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The mutual restraint effect between the expansion of Alternanthera philoxeroides (Mart.) Griseb and cadmium mobility in aquatic environment
Ecotoxicology and Environmental Safety 148 (2018) 237–243
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
The mutual restraint effect between the expansion of Alternanthera philoxeroides (Mart.) Griseb and cadmium mobility in aquatic environment ⁎
Jian Lia,b, Zhiwei Dua, Chris B. Zouc, Zhicong Daia, Daolin Dua, , Chongling Yanb, a b c
T
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Institute of Environment and Ecology, School of the Environment and Safety Engineering, Jiangsu University, Zhenjiang 212013, China State Key Lab of Marine Environmental Science, Xiamen University, Xiamen 361102, China Department of Natural Resource Ecology and Management, Oklahoma State University, Stillwater, OK 74078, USA
A R T I C L E I N F O
A B S T R A C T
Keywords: Hydrophyte Heavy metal Iron plaque Phytoremediation
Alternanthera philoxeroides (Mart.) Griseb is one of the most malignant weeds in its invision habitats. While in the cadmium-contaminated aquatic environment, does A. philoxeroides possess good tolerance and adaptability? To demonstrate the effects of cadmium on A. philoxeroides in the polluted water bodies, a hydroponic stress experiment was conducted over a gradient of Cd concentrations (0, 2.5 and 5 mg/l) in triplicate. The seedlings were cultured in a greenhouse and harvested on days 0, 10, 20, 30 and 40, respectively. The results showed the effects of mutual restraint between Cd and A. philoxeroides. The A. philoxeroides seedlings were enriched with large amounts of Cd, and the toxicity of Cd inhibited the rapid growth of A. philoxeroides and induced the rapid degradation of chlorophylls in its tissues. Furthermore, the use of iron plaque effectively immobilized Cd of 1123–2883 mg/kg·DW on the root surface, thus it decreased the transferability of Cd in the aquatic environment. Due to its extensive adaptability, good Cd tolerance and the immobilization of Cd predominantly in the roots (the highest Cd concentration enriched was 7588.65 ± 628.90 mg/kg·DW in roots). A. philoxeroides effectively restrained the translocation of Cd and partitioned Cd in the roots within water bodies. Capsule: The antagonistic effect exists between the invasion of A. philoxeroides and cadmium mobility in aquatic environments.
1. Introduction Heavy metal contamination is a major problem in aquatic ecosystems within human-dominated regions (William, 2007). In hydrophytes, heavy metals induce oxidative stress and inhibit photosynthesis and growth, leading to cell membrane alteration and malondialdehyde (MDA) content increase via lipoperoxidation (Ding et al., 2007; Krayem et al., 2016). Heavy metals are enriched from primary producers to consumers, and organisms achieve high Cd levels in vivo through food chains or food webs. As a result, heavy metal contamination in water bodies not only threatens the development of aquatic ecosystems but is also a human health hazard (Krayem et al., 2016). Heavy metals in the aquatic environment can be removed using a variety of approaches, such as chemical or physical adsorption, physical precipitation and phytoremediation (Liu et al., 2007; Li et al., 2016). Due to its low cost and low environmental impact, the use of phytoremediation to remove heavy metals has been a focus of researchers and environmental managers (Jasrotia et al., 2015; Rezania et al., 2016). Previous studies indicate that a good metal-accumulating wetland plant species can absorb more than 0.5% of its dry weight (DW) of a given ⁎
Corresponding authors. E-mail addresses: [email protected] (D. Du), [email protected] (C. Yan).
http://dx.doi.org/10.1016/j.ecoenv.2017.10.032 Received 20 July 2017; Received in revised form 6 October 2017; Accepted 13 October 2017 Available online 06 November 2017 0147-6513/ © 2017 Elsevier Inc. All rights reserved.
element and bio-concentrate the element in its tissue to up to 1,000-fold the initial elemental concentration in the water body (Zayed et al., 1998). In metal-scavenging hydrophytes, heavy metal uptake mechanism includes accumulation, exclusion, translocation, osmoregulation and distribution (Rezania et al., 2016). As a result, heavy metalscavenging hydrophytes play a critical role in cleaning up heavy metal pollution in wetlands. For example, Alternanthera philoxeroides (Mart.) Griseb, an amphibious species, is abundant worldwide and has a high tolerance for heavy metal-polluted environments. This plant can act as a pioneer species and can thrive in mine tailings and polluted lands or aquatic environments (typically along banks) (Naqvi and Rizvi, 2000; Minchinton et al., 2006). A record Mn concentration of 19,300 mg/kg was reported for A. philoxeroides, demonstrating its ability as a hyperaccumulator of Mn (Xue et al., 2003). In heavy metal-contaminated areas, A. philoxeroides has evolved into a cumulative ecotype under heavy metal stress (Hu et al., 2013). Baker et al. (1989) proposed some criteria for classifying a plant as a hyperaccumulator: it should be capable of accumulating more than 10,000 mg/kg for Mn and Zn and more than 100 mg/kg for Cd in their shoots, and the TF should be more than 1 (Baker et al., 1989). Based on
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Chlorophyll a (mg/g FW ) = (12.7 A663 − 2.69 A645 ) × V /(1000 × FW )
data from Hu et al. (2013), A. philoxeroides meets these criteria and can be considered a potential species for the phytoremediation of soils, sediments and aquatic environments polluted with Cd, Mn and Zn. They showed that the Cd and Zn concentrations in A. philoxeroides were 21 and 43,154 mg/kg in the roots, respectively, and 155 and 13,784 mg/ kg in the stems, respectively. In addition, the bioaccumulation factors (BCFs) of Cd and Zn were 36.78 and 49.23, respectively, and the translocation factors (TFs) were 7.38 and 3.19, respectively. However, the Cd accumulation and extraction capabilities of A. philoxeroides are debated. Liu et al. (2007) investigated the uptake and distribution of Cd, Pb and Zn in 19 wetland plant species from constructed wetlands, and they reported that the Cd concentrations in A. philoxeroides were 20.56 and 96.66 mg/kg in the aboveground and underground parts, respectively. The TF was less than 1, although the Cd accumulated mass was 28.17 mg/kg in the entire plant, representing the highest value among all of the studied species. These different results might be due to study differences in the growth media and the Cd concentration that was bioavailable to A. philoxeroides. Cd can be absorbed through symplast and non-symplast transport in the roots. During symplast transport, Cd is transported through selective or non-selective ionophores and channel proteins (e.g., Ca2+, Mg2+ and Fe2+ channel proteins) into the roots (Welch et al., 1999; Zhang et al., 2015). Plants absorb Cd through Mg, Ca or Fe channels because of their high biological activities and the bioavailability of Cd. Fe is important to plant metabolism and growth. Absorbed Cd competes with Fe in the plant metabolism and can interrupt the Fe balance directly or indirectly, causing iron deficiency (Gao et al., 2011). Fe deficiency causes a reduction in enzyme activities, chlorophyll synthesis and plant growth (Li et al., 2017a). As a result, Cd is one of the primary heavy metal pollutants, and it has received extensive attention in the field of phytoremediation. Here, we report a hydroponics experiment conducted in a controlled laboratory environment to understand the interactive effects of Cd concentration and stress time on A. philoxeroides. The aims of this study were to 1) reveal the tolerance of A. philoxeroides to Cd, 2) explore the tolerance mechanisms of A. philoxeroides to Cd and 3) assess the potential of A. philoxeroides as a phytoremediation species for Cd.
