in plant cells (Li et al., 1998; Wang, 1999). Increased activities of SOD, CAT, and APX in the presence of
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0 l
Relative expression level
Fig. 4. Changes of endogenous JA concentrations in leaves of K. obovata seedlings after 9 days treatment of Cd (200 mmol L 1) and different concentrations of MeJA (0.1, 1, and 10 μmol L 1 for MeJA 1, 2 and 3, respectively; Values are mean and SD; data with different letters are significantly different at Pr 0.05).
Fig. 5. The relative KoMT2 gene expression level (expressed by 2 tissues of K. obovata, values are mean and SD.
(ΔΔCt)
) in leaf
10 to 100 mmol L 1 MeJA were observed in soybean plant (Glycine max L.) under Cd stress (Keramat et al., 2009). JA at 0.1 mmol L 1 also activates the enzymatic (CAT, APX, and NADH POD) and nonenzymatic antioxidant (AsA and GSH) systems of W. arrhiza (Piotrowska et al., 2009). Similarly, the present study found that MeJA treatment at the concentration range of 0.1 mmol L 1 to 1 mmol L 1 significantly increased the activities of CAT and APX in the leaves of K. obovata, accompanied by low quantities of Cd uptake. 0.1 μmol L 1 MeJA also increased the concentrations of AsA, which is one of the major antioxidants that typically reacts with oxidants of the ROS, such as the hydroxyl radical, and helps to protect nucleic acids, proteins, and lipids. These finding suggested that low MeJA concentrations (0.1 to 1 mmol L 1) could help K. obovata seedlings alleviate the toxicity of Cd by stimulating antioxidative enzymes activities and increasing of antioxidant content in plant cells. The upregulation of AsA by exogenous MeJA was also reported in Arabidopsis and tobacco plants, and it was suggested the stimulation of AsA by MeJA coincides with enhanced transcription of two late methyl jasmonate-responsive genes that encoding enzymes for vitamin C (AsA) biosynthesis (Wolucka et al., 2005). The exact mechanism of the regulation of exogenous MeJA on antioxidative enzyme activity has not been elucidated yet. However, the accumulation of endogenous JA induced by MeJA could provide some insights into this mechanism. In the present study, the endogenous JA concentrations of the seedlings under combined treatments of Cd and different MeJA concentrations increased with increasing exogenous MeJA levels. Endogenous JA
is suggested to be involved in the cellular response to metal toxicity (Maksymiec and Krupa, 2002). MeJA treatment of plants elicits endogenous JA burst, which can be induced either from de novo biosynthesis or demethylation of MeJA intake (Ziegler et al. 2001; Wu et al., 2008). Increases in endogenous JA were also observed in A. thaliana and P. coccineus treated with Cu and Cd (Maksymiec et al., 2005), pea plants treated with Cd (RodriguezSerrano et al., 2009), rice seedlings treated with Cu (Koeduka et al. 2005), and mangrove seedlings, such as K. obovata, Acanthus ilicifolius, and Excoecaria agallocha, treated with Pb (Yan and Tam, 2013a, 2013b). As stress hormones, the changes of JA in plants subject to abiotic stresses were found to be transient and dynamic (Maksymiec et al., 2005), and its changes in plants along the time course of stress were found to be species-specific (Yan and Tam, 2013a). It was found the accumulation of JA in plants not only increased significantly at very early stages (7–24 h), but also kept at high levels after much longer time (5–49 days) of heavy metal stress (Maksymiec et al., 2005; Yan and Tam, 2013a). In the case of the present study, the changes of endogenous JA at the early stages (such as several hours to 2 days) after perceiving Cd and MeJA treatments are also an interesting problem, which deserve further studies. Plant MT genes are divided into four types, in which type-2 MT genes are mainly expressed in the leaves other than the roots (Cobbett and Goldsbrough, 2002). In the present study, the expression of type-2 MT genes in leaves of K. obovata was significantly upregulated by 200 mmol L 1 Cd on day 9. This result indicates that MTs are part of the heavy metal tolerance mechanism in aerial tissues (leaves) of this species. Considering the notable levels of Cd accumulation in K. obovata leaves (Fig. 2), the upregulation of the KoMT2 gene might have a function in the detoxification of heavy metals and mitigation of oxidative stress. This finding is in agreement with earlier reports in other 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, Cd (Huang et al., 2012), at varying treatment times (3 d to 11 d). Exogenous MeJA downregulated the expression of KoMT2 genes in seedlings under Cd stress (Fig. 5). This downregulation is in accordance with the decreases in leaf Cd accumulation induced by MeJA, suggesting that MeJA may have no direct regulatory effects on KoMT2 expression in this species. Besides MTs, plants are also 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). These above findings suggest that exogenous JA/MeJA might enhance the synthesis of PCs on transcript level, and thus help to alleviate the Cd toxicity. MeJA possibly exert different regulatory effect on the synthesis of MTs and PCs in higher plants, which awaits further studies.
