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The effects of exogenous salicylic acid on alleviating cadmium toxicity in Nymphaea tetragona Georgi
South African Journal of Botany 114 (2018) 267–271
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South African Journal of Botany journal homepage: www.elsevier.com/locate/sajb
The effects of exogenous salicylic acid on alleviating cadmium toxicity in Nymphaea tetragona Georgi C.-S. Gu a,b, Y.-H. Yang a, Y.-F. Shao a, K.-W. Wu a, Z.-L. Liu a,⁎ a b
College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China The Jiangsu Provincial Platform for Conservation and Utilization of Agricultural Germplasm, Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing 210014, China
a r t i c l e
i n f o
Article history: Received 17 May 2017 Received in revised form 25 September 2017 Accepted 17 November 2017 Available online xxxx Edited by M Vaculik Keywords: Accumulation Antioxidative system Cadmium Nymphaea tetragona Salicylic acid
a b s t r a c t The effects of salicylic acid pretreatments on lipid peroxidation, photosynthetic pigments, the antioxidative system and cadmium (Cd) accumulation in waterlily under Cd stress were studied. A salicylic acid pretreatment lowered the concentrations of malondialdehyde and proline, but increased the contents of photosynthetic pigments, glutathione, non-protein thiol and phytochelatins. Salicylic acid pretreatments enhanced the activities of antioxidant enzymes, such as superoxide dismutase, peroxidase and ascorbate peroxidase. Furthermore, SA pretreatments also decreased the Cd concentrations in different plant organs. Thus, exogenous SA treatments could attenuate the toxic effects of Cd in Nymphaea tetragona. © 2017 SAAB. Published by Elsevier B.V. All rights reserved.
1. Introduction Heavy metal pollution has become a dangerous global environmental pollution problem. Cadmium (Cd) is a widely distributed and highly toxic heavy metal. Cd can enter plants through contaminated soil and water, not only polluting the environment, but also resulting in declines in crop yield and quality. It is a threat to humans and others in the food chain. Cd pollution can be decreased by physical–chemical remediation, phytoremediation and microbial remediation methods (Liu et al., 2012). Phytoremediation refers to the use of plants to remove heavy metals from contaminated soil and waste water, and it has advantages of ‘green’ purification, producing no secondary pollutants, low investment costs and ease of use (Clemens and Ma, 2016). Salicylic acid (SA) is a kind of small-molecule phenolic compound prevalent in plants that can affect many physiological processes (Miura and Tada, 2014). It is also a plant endogenous signal molecule (Yan and Dong, 2014). SA has roles in improving plant disease resistance (Delaney et al., 1994), salt tolerance (Borsani et al., 2001), drought resistance (Saruhan et al., 2012) and cold resistance (Miura and Tada, 2014). Moreover, SA has alleviation effects on plants under heavy metal stress (Agami and Mohamed, 2013; Belkadhi et al., 2015) and can alleviate Cd toxicity in wheat seedlings (Agami and Mohamed,
⁎ Corresponding author. E-mail address: [email protected] (Z.-L. Liu).
https://doi.org/10.1016/j.sajb.2017.11.012 0254-6299/© 2017 SAAB. Published by Elsevier B.V. All rights reserved.
2013). Moreover, exogenous SA protects phospholipids against Cd stress in flax (Belkadhi et al., 2015). It is important to develop aquatic ornamental plants with characteristics of high ornamental value, fast growth and high biomass in phytoremediation technology in heavy metal polluted water bodies. In a previous study, physiological responses and bioaccumulation in Nymphaea tetragona Georgi under Cd exposure were investigated (Yang et al., 2015). Here, SA was used as a regulatory factor to explore its protective effects on the toxicity of Cd in N. tetragona Georgi and to provide foundations for understanding the alleviation of the stress effects of heavy metals on plants and the ecological defenses of heavy metal-contaminated plants. 2. Materials and methods 2.1. Plant material and growth conditions The test material N. tetragona Georgi cv ‘Masaniello’, was taken from Nanjing Yilian Court, Jiangsu Province. The experiments were performed in the Flower Research Institute of Nanjing Agricultural University. The rhizome explants of N. tetragona were watered with halfstrength Hoagland's nutrient solution in a turnover box (length, 50 cm; width, 38 cm; height, 29 cm) in with a 28 °C 14-h light/22 °C 10-h dark cycle and light intensity of 120 μmol m−2 s−1 (Yang et al., 2015). The full Hoagland's nutrient solution contained the following compositions: 5 mM KNO3, 5 mM Ca(NO3)2, 2 mM MgSO4, 1 mM
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KH2PO4, 1 mM FeSO4-EDTA, 46 μM H3BO4, 9.1 μM MnCl2.4H2O, 0.32 μM CuSO4.5H2O, 0.76 μM ZnSO4.7H2O, 0.5 μM H2MoO4·H2O. The hydroponic culture was adjusted to pH 6.0. When the plants had grown for ~7–8 weeks, healthy plants were selected. The following independent four treatments were used: (1) control (CK; exposed to neither SA nor Cd) for 9 d; (2) 20 μM SA treatment for 9 d; (3) 150 mg·l−1 Cd (CdCl2) treatment for 9 d; (4) plants were grown in 20 μM SA for 1 d and then transferred to half-strength Hoagland's nutrient solution containing 150 mg·l−1 Cd for 9 d. The relative physiological indices, including malondialdehyde (MDA) content, relative conductivity, proline content, antioxidant enzyme activities, glutathione (GSH), non-protein thiol (NPT) and phytochelatins (PCs), were measured in leaves sampled after 1, 5 and 9 d. After 9 d, the chlorophyll content and the accumulation of Cd in different plant parts (roots, rhizomes, petioles and leaves) were determined.
as follows: 2.75 ml of 50 mM potassium phosphate buffer (pH 6.1), 0.1 ml of 0.4% (v/v) H2O2, 0.1 ml of 1% (w/v) guaiacol and 0.05 ml of enzyme extract. One unit of POD activity was defined as the amount of enzyme causing the change in absorbance per minute. It is expressed as U g−1 FW. The ascorbic acid peroxidase activity (APX) was determined in a 3 ml reaction mixture containing 1.8 ml of 50 mM potassium phosphate buffer (pH 7.0), 0.1 ml of 15 mM ascorbate, 1 ml of 0.3 mM H2O2 and 0.1 ml of enzyme extract. The absorbance was determined at 290 nm for 60 s with New century T6 spectrophotometer (New century, Beijing, China). One unit of APX activity was defined as the amount of enzyme decomposing 1 μ mol of substrate min−1. It is expressed as U g−1 FW (Chen et al., 2014).
