Dynamic changes of rhizosphere properties and antioxidant enzyme responses of wheat plants (Triticum aestivum L.) grown in mercury-contaminated soils

Chemosphere xxx (2013) xxx–xxx

Contents lists available at SciVerse ScienceDirect

Chemosphere journal homepage: www.elsevier.com/locate/chemosphere

Dynamic changes of rhizosphere properties and antioxidant enzyme responses of wheat plants (Triticum aestivum L.) grown in mercury-contaminated soils Yonghua Li a,⇑, Hongfei Sun a,b, Hairong Li a, Linsheng Yang a, Bixiong Ye a, Wuyi Wang a a b

Institute of Geographical Sciences and Natural Resources Research, Chinese Academy of Sciences, Beijing 100101, China College of Environment and Plant Protection, Hainan University, Haikou 570228, China

h i g h l i g h t s  Non-destructive micro-technique is used for the collection of rhizosphere solution.  Acidification is observed in the rhizosphere soil solution during growth period.  Enhanced DOC and antioxidant enzyme activity are involved in Hg tolerance in plant.  Wheat plant (Triticum aestivum L.) can positively adapt to environmental Hg stress.

a r t i c l e

i n f o

Article history: Received 14 September 2012 Received in revised form 6 May 2013 Accepted 25 May 2013 Available online xxxx Keywords: Mercury Rhizobox Rhizosphere soil solution Antioxidants Triticum aestivum L.

a b s t r a c t A pot experiment was conducted to investigate the dynamic changes in the rhizosphere properties and antioxidant enzyme responses of wheat plants (Triticum aestivum L.) grown in three levels of Hg-contaminated soils. The concentrations of soluble Hg and dissolved organic carbon (DOC) in the rhizosphere soil solutions of the wheat plants were characterised by the sequence before sowing > trefoil stage > stooling stage, whereas the soil solution pH was found to follow an opposite distribution pattern. The activities of antioxidant enzymes in wheat plants under Hg stress were substantially altered. Greater superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) activities were observed in the wheat plants grown in a highly polluted soil than in a slightly polluted soil (with increases of 11–27% at the trefoil stage and 26–70% at the stooling stage); however, increasing concentrations of Hg up to seriously polluted level led to reduced enzyme activities. The present results suggest that wheat plants could positively adapt to environmental Hg stress, with rhizosphere acidification, the enhancement of DOC production and greater antioxidant enzyme activities perhaps being three important mechanisms involved in the metal uptake/tolerance in the rhizospheres of wheat plants grown in Hg-contaminated soils. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Due to the continuous exploitation of mineral resources and the widespread application of fertilisers, sewage sludge and Hg-containing fungicides in the production of horticultural crops and vegetables, the annual introduction of mercury into agricultural lands has become an increasingly important concern (Patra and Sharma, 2000; Zhou et al., 2008; Feng and Qiu, 2008; Fu et al., 2010). It has been estimated that the average Hg level in the global arable lands was 39 kg km 2 in 2000 (Han et al., 2002). In China, 20 million ha, accounting for approximately 20% of the total area

⇑ Corresponding author. Address: Institute of Geographic Sciences and Natural Resources Research, Chinese Academy of Sciences, 11A Datun Road, Chaoyang District, Beijing 100101, China. Tel.: +86 10 64889198; fax: +86 10 64856504. E-mail address: [email protected] (Y. Li).

under cultivation, is contaminated by Hg and other heavy metals from various human activities (Zeng et al., 2006). Increasing evidence has shown that Hg2+, the predominant form of Hg in agricultural soils (Fu et al., 2010), can readily accumulate in higher plants (Wang and Greger, 2004; Israr et al., 2006). Unfortunately, Hg is a well-documented non-essential element in most higher plants (Salt et al., 1995; Rellán-Álvarez et al., 2006), and the accumulation of Hg by plants may disrupt many cellular functions and reduce growth (Hall, 2002; Feng and Qiu, 2008; Gao et al., 2010; Costa et al., 2011; Sahu et al., 2012). Meanwhile, the intake of relatively low doses of Hg over a long period leads to the malfunction of human organs and may cause chronic human toxicity (WHO, 1979). The large input of Hg into arable lands affects the growth of crops and the quality of the agricultural products and also poses serious threats to human health through the food chain. Therefore, it is of great importance to investigate

0045-6535/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.chemosphere.2013.05.063

