Ecotoxicology and Environmental Safety 102 (2014) 55–61

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Dynamics of rhizosphere properties and antioxidative responses in wheat (Triticum aestivum L.) under cadmium stress Yonghua Li a,n, Li Wang a,b, Linsheng Yang a, Hairong Li a,nn a b

Institute of Geographical Sciences and Natural Resources Research, Chinese Academy of Sciences, 11A Datun Road, Chaoyang District, Beijing 100101, China Department of International Health, Faculty of Health, Medicine and Life Sciences, Maastricht University, 6200 MD, Maastricht, The Netherlands

art ic l e i nf o

a b s t r a c t

Article history: Received 3 September 2013 Received in revised form 8 January 2014 Accepted 8 January 2014 Available online 1 February 2014

In this study, we performed a rhizobox experiment to examine the dynamic changes in the rhizosphere properties and antioxidant enzyme responses of Triticum aestivum L. under three levels of cadmium stress. A set of micro-techniques (i.e., Rhizobox and Rhizon SMS) were applied for the dynamically nondestructive collection of the rhizosphere soil solution to enable the observation at a high temporal resolution. The dynamics of soluble cadmium and dissolved organic carbon (DOC) in the rhizosphere soil solutions of the Triticum aestivum L. were characterised by the sequence week 0 after sowing (WAS0) o 3 weeks after sowing (WAS3) o10 weeks after sowing (WAS10), whereas the soil solution pH was found to follow an opposite distribution pattern. Systematically, both superoxide dismutase (SOD) and catalase (CAT) activities in the leaves of the Triticum aestivum L. increased concomitantly with increasing cadmium levels (p40.05) and growth duration (po0.05), whilst ascorbate peroxidase (APX) activity was induced to an elevated level at moderate cadmium stress with a decrease at high cadmium stress (p40.05). These results suggested the enhancement of DOC production and the greater antioxidant enzyme activities were two important protective mechanisms of Triticum aestivum L. under cadmium stress, whereas rhizosphere acidification might be an important mechanism for the mobilisation of soil cadmium. The results also revealed that plant–soil interactions strongly influence the soil solution chemistry in the rhizosphere of Triticum aestivum L., that, in turn, can stimulate chemical and biochemical responses in the plants. In most cases, these responses to cadmium stress were sensitive and might allow us to develop strategies for reducing the risks of the cadmium contamination to crop production. & 2014 Elsevier Inc. All rights reserved.

Keywords: Heavy metal pollution Rhizobox experiment Non-destructive sampling Plant–soil interaction Protective mechanism

1. Introduction Areas of agricultural soil contaminated by cadmium (Cd) have been dramatically increased worldwide as a result of anthropogenic activities, such as disposal of industrial effluent, mining waste, and abuse of phosphate fertilizers, pesticides and sewage sludge (Gil et al., 2004; Page et al., 2006; Williams et al., 2009; Yang et al., 2010; Zhang et al., 2010; Nabulo et al., 2011, 2012; Wang et al., 2011; Gallego et al., 2012; Khan et al., 2013; Sun et al., 2013). Moreover, the problems are being aggravated in developing and underdeveloped countries with rapid industrialisation and urbanisation where raw effluents or effluents with little treatment are random disposed on the soils (Lal et al., 2008; McBeath and McBeath, 2009). In China, more than 20 million ha, accounting for approximately 20 per cent of the total area under cultivation, is contaminated by cadmium and other heavy metals from various anthropogenic activities (Zeng et al., 2006), which causes considerable losses in both quantity and quality

n

Corresponding author. Fax: þ 86 10 64856504. Corresponding author. Fax: þ 86 10 64889198. E-mail addresses: [email protected] (Y. Li), [email protected] (H. Li).

nn

0147-6513/$ - see front matter & 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ecoenv.2014.01.004

of agricultural products and therefore imposes serious threats to human health through the food chain (Cui et al., 2004; Li et al., 2010; Wang et al., 2011; Yang et al., 2012; Chen et al., 2013; Niu et al., 2013; Sun et al., 2013; Li et al., 2014). Cadmium has no biological function and is not even essential for plant growth (Tuteja and Gill, 2013). Being water soluble, Cd2 þ ion can be easily absorbed in tissues and can cause various phytotoxic visible symptoms (Singh et al., 2008; Hu et al., 2009; Valentovičová et al. 2010). Despite cadmium toxicity, plants have evolved a complex array of mechanisms to maintain optimal cadmium levels and avoid the detrimental effects of excessively high concentrations. Among the mechanisms, the activity of ROS-scavenging enzymes, including superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX), is considered as the most important protective mechanism to minimise the metalinduces oxidative damage in several plants (Tiryakioglu et al., 2006; Kafel et al., 2010; Parlak and Yilmaz, 2013; Tuteja and Gill, 2013). To date, most of our knowledge on the mechanisms stems from studies using techniques where plants are grown in rather artificial conditions such as hydroponic solution or agar gels or soil extracts; moreover, most studies on the metal tolerance in target plants are based on total soil cadmium concentrations, and rarely

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Y. Li et al. / Ecotoxicology and Environmental Safety 102 (2014) 55–61