5. Conclusions Deleterious effects, including decreased leaf chlorophyll content and increased leaf malondialdehyde concentration, were observed on the chlorophyll and cell membrane after 9 d of 200 mmol L 1 Cd treatment. The addition of MeJA at the concentration range of 0.1 to 1 mmol L 1 significantly increased the
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activities of CAT and APX in the leaves of K. obovata, accompanied by low quantities of Cd uptake. This finding suggested that low MeJA concentrations (0.1 to 1 mmol L 1) can help K. obovata seedlings alleviate Cd toxicity by increasing the activities of antioxidants in cells. The concentration of endogenous JA in the leaves of K. obovata was decreased by Cd but was positively stimulated by the addition of exogenous MeJA. Treatment of 200 mmol L 1 Cd for 9 d enhanced the expression of KoMT2 in the leaves, whereas application of exogenous MeJA significantly restored the expression levels to control values. This result indicated that MeJA might have no direct regulatory effect on the expression of KoMT2. Exogenous MeJA also inhibited the uptake of Cd to the aboveground sensitive part (leaves) of the seedlings. The reduced uptake of Cd might be a result of stomatal closure and decreased transpiration by exogenous MeJA.
Acknowledgments The work described in this paper was supported by National Nature Science Foundation of China (No. 41201525), SKLEC2012RCDW02 and National Nature Science Foundation of China (No. 41271065).
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Contents lists available at ScienceDirect
Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Effect of methyl jasmonate on cadmium uptake and antioxidative capacity in Kandelia obovata seedlings under cadmium stress Jun Chen, Zhongzheng Yan n, Xiuzhen Li n State Key Laboratory of Estuarine and Coastal Research, East China Normal University, Shanghai, China
art ic l e i nf o
a b s t r a c t
Article history: Received 9 November 2013 Received in revised form 11 January 2014 Accepted 19 January 2014
This study investigated the effects of methyl jasmonate (MeJA) on chlorophyll concentration, lipid peroxidation, Cd uptake, antioxidative capacity, and type-2 metallothionein gene (KoMT2) expression in the leaves of Kandelia obovata seedlings exposed to Cd stress. Deleterious effects, including decreased chlorophyll content and increased malondialdehyde concentration, were observed in leaves of K. obovata after 9 d of 200 μmol L 1 Cd treatment. Application of MeJA (0.1 to 1 μmol L 1) increased the concentration of ascorbic acid and the activities of catalase and ascorbate peroxidase in the leaves of K. obovata, which helped alleviate the oxidative damage induced by Cd stress. The concentration of endogenous jasmonic acid in the leaves of K. obovata was decreased by Cd but was positively stimulated by exogenous MeJA. The expression of KoMT2 in the leaves was enhanced after 9 d of 200 μmol L 1 Cd treatment, while the exogenous application of MeJA significantly restored the expression of KoMT2. Exogenous MeJA also inhibited the uptake of Cd to the aboveground part (leaves) of the seedlings, which helped reduce direct damages of Cd to the photosynthetic organ of the plant. The reduced uptake of Cd might be a result of stomatal closure and decreased transpiration by exogenous MeJA. & 2014 Elsevier Inc. All rights reserved.
Keywords: Jasmonic acid Mangrove Metallothionein Heavy metals Stress
1. Introduction Mangroves are under increasing heavy metal pollution pressure from human activities because of the rapid industrialization and urbanization in coastal areas (Harbison, 1986; MacFarlane, 2002; Tam, 2006). Metals at environmental concentrations exceeding the maximum tolerable amount damage the physiological functions of plants, thereby resulting in photosynthetic output reduction and growth degeneration (Prasad, 2004). The metals Cd, Pb, and Hg are strongly phytotoxic; this phytotoxicity can be partly attributed to the generation of reactive oxygen species (ROS), such as superoxide radical (O2d ), hydroxyl (∙OH), and hydrogen peroxide (H2O2) (Prasad, 2004). High levels of ROS usually damage cellular components, such as membranes, nucleic acids, and chloroplast pigments, resulting in lipid peroxidation (Tewari et al., 2002). Plants, including mangroves, develop a series 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 (MacFarlane, 2002; Prasad, 2004). Aside from their antioxidative defense systems, mangroves, similar to other
n
Corresponding authors. Fax: þ 86 21 62546441. E-mail addresses: [email protected] (Z. Yan), [email protected] (X. Li).