Please cite this article in press as: Li, Y., et al. Dynamic changes of rhizosphere properties and antioxidant enzyme responses of wheat plants (Triticum aestivum L.) grown in mercury-contaminated soils. Chemosphere (2013), http://dx.doi.org/10.1016/j.chemosphere.2013.05.063

2

Y. Li et al. / Chemosphere xxx (2013) xxx–xxx

both toxicological processes and adaptive mechanisms to minimise Hg accumulation in crop plants (Elbaz et al., 2010; Chen and Yang, 2012). It is well documented that the root-soil interactions in the rhizosphere play a key role in controlling the metal bioavailability to plants (Huynh et al., 2010; Li et al., 2011). The soil solution is the medium through which the beneficial and/or potentially toxic elements exert their effects on organisms (Knight et al., 1998). The concentration of Hg in the rhizosphere soil solution is considered to accurately reflect the plant-available concentration of Hg in the soil, which is influenced by such soil properties as the dissolved organic carbon (DOC) and pH, as they are closely connected to the chemical processes of precipitation, sorption and complexation (Sauvé et al., 1996; Vijver et al., 2003). Therefore, the analysis of the rhizosphere soil solution can provide valuable information concerning the potential toxicity of the Hg in the soil. 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 (Tiensing et al., 2001; Clemente et al., 2008; Beesley et al., 2010). In contrast, obtaining the soil solution by soil sampling, followed by centrifugation or extraction with water is operationally strongly biased, laborious and time consuming. Furthermore, because the sampling is destructive, such methodology does not allow for long-term dynamic studies. Similar to other heavy metals, Hg is able to induce oxidative stress by triggering the generation of reactive oxygen species (ROS), e.g., superoxide radical ( O2 ), hydrogen peroxide (H2O2) and hydroxyl radical (OH), in plants (Cho and Park, 2000; Cargnelutti et al., 2006). To resist oxidative damage, plants have developed antioxidant protective mechanisms that enable them to counteract the production of ROS. These protective mechanisms include changes in the lipid composition, changes in the antioxidant enzyme activity, increased sugar or amino acid contents, and changes in the level of soluble proteins and gene expression (Miller et al., 2008; Gao et al., 2010). In several plants, it has been reported that the activities of ROS-scavenging enzymes, including superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX), are important protective mechanisms to minimise the oxidative damage due to the exposure to Hg toxicity (Israr et al., 2006; Feng and Qiu, 2008; Chen and Yang, 2012). In contrast to other heavy metals, such as Cd, Cu and Zn, whose regulatory mechanisms in plants have been well described (Luo et al., 2000; Yang et al., 2001; Pereira et al., 2002; Santibáñez et al., 2008; Sun et al., 2009), the biochemical mechanisms for Hg-induced toxicity and the modification of metabolic pathways under Hg stress in plants, particularly in crop plants, remain to be elucidated (Feng and Qiu, 2008; Elbaz et al., 2010; Costa et al., 2011). In the present study, we used wheat plants (Triticum aestivum L.) 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, wheat is frequently used as an ecotoxicological indicator (Song et al., 2007). A pot experiment was conducted to study the dynamic changes in the rhizosphere properties and antioxidant enzyme responses of wheat plants grown in three levels of Hg-contaminated soils. The specific objectives of this study were (1) to monitor the temporal variation in the bioavailable Hg in the rhizoshphere soil solution during the different

