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Subcellular distribution, chemical forms and physiological responses involved in cadmium tolerance and detoxification in Agrocybe Aegerita
Ecotoxicology and Environmental Safety 171 (2019) 66–74
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Subcellular distribution, chemical forms and physiological responses involved in cadmium tolerance and detoxification in Agrocybe Aegerita ⁎
Xuedan Lia, Hang Maa, LingLing Lia, Yufeng Gaoa, Yunzhen Lib, , Heng Xua, a b
T
⁎
Key Laboratory of Bio-Resource and Eco-Environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu 610065, Sichuan, PR China Institute of Soil and Groundwater Pollution Control of Sichuan Academy of Environmental Sciences, Chengdu 610065, Sichuan, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Subcellular distribution Chemical forms Cadmium Accumulation Detoxification Physiological responses
A pot experiment was conducted to investigate the detoxification mechanism of Agrocybe aegerita (A. aegerita). The physiological responses, subcellular distribution and chemical forms of cadmium (Cd) in A. aegerita grown in Cd stress were analyzed. The results showed that the biomass was decreased under Cd stress, while the production of malonaldehyde, thiols, and low-molecular-weight organic acids (LWMOAs) as well as the antioxidant enzymes in A. aegerita was increased compared with control group. The HPLC results showed that nine LWMOAs were found in A. aegerita with critic acid as the dominant and they played important role in the detoxification and accumulation of Cd in A. aegerita. More Cd was accumulated in pileus than in stipe. Differential centrifugation technique showed that the majority of Cd was compartmentalized in the soluble fraction (53–75%) and bound to the cell wall (19–42%). The proportion of Cd in the cell wall increased with the increase of the accumulation of Cd in the fruiting body, but in the soluble fraction showed an opposite trend. Furthermore, most of the Cd in A. aegerita was mainly in the forms of NaCl- (29–49%) and ethanol-extractable Cd (20–40%). The ethanol- and water-extractable Cd in stipe (60–66%) was higher than in pileus (43–49%). Thus intracellular detoxification mechanisms of Cd in A. aegerita is related to subcellular partitioning and chemical forms of Cd and well-coordinated physiological responses.
1. Introduction With the development of industrialization and the agricultural activity of human beings, soil pollution by heavy metals has become a serious problem to human beings. Over 80% of contaminated soils are polluted by excess metals, among which 7% was accounted by cadmium (Cd) in China (Chen et al., 2017). Cadmium, which has low mobility and degradability, is non-essential element for human beings, but it often enters humans body via food chain (Volpe et al., 2009). Due to the high toxicity of Cd, it poses a serious threat to human's health. Because of the ability of inhibiting apoptosis and DNA repair, Cd can cause prostate, breast, and lung cancer (Luevano and Damodaran, 2014). Many methods have been used to remedy the Cd-contaminated soil, such as chemical immobilization, soil washing and phytoremediation. However, chemical immobilization and soil washing are easy to cause secondary pollution for soil remediation (Murtaza et al., 2014). Phytoremediation is affected not only by the climate conditions but also by the hyper-accumulator plant species (Ebbs and Kochian, 1997; Wang et al., 2008). Fortunately, it has been reported that macrofungi have an enormous potential for bioaccumulation of Cd from soil
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(Damodaran et al., 2014). Previous research has shown that the accumulation of Cd in Agrocybe aegerita (A. aegerita) was 1.43–3.16 mg/kg when A. aegerita was planted in Cd-polluted soils (Li et al., 2016b). The mycelia of mushroom can spread over the available area and effectively accumulate heavy metals in mycelia and fruiting body. Mycoremediation is an important and promising repair method to remove heavy metals from contaminated soil, due to its low cost, rapid growth, and high biomass. However, the tolerance and detoxification mechanism of Cd within mushroom is still unclear. The subcellular distribution and chemical forms of Cd in organisms can influence the Cd accumulation and tolerance of organisms (Lu et al., 2017; Shi et al., 2016). When Cd is absorbed by organisms, organisms may produce a variety of responses to the harmful effect of Cd, such as enhancing their antioxidant abilities (Cen et al., 2012), binding with the Cd and compartmentalization of Cd (Lu et al., 2017). Previous researches have revealed that most of Cd was deposited in the cell wall and vacuole, such as Oryza sativa, Kandelia obovate, Phytolacca americana, and Lactuca sativa (Fu et al., 2011; Li et al., 2016a; Ramos et al., 2002; Weng et al., 2012). The toxicity of Cd in the cell wall and vacuole is lower than in the organelle fraction (Yang et al., 2018). Therefore,
Corresponding author. E-mail address: [email protected] (H. Xu).