0147-6513/$ - see front matter & 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ecoenv.2014.01.022
plants, relieve heavy metal stress by chelating various ligands, including metallothioneins (MTs) (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 Cd and Hg, in animals, plants, eukaryotic microorganisms, and prokaryotes (Cobbett and Goldsbrough, 2002; Huang and Wang, 2010). The black mangrove Avicennia germinans has can overexpress MT responsive genes, which constitute a coordinated detoxification response mechanism targeting nonessential metals (Gonzalez-Mendoza et al., 2007). Similar findings were also reported in mangroves species Bruguiera gymnorrhiza (Huang and Wang, 2009) and Avicennia marina (Huang and Wang, 2010) when subjected to Zn, Cu, or Pb stress. Jasmonic acid (JA) and methyl jasmonate (MeJA) are members of a family of cyclopentanone compounds synthesized from linolenic acid via the octadecanoic pathway; these compounds exhibit important functions in the signaling network of plants under various biotic and abiotic stresses (Fujita et al., 2006). Plants under heavy metal stresses are prone to adjust their endogenous phytohormone levels for adaptation (Santner and Estelle, 2009). Abiotic stresses, such as metal stress, stimulate endogenous JA in herbaceous plants, such as Arabidopsis thaliana, Phaseolus coccineus, and Oryza sativa L. (Koeduka et al., 2005; Maksymiec et al., 2005). Moreover, JA also exhibits protective effects on plants under various abiotic stresses, including heavy metals (Walia et al., 2007; Keramat et al., 2009). These protective effects mainly include
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enhancing antioxidant capacity and reducing malondialdehyde (MDA) and H2O2 contents as well as protecting photosynthetic pigments (Maksymiec and Krupa, 2002; Piotrowska et al., 2009; Kováčik et al., 2011b). The stress hormone abscisic acid (ABA) and the plant steroid hormone brassinolide (2,4-epibrassinolide) also protect plants from abiotic stresses by reducing the plant uptake of Cd (Salt et al., 1995; Zhao et al., 2006) and Ni (Kanwar et al., 2012). To date, studies on the mechanism of the alleviatory effects of MeJA on plants under metal stress remain insufficient, especially for mangrove species. Available studies mainly focused on model plants (A. thaliana) and herbaceous plants (Maksymiec and Krupa, 2002; Piotrowska et al., 2009; Kováčik et al., 2011b). Abiotic stresses, such as metal stress, stimulate the production of endogenous JA in herbaceous plants, such as A. thaliana, P. coccineus, and O. sativa L. (Koeduka et al., 2005; Maksymiec et al., 2005). Studies on the effect of exogenous MeJA treatment on endogenous JA responses in metal-stressed plants are insufficient, and the mechanism by which exogenous MeJA elicits protective effects remains unknown. Accordingly, this study aims to investigate whether or not exogenous MeJA also exerts protective effects on mangrove seedlings under heavy metal stress. The effect of different MeJA concentrations on the antioxidative capacity of toxic metal-stressed mangrove seedlings was examined, and variations in the uptake of toxic metals and in the expression levels of type 2 MT responsive genes were investigated. The annual perennial mangrove species, Kandelia obovata, was selected as the plant model. Cd was selected as the model heavy metal because it is a nonessential element that is very common in wetland sediment (Tam, 2006).
2. Materials and methods 2.1. Experimental setup and sample collection Healthy propagules of K. obovata were collected from mangrove swamp in Beihai, China, and the propagules were comparable in terms of size (length of 18.5 7 1.9 cm, fresh weight of 10.8 7 2.6 g) and were free of insect damage and fungal infections. The propagules were sown on sand in plastic pots [24 cm (open top) 30 cm (height) 20 cm (flat bottom)] with half-strength Japanese garden test nutrient solution (Hori, 1966). The planted pots were placed on a bench in an artificial climate chamber with a daily temperature of 25 1C, a relative humidity of 70 percent, 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 every day until the third pair of leaves completely unfolded. The pots were then randomly divided into nine groups each in triplicate. Five groups were used to determine the effects of MeJA on the responses of the seedlings under Cd treatments: (i) control treatment; (ii) 200 mmol L 1 Cd; (iii) 200 mmol L 1 Cd þ 0.1 mmol L 1 MeJA (MeJA1); (iv) 200 mmol L 1 Cd þ 1 mmol L 1 MeJA (MeJA2); and (v) 200 mmol L 1 Cd þ10 mmol L 1 MeJA (MeJA3). For the treatment of 200 mmol L 1 Cd together with different levels of MeJA, appropriate amounts of MeJA (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 200 mmol L 1 Cd (CdCl2). For the treatment with 200 mmol L 1 Cd only, 0.5 mL ethanol was supplemented to keep the consistency of the treatment. One group with appropriate amounts of ethanol served as the control group. 