growth stages (i.e., before sowing, the trefoil stage and the stooling stage) of the wheat plants, (2) to relate them with the dynamics of several physico-chemical parameters and (3) to investigate the changes in the activities of the antioxidant enzymes SOD, CAT and APX in the leaves of the wheat plants under different Hg stresses. The results of the study were anticipated to assist in the development of an appropriate method that can be used to estimate the degree of toxicity in Hg-contaminated soils. 2. Materials and methods 2.1. Soil sampling and characterisation Three agricultural soils (S1, S2 and S3) containing different Hg concentrations were used in the greenhouse pot experiment. S1 and S2 were collected from the centre and the boundary of a historic Hg mine located in southwestern China, respectively; the S3 soil was sampled at approximately 40 km north of the Hg mine. The three soil samples were typical fragiudalfs, which were derived dominantly from shale. The soil samples were collected from the surface layer (0–20 cm) using a plastic shovel and the soils were then sealed in polyethylene bags. 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 to ensure homogeneity in the mineral composition and stored in sealed polyethylene bags for the subsequent analysis. Care was taken to avoid cross contamination in the processes of sampling, drying and grinding. The soil pH (soil:deionised water = 1:2.5, w/v) and soil organic matter (SOM) were measured according to the conventional methods issued by Institute of Soil Science, Chinese Academy of Sciences (ISSCAS) (1978), and the soil grain size was determined by laserdiffraction analysis (Mastersizer 2000, Malvern, UK). The total Hg concentration in the soils was determined using 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 material (soil GBW07404) were used during the analyses. The analytical results of Hg (59.8 ± 6.2 in ng g 1, n = 7) in the certified reference material were in good agreement with the certified values (60.0 ± 6.0 in ng g 1), with RSD in range from 8.71% to 10.37%. The percentage of the recoveries of the spiked samples ranged from 87% to 110%. The detection limit for Hg using ICP-MS was 2 ng L 1. 2.2. Greenhouse pot experiment design The rhizobox used in this study was designed in a similar way to that of Li et al. (2011) (Fig. 1). 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). The root growth was limited to the rhizosphere compartment. Approximately 0.5 kg of soil was placed in the rhizosphere compartment, and 3.0 kg was placed in the non-rhizosphere compartment. Initially, a 50% water-holding capacity (WHC) was generated using deionised water; 2 weeks prior to sowing, the level was increased to 75% WHC. Ten seeds of wheat 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

Please cite this article in press as: Li, Y., et al. Dynamic changes of rhizosphere properties and antioxidant enzyme responses of wheat plants (Triticum aestivum L.) grown in mercury-contaminated soils. Chemosphere (2013), http://dx.doi.org/10.1016/j.chemosphere.2013.05.063

3

Y. Li et al. / Chemosphere xxx (2013) xxx–xxx

2.5. Extraction of proteins and enzymes Fresh leaf tissues were homogenised (1:5 w/v) separately in a cold mortar using 50 mM potassium phosphate buffer (pH 7.0) containing 1 mM ethylenediaminetetraacetate (EDTA), 1% polyvinylpyrrolidone (PVP), and 1 mM ascorbate (in the case of the APX assay). The homogenate was centrifuged at 15 000g for 15 min at 4 °C, 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. 2.6. Assay of enzyme activities

Fig. 1. Scheme of the rhizobox and the soil solution sampling: (A) rhizosphere compartment, (B) non-rhizosphere compartment, (C) rhizon soil solution sampler.

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% relative humidity and a temperature of 25–30 °C), and the positions of the rhizoboxes were rotated regularly to ensure uniform growing conditions.

2.3. Rhizosphere soil solution and wheat leaf sampling Rhizon SMS samplers (Rhizosphere Research Products, Wageningen, the Netherlands) were used to extract the rhizosphere soil solution around the cluster roots; the extractions were performed in triplicate. The samplers consist of 5 cm of inert porous plastic tubing (2.5 mm diameter), capped with nylon wire at one end and connected to 30 cm-long PVC/PE tubing joined to a female lock. The capped end was inserted into each rhizosphere compartment during the addition of the soil. Two days before the sampling, the soil humidity was maintained at 100% of the WHC. After 48 h of water equilibration, a syringe needle was connected to the female lock and inserted into a 10 mL metal-free glass vacuum tube to extract the soil solution by vacuum. In this way, approximately 9 mL of soil solution was extracted overnight. Three batches of rhizosphere soil solution were obtained. The first batch was collected before sowing the wheat seeds, and the second and the third batches were collected when the growing wheat plants were at the trefoil stage and stooling stage, respectively. When the soil solution of these pots had been collected, approximately 2 g of fresh wheat leaves from each pot were harvested, rinsed with deionised water, blotted and immediately frozen in liquid nitrogen or stored at 80 °C for the protein and enzyme activity assays.

2.4. Rhizosphere soil solution analysis The extracted rhizosphere soil solutions were separated into several sub-samples for analysis. The pH and DOC were promptly analysed after sample collection. The pH was determined using a calibrated Thermo Orion 868 pH Meter (Thermo Orion Inc., USA), and the DOC was measured using a TOC-5050A TOC analyser (Shimadzu, Japan). The concentration of Hg in the soil solutions was determined using a Model 9000 ICP-MS (PerkinElmer, USA).