https://doi.org/10.1016/j.ecoenv.2018.12.063 Received 28 June 2018; Received in revised form 19 December 2018; Accepted 21 December 2018 0147-6513/ © 2018 Elsevier Inc. All rights reserved.
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holding capacity, 11.98 ± 0.68%; Alkeline-N (ALN), 46 ± 1.70 mg/ kg; Olsen-P (OSP), 32 ± 1.20 mg/g; Olsen-K (OSK), 45.40 ± 2.30 mg/ kg and pH, 8.26 ± 0.08. The CdCl2·2.5H2O was added to the unpolluted soil to obtain 10 mg/kg (T1) and 20 mg/kg (T2) Cd-polluted soil. The unpolluted soil was marked as CK. Each treatment included three replicates. The spiked soil samples were equilibrated for three months prior to the experiment. Each pot contained 2.0 kg of the as-prepared soil and 100 g of the A. aegerita mycelia. The experiments were conducted in a green-house to keep a constant temperature at 19 ± 3 ℃ and avoid direct sunshine. All pots received unified water to maintain 60% moisture of its water holding capacity till harvesting. About 20 days later, the matured fruiting bodies at the growth time of 3 days after emergence of fruiting bodies were successively collected.
the vacuolar compartmentalization of Cd and the affinity of cell wall for Cd synchronously take part in the detoxification of Cd. The subcellular distribution of Cd depends on the species, according to previous researches that the greatest amount of Cd was in the cell wall for Koelreuteria paniculata (Yang et al., 2018) and the highest concentration of Cd in Phytolacca americana L. was in the soluble fraction (Fu et al., 2011). The subcellular distribution of Cd in mushroom is unclear, which is necessary to research. Chemical forms of Cd indicate distinct bioavailability and toxicity, relating to different biological functions (Wei et al., 2014). Some researches have suggested that the bioavailability, migration, and toxicity of water-soluble Cd in the inorganic and organic form were higher than pectate and protein integrated Cd, nevertheless the insoluble Cd-phosphate complexes and Cd-oxalate with little toxicity (Fu et al., 2011; Qiu et al., 2011). The proportion of pectate and protein integrated Cd and inorganic Cd was highest in peanut cultivars, Haihua 1 (Shi et al., 2016), whereas, the highest amount of Cd for Siegesbeckia orientalis was bound to oxalate, pectate and protein (Xu et al., 2018). In the Koelreuteria paniculata, protein and pectates integrated Cd and undissolved Cd phosphate are the predominant forms (Yang et al., 2018). Given the above, the major chemical form of Cd in organisms is species dependent. The dominant chemical forms of Cd accumulated in mushroom are largely unknown. Therefore, detailed investigation is needed to elucidate the chemical forms of Cd in mushroom. The low-molecular-weight organic acids (LMWOAs) take part in the metal chelation, transportation, sequestration, and accumulation in organism (Kutrowska and Szelag, 2014). When plants grown in Cdpolluted soil, organic acids secreted by plants can bond to Cd adsorbed by the soil particles, consequently promoting the absorption of Cd into the plant (D'Alessandro et al., 2013). In addition, LMWOAs can be involved in the intracellular detoxification via Cd-ligand complexation and compartmentation of the complexation into the vacuole when Cd enters into the cell (Dresler et al., 2014). To our knowledge, macrofungi contain a lot of organic acids (Barros et al., 2013; Fujita et al., 2010), but the relation between cadmium and LMWOAs in mushrooms has not be elucidated. Agrocybe aegerita (A. aegerita) is a very common mushroom (an edible mushroom and has extraordinary nutritional value) with the most effective components of cyclooxygenase inhibitory and antioxidant compounds, which is able to accumulate Cd from Cd-polluted soil (Li et al., 2016b). However, the mechanism of Cd tolerance, detoxification, and accumulation within A. aegerita is absent. The aims of this study were to research the subcellular distribution and chemical forms of Cd in A. aegerita and to analyze their influence on the Cd tolerance and detoxification, as well as to evaluate the relationship between Cd accumulation and LMWOA content in A. aegerita. The physiological changes of A. aegerita under Cd stress were also analyzed.