2.2. Malondialdehyde (MDA)
In brief, leaves (0.5 g) were ground in 4 ml of 3% sulfosalicylic acid. After boiling the samples for 15 min, homogenates were centrifuged at 4000 × g at 4 °C for 20 min. 2 ml of supernatant was mixed with 2 ml of acetic acid and 3 ml of 2.5% acid ninhydrin. After boiling for 40 min and cooling, 4 ml of toluene was added. Absorbance was determined at 520 nm (New century, Beijing, China) using toluene as blank.
In brief, 0.5 g of leaves were homogenized with 10% trichloroacetic acid (TCA) and centrifuged at 4000×g for 20 min. 2 ml of the supernatant was then mixed with 2 ml of 0.6% thiobarbituric acid solution (TBA). After heating at 100 °C for 30 min, the mixtures were centrifuged at 4000×g for 15 min after quickly being cooled down. The absorbance of the supernatant was recorded at 532, 600 and 400 nm with New century T6 spectrophotometer (New century, Beijing, China). Amounts of MDA were expressed as μmol g−1 fresh weight using the following formula: 6.45(A532 − A600) − 0.56A450. 2.3. Relative conductivity In brief, 0.2 g disks (6 mm diameter each) from fresh leaves were rinsed with deionized water for 3 min. After dried with filter paper, samples were immersed in a tube with 10 ml of deionized water and gently shaken for 3 h at 25 °C. After the incubation, conductivity (C1) was measured using conductivity meter (Leici-DDS-307A, Shanghai, China). Then conductivity (C2) was measured after boiling the samples for 20 min. Relative conductivity (%) was defined as (C1 / C2) × 100. 2.4. Chlorophyll (Chl) content In brief, leaves (0.2 g) were ground in 80% acetone. Then the supernatant was used to measure chlorophyll content. The optical density (OD) was recorded at 470, 647 and 665 nm with New century T6 spectrophotometer (New century, Beijing, China) against 80% acetone used as blank. Chlorophyll content was calculated using the following formula: Chl a = 13.95 ∗ OD665 − 6.88 ∗ OD470; Chl b = 24.96 ∗ OD647 − 7.32 ∗ OD665; Total chlorophyll = Chl a + Chl b. 2.5. Antioxidant enzyme activity In brief, leaves (0.2 g) were ground in 5 ml of 50 mM phosphate buffer (pH 7.8 for SOD and POD activities, pH 7.0 for APX activity) containing 1% (w/v) polyvinylpyrrolidone (PVP) and centrifuged at 4000×g at 4 °C for 20 min. The supernatant was then used to measure the SOD, POD and APX activities. Total superoxide dismutase (SOD) activity was assayed by nitro blue tetrazolium (NBT) photoreduction method. In brief, the reaction mixture (3 ml) was as follows: 1.7 ml of 50 mM phosphate buffer saline (PBS) (pH 7.8), 0.3 ml of 130 mM methionine, 0.3 ml of 0.75 mM nitroblue tetrazolium (NBT), 0.3 ml of 1 mM EDTA, 0.3 ml of 20 μM riboflavin and 0.1 ml of enzyme extract. The reaction was conducted for 20 min under 4000 lx fluorescent lamp. One unit of SOD activity was defined as the amount of enzyme needed to inhibit NBT photoreduction by 50%. It is expressed as U g−1 FW. Total peroxidase activity (POD) was determined with guaiacol method with modifications. In brief, the reaction mixture (3 ml) was
2.6. Proline content
2.7. GSH, NPT and PCs contents GSH content was assayed as described previously (Hissin and Hilf, 1976). In brief, leaves (0.5 g) were ground in 1 ml of 25% HPO3 and 3 ml of 0.1 M sodium phosphate–EDTA buffer (pH 8.0) and centrifuged at 4000 × g for 20 min. The assay mixture for GSH estimation was as follows: 3.6 ml of phosphate-EDTA buffer, 0.2 ml of O-phthalaldehyde solution and 0.2 ml of supernatant extract. After mixing at room temperature for 15 min, fluorescence at 420 nm was measured with the excitation at 350 nm. NPT content was determined using the method of Rama Devi and Prasad (1998). In brief, leaves (0.5 g) were ground in 4 ml of 5% sulfosalicylic acid and centrifuged at 4000×g for 20 min. The assay mixture was as follows: 2 ml of 0.2 M Tris-HCl (pH 8.2), 0.15 ml of 5,5′Dithio-bis-(2-nitrobenzoic acid) (DTNB) and 0.2 ml of supernatant extract. After 20 min, absorbance was determined at 412 nm. PCs content was measured as described by Bhargava et al. (2005). Phytochelatin was measured by using formula: total thiol-GSH. 2.8. Cd accumulation After washing with distilled water, the tissues (root, rhizome, petiole and lamina) were dried at 80 °C for 48 h. The solubilized samples were a mixed of nitric-perchloric acid (4:1, v/v), diluted with 5 ml with 2.5% HNO3. Cd accumulation was measured by atomic absorption spectrophotometry (Model 3300, Perkin-Elmer, USA) (Gu et al., 2014). 2.9. Statistical treatment SPSS v17.0 software (SPSS Inc., Chicago, IL, USA) was used for the statistical analysis. A one-way analysis of variance followed by the Duncan's multiple range test (P ≤ 0.05) was carried out to determine the differences between mean values from at least three independent experiments. 3. Results 3.1. Effects of SA on MDA and relative conductivity under cd stress MDA, which is an important product of lipid peroxidation, indicates the peroxidation of the cell membrane. Under the 150 mg l−1 Cd stress, the content of MDA in leaves of N. tetragona increased significantly, and the content increased over the time. The highest content was 88% more
C.-S. Gu et al. / South African Journal of Botany 114 (2018) 267–271
than the control after 9 d (2.10 μmol mg−1 fresh weight) (Fig. 1a). However, the MDA content decreased significantly after the SA pretreatment, and compared with Cd stress alone, the MDA content at the three time points decreased by 16.4%, 18.2% and 9.4%, respectively (Fig. 1a). The MDA content was also increased by SA exposure alone, but there was no significant difference compared with the content of the control (Fig. 1a). Relative conductivity is an important indicator of plasma membrane permeability, reflecting the degree of plasma membrane injury. Relative conductivity increased after Cd stress, and the degree of plasma membrane injury increased along with the treatment time. The conductivity reached its greatest value after 9 d of SA, Cd and SA + Cd treatments (Fig. 1b). Moreover, the conductivity of SA + Cd was less than that of Cd, which indicated that SA could alleviate the effects of Cd on the plasma membrane in N. tetragona leaves and maintain the integrity of the membrane system. 3.2. Effects of SA on the chlorophyll content under cd stress