The SOD (EC 1.1.5.1.1) assay was performed according to Beauchamp and Fridovixh (1971). The 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 lL 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 determined by the consumption of H2O2, as the absorbance at 240 nm, according to the method of Aebi (1984). The 3 mL reaction mixture contained 50 mM sodium phosphate buffer (pH 7.0), 10 mM H2O2 and 10 lL 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 lL enzyme extract. The absorbance was measured at 290 nm. The activity was calculated using the extinction coefficient 2.8 mM 1 cm 1. 2.7. 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 Student’s t-test methods.

3. Results and discussion 3.1. Properties of the tested soils The general properties and metal concentrations of the soils under study are presented in Table 1. Only minor changes occurred in the soil organic matter and soil texture for the three soils. However, the Hg concentrations varied greatly (between 0.32 and 30.00 mg kg 1), reflecting the mining origins of some of the samples.

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

a b

Soil

pHa

SOMb (g kg 1)

Sand (%)

Silt (%)

Clay (%)

Total Hg (mg kg 1)

S1 S2 S3

7.54 6.09 6.32

43.6 42.9 48.4

39.0 40.0 26.8

47.3 44.5 56.9

13.2 15.5 16.3

30.00 10.90 0.32

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

Please cite this article in press as: Li, Y., et al. Dynamic changes of rhizosphere properties and antioxidant enzyme responses of wheat plants (Triticum aestivum L.) grown in mercury-contaminated soils. Chemosphere (2013), http://dx.doi.org/10.1016/j.chemosphere.2013.05.063

Y. Li et al. / Chemosphere xxx (2013) xxx–xxx

3.2. Soluble Hg in the rhizosphere soil solution

0.5 -1

The index of geoaccumulation (Igeo) developed by Müller (1969) was adopted to assess the soil quality contaminated by Hg: Igeo = log2(Cn/1.5Bn), where Cn was the measured concentration of the element in the soils, Bn was the content of the element in the average shale, and factor 1.5 was introduced to include the possible differences in the background values due to lithological variations. The Müller Igeo consists of seven grades ranging from uncontaminated to very seriously polluted. Igeo of Hg in soils at levels of 0, 0–1, 1–2, 2–3, 3–4, 4–5 and 5, representing uncontaminated, slightly polluted, moderately polluted, moderately to highly polluted, highly polluted, highly to very highly polluted and very seriously polluted, respectively. In our calculation of Igeo, Bn was the crustal average Hg content of 0.08 mg kg 1, as suggested by Habashi (1997). The corresponding Igeo and contamination levels of Hg in S1, S2, and S3 are 5.52, 4.86, and 0.98, indicating very seriously polluted, highly to very highly polluted, and slightly polluted, respectively. Therefore, in this pot experiment, S1, S2, and S3 represent very seriously Hg-polluted soil, highly to very highly Hg-polluted soil and slightly Hgpolluted soil, respectively.

Soluble Hg (µg L )

4

0.4

Before sowing Trefoil stage Stooling stage

a a

0.3 0.2 b 0.1

a

b c

c

S1

S2

3.3. Changes in the pH and DOC in the rhizosphere soil solution The temporal changes in the pH and DOC in the rhizosphere soil solution of the wheat plants for the three Hg-contaminated soils are illustrated in Fig. 3. In general, no significant changes in either the pH or DOC were observed in the rhizosphere soil solution among the different growth stages. However, a general soil solution pH trend was observed to follow the sequence before sowing > trefoil stage > stooling stage (Fig. 3A), whereas the concentration of DOC followed the opposite sequence before sowing < trefoil stage < stooling stage (Fig. 3B). When compared to the rhizosphere soil solution of the wheat plants before sowing, the soil solution in the trefoil and stooling stages were reduced 0.1–0.2 and 0.4–0.9 pH units, respectively, and the reduction in the heavily to very highly Hg-contaminated soils was greater than that in the slightly Hg-contaminated soil (Fig. 3A). Most previous work on the soil-root interface shows an

c

S3

Fig. 2. Hg concentrations in the rhizosphere soil solution of wheat plants before sowing and at the trefoil and stooling stages in three Hg-contaminated soils. S1, S2, and S3 represent a very seriously polluted soil, highly to very highly polluted soil and slightly polluted soil, respectively. The bars represent the standard deviations of four replicates. The bars with different letters among the different stages are significantly different at p < 0.05 by Student’s t-test.