2.3. Superoxide dismutase (SOD), guaiacol peroxidase (POD), and catalase (CAT) activities analysis The fresh tissue samples (1 g) were ground on the ice with 0.05 M phosphate buffers (pH 7.8) containing 1% PVP (5 mL) and then the homogenates were centrifuged (4 °C, 25 min, 15,000 rpm) to collect the supernatants. The supernatants were used immediately for measuring the activities of antioxidant enzymes. The activity of SOD (EC 1.15.1.1) was determined by measuring its ability to inhibit the photochemical reduction of nitroblue tetrazolium (NBT) at 560 nm (METASH, UV5500PC) (Xu et al., 2010; Li et al., 2017). The reaction medium consisted of 1.5 mL of 50 mM phosphate buffer (pH 7.8), 300 µL of methionine (130 mM), 300 µL of NBT (750 µM), 300 µL riboflavin (20 µM), 300 µL EDTA-Na2 (100 µM), and 50 µL of enzyme extract. One unit of SOD was defined as the amount of enzyme which causes 50% inhibition of the initial reduction of NBT under light. The activity of POD (EC 1.11.1.7) was spectrophotometrically following the increase in absorbance at 470 nm due to the oxidation of guaiacol (METASH, UV5500PC) (Zhu et al., 2004). The reaction mixture in a total volume of 50 mL 100 mM phosphate buffer (pH 6.0) containing 19 µL H2O2 (30%), 28 µL guaiacol was prepared immediately before use. Then 0.1 mL enzyme extract with 0.9 mL phosphate buffer was added to 3 mL reaction mixture. One unit of POD activity was defined as an absorbance change of 0.01 units/min. Catalase activity (EC1.11.1.6) was determined as described by Beers and Sizer (1952). Phosphate buffer (2.5 mL, pH 7.5) and 1% H2O2 (0.1 mL) were mixed in an ice bath and enzyme extract (0.1 mL) was added immediately. The absorption was determined spectrophotometrically at a wavelength of 240 nm (METASH, UV-5500PC). One unit of CAT activity was defined as an absorbance change of 0.01 units/min. 2.4. Measurement of total thiol content (T-SH), reduced glutathione (GSH) and malondialdehyde (MDA)
2. Materials and methods The tissue sample was homogenized in 0.02 M EDTA on ice and the total thiol content was measured by the spectrophotometer at 412 nm using Ellman's reagent, 5,5′-dithio-bis-(2-nitrobenzoicacid) (DTNB), according Aravind and Prasad (Aravind and Prasad, 2005). The content of GSH was measured using a kit which purchased from Nanjing Jian Cheng Bioengineering Institute (China), according to the instruction of the kit. The concentration of MDA was determined spectrophotometrically by reaction with thiobarbituric acid (TBA) and was calculated using 155 per mM per cm as extinction coefficient (Amor et al., 2005).
