Effect of Cd on growth, physiological response, Cd subcellular distribution and chemical forms of Koelreuteria paniculata

Ecotoxicology and Environmental Safety 160 (2018) 10–18

Contents lists available at ScienceDirect

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

Effect of Cd on growth, physiological response, Cd subcellular distribution and chemical forms of Koelreuteria paniculata ⁎

T

⁎

Lan Peng Yanga, Jian Zhua, , Ping Wanga, , Jing Zengb, Rong Tana, Yu Zhong Yanga, Zhi Ming Liuc a

College of Environmental Science and Engineering, Central South University of Forestry and Technology, Changsha 410004, PR China College of Life Science and Technology, Central South University of Forestry and Technology, Changsha 410004, PR China c Department of Biology, Eastern New Mexico University, Portales, NM 88130, USA b

A R T I C LE I N FO

A B S T R A C T

Keywords: Koelreuteria paniculata Cadmium Subcellular distribution Chemical forms Tolerance Accumulation

Koelreuteria paniculata were cultivated in nutrient solution with different concentrations of Cd (0, 50, 150, 250 and 500 µM) and sampled after 90 days. The resistance, translocation, accumulation and stress responses in Koelreuteria paniculata were investigated by hydroponic experiments. The results showed that Koelreuteria paniculata is an efficient Cd excluder that can tolerate high concentrations of Cd (up to 150–250 µM of Cd). The concentration of Cd never exceeds 5 ppm in leaves and 10 ppm in roots. The high concentration of Cd (≥ 250 µM) had a toxic effect on K. paniculata and significantly restricted the plant growth. The accumulation ability of Cd by different plant tissues followed the sequence of roots > leaves > stems. The bioconcentration factors and translocation factors both were less than 1. Cd has the highest content in the cell wall and is migrated to soluble fractions and organelles at high concentrations. Undissolved Cd phosphate, pectates and proteinbound Cd were the predominant forms. The low concentration of Cd (≤150 µM) promoted the synthesis of soluble proteins, AsA and GSH, while high concentration of Cd clearly inhibited the physiological and biochemical process, caused membrane lipid peroxidation and severe membrane damages, and increased MDA and H2O2 contents. POD, CAT and SOD exhibited positive and effective responses to low concentration Cd stress, but could not remove the toxicity caused by high concentration Cd stress. The content of IAA, GA and ZT decreased and ABA content was significantly increased under high-concentration Cd stress.

1. Introduction The heavy use of cadmium (Cd) containing fertilizers, and mining exploitations has led to a gradual concentrations of cadmium in the soil environment. Cd cannot be microbially degrade in soil, with the property of persistence and bioaccumulation (Azevedo et al., 2012; Dhir et al., 2009; Xin et al., 2013). Cd is not an essential element for biological growth. It is highly toxic and poses a threat to living organisms, from microorganisms to animals, affecting its normal growth and causing death (Dalcorso et al., 2010; Alessandro et al., 2012). For the heavy metal pollution in soil, phytoremediation is safe and reliable, with good economic and ecological benefits (Xue et al., 2014; He et al., 2014; Bjelková et al., 2011). In addition, phytoremediation can prevent soil erosion and water loss, improve the condition of landscape. On this foundation, phytoremediation is considered a sustainable way for the wide application in environment remediation of heavy metal contaminants (Cluis, 2004; Ghosh and Singh, 2005). Many herbaceous species including Typha latifoia, Thlaspi caerulescens, Arabidopsis halleri, Miscanthus floridulus (Labill) Warb, Eremochloa ciliaris L., Sedum alfredii ⁎

Corresponding authors. E-mail addresses: [email protected] (J. Zhu), [email protected] (P. Wang).

https://doi.org/10.1016/j.ecoenv.2018.05.026 Received 2 April 2018; Received in revised form 9 May 2018; Accepted 11 May 2018 0147-6513/ © 2018 Published by Elsevier Inc.

H. and Solanum nigrum L. have been confirmed to be able to accumulate and transfer heavy metals such as Pb, Cd and Mn in mine areas. Herbaceous plants are widely used in the soil restoration of heavy metal contaminated areas. However, each species produces a low biomass. Additionally, shallow root systems for these plants limit the removal of heavy metal from deep soil (Keller et al., 2003). There are also some non-hyper-accumulator plants, such as willows, eucalypts, poplars, Brassica napus and sunflower (Iori et al., 2017; Guo et al., 2015; Ehsan et al., 2014), can be used for phytoremediation because they have large biomass, rapid growth and deep root system (He et al., 2013). However, these plants are generally less resistant to Cd, which limits their repair efficiency. Woody plants with great biomass and high tolerance to heavy metals may be an alternative. (Pulford and Watson, 2003; Mukhopadhyay and Maiti, 2010). Koelreuteria paniculata is a deciduous species of the sapindaceae family native to China, Japan, and Korea. It can resist salinity, drought and short-term flooding. This species have a deep root system, and is strongly adaptable to the environment (Zhang et al., 2017). These characteristics make it a good tree species for phytoremediation in

Ecotoxicology and Environmental Safety 160 (2018) 10–18

L.P. Yang et al.

2.3. Subcellular fractions of Cd in leaves, stems and roots

heavy metal contaminated areas. In 2004, a three-square-kilometer base with K. paniculata ecological restoration was established in Xiangtan Manganese Mine, Hunan, China. It is a successful case of using K. paniculata to repair waste mining area (Tian et al., 2009). It tentatively confirmed that K. paniculata is highly resistant to various heavy metals and has certain potential of phytoremediation for heavy metal contaminated soils. So far, the reports of K. paniculata mainly focus on biological characteristics, pharmacology and chemical composition (Huang et al., 2015; Luo et al., 2015; Pipinis et al., 2015; Mostafa et al., 2014). The mechanism of plant tolerance to Cd has been reported quite a lot, but relatively few reports on woody plants and even more rarely reported on K. paniculata. Koelreuteria paniculata is known to grow on metal contaminated sites and thus might be involved in phytoremediation process. But its response to metal exposure (notably Cd exposure) has never been described so far. So, the resistance, translocation, accumulation and stress responses of K. paniculata to different concentrations Cd has yet to be studied thoroughly. Therefore, the present research is intended to: (1) investigate the effects of Cd on the growth of K. paniculata; (2) explore the subcellular distribution and chemical forms of Cd in K. paniculata; (3) examine the effects of Cd on physiological and biochemical indexes of K. paniculata, including chlorophyll, malondialdehyde, soluble protein, hormone contents, and antioxidant enzymes in order to uncover the potential mechanisms with regard to the uptake, accumulation, translocation and tolerance of Cd in K. paniculata. The results of this study are expected to provide the theoretical basis for the application and improvement of phytostabilization of K. paniculata in heavy metal contaminated soil.

The plant cells in fresh tissues of leaves, stems and roots were separated into three parts (cell wall fraction, organelle fraction and soluble fraction) on the basis of the method depicted by Y. Wang et al. (2012) and C. Wang et al. (2012). Plant tissues were homogenized in cooled extraction buffer [250 mM sucrose, 1.0 mM DTT (C4H10O2S2) and 50 mM Tris-HCl, pH 7.5] with a cooled mortar. The homogenate was sifted through nylon cloth (80 µM). As residue included mainly cell walls and cell wall debris, it was named the ‘cell wall fraction’. The filter liquor was centrifuged at 12,000 ×g for 40 min. The subsidence was designated as the ‘organelle fraction’ and the supernatant was the ‘soluble fraction’. All procedures were carried out at 4 ℃. 2.4. Chemical forms of Cd in leaves, stems and roots Cd chemical forms were extracted from the following steps: (1) 80% ethanol, which extracts inorganic Cd giving priority to nitrate/nitrite, chloride, and aminophenol Cd; (2) deionized water, which extracts Cdorganic acid complexes; (3) 1 M NaCl, which extracts pectate and protein-integrated Cd; (4) 2% acetic acid (HAc), which extracts undissolved Cd phosphate; (5) 0.6 M HCl, which extracts Cd oxalate; and (6) the residual Cd form (Fu et al., 2011). The frozen plant tissues were homogenized in an extraction solution with a mortar, then diluted in a rate of 1:100 (W/V), shaken for 22 h at 25 ℃. The homogenate was then centrifuged at 5000 ×g for 10 min to obtain the first supernatant in a centrifuge tube. The precipitate was suspended twice in the extractant, shaken for 2 h and centrifuged at 5000 ×g for 10 min at 25 ℃. The supernatants of the three suspensions were then combined. Using the above procedure, the residue was extracted sequentially with the next extractant. Each of the solution was evaporated to constant weight on an electroplated plate at 70 ℃.