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Table 1 Effects of exogenous SA on the chlorophyll contents in leaves of Nymphaea tetragona under Cd stress. Treatment
Chlorophyll a
Chlorophyll b
Total chlorophyll
CK SA Cd SA + Cd
1.839 ± 0.026a 1.822 ± 0.044a 1.405 ± 0.103c 1.609 ± 0.131b
0.701 ± 0.048a 0.694 ± 0.052a 0.598 ± 0.074c 0.641 ± 0.071b
2.545 ± 0.068a 2.516 ± 0.104a 2.003 ± 0.076c 2.252 ± 0.089b
Note: Chlorophyll is expressed in mg g−1 fresh weight (FW). Values are the means ± SD of three replicate measurements (n = 3). Different letters in the same column indicate significant differences at the 5% level. Labels denote the following: CK control, SA salicylic acid, Cd cadmium.
pretreatment with SA, SOD activities were increased 49.4%, 58.6% and 40.5% at 1, 5 and 9 d (Fig. 2a). The SOD activity also significantly increased under SA treatment alone (Fig. 2a). The trends of POD and APX activities were similar to that of SOD activity (Fig. 2b, c). The SA pretreatment increased the activity levels of
The chlorophyll content in plant leaves can be used as an indicator of the photosynthetic capacity and can also be used to characterize the damage to plants under adverse conditions. Cd stress significantly reduced the chlorophyll a and b contents in the leaves of the waterlilies, by 23.6% and 14.7%, respectively (Table 1). After a pretreatment with SA, the chlorophyll a and b contents significantly increased, indicating that SA significantly relieved the decrease in the chlorophyll content under Cd stress. SA exposure alone had no significant effect on the chlorophyll content (Table 1). 3.3. Effects of SA on antioxidant enzyme activities under cd stress After a 1 d treatment with 150 mg l−1 Cd, the SOD activity significantly increased to 39.2% higher than that of the control. The SOD activity decreased with a prolonged of Cd-treatment time (Fig. 2a). After a
Fig. 1. Effects of exogenous SA on MDA and relative conductivity in leaves of Nymphaea tetragona under Cd stress. Values are the means ± SD of three replicate measurements (n = 3). Different letters in the same column indicate significant differences at the 5% level.
Fig. 2. Effects of exogenous SA on the SOD, POD and APX activities in leaves of Nymphaea tetragona under Cd stress. Values are the means ± SD of three replicate measurements (n = 3). Different letters in the same column indicate significant differences at the 5% level.
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POD and APX under Cd stress. However, the effects were not correlated with an increase in processing time. The SA treatment alone had little effect on POD activity, but significantly increased APX activity (Fig. 2b, c). 3.4. Effects of SA on proline content under Cd stress Proline accumulation is an adaptive response of plants to stress. Cd stress significantly increased the proline content (Fig. 3), with a maximum increase of 2.2 times that of the control after 9 d. Compared with the Cd treatment alone, the SA pretreatment significantly decreased the proline content under Cd stress. The SA treatment alone had little effect on the proline content (Fig. 3). 3.5. Effects of SA on GSH, NPT and PCs contents under Cd stress The Cd treatment significantly increased the NPT content. After 1 d, the NPT content increased by 52.8% compared with the control. After 9 d, the proportion increased to 88.6%. The SA pretreatment further increased the NPT content under Cd stress, with the maximum value reaching 0.78 μmol g−1 fresh weight after 5 d of treatment, which was about 2.3 times that of the control (Fig. 4a). There was no difference between the effects of independent SA and Cd treatments on the GSH content after 1 d of treatment (Fig. 4b). Compared with the Cd treatment alone, the SA pretreatment increased the GSH content under Cd stress, and the GSH content increased by 61.5% compared with the control group after 5 d (Fig. 4b). The PCs content significantly increased under the Cd treatment (Fig. 4c) but no such trend was observed for the SA treatment alone. After 5 d and 9 d treatments, the PC contents in the SA plus Cd-treated group were 33.3% and 17.3% greater, respectively, than those in the Cd treatment alone (Fig. 4c). 3.6. Effects of SA on the Cd concentrations in different parts of N. tetragona under Cd stress After 9 d of 150 mg l−1 Cd treatments, the organs of N. tetragona accumulated large amounts of Cd (Table 2). After the SA pretreatment, the Cd concentration was significantly reduced in each organ. The accumulations in the underground (root and rhizome) and shoot (petiole and lamina) tissues decreased by ~20.0% and 15.7%, respectively, compared with that of Cd exposure alone (Table 2). 4. Discussion Cd stress causes significant damage to normal plant metabolism, disturbing the balance of active oxygen production and removal (Sytar et al., 2013). MDA, a membrane lipid peroxidation product, can cause serious damage to the membrane structure under Cd stress (Guo
Fig. 4. Effects of exogenous SA on the GSH, NPT and PCs contents in leaves of Nymphaea tetragona under Cd stress. Values are the means ± SD of three replicate measurements (n = 3). Different letters in the same column indicate significant differences at the 5% level.
et al., 2007). In our study, an SA pretreatment significantly reduced the MDA content in leaves of N. tetragona and significantly decreased plasma membrane permeability. Exogenous SA could alleviate the membrane peroxidation induced by Cd stress, stabilize the cell
Table 2 Effects of exogenous SA on the Cd concentrations in leaves of Nymphaea tetragona under Cd stress.
