Alleviation of cadmium toxicity in Lemna minor by exogenous salicylic acid

Ecotoxicology and Environmental Safety 147 (2018) 500–508

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Alleviation of cadmium toxicity in Lemna minor by exogenous salicylic acid Qianqian Lu, Tingting Zhang, Wei Zhang, Chunlei Su, Yaru Yang, Dan Hu, Qinsong Xu

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MARK

College of Life Science, Nanjing Normal University, Nanjing 210023, China

A R T I C L E I N F O

A B S T R A C T

Keywords: Salicylic acid L. minor Cadmium toxicity Oxidative damage Mineral status Hsp70

Cadmium (Cd) is a significant environmental pollutant in the aquatic environment. Salicylic acid (SA) is a ubiquitous phenolic compound. The goal of this study was to assess the morphological, physiological and biochemical changes in duckweed (L. minor) upon exposure to 10 μM CdCl2, 10 μM CdCl2 plus 50 μM SA, or 50 μM SA for 7 days. Reversing the effects of Cd, SA decreased Cd accumulation in plants, improved accumulation of minerals (Ca, Mg, Fe, B, Mo) absorption, increased endogenous SA concentration, and phenylalanine ammonialyase (PAL) activity. Chlorosis-associated symptoms, the reduction in chlorophyll content, and the overproduction of reactive oxygen species induced by Cd exposure were largely reversed by SA. SA significantly decreased the toxic effects of Cd on the activities of the superoxide dismutase, peroxidase, catalase, ascorbate peroxidase, and glutathione reductase in the fronds of L. minor. Furthermore, SA reversed the detrimental effects of Cd on total ascorbate, glutathione, the ascorbic acid/oxidized dehydroascorbate and glutathione/glutathione disulphide ratios, lipid peroxidation, malondialdehyde concentration, lipoxygenase activity, and the accumulation of proline. SA induced the up-regulation of heat shock proteins (Hsp70) and attenuated the adverse effects of Cd on cell viability. These results suggest that SA confers tolerance to Cd stress in L. minor through different mechanisms.

1. Introduction Heavy metal contamination has become an increasing ecological and environmental problem worldwide. Cd is one of the most toxic heavy metals. It is mainly derived from mining, smelting, and manufacturing; it enters agricultural and ecological systems via, sewage, irrigation, use of sludge, and use of Cd-containing phosphate fertilizers (Zhao et al., 2011). Through bioaccumulation, Cd is not only detrimental to terrestrial and aquatic ecosystems, but also to human health (Duruibe et al., 2007; Thijssen et al., 2007). The toxicity of Cd has been related to reduced absorption of nutrients (Andresen et al., 2016), increased oxidative stress, changes in the activity of antioxidant enzymes (Razinger et al., 2008), and modifications to gene expression (Herbette et al., 2006). In plants, Cd promotes the accumulation of reactive oxygen species (ROS) leading to decreased growth, roll, chlorosis of leaves, and necrosis of roots (Schützendübel et al., 2001). After exposure to Cd, plants activate antioxidant defense mechanisms, and change cellular metabolism, to maintain the cellular redox homeostasis (Zeng et al., 2011). Antioxidative enzymes (e.g. superoxide dismutase [SOD], catalase [CAT], and peroxidase [GPX]) are involved in the detoxification of O2•− and H2O2. Ascorbate peroxidase (APX), glutathione reductase (GR), and glutathione and ascorbate acid are important components of the ascorbate-glutathione (AsA-GSH) cycle. These

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enzymes are responsible for removal of H2O2 in different cellular compartments (Rodríguez-Serrano et al., 2009). Heat shock proteins (Hsps) are a family of proteins that are activated in plants under adverse conditions (Wang et al., 2004). Hsp70s are the most abundant chaperones in living cells. Different organisms produce distinct and variable isoforms of Hsp70s that are highly conserved (Basile et al., 2015). Upon exposure to heavy metals, plants tend to up-regulate the synthesis of Hsp70 (Wang et al., 2011; Basile et al., 2015). In order to maintain homeostasis, to protect normal cell functioning, and to adapt to various types of stress, Hsp70s are up-regulated so that they can repair denatured proteins, or accelerate their disposal of denatured proteins (Wang et al., 2004). The increase of Hsp70 expression in response to various types of stress has led to their use as biomarkers of environmental stress (Ireland et al., 2004; Saidi et al., 2007). For instance, western blotting for Hsp70s has recently been proposed as a method to identify the effects of heavy metals (Cd, Cu, Pb, Zn, and Cr) on plants (Wang et al., 2011; Basile et al., 2013, 2015). Salicylic acid (SA) is a regulator of metabolism and of various physiological processes. It is involved in both local and systemic defense responses in plants (Kawano and Bouteau, 2013). It has a fundamental role in the regulation of plant growth, and in the development and immune responses (An and Mou, 2011). SA is also involved in signaling during abiotic stress, such as plant responses to heavy metals.