2. Materials and methods 2.1. Plant cultivation K. paniculata seedlings were obtained from Central South University of Forestry and Technology, Changsha, PR China. All seedlings were 1year-old and of similar size: 50 cm high, with a diameter of 0.5 cm. They were grown in 20% Hoagland-Arnon nutrient solution and cut off all branches. The seedlings of K. paniculata were precultured for 4 weeks for the initiation of the new roots before they were exposed to Cd stress. Then they were used for pot experiment. Pot experiment was conducted by means of hydroponics in intelligent incubator at 25/20 ℃, a 16/8 h photoperiod, 65 ± 5% relative humidity, and irradiation of 350 µmol m−2 s−1. The selected plants were cultivated in Cd treatment solutions (0, 50, 150, 250, 500 µM), supplied as CdCl2·2H2O, with four replicates for each treatment. Plants were cultivated with deionized water daily to maintain volume of culture medium, and supplied 20% Hoagland-Arnon nutrient solution every week. After a growth period of 90 days all plants were thoroughly washed with deionized water to eliminate any particles attached to roots surface and used for next experimental treatment. Initial individual explant dry weight (grams), branch numbers, maximum root lengths (cm) and plant height (cm) were recorded. Each plant is labeled and the percent growth rate of entire plant (%GR) was calculated as change in fresh weight at 90 d. Tolerance Index (TI) were calculated as 100[root lengthCd-exposed/root lengthcontrol] (Clabeaux et al., 2011).

2.5. Analysis of chlorophyll, malondialdehyde, soluble protein, H2O2, glutathione, ascorbic acid and antioxidant enzyme The method of Porra (2002), was used to determine total chlorophyll, chlorophyll a and b. The degree of lipid peroxidation was estimated as malondialdehyde (MDA) content in leaves, stems and roots following the method of Esposito and Domingos (2014). Soluble protein content in leaves, stems and roots was following the method of Vassilev and Lidon (2011). Hydrogen peroxide (H2O2) in leaves, stems and roots was measured spectrophotometrically according to Alexieva et al. (2001). Determination of glutathione (GSH) content in leaves, stems and roots follows the method of Aravind and Prasad (2005). Ascorbic acid (AsA) content in leaves, stems and roots was measured according to the method of Hodges and Forney (2000). Superoxide dismutase activity (SOD) in leaves was estimated as 50% reduction of nitroblue tetrazolium (NBT) was determined by the method of Fatima and Ahmad (2005). Peroxidase activity (POD) in leaves was measured by the method of Wu et al. (2016). Catalase activity (CAT) in leaves was measured by the method of Apodaca et al. (2017). 2.6. Analysis of hormone Approximately 2 g of the freeze-dried leaf sample was weighed and then milled in an ice bath with the pre-cooled methanol solution (80%). The homogenate was immersed in a freezer (4 ℃) for 15 h, then centrifuged at 5000 ×g for 10 min. The supernatant was concentrated to half with a rotary evaporator at 40 ℃ and transferred to a separatory funnel. 10 mL petroleum ether was added to the funnel, separated the layers after the shock, then discard the upper liquid (repeat 3 times). The remaining liquid was concentrated to 5 mL with a rotary evaporator, and the pH of liquid was adjusted to 2.8 with 0.1 mM HCl. The remaining liquid was extracted twice with an equal volume of ethyl acetate in a separatory funnel, and the ethyl acetate phase was merged.

2.2. Plant Cd uptake The plant roots were washed in 20 mM Na2-EDTA for 20 min to eliminate Cd adsorbing to the roots surfaces. The whole plants were rinsed with deionized water and separated into roots, stems and leaves. All plant samples were frozen immediately in liquid nitrogen and divided into three sections for analysis of total Cd concentrations, subcellular distribution and chemical forms. The bioconcentration factor (BCF) was equal to the ratio of Cd concentrations in plant to soil Cd concentrations. The translocation factor (TF) was calculated as the Cd concentrations in the shoot divided by that in the root. 11

Ecotoxicology and Environmental Safety 160 (2018) 10–18

L.P. Yang et al.

fractions of leaves, stems and roots, Cd concentrations increased with the increase of Cd concentrations in nutrient solution (p < 0.05). Total Cd concentrations increased with the increase of Cd concentrations both in roots, stems and leaves (p < 0.05). The accumulation abilities of Cd for different plant tissues follow the sequence of roots > leaves > stems. The values of bioconcentration factor and translocation factor weaken with the increase of Cd concentrations, and no more than 1.

The ethyl acetate phase was evaporated to dryness and dissolved in 5 mL of chromatographic methanol. The hormones of plant were determined by High Performance Liquid Chromatography (HPLC, Agilent 1260, USA). The chromatographic conditions are as follows: Chromatographic column: Eclipse XDB-C18 (250 × 4.6 mm, 5 µM, Agilent); Mobile phase: 1% acetic acid-methanol (60:40); Current Speed: 1 mL min−1; Sample size: 5 μL; Detection wavelength: 254 nm. Standards include ZT (zeatin), IAA (auxin), ABA (abscisic acid), GA (Gibberellin) (both purchased at sigma, dissolved in methanol).

3.3. Chemical forms 2.7. Analysis of Cd

All results were tested by one-way ANOVA using the SPSS 20 statistical package. All figures were drawn using the Origin 8.5 statistical package. The Duncan test at 5% probabilities was performed for later comparison to test for treatment differences

When the Cd concentrations in the solution increased, the content of six chemical forms of Cd in plant leaves, stems and roots increased. In the leaves and stems, undissolved Cd phosphate (extracted with HAc) (42–68%) took a predominant role. In the roots, undissolved Cd phosphate (extracted with HAc) (36–43%), protein and pectates integrated Cd (extracted with NaCl) (37–41%) were the major forms. With the increase of Cd concentrations, the percentage of Cd-organic acid complexes (extracted with d-H2O) and inorganic Cd (extracted with ethanol) tended to rise in roots, stems and leaves. The percentage of undissolved Cd phosphate (extracted with HAc) and cadmium oxalate (extracted with HCl) have a tendency to reduce in leaves, stems and roots. The overall change in percentage of protein and pectates integrated Cd (extracted with NaCl) is small in each Cd concentrations (Fig. 1(b)).

3. Results

3.4. Physiological and biochemical indicators

3.1. Plant growth parameters

When the Cd concentrations was less than 150 µM, the total chlorophyll contents did not change significantly. Total chlorophyll contents decreased in plants in 250 µM added Cd (p < 0.05) (Fig. 2(a)). When the Cd concentrations was higher than 250 µM, the chlorophyll a/b value decreased rapidly, indicating that the leaf experienced accelerated aging in this concentrations range. Malondialdehyde contents increased in plants in 150 µM and tended to reached a balance at 250 µM (p < 0.05) (Fig. 2(b)). Soluble protein content reached the maximum when Cd concentrations was 50 µM, and then began to decrease (p < 0.05) (Fig. 2(c)). The content of soluble protein decreased by 23.3% compared to the control group at the Cd concentrations of 500 µM. SOD and CAT activity increased in plants in 50 µM added Cd, then began to decrease in 150 µM (p < 0.05) (Fig. 2(d)). POD activity increased in plants in 150 µM added Cd, then began to decrease in 250 µM (p < 0.05) (Fig. 2(d)). In roots and leaves, the GSH content increased significantly when the Cd concentrations reached 50–250 µM and then decreased (p < 0.05) (Fig. 2(e)). The AsA content have the same trend (p < 0.05) (Fig. 2(f)). With the increase of cadmium concentration in the culture solution, the content of H2O2 in leaves, stems and roots increased significantly (Fig. 2(g)). The effect of Cd stress on physiological and biochemical indexes of stems was less than that of roots and leaves. With the increase of Cd concentrations, the content of IAA in plants increased first and then decreased (p < 0.05) (Table 3). When the Cd concentrations reached 150 µM, the IAA content reached the highest level, and then began to decrease (p < 0.05) (Table 3). The

Plant samples were oven-dried at 70 ℃ for at least 48 h. All samples were digested with a mixture acid of HNO3: HClO4 (4:1) in a microwave digestion device, and the Cd concentrations were measured using Flame Atomic Absorption Spectrophotometer (FAAS, AA-7002, Thermo Fisher Scientific, USA). The Cd concentrations in each sample were measured in triplicate. Standard deviation for each sample was less than 5%. 2.8. Statistics analysis