Fig. 3. Effects of exogenous SA on the proline content in leaves of Nymphaea tetragona under Cd stress. Values are the means ± SD of three replicate measurements (n = 3). Different letters in the same column indicate significant differences at the 5% levels.
Treatment
Root
Rhizome
Petiole
Lamina
CK SA Cd SA + Cd
42.4 ± 5.6c 38.3 ± 4.4c 2899.6 ± 189.7a 2320.8 ± 157.4b
10.4 ± 2.4c 9.1 ± 2.5c 108.3 ± 17.9a 87.3 ± 14.8b
7.5 ± 2.8c 7.8 ± 1.9c 595.2 ± 53.3a 532.5 ± 78.4b
19.6 ± 3.5c 20.2 ± 4.2c 1241.6 ± 184.7a 1012.1 ± 169.6b
Note: The Cd concentration is expressed in μg g−1 dry weight (DW). Values are the means ± SD of three replicate measurements (n = 3). Different letters in the same column indicate significant differences at the different Cd levels (P b 0.05). Labels denote the following: CK control, SA salicylic acid, Cd cadmium.
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membrane structure and alleviate damage to the plasma membrane, thereby improving plant Cd resistance. Chlorophyll is the main pigment of photosynthesis in plants. The peroxidation caused by Cd stress can destroy the structure and function of the chloroplast membrane, which leads to a decrease in the chlorophyll content (Lagriffoul et al., 1998). In this study, a pretreatment with SA could increase the chlorophyll content of leaves. Cd stress can interfere with the normal operations of the antioxidant system in plants, resulting in the accumulation of a large number of reactive oxygen free radicals, which have strong oxidative activities, causing structural membrane changes that result in oxidative damage (Hayat et al., 2010). After pretreatment with SA, the activities of SOD, POD and APX were increased in N. tetragona. In ryegrass and Kentucky bluegrass, the activities of SOD and POD were also increased after pretreatments with SA (Chunyu, 2012; Guo et al., 2013). However, SA pretreatments could decrease SOD activity in Brassica juncea (Ahmad et al., 2011) and POD activity in Zea mays (Krantev et al., 2008). The opposite result was found for APX activity. In Hordeum vulgare and B. juncea, APX activities decreased in Cd-treated SA seedlings (Ahmad et al., 2011;Metwally et al., 2003). This may be due to the different periods of sensitivity to SA and heavy metals in different plants or organs. Proline, which is a stress signal, is an important osmotic regulator in plants. Its content can be used as a physiological and biochemical index of plant stress. The proline content in N. tetragona leaves significantly increased under Cd stress, while the SA pretreatment decreased the proline content, which indicated that N. tetragona pretreated with SA was more resistant to Cd stress. GSH, NPT and PCs, which were mercapto-enriched compounds, can complex Cd and reduce the activity level of Cd in the cells, thereby reducing the toxicity of Cd to plants (Liu et al., 2012). There were no significant differences in the GSH content among CK, SA- and Cd-stressed plants after 1 d. This may have been due to the large amount of PC synthesis at this stage. After 5 and 9 d, plant self-regulation increased the synthesis of GSH, and this increase helped to alleviate the oxidative damage caused by Cd stress. Additionally, the NPT and PC contents increased significantly, and the SA pretreatment coupled with the Cd treatment had a synergistic effect. The SA pretreatment also increased the contents of GSH and NPT in rice roots under Cd stress, which was consistent with the results of this study (Guo et al., 2007). The Cd concentrations of four parts (root, rhizome, petiole and lamina) in N. tetragona were all decreased under SA pretreatment. This may be that SA alleviated root Cd absorption, and Cd content transported to other parts was also reduced accordingly. The SA pretreatment reduced the accumulation of Cd in N. tetragona, and this was also found in pea and wheat (Popova et al., 2009; Moussa and El-Gamal, 2010). However, in soybean and barley, which are in the same families as pea and wheat, respectively, even though the SA treatment decreased Cd damage to plants, it did not reduce the plant's Cd uptake (Metwally et al., 2003; Drazic and Mihailovic, 2005). Furthermore, exogenous applications of SA showed inconsistent effects with respect to the Cd concentrations in shoot and root in mustard (Ahmad et al., 2011). This difference may be due to the plant species, growth period, treatment conditions or other factors. Here, we studied the mitigatory effects of exogenous SA on the toxicity of Cd in N. tetragona, but the molecular mechanisms of SA that alleviate Cd toxicity require further study.
5. Conclusion In summary, the MDA and proline contents in leaves of N. tetragona decreased after an SA pretreatment, while the contents of chlorophyll, GSH, NPT and PCs contents increased. Additionally, the activities of antioxidant enzymes (SOD, POD and APX) were enhanced. An SA pretreatment reduced the Cd accumulation in various organs of N. tetragona. Thus, exogenous SA pretreatments could relieve the toxic effects of the heavy metal Cd on N. tetragona.