A 8.0

a

a

Before sowing Trefoil stage Stooling stage a a a

a 7.0

pH

a 6.0

a b

5.0

4.0 S1

B

S2

S3

60 50

-1

DOC (mg kg )

The water-soluble Hg in the rhizosphere soil solutions of the wheat plants was determined at three different stages, namely, before sowing, the trefoil stage and the stooling stage (Fig. 2). The concentrations of the soluble Hg in the rhizosphere soil solution at both the trefoil and stooling stages for the three Hg-contaminated soils significantly decreased compared to their initial values (before sowing), with the decrease at the stooling stage being much greater than the trefoil stage (p < 0.05). When compared to their initial values, the soluble Hg levels of S1, S2 and S3 were 33%, 22%, and 40% lower at the trefoil stage and 7%, 7%, and 24% lower at the stooling stage, respectively. However, in contrast to the slightly Hg-polluted soil (S3), the highly to very highly Hg-polluted soil (S2) and the very seriously Hg-polluted soil (S1) showed more drastic decreases in the water-soluble Hg concentration after the growth of the wheat plants. Consequently, the dynamics of Hg in the rhizosphere soil solutions of the wheat plants over the culture period were characterised by a decrease in the three soils over time. The mechanism may be interpreted as an impact of the plant activity that may affect the fate of the Hg in the soil, notably through the uptake of nutrients from the rhizosphere. This interpretation echoes the finding by Luo et al. (2000) who observed that the soil solution Zn in the rhizosphere of Thlaspi caerulescens grown in a Zn-contaminated soil dropped from 23 to 2 mg L 1 during the period from 10 d to 84 d after transplanting.

b

0.0

b

40 30

a

a

a

a

Before sowing Trefoil stage Stooling stage a a a

a

20 10 0 S1

S2

S3

Fig. 3. The pH (A) and dissolved organic carbon (DOC) concentrations (B) in the rhizosphere soil solution of wheat plants before sowing and at the trefoil and stooling stages in three Hg-contaminated soils. S1, S2, and S3 represent a very seriously polluted soil, highly to very highly polluted soil and slightly polluted soil, respectively. The bars represent the standard deviations of four replicates. The bars with different letters among the different stages are significantly different at p < 0.05 by Student’s t-test.

acidification of the rhizosphere (Dessureault-Rompré et al., 2006; Li et al., 2011), though alkalinisation is also observed (Luo et al., 2000). Within this context, it can be considered that the present results are in good agreement with the literature. The plantmediated changes in the pH may be caused by an imbalance in the release and/of uptake of cations or anions (Nye, 1981). To maintain the electroneutrality at the root–soil interface, plants will compensate by releasing OH or H+ depending on whether anions or cations, respectively, are taken up in excess. According to this process, an acidification of the rhizosphere will occur when the uptake imbalance favours cations. Moreover, the release of CO2 from root respiration may accelerate the reduction in the pH of

Please cite this article in press as: Li, Y., et al. Dynamic changes of rhizosphere properties and antioxidant enzyme responses of wheat plants (Triticum aestivum L.) grown in mercury-contaminated soils. Chemosphere (2013), http://dx.doi.org/10.1016/j.chemosphere.2013.05.063

Y. Li et al. / Chemosphere xxx (2013) xxx–xxx

the solution (Padmavathiamma and Li, 2012). Given that the soil solution pH is one of the most important chemical factors controlling the availability of heavy metals and that a reduction of the rhizosphere pH increases the chemical activity of most metals, rhizosphere acidification may, thus, be an important mechanism for the mobilisation of Hg by wheat plants in Hgcontaminated soils. The soil solution DOC increased over the course of the growth period, probably due to the increased release of root exudates, in particular, low molecular weight organic acids, a finding that was consistent with the reduced pH values in the rhizosphere soil solution. The presence of these acids has been found to play an important role in the mobilisation and uptake of nutrients and the detoxification of harmful substances (Dessureault-Rompré et al., 2006). Therefore, we suggest that it was plausible to presume that the rhizosphere acidification and exudation of high amounts of DOC were the important mechanisms involved in the observed mobilisation and uptake of Hg in the rhizosphere of the wheat plants grown in the Hg-contaminated soils.