Corresponding author. E-mail address: [email protected] (Q. Xu).

http://dx.doi.org/10.1016/j.ecoenv.2017.09.015 Received 17 March 2017; Received in revised form 5 September 2017; Accepted 7 September 2017 0147-6513/ © 2017 Elsevier Inc. All rights reserved.

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390.0 nm and the concentration of H2O2 was extrapolated from a standard curve. Superoxide radical (O2•−) was measured according to Oracz et al. (2007). The incubation mixture contained the homogenized supernatant, 65.0 mM PBS (pH 7.8), 10.0 mM hydroxylamine hydrochloride, 17.0 mM sulfanilamide, and 7.0 mM α-naphthylamine. The absorbance was read at 530.0 nm. A calibration curve was established using sodium nitrite. H2O2 and O2•− were localized histochemically by staining fronds with 1% 3, 3-diaminobenzidine and 0.1% nitrobluetetrazolium (NBT) solution, respectively (Chen et al., 2010).

A great deal of work has been done to explore the role of SA in alleviating the toxicity of heavy metals in plants. SA was able to protect barley seedlings against Cd toxicity by inducing the synthesis of phytochelatins (PCs), and other low molecular mass metabolites and proteins (Metwally at el, 2003). In rice roots, increased levels of SA, induced by Cd, act directly as antioxidants to scavenge ROS. Also, increased levels of SA indirectly modulate the redox balance through the activation of antioxidant responses (Krantev et al., 2008; Guo et al., 2009). In soybean seedlings, SA leads to Cd passivation, combining it with other molecules, or through interaction with the PCs (Drazic and Mihailovic, 2005). In flax, SA alleviates Cd toxicity through indirect maintenance of ionic homeostasis (Drazic et al., 2006) and stabilization of membrane integrity (Belkadhi et al., 2015). Duckweed (Lemna minor) is a common widespread aquatic plant residing in lakes, streams, ponds, and other water bodies. Because of its small size, simple structure and morphology, rapid growth rate, short lifespan, and sensitivity to environmental pollutants, duckweed has been commonly used as a model hydrophyte in ecotoxicological studies (Balen et al., 2011). In particular, L. minor has been reported to accumulate toxic metals and therefore is being used as an experimental model system to investigate the accumulation of heavy metals (e.g. Cd, Cu, Zn, Ni, and Co) and their toxicity (Kanoun-Boulé et al., 2009; Appenroth et al., 2010; Balen et al., 2011; Sree et al., 2015). In the present study, the influence of SA on Cd-induced changes of various antioxidants and Cd, Ca, Mg, Fe, B and Mo, as well as on phenylalanine ammonialyase (PAL) activity and Hsp70 induction in L. minor has been assayed. Our results support the idea that exogenous SA alleviates Cd toxicity by reducing Cd uptake, suppressing ROS production and oxidative stress by re-establishing redox homeostasis, and maintaining elemental balance. The results of present study are novel in being the first to demonstrate that exogenous application of SA modulates the PAL activity and Hsp70 induction to confer resistance against Cd stress.

2.5. Lipid peroxidation The level of lipid peroxidation products was expressed as malondialdehyde (MDA) content and was determined by measuring 2thiobarbituric acid-reactive metabolites (Razinger et al., 2008). The concentration of MDA was expressed as nmol g−1 fresh weight (FW). 2.6. Enzyme extraction and gel electrophoresis Fresh fronds were homogenized in 50.0 mM PBS (pH 7.8) containing 0.2 mM EDTA and 2% (w/v) polyvinylpyrrolidone. The homogenate was centrifuged for 20 min at 10,000 g at 4 °C, and the supernatant was used for determination of LOX, SOD, GPX, CAT, APX, and GR activities. Plant extracts containing equal amounts of protein (20 μg/well) were subjected to non-denaturing gel electrophoresis (stacking gel 3% and separating gel 10%). After electrophoresis, gels were rinsed and washed with PBS (pH 7.0), and used for detection of LOX, SOD, GPX, CAT, APX, and GR isozymes. 2.6.1. Lipoxygenase activity and gel electrophoresis Extract for lipoxygenase (LOX) was prepared according to the method described by Cui et al. (2013). The reaction mixture contained 50.0 μL of enzyme extract (in 1.5 mL total volume), 200.0 mM borate buffer (pH 6.0), 0.25% linoleic acid, and 0.25% Tween-20. Enzyme activity was calculated by the increase in absorbance at 234.0 nm min−1 g−1 fresh weight. The gels were stained for at least 6 h in 50.0 mM PBS (pH 6.0) solution containing 0.1% linoleic acid, 0.02% o-dianisidine, and 6.6% ethanol (Wang et al., 2009).