Chlorophyll b (mg/g FW ) = (22.9 A645 − 4.68 A663 ) × V /(1000 × FW ) Total chlorophyll(mg/g FW ) = (20.2 A645 + 8.02 A663 ) × V /(1000 × FW ) where A663 and A645 are the absorbance at 663 nm and 645 nm, respectively; V is the volume of extracting solution (ml); and FW is the fresh weight of the sample (g). 2.3. 2-thiobarbituric acid reactive substances (TBARSs) concentration The equivalent of MDA concentration was expressed as the rate of oxidative damage, which was evaluated by analysing the concentration of 2-thiobarbituric acid reactive substances (TBARSs) in leaves and roots of A. philoxeroides. For the measurement of lipid peroxidation in plants, the TBA (2-thiobarbituric acid) test which determines MDA as an end product of lipid peroxidation was used (Cakmak and Horst, 2006). The TBARS concentration was estimated with the method of Cakmak and Horst (2006) with slight modifications. Samples of 0.5 g (FW) were homogenized (with silica sand added as appropriate) in 5 ml 5% (w/v) trichloroacetic acid (TCA) and then centrifuged at 8000g for 10 min. Next, 2 ml supernatant was added to 2 ml 0.6% (w/v) 2-thiobarbituric acid (TBA) in 5% (w/v) TCA. Samples were incubated at 100 °C for 10 min, with the reaction stopped by ice bath. Then, the samples were centrifuged at 6000g for 15 min. The absorbance of the supernatant was measured at 532 nm using a UV–visible spectrophotometer. The reading was corrected for non-specific turbidity by subtracting the absorbance at 600 nm. The TBARS concentration was calculated using the following equation:
⎛TBARS concentration(nmol·g−1FW) = (A532 − A 600)V ⎞ ε × FW ⎝ ⎠ where A532 and A600 are the absorbance at 532 nm and 600 nm, respectively; V is the volume of crushing medium (ml); ε is the specific extinction coefficient (155 mm cm−1); and FW is the fresh weight of the sample (g). 2.4. Extraction of iron plaque
2. Materials and methods The iron plaque on the fresh root surfaces of A. philoxeroides was extracted according to the DCB technique (Hu et al., 2007). A dithionite-citrate-bicarbonate solution was used to extract the iron plaque for 60 min at room temperature. The resulting solution was filled up to 100 ml with deionized water, defined as DCB extracts. The extractable Fe and Cd concentrations in the DCB extracts were determined by inductively coupled plasma mass spectrometry (ICP-MS, XSeries II, Thermo, USA). After DCB extraction, the roots were oven dried at 70 °C to a constant weight.
2.1. Experimental design and sample culture Fresh A. philoxeroides shoots with two nodes were collected from the Yangtze River Valley in Zhenjiang City, Jiangsu Province, China. The shoots were cultured in a greenhouse at a temperature of 25–28 °C (60–80% relative humidity, and 12 h light/dark) until the roots and leaves were fully developed. One week before the Cd stress treatment, the plants were transferred to 1/10 modified Hoagland culture solution to undergo an adaptation period (Li et al., 2015). The seedlings were then treated with Cd (CdCl2) at 0, 2.5 or 5.0 mg/l in triplicate, and defined as the control, medium-dose treatment and high-dose treatment, respectively. Five seedlings were inserted into each pot containing 5 l of culture solution with or without Cd, with a total of 45 pots. The culture solution was renewed every 3 days. The plants were harvested on days 0, 10, 20, 30 and 40 in each tretment, and their physiological and biochemical indexes were determined immediately.
2.5. Analysis of Cd concentration in tissues of A. philoxeroides by ICP-MS Dry plant samples (approximately 0.5 g) were digested as described by Soto-Jimenez and Paez-Osuna (2001). Reagent blanks and standard references of plant material (GBW-07603) (from the National Research Center for Standards in China) were included to verify the accuracy and precision of the analysis procedure (Li et al., 2017b). All of the reagents were Merck analytical grade or Suprapur quality, and all of the materials (bottles, beakers, glass funnels, measuring cylinders, filters and digestion tanks) were acid-cleaned (14% (v/v) nitric acid) and rinsed with deionized water prior to use. Fe and Cd concentrations in the samples were detected by ICP-MS (XSeries II, Thermo, USA).
2.2. Chlorophyll assay Fresh leaf samples weighing 0.5 g were ground in 95% alcohol. The homogenate was filtered in darkness. The extract was diluted with 95% alcohol to 25 ml. The absorbance of the supernatant at 645 and 663 nm was recorded by a UV–visible spectrophotometer. The concentrations (mg l−1) of chlorophyll a (chl a), chlorophyll b (chl b) and total chlorophyll (total chl) in the supernatant were calculated using the following equations (Li, 2000):
2.6. Statistical analysis All of the results presented and discussed here are based on the mean values and standard deviation (S.D.) of three replicates. One-way 238
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Fig. 1. The biomasses of the roots, stems and leaves of A. philoxeroides under Cd stress over time. Values with the same letters are equivalent; different letters denote significant differences (p < 0.05) based on one-way ANOVAs (n=3) within stress time or two-way ANOVAs (n=12) under different Cd concentration and period stresses.