relate any observed effects to cadmium concentrations in soil solution (Chaudri et al., 2000; Ait Ali et al., 2004; Zhang et al., 2010). However, plants in hydroponic systems behave differently compared to natural conditions in soils. Additionally, scarce research is related to the plant growth stages, thus a comprehensive understanding of the plant protective mechanisms remains elusive. It is well documented that the soil–plant interactions in the rhizosphere play a key role in controlling the metal bioavailability to plants (Huynh et al., 2010; Li et al., 2011; Motaghian and Hosseinpur, 2013). Plants have the ability to transform metal fractions for easier uptake through root exudation or pH changes in the rhizosphere (Wang et al., 2002; Hinsinger et al., 2003). Given that the rhizosphere soil solution is the medium through which the cadmium exerts its effects on plants; therefore the analysis of the rhizosphere soil solution can provide valuable information concerning the potential toxicity of the cadmium in the soil. Accordingly, there is a great need for continuous in situ studies in plant–soil systems. The most traditional techniques for collecting soil solution are water displacement, centrifugation or extraction after the destructive sampling of rhizosphere soils (Grossman and Udluft, 1991; Knight et al., 1998). Recently, the use of micro-techniques for the collection of the soil solution enables the non-destructive in situ observation of soil solution chemistry at a high temporal resolution. For example, Rhizon soil moisture samplers (Rhizon SMS), which can extract a 5–10 ml volume of interstitial soil pore water without significantly disturbing the structure, chemistry or biology of the soil, are being used for this purpose (Luo et al., 2003; Cattani et al., 2006) and have been proposed as a valid tool for monitoring and assessing eco-toxicity in soils (Clemente et al., 2008; Beesley et al., 2010). Furthermore, because the sampling is non-destructive, such methodology does allow for long-term dynamic studies. Wheat plants (Triticum aestivum L.) was selected as a test crop plant because it is one of the most important economic crops worldwide and is cultured on a global scale. Additionally, as an important crop, Triticum aestivum L. is frequently used as an ecotoxicological indicator (Song et al., 2007; Pérez and Anderson, 2009). In this study, a rhizobox experiment was conducted to investigate the dynamic changes in the rhizosphere properties and antioxidant enzyme responses of Triticum aestivum L. grown in three levels of cadmium-contaminated agricultural soils. We used micro-techniques (i.e., Rhizobox and Rhizon SMS) for the collection of the soil solution of the Triticum aestivum L. at different growth stages, thus enabling the non-destructive observation of the rhizosphere soil properties at a high temporal resolution.

2. Materials and methods 2.1. Soil sampling and preparation Three agricultural topsoils (S1, S2 and S3) containing different cadmium concentrations were used in the rhizobox experiment. S1 and S2 were collected from the centre and the boundary of a lead mine, respectively; the S3 was collected from an agricultural town situated about 40 km north of the mine. The three soil samples were typical fragiudalfs, which were derived dominantly from shale and limestone. In this study, S1, S2 and S3 were taken as high cadmium stress soil (HS), moderate cadmium stress soil (MS) and control soil (CK), respectively. In the laboratory, the soil samples were air-dried and passed through a 2 mm nylon sieve for the soil pH determination, soil grain size analyses and pot experiment. Subsamples (15 g) were ground in an agate vibrating-cup mill to pass through a 0.16-mm nylon sieve for the chemical analysis. Each ground powder was then thoroughly mixed and stored in sealed polyethylene bags for the subsequent analysis. 2.2. Greenhouse pot experiment design The rhizobox was made from polyvinylchloride (PVC), with dimensions of 140  140  200 (length  width  height in mm). Each box was divided into three vertical sections: a rhizosphere compartment (20 mm in width), which was

surrounded with nylon mesh (300 mesh), and left and right non-rhizosphere compartments (60 mm in width) (Supplementary Fig. 1). Approximately 0.5 kg of soil was placed in the rhizosphere compartment, and 3.0 kg was placed in the nonrhizosphere compartment. Initially, a 50 per cent water-holding capacity (WHC) was generated using deionised water; 2 weeks prior to sowing, the level was increased to 75 per cent WHC. Triticum aestivum L. was grown in the rhizosphere compartment according to Greger and Landberg (2008). Briefly, ten seeds were germinated in each rhizosphere compartment for 7 d, and each treatment was prepared with four replications. At the end of this period, five healthy seedlings of approximately the same size per pot were selected and allowed to grow for experimental purposes. During the experiment, the rhizoboxes were arranged in a greenhouse (natural light, 60–80 per cent relative humidity and a temperature of 25–30 1C), and the positions of the rhizoboxes were rotated regularly to ensure uniform growing conditions.

2.3. Rhizosphere soil solution and wheat leaf sampling Rhizosphere soil solution samples were collected through the rhizoboxes using the Rhizon SMS samplers (Rhizosphere Research Products, Wageningen, the Netherlands) as described by Li et al. (2013b). Rhizosphere solution sampling started immediately after sowing (WAS0), and was performed in week 3 after sowing (WAS3) and week 10 after sowing (WAS10). When the soil solution of these pots had been collected, approximately 4 g of fresh wheat leaf from each pot were harvested and rinsed thoroughly with running tap water followed by deionised water. An approximately 2 g leaf was immediately frozen in liquid nitrogen for the protein and enzyme activity assays. 2 g leaf was dried at 80 1C for 48 h and then ground and sieved through a 1-mm nylon sieve for the content of cadmium analysis.

2.4. Extraction of proteins and enzymes Fresh leaf tissues were homogenised under liquid nitrogen in a mortar according to Li et al. (2013b). The homogenate was then centrifuged at 15,000g for 15 min at 4 1C, and the supernatant (crude extract of leaves) was used to determine the protein levels and enzyme activities. The total soluble protein was estimated according to the method of Bradford (1976) using bovine serum albumin (BSA) as the standard.

2.5. Assay of enzyme activities The activity of SOD (EC 1.1.5.1.1) was assayed by the method of Beauchamp and Fridovich (1971). 3 ml reaction mixture contained 50 mM sodium phosphate buffer (pH 7.8), 10 mM methionine, 1.17 mM riboflavin, 56 mM nitro-blue tetrazolium (NBT) and 50 ml enzyme extract. The absorbance was determined at 560 nm using a UV/vis spectrophotometer (TU-1901, Purkinje General, Beijing, China). One unit of SOD was defined as the amount of enzyme causing the half-maximal inhibition of NBT reduction under the assay conditions. The CAT (EC 1.11.1.6) activity was measured by the consumption of H2O2, as the absorbance at 240 nm, according to the method of Aebi (1984). 3 ml reaction mixture contained 50 mM sodium phosphate buffer (pH 7.0), 10 mM H2O2 and 10 ml enzyme extract. The activity was calculated using the extinction coefficient 0.036 mM  1 cm  1. The APX (EC 1.11.1.1) activity was determined according to the method of Nakano and Asada (1981) using a 3 ml reaction solution containing 50 mM sodium phosphate buffer (pH 7.0), 0.5 mM ascorbate, 0.1 mM H2O2 and 10 ml enzyme extract. The absorbance was measured at 290 nm. The activity was calculated using the extinction coefficient 2.8 mM  1 cm  1.