2. Materials and methods 2.1. Plant material and experimental design L. minor was collected from Nanjing, China and cultivated under laboratory conditions (114 μmol m−2 s−1 light irradiance, 14 h photoperiod, 25 °C/20 °C day/night temperature) in 1/10 Hoagland solution. After growing for at least one week, similar fronds of duckweed were cultivated in 1/10 Hoagland solution for 7 days while being exposed to: (1) left untreated (control), (2) 10 μM Cd2+, (3) 10 μM Cd2+ plus 50 μM SA, (4) 50 μM SA. All solutions were renewed every 2 day. Three replicates were used for each treatment, and analyzed independently.

2.6.2. SOD activity and gel electrophoresis SOD activity was analyzed using the method reported by Jin et al. (2008). The reaction mixture contained the enzyme extract, 2.0 mM riboflavin, 75.0 mM NBT, 13.0 mM methionine, 0.1 mM EDTA, and 50.0 mM PBS (pH 7.8). One unit of the activity was defined as the amount of enzyme required to inhibit 50% of the initial NBT reduction under light. SOD isozymes were separated by electrophoresis and stained with NBT and riboflavin (Liu et al., 2011). The gels were incubated in the dark for 30 min at room temperature in an assay mixture containing 50.0 mM PBS (pH 7.8), 1.0 mM EDTA, 0.05 mM riboflavin, 0.1 mM NBT, and 0.3% N, N, N′′, N′′-tetramethylethylenediamine (TEMED). After that, the gels were rinsed with water and developed on a light box for 10 min at room temperature, after which the colorless bands of SOD activity on a purple-stained gel were visible.

2.2. Metal uptake The content of Cd and nutrient elements (Fe, Mg, Ca, B, and Mo) was analyzed by inductively-coupled plasma atomic emission spectrometry (Leeman Labs, Prodigy, Hudson, NH, USA) after decomposition with HNO3 and HClO4. 2.3. Photosynthetic pigments and visible injury Chlorophylls (Chl) and carotenoids (Car) were extracted from 0.4 g leaves of L. minor with 80% acetone. The Chl and Car contents were calculated according to Lichtenthaler (1987). The fronds were scanned with an Epson Perfection V700 Photo (J221A, Tokyo, Japan).

2.6.3. GPX activity and gel electrophoresis GPX activity was determined with guaiacol (Jin et al., 2008). The reaction mixture consisted of the enzyme extract in 50.0 mM PBS (pH 6.5) containing 1% guaiacol, and 0.4% H2O2. Enzyme activity was calculated by the increment in the absorbance at 470.0 nm min−1 g−1 (FW) at 25 °C. To detect the activity of GPX isozymes, the gel was stained in 1.0 mM 3, 3-diaminobenzidine, and 0.03% H2O2 (Adam et al., 1995).

2.4. Analysis of H2O2 and O2•− H2O2 measurements were performed according to Jin et al. (2008). Fronds were homogenized with 0.1% (w/v) TCA. 1.0 mL of the supernatant was mixed with 1.0 mL 10.0 mM phosphate-buffered saline (PBS) and 2.0 mL 1.0 M KI. The absorbance of supernatant was read at 501

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supernatant was collected and the extraction was repeated. Pooled supernatants were dried under N2; residues were redissolved in 2.0 mL of 0.05% HOAc in H2O-MeCN (85:15, v/v), and finally filtered with a 0.25-mm nylon syringe pre-filter for quantitative analysis. Quantitative analysis of SA in solution was conducted using a Waters ACQUITY TQD Tandem Quadrupole UPLC/MS/MS System. A BEHC18 (Waters, Co., Milford, MA, USA) column (50 mm × 2.1 mm, 1.7 mm) was used at 45 °C and the injected volume was 5.0 μL. The elution gradient was carried out using a binary solvent system consisting of 0.2% HOAc in H2O and MeOH at a constant flow rate of 300 mL/min. A linear gradient profile with the following proportions (v/v) of MeOH was applied. MS settings of ionization were ESI negative for SA. Nitrogen was used as the desolution gas (400 L, h−1). We used the multiple reactions monitoring (MRM): MRM transition 137 > 93, dwell time 0.1 s, cone voltage 36 V, and collision energy 16 eV. Quantification of AS was done by the standard addition method, spiking control samples with authentic SA. PAL activity was assayed by monitoring the production of t-cinnamic acid at 290.0 nm (Chen et al., 2015). Fronds were ground with liquid nitrogen and homogenized in 50.0 mM Tris-HCl (pH 8.8) containing 0.5 mM EDTA. The reaction mixture contained 50.0 mM TrisHCl buffer (pH 8.8), 20.0 mM L-phenylalanine, and the enzyme extract. Absorbance at 290.0 nm was measured after 30 min. One unit of enzyme activity represents the amount of enzyme causing the decrease in absorbance of 0.01 per min. PAL activity was expressed as U min−1 g−1 FW.