Cd) at the first 10 days (p > 0.05, n=3) (Fig. 3). However, the chl a concentration was significantly reduced under this Cd treatment on days 20, 30 and 40 (p < 0.01, n=3). Under medium-dose Cd exposure, a distinct decrease in chl a concentration was observed only on day 40 (p < 0.05, n=3), whereas the chl a concentration under high-dose Cd stress (5 mg/l) was significantly lower than it was under medium-dose Cd stress (p < 0.05, n=15) and in the control treatment (p < 0.01, n=15). In addition, the chl a concentration decreased over time under Cd stress (p < 0.05, n=30). The chl a concentration determined for the A. philoxeroides leaves showed a negative correlation with the Cd gradient concentrations (p < 0.01, n=45, Fig. 3). As shown in Fig. 3, the chl b and total chl concentrations were significantly lower under highdose and medium-dose Cd stresses than they were in the control treatment (p < 0.01, n=15), and there was a significant negative correlation between each of chl b and total chl concentration and Cd concentration (p < 0.01, n=45, Fig. 3). The concentration of Cd that precipitated in iron plaque increased with the Cd concentration in the culture medium and the exposure time, and it exhibited a positive correlation with the Cd treatment concentration and stress time (p < 0.05, n=45) (Fig. 4). Cd concentrations showed 1123 ± 151.68 − 2883 ± 144.69 mg/kg DW in iron plaques of the root surface. The Fe concentration showed similar behaviour to that of the Cd in the DCB extracts, and it increased with Cd concentration and exposure time. There was a positive correlation between the Fe and Cd concentrations in the DCB extracts (p < 0.01, n=45). In A. philoxeroides, Cd induced an increase in TBARS concentration
and two-way analysis of variance was carried out. All of the statistical analyses were performed using SPSS version 13.0 statistical software, and statistically significant differences between groups were tested using Duncan's and least significant difference (LSD) multiple comparison tests. 3. Results The biomasses of the roots, stems and leaves of A. philoxeroides were greatly inhibited by Cd stress (Fig. 1), and no ramet was found. When A. philoxeroides was exposed to Cd, the roots stopped growing after day 10, although slow growth was observed for the stems and leaves at the first 30 days. In addition, the biomasses of the stems and leaves were higher at day 30 than that at day 10 under Cd stress (p < 0.05, n=3). Cd concentration was detected in the A. philoxeroides tissues. Cd was accumulated in a dose-dependent manner in the A. philoxeroides plants at Cd concentrations up to 5 mg/l in the culture solution (Fig. 2). The Cd concentration was found to increase with the Cd stress concentration and exposure time in the roots, stems and leaves of A. philoxeroides (Fig. 2). Significant positive correlations were found between the Cd concentration in the plants and each of Cd stress concentration (p < 0.01, n=45) and exposure time (p < 0.01, n=45) in the roots, stems and leaves. The Cd concentration in A. philoxeroides was the highest in the roots, ranged from 3036.10 ± 308.33 to 7588.65 ± 628.90 mg/kg·DW, followed by the stems and leaves. The medium-dose Cd stress treatment (2.5 mg/l) had little impact on the chl a concentration compared with the control treatment (0 mg/l 239
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Fig. 2. Tissue distribution of Cd in A. philoxeroides under Cd stress over time. Values with the same letters are equivalent; different letters denote significant differences (p < 0.05) based on one-way(n=3) or two-way (n=9) ANOVAs under different Cd concentration and period stresses. “IV” shows the percentage of Cd concentration in tissues.
concentration in the culture medium. For example, Simmons et al. (2007) found that the Pb and As concentrations in A. philoxeroides were directly proportional to the concentrations in the culture medium under a hydroponic laboratory setting (Simmons et al., 2007). They suggested that the total metal partitioned in the plants ranged from 50% to 65% for Pb exposure and from 24% to 40% for As exposure in the culture medium. However, the metal concentrations in the roots were much higher than they were in the stems or leaves. Similarly, in the present study, Cd was preferentially accumulated in the roots of A. philoxeroides, with less enrichment in the stems and leaves (Fig. 2). The Cd concentration ratio in the roots accounted for 78.53–94.59% of the total Cd concentration in the roots, stems and leaves. In many other plants, one detoxification mechanism is the sequestration of metals by phytochelatins and the restraint of toxic element transport from the roots (Mazhoudi et al., 1997; Mazej and Germ, 2009; Krayem et al., 2016). The physical root barrier for toxic elements might be provided by sclerenchymatous fibres with thick secondary walls, densely packed cells, suberin deposits and lignification in the outer cell layers of the cortex (Deng et al., 2009). This barrier restricts the mobility of metals to the aboveground portions of the plants. Therefore, we believe that the root system is a barrier that immobilizes Cd and impedes the transport of Cd to the aboveground portion and that this system is one of the tolerance mechanisms of A. philoxeroides. Combining the inhibition of Cd to A. philoxeroides growth, they suggests the possibility of mutual restraint between heavy metal pollution and species expansion in this plant The presence of heavy metals can cause damage to the chloroplast
only under a high-dose Cd supply (p < 0.05, n=3) and not under the medium-dose Cd supply (p > 0.05, n=3) compared with the control treatment at the first 10 days (Fig. 5). The TBARS concentrations reacting with the extractives of roots and leaves increased with increasing Cd concentration in the culture solution (p < 0.05, n=15), and a significant linear positive correlation was found between the Cd and TBARS concentrations after 20 days of stress (p < 0.01, n=27). As shown in Fig. 5, the TBARS concentration reacting with the extractive of roots was lower than that of leaves under the same stress concentration except for in the longest stress period (p < 0.01, n=3).
4. Discussion Cd toxicity may interfere with photosynthetic electron transport and have effects on photosynthetic processes in A. philoxeroides, thus negatively influencing plant growth and development (Fig. 1). A. philoxeroides is known as a clonal plant, but when it is exposed to high levels of heavy metals, the survival rate and growth measures of the distal ramets are substantially lower than those of control plants (Guo and Hu, 2012). The current results indicated that the growth of A. philoxeroides was inhibited by medium-dose and high-dose Cd. Clonal integration plays a limited role in the growth of this hydrophyte (Guo and Hu, 2012), and no ramet was found. Thus, the rapid growth of A. philoxeroides was greatly impeded by the toxic Cd element in the aquatic environment. In most previous studies, the heavy metal concentration in plants was reported to be linearly proportional to the heavy metal 240
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Fig. 3. The concentrations of chlorophyll a, chlorophyll b and total chlorophylls in the A. philoxeroides leaves under Cd stress over time. Values with the same letters are equivalent; different letters denote significant differences (p < 0.05) based on one-way ANOVAs (n=3) within stress time or two-way ANOVAs (n=12) under different Cd concentration and period stresses.