In order to determine the tolerance of K. paniculata to Cd, we measured the effect of Cd on growth parameters of K. paniculata. Plants can tolerate up to 150 µM added Cd with no obvious change in any growth parameter (P > 0.05) (Table 1). Root lengths decreased at 250 µM added Cd, relative to the plants unexposed to Cd (P < 0.05) (Table 1). The number of new branches generated at top and base nodes was higher in plants with 150 µM added Cd than those with 250 µM added Cd (p < 0.05) (Table 1). Plant growth rates decreased in nutrient solution with 250 µM added Cd. Plant dry weight shows the same trend as the growth rate. The height of the plant did not change significantly until the Cd concentration reached 150 µM, and it decreased when the Cd concentration exceeded 250 µM (P < 0.05) (Table 1). Tolerance index were not different from 100% in cultures up to 150 µM added Cd (P > 0.05), indicating tolerance. 3.2. Cd accumulation and subcellular distribution Total Cd concentrations and its subcellular distribution of leaves, stems and roots tissues varied with different Cd concentrations in the culture solution. As show in Table 2, the majority of Cd was distributed in the cell wall fraction (45–77%) then distributed in the soluble fraction (20–45%) and organelle fraction (2–11%), both for leaves, stems and roots. For cell wall fractions, soluble fractions and organelle

Table 1 Growth responses of K. paniculata seedlings to Cd. Data are means of four replicates ± SE (n = 4). Different letters in the same column represent statistically significant differences between treatments within the different Cd concentrations (p < 0.05). Cd treatment (µM)

Growth parameter Root length (cm)

0 50 150 250 500

6.47 6.84 5.67 3.71 2.69

± ± ± ± ±

a

1.04 1.13a 0.98a 0.57b 0.35b

Branches number 5.43 5.67 4.72 3.51 2.31

± ± ± ± ±

a

0.86 0.44a 0.62a 0.45b 0.51c

Growth rate (%) 30.98 32.07 31.55 25.21 15.25

± ± ± ± ±

12

a

2.75 3.05a 2.64a 1.67b 1.25c

Dry weight (g) a

11.04 ± 0.28 11.43 ± 0.31a 10.84 ± 0.23a 9.58 ± 0.22b 8.69 ± 0.16c

Plant height (cm) 73.23 75.54 71.21 59.87 55.95

± ± ± ± ±

a

3.27 3.51a 3.75a 2.12b 1.78c

Tolerance index (%) 100 106.03 ± 8.87a 87.63 ± 9.78a 67.54 ± 7.45b 41.58 ± 3.61c

Ecotoxicology and Environmental Safety 160 (2018) 10–18

L.P. Yang et al.

Table 2 Cd concentrations and its subcellular distribution of Cd in leaves, stems and roots of K. paniculata seedlings. Data are means of four replicates ± SE (n = 4). Different letters in the same row represent statistically significant differences between treatments within the different Cd concentrations (p < 0.05). F1, F2 and F3 refer to the cell wall, soluble and organelle fractions. Cd Concentrations (mg kg−1 DW)

Stage

Leaves 50 µM Cd

150 µM Cd

250 µM Cd

500 µM Cd

Stems c

F1 F2 F3 Total F1 F2 F3 Total F1 F2 F3 Total F1 F2 F3 Total

Relative Cd Allocation (%) Roots d

0.583 ± 0.06 0.165 ± 0.03d 0.058 ± 0.02c 0.94 ± 0.12c 0.853 ± 0.12c 0.333 ± 0.05c 0.105 ± 0.03b 1.31 ± 0.21b 1.191 ± 0.14b 0.517 ± 0.06b 0.148 ± 0.04b 1.95 ± 0.25a 1.469 ± 0.16a 0.780 ± 0.11a 0.233 ± 0.06a 2.31 ± 0.34a

0.962 ± 0.12 0.480 ± 0.08d 0.077 ± 0.03d 1.68 ± 0.2d 1.197 ± 0.17b 0.958 ± 0.12c 0.127 ± 0.04c 2.25 ± 0.34c 1.503 ± 0.22a 1.490 ± 0.22b 0.292 ± 0.08b 3.43 ± 0.52b 1.755 ± 0.34a 1.780 ± 0.27a 0.402 ± 0.14a 4.04 ± 0.74a

Leaves c

1.890 ± 0.14 0.540 ± 0.6d 0.052 ± 0.02c 2.43 ± 0.23d 3.090 ± 0.27b 0.819 ± 0.14c 0.119 ± 0.04c 3.95 ± 0.45c 4.176 ± 0.45a 1.461 ± 0.21b 0.622 ± 0.05b 6.33 ± 0.72b 4.908 ± 0.41a 1.933 ± 0.22a 0.869 ± 0.12a 8.48 ± 0.77a

Hormone content (µg kg−1 FW) IAA

0 50 150 250 500

1.73 1.90 3.23 1.81 1.39

GA ± ± ± ± ±

b

0.39 0.42b 0.67a 0.32b 0.46c

32.70 31.48 28.89 25.83 23.74

ZT ± ± ± ± ±

a

3.12 2.65a 2.11b 2.25c 1.86d

1.26 1.01 0.99 0.80 0.48

TF

Roots

a

63 ± 8 32 ± 4b 5 ± 2b

a

72 ± 7 21 ± 3b 7 ± 1a

76 ± 6a 22 ± 3a 2 ± 1b

0.318 ± 0.05a

0.705 ± 0.08a

52 ± 7b 42 ± 5a 6 ± 2b

66 ± 9a 26 ± 4a 8 ± 2a

77 ± 6a 20 ± 3a 3 ± 1b

0.135 ± 0.03b

0.659 ± 0.06b

46 ± 8b 45 ± 6a 9 ± 2a

64 ± 8a 28 ± 3a 8 ± 2a

67 ± 7a 23 ± 3a 10 ± 1a

0.089 ± 0.02c

0.501 ± 0.06c

45 ± 9b 45 ± 7a 10 ± 4a

59 ± 6b 31 ± 4a 9 ± 2a

64 ± 5b 25 ± 3a 11 ± 2a

0.052 ± 0.01d

0.464 ± 0.05d

caerulescens can tolerate 300 µM of Cd (Wójcik et al., 2005a, 2005b), and Arabidopsis halleri can tolerate at least 100 µM of Cd (Zhao et al., 2006). In the soil culture test, the tolerance range of Cd is much higher than that. K. paniculata's ability to tolerate up to 150–250 µM Cd supports its potential utility for phytostabilization of contaminated soil containing up to this concentrations of Cd.

Table 3 Effect of cadmium stress on hormone in leaves. Data are means of four replicates ± SE (n = 4). Different letters in the same column represent statistically significant differences between treatments within the different Cd concentrations (p < 0.05). Cd treatment (µM)

Stems

BCF

ABA ± ± ± ± ±

a

0.24 0.16b 0.19b 0.14c 0.13d

0.21 0.27 0.28 0.31 0.35

± ± ± ± ±

4.2. Accumulation and distribution of Cd in K. paniculata

0.06c 0.05b 0.04b 0.06a 0.08a

Cd is mainly concentrated in the roots of K. paniculata, followed by leaves and stems. The concentration of Cd in plant tissues remains low, even in plants exposed to very high concentrations of Cd. Indeed, the concentration of Cd never exceeds 5 ppm in leaves and 10 ppm in roots even in plants exposed to 500 µM Cd. This makes Koelreuteria paniculata an efficient Cd excluder. In contaminated plants, most of the cadmium are present in the cell wall and soluble fractions, with few in the organelle fraction. This result was the same to those studies on rice (Li et al., 2016), edible seaweed (zhao et al., 2015), pokeweed (Fu et al., 2011) and raime (Wang et al., 2008). Under each Cd concentrations (50–500 µM), cell wall fractions accounted for major proportion of Cd in roots and stems (44.6–76.7%), followed by soluble fractions (20.3–45.4%). So, the protective role of cell wall is important under Cd stress. Cell wall separation of cadmium is one of the mechanisms to inhibit Cd transport in plants (Qiu et al., 2011). Plant cell walls consisted of proteins and polysaccharides, including cellulose hemicellulose and pectin. It has provided many potential ligands such as hydroxyl, carboxyl, aldehyde and amino. These ligands could participate in various reactions, including adsorption, complexation, ion exchange, crystallization and precipitation, which could bind Cd actions and limit their entry into the cell membrane (J. Wang et al., 2009; C. Wang et al., 2009). So the cell wall was served as the prime protective screen to protect the plant cells from the heavy metal poisoning effects (Weng et al., 2012). With the increase of Cd concentrations, the proportion of Cd of soluble fractions increased gradually and the proportion of cell wall fractions decreased (p < 0.05) (Table 2). The severe toxicity caused by the high Cd concentrations stress possibly caused some serious damage to the cell wall, resulting in increased Cd transferred from cell wall fractions to the soluble fractions. The soluble fraction was mainly composed of vacuoles, acting as the secondary site of preferential Cd binding in leaves, stems and roots (Guan et al., 2018). This way could reduce the amount of Cd entering the organelles (Zhao et al., 2015). It can be presumed from the above results that the protection of cell wall fractions played the most important role under low