2.1. Materials The A. aegerita was purchased from Sichuan Academy of Agricultural Sciences (China). The LMWOAs standards (oxalic, citric, malic, tartaric, formic, succinic, malonic, lactic and acetic acids) with certified standard grade and the chromatographic-grade reagents (H3PO4, KH2PO4 and methanol) were purchased from Solarbio (China) and Aladdin (China), respectively. The rest of the chemicals in our experiment were analytical reagents bought from Chengdu Kelong Corp (China).
2.5. Extraction of Cd subcellular distribution 2.2. Soil preparation and pot experiment The fresh sample (1 g) was homogenized in 50 mL precooled extraction solution (250 mM sucrose, 20 mM Tris-HCl pH 7.5, 1 mM DLdithioerythritol) on ice. Then the homogenate was centrifuged at 300 g for 1 min and the solid residue was defined as cell wall fraction. The
Unpolluted soil without Cd was collected from Sichuan University, China, at a depth of 5–20 cm. The properties of soil are as follows: CEC, 10.41 ± 0.92 cmol/kg; organic carbon, 11.70 ± 0.27 g/kg; water 67
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Fig. 1. Biomass (a), MDA contents (b), Cd concentration (c) and Cd accumulation (d) of A. aegerita. The values are represented as the means ± standard. The letters in same pattern column indicate significant differences among different treatments (P < 0.05). CK: without the addition of Cd in the soil; T1 and T2: Cd concentrations of 10 mg/kg and 20 mg/kg respectively in soil.
digested using HNO3-HClO4 (3:1, v/v).
supernatant was centrifuged at 15,000 g for 45 min. The precipitate and the supernatant were designated as organelle fraction and a soluble fraction, respectively (Su et al., 2014). All centrifugations were done at 4 °C. The three fractions were evaporated and digested using HNO3HClO4 (3:1, v/v).
2.7. Analysis of low-molecular-weight organic acids (LMWOAs) The mushroom sample was dried at 50 °C for 96 h and powered. Then 8 mL 1% H3PO4 was added to suspend the power (1 g) and then the homogenate subjected to ultrasound action (400 W, 30 min, 25 °C) to extract LMWOAs. The solution was centrifuged at 2500g for 10 min, and the supernatant was filtered through a 0.45 µm cellulose membrane for analysis (Mleczek et al., 2016). The content of LMWOAs (oxalic, citric, malic, tartaric, formic, succinic, malonic, lactic and acetic acids) ware analyzed by high-performance liquid chromatography (HPLC, Shimadzu CBM-20A, Japan) with a SWELL Chromplus C18 column (250 mm × 4.6 mm × 5 µm). HPLC conditions were as follows: mobile phase, 20 mM KH2PO4 (pH 2.2) and methanol (98:2, v/v); flow rate, 1 mL/min; temperature, 25 ℃; UV detection wavelength, 210 nm (Mleczek et al., 2016).
2.6. Extraction of Cd in chemical forms The chemical forms of Cd were analyzed using successively extracted by designated solutions in the following order: (1) 80% ethanol, extracting inorganic Cd; (2) deionized water, extracting water-soluble organic Cd; (3) 1 M NaCl, extracting pectate and protein-integrated Cd; (4) 2% acetic acid, extracting insoluble Cd-phosphate complexes; (5) 0.6 M HCl, extracting Cd-oxalate and (6) the residual Cd. The fresh sample (4 g) was homogenized in 80 mL extraction solution, and shaken for 18 h at 25 °C, and then the solution was centrifuged at 4000g for 10 min, and the first supernatant was collected in a conical beaker. The precipitate was suspended twice in the extraction solution, shaken for 2 h at 25 °C, and centrifuged. The supernatants of the three centrifugations were pooled and the solid residue was subjected to the next extraction in the indicated order with the same steps mentioned above (Lu et al., 2017). The five extraction solutions and the residue were evaporated on an electroplated plate at 70 °C to a constant weight and
2.8. Cd concentration analysis Dried and powered mushroom samples were digested with a mixture of HNO3 and H2O2 (5:2, v/v) using a microwave oven (SINEO, MDS-6) following the procedure of Tuzen et al. (2007). The total 68
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Fig. 2. Activities of SOD (a), CAT (b) and POD (c), and concentration of GSH and T-SH (d) of A. aegerita. The values are represented as the means ± standard. The letters in same pattern column indicate significant differences among different treatments (P < 0.05). CK: without the addition of Cd in the soil; T1 and T2: Cd concentrations of 10 mg/kg and 20 mg/kg respectively in soil.