philoxeroides root (Deng et al., 2009). This loss results in large deposits of Fe on A. philoxeroides root surfaces as an ochreous plaque (Fig. 4). Iron plaque strongly adsorbs Cd in the culture medium. The greatest adsorption of Cd by iron plaque occurs on day 40 under the high-dose Cd provided at 2883.16 mg/kg, which is much higher than the adsorption by iron plaque in rice, an aquatic plant (Dong et al., 2016), and in Avicennia marina (Li et al., 2016). As a “barrier”, the iron plaque increased in thickness with increasing stress gradient concentration and stress duration, and it was positively correlated with Cd concentration in the DCB extracts (Fig. 4). The thick iron plaque strongly adsorbed the Cd and effectively prevented it from being absorbed onto the roots of A. philoxeroides. This might represent a critical mechanism in A. philoxeroides for tolerating Cd toxicity. As the primary by-product of lipid peroxidation, MDA determined by TBARS concentration did not increase significantly in concentration under the medium-dose Cd condition at the first 10 days in A. philoxeroides (Fig. 5). It is possible that the oxidative stress generated by Cd was mitigated by the enzymatic and non-enzymatic defence systems of the plant, which protected the cytomembranes from alteration and peroxidation by reactive oxygen species (Srivastava et al., 2006; Krayem et al., 2016). According to the TBARS concentration, the MDA in the roots were significantly lower than they were in the leaves during the first 30 days (Fig. 5). This finding indicated that the roots were the “first door” and “barrier” to Cd absorption, and they had greater Cd tolerance than did the leaves, which might reflect a strategy for selfprotection, preventing the plant nutrient source from being poisoned by toxic elements. However, the MDA concentration in the roots showed a
ultrastructure, such as the shape of the chloroplasts, resulting in the structural disarrangement of thylakoids and stroma, and it can affect photosynthesis in the leaves (Mangabeira et al., 2011). Cd inhibits protochlorophyllide reductase and affects the synthesis of aminolaevulinic acid in plants (Stobart et al., 1985). Exposure to this metal has resulted in synthesis failure in chlorophylls, and chlorophyll concentrations are reduced with increasing plant Cd concentration (Fig. 3). In the present study, the primary variables that were affected by Cd in A. philoxeroides leaves were chl a and chl b concentrations. A similar finding was observed in previous research, in which Cd exposure resulted in a significant loss of chlorophyll concentration in A. philoxeroides leaves in a water body polluted by Cd (Ding et al., 2007). Although more rapid degradation was reported for chl a than for chl b and carotenoids in that study, more rapid degradation of chl b than of chl a or total chl was found in the current study. This degradation might be involved in the photooxidative mechanisms under Cd-induced damage (Wieckowski and Waloszek, 1993). This result also indicates that chl b was more sensitive than was chl a to Cd stress in our experimental setting. Aquatic macrophytes have been used to monitor water contaminated by heavy metals. Rapid degradation of chlorophylls was detected in A. philoxeroides, suggesting a possible application of this species as a biomarker for Cd contamination in water bodies (Mangabeira et al., 2011). Cd tolerance has been suggested to be related to the root's ability to oxidize Fe to produce iron plaque on root surfaces in wetland plants (Li et al., 2016). As the essential condition for iron plaque formation, high radial oxygen loss (ROL) occurs along the entire length of the A. 241
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Fig. 4. The Cd and Fe concentrations in the iron plaques from A. philoxeroides roots under Cd stress over time. Values with the same letters are equivalent and different letters denote significant differences (p < 0.05) based on one-way ANOVAs (n=3) within stress concentration or two-way ANOVAs (n=9) under different Cd concentration and period stresses.
Fig. 5. Concentrations of 2-thiobarbituric acid reactive substance (TBARS) in the roots and leaves of A. philoxeroides under Cd stress over time. Values with the same letters are equivalent and different letters denote significant differences (p < 0.05) based on oneway ANOVAs (n=3) within stress time. TBARS concentrations in both roots and leaves showed significant difference based on two-way ANOVAs (p < 0.05, n=12) under different Cd concentration and period stresses.
dramatic increase after 30 days, exceeding the concentration in the leaves. This result might be due to the loss of the intrinsic balance of the protective enzymes of the A. philoxeroides roots after treatment for 30 days, with their protective functions reaching their limit after this time (Ding et al., 2007). As a biomarker of lipid peroxidation, MDA concentration has been considered an indicator of oxidative damage (Krayem et al., 2016). Manikandan et al. (2016) detected the MDA concentration of Vetiveria zizanioides (L.) Nash under 5 mg/l Cd stress in a hydroponic system. At the 16th day, they found that the MDA concentrations were 0.26 μmol/ g·FW and 0.32 μmol/g·FW in the stems and roots, respectively (Manikandan et al., 2016). Similarly, the macrophyte Myriophyllum alterniflorum DC. produced MDA at 13.93 ± 1.63 mol/g·FW and 16.44 ± 2.74 mol/g·FW for 100 μg/L Cu and As exposure, respectively, at 21 days (Krayem et al., 2016). Although the stresses in those studies were from different heavy metals and for different times than in the present study, the two macrophytes both had higher degrees of lipid peroxidation than did A. philoxeroides in the present study (Fig. 5). Similarly, under Zn treatment, Arabidopsis thaliana (L.) Heynh. and rice produced higher MDA concentrations than did A. philoxeroides over the same stress duration (Wang et al., 2016). Because of the degree of lipid peroxidation observed in A. philoxeroides, we suggest that this species might possess more extensive adaptability and tolerance than possessed by other plants in an adverse environment. As the MDA equivalent, the TBARS results provide further evidence to support the use of A.
philoxeroides in the phytoremediation of aquatic environments contaminated by heavy metals.
5. Conclusion A mutual restraint effect was found between Cd and A. philoxeroides in the present research. A large amount of Cd was enriched and immobilized in A. philoxeroides. In addition, the toxicity of Cd inhibited the rapid growth of A. philoxeroides and induced the rapid degradation of chlorophylls in its tissues. Therefore, A. philoxeroides should be considered a possible biomarker for Cd contamination in water bodies. Furthermore, iron plaque effectively immobilized Cd on the root surface and decreased both the transferability of Cd in water bodies. This iron plaque can be considered an important detoxification mechanism for A. philoxeroides in water bodies that are subject to Cd pollution. Low MDA concentrations combined with extensive adaptability provided A. philoxeroides with high tolerance to Cd toxicity. With the immobilization of most of the Cd in the roots (78.53–94.59%), A. philoxeroides had high efficiency at restraining the translocation of Cd, and it showed a high degree of Cd partitioning in the plant roots. In conclusion, A. philoxeroides is a good candidate for use in the Cd-phytoremediation of invaded aquatic environments, and it could act a biomarker of aquatic environments contaminated with Cd. 242