2.6. Soil and plant analysis The soil pH (soil:deionised water¼ 1:2.5, w/v) and soil organic matter (SOM) were measured according to the conventional methods issued by ISSCAS (Institute of Soil Science, Chinese Academy of Sciences) (1978), and the soil grain size was determined by laser-diffraction analysis (Mastersizer 2000, Malvern, UK). The content of cadmium and lead in the soil and wheat leaf samples was determined by inductively coupled plasma-mass spectrometry (ICP-MS; ELAN 9000, PerkinElmer, Waltham, MA, USA) after microwave digestion with concentrated nitric acid (Clemente et al., 2010). For quality assurance and quality control (QA/QC), blank spikes and certified reference materials (soil GBW07404 and plant GBW08505) were used during the analyses. The analytical results of cadmium and lead in the certified reference material were in good agreement with the certified values, with RSDs ranging from 2 per cent to 13 per cent. The percentage of the recoveries of the spiked samples ranged from 93 per cent to 110 per cent for the elements. Some physico-chemical parameters including soil pH, soil organic matter (SOM), soil grain size, cadmium and lead concentrations of the tested soils (CK, MS and HS) were listed in Table 1.

Y. Li et al. / Ecotoxicology and Environmental Safety 102 (2014) 55–61

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c

Table 1 General properties of the soils used in the pot experiment. Soil

pHa

SOMb (g kg  1)

Sand (%)

Silt (%)

Clay (%)

Cadmium (mg kg  1)

Lead (mg kg  1)

CK MS HS

6.32 5.51 7.90

48.40 38.37 49.12

26.8 45.71 45.95

56.9 39.68 41.17

16.3 14.61 12.88

0.59 2.69 9.48

42 343 947

a b

1:2.5 Soil/deionised water ratio. Soil organic matter.

2.7. Rhizosphere soil solution analysis The extracted rhizosphere soil solutions were separated into several subsamples for analysis. The pH and DOC were promptly analysed after sample collection. The pH was determined using a calibrated Thermo Orion 868 pH Metre (Thermo Orion Inc., USA), and the DOC was measured using a TOC-5050A TOC analyser (Shimadzu, Japan) (Li et al., 2013b). The concentration of cadmium in the soil solutions was determined using a Model 9000 ICP-MS (PerkinElmer, USA).

b

c b a

a a

b

c

Fig. 1. Cadmium concentrations in the rhizosphere soil solution of Triticum aestivum L. at the stage of immediately after sowing (WAS0), week 3 after sowing (WAS3) and week 10 after sowing (WAS10) in control soil (CK), moderate cadmium stress soil (MS) and high cadmium stress soil (HS). The bars represent the standard deviations of four replicates. The bars with different letters among the different stages are significantly different at p o 0.05 by Student0 s t-test.

a 2.8. Statistical methods The statistical analysis was conducted using STATISTICA version 6.0 (StatSoft, Inc., Tulsa, USA). The significance differences between treatments were statistically evaluated by the standard deviation (SD) and Student0 s t-test methods.

b a

a

a a

3. Results and discussion

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a

a

3.1. Soluble cadmium in the rhizosphere soil solution Soluble metals are of particular interest, as they constitute the most readily available chemical form to plants. The water soluble cadmium in the rhizosphere soil solutions of the Triticum aestivum L. was determined at three different stages, namely, WAS0, WAS3 and WAS10 (Fig. 1). The concentrations of the soluble cadmium in the rhizosphere soil solution at both WAS3 and WAS10 for the three soils (namely, CK, MS and HS) significantly increased compared to their initial values at WAS0, with the increase at WAS10 being much greater than WAS3 (p o0.05). When compared to their initial values, the soluble cadmium levels of CK, MS and HS were 2.14, 1.72, and 3.73 times higher at WAS3 and 4.88, 2.66, and 5.16 times higher at WAS10, respectively. Consequently, the dynamics of cadmium in the rhizosphere soil solutions of the Triticum aestivum L. over the culture period were characterised by an increase in the three soils over time. However, there is currently no consensus concerning the impact of culture period and associated root activities on metal concentrations and speciation. For instance, Page et al. (2006) who observed that the content of 109Cd in the rhizosphere soil of Triticum aestivum L. grown on rhizoboxes increased steadily from 4 to 28 days after transplanting and then dropped from 28 to 50 days after transplanting. 3.2. Root-induced changes of pH in the rhizosphere soil solution The temporal changes of the pH in the rhizosphere soil solution of the Triticum aestivum L. for the three soils were illustrated in Fig. 2. In general, no significant change of the pH was observed in the rhizosphere soil solution among different stages. However, a general soil solution pH trend could be observed for the three soils following the sequence WAS0 4WAS3 4WAS10. When compared to the rhizosphere soil solution of the Triticum aestivum L. at WAS0, the soil solution at WAS3 and WAS10 stages were reduced 0.20 and 0.36 pH unit in CK, 0.13 and 0.35 pH unit in MS and 0.21 and 0.60 pH unit in HS, respectively; and the reductions in the high cadmium stress soil were greater than those in moderate cadmium stress soil (Fig. 2). Most previous work on the soil–root

Fig. 2. The pH in the rhizosphere soil solution of Triticum aestivum L. at the stage of immediately after sowing (WAS0), week 3 after sowing (WAS3) and week 10 after sowing (WAS10) in control soil (CK), moderate cadmium stress soil (MS) and high cadmium stress soil (HS). The bars represent the standard deviations of four replicates. The bars with different letters among the different stages are significantly different at p o0.05 by Student0 s t-test.