concentration of Cd in mushroom samples was determined by flame atomic adsorption spectrometry (FAAS, VARIAN, SpectrAA-220Fs). The conditions were as follows: acetylene flow rate, 2.0 L/min; the air flow rate, 13.5 L/min; wave length, 228.8 nm; Cd lamp current, 4.0 nm; slit width, 0.5 nm. The Cd concentrations in different fractions (subcellular distribution and chemical forms) were determined by inductively coupled plasma mass spectrometry (ICP-MS, aurora M90). The isotopes selected for measurement were 113Cd with internal standard 103Rh. The conditions were as follows: cooling air flow rate, 18 L/min; auxiliary gas flow rate, 1.8 L/min; atomizing gas flow rate, 1 L/min; sample depth, 6.5 mm; radio frequency (RF) power, 1400 W.
following formula:
TF = Concentration of Cd in pileus / Concentration of Cd in stipe 3. Results and discussions 3.1. Biomass, MDA, Cd concentration and accumulation The biomass of A. aegerita decreased with an increase in the concentration of Cd (Fig. 1a). The fresh weights (FW) of pileus and stipe of A. aegerita in T1 had decreased by 16% and 20% respectively compared to CK. Furthermore, the biomass of pileus and stipe in T2 were 73% and 71% of CK, respectively. According to the tolerance index, the A. aegerita belonged to high tolerance (Tolerance index > 70%) in the polluted soil containing 20 mg/kg Cd (Yang et al., 2018). Biomass is a significant indicator of stress response for mushroom. It is reported that Cd can inhibit metabolic enzyme activities and influence auxin homeostasis, as well as affect the synthesis of protein (Luevano and Damodaran, 2014; Thomet et al., 1999; Yu et al., 2015), which may explain why accumulation of Cd in A. aegerita caused the decrease of biomass. The addition of Cd greatly influenced the MDA concentrations in pileus and stipe of A. aegerita (Fig. 1b). The contents of MDA in pileus and stipe of T1 were 41% and 36% higher than the control,
2.9. Statistical analysis All results were tested by one-way ANOVA using the SPSS statistics 17.0 package. All data were the means ± SD of three independent replicates and the significance of the difference between different treatments were compared using least significant difference (LSD) calculated at a significance level of P < 0.05. All figures were performed using OriginPro 8. The tolerance index (TI), which was defined as the ratio of biomass exposed to Cd to the control, was calculated to determine the tolerance of A. aegerita. The translocation factor (TF) was used to describe the transport of Cd in fruiting body of A. aegerita, calculated by the 69
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through secreting antioxidant enzymes (Zhang et al., 2012). SOD is the first line of defense against the ROS, converting the superoxide radical to molecular oxygen and H2O2 (Irfan et al., 2014). CAT participates in the decrease of H2O2 concentration in the cell, which breaks H2O2 to H2O and oxygen (Irfan et al., 2014). The H2O2 can also be scavenged by SOD using chlorogenic acid as a substrate (Takahama and Oniki, 1997). A large amounts of active oxygen were produced when fungi were exposed in Cd stress (Li et al., 2017). The generation of active oxygen can result in high toxicity to organisms. Organisms have an effective system to counter this toxicity. Previous research showed that Cd stress can induce the response of SOD, CAT and POD in fungi (Li et al., 2017). In this study, the activities of SOD, CAT and POD were increased under Cd stress. These results suggested that antioxidant