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 two days to prevent depletion of nutrients and changes in the concentrations of Cd and MeJA concentrations. Samples were collected at 9 d after the treatment. The treatment solutions were also collected at the end of the experiments, and the exposure concentrations of Cd and MeJA were investigated. Results showed that the nominal and real applied Cd doses were not significantly different according to t-test; therefore, we used the nominal exposure concentrations in the analysis. Changes in CAT, APX, and POD activities and AsA content at different times after the treatments were determined by setting four treatments as follows: (i) control group; (ii) 200 mmol L 1 Cd; (iii) 1 mmol L 1 MeJA; and (iv) 200 mmol L 1 Cdþ 1 mmol L 1 MeJA. The seedlings were collected at 1, 5, and 11 d after the treatment. For sample collection, the 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 1C until further analyses. Parts of the leaf and root samples were collected and then dried in an oven at 70 1C for 48 h. 2.2. Determinations 2.2.1. Chlorophyll concentration Leaf chlorophyll concentration was determined according to the method described by Wang et al. (2009). In brief, 0.1 g of fresh leaf sample was cut from a mature leaf and then 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 1C for 2 d. The absorbance readings of the extract were recorded at 645 and 663 nm, and the concentrations (mg L 1) of chlorophyll a (chl a), chlorophyll b (chl b), and total chlorophyll (total chls) in the extract were calculated according to method described by Arnon (1949). 2.2.2. Cd concentration in plant tissue Cd concentration in plant tissues was determined according to the method described by Wong et al. (1993). Approximately 0.2 g to 0.3 g of oven-dried plant sample was charred on a hot plate for approximately 1 h and then ashed in a muffle furnace at 500 1C for 6 h. The ash was digested and diluted in 10 mL of 1 percent nitric acid. Cd in the digested solutions was analyzed with an atomic absorption spectrometer (PerkinElmer AAnalyst 800). The accuracy of the method was determined in terms of the recovery of spiked Cd standards (as CdCl2) in homogenized plant tissue samples at 10 mg L 1. The average recoveries and SE of Cd were 91.579.9 (n¼ 3), and the limit of detection for Cd was 0.05 mg kg 1 DW. 2.2.3. MDA concentration MDA concentration in the leaf was determined according to the method described by Wang and Jin (2005) with modifications. In a typical procedure, 0.2 g of fresh plant sample was homogenized using a mortar and pestle with 4 mL of 20 percent trichloroacetic acid (TCA) (w/v). The homogenate was centrifuged at 9000 g for 5 min. Then, 1 mL of the supernatant was mixed with an equal volume of 0.6 percent (w/v) thiobarbituric acid solution comprising 10 percent 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 5000 g for 10 min at 25 1C, and the absorbance readings of the supernatant at 450, 532, and 600 nm were recorded. The MDA concentration (CMDA) was calculated according to the following equation: C MDA ¼ 6:45ðA532 A600Þ 0:56A450:
2.2.4. AsA concentration AsA concentration was determined according to the method described by Law et al. (1983). The assay is based on the reduction of Fe3 þ to Fe2 þ by ascorbic acid and on the formation of a pink color complex between Fe2 þ and 2, 20 -bipyridyl, with a maximum absorption at 525 nm. In brief, 0.2 g of fresh leaf sample was extracted by grinding fresh leaf tissue in 5 percent TCA, and the homogenate was centrifuged at 9000 g for 10 min. Then, 0.2 mL of the supernatant was mixed with equal volumes of 150 mmol L 1 phosphate buffer (pH 7.4) and deionized water. The mixture was maintained at ambient room temperature for 30 s. Then, 400 mL of 10 percent TCA, 400 mL of 44 percent (v:v) phosphate acid, 400 mL of 4 percent 2,20 bipyridyl (w:v, in 70 percent ethanol), and 200 mL of 3 percent (w/v) FeCl3 were added, mixed, and then incubated in water bath at 37 1C for 60 min. The mixture was centrifuged at 9000 g at 4 1C for 5 min, and the absorbance readings of the supernatant at 525 nm were recorded. L-AsA was used as standard. 2.2.5. Endogenous JA concentration Endogenous JA concentration was determined using an ELISA kit (Rapidbio, USA) according to the manufacturer's instructions. About 100 mg tissue was rinsed with 1X 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), 1.46 mmol L 1 potassium dihydrogen phosphate (KH2PO4), then homogenized in 1 mL of 1 PBS and stored overnight at 20 1C. After two freeze-thaw cycles were performed to break the cell membranes, the homogenates were centrifuged for 5 min at 9000 g at 4 1C. 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. 2.2.6. Antioxidative enzyme activity 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 9000 g for 10 min at 4 1C, and the supernatant was used for the enzyme assay.
3. Results 3.1. Growth of the seedlings and chlorophyll and MDA concentrations The growth of the seedling was not affected after 9 days treatments of Cd and MeJA (Fig. 1a). The concentrations of chl a, chl b, and total chls significantly reduced after 9 d of 200 mmol L 1 Cd treatment (Fig. 1b). However, 200 mmol L 1 Cd treatment with 0.1 mmol L 1 MeJA, significantly restored the chlorophyll content reduced by Cd stress (Fig. 1a). The concentration of MDA in the
total chl chl a chl b ab
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The mean and standard deviation of the three replicates for each treatment were calculated. One-way ANOVA and post-hoc multiple comparison (Tukey's test) were conducted to determine significant differences in the detected parameters among the different levels of Cd treatments. Two-way multivariate ANOVA (MANOVA), with treatment and treatment time as the independent variables, was applied to examine the significant interactive effects and differences between K. obovata seedlings based on different variables (i.e., AsA content and APX, CAT, and POD activities in the leaves). Statistical analyses were performed in SPSS version 16.0.