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Acknowledgments This study is supported by the National Natural Science Foundation of China (Grant No. 31071820), the Program for Hi-Tech Research, Jiangsu, China, Grant (No. BE2010303), the Fundamental Research Funds for the Central Universities (KYJ 200907) and the Program of Innovation Capacity Construction of Jiangsu Province (BM2015019). References Agami, R.A., Mohamed, G.F., 2013. Exogenous treatment with indole-3-acetic acid and salicylic acid alleviates cadmium toxicity in wheat seedlings. Ecotoxicology and Environmental Safety 94, 164–171. Ahmad, P., Nabi, G., Ashraf, M., 2011. Cadmium-induced oxidative damage in mustard [Brassica juncea (L.) Czern. & Coss.] plants can be alleviated by salicylic acid. South African Journal of Botany 77, 36–44. Belkadhi, A., De Haro, A., Obregon, S., Chaïbi, W., Djebali, W., 2015. Exogenous salicylic acid protects phospholipids against cadmium stress in flax (Linum usitatissimum L.). Ecotoxicology and Environmental Safety 120, 102–109. 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South African Journal of Botany journal homepage: www.elsevier.com/locate/sajb
The effects of exogenous salicylic acid on alleviating cadmium toxicity in Nymphaea tetragona Georgi C.-S. Gu a,b, Y.-H. Yang a, Y.-F. Shao a, K.-W. Wu a, Z.-L. Liu a,⁎ a b
College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China The Jiangsu Provincial Platform for Conservation and Utilization of Agricultural Germplasm, Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing 210014, China
a r t i c l e
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Article history: Received 17 May 2017 Received in revised form 25 September 2017 Accepted 17 November 2017 Available online xxxx Edited by M Vaculik Keywords: Accumulation Antioxidative system Cadmium Nymphaea tetragona Salicylic acid
a b s t r a c t The effects of salicylic acid pretreatments on lipid peroxidation, photosynthetic pigments, the antioxidative system and cadmium (Cd) accumulation in waterlily under Cd stress were studied. A salicylic acid pretreatment lowered the concentrations of malondialdehyde and proline, but increased the contents of photosynthetic pigments, glutathione, non-protein thiol and phytochelatins. Salicylic acid pretreatments enhanced the activities of antioxidant enzymes, such as superoxide dismutase, peroxidase and ascorbate peroxidase. Furthermore, SA pretreatments also decreased the Cd concentrations in different plant organs. Thus, exogenous SA treatments could attenuate the toxic effects of Cd in Nymphaea tetragona. © 2017 SAAB. Published by Elsevier B.V. All rights reserved.
1. Introduction Heavy metal pollution has become a dangerous global environmental pollution problem. Cadmium (Cd) is a widely distributed and highly toxic heavy metal. Cd can enter plants through contaminated soil and water, not only polluting the environment, but also resulting in declines in crop yield and quality. It is a threat to humans and others in the food chain. Cd pollution can be decreased by physical–chemical remediation, phytoremediation and microbial remediation methods (Liu et al., 2012). Phytoremediation refers to the use of plants to remove heavy metals from contaminated soil and waste water, and it has advantages of ‘green’ purification, producing no secondary pollutants, low investment costs and ease of use (Clemens and Ma, 2016). Salicylic acid (SA) is a kind of small-molecule phenolic compound prevalent in plants that can affect many physiological processes (Miura and Tada, 2014). It is also a plant endogenous signal molecule (Yan and Dong, 2014). SA has roles in improving plant disease resistance (Delaney et al., 1994), salt tolerance (Borsani et al., 2001), drought resistance (Saruhan et al., 2012) and cold resistance (Miura and Tada, 2014). Moreover, SA has alleviation effects on plants under heavy metal stress (Agami and Mohamed, 2013; Belkadhi et al., 2015) and can alleviate Cd toxicity in wheat seedlings (Agami and Mohamed,
⁎ Corresponding author. E-mail address: [email protected] (Z.-L. Liu).
https://doi.org/10.1016/j.sajb.2017.11.012 0254-6299/© 2017 SAAB. Published by Elsevier B.V. All rights reserved.
2013). Moreover, exogenous SA protects phospholipids against Cd stress in flax (Belkadhi et al., 2015). It is important to develop aquatic ornamental plants with characteristics of high ornamental value, fast growth and high biomass in phytoremediation technology in heavy metal polluted water bodies. In a previous study, physiological responses and bioaccumulation in Nymphaea tetragona Georgi under Cd exposure were investigated (Yang et al., 2015). Here, SA was used as a regulatory factor to explore its protective effects on the toxicity of Cd in N. tetragona Georgi and to provide foundations for understanding the alleviation of the stress effects of heavy metals on plants and the ecological defenses of heavy metal-contaminated plants. 2. Materials and methods 2.1. Plant material and growth conditions The test material N. tetragona Georgi cv ‘Masaniello’, was taken from Nanjing Yilian Court, Jiangsu Province. The experiments were performed in the Flower Research Institute of Nanjing Agricultural University. The rhizome explants of N. tetragona were watered with halfstrength Hoagland's nutrient solution in a turnover box (length, 50 cm; width, 38 cm; height, 29 cm) in with a 28 °C 14-h light/22 °C 10-h dark cycle and light intensity of 120 μmol m−2 s−1 (Yang et al., 2015). The full Hoagland's nutrient solution contained the following compositions: 5 mM KNO3, 5 mM Ca(NO3)2, 2 mM MgSO4, 1 mM
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KH2PO4, 1 mM FeSO4-EDTA, 46 μM H3BO4, 9.1 μM MnCl2.4H2O, 0.32 μM CuSO4.5H2O, 0.76 μM ZnSO4.7H2O, 0.5 μM H2MoO4·H2O. The hydroponic culture was adjusted to pH 6.0. When the plants had grown for ~7–8 weeks, healthy plants were selected. The following independent four treatments were used: (1) control (CK; exposed to neither SA nor Cd) for 9 d; (2) 20 μM SA treatment for 9 d; (3) 150 mg·l−1 Cd (CdCl2) treatment for 9 d; (4) plants were grown in 20 μM SA for 1 d and then transferred to half-strength Hoagland's nutrient solution containing 150 mg·l−1 Cd for 9 d. The relative physiological indices, including malondialdehyde (MDA) content, relative conductivity, proline content, antioxidant enzyme activities, glutathione (GSH), non-protein thiol (NPT) and phytochelatins (PCs), were measured in leaves sampled after 1, 5 and 9 d. After 9 d, the chlorophyll content and the accumulation of Cd in different plant parts (roots, rhizomes, petioles and leaves) were determined.
as follows: 2.75 ml of 50 mM potassium phosphate buffer (pH 6.1), 0.1 ml of 0.4% (v/v) H2O2, 0.1 ml of 1% (w/v) guaiacol and 0.05 ml of enzyme extract. One unit of POD activity was defined as the amount of enzyme causing the change in absorbance per minute. It is expressed as U g−1 FW. The ascorbic acid peroxidase activity (APX) was determined in a 3 ml reaction mixture containing 1.8 ml of 50 mM potassium phosphate buffer (pH 7.0), 0.1 ml of 15 mM ascorbate, 1 ml of 0.3 mM H2O2 and 0.1 ml of enzyme extract. The absorbance was determined at 290 nm for 60 s with New century T6 spectrophotometer (New century, Beijing, China). One unit of APX activity was defined as the amount of enzyme decomposing 1 μ mol of substrate min−1. It is expressed as U g−1 FW (Chen et al., 2014).