2.6.4. CAT activity and gel electrophoresis CAT activity was measured at 405.0 nm through a hydrogen peroxide-dependent assay, based on the formation of a stable complex with ammonium molybdate (Goth, 1991). One unit of CAT activity represents the decomposition of 1.0 μM of hydrogen peroxide per minute. CAT isozymes were detected on non-denaturing gels by soaking the gels in H2O2, rinsing them with water, and staining in 1% potassium ferricyanide plus 1% ferric chloride solution (Liu et al., 2011). 2.6.5. APX activity and gel electrophoresis APX activity was determined by monitoring the rate of ascorbate oxidation at 290.0 nm (Jin et al., 2008). The protein extract was added to the reaction solution, 50.0 mM PBS (pH 7.0) containing 0.2 mM EDTA and 0.5 mM ascorbic acid (AsA). The reaction was started by adding 50.0 μL of 200.0 mM H2O2. The decrease in absorbance at 290.0 nm was monitored, and enzyme activity was calculated using an absorbance coefficient for AsA of 2.6 mM−1 cm−1. One unit of ascorbate peroxidase represents the oxidation of 1 μM min−1 at 25 °C. The APX gel was washed in the buffer for 1 min and submerged in a solution of 50.0 mM PBS (pH 7.8) containing 28.0 mM TEMED and 2.45 mM NBT for 10–20 min with gentle agitation in the presence of light (Liu et al., 2011). 2.6.6. GR activity and gel electrophoresis GR activity was estimated by measuring the decrease of absorbance at 340.0 nm (Foyer and Halliwell, 1976). The reaction mixture contained 150.0 μL enzyme in 3.0 mL of 25.0 mM PBS (pH 7.0) containing 0.1 mM EDTA, 0.5 mM GSSG, and 0.12 mM NADPH. GR isozymes were detected by incubating the gel in 50.0 mL 0.25 M Tris-HCl buffer (pH 7.5) containing 10.0 mg of 3-(4, 5-dimethylthiazol2–4)-2, 5-diphenyl tetrazolium bromide, 10.0 mg of 2, 6-dichlorophenolindophenol, 3.4 mM GSSG, and 0.5 mM NADPH (Liu et al., 2011).

2.10. Western blotting of Hsp70 products Plant material was homogenized and extracted with 50.0 mM pH 7.6 Tris-HCl, 5.0 mM MgCl2, 10.0 mM NaCl, 0.4 M sucrose, and 0.1% BSA in ice-cold extraction buffer according to the protocol of Kügler et al. (1997). For sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE) (separating gel 12% and stacking gel 5%), 20.0 μg of total proteins were loaded onto each lane. After electrophoresis, the proteins were transferred to PVDF membranes and immunoblotted with anti-Hsp70 antibody (1:5000) purchased from Agrisera AB (Vannas, Sweden). A chemiluminescence detection system (ECL, Sigma) was used for antibody detection.

2.7. Ascorbate and glutathione AsA content was measured according to Tewari et al. (2006). This assay is based on the reduction of Fe3+ to Fe2+ by AsA and the subsequent formation of Fe2+ and bipyridyl complexes that absorb light at 525.0 nm (pink color). The amount of AsA was calculated using a standard curve that was prepared with known concentrations of AsA. Total ascorbate (ASC) was measured after reducing dehydroascorbate (DHA) to AsA by dithiothreitol in the supernatant. DHA content was calculated from the difference between ASC and AsA. Total glutathione and GSSG were determined by the 5, 5-dithiobis(2-nitrobenzoic acid)-GR recycling procedure (Nagalakshmi and Prasad, 2001). Changes in absorbance of the reaction mixtures were measured at 412.0 nm and the total glutathione content was calculated from a standard curve with GSH. GSSG was determined after removal of GSH by 2-vinylpyridine derivatization. A specific standard curve with GSSG was used. GSH was determined by subtraction of GSSG from the total glutathione content.

2.11. Cell death Cell death was measured according to the protocol of Guo et al. (2009). Fronds were stained with 0.025% (w/v) Evans blue, and then washed twice in distilled water. The fronds were ground in a mortar with 50% (v/v) MeOH and 1% (w/v) SDS. The homogenates were incubated in a water bath at 50 °C, and then centrifuged at 10,000 g for 15 min. Absorbance of the supernatant was measured at 600.0 nm. 2.12. Statistical analysis For each assay, all the experiments were repeated at least three times. The results are presented as means ± SD. For statistical analysis, we performed ANOVA tests using SPSS 16.0 software package. Differences between the treatments were tested by the least significant difference test at a 0.05 probability level.

2.8. Proline Proline content was determined according to the protocol reported by Bates et al. (1973). The reaction mixture consisted of 2.0 mL of the supernatant, 2.0 mL acid ninhydrin, and 2.0 mL glacial acetic acid; the reaction mixture was then extracted with 4.0 mL of toluene. The proline content was expressed as μg g−1 FW.