351
a
nt
2.3. Statistical analyses
2.0
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2.2.7. RT-PCR expression of type-2 MT gene Total RNA of leaf tissue was extracted using Trizol reagent (Invitrogen). Briefly, 50 mg to 100 mg of fresh leaf sample was sufficiently ground with liquid nitrogen, added with 1 mL of Trizol reagent, 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 12,000 g for 10 min at 4 1C. The supernatants were transferred to new RNase-free tubes, mixed with 0.5 mL of isopropanol, and then centrifuged at 12,000 g (4 1C) for 10 min after 30 min of incubation. The supernatants were removed, and the precipitate was washed with 1 ml of 75 percent ethanol (diluted in DEPC water) and then centrifuged at 7500 g for 5 min at 4 1C. The supernatant was removed, the tubes were air dried at room temperature, and the RNA was dissolved by RNase-free H2O. First-strand cDNA was synthesized using an AMV First-Strand cDNA Synthesis kit by Sangon Biotech, Shanghai, China. Real-time quantitative PCR was performed on an ABI StepOne Plus analyzer with ABI SybrGreen PCR master mix. The K. candel complete MT cDNA sequence submitted to GenBank (Accession No. DQ414691) was used to design primers for RT-PCR. The forward and reverse primers were 50 TCTTGCTGTGGTGGAAACTG-30 and 50 -ATCTCGGCTCCCTCAAAGT-30 , respectively. 18S rRNA was used as the reference gene to normalize the expression levels between the samples, and the forward and reverse primers were F: 50 -CCCGTTGCTGCGATGAT-30 and R: 50 -GCTGCCTTCCTTGGATGTG-30 , 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 KoMT2 mRNA expression relative to the control was determined by the standard 2 ΔΔCT method (Livak and Schmittgen, 2001; Huang and Wang, 2010).
MDA conc. (µmol g-1 fw)
CAT activity was determined according to the method described by Beer and Sizer (1952) with modifications. The reaction mixture (2.5 mL) consisted of 50 mmol L 1 phosphate buffer (pH 7.4), 0.1 mmol L 1 EDTA-Na2, 20 mmol L 1H2O2, and 0.5 mL of enzyme extract. The reaction was initiated by adding the extract. The decrease in H2O2 was monitored at 240 nm for 2 min and quantified based on its molar extinction coefficient (36 mol L 1 cm 1). One unit (U) of CAT was defined as the amount of enzyme that decomposes 1 mmol of H2O2 per minute, and the activity was expressed as U mg 1 protein 1. Guaiacol POD activity was determined according to the guaiacol method (Fielding and Hall, 1978). Approximately 100 mL of enzyme extract was mixed with 3 mL of 50 mmol L 1 phosphate buffer (pH 7.4) containing 0.2 percent guaiacol (v:v). The reaction was initiated with 1 mL of 0.3 percent H2O2, and guaiacol oxidation was determined based on an increase in the absorbance at 470 nm for 2 min. POD activity was expressed as units of enzyme activity (U), and the units were calculated based on the molar extinction coefficient of tetraguaiacol (26.6 mM 1 cm 1). One unit was defined as the amount of enzyme catalyzing the formation of 1 μmol tetraguaiacol per minute, and the activity was expressed as U mg 1 protein 1. APX activity was estimated 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 1H2O2. 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.
chlorophyll conc. mg g-1FW
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Fig. 1. (a) Exterior appearance of K. obovata seedlings, changes of (b) chlorophyll a, chlorophyll b and total chlorophylls, and (c) MDA concentrations in leaves of K. obovata seedlings after 9 days treatment of Cd (200 mmol L 1) and different concentrations of MeJA (0.1, 1, and 10 μmol L 1 for MeJA 1, 2 and 3, respectively; Values are mean and SD; data with different letters are significantly different at Pr0.05).
leaves of K. obovata significantly increased by 200 mmol L 1 Cd. However, exogenous MeJA, especially at 1 mmol L 1 concentration, alleviated this Cd stress-induced increase in MDA (Fig. 1c). 3.2. Cd accumulation in leaves and roots After 9 d of 200 mmol L 1 Cd treatment, the leaves and roots of K. obovata accumulated 248.5 and 384.0 mg g 1 DW of Cd, respectively, which are significantly higher than the Cd content
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1000
leaf root
bc bc
800
bc
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alone (Fig. 3g). However, MeJA treatment alone did not stimulate the concentration of AsA in the leaves at 1 and 5 d after the treatment (Fig. 3h). MANOVA, followed by Wilk's lambda test, showed that treatment and treatment time as well as their interactions significantly affected the production of AsA in the leaves of K. obovata (Table S1 of supporting information).