contents of ZT and GA obviously decreased with the increase of Cd concentrations (p < 0.05) (Table 3). The content of ABA increased with the increase of Cd concentrations (p < 0.05) (Table 3). 4. Discussion 4.1. Cd effect on growth Cadmium is highly toxic to plants (Li et al., 2010). Some studies have reported that, Cd stress inhibits plant growth and biomass (Ali et al., 2014; Mostofa et al., 2015), and also affects plant uptake and translocation of essential nutrients by competition (Liu et al., 2003; Sun et al., 2012; Pereira et al., 2017). In our study, plants tolerated up to 150 µM added Cd with no obvious change in any growth parameter (p > 0.05) (Table 1). When the Cd concentrations was higher than 150 µM, this parameter significantly decreased. This showed that the critical concentrations of tolerance of plants to Cd was 150 µM. At any concentrations below this standard, plants could effectively respond to the toxicity of Cd. When the Cd concentrations exceeded the critical concentrations, plants were affected by Cd toxicity, inhibiting the growth of root and shoot, and growth rate decreased significantly (Gallego et al., 2012). The tolerance index can reflect the degree of plant's resistance to heavy metals (Lux et al., 2004). On the basis of the tolerance index, plants can be divided into high tolerance (TI > 60%), moderate tolerance (35% ≤ TI ≤ 60%) and sensitive tolerance (TI < 35%). According to this criterion, when the Cd concentrations do not exceed 250 µM, K. paniculata belongs to high-tolerant plants of Cd. In hydroponics experiment, Cd hyperaccumulators Sedum alfredii Hance can tolerate 200–400 µM of Cd (Yang and Stoffella, 2004), Thlaspi 13

Ecotoxicology and Environmental Safety 160 (2018) 10–18

L.P. Yang et al.

(a)

Cd concentrations stress, while the soluble fractions relieve the toxicity of Cd under high Cd stress. Organelle fractions accounted for the least proportion. However, when the Cd concentrations exceeded 250 µM, the proportion increased significantly (p < 0.05) (Table 2). This indicated that under high Cd concentrations stress, Cd ions entered the organelles, causing severe damage to plant cells (Belleghem et al., 2007). The total amount of Cd in leaves, stems and roots of plants increased linearly with the increase of Cd concentrations in culture solution. None of the plants at any Cd concentrations had bioconcentration factor and translocation factor higher than 1. This indicated the weak ability of transport and accumulation of K. paniculata to Cd. The declination in BCF and TF is not in proportion to the increase of Cd concentration in the solution. This is because the plant's defensive mechanism against Cd will prevent K. paniculata from absorbing and transporting Cd. (Utmazian and Wenzel, 2007; Redovniković et al., 2017). And the increased cadmium in the culture solution that may force toxicity in plants and hampers cadmium uptake by the roots and shoots (Sidhu et al., 2017). Heavy metals were absorbed and transported by plants and present in different organ tissues in different chemical forms to limit the movement of heavy metals in plants, thereby reducing their toxicity to plants (Wang et al., 2008). With the sequence of each extractant ethanol, deionized water, sodium chloride, acetic acid and hydrochloric acid, the polarity of extractant increases. The higher the polarity of the extractant, the lower the activity of the extracted heavy metal and the closer the integration with the plant matrix (Fu et al., 2011). Cadmium in inorganic forms (extracted with ethanol) and in water-soluble forms (extracted with H2O) had the highest activity, followed by pectates and protein-bound Cd (extracted with NaCl) and undissolved Cd phosphates (extracted with HAc), and the cadmium oxalate (extracted with HCl) and residues had the lowest activity (Qiu et al., 2011). In this study, the majority of Cd was accounted for the pectates and protein-bound Cd and undissolved Cd, both in leaves, stems and roots (Fig. 1). Similar results were found by Li et al. (2016). Cd is mainly present in the form of low activity in K. paniculata (Zhao et al., 2015). When the concentration of Cd is high, the proportion of water soluble Cd-organic and inorganic Cd increased, suggesting that at higher concentrations of Cd the ability of the plant to bind to Cd is limited. Cd exists in a highly active form, witch can cause serious damage to the plant (C. Wang et al., 2012; Y. Wang et al., 2012). It was hypothesized that when the Cd concentrations in substrate was low, Cadmium chelated some specific polar substances, such as carboxyl or hydroxyl, to form a nontoxic complex, resulting in reduced toxicity (Fu et al., 2011). Considering the subcellular distribution and chemical forms of Cd, the cell wall of K. paniculata can combine Cd into pectates and protein-bound Cd and undissolved Cd phosphates with the state of low bioavailability to reduce the amount of Cd into the cytoplasm (Bais et al., 2006; Nedelkoska and Doran, 2000). Besides, vacuoles contain many proteins, sugars and organic acids, which can be combined with Cd to reduce their effectiveness (Verbruggen et al., 2009; Wójcik et al., 2005a, 2005b; Gallego et al., 2012). The Cd in the cell wall of the cadmium-tolerant plant occupies a greater proportion, while the cadmium in the vacuole of the cadmium-sensitive plant occupies a greater proportion (Uraguchi et al., 2009). This conclusion proves that K. paniculata does have the characteristics of cd-tolerant plants. Fixation of cell wall and compartmentalization of vacuole is one of the mechanisms of tolerance to Cd stress in K. paniculata.

10

-1

Cd concentration (mg kg )

9

residue NaCl

HAc ethanol

8 7 6 5 4 3 2 1 0

50 150 250 500

leaves

(b) proportion of Cd in different fractions (%)

HCl H2 O

100

50 150 250 500

50 150 250 500

stems -1 Cd added (μmol L )

residue NaCl

HCl H2 O

roots

HAc ethanol

90 80 70 60 50 40 30 20 10 0

50 150 250 500

leaves

50 150 250 500

stems -1 Cd added (μmol L )

50 150 250 500

roots

Fig. 1. Different chemical forms of Cd and its proportion in leaves, stems and roots of K. paniculata seedlings.

a/b values increased first, and then decreased with the increase of Cd concentrations (p < 0.05) (Fig. 2), indicating that high-concentration Cd stress inhibited photosynthesis and accelerated leaf aging, and ultimately showed a serious decline in biomass. The decrease of chlorophyll content may be due to the absorption of Cd by the plant, the combination of Cd and the mercapto group of the chloroplast (Jiang et al., 2007). This would inhibit the synthesis of chlorophyll and the related enzyme activity of chlorophyll biosynthesis. But it may also be affected by the role of reactive oxygen species under Cd stress, in which case the structure and function of the chloroplast was destroyed or the chlorophyll was decomposed (Bhaduri and Fulekar, 2012). MDA was the final decomposition product of membrane lipid peroxidation. Accumulation of MDA would cause severe damage to the membrane and cells. The environmental stress of plant cells could be reflected by the MDA content to a certain extent (Li et al., 2013). The results showed that MDA content increased with the increase of Cd concentrations (p < 0.05) (Fig. 2), indicating that Cd stress caused significant membrane lipid peroxidation. This resulted in the cell membrane damage, intracellular osmotic pressure loss of balance. Further, this would cause toxic effects on the chloroplast, mitochondria, and other organelles, interfere with the normal physiological processes of plants, and inhibit plant growth (Celekli et al., 2013). The content of soluble protein increased initially and then decreased with the increase of Cd concentrations (p < 0.05) (Fig. 2). Under low-concentration Cd stress, the increase of soluble protein content may be a kind of adaptation by the plant to Cd, and it could weaken the damage of Cd to the plant by synthesizing the specific protein or polypeptide bound to Cd (Liu et al.,

4.3. Physiological and biochemical indicators Soil environments are at increasing risk of becoming toxic due to industrial and agricultural pollutants, leading to potentially lethal poisoning of plants. Plants can relieve the toxicity of heavy metals through a series of physiological reactions (Clemens, 2006; Pant, 2014). The level of chlorophyll content reflected the level of plant photosynthesis. The results showed that chlorophyll content and chlorophyll 14

Ecotoxicology and Environmental Safety 160 (2018) 10–18

L.P. Yang et al.