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Acknowledgements This work was supported by the National Key Research Development Program of China (2017YFC1200100), the National Natural Science Foundation of China (31570414), the Natural Science Foundation of Jiangsu Province (BK20170540), the China Postdoctoral Science Foundation (2016M591773) and the Jiangsu Planned Projects for Postdoctoral Research Funds (1501028B). References Baker, A.J.M., Brooks, R., Baker, N.A., 1989. Terrestrial higher plants which hyperaccumulate metallic elements. A review of their distribution, ecology and phytochemistry. Biorecovery 1, 81–126. Cakmak, I., Horst, W.J., 2006. Effect of aluminium on lipid peroxidation, superoxide dismutase, catalase, and peroxidase activities in root tips of soybean (Glycine max). Physiol. Plant. 83, 463–468. Deng, H., Ye, Z.H., Wong, M.H., 2009. Lead, zinc and iron (Fe2+) tolerances in wetland plants and relation to root anatomy and spatial pattern of ROL. Environ. Exp. Bot. 65, 353–362. Ding, B., Shi, G., Xu, Y., Hu, J., Xu, Q., 2007. Physiological responses of Alternanthera philoxeroides (Mart.) Griseb leaves to cadmium stress. Environ. Pollut. 147, 800–803. Dong, M.F., Feng, R.W., Wang, R.G., Sun, Y., Ding, Y.Z., Xu, Y.M., Fan, Z.L., Guo, J.K., 2016. Inoculation of Fe/Mn-oxidizing bacteria enhances Fe/Mn plaque formation and reduces Cd and As accumulation in Rice Plant tissues. Plant Soil 404, 75–83. Gao, C., Wang, Y., Xiao, D.-S., Qiu, C.-P., Han, D.-G., Zhang, X.-Z., Wu, T., Han, Z.-H., 2011. Comparison of cadmium-induced iron-deficiency responses and genuine irondeficiency responses in Malus xiaojinensis. Plant Sci. 181, 269–274. Guo, W., Hu, Z., 2012. Effects of stolon severing on the expansion of Alternanthera philoxeroides from terrestrial to contaminated aquatic habitats. Plant Species Biol. 27, 46–52. Hu, N., Zheng, J., Ding, D., Li, G., Yin, J., Chen, X., Yu, J., 2013. Screening of native Hyperaccumulators at the Huayuan River contaminated by heavy metals. Bioremediation J. 17, 21–29. Hu, Z.Y., Zhu, Y.G., Li, M., Zhang, L.G., Cao, Z.H., Smith, E.A., 2007. Sulfur (S)-induced enhancement of iron plaque formation in the rhizosphere reduces arsenic accumulation in rice (Oryza sativa L.) seedlings. Environ. Pollut. 147, 387–393. Jasrotia, S., Kansal, A., Mehra, A., 2015. Performance of aquatic plant species for phytoremediation of arsenic-contaminated water. Appl. Water Sci. 1–8. Krayem, M., Baydoun, M., Deluchat, V., Lenain, J.F., Kazpard, V., Labrousse, P., 2016. Absorption and translocation of copper and arsenic in an aquatic macrophyte Myriophyllum alterniflorum DC. in oligotrophic and eutrophic conditions. Environ. Sci. Pollut. Res. 23, 11129–11136. Li, H., 2000. Principles and Techniques of Plant Physiological Biochemical Experiment. Higher Education Press, Beijing. Li, J., Liu, J., Lu, H., Jia, H., Yu, J., Hong, H., Yan, C., 2016. Influence of the phenols on the biogeochemical behavior of cadmium in the mangrove sediment. Chemosphere 144, 2206–2213. Li, J., Lu, H., Liu, J., Hong, H., Yan, C., 2015. The influence of flavonoid amendment on the absorption of cadmium in Avicennia marina roots. Ecotoxicol. Environ. Saf. 120, 1–6. Li, J., Yan, C., Du, D., Lu, H., Liu, J., 2017a. Accumulation and speciation of Cd in Avicennia marina tissues. Int. J. Phytoremediat. http://dx.doi.org/10.1080/ 15226514.2017.1303817.
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The mutual restraint effect between the expansion of Alternanthera philoxeroides (Mart.) Griseb and cadmium mobility in aquatic environment ⁎
Jian Lia,b, Zhiwei Dua, Chris B. Zouc, Zhicong Daia, Daolin Dua, , Chongling Yanb, a b c
T
⁎
Institute of Environment and Ecology, School of the Environment and Safety Engineering, Jiangsu University, Zhenjiang 212013, China State Key Lab of Marine Environmental Science, Xiamen University, Xiamen 361102, China Department of Natural Resource Ecology and Management, Oklahoma State University, Stillwater, OK 74078, USA
A R T I C L E I N F O
A B S T R A C T
Keywords: Hydrophyte Heavy metal Iron plaque Phytoremediation
Alternanthera philoxeroides (Mart.) Griseb is one of the most malignant weeds in its invision habitats. While in the cadmium-contaminated aquatic environment, does A. philoxeroides possess good tolerance and adaptability? To demonstrate the effects of cadmium on A. philoxeroides in the polluted water bodies, a hydroponic stress experiment was conducted over a gradient of Cd concentrations (0, 2.5 and 5 mg/l) in triplicate. The seedlings were cultured in a greenhouse and harvested on days 0, 10, 20, 30 and 40, respectively. The results showed the effects of mutual restraint between Cd and A. philoxeroides. The A. philoxeroides seedlings were enriched with large amounts of Cd, and the toxicity of Cd inhibited the rapid growth of A. philoxeroides and induced the rapid degradation of chlorophylls in its tissues. Furthermore, the use of iron plaque effectively immobilized Cd of 1123–2883 mg/kg·DW on the root surface, thus it decreased the transferability of Cd in the aquatic environment. Due to its extensive adaptability, good Cd tolerance and the immobilization of Cd predominantly in the roots (the highest Cd concentration enriched was 7588.65 ± 628.90 mg/kg·DW in roots). A. philoxeroides effectively restrained the translocation of Cd and partitioned Cd in the roots within water bodies. Capsule: The antagonistic effect exists between the invasion of A. philoxeroides and cadmium mobility in aquatic environments.
1. Introduction Heavy metal contamination is a major problem in aquatic ecosystems within human-dominated regions (William, 2007). In hydrophytes, heavy metals induce oxidative stress and inhibit photosynthesis and growth, leading to cell membrane alteration and malondialdehyde (MDA) content increase via lipoperoxidation (Ding et al., 2007; Krayem et al., 2016). Heavy metals are enriched from primary producers to consumers, and organisms achieve high Cd levels in vivo through food chains or food webs. As a result, heavy metal contamination in water bodies not only threatens the development of aquatic ecosystems but is also a human health hazard (Krayem et al., 2016). Heavy metals in the aquatic environment can be removed using a variety of approaches, such as chemical or physical adsorption, physical precipitation and phytoremediation (Liu et al., 2007; Li et al., 2016). Due to its low cost and low environmental impact, the use of phytoremediation to remove heavy metals has been a focus of researchers and environmental managers (Jasrotia et al., 2015; Rezania et al., 2016). Previous studies indicate that a good metal-accumulating wetland plant species can absorb more than 0.5% of its dry weight (DW) of a given ⁎
Corresponding authors. E-mail addresses: [email protected] (D. Du), [email protected] (C. Yan).
http://dx.doi.org/10.1016/j.ecoenv.2017.10.032 Received 20 July 2017; Received in revised form 6 October 2017; Accepted 13 October 2017 Available online 06 November 2017 0147-6513/ © 2017 Elsevier Inc. All rights reserved.