2.2. Malondialdehyde (MDA)
In brief, leaves (0.5 g) were ground in 4 ml of 3% sulfosalicylic acid. After boiling the samples for 15 min, homogenates were centrifuged at 4000 × g at 4 °C for 20 min. 2 ml of supernatant was mixed with 2 ml of acetic acid and 3 ml of 2.5% acid ninhydrin. After boiling for 40 min and cooling, 4 ml of toluene was added. Absorbance was determined at 520 nm (New century, Beijing, China) using toluene as blank.
In brief, 0.5 g of leaves were homogenized with 10% trichloroacetic acid (TCA) and centrifuged at 4000×g for 20 min. 2 ml of the supernatant was then mixed with 2 ml of 0.6% thiobarbituric acid solution (TBA). After heating at 100 °C for 30 min, the mixtures were centrifuged at 4000×g for 15 min after quickly being cooled down. The absorbance of the supernatant was recorded at 532, 600 and 400 nm with New century T6 spectrophotometer (New century, Beijing, China). Amounts of MDA were expressed as μmol g−1 fresh weight using the following formula: 6.45(A532 − A600) − 0.56A450. 2.3. Relative conductivity In brief, 0.2 g disks (6 mm diameter each) from fresh leaves were rinsed with deionized water for 3 min. After dried with filter paper, samples were immersed in a tube with 10 ml of deionized water and gently shaken for 3 h at 25 °C. After the incubation, conductivity (C1) was measured using conductivity meter (Leici-DDS-307A, Shanghai, China). Then conductivity (C2) was measured after boiling the samples for 20 min. Relative conductivity (%) was defined as (C1 / C2) × 100. 2.4. Chlorophyll (Chl) content In brief, leaves (0.2 g) were ground in 80% acetone. Then the supernatant was used to measure chlorophyll content. The optical density (OD) was recorded at 470, 647 and 665 nm with New century T6 spectrophotometer (New century, Beijing, China) against 80% acetone used as blank. Chlorophyll content was calculated using the following formula: Chl a = 13.95 ∗ OD665 − 6.88 ∗ OD470; Chl b = 24.96 ∗ OD647 − 7.32 ∗ OD665; Total chlorophyll = Chl a + Chl b. 2.5. Antioxidant enzyme activity In brief, leaves (0.2 g) were ground in 5 ml of 50 mM phosphate buffer (pH 7.8 for SOD and POD activities, pH 7.0 for APX activity) containing 1% (w/v) polyvinylpyrrolidone (PVP) and centrifuged at 4000×g at 4 °C for 20 min. The supernatant was then used to measure the SOD, POD and APX activities. Total superoxide dismutase (SOD) activity was assayed by nitro blue tetrazolium (NBT) photoreduction method. In brief, the reaction mixture (3 ml) was as follows: 1.7 ml of 50 mM phosphate buffer saline (PBS) (pH 7.8), 0.3 ml of 130 mM methionine, 0.3 ml of 0.75 mM nitroblue tetrazolium (NBT), 0.3 ml of 1 mM EDTA, 0.3 ml of 20 μM riboflavin and 0.1 ml of enzyme extract. The reaction was conducted for 20 min under 4000 lx fluorescent lamp. One unit of SOD activity was defined as the amount of enzyme needed to inhibit NBT photoreduction by 50%. It is expressed as U g−1 FW. Total peroxidase activity (POD) was determined with guaiacol method with modifications. In brief, the reaction mixture (3 ml) was
2.6. Proline content
2.7. GSH, NPT and PCs contents GSH content was assayed as described previously (Hissin and Hilf, 1976). In brief, leaves (0.5 g) were ground in 1 ml of 25% HPO3 and 3 ml of 0.1 M sodium phosphate–EDTA buffer (pH 8.0) and centrifuged at 4000 × g for 20 min. The assay mixture for GSH estimation was as follows: 3.6 ml of phosphate-EDTA buffer, 0.2 ml of O-phthalaldehyde solution and 0.2 ml of supernatant extract. After mixing at room temperature for 15 min, fluorescence at 420 nm was measured with the excitation at 350 nm. NPT content was determined using the method of Rama Devi and Prasad (1998). In brief, leaves (0.5 g) were ground in 4 ml of 5% sulfosalicylic acid and centrifuged at 4000×g for 20 min. The assay mixture was as follows: 2 ml of 0.2 M Tris-HCl (pH 8.2), 0.15 ml of 5,5′Dithio-bis-(2-nitrobenzoic acid) (DTNB) and 0.2 ml of supernatant extract. After 20 min, absorbance was determined at 412 nm. PCs content was measured as described by Bhargava et al. (2005). Phytochelatin was measured by using formula: total thiol-GSH. 2.8. Cd accumulation After washing with distilled water, the tissues (root, rhizome, petiole and lamina) were dried at 80 °C for 48 h. The solubilized samples were a mixed of nitric-perchloric acid (4:1, v/v), diluted with 5 ml with 2.5% HNO3. Cd accumulation was measured by atomic absorption spectrophotometry (Model 3300, Perkin-Elmer, USA) (Gu et al., 2014). 2.9. Statistical treatment SPSS v17.0 software (SPSS Inc., Chicago, IL, USA) was used for the statistical analysis. A one-way analysis of variance followed by the Duncan's multiple range test (P ≤ 0.05) was carried out to determine the differences between mean values from at least three independent experiments. 3. Results 3.1. Effects of SA on MDA and relative conductivity under cd stress MDA, which is an important product of lipid peroxidation, indicates the peroxidation of the cell membrane. Under the 150 mg l−1 Cd stress, the content of MDA in leaves of N. tetragona increased significantly, and the content increased over the time. The highest content was 88% more
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than the control after 9 d (2.10 μmol mg−1 fresh weight) (Fig. 1a). However, the MDA content decreased significantly after the SA pretreatment, and compared with Cd stress alone, the MDA content at the three time points decreased by 16.4%, 18.2% and 9.4%, respectively (Fig. 1a). The MDA content was also increased by SA exposure alone, but there was no significant difference compared with the content of the control (Fig. 1a). Relative conductivity is an important indicator of plasma membrane permeability, reflecting the degree of plasma membrane injury. Relative conductivity increased after Cd stress, and the degree of plasma membrane injury increased along with the treatment time. The conductivity reached its greatest value after 9 d of SA, Cd and SA + Cd treatments (Fig. 1b). Moreover, the conductivity of SA + Cd was less than that of Cd, which indicated that SA could alleviate the effects of Cd on the plasma membrane in N. tetragona leaves and maintain the integrity of the membrane system. 3.2. Effects of SA on the chlorophyll content under cd stress
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Table 1 Effects of exogenous SA on the chlorophyll contents in leaves of Nymphaea tetragona under Cd stress. Treatment
Chlorophyll a
Chlorophyll b
Total chlorophyll
CK SA Cd SA + Cd
1.839 ± 0.026a 1.822 ± 0.044a 1.405 ± 0.103c 1.609 ± 0.131b
0.701 ± 0.048a 0.694 ± 0.052a 0.598 ± 0.074c 0.641 ± 0.071b
2.545 ± 0.068a 2.516 ± 0.104a 2.003 ± 0.076c 2.252 ± 0.089b
Note: Chlorophyll is expressed in mg g−1 fresh weight (FW). Values are the means ± SD of three replicate measurements (n = 3). Different letters in the same column indicate significant differences at the 5% level. Labels denote the following: CK control, SA salicylic acid, Cd cadmium.