(a) 6

50 150 250 -1 Cd added (μmol L )

a

70 60

leaves a

c

b

b

40 30 a

a

b

10 0

b

b

50 150 250 -1 Cd added (μmol L )

leaves a

stems

200 150

-1

a

250

a

b

c

c

b

b c

100 a

50 0

a

a

0

50

a

150

a

250

a

a

250

500

a

b

b

0

50

150

SOD

1200 1000

POD

a a

a

CAT

a

a b

800 600

c

b

400

a

a

b

200 0

c b

d

50 150 250 -1 Cd added (μmol L )

b 500

240

a

160 120

stems

roots

a

200

b

b

b

b

b

c

c

80 a

a

0

50

d

a

40 0

500

150

a

a

250

500

-1

-1

Cd added (μmol L )

Cd added (μmol L )

(g)

c

leaves

a

300

200

a

b c

d

roots Ascorbic acid (μmol g FW)

350

d

400

0

500

(f)

(e)

c

(d) 1400

roots

b

20

600

-1

b b

a

b

0

a

50

roots a

Cd added (μmol L )

stems

a

stems

800

-1

2

500

-1

90

3

Enzymatic activity (U g FW)

0

Malondialdehyde (nmol g FW)

c

Chlorophyll a/b

-1

Chlorophyll (mg g FW)

-1

Soluble protein (mg g FW)

b

b

3

0

-1

4 b

80

Glutathione (nmol g FW)

a

a

a

2

(c)

5

a a

leaves

Chlorophyll a/b a

5

4

(b)

6 Total chlorophyll

250 leaves

stems

roots

a

-1

H2O2 (nmol g FW)

200 b

b 150

c

100 50 0

d

b b

c

a a

d

a

a

a

b

0

50

150

250

500

-1

Cd added (μmol L ) Fig. 2. Effects of Cd stress on chlorophyll (a), malondialdehyde (b), soluble protein (c), gutathione (e), ascorbic acid (f), H2O2 (g) contents and antioxidant enzyme activities (in leaves) (d) in K. paniculata seedlings. Different letters represent statistically significant differences between treatments within the different Cd concentrations (p < 0.05).

15

Ecotoxicology and Environmental Safety 160 (2018) 10–18

L.P. Yang et al.

of H2O2 usually leads to increased SOD activity (Arasimowicz-Jelonek et al., 2012; Chmielowska-Bak et al., 2014). The accumulation of H2O2 is due to Cd-induced GSH depletion and inhibition of antioxidant enzymes (Schützendübel and Polle, 2002). And the Cd bind and compete for the binding sites that finally disturbs and changes the function of target proteins, which can also cause H2O2 accumulation in the plant (Zhang et al., 2009). Cd stress can significantly change the level of endogenous hormones in plants, as well as break the hormonal balance, and thus affect the normal growth and development of plants (C. Wang et al., 2012; Y. Wang et al., 2012). Low Cd concentrations stress increased the IAA content, while the high-concentration Cd stress reduced the IAA content (p < 0.05) (Table 3). Tamás et al. (2012) argued that IAA can effectively alleviate the accumulation of H2O2 caused by Cd. Increased IAA at low Cd concentrations Substrates can persist with oxidative damage caused by Cd. In the high-concentration Cd stress, membrane lipid peroxidation increased, IAAO (indole acetic acid oxidase) activity increased, which accelerated the IAA decomposition. Cd stress reduces the GA content in plants (p < 0.05) (Table 3). Similar results were also observed by Atici et al. (2005). Zhu et al. (2012) argued that GA can reduce the content of endogenous NO and inhibit the synthesis of Cd transporters, thereby reducing Cd uptake and reducing Cd toxicity. The decrease of ZT content may result from the situation where the activity of cytokinin oxidase and the oxidation of cytokinin increased by cadmium stress, which was an important pathway for its inactivation (Thomas et al., 2010; Kamínek et al., 1997). ABA content increased with the increase of Cd concentrations (p < 0.05) (Table 3), and similar results were also observed by Chaca et al. (2014). Sharma and Kumar (2002) argued that the plant went through the increase of ABA. As a result, plants can maintain water balance by closeing stomata. The root of the direct absorption of Cd would be reduced, so did the concentrations of Cd in the body, thereby reducing the toxicity. ABA plays a important role in plants under Cd stress. Li et al. (2014) argued that ABA relieves Cd stress through regulating antioxidant system. Stroiński et al. (2012) and Hayward et al. (2013) pointed out that ABA can regulate plant chelating peptides synthesis relieves Cd stress. The regulation of endogenous hormones is an important way for plants to positively respond to heavy metal stress, but the deep-seated mechanism needs further study.

2007). At the same time, the increase of soluble protein content could increase the number of functional proteins and cell osmotic concentrations. This behavior helps maintain the osmotic pressure imbalance caused by the destruction of membrane structure under Cd stress. Therefore, the normal metabolism and cell ultrastructure stability could be maintained, and the damage caused by reactive oxygen species could be removed (Hou et al., 2007; Jie et al., 2009). Under high-concentration Cd stress, the decrease of soluble protein content may result from the destruction of the protein synthesis system by Cd. It is possible that the participation of Mg2+ was required in the synthesis phase of the protein, and Mg2+ might exchange with Cd2+ under high Cd stress, leading to the inability of protein synthesis to start. It is also possible that high-concentration Cd2+ inhibited the synthesis of DNA and increased the activity of the associated protease to accelerate the decomposition of the protein, resulting in a decrease in its content (Vassilev and Lidon, 2011). SOD, POD and CAT activities increased initially and then decreased rapidly with the increase of Cd concentrations (p < 0.05) (Fig. 2). That is because the antioxidant enzyme system could basically remove the excessive reactive oxygen generated by Cd stress(≤150 µM), so as to maintain the free metabolism of free radicals in plant (Limónpacheco and Gonsebatt, 2009). When the Cd concentrations was too high (> 150 µM), the activity of the three antioxidant enzymes began to decline sharply, indicating that the reactive oxygen species in plants have exceeded the antioxidant enzyme scavenging ability. So, the toxic effects on cells began to appear, and the synthesis of antioxidant enzymes was inhibited. A similar result was obtained by Wu et al. (2003). The final decrease in enzyme activities probably due to the inhibition of Cd-mediated enzyme synthesis or changes in the accumulation of enzyme subunits caused by ROS entry (Sidhu et al., 2017). GSH is the most abundant low-molecular-mass thiol compound in cells. It can directly eliminate active oxygen in plants and maintain intracellular redox balance. At the same time, it can complex Cd to reduce the bioavailability of Cd, and it is the precursor of synthetic plant phytochelatins (PCs) (Nagalakshmi and Prasad, 2001). When the cadmium concentration reaches 50–250 µM, the GSH content increases significantly, which increases the plant's ability to clear active oxygen free radicals and increase the detoxification ability of Cd. The increase of GSH content can also effectively complex Cd and promote the synthesis of PCs, which can reduce the bioavailability of Cd in plant. Studies have shown that glutathione play an important role in improving the tolerance of Cd. GSH contributes to the alleviation of Cd-induced oxidative stress (Lin and Aarts, 2012; Ye et al., 2016; Jia et al., 2016). The depletion of GSH is apparently a crucial step in cadmium sensitivity since plants with improved capacities for GSH synthesis displayed higher Cd tolerance (Schützendübel et al., 2001). Under prolonged exposure to cadmium stress, GSH content increased. The reason is that under such environment the plant's need for sulfur increases, which leads to increased expression of a high affinity sulfate transporter, thereby increasing sulfate absorption (Nocito et al., 2002). However, this ability in K. paniculata is limited when the Cd concentration reaches 500 µM. AsA is one of the most abundant antioxidants in plants. It can directly remove reactive oxygen species and play an important role in plant antioxidant stress. When the cadmium concentration reaches 50 µM, the AsA content increases significantly, which increases the antioxidant capacity of the plant. The increase in AsA and GSH promotes the AsAGSH cycle, which is one of the ways in which plant cells effectively clear H2O2 (Drążkiewicz et al., 2003; Gill and Tuteja, 2011). However, this function is also destroyed when the Cd concentration increases. Excessive heavy metals can cause H2O2 accumulation in plants by oxidative stress (Remans et al., 2012; C. Wang et al., 2009; J. Wang et al., 2009; Jin et al., 2013). We found that Cd stress promotes the accumulation of H2O2 in plant leaves, stems and roots, which can cause damage to plant cells. At the same time H2O2 is an important signaling molecule involved in sensing and responding to heavy metal stress (Wrzaczek et al., 2013). In heavy metal-treated plants, overproduction

5. Conclusions The resistance, translocation, accumulation and stress responses of Koelreuteria paniculata are reported in this paper for the first time. Koelreuteria paniculata is an efficient Cd excluder that can tolerate high concentrations of Cd (up to 150–250 µM of Cd). Under low Cd concentrations stress (≤150 µM), fixation of cell wall and compartmentalization of vacuole appear to play an important role in reducing the toxicity of Cd. Antioxidant enzyme system, hormone and AsA-GSH cycle also play an irreplaceable role in the detoxification of Cd. Under high Cd concentration stress (> 15 µM), the biomass of the plant significantly decreased. Cd crossed the cell wall and entered the soluble fractions and organelle fractions, and transformed into the highly-migratory forms. The physiological and biochemical processes were interfered. In conclusion, our results indicated that Koelreuteria paniculata has good adaptability to Cd stress, showing its promising potential to be applied to the ecological restoration of general Cd-contaminated mining area. These findings provide a new perspective on the role of plant in restoration of cadmium contaminated sites, and provide a theoretical basis for its application and improvement. Acknowledgements This work was supported by the Young Scientists Fund of the National Natural Science Foundation of China (21707169), the National Key Technology Research and Development Program of the 16

Ecotoxicology and Environmental Safety 160 (2018) 10–18

L.P. Yang et al.