interface shows an acidification of the rhizosphere (Séguin et al., 2004; Li et al., 2011). Within this context, the present results are in good agreement with the literature. Changes in pH variations owing to plants may be caused by an imbalance in the release or uptake of cations or anions. To maintain the electroneutrality at the root-soil interface, plants will compensate by releasing OH  or H þ depending on whether anions or cations, respectively, are taken up in excess (Nye, 1981). Moreover, the release of CO2 from root respiration may accelerate the reduction in the pH of the solution. A similar phenomenon was observed by Li et al. (2013a) when they investigated the effects of elevated CO2 on rhizosphere characteristics of Cd/Zn hyperaccumulator Sedum alfredii. In addition, the effect of rhizosphere microorganisms on pH should also be taken into account, since rhizosphere is a unique environment where microorganisms play an important role, contributing in most cases to changes on rhizosphere soil solution chemistry (Robinson et al., 2001; Hinsinger et al., 2006; Cloutier-Hurteau et al., 2008; Majewska and Kurek, 2011). It has frequently been reported that soil pH has the potential to modify metal solubility in several ways, including dissolution/precipitation reactions, regulating the ionisation of pH-dependent exchange sites on organic matter and oxide clay minerals, reducing the partitioning of metal to the solid phase (i.e. decreasing the negative surface charge on soil particles) and influencing metal speciation in soils (Adriano et al., 2004; Conesa et al., 2006; Padmavathiamma and Li, 2012). The decreases in pH in the rhizosphere soil solutions may have resulted in the increase in the soluble cadmium in the rhizosphere soil solution as mentioned above. Given that the soil solution pH is one of the

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Y. Li et al. / Ecotoxicology and Environmental Safety 102 (2014) 55–61

Table 2 Cadmium concentrations in leaves of Triticum aestivum L. at different growth stages.

a

a

b

b a

b b

Soil

WAS3 (mg kg  1)

WAS10 (mg kg  1)

CK MS HS

0.45 7 0.10 1.647 0.31 2.42 7 0.66

0.58 70.13 3.51 70.72 6.57 71.27

b

a

Fig. 3. The dissolved organic carbon (DOC) concentrations in the rhizosphere soil solution of Triticum aestivum L. at the stage of immediately after sowing (WAS0), week 3 after sowing (WAS3) and week 10 after sowing (WAS10) in control soil (CK), moderate cadmium stress soil (MS) and high cadmium stress soil (HS). The bars represent the standard deviations of four replicates. The bars with different letters among the different stages are significantly different at p o 0.05 by Student0 s t-test.

most important parameters affecting the solubility and mobility of metal fractions and that a reduction of the rhizosphere pH increases the chemical activity of most metals, rhizosphere acidification may be an important mechanism for the mobilisation of cadmium by Triticum aestivum L. in the soils.

b

a

a

a

b

b

3.3. Temporal variability in DOC in the rhizosphere soil solution The influence of plant growth on DOC in the rhizosphere soil solution is given in Fig. 3. As can be seen, the concentration of DOC in the rhizosphere soil solution for the three soils followed the sequence WAS0 oWAS3 o WAS10. For a given soil, the differences between growth stages, although significant in most cases, were not very pronounced (p4 0.05). When compared to the DOC concentration in the rhizosphere soil solution of the Triticum aestivum L. at WAS0, the DOC concentration at WAS3 and WAS10 stages were 1.06 and 1.16 times higher in CK, 1.46 and 1.56 times higher in MS and 1.30 and 1.44 times higher in HS, respectively (Fig. 3). The soil solution DOC increased over the course of the growth duration, probably due to the increased release of root exudates, in particular, CO2 and low molecular weight organic acids, a finding that was consistent with the reduced pH values in the rhizosphere soil solution. This result is in accordance with Kim and Kang (2011) who reported that DOC concentrations in the rhizosphere of pine increased by elevated CO2 and in accordance with Guo et al. (2011) who proposed that elevated CO2 increase exudation of lowmolecular-weight organic compounds by the roots of rice. It is well documented that organic matter content, as well as pH, is a key factor influencing the concentrations of metals. Organic matter, both in the dissolved and solid states, has a large specific surface area and an elevated negative charge, thus attracting metals. Many strong bonds can then be established to bind metals to the organic matter (McBride, 1994). Given that the availability of cadmium for plants is greater in acid condition (Kirkham, 2006) and its availability can be reduced by DOC (Vaughan et al., 1993), the exudation of high amounts of DOC by Triticum aestivum L. is an important protective mechanism under cadmium stress. 3.4. Dynamic changes in the antioxidant enzyme activity Concentrations of cadmium in leaves of Triticum aestivum L. exposed to three different levels of cadmium at different growth stages are listed in Table 2. As can be seen, cadmium concentration in the leaves increased when there was an elevated cadmium concentration in the medium. The responses of antioxidant enzymes in the Triticum aestivum L. under different levels of

a

b

b

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a

a a

a b

b

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Fig. 4. Activity of SOD (A), CAT (B) and APX (C) in leaves of Triticum aestivum L. at the stage of week 3 after sowing (WAS3) and week 10 after sowing (WAS10) in control soil (CK), moderate cadmium stress soil (MS) and high cadmium stress soil (HS). The bars represent the standard deviations of four replicates. The bars with different letters between WAS3 and WAS10 stages are significantly different at po 0.05 by Student0 s t-test.

cadmium stress are shown in Fig. 4. Generally, SOD activity in the leaves of the Triticum aestivum L. increased concomitantly with increasing cadmium stress levels (p 40.05) (Fig. 4A). When compared with the control, the activity of SOD at WAS3 under moderate cadmium stress and high cadmium stress increased by 40 per cent and 57 per cent, respectively; while at WAS10, the activity of SOD increased by 4% at moderate cadmium stress and 11 per cent at high cadmium stress as compared to the control. CAT activity exhibited a similar pattern of that of SOD activity