160 b

b

Trefoil stage Stooling stage

120

b

-1

SOD activity (U mg protein)

A

80

a a

a 40

0 S1

-1

CAT activity (U mg protein)

B

S2

S3

300 b 250

b

Trefoil stage Stooling stage b

200 150

a

a

a

100 50 0 S1

3.4. Changes in the antioxidant enzyme activity The effects of Hg on the antioxidant enzyme activities in the leaves of the wheat plants at three levels of Hg-contaminated soils at the trefoil and stooling stages are shown in Fig. 4. As indicated, the activities of the antioxidant enzymes in the wheat plants changed substantially under Hg stress. When compared to the slightly Hg-polluted soil (S3), the activities of all of the tested antioxidant enzymes, SOD (Fig. 4A), CAT (Fig. 4B) and APX (Fig. 4C), were significantly enhanced in the highly to very highly Hg-polluted soil (S2) (p < 0.05), with increases ranging from 11% to 27% at the trefoil stage and from 26% to 70% at the stooling stage. Greater Hg concentrations in the potting soil resulted in greater antioxidant enzyme activities in the wheat plants, indicating that the Hg induced the wheat plants to increase their antioxidant enzyme activities, consequently increasing their Hg uptake and tolerance. However, increasing the concentration of Hg up to the seriously polluted level led to decreased activities. When compared to the highly to very highly Hg-polluted soil (S2), the activities of SOD, CAT and APX decreased clearly but non-statistically significant in the very seriously Hg polluted soil (S1), with reductions of 5–15% at the trefoil stage and 5–2% at the stooling stage. Consequently, it was possible that the antioxidant enzymes might be impaired in the soils seriously suffering from Hg pollution. The reason for the decrease in the antioxidant enzyme activities might be the increase in the ROS (e.g.,  O2 , H2O2, OH) induced by the relatively higher level of Hg that, in turn, inactivated the activities of these antioxidant enzymes. Simultaneously, it was observed that the activities of both SOD and CAT in the leaves of the wheat plants significantly increased from the trefoil stage to the stooling stage (p < 0.05), regardless of the levels of initial Hg in the potting soils; in contrast, the APX activity decreased from the trefoil stage to the stooling stage. The SOD and CAT activities of the wheat plants at the stooling stage in S1, S2 and S3 were 2.34, 2.09, and 2.10 times higher and 1.63, 1.72, and 1.53 times higher, respectively, when compared to the trefoil stage. Conversely, the APX activities of the wheat plants at the stooling stage in S1, S2 and S3 were 0.66, 0.91 and 0.63 times lower compared to the trefoil 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. Nonetheless, the enhanced SOD and CAT activities in the wheat plants during their growth and development under Hg stress are circumstantial evidence for the tolerance mechanisms developed by this plant.

S3

4. Conclusion 80 a

a

a

60

Trefoil stage Stooling stage a

a

-1

APX activity (U mg protein)

C

S2

5

40

b

20

0 S1

S2

S3

Fig. 4. Activity of SOD (A), CAT (B) and APX (C) in the leaves of wheat plants at the trefoil and stooling stages in three Hg-contaminated soils. S1, S2, and S3 represent a very seriously polluted soil, highly to very highly polluted soil and slightly polluted soil, respectively. The bars represent the standard deviations of four replicates. The bars with different letters between the trefoil and stooling stages are significantly different at p < 0.05 by Student’s t-test.

Plant–soil interactions strongly influence the soil solution chemistry in the rhizosphere, which, in turn, can stimulate biochemical responses in the plant. To date, most of our knowledge on the changes in the rhizosphere properties stems from studies using hydroponic systems or soil extracts. 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 uptake/tolerance mechanisms remains elusive. Accordingly, there is a great need for continuous in situ studies in plant–soil systems. In the present study, we used micro-techniques (i.e., rhizobox and rhizon soil solution sampler) for the collection of the soil solution of the wheat plants at the stages of before sowing, the trefoil stage and the stooling stage in three levels of Hg-contaminated soils, thus enabling the non-destructive in situ observation of the rhizosphere soil properties at a high temporal resolution.

Please cite this article in press as: Li, Y., et al. Dynamic changes of rhizosphere properties and antioxidant enzyme responses of wheat plants (Triticum aestivum L.) grown in mercury-contaminated soils. Chemosphere (2013), http://dx.doi.org/10.1016/j.chemosphere.2013.05.063