3. Results 3.1. Mineral nutrients and Cd content Cd treatment decreased the plant content of Fe (49% of the content of samples without Cd), Mg (70% of the content of samples without Cd), Ca (77% of the content of samples without Cd), B (83% of the content of samples without Cd) and Mo (78% of the content of samples without Cd). Inversely, as compared to the controls, SA (50 μM) added to the

2.9. Endogenous salicylic acid and PAL activity SA in L. minor was prepared for quantitative analysis according to the protocol by Segarra et al. (2006). Fronds were extracted with 3.0 mL of a mixture of MeOH-H2O-HOAc (90:9:1, v/v/v). The 502

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Table 1 Effects of SA on Cd and nutrient element contents in the fronds of L. minor treated with (1) control (CK), (2) 10 μM Cd (Cd), (3) 10 μM Cd + 50 μM SA (Cd + SA), (4) 50 μM SA (SA) for 7d. Data are mean ± SD, n = 3. Different letters in the same line indicate significant differences at P < 0.05. Element content (μg g−1FW)

CK

Cd

Cd + SA

SA

Cd Ca Mg Fe B Mo

N.D. 1950b ± 21.2 269a ± 3.29 60.2b ± 0.232 1.74a ± 0.135 0.368ab ± 0.0113

125a ± 0.829 1490d ± 6.44 191c ± 6.96 29.6d ± 4.69 0.768c ± 0.0629 0.288c ± 0.0163

77.8b ± 7.93 1870c ± 17.7 231b ± 8.04 51. 9c ± 5.44 1.05b ± 0.0255 0.336b ± 0.00778

N.D. 2180a ± 37.9 274a ± 8.01 76.4a ± 4.12 1.71a ± 0.0396 0.384a ± 0.0205

alone had no significant effect on SOD activity under non-stress condition; however, it notably increased the SOD activity of Cd-stressed L. minor. Non-denaturing PAGE analysis revealed six SOD isoenzymes in duckweed plants (Fig. 2B). The activity staining for SOD isoenzymes confirmed that, upon exposure to Cd, the activity of SOD II isozymes disappeared completely, while the SOD III isozyme had an obvious decrease in activity. These effects were reversed in plants exposed to Cd and SA together. Exogenous SA influenced SOD II and III to increase total SOD activity. After exposure to Cd, GPX activity was 23% less than the control (Fig. 2C). However, in Cd stressed fronds supplemented with SA, GPX activity was 17% lower than the control. No significant effect was noticed on GPX activity in L. minor treated with SA alone. Native gel electrophoresis revealed five GPX isoenzymes in L. minor (Fig. 2D). GPX V disappeared completely upon exposure of plants to Cd. The activities of GPX II, III, IV (thus total GPX activity) increased in plants exposed to Cd in combination to SA, compared to plants exposed to Cd alone. Exposure to Cd strongly inhibited the activity of CAT in the fronds; this activity was 47% lower than the control (Fig. 2E). However, this effect was significantly reversed by the addition of SA to fronds exposed to Cd (P < 0.05). Application of SA alone had no significant effect on CAT activity in comparison to the control. The activities of CAT I and II isoenzymes were decreased in plants exposed to Cd compared to the control (Fig. 2F). Exogenous SA reversed this effect and induced the activity of two new isozymes CAT III and IV, resulting in increased total CAT activity. Under Cd stress, the activity of APX increased by 287% as compared to the control (Fig. 2G). However, exogenous application of SA to Cd stressed fronds notably enhanced APX activity (185% over the untreated controls) (P < 0.05). SA treatment alone had on significant effect on APX activity. Cd enhanced the activities of APX I and II (particularly of APX I) (Fig. 2H). This effect was reversed when SA was present in the Cd medium, although APX activity was still higher compared with Cd-free plants. Fig. 2I shows that exposure to Cd reduced GR activity; this activity was 16% lower than in the control. The presence of SA counteracted the effect of Cd, and there was only a 12% decrease in GR activity compared to the control. Application of SA alone showed a significant increase in GR activity over the control (P < 0.05). Cd decreased the activity of GR I, II, and III, while exogenous SA reversed this effect for all GR isozymes (Fig. 2J).