Ministry of Science and Technology of China (2016YFD0800805-4), the Hunan Provincial Innovation Foundation for Postgraduate (CX2017B404), Innovation Foundation for Postgraduate of Central South University of Forestry and Technology (CX2017B13)

Guo, B., Dai, S., Wang, R., Guo, J., Ding, Y., Xu, Y., 2015. Combined effects of elevated CO2 and Cd-contaminated soil on the growth, gas exchange, antioxidant defense, and Cd accumulation of poplars and willows. Environ. Exp. Bot. 115, 1–10. Hayward, A.R., Coates, K.E., Galer, A.L., Hutchinson, T.C., Emery, R.J.N., 2013. Chelator profiling in Deschampsia cespitosa (L.) Beauv. reveals a Ni reaction, which is distinct from the ABA and cytokinin associated response to Cd. Plant Physiol. Biochem. 64, 84–91. He, J., Ma, C., Ma, Y., Li, H., Kang, J., Liu, T., et al., 2013. Cadmium tolerance in six poplar species. Environ. Sci. Pollut. Res. 20, 163–174. He, J., Ren, Y., Chen, X., Chen, H., 2014. Protective roles of nitric oxide on seed germination and seedling growth of rice (Oryza sativa L.) under cadmium stress. Ecotox. Environ. Safe. 108, 114–119. Hodges, D.M., Forney, C.F., 2000. The effects of ethylene, depressed oxygen and elevated carbon dioxide on antioxidant profiles of senescing spinach leaves. J. Exp. Bot. 51, 645–655. Hou, W., Chen, X., Song, G., Wang, Q., Chi, C.C., 2007. Effects of copper and cadmium on heavy metal polluted waterbody restoration by duckweed (Lemna minor). Plant Physiol. Biochem. 45, 62–69. Huang, Z., Xiang, W., Ma, Y., Lei, P., Tian, D., Deng, X., Yan, W., Fang, X., 2015. Growth and heavy metal accumulation of Koelreuteria paniculata seedlings and their potential for restoring manganese mine wastelands in Hunan, China. Int. J. Environ. Res. Public Health 12, 1726–1744. Iori, V., Pietrini, F., Bianconi, D., Mughini, G., Massacci, A., Zacchini, M., 2017. Analysis of biometric, physiological, and biochemical traits to evaluate the cadmium phytoremediation ability of eucalypt plants under hydroponics. iForest 10, 416. Jia, H., Wang, X., Dou, Y., Liu, D., Si, W., Fang, H., Zhao, C., Chen, S., Xi, J., Li, J., 2016. Hydrogen sulfide-cysteine cycle system enhances cadmium tolerance through alleviating cadmium-induced oxidative stress and ion toxicity in Arabidopsis roots. Sci. Rep. 6, 39702. Jiang, H.M., Yang, J.C., Zhang, J.F., 2007. Effects of external phosphorus on the cell ultrastructure and the chlorophyll content of maize under cadmium and zinc stress. Environ. Pollut. 147, 750–756. Jie, X., An, L.Y., Han, L., Cheng, Z., 2009. Exogenous nitric oxide enhances cadmium tolerance of rice by increasing pectin and hemicellulose contents in root cell wall. Planta 230, 755–765. Jin, C.W., Mao, Q.Q., Luo, B.F., Lin, X.Y., Du, S.T., 2013. Mutation of mpk6 enhances cadmium tolerance in Arabidopsis plants by alleviating oxidative stress. Plant Soil. 371, 387–396. Kamínek, M., Motyka, V., Vanková, Radomíra, 1997. Regulation of cytokinin content in plant cells. Physiol. Plant. 101, 689–700. Keller, C., Hammer, D., Kayser, A., Richner, W., Brodbeck, M., Sennhauser, M., 2003. Root development and heavy metal phytoextraction efficiency: comparison of different plant species in the field. Plant Soil. 249, 67–81. Li, H., Luo, N., Zhang, L., Zhao, H., Li, Y., Cai, Q., Wong, M., Mo, C., 2016. Do arbuscular mycorrhizal fungi affect cadmium uptake kinetics, subcellular distribution and chemical forms in rice? Sci. Total Environ. 571, 1183–1190. Limónpacheco, J., Gonsebatt, M., 2009. The role of antioxidants and antioxidant-related enzymes in protective responses to environmentally induced oxidative stress. Mutat. Res. 674, 137–147. Li, J.Y., Fu, Y.L., Pike, S.M., Bao, J., Tian, W., Zhang, Y., 2010. The Arabidopsis nitrate transporter nrt 1.8 functions in nitrate removal from the xylem sap and mediates cadmium tolerance. Plant Cell. 22, 1633–1646. Lin, Y.F., Aarts, M.G., 2012. The molecular mechanism of zinc and cadmium stress response in plants. Cell Mol. Life Sci. 69, 3187–3206. Liu, X., Zhang, S., Shan, X.Q., Christie, P., 2007. Combined toxicity of cadmium and arsenate to wheat seedlings and plant uptake and antioxidative enzyme responses to cadmium and arsenate co-contamination. Ecotoxicol. Environ. Safe. 68, 305–313. Liu, J.G., Liang, J.S., Li, K.Q., Zhang, Z.J., Yu, B.Y., Lu, X.L., 2003. Correlations between cadmium and mineral nutrients in absorption and accumulation in various genotypes of rice under cadmium stress. Chemosphere 52, 1467–1473. Li, Y., Zhang, S., Jiang, W., Liu, D., 2013. Cadmium accumulation, activities of antioxidant enzymes, and malondialdehyde (MDA) content in Pistia stratiotes, L. Environ. Sci. Pollut. Res. Int. 20, 1117–1123. Li, S.W., Leng, Y., Feng, L., Zeng, X.Y., 2014. Involvement of abscisic acid in regulating antioxidative defense systems and iaa-oxidase activity and improving adventitious rooting in mung bean [Vigna radiata (l.) Wilczek] seedlings under cadmium stress. Environ. Sci. Pollut. R. 21, 523–537. Luo, Z., Tian, D., Ning, C., Yan, W., Xiang, W., Peng, C., 2015. Roles of Koelreuteria bipinnata, as a suitable accumulator tree species in remediating mn, zn, pb, and cd pollution on mn mining wastelands in southern china. Environ. Earth Sci. 74, 4549–4559. Lux, A., SottnãKovã, A., Opatrnã, J., Greger, M., 2004. Differences in structure of adventitious roots in salix clones with contrasting characteristics of cadmium accumulation and sensitivity. Physiol. Plant. 120, 537–545. Mostafa, A., El-Hela, A., Mohammad, A., Cutler, S., Ross, S., 2014. Biologically active secondary metabolites isolated from Koelreuteria paniculata cultivated in Egypt. Planta Med. 80 (798–798). Mostofa, M.G., Rahman, A., Ansary, M.M.U., Watanabe, A., Fujita, M., Tran, L.S.P., 2015. Hydrogen sulfide modulates cadmium-induced physiological and biochemical responses to alleviate cadmium toxicity in rice. Sci. Rep. 5 (14078), 14078. Mukhopadhyay, S., Maiti, S.K., 2010. Phytoremediation of metal enriched mine waste: a review. Glob. J. Environ. Res. 4, 135–150. Nagalakshmi, N., Prasad, M.N., 2001. Responses of glutathione cycle enzymes and glutathione metabolism to copper stress in Scenedesmus bijugatus. Plant Sci. 160, 291–299. Nedelkoska, T.V., Doran, P.M., 2000. Hyperaccumulation of cadmium by hairy roots of