6

Y. Li et al. / Chemosphere xxx (2013) xxx–xxx

The soluble Hg in the rhizosphere soil solutions of the wheat plants over the culture period were characterised by a decrease over time, and the reduction was more drastic in the highly Hgpolluted soil and seriously Hg-polluted soil than the slightly Hgpolluted soil. Simultaneously, a general increasing trend in the soil solution pH during the growth of the wheat plants was observed, whereas the concentration of the DOC followed the opposite distribution pattern. The greater SOD, CAT, and APX activities in the wheat plants grown in the highly polluted soil than in the slightly polluted soil suggested an increased tolerance and capacity of the plant to protect itself from the oxidative damage induced by Hg stress. However, increasing the concentration of Hg to the seriously polluted level led to decreased activities. Taken together, the present results allow us to conclude that the wheat plants showed a positive response to Hg stress. Rhizosphere acidification, the enhancement of DOC production and the greater antioxidant enzyme activities were three important mechanisms involved in the metal uptake/tolerance in the rhizosphere of wheat plants grown in Hg-contaminated soils. In most cases, these chemical and biochemical responses to Hg were sensitive and, therefore, may allow us to develop strategies for monitoring and/or reducing the risks of Hg contamination in wheat plants and its agroecosystem. Acknowledgments This work was supported by the National Science Foundation of China (No. 41171082; No. 41571008), the Key Project of the Knowledge Innovation Program of IGSNRR (2012ZD002) and the Special Fund for Agro-scientific Research in the Public Interest (No. 201203012-6). References Aebi, H., 1984. Catalase in vitro. Methods Enzymol. 105, 121–126. Beauchamp, C.H., Fridovixh, I., 1971. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal. Biochem. 44, 276–287. 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. 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. Cargnelutti, D., Tabaldi, L.A., Spanevello, R.M., Jucoski, G.O., Battisti, V., Redin, M., Linares, C.E.B., Dressler, V.L., Flores, M.M., Nicoloso, F.T., Morsch, V.M., Schetinger, M.R.C., 2006. Mercury toxicity induces oxidative stress in growing cucumber seedlings. Chemosphere 65, 999–1006. 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. Chen, J., Yang, Z.M., 2012. Mercury toxicity, molecular response and tolerance in higher plants. Biometals 25, 847–857. Cho, U.H., Park, J.O., 2000. Mercury-induced oxidative stress in tomato seedlings. Plant Sci. 156, 1–9. 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. 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. Costa, S., Crespo, D., Henriques, B., Pereira, E., Duarte, A., Pardal, M., 2011. Kinetics of mercury accumulation and its effects on Ulva lactuca growth rate at two salinities and exposure conditions. Water Air Soil Pollut. 217, 689–699. Dessureault-Rompré, J., Nowack, B., Schulin, R., Luster, J., 2006. Modified microsuction cup/rhizobox approach for the in situ detection of organic acids in rhizosphere soil solution. Plant Soil 286, 99–107. Elbaz, A., Wei, Y.Y., Meng, Q., Zheng, Q., Yang, Z.M., 2010. Mercury-induced oxidative stress and impact on antioxidant enzymes in Chlamydomonas reinhardtii. Ecotoxicology 19, 1285–1293. Feng, X., Qiu, G., 2008. Mercury pollution in Guizhou, China an overview. Sci. Total Environ. 400, 227–237. Fu, X., Feng, X., Zhu, W., Rothenberg, S., Yao, H., Zhang, H., 2010. Elevated atmospheric deposition and dynamics of mercury in a remote upland forest of Southwestern China. Environ. Pollut. 158, 2324–2333.

