Chemosphere xxx (2013) xxx–xxx
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Chemosphere journal homepage: www.elsevier.com/locate/chemosphere
Accumulation of heavy metals using Sorghum sp. Petr Soudek a, Šarka Petrová a, Radomíra Vanˇková b, Jing Song c, Tomaš Vaneˇk a,⇑ a
Laboratory of Plant Biotechnologies, Institute of Experimental Botany AS CR, v.v.i., Rozvojová 263, 165 02 Prague 6 – Lysolaje, Czech Republic Laboratory of Hormonal Regulations in Plants, Institute of Experimental Botany AS CR, v.v.i., Rozvojová 263, 165 02 Prague 6 – Lysolaje, Czech Republic c Key Laboratory of Soil Environment and Pollution Remediation, Institute of Soil Science, Chinese Academy of Sciences, No. 71 East Beijing Road, 210008 Nanjing, China b
h i g h l i g h t s Cd and Zn accumulated primarily in the roots of sorghum plants. Toxic effects of metals in the shoots caused growth reduction of the leaves. Increase of metal concentration in the shoots led to an increase of Chl a/b ratio. Increase of metal concentration in the roots reduced the activities of POX and GST enzymes. Glutathione addition significantly increased the accumulation of cadmium in the roots.
a r t i c l e
i n f o
Article history: Received 22 April 2013 Received in revised form 13 September 2013 Accepted 23 September 2013 Available online xxxx Keywords: Sorghum Cadmium Zinc Accumulation Oxidative stress
a b s t r a c t The essential requirement for the effective phytoremediation is selection of a plant species which should be metal tolerant, with high biomass production and known agronomic techniques. The above mentioned criteria are met by crop plant sorghum (Sorghum bicolor). The response of hydroponically grown S. bicolor plants to cadmium and zinc stress was followed. The impact of metal application on physiological parameters, including changes in chlorophylls contents and antioxidative enzymes activities, was followed during the stress progression. Cadmium and zinc were accumulated primarily in the roots of sorghum plants. However, elevation of metal concentrations in the media promoted their transfer to the shoots. Toxic effects of metals applied at lower concentrations were less serious in the shoots in comparison with their influence to the roots. When applied at higher concentrations, transfer of the metals into the leaves increased, causing growth reduction and leading to Chl loss and metal-induced chlorosis. Moreover, higher metal levels in the roots overcame the quenching capacity of peroxidase and glutathione transferase, which was associated with reduction of their activities. Fortification of antioxidant system by addition of glutathione significantly increased the accumulation of cadmium in the roots as well as in the shoots at the highest cadmium concentration applied. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Heavy metal pollution of soil is usually related to human activities. Sites near mining activities or heavy industry are often highly contaminated with toxic metals. Besides, application of low-quality fertilizers contaminated with heavy metals, e.g. cadmium, increased metal content in top-soils (Lado et al., 2008). Such highly polluted soils are hardly usable for agricultural purposes, because the pollution can be transferred into the food chain. Heavy metals bounded in the soil are leachable, they can be also spread via ground water. To avoid the danger of contaminant spread, it is possible to use phytoremediation techniques, which can immobilize pollutant, decreasing the soil or water pollution (Salt et al., 1998). Plants are able to immobilize metals in soil by formation ⇑ Corresponding author. Tel.: +420 225 106 832. E-mail address: [email protected] (T. Vaneˇk).
of insoluble compounds as a result of interactions of contaminants with plant exudates in rhizosphere or by adsorption to root system (Kidd et al., 2009). Some plant species are also able to accumulate heavy metals in their tissues so the contaminant can be removed from locality by plant harvest (McGrath and Zhao, 2003; Van Nevel et al., 2007; Maestri et al., 2010). Many researchers studied heavy metal uptake and transport within the plants (Clemens, 2006; de Livera et al., 2011; Gallego et al., 2012; Bhargava et al., 2012). The most widely studied toxic metal has been cadmium. It enters plant cells via transport system for essential cations (such as Zn2+). It is translocated from roots to the shoots by the metal-sequestering pathways (Clemens, 2006). In contrast to cadmium, zinc is an essential micronutrient for plant growth. Common zinc/cadmium transporters were identified in Brassicaceae plants (Eren et al., 2007; Ueno et al., 2008). Zn absorbed by roots was found to be readily available for loading into the xylem of hyperaccumulator Thlaspi caerulescens, while it was stored
0045-6535/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.chemosphere.2013.09.079
Please cite this article in press as: Soudek, P., et al. Accumulation of heavy metals using Sorghum sp. Chemosphere (2013), http://dx.doi.org/10.1016/ j.chemosphere.2013.09.079
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P. Soudek et al. / Chemosphere xxx (2013) xxx–xxx