Y. Li et al. / Ecotoxicology and Environmental Safety 102 (2014) 55–61

(Fig. 4B). When compared with the control, CAT activity at WAS3 under moderate cadmium stress and high cadmium stress increased by 2 per cent and 17 per cent, respectively; while at WAS10, CAT activity increased by 8 per cent at moderate cadmium stress and 14 per cent at high cadmium stress as compared to the control. However, the trend observed for APX was different of those observed for SOD and CAT mentioned above (Fig. 4C). APX activity in the leaves of the Triticum aestivum L. initially increased about 9–10 per cent at moderate cadmium stress then decreased about 1–18 per cent at high cadmium stress as compared to the control. Simultaneously, it was observed that the activities of both SOD and CAT in the leaves of the Triticum aestivum L. significantly increased from WAS3 stage to WAS10 stage (p o0.05), regardless of the levels of initial cadmium stress in the potting soils; in contrast, the APX activity decreased from WAS3 stage to WAS10 stage (p o0.05). The SOD and CAT activities in the leaves of the Triticum aestivum L. at WAS10 stage in CK, MS and HS were 2.78, 2.07, and 1.96 times higher and 1.63, 1.73, and 1.59 times higher, respectively, when compared to WAS3 stage. Conversely, the APX activities in the leaves of the Triticum aestivum L. at WAS10 stage in CK, MS and HS were 0.56, 0.56 and 0.68 times lower compared to WAS3 stage, a result that was similar to the finding of Shao et al. (2005) who observed that the APX activities decreased gradually from the seeding stage to maturation stage. The results obtained in the present study demonstrated significant upregulation of SOD and CAT activity in leaves of the Triticum aestivum L. under cadmium stress. SOD is considered as first defence against ROS as it acts on superoxide radicals, which are produced in different compartments of the cell and act as precursor to other ROS (Alscher et al., 2002). SOD dismutates two superoxide radicals to H2O2 and oxygen and thus maintains superoxide radicals in steady state level. Increase in SOD activity is attributed to increase in superoxide radical concentration. CAT is present in peroxisomes and mitochondria where it converts H2O2 to water and molecular oxygen. Increase in CAT activity can be explained by increase in its substrate i.e. to maintain the level of H2O2 as an adaptive mechanism of the plants (Reddy et al., 2005). In present study, the increased CAT activity in cadmium exposed plants may be due to the activation of its gene by increased H2O2 produced by significantly elevated SOD, as observed in the cadmium stressed plants. APX, as well as ascorbate (AsA) and glutathione (GSH), is an important component of the ascorbate-glutathione cycle -(AsA–GSH cycle) responsible for the removal of H2O2 in different cellular compartments (Gill and Tuteja, 2010) and therefore efficiently protects plants against oxidative damage. APX activity in present study was induced to an elevated level at moderate cadmium stress with decrease at high cadmium stress. Decrease in APX activity at high cadmium stress might be attributed to inactivation of enzyme by high levels of ROS, decrease in synthesis of enzyme or change in assembly of its subunits (Verma and Dubey, 2003). Nonetheless, the enzymes SOD and CAT are involved in the detoxification of Od2  and H2O2, respectively, thereby preventing the formation of OH  radicals (Gill and Tuteja, 2010). Hence, the enhanced SOD and CAT activities in the Triticum aestivum L. during their growth and development under cadmium stress suggest that Triticum aestivum L. is equipped with an efficient antioxidant mechanism against cadmium induced oxidative stress which protects the plant0 s from damage. This is in agreement with Parlak and Yilmaz (2013), who carried out an experiment to study the process of stress adaptation in Lemna gibba L. grown under cadmiums tress (0–20 mg Cd L  1) and found that the activities of antioxidants such as SOD and CAT increased linearly with increasing cadmium levels, which in turn favors the ecophysiological tolerance of Lemna gibba L. exposed to cadmium.

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4. Conclusion The micro-techniques (i.e., Rhizobox and Rhizon SMS) presented in this study allow new insights into the plant–soil interactions, especially with regard to the dynamics of root induced changes of the pH and DOC in the rhizosphere. These techniques provide new results for further understanding of the dynamic interplay between rhizosphere soil solution chemistry and the ecophysiological response in the cultivated plants under more realistic and non-destructive conditions. For example, the present study reveal that the enhancement of DOC production and the greater antioxidant enzyme activities are two important cadmium protective mechanisms of Triticum aestivum L. under cadmium stress, whereas rhizosphere acidification is an important mechanism for the mobilisation of cadmium by Triticum aestivum L. in the cadmium contaminated soils. Generally, these chemical and biochemical responses to cadmium stress are sensitive and may allow us to develop strategies for reducing the risks of the cadmium contamination to crop production. However, mechanisms affecting the cadmium tolerance of crop plants are difficult to determine due to multiple factors involved. The present study only determined the activity of the three antioxidant enzymes (SOD, CAT and APX), but the antioxidant system is very complex and it involves other enzymatic and non-enzymatic mechanisms (e.g. glutathione, ascorbate, proline, malondialdehyde and phytochelatins). These metal tolerance mechanisms, together with whether cadmium influences the activities of the antioxidants differently at low and high doses, are areas worthy of further research.

Acknowledgments This work was supported by the Special Fund for Agro-scientific Research in the Public Interest (No. 201203012-6) and the National Natural Science Foundation of China (Nos. 40571008, 41040014).

Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ecoenv.2014.01.004. References Adriano, D.C., Wenzel, W.W., Vangronsveld, J., Bolan, N.S., 2004. Role of assisted natural remediation in environmental cleanup. Geoderma 122, 121–142, http: //dx.doi.org/10.1016/j.geoderma.2004.01.003. Aebi, H., 1984. Catalase in vitro. Methods Enzymol. 105, 121–126. Ait Ali N.M., Bernal, P., Ater, M., 2004. Tolerance and bioaccumulation of cadmium by Phragmites australis grown in the presence of elevated concentrations of cadmium, copper, and zinc. Aquat. Bot. 80, 163–176. 10.1016/j.aquabot.2004.08.008. Alscher, R.G., Erturk, N., Heath, L.S., 2002. Role of superoxide dismutases (SODs) in controlling oxidative stress in plants. J. Exp. Bot. 53, 1331–1341, http://dx.doi. org/10.1093/jexbot/53.372.1331. Beauchamp, C.H., Fridovich, I., 1971. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal. Biochem. 44, 276–287, http://dx.doi. org/10.1016/0003-2697(71)90370-8. Beesley, L., Moreno-Jimenez, E., Clemente, R., Lepp, N., Dickinson, N., 2010. Mobility of arsenic, cadmium and zinc in a multi-element contaminated soil profile assessed by in-situ soil pore water sampling, column leaching and sequential extraction. Environ. Pollut. 158, 155–160, http://dx.doi.org/10.1016/j.envpol.2009.07.021. Bradford, M., 1976. A rapid and sensitive method for quantification of microgram quantities of protein utilizing the principle of proteindye binding. Anal. Biochem 72, 248–254, http://dx.doi.org/10.1016/0003-2697(76)90527-3. Cattani, I., Fragoulis, G., Boccelli, R., Capri, E., 2006. Copper bioavailability in the rhizosphere of maize (Zea mays L.) grown in two Italian soils. Chemosphere 64, 1972–1979, http://dx.doi.org/10.1016/j.chemosphere.2006.01.007. Chaudri, A.M., Allain, C.M.G., Barbosa-Jefferson, V.L., Nicholson, F.A., Chambers, B.J., McGrath, S.P., 2000. A study of the impacts of Zn and Cu on two rhizobial species in soils of a long-term field experiment. Plant. Soil. 221, 167–179, http: //dx.doi.org/10.1023/A:1004735705492. Chen, Y., Hu, W., Huang, B., Weindorf, D.C., Rajan, N., Liu, X., Niedermann, S., 2013. Accumulation and health risk of heavy metals in vegetables from harmless and