growth medium, Fe, Mg, Ca, and Mo increased by 27%, 2%, 12%, 4%, respectively, and B was slightly decreased by 1%. Plant exposure to Cd in combination with SA reversed the reduction of Fe (86% of the content of samples without Cd), Mg (85% of the content of samples without Cd), Ca (96% of the content of samples without Cd), B (95% of the content of samples without Cd) and Mo (91% of the content of samples without Cd) induced by Cd. Plant exposure to Cd in combination with SA reduced Cd accumulation by 38% in the fronds of L. minor (P < 0.05, Table 1). 3.2. Photosynthetic pigments and apparent injury Compared with control, when the growth medium was supplemented with SA, the Chl a and Car concentrations increased by 5% and 6%, respectively, whereas Chl b decreased by 14%. Exposure to Cd caused reduced Chl a (33%), Chl b (51%) and Car (15%). Upon exposure to Cd in combination with SA there were minor reductions in the levels of Chl a (7%), Chl b (11%) and Car (8%) (Fig. 1A). The edges and the central area of the fronds showed chlorosis upon Cd treatment, while SA amendment to the Cd-treated plants decreased the area and the degree of chlorosis; only the edges of leaves exhibited chlorosis (Fig. 1B). 3.3. MDA concentration and LOX activity MDA content was 23% higher in the fronds of L. minor exposed to Cd, than in the un-exposed control. Inversely, application of SA separately in the culture solution in the absence of Cd reduced the MDA content and it significantly alleviated this increment, being only 12% higher than the control (P < 0.05) (Fig. 1C). Increased MDA content induced by Cd has been associated with increased LOX activity. Cd significantly enhanced the activity of LOX (136% with respect to the control) (P < 0.05). Application of SA alone had no significant effect on LOX activity; however, it significantly decreased LOX activity in Cd-affected plants (125% with respect to the control) (P < 0.05, Fig. 1D). Analysis of LOX isoenzymes indicated that the addition of SA to plants exposed to Cd significantly decreased LOX I, II, III activity (Fig. 1E). 3.4. ROS production Cd caused a slightly increase in the levels of O2•− (119%) and H2O2 (106%) (P < 0.05), as compared with the controls. Application of SA alone to the nutrient medium did not notably affect ROS production in the fronds of L. minor, however, addition of SA to the Cd medium reversed the effects of Cd on the levels of O2•− and H2O2 (Fig. 1F and G). Histochemical staining showed overproduction of O2•− and H2O2 in plants exposed to Cd. Addition of SA to L. minor plants exposed to Cd reduced ROS content (Fig. 1H and I).

3.6. Ascorbate and glutathione Exposure to Cd increased total ascorbate and total glutathione significantly (P < 0.05), but the AsA/DHA and GSH/GSSG ratios declined 43% and 20% with respect to the control (Table 2). Further experiments showed that the effect of Cd was reversed by SA; total ascorbate and total glutathione were significantly reduced (P < 0.05), and the ratios of AsA/DHA and GSH/GSSG were only 11% and 9% higher than the control. No significant effects were noticed on total ascorbate, total glutathione, AsA/DHA and GSH/GSSG ratios in fronds treated with SA alone.

3.5. Antioxidant enzyme activities As shown in Fig. 2A, SOD activity decreased significantly (P < 0.05) upon the exposure to 10 μM Cd. Compared to control, SA amendment 503

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Fig. 1. Effects of SA on photosynthetic pigments contents and visible injury, MDA content, LOX activity, and ROS accumulation in the fronds of L. minor exposed to four treatments (1) control (CK), (2) 10 μM Cd (Cd), (3) 10 μM Cd + 50 μM SA (Cd + SA), (4) 50 μM SA (SA) for 7d. (A) Photosynthetic pigments contents; (B) visible injury; (C) MDA content; (D) LOX total activity; (E) the activity staining for LOX isoenzymes; (F) O2•− accumulation; (G) H2O2 accumulation; (H) histochemical staining of O2•−; (I) histochemical staining of H2O2. Data are mean ± SD, n = 3. Different letters indicate significant differences at P < 0.05.

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Fig. 2. Changes in enzymatic activities of SOD, GPX, CAT, APX and GR in the fronds L. minor treated with (1) control (CK), (2) 10 μM Cd (Cd), (3) 10 μM Cd + 50 μM SA (Cd + SA), (4) 50 μM SA (SA) for 7d. (A) SOD total activity; (B) staining for SOD isoenzymes; (C) GPX total activity; (D) staining for GPX isoenzymes; (E) CAT total activity; (F) staining for CAT isoenzymes; (G) APX total activity; (H) staining for APX isoenzymes; (I) GR total activity; (J) staining for APX isoenzymes. Data are mean ± SD, n = 3. Different letters indicate significant differences at P < 0.05.