b a
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Fig. 2. Accumulation of Cd in roots and leaves of K. obovata seedlings after 9 days treatment of Cd (200 mmol L 1) and different concentrations of MeJA (0.1, 1, and 10 μmol L 1 for MeJA 1, 2 and 3, respectively) (Values are mean and SD; For each plant parts, data with different letters are significantly different at Pr 0.05; ND indicates not detected).
in the control group (not detected under the detection limits of 0.05 μg g 1 DW) (Fig. 2). By contrast, Cd treatment with different MeJA concentrations significantly inhibited the translocation of Cd to the leaves. The concentration of Cd in the leaves under MeJA treatment ranged from 22.6 mg g 1 to 98.4 mg g 1 DW, which is much lower than that in roots (582.6–757.7 μg g 1 DW). However, the addition of different MeJA concentrations did not affect the root uptake of Cd Fig. 2. 3.3. Changes in antioxidants In this study, 9 d of Cd treatment did not affect APX activity in the leaves, whereas Cd treatment with different MeJA concentrations significantly increased APX activity in the leaves compared with the seedling leaves under control and Cd treatments (Fig. 3a). However, 1 mmol L 1 MeJA did not significantly stimulate APX activity in the leaves on days 1, 5, and 11 (Fig. 3b). Two-way MANOVA analyses showed that the influences of the two factors as well as their interactions significantly affected the activity of APX (Table S1 of supporting information). Cd stress stimulated CAT activity in the leaves of K. obovata from day 5 after the treatment. The stimulations were negative on day 1 and became positive on days 5 and 11 (Fig. 3d). Treatment with MeJA and 200 mmol L 1 Cd for 9 d significantly enhanced CAT activity in the leaves (Fig. 3c); similar effects were also observed on days 5 and 11. In contrast to APX, leaf CAT activity showed positive stimulation at low levels of MeJA (0.1 and 1 mmol L 1) but not at the highest level (10 mmol L 1, Fig. 3c). Guaiacol POD detoxifies a large family of peroxides, including H2O2. In this study, POD activity showed no changes at 1, 5, 9, and 11 d after 200 mmol L 1 Cd treatment (Fig. 3e and f). In general, POD activity increased with increasing exogenous MeJA concentration, and POD activity only significantly increased compared with the control group at the highest MeJA level (Fig. 3e). Exogenous MeJA at 1 mmol L 1 significantly enhanced POD activity on day 5 only (Fig. 3f). Treatment and treatment time as well as their interactions significantly affected CAT and POD activities (Table S1 of supporting information). Cd significantly reduced AsA concentration in the leaves of K. obovata at 1 d to 5 d after the treatment (Fig. 3h). However, this effect was not observed at 9 and 11 d after the treatment (Fig. 3g and h). The addition of different levels of MeJA significantly restored the concentration of AsA in the leaves compare to that under treatment of 200 mmol L 1 Cd (Fig. 3g). The addition of 1 μmol L 1 MeJA also significantly increased the concentration of AsA in the leaves compared with the leaves under Cd treatment
3.4. Changes in endogenous JA The level of endogenous JA in the leaves of K. obovata was significantly decreased by Cd at 200 mmol L 1. This reduction effect was alleviated by the addition of MeJA, and endogenous JA concentration increased with increasing exogenous MeJA concentration (Fig. 4). 3.5. mRNA expression of KoMT2 The mRNA expression of KoMT2 mRNA in the leaves of K. obovata seedlings increased by fivefold (Po0.01) under 200 mmol L 1 Cd treatment compared with that under the control treatment (Fig. 5). However, this amplification of KoMT2 gene expression was abated or even restored by the addition of exogenous MeJA at 1 and 10 μmol L 1.
4. Discussion Most plants are sensitive to low Cd concentrations, which inhibit plant growth as a consequence of alterations in photosynthetic rate (Sandalio et al., 2001; Benavides et al., 2005; RodriguezSerrano et al., 2009). In the present study, the concentrations of chl a, chl b, and total chls in the leaves of K. obovata significantly decreased after 9 d of 200 mmol L 1 Cd treatment. This result indicates that the synthesis/accumulation of the pigments was interfered by Cd. The inhibition in photosynthetic pigment accumulation in response to heavy metal stress might be a consequence of chloroplast membrane peroxidation through enhanced rates of H2O2 production and lipid peroxidation in chloroplast membranes (Piotrowska-Niczyporuk et al., 2012). Low concentrations of MeJA (normally within the levels of 0.1 to 1 mmol L 1) usually exert protective effect on chlorophylls of plant under toxic metal treatment (Maksymiec and Krupa 2002; Keramat et al., 2009; Piotrowska et al., 2009; Kovač́ ik et al., 2011b). Similar results were observed in the present study. That is, low level (0.1 mmol L 1) of MeJA significantly restored the impairment induced by Cd in leaf chlorophylls, especially chl a (under 0.1 mmol L 1 MeJAþ 200 mmol L 1 Cd treatment). These results suggested that MeJA elicited protective effects during leaf photosynthesis under Cd stress. Other studies also demonstrated similar findings in Wolffia arrhiza treated with 0.1 mmol L 1 JA and Pb (Piotrowska et al., 2009) as well as in soybean plants treated with 10 mmol L 1 MeJA and Cd (Keramat et al., 2009). Cd stress indirectly induces the overproduction of different ROS, such as H2O2, O2d , and dOH, resulting in membrane lipid peroxidation and MDA accumulation (Olmos et al., 2003). In the present study, significant increases in MDA content also indicated oxidative impairment in the leaves of K. obovata. The MDA content in the leaves treated with 200 mmol L 1 Cdþ 1 mmol L 1 MeJA was significantly lower than that in the leaves treated with 200 mmol L 1 Cd alone. Therefore, the low level of MeJA exhibited protective effects on the cell membrane lipid by alleviating lipid peroxidation in K. obovata seedlings under Cd stress. The protective effect of MeJA on leaf chlorophylls under metal stress may be attributed to the mitigated oxidative damage induced by the exogenous MeJA.