enzymes, such as SOD, CAT and POD, take part in the detoxification of Cd. The effect of Cd on glutathione (GSH) and total thiol content (T-SH) in A. aegerita are showed in Fig. 2d. The concentrations of GSH in pileus and stipe were significantly increased compared to those in CK and reached their maximum level of 60.82 and 55.25 μmol/g FW respectively at T2. The concentrations of GSH in pileus and stipe of T1 were improved by 129% and 117% over CK levels, respectively. Furthermore, the contents of T-SH in pileus and stipe were also dramatically increased compared to those in CK. The maximum concentrations of TSH in pileus and stipe at T2 were 1.51 and 1.38 times higher than those of CK, respectively. In general, the contents of GSH and T-SH in pileus were higher than those in stipe, but the difference between pileus and stipe was not significant (P > 0.05). The complexation of Cd with thiol compounds is another intracellular detoxification of Cd (Weng et al., 2012). GSH is the main non-protein thiols in the cells and can to relieve Cd stress using various mechanisms, such as the activation of antioxidation mechanism and chelation of cadmium ions (Xu et al., 2011; Zhuo et al., 2017). On the one hand, GSH is an important antioxidation molecule, which can take part in the removal of active oxygen induced by Cd stress (Xu et al., 2018). On the other hand, GSH is the precursors of phytochelatins (PCs) which can bind to Cd2+ forming Cd-PC complexes that facilitate the sequestration of Cd in the vacuoles (Rahman et al., 2017). The thiol groups of T-SH can bind to Cd leading to the sequestration of Cd in the vacuole and reducing the damage of Cd to A. aegerita. Therefore, the increase of GSH and T-SH contents in A. aegerita under Cd stress revealed that they play an important role in reducing the toxic effect of Cd on mushrooms.
respectively. The MDA contents in pileus and stipe at T2 were increased by 1.52 and 1.92 times compared to CK, respectively. MDA is an indicator of lipid peroxidation to reflect the peroxidation degree, indicating the toxic effects of Cd to mushroom (Li et al., 2017; Zhao et al., 2012). In this research, the result indicated that the membrane lipids of A. aegerita were oxidized by Cd stress. Cadmium concentration in pileus and stipe increased with the increase of Cd level in the soils (Fig. 1c). On the whole, the contents of Cd in pileus were higher than in stipe, with a TF of 1.38–1.90. Similarly, the accumulation of Cd in pileus was higher than in stipe (Fig. 1d). Cadmium accumulation in pileus was 85–88% of the total. Although the ability of accumulating Cd for A. aegerita is limited, the A. aegerita possesses abilities of rapid growth and high biomass, and can be cultivated from February to November, which is a eurythermic species. Therefore, A. aegerita can be used as an effective accumulator for bioremediation of the Cd-polluted soil. Despite the biomass of A. aegerita decreased with an increase in the concentration of Cd, the accumulation of Cd in A. aegerita had improved. A. aegerita may employ some measures to detoxify Cd ions within the cell and to enhance the capacity of Cd tolerance and accumulation. 