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plants (Drazic et al., 2006; Krantev et al., 2008; Chen et al., 2010; Belkadhi et al., 2015). In the present study, we confirmed that addition of SA had a protective role in L. minor exposed to Cd. Cd leads to a deficiency of macro and micronutrients, which may be a cause of inhibition of photosynthetic electron transport, chlorophyll synthesis, and of the plasma membrane permeability changes (Ramos et al., 2002; Carvalho Bertoli et al., 2012; Xu et al., 2015). In the present study, Cd exposure significantly reduced the contents of Fe, Mg, Ca, B, and Mo in L. minor, but the application of SA significantly reversed this effect, while also reducing Cd accumulation. The lower level of Cd accumulation might be a tolerance strategy of L. minor. Similar effects of exogenous SA on Cd accumulation have been reported in Arabidopsis (Guo et al., 2016). Mg and Fe are key elements in chlorophyll synthesis. Chlorosis caused by Cd has been attributed to the deficiency of these elements in maize (Siedlecka and BaszyńAski, 1993), and soybean (Xu et al., 2015). In our work, the protective action of SA on chlorosis and Chl content could result from increased Mg and Fe levels (Fig. 1A and B). H+-ATPase in plasma membrane plays an important role in the transport of multiple ions (Palmgren and Harper, 1999). SA induces H+-ATPase activity (Metwally et al., 2003; Eichhorn et al., 2006), which might promote increased absorption of Ca, Mg, Fe, B, and Mo. SA might thus play an important role in reducing Cd uptake and maintaining ionic homeostasis in L. minor, thereby improving the resistance to stress by Cd. Hayat et al. (2010) proposed that the protective mechanisms of SA against toxicity by metals were related to its effect on absorption of heavy metal, ions, and nutrient elements. Cd uptake leads to overproduction of ROS and subsequent oxidative stress (Sharma and Dietz, 2009). To reduce detrimental effects of ROS induced by Cd, plants employ ROS-detoxifying antioxidant defense mechanisms (Schützendübel et al., 2001). In this study, SA prevented ROS over production (Fig. 1F–I) and cell death (Fig. 4C). Lipid peroxidation and proline content levels of SA-treated plants were lower than those of plants exposed to Cd (Figs. 1C–E and 3). Similar results were also observed in rice and pea (Guo et al., 2009; Popova et al., 2009). Activities of SOD, GPX, and CAT were augmented in SA-treated plants that were exposed to Cd (Fig. 2). Thus, SA may increase SOD (possibly Cu/Zn-SOD and Mn-SOD), GPX, and CAT activities to reduce the generation of ROS. Similar results were obtained in maize (Krantev et al., 2008) and rice exposed to Cd (Guo et al., 2009). Ascorbate and glutathione are associated with cellular redox balance. The ratios of AsA/DHA and GSH/GSSG may function as signals for the regulation of antioxidant mechanisms (Mittler, 2002). APX and GR are two key enzymes in the AsA-GSH cycle. In this cycle AsA is catalyzed by APX to form monodehydroascorbate by consuming H2O2. GSSG is reduced by GR to form GSH in the presence of NADPH (Apel and Hirt, 2004). In this study APX activity decreased and GR activity increased (Fig. 2I and J) in the presence of SA and Cd, in comparison to Cd treatment alone. AsA/DHA and GSH/GSSG ratios decreased in response to Cd and recovered by addition of SA (Table 2). These results indicate that SA-dependent reduction of the oxidative stress caused by Cd was related to homeostatic balance and to the activities of antioxidative enzymes in the AsA-GSH cycle. Jozefczak et al. (2014) suggested that the balance of AsA/DHA and GSH/GSSG ratios maintained by SA in L. minor upon Cd exposure was due to decreased APX activity and increased GR activity. In agreement with our results, the study of Ali et al. (2015) indicated that SA increased the activity of GR in Brassica napus exposed to Cd. Shi and Zhu (2008) also reported that SA pretreatment inhibited APX activity and promoted GR activity in cucumber. In contrast, the change of APX activity was different from the previous reports (Guo et al., 2009; Wang et al., 2013; Ali et al., 2015). The enhanced Hsp70 and antioxidant enzymes constitute an integrative defense system against Cd-induced oxidative stress (Wang et al., 2012). In our experiments, Cd stimulated the biosynthesis of Hsp70, which was further enhanced by SA (Fig. 4D and E). This suggested that the protective role of SA against oxidative stress induced by

Table 2 SA re-established glutathione and ascorbate homeostasis in the fronds of L. minor treated with (1) control (CK), (2) 10 μM Cd (Cd), (3) 10 μM Cd + 50 μM SA (Cd + SA), (4) 50 μM SA (SA) for 7d. Data are mean ± SD, n = 3. Different letters in the same line indicate significant differences at P < 0.05. Treatment

Total glutathione (μmol g−1FW)

GSH/GSSG

Total ascorbate (μmol g−1FW)

AsA/DHA

CK Cd Cd+SA SA

125c ± 2.63 198a ± 4.97 162b ± 3.47 126c ± 2.51

1.74a ± 0.0375 1.39c ± 0.0651 1.58b ± 0.0621 1.64ab ± 0.0318

17.3c ± 0.239 26.3a ± 0.109 19.5b ± 0.539 17.9c ± 0.162

4.47a ± 0.622 2.573c ± 0.143 3.99b ± 0.871 5.01a ± 0.552

Fig. 3. Effects of SA on proline concentration in L. minor fronds treated with (1) control (CK), (2) 10 μM Cd (Cd), (3) 10 μM Cd + 50 μM SA (Cd + SA), (4) 50 μM SA (SA) for 7d. Data are mean ± SD, n = 3. Different letters indicate significant differences at P < 0.05.