mainly in root vacuoles in non-hyperaccumulator Thlaspi arvense (Lasat et al., 1998). Cadmium was reported to accumulate preferentially in the roots (Verbruggen et al., 2009; Lux et al., 2011). Therefore, different ways of enhancement of cadmium content in shoots were tested using various amendments. Chemically-assisted phytoextraction was initially based on EDTA (ethylenediamine tetraacetic acid) application. The addition of 0.5 or 2 g kg 1 EDTA increased Cd content in shoots of Populus sp. plants (Robinson et al., 2000). Unfortunately, addition of 2 g kg 1 EDTA caused a significant growth reduction, as well as leaf abscission. In order to avoid EDTA toxic effects, other efficient and environment friendly amendments were tested. One of the promising alternatives was a mixture of humic substances, such as humic acid, fulvic acid, and humin (Evangelou et al., 2007). Humic acids were found to cause increased accumulation of metals in Helianthus annuus and Zea mays plants (Turan and Angin, 2004). Effective phytoremediation requires to select an appropriate plant species, metal tolerant, with high biomass production and known agronomic techniques. The above mentioned conditions are met by woody plants (Licht and Isebrands, 2005), grasses (Gupta et al., 2008), and crop plants (Shi and Cai, 2009). In case of biomass crops, the primary interest concerning is now focused on energy crops i.e. Miscanthus giganteus (Kocon´ and Matyka, 2012), Salix sp. (Pulford et al., 2002; Šyc et al., 2012), Populus sp. (Fernàndez et al., 2012), Zea mays (Meers et al., 2010), and Sorghum sp. (Angelova et al., 2011). Sorghum bicolor is C4 grass widely used as a forage crop. It is the fifth most important cereal in the world. Sorghum plants are multipurpose cereals of potential interest for several non-food uses, especially as energy crops (Barbanti et al., 2006; Meki et al., 2013). The crop is resistant to drought, heat stress, and toxic pollution. It was shown that sorghum plants were able to accumulate large quantities of Cd, Cu, Pb, and Zn in the shoots and their biomass production was higher than that of sunflower or corn (Epelde et al., 2009; Zhuang et al., 2009). Moreover, other studies demonstrated that sorghum plants were highly tolerant to metal pollution and able to reach high biomass, even in the presence of heavy metals (Pinto et al., 2004; Hernández-Allica et al., 2008; Angelova et al., 2011). Only few studies have dealt with practical applications. A field trial with sorghum plants was located near the lead and zinc mining site in China (Zhuang et al., 2009). Another field trial was in Bulgaria near non-ferrous-metal works. All plants accumulated heavy metals primarily in roots, however, relatively high amount of Cd was found in stems of Sudan grass (Angelova et al., 2011). Experiments were also focused on heavy metal polluted marginal land. In a pot experiment, the effect of plant-growth-promoting endophyte on sorghum biomass production and metal accumulation was tested. Significant increase of biomass production and also an increase of Mn and Cd contents were observed in shoots of sorghum plants upon the endophyte addition (Luo et al., 2012). The elucidation of the interactions between metal, soil, and plants is essential to reduce the risk of food chain contamination. Significant decrease of Cd concentration in Sorghum roots may be achieved by application of organic matter (mainly fulvic acid). However, the addition of fulvic acid caused an enhanced translocation of Cd into shoots (Pinto et al., 2004). When biomass production of sorghum was tested in polymer-amended soil, the application of polyacrylate polymers reduced Cd bioavailability, thereby reducing Cd toxicity and also concentration of Cd in shoots (Guiwei et al., 2010). Cd exposure can inactivate many important enzymes, due to its high affinity to thiol and hydroxyl groups and nitrogen-containing ligands (Torres et al., 2000). Thiol groups which can sequester the metal ions were increased in sorghum plants upon metal stress (Pinto et al., 2006), however, metal toxicity led to an increase of
reactive oxygen species (ROS) production, too. This stimulates activity of antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX), which are able to eliminate ROS. The intensity of the response to Cd stress is species specific. Low concentrations of Cd (below 10 mg/kg) stimulated activities of glutathione-S-transferase (GST), SOD, peroxidase (POD), and CAT in leaves (Liu et al., 2010). Authors suggested that low concentrations of Cd may be sensed by sorghum plants as the oxidative stress, which may stimulate antioxidant system resulting in decrease of the ROS and reduction of the oxidative damage. When Cd stress overcame the capacity of protective enzymes, their activities declined rapidly, simultaneously with cell damage. Zn as an essential micronutrient is a cofactor of many enzymes, including those involved in chlorophyll synthesis (Hänsch and Mendel, 2009). However, high zinc levels may induce similar toxic effects as other heavy metals. Increasing Zn concentrations significantly inhibited plant height, biomass, content of a, b, and total chlorophyll (Mirshekali et al., 2012). In fact, Zn was reported to increase POD activity in bean plants (Chaoui et al., 1997). The aim of this work was to elucidate protection mechanisms involved in the response of sorghum plants to Cd and Zn stress. The metal accumulation was followed in roots and shoots of hydroponically grown S. bicolor plants. The impact of heavy metal stress on the physiological parameters, including chlorophylls contents and antioxidative enzymes activities was determined during the stress progression. 2. Material and methods 2.1. Plant material Sorghum bicolor L. plants (cultivars Honey Graze, Express, DSM 14-535, Nutri Honey, Sweet Virginia and Sucrosorgo 506) (obtained from SEED SERVICE s.r.o., Czech Republic) were cultivated
(A)
(B)
Fig. 1. Cadmium and zinc concentrations (mg/g) in shoots (A) and roots (B) of S. bicolor after 7 d of growth in solution with 0, 50, 100, 200, 500, 1000, 2000 or 5000 (lM) concentration; standard deviation is represented as ±SD (n = 3).