element and bio-concentrate the element in its tissue to up to 1,000-fold the initial elemental concentration in the water body (Zayed et al., 1998). In metal-scavenging hydrophytes, heavy metal uptake mechanism includes accumulation, exclusion, translocation, osmoregulation and distribution (Rezania et al., 2016). As a result, heavy metalscavenging hydrophytes play a critical role in cleaning up heavy metal pollution in wetlands. For example, Alternanthera philoxeroides (Mart.) Griseb, an amphibious species, is abundant worldwide and has a high tolerance for heavy metal-polluted environments. This plant can act as a pioneer species and can thrive in mine tailings and polluted lands or aquatic environments (typically along banks) (Naqvi and Rizvi, 2000; Minchinton et al., 2006). A record Mn concentration of 19,300 mg/kg was reported for A. philoxeroides, demonstrating its ability as a hyperaccumulator of Mn (Xue et al., 2003). In heavy metal-contaminated areas, A. philoxeroides has evolved into a cumulative ecotype under heavy metal stress (Hu et al., 2013). Baker et al. (1989) proposed some criteria for classifying a plant as a hyperaccumulator: it should be capable of accumulating more than 10,000 mg/kg for Mn and Zn and more than 100 mg/kg for Cd in their shoots, and the TF should be more than 1 (Baker et al., 1989). Based on
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Chlorophyll a (mg/g FW ) = (12.7 A663 − 2.69 A645 ) × V /(1000 × FW )
data from Hu et al. (2013), A. philoxeroides meets these criteria and can be considered a potential species for the phytoremediation of soils, sediments and aquatic environments polluted with Cd, Mn and Zn. They showed that the Cd and Zn concentrations in A. philoxeroides were 21 and 43,154 mg/kg in the roots, respectively, and 155 and 13,784 mg/ kg in the stems, respectively. In addition, the bioaccumulation factors (BCFs) of Cd and Zn were 36.78 and 49.23, respectively, and the translocation factors (TFs) were 7.38 and 3.19, respectively. However, the Cd accumulation and extraction capabilities of A. philoxeroides are debated. Liu et al. (2007) investigated the uptake and distribution of Cd, Pb and Zn in 19 wetland plant species from constructed wetlands, and they reported that the Cd concentrations in A. philoxeroides were 20.56 and 96.66 mg/kg in the aboveground and underground parts, respectively. The TF was less than 1, although the Cd accumulated mass was 28.17 mg/kg in the entire plant, representing the highest value among all of the studied species. These different results might be due to study differences in the growth media and the Cd concentration that was bioavailable to A. philoxeroides. Cd can be absorbed through symplast and non-symplast transport in the roots. During symplast transport, Cd is transported through selective or non-selective ionophores and channel proteins (e.g., Ca2+, Mg2+ and Fe2+ channel proteins) into the roots (Welch et al., 1999; Zhang et al., 2015). Plants absorb Cd through Mg, Ca or Fe channels because of their high biological activities and the bioavailability of Cd. Fe is important to plant metabolism and growth. Absorbed Cd competes with Fe in the plant metabolism and can interrupt the Fe balance directly or indirectly, causing iron deficiency (Gao et al., 2011). Fe deficiency causes a reduction in enzyme activities, chlorophyll synthesis and plant growth (Li et al., 2017a). As a result, Cd is one of the primary heavy metal pollutants, and it has received extensive attention in the field of phytoremediation. Here, we report a hydroponics experiment conducted in a controlled laboratory environment to understand the interactive effects of Cd concentration and stress time on A. philoxeroides. The aims of this study were to 1) reveal the tolerance of A. philoxeroides to Cd, 2) explore the tolerance mechanisms of A. philoxeroides to Cd and 3) assess the potential of A. philoxeroides as a phytoremediation species for Cd.
Chlorophyll b (mg/g FW ) = (22.9 A645 − 4.68 A663 ) × V /(1000 × FW ) Total chlorophyll(mg/g FW ) = (20.2 A645 + 8.02 A663 ) × V /(1000 × FW ) where A663 and A645 are the absorbance at 663 nm and 645 nm, respectively; V is the volume of extracting solution (ml); and FW is the fresh weight of the sample (g). 2.3. 2-thiobarbituric acid reactive substances (TBARSs) concentration The equivalent of MDA concentration was expressed as the rate of oxidative damage, which was evaluated by analysing the concentration of 2-thiobarbituric acid reactive substances (TBARSs) in leaves and roots of A. philoxeroides. For the measurement of lipid peroxidation in plants, the TBA (2-thiobarbituric acid) test which determines MDA as an end product of lipid peroxidation was used (Cakmak and Horst, 2006). The TBARS concentration was estimated with the method of Cakmak and Horst (2006) with slight modifications. Samples of 0.5 g (FW) were homogenized (with silica sand added as appropriate) in 5 ml 5% (w/v) trichloroacetic acid (TCA) and then centrifuged at 8000g for 10 min. Next, 2 ml supernatant was added to 2 ml 0.6% (w/v) 2-thiobarbituric acid (TBA) in 5% (w/v) TCA. Samples were incubated at 100 °C for 10 min, with the reaction stopped by ice bath. Then, the samples were centrifuged at 6000g for 15 min. The absorbance of the supernatant was measured at 532 nm using a UV–visible spectrophotometer. The reading was corrected for non-specific turbidity by subtracting the absorbance at 600 nm. The TBARS concentration was calculated using the following equation:
⎛TBARS concentration(nmol·g−1FW) = (A532 − A 600)V ⎞ ε × FW ⎝ ⎠ where A532 and A600 are the absorbance at 532 nm and 600 nm, respectively; V is the volume of crushing medium (ml); ε is the specific extinction coefficient (155 mm cm−1); and FW is the fresh weight of the sample (g). 2.4. Extraction of iron plaque
2. Materials and methods The iron plaque on the fresh root surfaces of A. philoxeroides was extracted according to the DCB technique (Hu et al., 2007). A dithionite-citrate-bicarbonate solution was used to extract the iron plaque for 60 min at room temperature. The resulting solution was filled up to 100 ml with deionized water, defined as DCB extracts. The extractable Fe and Cd concentrations in the DCB extracts were determined by inductively coupled plasma mass spectrometry (ICP-MS, XSeries II, Thermo, USA). After DCB extraction, the roots were oven dried at 70 °C to a constant weight.
2.1. Experimental design and sample culture Fresh A. philoxeroides shoots with two nodes were collected from the Yangtze River Valley in Zhenjiang City, Jiangsu Province, China. The shoots were cultured in a greenhouse at a temperature of 25–28 °C (60–80% relative humidity, and 12 h light/dark) until the roots and leaves were fully developed. One week before the Cd stress treatment, the plants were transferred to 1/10 modified Hoagland culture solution to undergo an adaptation period (Li et al., 2015). The seedlings were then treated with Cd (CdCl2) at 0, 2.5 or 5.0 mg/l in triplicate, and defined as the control, medium-dose treatment and high-dose treatment, respectively. Five seedlings were inserted into each pot containing 5 l of culture solution with or without Cd, with a total of 45 pots. The culture solution was renewed every 3 days. The plants were harvested on days 0, 10, 20, 30 and 40 in each tretment, and their physiological and biochemical indexes were determined immediately.
2.5. Analysis of Cd concentration in tissues of A. philoxeroides by ICP-MS Dry plant samples (approximately 0.5 g) were digested as described by Soto-Jimenez and Paez-Osuna (2001). Reagent blanks and standard references of plant material (GBW-07603) (from the National Research Center for Standards in China) were included to verify the accuracy and precision of the analysis procedure (Li et al., 2017b). All of the reagents were Merck analytical grade or Suprapur quality, and all of the materials (bottles, beakers, glass funnels, measuring cylinders, filters and digestion tanks) were acid-cleaned (14% (v/v) nitric acid) and rinsed with deionized water prior to use. Fe and Cd concentrations in the samples were detected by ICP-MS (XSeries II, Thermo, USA).