60

Y. Li et al. / Ecotoxicology and Environmental Safety 102 (2014) 55–61

organic vegetable production systems of China. Ecotox. Environ. Safe 98, 324–330. Clemente, R., Dickinson, N.M., Lepp, N.W., 2008. Mobility of metals and metalloids in a multi-element contaminated soil 20 years after cessation of the pollution source activity. Environ. Pollut. 155, 254–261, http://dx.doi.org/10.1016/j. envpol.2007.11.024. Clemente, R., Hartley, W., Riby, P., Dickinson, N.M., Lepp, N.W., 2010. Trace element mobility in a contaminated soil two years after field-amendment with a greenwaste compost mulch. Environ. Pollut. 158, 1644–1651, http://dx.doi. org/10.1016/j.envpol.2009.12.006. Cloutier-Hurteau, B., Sauvé, S., Courchesne, F., 2008. Influence of microorganisms on Cu speciation in the rhizosphere of forest soils. Soil Biol. Biochem. 40, 2441–2451 (doi:0.1016/j.soilbio.2008.06.006). Conesa, H.M., Faz, Á., Arnaldos, R., 2006. Heavy metal accumulation and tolerance in plants from mine tailings of the semiarid Cartagena-La Unión mining district (SE Spain). Sci. Total Environ. 366, 1–11, http://dx.doi.org/10.1016/j. scitotenv.2005.12.008. Cui, Y.J., Zhu, Y.G., Zhai, R.H., Chen, D.Y., Huang, Y.Z., Qiu, Y., Liang, J.Z., 2004. Transfer of metals from soil to vegetables in an area near a smelter in Nanning, China. Environ. Int. 30, 785–791, http://dx.doi.org/10.1016/j. envint.2004.01.003. Gallego, S.M., Pena, L.B., Barcia, R.A., Azpilicueta, C.E., Iannone, M.F., Rosales, E.P., Zawoznik, M.S., Groppa, M.D., Benavides, M.P., 2012. Unravelling cadmium toxicity and tolerance in plants: insight into regulatory mechanisms. Environ. Exp. Bot. 83, 33–46, http://dx.doi.org/10.1016/j.envexpbot.2012.04.006. Gill, S.S., Tuteja, N., 2010. Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiol. Biochem. 48 (12), 909–930, http://dx.doi.org/10.1016/j.plaphy.2010.08.016. Gil, C., Boluda, R., Ramos, J., 2004. Determination and evaluation of cadmium, lead and nickel in greenhouse soils of Almería (Spain). Chemosphere 55, 1027–1034, http://dx.doi.org/10.1016/j.chemosphere.2004.01.013. Greger, M., Landberg, T., 2008. Role of rhizosphere mechanisms in Cd uptake by various wheat cultivars. Plant. Soil. 312, 185–205, http://dx.doi.org/10.1007/ s11104-008-9725-y. Grossman, J., Udluft, P., 1991. The extraction of soil water by the suction-cup method: a review. J. Soil Sci. 42, 83–93, http://dx.doi.org/10.1111/j.13652389.1991.tb00093.x. Guo, H.Y., Zhu, J.G., Zhou, H., Sun, Y.Y., Yin, Y., Pei, D.P., Ji, R., Wu, J.C., Wang, X.R., 2011. Elevated CO2 levels affects the concentrations of copper and cadmium in crops grown in soil contaminated with heavy metals under fully open-air field conditions. Environ. Sci. Technol. 45, 6997–7003, http://dx.doi.org/10.1021/ es2001584. Hinsinger, P., Plassard, C., Jaillard, B., 2006. Rhizosphere: a new frontier for soil biogeochemistry. J. Geochem. Explor. 88, 210–213, http://dx.doi.org/10.1016/j. gexplo.2005.08.041. Hinsinger, P., Plassard, C., Tang, C., Jaillard, B., 2003. Origins of root-mediated pH changes in the rhizosphere and their responses to environmental constraints: a review. Plant. Soil. 248, 43–59, http://dx.doi.org/10.1023/A:1022371130939. Huynh, T.T., Zhang, H., Laidlaw, W.C., Singh, B., Baker, A.J.M., 2010. Plant-induced changes in the bioavailability of heavy metals in soil and biosolids assessed by DGT measurements. J. Soils Sediments 10, 1131–1141, http://dx.doi.org/10.1007/ s11368-010-0228-0. Hu, P.J., Qiu, R.L., Senthilkumar, P., Jiang, D., Chen, Z.W., Tang, Y.T., Liu, F.J., 2009. Tolerance, accumulation and distribution of zinc and cadmium in hyperaccumulator Potentilla griffithii. Environ. Exp. Bot. 66, 317–325, http://dx.doi.org/ 10.1016/j.envexpbot.2009.02.014. ISSCAS (Institute of Soil Science, Chinese Academy of Sciences), 1978. Physical and Chemical Analysis of SoilsShanghai Science Press, Shanghai(in Chinese) Kafel, A., Nadgórska-Socha, A., Gospodarek, J., Babczyńska, A., Skowronek, M., Kandziora, M., Rozpędek, K., 2010. The effects of Aphis fabae infestation on the antioxidant response and heavy metal content in field grown Philadelphus coronarius plants. Sci. Total Environ. 408, 1111–1119, http://dx.doi.org/10.1016/j. scitotenv.2009.11.013. Kirkham, M.B., 2006. Cadmium in plants on polluted soils: effects of soil factors, hyperaccumulation, and amendments. Geoderma 137, 19–32, http://dx.doi.org/ 10.1016/j.geoderma.2006.08.024. Khan, K., Lu, Y., Khan, H., Ishtiaq, M., Khan, S., Waqas, M., Wei, L., Wang, T., 2013. Heavy metals in agricultural soils and crops and their health risks in Swat District, northern Pakistan. Food Chem. Toxicol. 58, 449–458, http://dx.doi.org/ 10.1016/j.fct.2013.05.014. Kim, S., Kang, H., 2011. Effects of elevated CO2 and Pb on phytoextraction and enzyme activity. Water Air Soil Pollut. 19, 365–375, http://dx.doi.org/10.1007/ s11270-010-0713-5. Knight, B.P., Chaudri, A.M., McGrath, S.P., Giller, K.E., 1998. Determination of chemical availability of cadmium and zinc in soils using inert soil moisture samplers. Environ. Pollut. 99, 293–298, http://dx.doi.org/10.1016/S0269-7491 (98)00021-9. Lal, K., Minhas, P.S., Chaturvedi, S.R.K., Yadav, R.K., 2008. Extraction of cadmium and tolerance of three annual cut flowers on Cd-contaminated soils. Bioresour. Technol. 99, 1006–1011, http://dx.doi.org/10.1016/j.biortech.2007.03.005. Li, F., Ni, L., Yuan, J., Sheng, G.D., 2010. Cultivation practices affect heavy metal migration between soil and Vicia faba (broad bean). Chemosphere 80, 1393–1398, http://dx.doi.org/10.1016/j.chemosphere.2010.06.001. Li, T., Di, Z., Islam, E., Jiang, H., Yang, X., 2011. Rhizosphere characteristics of zinc hyperaccumulator Sedum alfredii involved in zinc accumulation. J. Hazard. Mater. 185, 818–823, http://dx.doi.org/10.1016/j.jhazmat.2010.09.093.