pretreatment with SA, SOD activities were increased 49.4%, 58.6% and 40.5% at 1, 5 and 9 d (Fig. 2a). The SOD activity also significantly increased under SA treatment alone (Fig. 2a). The trends of POD and APX activities were similar to that of SOD activity (Fig. 2b, c). The SA pretreatment increased the activity levels of
The chlorophyll content in plant leaves can be used as an indicator of the photosynthetic capacity and can also be used to characterize the damage to plants under adverse conditions. Cd stress significantly reduced the chlorophyll a and b contents in the leaves of the waterlilies, by 23.6% and 14.7%, respectively (Table 1). After a pretreatment with SA, the chlorophyll a and b contents significantly increased, indicating that SA significantly relieved the decrease in the chlorophyll content under Cd stress. SA exposure alone had no significant effect on the chlorophyll content (Table 1). 3.3. Effects of SA on antioxidant enzyme activities under cd stress After a 1 d treatment with 150 mg l−1 Cd, the SOD activity significantly increased to 39.2% higher than that of the control. The SOD activity decreased with a prolonged of Cd-treatment time (Fig. 2a). After a
Fig. 1. Effects of exogenous SA on MDA and relative conductivity in leaves of Nymphaea tetragona under Cd stress. Values are the means ± SD of three replicate measurements (n = 3). Different letters in the same column indicate significant differences at the 5% level.
Fig. 2. Effects of exogenous SA on the SOD, POD and APX activities in leaves of Nymphaea tetragona under Cd stress. Values are the means ± SD of three replicate measurements (n = 3). Different letters in the same column indicate significant differences at the 5% level.
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POD and APX under Cd stress. However, the effects were not correlated with an increase in processing time. The SA treatment alone had little effect on POD activity, but significantly increased APX activity (Fig. 2b, c). 3.4. Effects of SA on proline content under Cd stress Proline accumulation is an adaptive response of plants to stress. Cd stress significantly increased the proline content (Fig. 3), with a maximum increase of 2.2 times that of the control after 9 d. Compared with the Cd treatment alone, the SA pretreatment significantly decreased the proline content under Cd stress. The SA treatment alone had little effect on the proline content (Fig. 3). 3.5. Effects of SA on GSH, NPT and PCs contents under Cd stress The Cd treatment significantly increased the NPT content. After 1 d, the NPT content increased by 52.8% compared with the control. After 9 d, the proportion increased to 88.6%. The SA pretreatment further increased the NPT content under Cd stress, with the maximum value reaching 0.78 μmol g−1 fresh weight after 5 d of treatment, which was about 2.3 times that of the control (Fig. 4a). There was no difference between the effects of independent SA and Cd treatments on the GSH content after 1 d of treatment (Fig. 4b). Compared with the Cd treatment alone, the SA pretreatment increased the GSH content under Cd stress, and the GSH content increased by 61.5% compared with the control group after 5 d (Fig. 4b). The PCs content significantly increased under the Cd treatment (Fig. 4c) but no such trend was observed for the SA treatment alone. After 5 d and 9 d treatments, the PC contents in the SA plus Cd-treated group were 33.3% and 17.3% greater, respectively, than those in the Cd treatment alone (Fig. 4c). 3.6. Effects of SA on the Cd concentrations in different parts of N. tetragona under Cd stress After 9 d of 150 mg l−1 Cd treatments, the organs of N. tetragona accumulated large amounts of Cd (Table 2). After the SA pretreatment, the Cd concentration was significantly reduced in each organ. The accumulations in the underground (root and rhizome) and shoot (petiole and lamina) tissues decreased by ~20.0% and 15.7%, respectively, compared with that of Cd exposure alone (Table 2). 4. Discussion Cd stress causes significant damage to normal plant metabolism, disturbing the balance of active oxygen production and removal (Sytar et al., 2013). MDA, a membrane lipid peroxidation product, can cause serious damage to the membrane structure under Cd stress (Guo
Fig. 4. Effects of exogenous SA on the GSH, NPT and PCs contents in leaves of Nymphaea tetragona under Cd stress. Values are the means ± SD of three replicate measurements (n = 3). Different letters in the same column indicate significant differences at the 5% level.
et al., 2007). In our study, an SA pretreatment significantly reduced the MDA content in leaves of N. tetragona and significantly decreased plasma membrane permeability. Exogenous SA could alleviate the membrane peroxidation induced by Cd stress, stabilize the cell
Table 2 Effects of exogenous SA on the Cd concentrations in leaves of Nymphaea tetragona under Cd stress.