Please cite this article in press as: Soudek, P., et al. Accumulation of heavy metals using Sorghum sp. Chemosphere (2013), http://dx.doi.org/10.1016/ j.chemosphere.2013.09.079
P. Soudek et al. / Chemosphere xxx (2013) xxx–xxx
Fig. 2. Root length (cm) of S. bicolor after 14, 21 and 28 d of growth in solution with 100, 200 or 500 (lM) cadmium concentration or with 200, 500, 1000 or 5000 (lM) zinc concentration; standard deviation is represented as ±SD (n = 4).
3
supplementation into modified Hoagland’s medium, both substances were applied at concentration 100 lM. Six additional sorghum cultivars (Honey Graze, Express, DSM 14-535, Nutri Honey, Sweet Virginia and Sucrosorgo 506) (Seed Servis, Ltd., Czech Republic) were grown in the same way as described for cv. ‘‘Nutri Honey’’. Cadmium was applied at 100 or 200 lM concentration, zinc at 2000 or 5000 lM concentration. Samples were harvest after 28 d of cultivation. The roots of plants were washed subsequently with doubledistiled water, 0.1 M EDTA solution, and double-distiled water again. Before storage, the roots were analysed by scanner. All leaf and root samples were frozen in liquid nitrogen and stored at 80 °C. The frozen samples were used for enzyme and photosynthetic pigment analysis or they were lyophilized. Lyophilized plant samples were used for heavy metal determination. Four replications were done for each treatment and each metal concentration. 2.3. Heavy metal determination Dried leaf and root tissues were ground to a powder and samples (ca 0.125 g DW) were digested in 5 mL mixture HClO4/HNO3 (15/85%, v/v) in digestion glass tubes overnight. Digestion was completed by gradual increase of temperature from 60 to 195 °C according Zhao et al. (1994). Digestion protocol was as follows: 3 h, 60 °C; 1 h, 100 °C; 1 h, 120 °C; 3 h, 195 °C. After cooling, 2.5 mL 20% HCl was added, whirl mixed and warmed to 80 °C for 1 h. The final volume was brought to 10 mL accurately. Heavy metal content was measured by AAS (SensAA, GBS, Australia). 2.4. Chlorophyll measurement
Fig. 3. Number of leaves at sampling day and number of leaves at start of experiment ratio of S. bicolor after 14, 21 and 28 d of growth in solution with 100, 200 or 500 (lM) cadmium concentration or with 200, 500, 1000 or 5000 (lM) zinc concentration. (n = 4).
Leaf samples (10–20 mg FW) were extracted with 10 mL methanol overnight. The absorbance of methanolic extract was measured 470, 652.4 and 665.2 nm using UV mini 1240 Shimadzu spectrophotometer. Chlorophyll and carotenoid contents were calculated according to Lichtenhaler (1987).
hydroponically in modified Hoagland’s solution (Hoagland, 1920), pH 5.5, at 16 h photoperiod, at 72 lmol cm 2 s 1, 23 °C and relative humidity about 60%. Four-week old plants were used for experiments.
2.5. Protein extraction
2.2. Experiment designs Sorghum seedlings cv. ‘‘Nutri Honey’’ were cultivated in Erlenmeyer flasks (each plant per one flask) in 300 mL of modified Hoagland’s medium supplemented with heavy metals (0, 50, 100, 200, 500, 1000, 2000 or 5000 lM). Cd(NO3)2 (Penta, Czech Republic) and Zn(NO3)2 (Lach-Ner, Ltd., Czech Republic) were used. Samples were harvested after 0, 7, 14, 21 or 28 d of cultivation. In case of EDTA (Penta, Czech Republic) or glutathione (Sigma–Aldrich)
Plant tissues were homogenised with mortar and pestle in cold 0.1 M Tris/HCl buffer (supplemented with 5 mM EDTA, 1% PVP K30, 5 mM DTE, 1% Nonidet P40) at pH 7.8 (10 mL of extraction buffer per 1 g FW). The homogenate was centrifuged at 20 000 rpm at 4 °C for 30 min. Samples were filtered with Miracloth Filter. The first precipitation was done with ammonium sulphate (40% saturation). After agitation for 30 min, suspension was centrifuged at 20 000 rpm at 4 °C for 30 min. Samples were filtered through Miracloth Filter (Calbiochem). The second precipitation with ammonium sulphate was done to 80% saturation. After 30 min agitatation, suspension was centrifuged at 20 000 rpm at 4 °C for
Fig. 4. Chl a/b ratio in the leaves of S. bicolor after 7 d of growth in solution with 0, 50, 100, 200, 500, 1000, 2000, or 5000 (lM) concentration.
Please cite this article in press as: Soudek, P., et al. Accumulation of heavy metals using Sorghum sp. Chemosphere (2013), http://dx.doi.org/10.1016/ j.chemosphere.2013.09.079
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P. Soudek et al. / Chemosphere xxx (2013) xxx–xxx
(A) β β
(B)
β
β
(C)
β β
β
(D)
β
β
Fig. 5. Peroxidase (A), glutathione-S-transferase (B), catalase (C) and ascorbat peroxidase (D) activity (lkat mg protein) in root and shoot of S. bicolor after 7 d of growth in solution with 0, 100, 500, 1000 or 5000 (lM) concentration; standard deviation is represented as ±SD (n = 6).