2.2. Chlorophyll assay Fresh leaf samples weighing 0.5 g were ground in 95% alcohol. The homogenate was filtered in darkness. The extract was diluted with 95% alcohol to 25 ml. The absorbance of the supernatant at 645 and 663 nm was recorded by a UV–visible spectrophotometer. The concentrations (mg l−1) of chlorophyll a (chl a), chlorophyll b (chl b) and total chlorophyll (total chl) in the supernatant were calculated using the following equations (Li, 2000):
2.6. Statistical analysis All of the results presented and discussed here are based on the mean values and standard deviation (S.D.) of three replicates. One-way 238
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Fig. 1. The biomasses of the roots, stems and leaves of A. philoxeroides under Cd stress over time. Values with the same letters are equivalent; different letters denote significant differences (p < 0.05) based on one-way ANOVAs (n=3) within stress time or two-way ANOVAs (n=12) under different Cd concentration and period stresses.
Cd) at the first 10 days (p > 0.05, n=3) (Fig. 3). However, the chl a concentration was significantly reduced under this Cd treatment on days 20, 30 and 40 (p < 0.01, n=3). Under medium-dose Cd exposure, a distinct decrease in chl a concentration was observed only on day 40 (p < 0.05, n=3), whereas the chl a concentration under high-dose Cd stress (5 mg/l) was significantly lower than it was under medium-dose Cd stress (p < 0.05, n=15) and in the control treatment (p < 0.01, n=15). In addition, the chl a concentration decreased over time under Cd stress (p < 0.05, n=30). The chl a concentration determined for the A. philoxeroides leaves showed a negative correlation with the Cd gradient concentrations (p < 0.01, n=45, Fig. 3). As shown in Fig. 3, the chl b and total chl concentrations were significantly lower under highdose and medium-dose Cd stresses than they were in the control treatment (p < 0.01, n=15), and there was a significant negative correlation between each of chl b and total chl concentration and Cd concentration (p < 0.01, n=45, Fig. 3). The concentration of Cd that precipitated in iron plaque increased with the Cd concentration in the culture medium and the exposure time, and it exhibited a positive correlation with the Cd treatment concentration and stress time (p < 0.05, n=45) (Fig. 4). Cd concentrations showed 1123 ± 151.68 − 2883 ± 144.69 mg/kg DW in iron plaques of the root surface. The Fe concentration showed similar behaviour to that of the Cd in the DCB extracts, and it increased with Cd concentration and exposure time. There was a positive correlation between the Fe and Cd concentrations in the DCB extracts (p < 0.01, n=45). In A. philoxeroides, Cd induced an increase in TBARS concentration
and two-way analysis of variance was carried out. All of the statistical analyses were performed using SPSS version 13.0 statistical software, and statistically significant differences between groups were tested using Duncan's and least significant difference (LSD) multiple comparison tests. 3. Results The biomasses of the roots, stems and leaves of A. philoxeroides were greatly inhibited by Cd stress (Fig. 1), and no ramet was found. When A. philoxeroides was exposed to Cd, the roots stopped growing after day 10, although slow growth was observed for the stems and leaves at the first 30 days. In addition, the biomasses of the stems and leaves were higher at day 30 than that at day 10 under Cd stress (p < 0.05, n=3). Cd concentration was detected in the A. philoxeroides tissues. Cd was accumulated in a dose-dependent manner in the A. philoxeroides plants at Cd concentrations up to 5 mg/l in the culture solution (Fig. 2). The Cd concentration was found to increase with the Cd stress concentration and exposure time in the roots, stems and leaves of A. philoxeroides (Fig. 2). Significant positive correlations were found between the Cd concentration in the plants and each of Cd stress concentration (p < 0.01, n=45) and exposure time (p < 0.01, n=45) in the roots, stems and leaves. The Cd concentration in A. philoxeroides was the highest in the roots, ranged from 3036.10 ± 308.33 to 7588.65 ± 628.90 mg/kg·DW, followed by the stems and leaves. The medium-dose Cd stress treatment (2.5 mg/l) had little impact on the chl a concentration compared with the control treatment (0 mg/l 239
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Fig. 2. Tissue distribution of Cd in A. philoxeroides under Cd stress over time. Values with the same letters are equivalent; different letters denote significant differences (p < 0.05) based on one-way(n=3) or two-way (n=9) ANOVAs under different Cd concentration and period stresses. “IV” shows the percentage of Cd concentration in tissues.
concentration in the culture medium. For example, Simmons et al. (2007) found that the Pb and As concentrations in A. philoxeroides were directly proportional to the concentrations in the culture medium under a hydroponic laboratory setting (Simmons et al., 2007). They suggested that the total metal partitioned in the plants ranged from 50% to 65% for Pb exposure and from 24% to 40% for As exposure in the culture medium. However, the metal concentrations in the roots were much higher than they were in the stems or leaves. Similarly, in the present study, Cd was preferentially accumulated in the roots of A. philoxeroides, with less enrichment in the stems and leaves (Fig. 2). The Cd concentration ratio in the roots accounted for 78.53–94.59% of the total Cd concentration in the roots, stems and leaves. In many other plants, one detoxification mechanism is the sequestration of metals by phytochelatins and the restraint of toxic element transport from the roots (Mazhoudi et al., 1997; Mazej and Germ, 2009; Krayem et al., 2016). The physical root barrier for toxic elements might be provided by sclerenchymatous fibres with thick secondary walls, densely packed cells, suberin deposits and lignification in the outer cell layers of the cortex (Deng et al., 2009). This barrier restricts the mobility of metals to the aboveground portions of the plants. Therefore, we believe that the root system is a barrier that immobilizes Cd and impedes the transport of Cd to the aboveground portion and that this system is one of the tolerance mechanisms of A. philoxeroides. Combining the inhibition of Cd to A. philoxeroides growth, they suggests the possibility of mutual restraint between heavy metal pollution and species expansion in this plant The presence of heavy metals can cause damage to the chloroplast
only under a high-dose Cd supply (p < 0.05, n=3) and not under the medium-dose Cd supply (p > 0.05, n=3) compared with the control treatment at the first 10 days (Fig. 5). The TBARS concentrations reacting with the extractives of roots and leaves increased with increasing Cd concentration in the culture solution (p < 0.05, n=15), and a significant linear positive correlation was found between the Cd and TBARS concentrations after 20 days of stress (p < 0.01, n=27). As shown in Fig. 5, the TBARS concentration reacting with the extractive of roots was lower than that of leaves under the same stress concentration except for in the longest stress period (p < 0.01, n=3).