Fig. 3. Effects of exogenous SA on the proline content in leaves of Nymphaea tetragona under Cd stress. Values are the means ± SD of three replicate measurements (n = 3). Different letters in the same column indicate significant differences at the 5% levels.
Treatment
Root
Rhizome
Petiole
Lamina
CK SA Cd SA + Cd
42.4 ± 5.6c 38.3 ± 4.4c 2899.6 ± 189.7a 2320.8 ± 157.4b
10.4 ± 2.4c 9.1 ± 2.5c 108.3 ± 17.9a 87.3 ± 14.8b
7.5 ± 2.8c 7.8 ± 1.9c 595.2 ± 53.3a 532.5 ± 78.4b
19.6 ± 3.5c 20.2 ± 4.2c 1241.6 ± 184.7a 1012.1 ± 169.6b
Note: The Cd concentration is expressed in μg g−1 dry weight (DW). Values are the means ± SD of three replicate measurements (n = 3). Different letters in the same column indicate significant differences at the different Cd levels (P b 0.05). Labels denote the following: CK control, SA salicylic acid, Cd cadmium.
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membrane structure and alleviate damage to the plasma membrane, thereby improving plant Cd resistance. Chlorophyll is the main pigment of photosynthesis in plants. The peroxidation caused by Cd stress can destroy the structure and function of the chloroplast membrane, which leads to a decrease in the chlorophyll content (Lagriffoul et al., 1998). In this study, a pretreatment with SA could increase the chlorophyll content of leaves. Cd stress can interfere with the normal operations of the antioxidant system in plants, resulting in the accumulation of a large number of reactive oxygen free radicals, which have strong oxidative activities, causing structural membrane changes that result in oxidative damage (Hayat et al., 2010). After pretreatment with SA, the activities of SOD, POD and APX were increased in N. tetragona. In ryegrass and Kentucky bluegrass, the activities of SOD and POD were also increased after pretreatments with SA (Chunyu, 2012; Guo et al., 2013). However, SA pretreatments could decrease SOD activity in Brassica juncea (Ahmad et al., 2011) and POD activity in Zea mays (Krantev et al., 2008). The opposite result was found for APX activity. In Hordeum vulgare and B. juncea, APX activities decreased in Cd-treated SA seedlings (Ahmad et al., 2011;Metwally et al., 2003). This may be due to the different periods of sensitivity to SA and heavy metals in different plants or organs. Proline, which is a stress signal, is an important osmotic regulator in plants. Its content can be used as a physiological and biochemical index of plant stress. The proline content in N. tetragona leaves significantly increased under Cd stress, while the SA pretreatment decreased the proline content, which indicated that N. tetragona pretreated with SA was more resistant to Cd stress. GSH, NPT and PCs, which were mercapto-enriched compounds, can complex Cd and reduce the activity level of Cd in the cells, thereby reducing the toxicity of Cd to plants (Liu et al., 2012). There were no significant differences in the GSH content among CK, SA- and Cd-stressed plants after 1 d. This may have been due to the large amount of PC synthesis at this stage. After 5 and 9 d, plant self-regulation increased the synthesis of GSH, and this increase helped to alleviate the oxidative damage caused by Cd stress. Additionally, the NPT and PC contents increased significantly, and the SA pretreatment coupled with the Cd treatment had a synergistic effect. The SA pretreatment also increased the contents of GSH and NPT in rice roots under Cd stress, which was consistent with the results of this study (Guo et al., 2007). The Cd concentrations of four parts (root, rhizome, petiole and lamina) in N. tetragona were all decreased under SA pretreatment. This may be that SA alleviated root Cd absorption, and Cd content transported to other parts was also reduced accordingly. The SA pretreatment reduced the accumulation of Cd in N. tetragona, and this was also found in pea and wheat (Popova et al., 2009; Moussa and El-Gamal, 2010). However, in soybean and barley, which are in the same families as pea and wheat, respectively, even though the SA treatment decreased Cd damage to plants, it did not reduce the plant's Cd uptake (Metwally et al., 2003; Drazic and Mihailovic, 2005). Furthermore, exogenous applications of SA showed inconsistent effects with respect to the Cd concentrations in shoot and root in mustard (Ahmad et al., 2011). This difference may be due to the plant species, growth period, treatment conditions or other factors. Here, we studied the mitigatory effects of exogenous SA on the toxicity of Cd in N. tetragona, but the molecular mechanisms of SA that alleviate Cd toxicity require further study.
5. Conclusion In summary, the MDA and proline contents in leaves of N. tetragona decreased after an SA pretreatment, while the contents of chlorophyll, GSH, NPT and PCs contents increased. Additionally, the activities of antioxidant enzymes (SOD, POD and APX) were enhanced. An SA pretreatment reduced the Cd accumulation in various organs of N. tetragona. Thus, exogenous SA pretreatments could relieve the toxic effects of the heavy metal Cd on N. tetragona.
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Acknowledgments This study is supported by the National Natural Science Foundation of China (Grant No. 31071820), the Program for Hi-Tech Research, Jiangsu, China, Grant (No. BE2010303), the Fundamental Research Funds for the Central Universities (KYJ 200907) and the Program of Innovation Capacity Construction of Jiangsu Province (BM2015019). References Agami, R.A., Mohamed, G.F., 2013. Exogenous treatment with indole-3-acetic acid and salicylic acid alleviates cadmium toxicity in wheat seedlings. Ecotoxicology and Environmental Safety 94, 164–171. Ahmad, P., Nabi, G., Ashraf, M., 2011. Cadmium-induced oxidative damage in mustard [Brassica juncea (L.) Czern. & Coss.] plants can be alleviated by salicylic acid. South African Journal of Botany 77, 36–44. Belkadhi, A., De Haro, A., Obregon, S., Chaïbi, W., Djebali, W., 2015. Exogenous salicylic acid protects phospholipids against cadmium stress in flax (Linum usitatissimum L.). Ecotoxicology and Environmental Safety 120, 102–109. 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