Gao, S., Ou-yang, C., Tang, L., Zhu, J.Q., Xu, Y., Wang, S.H., Chen, F., 2010. Growth and antioxidant responses in Jatropha curcas seedling exposed to mercury toxicity. J. Hazard. Mater. 182, 591–597. Grossman, J., Udluft, P., 1991. The extraction of soil water by the suction-cup method: a review. J. Soil Sci. 42, 83–93. Habashi, F., 1997. Handbook of Extractive Metallurgy. Wiley-VCH, Weinheim. Hall, J.L., 2002. Cellular mechanisms for heavy metal detoxification and tolerance. J. Exp. Bot. 53, 1–11. Han, F.X., Banin, A., Su, Y., Monts, D.L., Plodinec, M.J., Kingery, W.L., Triplett, G.B., 2002. Industrial age anthropogenic inputs of heavy metals into the pedosphere. Naturwissenschaften 89, 497–504. 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. Israr, M., Sahi, S., Datta, R., Sarkar, D., 2006. Bioaccumulation and physiological effects of mercury in Sesbania drummonii. Chemosphere 65, 591–598. ISSCAS (Institute of Soil Science, Chinese Academy of Sciences). 1978. Physical and Chemical Analysis of Soils. Shanghai Science Press, Shanghai (in Chinese). 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. 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. Luo, Y.M., Christie, P., Baker, A.J.M., 2000. Soil solution Zn and pH dynamics in nonrhizosphere soil and in the rhizosphere of Thlaspi caerulescens grown in a Zn/Cd contaminated soil. Chemosphere 41, 161–164. 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. Miller, G., Shulaev, V., Mitter, R., 2008. Reactive oxygen signaling and abiotic stress. Physiol. Plantarum. 133, 481–489. Müller, G., 1969. Index of geoaccumulation in sediments of the Rhine River. Geojournal 2, 108–118. Nakano, Y., Asada, K., 1981. Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant Cell Physiol. 22, 867–880. Nye, P.H., 1981. Changes of pH across the rhizosphere induced by roots. Plant Soil 61, 7–26. Padmavathiamma, P.K., Li, L.Y., 2012. Rhizosphere influence and seasonal impact on phytostabilisation of metals a field study. Water Air Soil Poll. 223, 107–124. Patra, M., Sharma, A., 2000. Mercury toxicity in plants. Bot. Rev. 66, 379–422. Pereira, G.J.G., Molina, S.M.G., Lea, P.J., Azevedo, R.A., 2002. Activity of antioxidant enzymes in response to cadmium in Crotalaria juncea. Plant Soil 239, 123–132. Rellán-Álvarez, R., Ortega-Villasante, C., Álvarez-Fernández, A., del Campo, F.F., Hernández, L.E., 2006. Stress responses of zea mays to cadmium and mercury. Plant Soil 279, 41–50. Sahu, G.K., Upadhyay, S., Sahoo, B.B., 2012. Mercury induced phytotoxicity and oxidative stress in wheat (Triticum aestivum L.) plants. Physiol. Mol. Biol. Plants 18, 21–31. Salt, D.E., Blaylock, M., Kumar, N.P.B.A., Dushenkov, V., Ensley, B.D., Chet, I., Raskin, I., 1995. Phytoremediation: a novel strategy for the removal of toxic metals from the environment using plants. Biotechnology 13, 468–474. Santibáñez, C., Verdugo, C., Ginocchio, R., 2008. Phytostabilization of copper mine tailings with biosolids: Implications for metal uptake and productivity of Lolium perenne. Sci. Total Environ. 395, 1–10. Sauvé, S., McBride, M.B., Norvell, W.A., Hendershot, W.H., 1996. Copper solubility and speciation of in situ contaminated soils: effects of copper level, pH and organic matter. Water Air Soil Pollut. 100, 133–149. 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. 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. Sun, Y., Zhou, Q., Wang, L., Liu, W., 2009. Cadmium tolerance and accumulation characteristics of Bidens pilosa L. as a potential Cd-hyperaccumulator. J. Hazard. Mater. 161, 808–814. Tiensing, T., Preston, S., Strachan, N., Paton, G.L., 2001. Soil solution extraction techniques for microbial ecotoxicity testing: a comparative evaluation. J. Environ. Monitor. 3, 91–96. Vijver, M., Jager, T., Posthuma, L., Peijnenburg, W., 2003. Metal uptake from soils and soil-sediment mixtures by larvae of Tenebrio molitor (L.) (Coleoptera). Ecotox. Environ. Safe. 54, 277–289. Wang, Y., Greger, M., 2004. Clonal differences in mercury tolerance, accumulation and distribution in willow. J. Environ. Qual. 33, 1779–1785. WHO, 1979. Environmental Health Criteria I Mercury, WHO, Geneva. Yang, H., Wong, J.W.C., Yang, Z.M., Zhou, L.X., 2001. Ability of Agrogyron elongatum to accumulation the single metal of cadmium, copper, nickel and root exudation of organic acids. J. Environ. Sci – China 13, 368–375. 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. Zhou, Z.S., Wang, S.J., Yang, Z.M., 2008. Biological detection and analysis of mercury toxicity to alfalfa (Medicago sativa) plants. Chemosphere 70, 1500–1509.

Please cite this article in press as: Li, Y., et al. Dynamic changes of rhizosphere properties and antioxidant enzyme responses of wheat plants (Triticum aestivum L.) grown in mercury-contaminated soils. Chemosphere (2013), http://dx.doi.org/10.1016/j.chemosphere.2013.05.063