Please cite this article in press as: Soudek, P., et al. Accumulation of heavy metals using Sorghum sp. Chemosphere (2013), http://dx.doi.org/10.1016/ j.chemosphere.2013.09.079
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(A)
(A) 2.0 1.8
0 days
c plant part (mg/g)
1.6
14 days
1.4
21 days
1.2
28 days
1.0 0.8 0.6 0.4 0.2 0.0 0
200
500
co nt ro l
1000
5000
Zn
csolution (μ μ M)
(B)
(B)
6
c plant part (mg/g)
5
0 days 14 days
4
21 days 28 days
3 2 1 0 0
200
c o nt rol
Fig. 6. Cadmium concentrations (mg/g) in shoots (A) and roots (B) of S. bicolor during 28 d of growth in solution with 0, 100, 200 or 500 (lM) concentration; standard deviation is represented as ±SD (n = 3).
30 min. The pellet was resuspended with 2.5 mL 25 mM Tris/HCl buffer (pH 7.8), filled into the columns PD 10, eluted with 3.5 mL of 25 mM Tris/HCl buffer (pH 7.8), frozen and stored at 80 °C.
500
1000
5000
Zn
c solution (μ M) Fig. 7. Zinc concentrations (mg/g) in shoots (A) and roots (B) of S. bicolor during 28 d of growth in solution with 0, 200, 500, 1000 or 5000 (lM) concentration; standard deviation is represented as ±SD (n = 3).
serum albumin as a standard. All enzyme activities are the mean values from four independent biological experiments. 2.7. Roots length measurement
2.6. Enzyme assays The enzyme assays were performed with a microplate reader TECAN Infinite N200. Peroxidase (POX) activity was detected using colour reaction with guaiacol. The reaction mixture contained guaiacol (3.4 mM, 0.6 mL), Tris/HCl buffer (50 mM, pH 6.0, 27 mL) and H2O2 (9 mM, 0.6 mL). The supernatant (0.01 mL) was added to 0.19 mL reaction mixture and POX activity was measured in microplate wells at 436 nm (e = 26.6 mM 1 cm 1) (modified from Drotar et al., 1985). Glutathione-S-transferase (GST) activity was detected using reaction with 1-chloro-2,4-dinitrobenzene (CDNB). The reaction mixture contained CDNB (0.06 mM, 1 mL), Tris/HCl buffer (100 mM, pH 6.4, 23.7 mL) and GSH (0.12 mM, 0.5 mL). The supernatant (0.04 mL) was added to 0.15 mL of reaction mixture and GST activity was measured in microplate wells at 340 nm (e = 9.6 mM 1 cm 1) (modified from Lyubenova and Schröder, 2011). Catalase (CAT) activity was detected via hydrogen peroxide consumption. The reaction mixture contained phosphate buffer (100 mM KH2PO4, pH 7.0, 30 mL) and H2O2 (200 mM, 12 mL). The supernatant (0.01 mL) was added to 0.14 mL reaction mixture and CAT activity was measured at 240 nm (e = 0.036 mM 1 cm 1) (modified from Verma and Dubey, 2003). Ascorbate peroxidase (APX) activity was detected via decrease of absorbance of ascorbate. The reaction mixture contained sodium ascorbate (60 mM, 0.01 mL), Tris/HCl buffer (50 mM, pH 6.0, 27 mL) and H2O2 (3%, 0.041 mL). The supernatant (0.02 mL) was added to 0.18 mL reaction mixture and APX activity was measured at 290 nm (e = 2.8 mM 1 cm 1) (modified from Vanacker et al., 1998). The enzyme activities were expressed in lkat/mg protein. The protein extraction was done according to Bradford (1976) using
Roots of each plant were washed, scanned on scanner EPSON PERFECTION V700 PHOTO. The root images were processed by software WinRHIZO (Regent Instruments, Inc., Canada). 2.8. Leaves counting Leaf number was determined at the beginning of the experiment and after14, 21 and 28 d of stress treatment. The ratio of a number of leaves at sampling day and a number of leaves at start of the experiment was calculated. 2.9. Data analysis Mean values were calculated from four replicates. Analysis of variances was used to estimate statistically significant differences between groups of samples. The significance of differences were determined using Student’s t-test for a P 0.05. The differences among treatments were tested by one-way ANOVA with Turkey HSD multiple comparison test. Significance level P = 0.05 was used for both analyses. Statistically insignificant values on the level of probability P < 0.05 are indicating by the same symbol above columns. Each treatment was represented by four biological replicates. STATISTICA 8 (StatSoft, Tulsa, OK, USA) software was used for all the computations. 3. Results and discussion Both Cd and Zn accumulation in the plants positively correlated with metal concentration in the cultivation medium (Fig. 1).
Please cite this article in press as: Soudek, P., et al. Accumulation of heavy metals using Sorghum sp. Chemosphere (2013), http://dx.doi.org/10.1016/ j.chemosphere.2013.09.079
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P. Soudek et al. / Chemosphere xxx (2013) xxx–xxx
(A)
(B)
Fig. 8. Cadmium concentrations (mg/g) in shoots (A) and roots (B) of six cultivars of S. bicolor after 28 d of growth in solution with 0, 200 or 500 (lM) cadmium concentration or with 0, 2000 or 5000 (lM) zinc; standard deviation is represented as ±SD (n = 5).