4. Discussion Cd toxicity may interfere with photosynthetic electron transport and have effects on photosynthetic processes in A. philoxeroides, thus negatively influencing plant growth and development (Fig. 1). A. philoxeroides is known as a clonal plant, but when it is exposed to high levels of heavy metals, the survival rate and growth measures of the distal ramets are substantially lower than those of control plants (Guo and Hu, 2012). The current results indicated that the growth of A. philoxeroides was inhibited by medium-dose and high-dose Cd. Clonal integration plays a limited role in the growth of this hydrophyte (Guo and Hu, 2012), and no ramet was found. Thus, the rapid growth of A. philoxeroides was greatly impeded by the toxic Cd element in the aquatic environment. In most previous studies, the heavy metal concentration in plants was reported to be linearly proportional to the heavy metal 240
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Fig. 3. The concentrations of chlorophyll a, chlorophyll b and total chlorophylls in the A. philoxeroides leaves under Cd stress over time. Values with the same letters are equivalent; different letters denote significant differences (p < 0.05) based on one-way ANOVAs (n=3) within stress time or two-way ANOVAs (n=12) under different Cd concentration and period stresses.
philoxeroides root (Deng et al., 2009). This loss results in large deposits of Fe on A. philoxeroides root surfaces as an ochreous plaque (Fig. 4). Iron plaque strongly adsorbs Cd in the culture medium. The greatest adsorption of Cd by iron plaque occurs on day 40 under the high-dose Cd provided at 2883.16 mg/kg, which is much higher than the adsorption by iron plaque in rice, an aquatic plant (Dong et al., 2016), and in Avicennia marina (Li et al., 2016). As a “barrier”, the iron plaque increased in thickness with increasing stress gradient concentration and stress duration, and it was positively correlated with Cd concentration in the DCB extracts (Fig. 4). The thick iron plaque strongly adsorbed the Cd and effectively prevented it from being absorbed onto the roots of A. philoxeroides. This might represent a critical mechanism in A. philoxeroides for tolerating Cd toxicity. As the primary by-product of lipid peroxidation, MDA determined by TBARS concentration did not increase significantly in concentration under the medium-dose Cd condition at the first 10 days in A. philoxeroides (Fig. 5). It is possible that the oxidative stress generated by Cd was mitigated by the enzymatic and non-enzymatic defence systems of the plant, which protected the cytomembranes from alteration and peroxidation by reactive oxygen species (Srivastava et al., 2006; Krayem et al., 2016). According to the TBARS concentration, the MDA in the roots were significantly lower than they were in the leaves during the first 30 days (Fig. 5). This finding indicated that the roots were the “first door” and “barrier” to Cd absorption, and they had greater Cd tolerance than did the leaves, which might reflect a strategy for selfprotection, preventing the plant nutrient source from being poisoned by toxic elements. However, the MDA concentration in the roots showed a
ultrastructure, such as the shape of the chloroplasts, resulting in the structural disarrangement of thylakoids and stroma, and it can affect photosynthesis in the leaves (Mangabeira et al., 2011). Cd inhibits protochlorophyllide reductase and affects the synthesis of aminolaevulinic acid in plants (Stobart et al., 1985). Exposure to this metal has resulted in synthesis failure in chlorophylls, and chlorophyll concentrations are reduced with increasing plant Cd concentration (Fig. 3). In the present study, the primary variables that were affected by Cd in A. philoxeroides leaves were chl a and chl b concentrations. A similar finding was observed in previous research, in which Cd exposure resulted in a significant loss of chlorophyll concentration in A. philoxeroides leaves in a water body polluted by Cd (Ding et al., 2007). Although more rapid degradation was reported for chl a than for chl b and carotenoids in that study, more rapid degradation of chl b than of chl a or total chl was found in the current study. This degradation might be involved in the photooxidative mechanisms under Cd-induced damage (Wieckowski and Waloszek, 1993). This result also indicates that chl b was more sensitive than was chl a to Cd stress in our experimental setting. Aquatic macrophytes have been used to monitor water contaminated by heavy metals. Rapid degradation of chlorophylls was detected in A. philoxeroides, suggesting a possible application of this species as a biomarker for Cd contamination in water bodies (Mangabeira et al., 2011). Cd tolerance has been suggested to be related to the root's ability to oxidize Fe to produce iron plaque on root surfaces in wetland plants (Li et al., 2016). As the essential condition for iron plaque formation, high radial oxygen loss (ROL) occurs along the entire length of the A. 241
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Fig. 4. The Cd and Fe concentrations in the iron plaques from A. philoxeroides roots under Cd stress over time. Values with the same letters are equivalent and different letters denote significant differences (p < 0.05) based on one-way ANOVAs (n=3) within stress concentration or two-way ANOVAs (n=9) under different Cd concentration and period stresses.
Fig. 5. Concentrations of 2-thiobarbituric acid reactive substance (TBARS) in the roots and leaves of A. philoxeroides under Cd stress over time. Values with the same letters are equivalent and different letters denote significant differences (p < 0.05) based on oneway ANOVAs (n=3) within stress time. TBARS concentrations in both roots and leaves showed significant difference based on two-way ANOVAs (p < 0.05, n=12) under different Cd concentration and period stresses.
dramatic increase after 30 days, exceeding the concentration in the leaves. This result might be due to the loss of the intrinsic balance of the protective enzymes of the A. philoxeroides roots after treatment for 30 days, with their protective functions reaching their limit after this time (Ding et al., 2007). As a biomarker of lipid peroxidation, MDA concentration has been considered an indicator of oxidative damage (Krayem et al., 2016). Manikandan et al. (2016) detected the MDA concentration of Vetiveria zizanioides (L.) Nash under 5 mg/l Cd stress in a hydroponic system. At the 16th day, they found that the MDA concentrations were 0.26 μmol/ g·FW and 0.32 μmol/g·FW in the stems and roots, respectively (Manikandan et al., 2016). Similarly, the macrophyte Myriophyllum alterniflorum DC. produced MDA at 13.93 ± 1.63 mol/g·FW and 16.44 ± 2.74 mol/g·FW for 100 μg/L Cu and As exposure, respectively, at 21 days (Krayem et al., 2016). Although the stresses in those studies were from different heavy metals and for different times than in the present study, the two macrophytes both had higher degrees of lipid peroxidation than did A. philoxeroides in the present study (Fig. 5). Similarly, under Zn treatment, Arabidopsis thaliana (L.) Heynh. and rice produced higher MDA concentrations than did A. philoxeroides over the same stress duration (Wang et al., 2016). Because of the degree of lipid peroxidation observed in A. philoxeroides, we suggest that this species might possess more extensive adaptability and tolerance than possessed by other plants in an adverse environment. As the MDA equivalent, the TBARS results provide further evidence to support the use of A.
philoxeroides in the phytoremediation of aquatic environments contaminated by heavy metals.
5. Conclusion A mutual restraint effect was found between Cd and A. philoxeroides in the present research. A large amount of Cd was enriched and immobilized in A. philoxeroides. In addition, the toxicity of Cd inhibited the rapid growth of A. philoxeroides and induced the rapid degradation of chlorophylls in its tissues. Therefore, A. philoxeroides should be considered a possible biomarker for Cd contamination in water bodies. Furthermore, iron plaque effectively immobilized Cd on the root surface and decreased both the transferability of Cd in water bodies. This iron plaque can be considered an important detoxification mechanism for A. philoxeroides in water bodies that are subject to Cd pollution. Low MDA concentrations combined with extensive adaptability provided A. philoxeroides with high tolerance to Cd toxicity. With the immobilization of most of the Cd in the roots (78.53–94.59%), A. philoxeroides had high efficiency at restraining the translocation of Cd, and it showed a high degree of Cd partitioning in the plant roots. In conclusion, A. philoxeroides is a good candidate for use in the Cd-phytoremediation of invaded aquatic environments, and it could act a biomarker of aquatic environments contaminated with Cd. 242
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