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Fig. 3. Changes of APX, CAT, POD activities and AsA concentration in leaves of K. obovata seedlings under different treatments of Cd (200 mg L 1) and MeJA (0.1, 1 and 10 μmol L 1 for MeJA 1, 2 and 3, respectively) at day 9, and (b) at different time after the treatments (Values are mean and SD; bars with different letters are significantly different at Pr 0.05; for time series changes of the parameters, statistical was made in separate groups of time, NS indicates not significant).
The translocation of Cd from roots to leaves of K. obovata under different MeJA concentrations (0.1 to 10 mmol L 1) was significantly reduced compared with that under Cd treatment alone. These results are in accordance with a previous finding that the application of JA at 0.1 mmol L 1 significantly inhibits Pb accumulation in W. arrhiza (Piotrowska et al., 2009). Other stress hormones, such as ABA and brassinolide (2,4-epibrassinolide), were also reported to reduce the uptake of toxic metals (Cd or Ni) in both xylem sap and shoot of hydroponically grown plants (Salt et al., 1995; Zhao et al., 2006; Kanwar et al., 2012). The reduced uptake of metals under ABA treatments is due to the reduction in transpiration rate and symplastic loading of Cd into the xylem (Salt et al., 1995). The xylem loading of Cd in the plant is mainly influenced by plant transpiration (Lux et al., 2011). Similar to ABA and brassinolide, MeJA also affects plant transpiration by promoting stomatal closure (Suhita et al., 2004; Hossain et al., 2011). In the
present study, the reduced uptake of Cd in the shoot of K. obovata might be a result of stomatal closure and decreased transpiration by exogenous MeJA. Besides transpiration, metal uptake in the plant can also be influenced by other factors such as accumulation/exudation of organic compounds, such as phenolic compounds. It was reported that the strong accumulation of phenolic compounds in aerial parts of Chamomile plants significantly repressed the uptake of Ni and Cd in shoot (Kovač́ ik et al., 2011a). Significant increases of phenolic compounds in plants to exogenous MeJA have been reported in many studies (Kim et al., 2007; Li et al., 2007). There also remains a possibility that MeJA treatments increased the phenolic compounds in leaves of K. obovata, which thereby repressed the uptake of Cd in leaves. MeJA affects the activity of stress enzymes, thereby causing alleviation of oxidative stress in plant cells (Li et al., 1998; Wang, 1999). Increased activities of SOD, CAT, and APX in the presence of
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JA conc. (pmol g-1 FW)
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Relative expression level
Fig. 4. Changes of endogenous JA concentrations in leaves of K. obovata seedlings after 9 days treatment of Cd (200 mmol L 1) and different concentrations of MeJA (0.1, 1, and 10 μmol L 1 for MeJA 1, 2 and 3, respectively; Values are mean and SD; data with different letters are significantly different at Pr 0.05).
Fig. 5. The relative KoMT2 gene expression level (expressed by 2 tissues of K. obovata, values are mean and SD.