Although, the distribution of individual metals in plant tissues differed (Figs. 1 and 2). Tested metals accumulated primarily in the roots. The restriction of metal absorption and translocation to the shoots may be related to the avoidance mechanism in the roots. El-Beltagi et al. (2010) tested cadmium accumulation in plant Raphanus sativus grown at the presence of Cd in concentration range 0–0.444 mM. They found an increase of cadmium content in radish plants but cadmium content in the roots was only twice higher than in the shoots. Also Izadiyar and Yargholi (2010) found minor differences in Cd content between roots and shoots in clover, alfalfa, sorghum and sainfoin cultivated on wastewater. Surprisingly, our data showed that cadmium content in the roots was max 280 times higher in case of low concentrations (
Contents lists available at ScienceDirect
Chemosphere journal homepage: www.elsevier.com/locate/chemosphere
Accumulation of heavy metals using Sorghum sp. Petr Soudek a, Šarka Petrová a, Radomíra Vanˇková b, Jing Song c, Tomaš Vaneˇk a,⇑ a
Laboratory of Plant Biotechnologies, Institute of Experimental Botany AS CR, v.v.i., Rozvojová 263, 165 02 Prague 6 – Lysolaje, Czech Republic Laboratory of Hormonal Regulations in Plants, Institute of Experimental Botany AS CR, v.v.i., Rozvojová 263, 165 02 Prague 6 – Lysolaje, Czech Republic c Key Laboratory of Soil Environment and Pollution Remediation, Institute of Soil Science, Chinese Academy of Sciences, No. 71 East Beijing Road, 210008 Nanjing, China b
h i g h l i g h t s Cd and Zn accumulated primarily in the roots of sorghum plants. Toxic effects of metals in the shoots caused growth reduction of the leaves. Increase of metal concentration in the shoots led to an increase of Chl a/b ratio. Increase of metal concentration in the roots reduced the activities of POX and GST enzymes. Glutathione addition significantly increased the accumulation of cadmium in the roots.
a r t i c l e
i n f o
Article history: Received 22 April 2013 Received in revised form 13 September 2013 Accepted 23 September 2013 Available online xxxx Keywords: Sorghum Cadmium Zinc Accumulation Oxidative stress
a b s t r a c t The essential requirement for the effective phytoremediation is selection of a plant species which should be metal tolerant, with high biomass production and known agronomic techniques. The above mentioned criteria are met by crop plant sorghum (Sorghum bicolor). The response of hydroponically grown S. bicolor plants to cadmium and zinc stress was followed. The impact of metal application on physiological parameters, including changes in chlorophylls contents and antioxidative enzymes activities, was followed during the stress progression. Cadmium and zinc were accumulated primarily in the roots of sorghum plants. However, elevation of metal concentrations in the media promoted their transfer to the shoots. Toxic effects of metals applied at lower concentrations were less serious in the shoots in comparison with their influence to the roots. When applied at higher concentrations, transfer of the metals into the leaves increased, causing growth reduction and leading to Chl loss and metal-induced chlorosis. Moreover, higher metal levels in the roots overcame the quenching capacity of peroxidase and glutathione transferase, which was associated with reduction of their activities. Fortification of antioxidant system by addition of glutathione significantly increased the accumulation of cadmium in the roots as well as in the shoots at the highest cadmium concentration applied. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Heavy metal pollution of soil is usually related to human activities. Sites near mining activities or heavy industry are often highly contaminated with toxic metals. Besides, application of low-quality fertilizers contaminated with heavy metals, e.g. cadmium, increased metal content in top-soils (Lado et al., 2008). Such highly polluted soils are hardly usable for agricultural purposes, because the pollution can be transferred into the food chain. Heavy metals bounded in the soil are leachable, they can be also spread via ground water. To avoid the danger of contaminant spread, it is possible to use phytoremediation techniques, which can immobilize pollutant, decreasing the soil or water pollution (Salt et al., 1998). Plants are able to immobilize metals in soil by formation ⇑ Corresponding author. Tel.: +420 225 106 832. E-mail address: [email protected] (T. Vaneˇk).