3.7. Proline Exposure to Cd increased the level of proline by up to 343% of the control. In the presence of SA, the content of proline was only 110% higher than the control (Fig. 3). No significant change in proline content was observed in fronds treated with SA alone. 3.8. Endogenous SA content and PAL activity As shown in Fig. 4A, the content of endogenous salicylic decreased upon exposure to Cd (P < 0.05), but it was 33% higher than the control when plants were exposed to Cd and SA. Endogenous SA increased by 251% as compared to the control in plants treated only with SA. The activity of PAL was 83% of the control upon exposure to Cd, but increased over the control by 16% in plants exposed to Cd and SA. The activity of PAL was highest (25% higher than the control) in plants treated only with SA (Fig. 4B). 3.9. Cell death As shown in Fig. 4C, Cd exposure induced cell death by 22% over the control (P < 0.05). In plants exposed to Cd in the presence of SA, cell death was reduced to only 9% over the control. Application of SA alone had no significant effect on cell death as compared to the control. 3.10. Hsp70 accumulation Exposure to Cd increased the accumulation of Hsp70 (revealed by immunoblotting) 50% over the control. The combination of Cd and SA caused a further increment of 92% over the control. SA alone caused an increment of Hsp70 that was 110% higher than the control (Fig. 4D and E). 4. Discussion Supplementation with SA has been shown to attenuate Cd toxicity in 506

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Fig. 4. Effects of SA on endogenous SA and PAL activity, cell death, Hsp70 accumulation in L. minor fronds treated with (1) control (CK), (2) 10 μM Cd (Cd), (3) 10 μM Cd + 50 μM SA (Cd + SA), (4) 50 μM SA (SA) for 7d. (A) Endogenous SA content; (B) PAL activity; (C) cell death; (D) Hsp70 accumulation is expressed as a percentage of the control values of 1; (E) immunodetection of Hsp70. Data are mean ± SD, n = 3. Different letters indicate significant differences at P < 0.05.

reduced cell death; (2) decreased levels of ROS and reestablishment of redox homeostasis (especially GSH/GSSH and AsA/DHA), (3) modulation of total and isozymatic antioxidant enzyme activities, and reduced lipid peroxidation. Moreover, exogenous SA augmented endogenous SA and Hsp70 contents, which regulate plant metabolism, and act as natural signaling molecules involved in both local and systemic plant defense responses. It is thus clear that exogenous SA plays a positive role in reducing Cd toxicity in L. minor.

Cd is probably related to Hsp70 biosynthesis. Stimulation of Hsp70 upon Cd exposure has been reported previously in this species (Basile et al., 2015). The exact mechanisms of Cd-induced Hsp70 biosynthesis and SA alleviation of Cd toxicity in L. minor need to be further investigated. The protective effect of exogenous SA on plants depends on numerous factors, possibly including endogenous SA levels (Horváth et al., 2007). SA accumulation in maize (Pál et al., 2005) and bean (Senaratna et al., 2000) exposed to Cd, and the protective effect of exogenous SA against Cd-induced stress, was also demonstrated in these plant species (Metwally et al., 2003; Krantev et al., 2008). However, reduced activity of PAL and accumulation of SA were detected in the Cd-challenged duckweed. Exogenous SA could stimulate endogenous SA synthesis and enhance PAL activity in our study (Fig. 4A and B). This difference could be attributed to the plant species or to duration of exposure. Biochemical studies have suggested that endogenous SA is synthesized from phenylalanine, with benzoate as the immediate precursor by PAL. PAL is a key regulator of the phenylpropanoid pathway and is induced under a variety of biotic and abiotic stress conditions (Kovács et al., 2014; Chen et al., 2015). The decline in PAL activity reduces SA content in wheat leaves exposed to Cd (Kovács et al., 2014). The positive relationship between PAL activity and endogenous SA content suggested that PAL may be a key regulatory of SA biosynthesis in L. minor.

Acknowledgments This research was supported by the National Natural Science Foundation of China (No. 30800055), the Priority Academic Pro-gram Development of the Jiangsu Higher Education Institutions (PAPD) and by the Qin-Lan Project. The metal contents were analyzed by Nanjing Normal University Center for Analysis and Testing. References Adam, A.L., Bestwick, C.S., Barna, B., Mansfield, J.W., 1995. Enzymes regulating the accumulation of active oxygen species during the hypersensitive reaction of bean to Pseudomonas syringae pv. Phaseolicola. Planta 197, 240–249. Ali, E., Maodzeka, A., Hussain, N., Shamsi, H., Jiang, L., 2015. The alleviation of cadmium toxicity in oilseed rape (Brassica napus) by the application of salicylic acid. Plant Growth Regul. 75, 641–655. An, C., Mou, Z., 2011. Salicylic acid and its function in plant immunity. J. Integr. Plant Biol. 53, 412–428. Andresen, E., Kappel, S., Stärk, H.J., Riegger, U., Borovec, J., Mattusch, J., Heinz, A., Schmelzer, C.E.H., Matoušková, Š., Dickinson, B., Küpper, H., 2016. Cadmium toxicity investigated at the physiological and biophysical levels under environmentally relevant conditions using the aquatic model plant Ceratophyllum demersum. New Phytol. 210, 1244–1258. Apel, K., Hirt, H., 2004. Reactive oxygen species: metabolism, oxidative stress, and signal

5. Conclusions In conclusion, it was observed that exogenous SA treatment resulted in the alleviation of Cd stress as revealed by: (1) increased photosynthetic pigments and mineral nutrients, reduced Cd accumulation, 507

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