Li, T., Tao, Q., Han, X., Yang, X., 2013a. Effects of elevated CO2 on rhizosphere characteristics of Cd/Zn hyperaccumulator Sedum alfredii. Sci. Total Environ. 454-455, 510–516, http://dx.doi.org/10.1016/j.scitotenv.2013.03.054. Li, Y., Sun, H., Li, H., Yang, L., Ye, B., Wang, W., 2013b. Dynamic changes of rhizosphere properties and antioxidant enzyme responses of wheat plants (Triticum aestivum L.) grown in mercury-contaminated soils. Chemosphere 93, 972–977, http://dx.doi.org/10.1016/j.chemosphere.2013.05.063. Li, Z., Ma, Z., van der Kuijp, T.J., Yuan, Z, Huang, L., 2014. A review of soil heavy metal pollution from mines in China: pollution and health risk assessment. Sci. Total Environ. 468–469, 843–853, http://dx.doi.org/10.1016/j.scitotenv.2013.08.090. Luo, Y., Qiao, X., Song, J., Christie, P., Wong, M., 2003. Use of a multi-layer column device for study on leachability of nitrate in sludge-amended soils. Chemosphere 52, 1483–1488, http://dx.doi.org/10.1016/S0045-6535(03)00486-7. Majewska, M., Kurek, E., 2011. Effect of Cd concentration in growth media on Secale cereal roots and Cd interaction with rhizosphere microorganisms originating from different parts of the grain. Eur. J. Soil Biol. 47, 95–101, http://dx.doi.org/ 10.1016/j.ejsobi.2010.12.005. McBeath, J., McBeath, J.H., 2009. Environmental stressors and food security in China. J. Chin. Polit. Sci. 14, 49–80, http://dx.doi.org/10.1007/s11366-008-9036-4. McBride, M.B., 1994. Environmental Chemistry of Soils. Oxford University Press, New York Motaghian, H.R., Hosseinpur, A.R., 2013. Copper desorption kinetics in wheat (Triticum aestivum L.) rhizosphere in some sewage sludge amended soils. Environ. Earth Sci.. 10.1007/s12665-013-2242-1. Nabulo, G., Black, C.R., Craigon, J., Young, S.D., 2012. Does consumption of leafy vegetables grown in peri-urban agriculture pose a risk to human health? Environ. Pollut. 162, 389–398, http://dx.doi.org/10.1016/j.envpol.2011.11.040. Nabulo, G., Black, C.R., Young, S.D., 2011. Trace metal uptake by tropical vegetables grown on soil amended with urban sewage sludge. Environ. Pollut. 159, 368–376, http://dx.doi.org/10.1016/j.envpol.2010.11.007. Nakano, Y., Asada, K., 1981. Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant. Cell. Physiol. 22, 867–880. Niu, L., Yang, F., Xu, C., Yang, H., Liu, W., 2013. Status of metal accumulation in farmland soils across China: from distribution to risk assessment. Environ. Pollut. 176, 55–62, http://dx.doi.org/10.1016/j.envpol.2013.01.019. Nye, P.H., 1981. Changes of pH across the rhizosphere induced by roots. Plant Soil. 61, 7–26, http://dx.doi.org/10.1007/BF02277359. Padmavathiamma, P.K., Li, L.Y., 2012. Rhizosphere influence and seasonal impact on phytostabilisation of metals  a field study. Water Air Soil Pollut. 223, 107–124, http://dx.doi.org/10.1007/s11270-011-0843-4. Page, V., Bayon, R.C.L., Urs Feller, U., 2006. Partitioning of zinc, cadmium, manganese and cobalt in wheat (Triticum aestivum) and lupin (Lupinus albus) and further release into the soil. Environ. Exp. Bot. 58, 269–278, http://dx.doi. org/10.1016/j.envexpbot.2005.09.005. Parlak, K.U., Yilmaz, D.D., 2013. Ecophysiological tolerance of Lemna gibba L. exposed to cadmium. Ecotox. Environ. Safe 91, 79–85, http://dx.doi.org/ 10.1016/j.ecoenv.2013.01.009. Pérez, A.L., Anderson, K.A., 2009. DGT estimates cadmium accumulation in wheat and potato from phosphate fertilizer applications. Sci. Total Environ. 407, 5096–5103, http://dx.doi.org/10.1016/j.scitotenv.2009.05.045. Reddy, A.M., Kumar, S.G., Jyonthsnakumari, G., Thimmanaik, S., Sudhakar, C., 2005. Lead induced changes in antioxidant metabolism of horsegram (Macrotyloma uniflorum (Lam.) Verdc.) and bengalgram (Cicer arietinum L.). Chemospere 60, 97–104, http://dx.doi.org/10.1016/j.chemosphere.2004.11.092. Robinson, B., Charles Russell, C., Hedley, M., Clothier, B., 2001. Cadmium adsorption by rhizobacteria: implications for New Zealand pastureland. Agr. Ecosyst. Environ. 87, 315–321. Séguin, V., Gagnon, C., Courchesne, F., 2004. Changes in water extractable metals, pH and organic carbon concentrations at the soil–root interface of forested soils. Plant Soil 260, 1–17, http://dx.doi.org/10.1023/B:PLSO.0000030170.49493.5f. Shao, H.B., Liang, Z.S., Shao, M.A., Sun, Q., 2005. Dynamic changes of anti-oxidative enzymes of 10 wheat genotypes at soil water deficits. Colloid Surf. B 42, 187–195, http://dx.doi.org/10.1016/j.colsurfb.2005.02.007. Singh, H.P., Batish, D.R., Kaur, G., Arora, K., Kohli, R.K., 2008. Nitric oxide (as sodium nitroprusside) supplementation ameliorates Cd toxicity in hydroponically grown wheat roots. Environ. Exp. Bot. 63, 158–167, http://dx.doi.org/10.1016/ j.envexpbot.2007.12.005. Song, N.H., Yin, X.L., Chen, G.F., Yang, H., 2007. Biological responses of wheat (Triticum aestivum) plants to the herbicide chlorotoluron in soils. Chemosphere 68, 1779–1787, http://dx.doi.org/10.1016/j.chemosphere.2007.03.023. Sun, C., Liu, J., Wang, Y., Sun, L., Yu, H., 2013. Multivariate and geostatistical analyses of the spatial distribution and sources of heavy metals in agricultural soil in Dehui, Northeast China. Chemosphere 92, 517–523, http://dx.doi.org/10.1016/j. chemosphere.2013.02.063. Tiryakioglu, M., Eker, S., Ozkutlu, F., Husted, S., Cakmak, I., 2006. Antioxidant defence system and cadmium uptake in barley genotypes differing in cadmium tolerance. J. Trace Elem. Med. Biol. 20, 181–189, http://dx.doi.org/10.1016/j. jtemb.2005.12.004. Tuteja, N., Gill, S.S., 2013. Crop improvement under adverse conditions. Springer, New York (Science þ Business Media) Valentovičová, K., Halušková, L., Huttová, J., Mistrík, I., Tamás, L., 2010. Effect of cadmium on diaphorase activity and nitric oxide production in barley root tips. J. Plant Physiol. 167, 10–14, http://dx.doi.org/10.1016/j.jplph.2009.06.018. Vaughan, D., Lumsdom, D.G., Linehan, D.J., 1993. Influence of dissolved organic matter on the bio-availability and toxicity of metals in soils and aquatic systems. Chem. Ecol. 8, 185–201, http://dx.doi.org/10.1080/02757549308035308.