of insoluble compounds as a result of interactions of contaminants with plant exudates in rhizosphere or by adsorption to root system (Kidd et al., 2009). Some plant species are also able to accumulate heavy metals in their tissues so the contaminant can be removed from locality by plant harvest (McGrath and Zhao, 2003; Van Nevel et al., 2007; Maestri et al., 2010). Many researchers studied heavy metal uptake and transport within the plants (Clemens, 2006; de Livera et al., 2011; Gallego et al., 2012; Bhargava et al., 2012). The most widely studied toxic metal has been cadmium. It enters plant cells via transport system for essential cations (such as Zn2+). It is translocated from roots to the shoots by the metal-sequestering pathways (Clemens, 2006). In contrast to cadmium, zinc is an essential micronutrient for plant growth. Common zinc/cadmium transporters were identified in Brassicaceae plants (Eren et al., 2007; Ueno et al., 2008). Zn absorbed by roots was found to be readily available for loading into the xylem of hyperaccumulator Thlaspi caerulescens, while it was stored
0045-6535/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.chemosphere.2013.09.079
Please cite this article in press as: Soudek, P., et al. Accumulation of heavy metals using Sorghum sp. Chemosphere (2013), http://dx.doi.org/10.1016/ j.chemosphere.2013.09.079
2
P. Soudek et al. / Chemosphere xxx (2013) xxx–xxx
mainly in root vacuoles in non-hyperaccumulator Thlaspi arvense (Lasat et al., 1998). Cadmium was reported to accumulate preferentially in the roots (Verbruggen et al., 2009; Lux et al., 2011). Therefore, different ways of enhancement of cadmium content in shoots were tested using various amendments. Chemically-assisted phytoextraction was initially based on EDTA (ethylenediamine tetraacetic acid) application. The addition of 0.5 or 2 g kg 1 EDTA increased Cd content in shoots of Populus sp. plants (Robinson et al., 2000). Unfortunately, addition of 2 g kg 1 EDTA caused a significant growth reduction, as well as leaf abscission. In order to avoid EDTA toxic effects, other efficient and environment friendly amendments were tested. One of the promising alternatives was a mixture of humic substances, such as humic acid, fulvic acid, and humin (Evangelou et al., 2007). Humic acids were found to cause increased accumulation of metals in Helianthus annuus and Zea mays plants (Turan and Angin, 2004). Effective phytoremediation requires to select an appropriate plant species, metal tolerant, with high biomass production and known agronomic techniques. The above mentioned conditions are met by woody plants (Licht and Isebrands, 2005), grasses (Gupta et al., 2008), and crop plants (Shi and Cai, 2009). In case of biomass crops, the primary interest concerning is now focused on energy crops i.e. Miscanthus giganteus (Kocon´ and Matyka, 2012), Salix sp. (Pulford et al., 2002; Šyc et al., 2012), Populus sp. (Fernàndez et al., 2012), Zea mays (Meers et al., 2010), and Sorghum sp. (Angelova et al., 2011). Sorghum bicolor is C4 grass widely used as a forage crop. It is the fifth most important cereal in the world. Sorghum plants are multipurpose cereals of potential interest for several non-food uses, especially as energy crops (Barbanti et al., 2006; Meki et al., 2013). The crop is resistant to drought, heat stress, and toxic pollution. It was shown that sorghum plants were able to accumulate large quantities of Cd, Cu, Pb, and Zn in the shoots and their biomass production was higher than that of sunflower or corn (Epelde et al., 2009; Zhuang et al., 2009). Moreover, other studies demonstrated that sorghum plants were highly tolerant to metal pollution and able to reach high biomass, even in the presence of heavy metals (Pinto et al., 2004; Hernández-Allica et al., 2008; Angelova et al., 2011). Only few studies have dealt with practical applications. A field trial with sorghum plants was located near the lead and zinc mining site in China (Zhuang et al., 2009). Another field trial was in Bulgaria near non-ferrous-metal works. All plants accumulated heavy metals primarily in roots, however, relatively high amount of Cd was found in stems of Sudan grass (Angelova et al., 2011). Experiments were also focused on heavy metal polluted marginal land. In a pot experiment, the effect of plant-growth-promoting endophyte on sorghum biomass production and metal accumulation was tested. Significant increase of biomass production and also an increase of Mn and Cd contents were observed in shoots of sorghum plants upon the endophyte addition (Luo et al., 2012). The elucidation of the interactions between metal, soil, and plants is essential to reduce the risk of food chain contamination. Significant decrease of Cd concentration in Sorghum roots may be achieved by application of organic matter (mainly fulvic acid). However, the addition of fulvic acid caused an enhanced translocation of Cd into shoots (Pinto et al., 2004). When biomass production of sorghum was tested in polymer-amended soil, the application of polyacrylate polymers reduced Cd bioavailability, thereby reducing Cd toxicity and also concentration of Cd in shoots (Guiwei et al., 2010). Cd exposure can inactivate many important enzymes, due to its high affinity to thiol and hydroxyl groups and nitrogen-containing ligands (Torres et al., 2000). Thiol groups which can sequester the metal ions were increased in sorghum plants upon metal stress (Pinto et al., 2006), however, metal toxicity led to an increase of
reactive oxygen species (ROS) production, too. This stimulates activity of antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX), which are able to eliminate ROS. The intensity of the response to Cd stress is species specific. Low concentrations of Cd (below 10 mg/kg) stimulated activities of glutathione-S-transferase (GST), SOD, peroxidase (POD), and CAT in leaves (Liu et al., 2010). Authors suggested that low concentrations of Cd may be sensed by sorghum plants as the oxidative stress, which may stimulate antioxidant system resulting in decrease of the ROS and reduction of the oxidative damage. When Cd stress overcame the capacity of protective enzymes, their activities declined rapidly, simultaneously with cell damage. Zn as an essential micronutrient is a cofactor of many enzymes, including those involved in chlorophyll synthesis (Hänsch and Mendel, 2009). However, high zinc levels may induce similar toxic effects as other heavy metals. Increasing Zn concentrations significantly inhibited plant height, biomass, content of a, b, and total chlorophyll (Mirshekali et al., 2012). In fact, Zn was reported to increase POD activity in bean plants (Chaoui et al., 1997). The aim of this work was to elucidate protection mechanisms involved in the response of sorghum plants to Cd and Zn stress. The metal accumulation was followed in roots and shoots of hydroponically grown S. bicolor plants. The impact of heavy metal stress on the physiological parameters, including chlorophylls contents and antioxidative enzymes activities was determined during the stress progression. 2. Material and methods 2.1. Plant material Sorghum bicolor L. plants (cultivars Honey Graze, Express, DSM 14-535, Nutri Honey, Sweet Virginia and Sucrosorgo 506) (obtained from SEED SERVICE s.r.o., Czech Republic) were cultivated
(A)
(B)
Fig. 1. Cadmium and zinc concentrations (mg/g) in shoots (A) and roots (B) of S. bicolor after 7 d of growth in solution with 0, 50, 100, 200, 500, 1000, 2000 or 5000 (lM) concentration; standard deviation is represented as ±SD (n = 3).