References Ali, B., Qian, P., Jin, R., Ali, S., Khan, M., Aziz, R., 2014. Physiological and ultra-structural changes in Brassica napus, seedlings induced by cadmium stress. Biol. Plant. 58, 131–138. Alexieva, V., Sergiev, I., Mapelli, S., Karanov, E., 2001. The effect of drought and ultraviolet radiation on growth and stress markers in pea and wheat. Plant Cell Environ. 24, 1337–1344. Apodaca, S., Tan, W., Dominguez, O., Hernandezviezcas, J., Peraltavidea, J., Gardeatorresdey, J., 2017. Physiological and biochemical effects of nanoparticulate copper, bulk copper, copper chloride, and kinetin in kidney bean (Phaseolus vulgaris) plants. Sci. Total Environ. s 599–600, 2085–2094. Atici, O., Ağar, G., Battal, P., 2005. Changes in phytohormone contents in chickpea seeds germinating under lead or zinc stress. Biol. Plant. 49, 215–222. Arasimowicz-Jelonek, M., Floryszak-Wieczorek, J., Deckert, J., Rucińska-Sobkowiak, R., Gzyl, J., Pawlak-Sprada, S., Abramowski, D., Jelonek, T., Gwozdz, E.A., 2012. Nitric oxide implication in cadmium-induced programmed cell death in roots and signaling response of yellow lupine plants. Plant Physiol. Biochem. 58, 124–134. Aravind, P., Prasad, M.N., 2005. Modulation of cadmium-induced oxidative stress in Ceratophyllum demersum by zinc involves ascorbate-glutathione cycle and glutathione metabolism. Plant Physiol. Biochem. 43, 107–116. Azevedo, R., Gratão, P., Monteiro, C., Carvalho, R.F., 2012. What is new in the research on cadmium-induced stress in plants? Food Energy Secur. 1, 133–140. Bais, H.P., Weir, T.L., Perry, L.G., Gilroy, S., Vivanco, J.M., 2006. The role of root exudates in rhizosphere interactions with plants and other organisms. Annu. Rev. Plant Biol. 57, 233–266. Belleghem, F.V., Cuypers, A., Semane, B., Smeets, K., Vangronsveld, J., D’Haen, J., 2007. Subcellular localization of cadmium in roots and leaves of Arabidopsis thaliana. New Phytol. 173, 495–508. Bhaduri, A., Fulekar, M., 2012. Antioxidant enzyme responses of plants to heavy metal stress. Rev. Environ. Sci. Biol. 11, 55–69. Bjelková, M., Genčurová, V., Griga, M., 2011. Accumulation of cadmium by flax and linseed cultivars in field-simulated conditions: a potential for phytoremediation of cdcontaminated soils. Ind. Crops Prod. 33, 761–774. Celekli, A., Kapı, M., Bozkurt, H., 2013. Effect of cadmium on biomass, pigmentation, malondialdehyde, and proline of Scenedesmus quadricauda var. longispina. Bull. Environ. Contam. Toxicol. 91, 571–576. Chaca, M.V.P., Vigliocco, A., Reinoso, H., Molina, A., Abdala, G., Zirulnik, F., 2014. Effects of cadmium stress on growth, anatomy and hormone contents in Glycine max, (l.) merr. Acta Physiol. Plant. 36, 2815–2826. Chmielowska-Bak, J., Gzyl, J., Rucinska-Sobkowiak, R., Arasimowicz-Jelonek, M., Deckert, J., 2014. The new insights into cadmium sensing. Front. Plant Sci. 5, 1–13. Clemens, S., 2006. Toxic metal accumulation, responses to exposure and mechanisms of tolerance in plants. Biochimie 88, 1707–1719. Cluis, C., 2004. Junk-greedy greens: phytoremediation as a new option for soil decontamination. Biol. Teach. J. 2, 61–67. Clabeaux, B.L., Navarro, D.A., Aga, D.S., Bisson, M.A., 2011. Cd tolerance and accumulation in the aquatic macrophyte, chara australis: potential use for charophytes in phytoremediation. Environ. Sci. Technol. 45, 5332–5338. Dalcorso, G., Farinati, S., Furini, A., 2010. Regulatory networks of cadmium stress in plants. Plant Signal. Behav. 5, 663–667. D'Alessandro, A., Zolla, L., 2012. We are what we eat: food safety and proteomics. J. Proteome Res. 11, 26–36. Dhir, B., Sharmila, P., Pardha Saradhi, P., 2009. Potential of aquatic macrophytes for removing contaminants from the environment. Environ. Sci. Technol. 39, 754–781. Drążkiewicz, M., Skórzyńska-Polit, E., Krupa, Z., 2003. Response of the ascorbate–glutathione cycle to excess copper in Arabidopsis thaliana (L.). Plant Sci. 164, 195–202. Ehsan, S., Ali, S., Noureen, S., Mahmood, K., Farid, M., Ishaque, W., et al., 2014. Citric acid assisted phytoremediation of cadmium by Brassica napus L. Ecotoxicol. Environ. Safe. 106, 164–172. Esposito, M., Domingos, M., 2014. Establishing the redox potential of Tibouchina pulchra (Cham.) Cogn., a native tree species from the Atlantic Rainforest in the vicinity of anoil refinery in SE Brazil. Environ. Sci. Pollut. Res. 21, 5484–5495. Fatima, A., Ahmad, M., 2005. Certain antioxidant enzymes of Allium cepa as biomarkers for the detection of toxic heavy metals in waste water. Sci. Total Environ. 346, 256–273. Fu, X., Dou, C., Chen, Y., Chen, X., Shi, J., Yu, M., Jie, X., 2011. Subcellular distribution and chemical forms of cadmium in Phytolacca americana L.. J. Hazard Mater. 186, 103–107. Gallego, S.M., Pena, L.B., Barcia, R.A., Azpilicueta, C.E., 2012. Unravelling cadmium toxicity and tolerance in plants: insight into regulatory mechanisms. Environ. Exp. Bot. 83, 33–46. Ghosh, M., Singh, S.P., 2005. A review on phytoremediation of heavy metals and utilization of it's by products. Asian J. Energy Environ. 6, 214–231. Gill, S.S., Tuteja, N., 2011. Cadmium stress tolerance in crop plants: probing the role of sulfur. Plant Signal. Behav. 6, 215–222. Guan, M.Y., Zhang, H.H., Pan, W., Jin, C.W., Lin, X.Y., 2018. Sulfide alleviates cadmium toxicity in Arabidopsis, plants by altering the chemical form and the subcellular distribution of cadmium. Sci. Total Environ. 627, 663–670.

17

Ecotoxicology and Environmental Safety 160 (2018) 10–18

L.P. Yang et al.