(ΔΔCt)
) in leaf
10 to 100 mmol L 1 MeJA were observed in soybean plant (Glycine max L.) under Cd stress (Keramat et al., 2009). JA at 0.1 mmol L 1 also activates the enzymatic (CAT, APX, and NADH POD) and nonenzymatic antioxidant (AsA and GSH) systems of W. arrhiza (Piotrowska et al., 2009). Similarly, the present study found that MeJA treatment at the concentration range of 0.1 mmol L 1 to 1 mmol L 1 significantly increased the activities of CAT and APX in the leaves of K. obovata, accompanied by low quantities of Cd uptake. 0.1 μmol L 1 MeJA also increased the concentrations of AsA, which is one of the major antioxidants that typically reacts with oxidants of the ROS, such as the hydroxyl radical, and helps to protect nucleic acids, proteins, and lipids. These finding suggested that low MeJA concentrations (0.1 to 1 mmol L 1) could help K. obovata seedlings alleviate the toxicity of Cd by stimulating antioxidative enzymes activities and increasing of antioxidant content in plant cells. The upregulation of AsA by exogenous MeJA was also reported in Arabidopsis and tobacco plants, and it was suggested the stimulation of AsA by MeJA coincides with enhanced transcription of two late methyl jasmonate-responsive genes that encoding enzymes for vitamin C (AsA) biosynthesis (Wolucka et al., 2005). The exact mechanism of the regulation of exogenous MeJA on antioxidative enzyme activity has not been elucidated yet. However, the accumulation of endogenous JA induced by MeJA could provide some insights into this mechanism. In the present study, the endogenous JA concentrations of the seedlings under combined treatments of Cd and different MeJA concentrations increased with increasing exogenous MeJA levels. Endogenous JA
is suggested to be involved in the cellular response to metal toxicity (Maksymiec and Krupa, 2002). MeJA treatment of plants elicits endogenous JA burst, which can be induced either from de novo biosynthesis or demethylation of MeJA intake (Ziegler et al. 2001; Wu et al., 2008). Increases in endogenous JA were also observed in A. thaliana and P. coccineus treated with Cu and Cd (Maksymiec et al., 2005), pea plants treated with Cd (RodriguezSerrano et al., 2009), rice seedlings treated with Cu (Koeduka et al. 2005), and mangrove seedlings, such as K. obovata, Acanthus ilicifolius, and Excoecaria agallocha, treated with Pb (Yan and Tam, 2013a, 2013b). As stress hormones, the changes of JA in plants subject to abiotic stresses were found to be transient and dynamic (Maksymiec et al., 2005), and its changes in plants along the time course of stress were found to be species-specific (Yan and Tam, 2013a). It was found the accumulation of JA in plants not only increased significantly at very early stages (7–24 h), but also kept at high levels after much longer time (5–49 days) of heavy metal stress (Maksymiec et al., 2005; Yan and Tam, 2013a). In the case of the present study, the changes of endogenous JA at the early stages (such as several hours to 2 days) after perceiving Cd and MeJA treatments are also an interesting problem, which deserve further studies. Plant MT genes are divided into four types, in which type-2 MT genes are mainly expressed in the leaves other than the roots (Cobbett and Goldsbrough, 2002). In the present study, the expression of type-2 MT genes in leaves of K. obovata was significantly upregulated by 200 mmol L 1 Cd on day 9. This result indicates that MTs are part of the heavy metal tolerance mechanism in aerial tissues (leaves) of this species. Considering the notable levels of Cd accumulation in K. obovata leaves (Fig. 2), the upregulation of the KoMT2 gene might have a function in the detoxification of heavy metals and mitigation of oxidative stress. This finding is in agreement with earlier reports in other 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, Cd (Huang et al., 2012), at varying treatment times (3 d to 11 d). Exogenous MeJA downregulated the expression of KoMT2 genes in seedlings under Cd stress (Fig. 5). This downregulation is in accordance with the decreases in leaf Cd accumulation induced by MeJA, suggesting that MeJA may have no direct regulatory effects on KoMT2 expression in this species. Besides MTs, plants are also 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). These above findings suggest that exogenous JA/MeJA might enhance the synthesis of PCs on transcript level, and thus help to alleviate the Cd toxicity. MeJA possibly exert different regulatory effect on the synthesis of MTs and PCs in higher plants, which awaits further studies.
5. Conclusions Deleterious effects, including decreased leaf chlorophyll content and increased leaf malondialdehyde concentration, were observed on the chlorophyll and cell membrane after 9 d of 200 mmol L 1 Cd treatment. The addition of MeJA at the concentration range of 0.1 to 1 mmol L 1 significantly increased the
J. Chen et al. / Ecotoxicology and Environmental Safety 104 (2014) 349–356
activities of CAT and APX in the leaves of K. obovata, accompanied by low quantities of Cd uptake. This finding suggested that low MeJA concentrations (0.1 to 1 mmol L 1) can help K. obovata seedlings alleviate Cd toxicity by increasing the activities of antioxidants in cells. The concentration of endogenous JA in the leaves of K. obovata was decreased by Cd but was positively stimulated by the addition of exogenous MeJA. Treatment of 200 mmol L 1 Cd for 9 d enhanced the expression of KoMT2 in the leaves, whereas application of exogenous MeJA significantly restored the expression levels to control values. This result indicated that MeJA might have no direct regulatory effect on the expression of KoMT2. Exogenous MeJA also inhibited the uptake of Cd to the aboveground sensitive part (leaves) of the seedlings. The reduced uptake of Cd might be a result of stomatal closure and decreased transpiration by exogenous MeJA.
Acknowledgments The work described in this paper was supported by National Nature Science Foundation of China (No. 41201525), SKLEC2012RCDW02 and National Nature Science Foundation of China (No. 41271065).
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