Please cite this article in press as: Soudek, P., et al. Accumulation of heavy metals using Sorghum sp. Chemosphere (2013), http://dx.doi.org/10.1016/ j.chemosphere.2013.09.079
P. Soudek et al. / Chemosphere xxx (2013) xxx–xxx
Fig. 2. Root length (cm) of S. bicolor after 14, 21 and 28 d of growth in solution with 100, 200 or 500 (lM) cadmium concentration or with 200, 500, 1000 or 5000 (lM) zinc concentration; standard deviation is represented as ±SD (n = 4).
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supplementation into modified Hoagland’s medium, both substances were applied at concentration 100 lM. Six additional sorghum cultivars (Honey Graze, Express, DSM 14-535, Nutri Honey, Sweet Virginia and Sucrosorgo 506) (Seed Servis, Ltd., Czech Republic) were grown in the same way as described for cv. ‘‘Nutri Honey’’. Cadmium was applied at 100 or 200 lM concentration, zinc at 2000 or 5000 lM concentration. Samples were harvest after 28 d of cultivation. The roots of plants were washed subsequently with doubledistiled water, 0.1 M EDTA solution, and double-distiled water again. Before storage, the roots were analysed by scanner. All leaf and root samples were frozen in liquid nitrogen and stored at 80 °C. The frozen samples were used for enzyme and photosynthetic pigment analysis or they were lyophilized. Lyophilized plant samples were used for heavy metal determination. Four replications were done for each treatment and each metal concentration. 2.3. Heavy metal determination Dried leaf and root tissues were ground to a powder and samples (ca 0.125 g DW) were digested in 5 mL mixture HClO4/HNO3 (15/85%, v/v) in digestion glass tubes overnight. Digestion was completed by gradual increase of temperature from 60 to 195 °C according Zhao et al. (1994). Digestion protocol was as follows: 3 h, 60 °C; 1 h, 100 °C; 1 h, 120 °C; 3 h, 195 °C. After cooling, 2.5 mL 20% HCl was added, whirl mixed and warmed to 80 °C for 1 h. The final volume was brought to 10 mL accurately. Heavy metal content was measured by AAS (SensAA, GBS, Australia). 2.4. Chlorophyll measurement
Fig. 3. Number of leaves at sampling day and number of leaves at start of experiment ratio of S. bicolor after 14, 21 and 28 d of growth in solution with 100, 200 or 500 (lM) cadmium concentration or with 200, 500, 1000 or 5000 (lM) zinc concentration. (n = 4).
Leaf samples (10–20 mg FW) were extracted with 10 mL methanol overnight. The absorbance of methanolic extract was measured 470, 652.4 and 665.2 nm using UV mini 1240 Shimadzu spectrophotometer. Chlorophyll and carotenoid contents were calculated according to Lichtenhaler (1987).
hydroponically in modified Hoagland’s solution (Hoagland, 1920), pH 5.5, at 16 h photoperiod, at 72 lmol cm 2 s 1, 23 °C and relative humidity about 60%. Four-week old plants were used for experiments.
2.5. Protein extraction
2.2. Experiment designs Sorghum seedlings cv. ‘‘Nutri Honey’’ were cultivated in Erlenmeyer flasks (each plant per one flask) in 300 mL of modified Hoagland’s medium supplemented with heavy metals (0, 50, 100, 200, 500, 1000, 2000 or 5000 lM). Cd(NO3)2 (Penta, Czech Republic) and Zn(NO3)2 (Lach-Ner, Ltd., Czech Republic) were used. Samples were harvested after 0, 7, 14, 21 or 28 d of cultivation. In case of EDTA (Penta, Czech Republic) or glutathione (Sigma–Aldrich)
Plant tissues were homogenised with mortar and pestle in cold 0.1 M Tris/HCl buffer (supplemented with 5 mM EDTA, 1% PVP K30, 5 mM DTE, 1% Nonidet P40) at pH 7.8 (10 mL of extraction buffer per 1 g FW). The homogenate was centrifuged at 20 000 rpm at 4 °C for 30 min. Samples were filtered with Miracloth Filter. The first precipitation was done with ammonium sulphate (40% saturation). After agitation for 30 min, suspension was centrifuged at 20 000 rpm at 4 °C for 30 min. Samples were filtered through Miracloth Filter (Calbiochem). The second precipitation with ammonium sulphate was done to 80% saturation. After 30 min agitatation, suspension was centrifuged at 20 000 rpm at 4 °C for
Fig. 4. Chl a/b ratio in the leaves of S. bicolor after 7 d of growth in solution with 0, 50, 100, 200, 500, 1000, 2000, or 5000 (lM) concentration.
Please cite this article in press as: Soudek, P., et al. Accumulation of heavy metals using Sorghum sp. Chemosphere (2013), http://dx.doi.org/10.1016/ j.chemosphere.2013.09.079
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Fig. 5. Peroxidase (A), glutathione-S-transferase (B), catalase (C) and ascorbat peroxidase (D) activity (lkat mg protein) in root and shoot of S. bicolor after 7 d of growth in solution with 0, 100, 500, 1000 or 5000 (lM) concentration; standard deviation is represented as ±SD (n = 6).