Y. Li et al. / Ecotoxicology and Environmental Safety 102 (2014) 55–61

Verma, S., Dubey, R.S., 2003. Lead toxicity induces lipid peroxidation and alters the activities of antioxidant enzymes in growing rice plants. Plant. Sci. 164, 645–655, http://dx.doi.org/10.1016/S0168-9452(03)00022-0. Wang, M.Y., Chen, A.K., Wong, M.H., Qiu, R.L., Cheng, H., Ye, Z.H., 2011. Cadmium accumulation in and tolerance of rice (Oryza sativa L.) varieties with different rates of radial oxygen loss. Environ. Pollut. 159, 1730–1736, http://dx.doi.org/ 10.1016/j.envpol.2011.02.025. Wang, Z., Shan, X.Q., Zhang, S., 2002. Comparison between fractionation and bioavailability of trace elements in rhizosphere and bulk soils. Chemosphere 46, 1163–1171, http://dx.doi.org/10.1016/S0045-6535(01)00206-5. Williams, P.N., Lei, M., Sun, G.X., Huang, Q., Lu, Y., Deacon, C., Meharg, A.A., Zhu, Y.G., 2009. Occurrence and partitioning of cadmium, arsenic and lead in mine impacted paddy rice: Hunan, China. Environ. Sci. Technol., 43; , pp. 637–642, http://dx.doi.org/10.1021/es802412r.

61

Yang, Y., Wei, X., Lu, J., You, J., Wang, W., Shi, R., 2010. Lead-induced phytotoxicity mechanism involved in seed germination and seedling growth of wheat (Triticum aestivum L.). Ecotox. Environ. Safe 73, 1982–1987, http://dx.doi.org/ 10.1016/j.ecoenv.2010.08.041. Yang, L.S., Zhang, X.W., Li, Y.H., Li, H.R., Wang, Y., Wang, W.Y., 2012. Bioaccessibility and risk assessment of cadmium from uncooked rice using an in vitro digestion model. Biol. Trace Elem. Res. 145, 81–86, http://dx.doi.org/10.1007/s12011-011-9159-x. Zeng, L.S., Liao, M., Chen, C.L., Huang, C.Y., 2006. Effects of lead contamination on soil microbial activity and rice physiological indices in soil–Pb–rice (Oryza sativa L.) system. Chemosphere 65, 567–574, http://dx.doi.org/10.1016/j. chemosphere.2006.02.039. Zhang, X., Zhang, S., Xu, X., Li, T., Gong, G., Jia, Y., Li, Y., Deng, L., 2010. Tolerance and accumulation characteristics of cadmium in Amaranthus hybridus L. J. Hazard. Mater. 180, 303–308, http://dx.doi.org/10.1016/j.jhazmat.2010.04.031.