Verbruggen, N., Hermans, C., Schat, H., 2009. Mechanisms to cope with arsenic or cadmium excess in plants. Curr. Opin. Plant Biol. 12, 364–372. Wang, C., Luo, X., Tian, Y., Xie, Y., Wang, S., Li, Y., Tian, L., Wang, X., 2012. Biphasic effects of lanthanum on Vicia faba L. seedlings under cadmium stress, implicating finite antioxidation and potential ecological risk. Chemosphere 86, 530–537. Wang, C., Zhang, S.H., Wang, P.F., Hou, J., Zhang, W.J., Li, W., Lin, Z.P., 2009. The effect of excess Zn on mineral nutrition and antioxidative response in rapeseed seedlings. Chemosphere 75, 1468–1476. Wang, J., Yuan, J., Yang, Z., Huang, B., Zhou, Y., Xin, J., Gong, Y., Yu, H., 2009. Variation in cadmium accumulation among 30 cultivars and cadmium subcellular distribution in 2 selected cultivars of water spinach (Ipomoea aquatica Forsk.). J. Agric. Food Chem. 57, 8942–8949. Wang, X., Liu, Y., Zeng, G., Chai, L., Song, X., Min, Z., Xin, X., 2008. Subcellular distribution and chemical forms of cadmium in Bechmeria nivea, (L.) gaud. Environ. Exp. Bot. 62, 389–395. Wang, Y., Huang, J., Gao, Y., 2012. Arbuscular mycorrhizal colonization alters subcellular distribution and chemical forms of cadmium in Medicago sativa L. and resists cadmium toxicity. PLos One 7, e48669. Weng, B., Xie, X., Weiss, D., Liu, J., Lu, H., Yan, C., 2012. Kandelia obovata (S., L.) Yong tolerance mechanisms to cadmium: subcellular distribution, chemical formsand thiol pools. Mar. Pollut. Bull. 64, 2453–2460. Wójcik, M., Vangronsveld, J., D’Haen, J., Tukiendorf, A., 2005a. Cadmium tolerance in Thlaspi caerulescens: II. Localization of cadmium in Thlaspi caerulescens. Environ. Exp. Bot. 53, 163–171. Wójcik, M., Vangronsveld, J., Tukiendorf, A., 2005b. Cadmium tolerance in Thlaspi caerulescens: i. Growth parameters, metal accumulation and phytochelatin synthesis in response to cadmium. Environ. Exp. Bot. 53, 151–161. Wrzaczek, M., Brosché, M., Kangasjärvi, J., 2013. ROS signaling loops-production, perception, regulation. Curr. Opin. Plant Biol. 16, 575–582. Wu, F., Zhang, G., Dominy, P., 2003. Four barley genotypes respond differently to cadmium: lipid peroxidation and activities of antioxidant capacity. Environ. Exp. Bot. 50, 67–78. Wu, J., Feng, Y.Q., Dai, Y.R., Cui, N.X., Anderson, B., Cheng, S.P., 2016. Biological mechanisms associated with triazophos (TPA) removal by horizontal subsurface flow constructed wetlands (HSFCW). Sci. Total Environ. 553, 13–19. Xin, J., Huang, B., Yang, Z., Yuan, J., Zhang, Y., 2013. Comparison of cadmium subcellular distribution in different organs of two water spinach (Ipomoea aquatica, Forsk.) cultivars. Plant Soil. 372, 431–444. Xue, D., Jiang, H., Deng, X., Zhang, X., Wang, H., Xu, X., 2014. Comparative proteomic analysis provides new insights into cadmium accumulation in rice grain under cadmium stress. J. Hazard Mater. 280, 269–278. Yang, X.E., Stoffella, P.J., 2004. Cadmium tolerance and hyperaccumulation in a new Znhyperaccumulating plant species (Sedum alfredii Hance). Plant Soil. 259, 181–189. Ye, X., Ling, T., Xue, Y., Xu, C., Zhou, W., Hu, L., Chen, J., Shi, Z., 2016. Thymol mitigates cadmium stress by regulating glutathione levels and reactive oxygen species homeostasis in tobacco seedlings. Molecules 21, 1339. Zhang, F.Q., Zhang, H.X., Wang, G.P., Xu, L.L., Shen, Z.G., 2009. Cadmium-induced accumulation of hydrogen peroxide in the leaf apoplast of Phaseolus aureus and Vicia sativa and the roles of different antioxidant enzymes. J. Hazard Mater. 168, 76–84. Zhang, H., Li, H., Pan, H., Liu, X., Yang, K., Huang, S., 2017. Efficient production of biodiesel with promising fuel properties from Koelreuteria integrifoliola, oil using a magnetically recyclable acidic ionic liquid. Energ. Convers. Manag. 138, 45–53. Zhao, F.J., Jiang, R.F., Dunham, S.J., Mcgrath, S.P., 2006. Cadmium uptake, translocation and tolerance in the hyperaccumulator Arabidopsis halleri. New Phytol. 172, 646–654. Zhao, Y., Wu, J., Shang, D., Ning, J., Zhai, Y., Sheng, X., Ding, H., 2015. Subcellular distribution and chemical forms of cadmium in the edible seaweed, Porphyra yezoensis. Food Chem. 168, 48–54. Zhu, X., Jiang, T., Wang, Z., Lei, G., Shi, Y., Li, G., Zheng, S., 2012. Gibberellic acid alleviates cadmium toxicity by reducing nitric oxide accumulation and expression of IRT1 in Arabidopsis thaliana. J. Hazard Mater. s 239–240, 302–307.

Thlaspi caerulescens. Biotechnol. Bioeng. 67, 607–615. Nocito, F.F., Pirovano, L., Coccuci, M., Sacchi, G.A., 2002. Cadmium induced sulfate uptake in maize roots. Plant Physiol. 129, 1872–1879. Pant, M., 2014. Cellular mechanisms of cadmium-induced toxicity: a review. Inter J. Environ. Heal Res 24, 378–399. Pereira, d.A.R., Aa, F.D.A., Silva, P.L., Pao, M., Olimpio, S.J., Pirovani, C.P., 2017. Photosynthetic, antioxidative, molecular and ultrastructural responses of young cacao plants to Cd toxicity in the soil. Ecotoxicol. Environ. Safe. 144, 148–157. Pipinis, E., Mavrokordopoulou, O., Milios, E., Diamanta, A., Kotili, I., Smiris, P., 2015. Effects of dormancy-breaking treatments on seed germination of Koelreuteria paniculata and Mahonia aquifolium. Dendrobiology 74, 149–155. Porra, J., 2002. The chequered history of the development and use of simultaneous equations for the accurate determination of chlorophylls a and b. Photosynth. Res. 73, 149–156. Pulford, I.D., Watson, C., 2003. Phytoremediation of heavy metal-contaminated land by trees-a review. Environ. Int. 29, 529–540. Qiu, Q., Wang, Y., Yang, Z., Yuan, J., 2011. Effects of phosphorus supplied in soil on subcellular distribution and chemical forms of cadmium in two Chinese flowering cabbage (Brassica parachinensis L.) cultivars differing in cadmium accumulation. Food Chem. Toxicol. 49, 2260–2267. Redovniković, I.R., Marco, A.D., Proietti, C., Hanousek, K., Sedak, M., Bilandžić, N., 2017. Poplar response to cadmium and lead soil contamination. Ecotox. Environ. Safe 144, 482–489. Remans, T., Opdenakker, K., Guisez, Y., Carleer, R., Schat, H., Vangronsveld, J., Cuypers, A., 2012. Exposure of Arabidopsis thaliana to excess Zn reveals a Zn-specific oxidative stress signature. Environ. Exp. Bot. 84, 61–71. Schützendübel, A., Polle, A., 2002. Plant responses to abiotic stresses: heavy metal-induced oxidative stress and protection by mycorrhization. J. Exp. Bot. 53 (372), 1351–1365. Schützendübel, A., Schwanz, P., Teichmann, T., Gross, K., Langenfeldheyser, R., Godbold, D.L., 2001. Cadmium-induced changes in antioxidative systems, hydrogen peroxide content, and differentiation in scots pine roots. Plant Physiol. 127, 887–898. Sharma, S., Kumar, V., 2002. Responses of wild type and abscisic acid mutants of Arabidopsis thaliana to cadmium. J. Plant Physiol. 159, 1323–1327. Sidhu, G.P., Singh, H.P., Batish, D.R., Kohli, R.K., 2017. Tolerance and hyperaccumulation of cadmium by a wild, unpalatable herb Coronopus didymus (L.) sm. (Brassicaceae). Ecotoxicol. Environ. Safe 135, 209–215. Stroiński, A., Giżewska, K., Zielezińska, M., 2012. Abscisic acid is required in transduction of cadmium signal to potato roots. Biol. Plant. 57, 121–127. Sun, L.I., Jingling, Y.U., Zhu, M., Zhao, F., Luan, S., 2012. Cadmium impairs ion homeostasis by altering K+ and Ca2+ channel activities in rice root hair cells. Plant Cell Environ. 35, 1998–2013. Thomas, J., Perron, M., Smigocki, A., 2010. Cytokinin and the regulation of a tobacco metallothionein-like gene during copper stress. Physiol. Plant. 123, 262–271. Tamás, L., Bočová, B., Huttová, J., Liptáková, Ľ., Mistrík, I., Valentovičová, K., Zelinová, V., 2012. Impact of the auxin signaling inhibitor p-chlorophenoxyisobutyric acid on short-term Cd-induced hydrogen peroxide production and growth response in barley root tip. J. Plant Physiol. 169, 1375–1381. Tian, D., Fan, L., Yan, Z., W, D., Fang, X., Xiang, W., Deng, H., X, W., 2009. Heavy metal accumulation by panicled golden rain tree (Koelreuteria paniculata) and common elaeocarpus (Elaeocarpus decipens) in abandoned mine soils in southern China. J. Environ. Sci. 21, 340–345. Uraguchi, S., Kiyono, M., Sakamoto, T., Watanabe, I., Kuno, K., 2009. Contributions of apoplasmic cadmium accumulation, antioxidative enzymes and induction of phytochelatins in cadmium tolerance of the cadmium-accumulating cultivar of black oat (Avena strigosa Schreb.). Planta 230, 267–276. Utmazian, M.N.D.S., Wenzel, W.W., 2007. Cadmium and zinc accumulation in willow and poplar species grown on polluted soils. J. Plant Nutr. Soil Sci. 170, 265–272. Vassilev, A., Lidon, F., 2011. Cd-induced membrane damages and changes in soluble protein and free amino acid contents in young barley plants. Emir. J. Food Agr. 23, 130–136.

18