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Fig. 6. Cadmium concentrations (mg/g) in shoots (A) and roots (B) of S. bicolor during 28 d of growth in solution with 0, 100, 200 or 500 (lM) concentration; standard deviation is represented as ±SD (n = 3).
30 min. The pellet was resuspended with 2.5 mL 25 mM Tris/HCl buffer (pH 7.8), filled into the columns PD 10, eluted with 3.5 mL of 25 mM Tris/HCl buffer (pH 7.8), frozen and stored at 80 °C.
500
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c solution (μ M) Fig. 7. Zinc concentrations (mg/g) in shoots (A) and roots (B) of S. bicolor during 28 d of growth in solution with 0, 200, 500, 1000 or 5000 (lM) concentration; standard deviation is represented as ±SD (n = 3).
serum albumin as a standard. All enzyme activities are the mean values from four independent biological experiments. 2.7. Roots length measurement
2.6. Enzyme assays The enzyme assays were performed with a microplate reader TECAN Infinite N200. Peroxidase (POX) activity was detected using colour reaction with guaiacol. The reaction mixture contained guaiacol (3.4 mM, 0.6 mL), Tris/HCl buffer (50 mM, pH 6.0, 27 mL) and H2O2 (9 mM, 0.6 mL). The supernatant (0.01 mL) was added to 0.19 mL reaction mixture and POX activity was measured in microplate wells at 436 nm (e = 26.6 mM 1 cm 1) (modified from Drotar et al., 1985). Glutathione-S-transferase (GST) activity was detected using reaction with 1-chloro-2,4-dinitrobenzene (CDNB). The reaction mixture contained CDNB (0.06 mM, 1 mL), Tris/HCl buffer (100 mM, pH 6.4, 23.7 mL) and GSH (0.12 mM, 0.5 mL). The supernatant (0.04 mL) was added to 0.15 mL of reaction mixture and GST activity was measured in microplate wells at 340 nm (e = 9.6 mM 1 cm 1) (modified from Lyubenova and Schröder, 2011). Catalase (CAT) activity was detected via hydrogen peroxide consumption. The reaction mixture contained phosphate buffer (100 mM KH2PO4, pH 7.0, 30 mL) and H2O2 (200 mM, 12 mL). The supernatant (0.01 mL) was added to 0.14 mL reaction mixture and CAT activity was measured at 240 nm (e = 0.036 mM 1 cm 1) (modified from Verma and Dubey, 2003). Ascorbate peroxidase (APX) activity was detected via decrease of absorbance of ascorbate. The reaction mixture contained sodium ascorbate (60 mM, 0.01 mL), Tris/HCl buffer (50 mM, pH 6.0, 27 mL) and H2O2 (3%, 0.041 mL). The supernatant (0.02 mL) was added to 0.18 mL reaction mixture and APX activity was measured at 290 nm (e = 2.8 mM 1 cm 1) (modified from Vanacker et al., 1998). The enzyme activities were expressed in lkat/mg protein. The protein extraction was done according to Bradford (1976) using
Roots of each plant were washed, scanned on scanner EPSON PERFECTION V700 PHOTO. The root images were processed by software WinRHIZO (Regent Instruments, Inc., Canada). 2.8. Leaves counting Leaf number was determined at the beginning of the experiment and after14, 21 and 28 d of stress treatment. The ratio of a number of leaves at sampling day and a number of leaves at start of the experiment was calculated. 2.9. Data analysis Mean values were calculated from four replicates. Analysis of variances was used to estimate statistically significant differences between groups of samples. The significance of differences were determined using Student’s t-test for a P 0.05. The differences among treatments were tested by one-way ANOVA with Turkey HSD multiple comparison test. Significance level P = 0.05 was used for both analyses. Statistically insignificant values on the level of probability P < 0.05 are indicating by the same symbol above columns. Each treatment was represented by four biological replicates. STATISTICA 8 (StatSoft, Tulsa, OK, USA) software was used for all the computations. 3. Results and discussion Both Cd and Zn accumulation in the plants positively correlated with metal concentration in the cultivation medium (Fig. 1).
Please cite this article in press as: Soudek, P., et al. Accumulation of heavy metals using Sorghum sp. Chemosphere (2013), http://dx.doi.org/10.1016/ j.chemosphere.2013.09.079
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Fig. 8. Cadmium concentrations (mg/g) in shoots (A) and roots (B) of six cultivars of S. bicolor after 28 d of growth in solution with 0, 200 or 500 (lM) cadmium concentration or with 0, 2000 or 5000 (lM) zinc; standard deviation is represented as ±SD (n = 5).
Although, the distribution of individual metals in plant tissues differed (Figs. 1 and 2). Tested metals accumulated primarily in the roots. The restriction of metal absorption and translocation to the shoots may be related to the avoidance mechanism in the roots. El-Beltagi et al. (2010) tested cadmium accumulation in plant Raphanus sativus grown at the presence of Cd in concentration range 0–0.444 mM. They found an increase of cadmium content in radish plants but cadmium content in the roots was only twice higher than in the shoots. Also Izadiyar and Yargholi (2010) found minor differences in Cd content between roots and shoots in clover, alfalfa, sorghum and sainfoin cultivated on wastewater. Surprisingly, our data showed that cadmium content in the roots was max 280 times higher in case of low concentrations (