3.2. Effect of Cd on the activities of SOD, POD and CAT as well as on the production of GSH and T-SH In this research, the effect of Cd on the activities of antioxidant enzymes showed an increasing tendency (Fig. 2a, b, c). The activities of SOD in T1 of pileus and stipe increased by 60% and 45% compared to those in CK, respectively. The SOD activities in T2 reached the maximum amount of 368–369 U/g FW. The activity of CAT increased in a Cd concentration dependent manner, and the maximum value of 254 U/g FW was appeared in stipe at T2. CAT activity in pileus of T2 was 208 U/g FW, but there was no significant difference in pileus and stipe at T2 according to statistical analysis (P > 0.05). The activities of CAT in T1 of pileus and stipe were 1.57 and 1.31 times higher than those of CK, respectively. POD activities under Cd stress were also higher compared with the CK. The soil spiked with 20 mg/kg Cd led to the maximal values of 230 and 240 U/g FW for POD in pileus and stipe, respectively. Cadmium can induce the production of reactive oxygen species (ROS) in organisms directly or indirectly, thereby disturbing the balance of the cellular redox status and seriously impairing the function of cells (Zhang et al., 2016). To resist the damage from ROS such as superoxide (O2-) and hydrogen peroxide (H2O2), organisms activate cellular immune system for the removal of cellular ROS, for example
Fig. 3. Subcellular distribution of Cd in A. aegerita grown in Cd polluted soil. The values are represented as the means ± standard. The letters in the same fraction means significant differences (P < 0.05) among different treatments and tissues. T1 and T2: Cd concentrations of 10 mg/kg and 20 mg/kg respectively in soil. 70
71
244.45 ± 2.84 C 364.71 ± 24.73 B 509.04 ± 24.55 A 0.908* 232.25 ± 9.27 c 313.01 ± 38.45 b 470.54 ± 17.52 a 0.846* 14.15 ± 2.88 B 42.14 ± 4.22 A 27.84 ± 6.20 AB 0.741 14.21 ± 0.47 b 19.65 ± 5.81 ab 31.95 ± 5.37 a 0.772 13.84 ± 1.67 C 62.52 ± 8.35 B 164.53 ± 1.95 A 0.811 6.79 ± 1.58 c 17.60 ± 1.27 b 83.38 ± 4.98 a 0.725 22.18 ± 5.82 A 28.60 ± 4.83 A 30.30 ± 7.44 A 0.634 9.79 ± 1.32 a 11.73 ± 1.81 a 13.46 ± 7.79 a 0.411 6.65 ± 2.09 A 9.96 ± 1.26 A 10.05 ± 1.06 A 0.83* 4.21 ± 1.11 a 5.17 ± 0.33 a 6.92 ± 0.45 a 0.79 1.52 ± 0.01 A 2.00 ± 0.26 A 2.78 ± 0.31 A 0.848* 1.04 ± 0.02 b 1.66 ± 0.67 b 1.80 ± 0.09 a 0.987** 23.42 ± 5.30 A 23.17 ± 2.37 A 27.53 ± 12.88 A 0.174 12.15 ± 4.90 a 12.36 ± 0.66 a 17.93 ± 0.419 a 0.525 8.95 ± 0.27 A 10.02 ± 2.43 A 11.27 ± 1.13 A 0.632 4.21 ± 0.58 b 2.95 ± 0.93 b 7.12 ± 0.99 a 0.36 129.70 ± 5.24 B 155.43 ± 3.85 A 154.52 ± 6.44 A 0.939** 167.71 ± 1.01 c 224.36 ± 23.64 b 281.31 ± 14.59 a 0.915*
Tartaric Malic Succinic Citric
Stipe
CK T1 T2 r CK T1 T2 r Pileus
Oxalic
Low-molecular-weight organic acid content (mg/g DW)
To understand the mechanisms of Cd detoxification, transportation and accumulation in A. aegerita, the chemical forms of Cd in A. aegerita were analyzed. Overall, the Cd concentrations in different chemical forms increased with the increase of Cd concentrations in the soil (Fig. 4a). The proportion of Cd in different chemical forms is shown in Fig. 4b. The ethanol- (40% and 34% in T1 and T2, respectively) and NaCl-extractable Cd (29% and 35% in T1 and T2, respectively) were predominant in stipe, followed by H2O-extractable Cd. Whereas, NaClextractable Cd occupied 46% and 49% of the total concentrations of Cd at pileus in T1 and T2, respectively, followed by ethanol- (26% and 20% in T1 and T2, respectively) and H2O-extractable Cd (24% and 22% in T1 and T2, respectively). The chemical forms of Cd have a great influence on the toxicity and migration capability of Cd in organisms, and Cd exists in organisms in different chemical forms to perform distinctive biological function (Yang et al., 2018). The migration ability of Cd in ethanol- and H2O-
Treatments
3.4. Chemical forms of Cd
Tissues
Table 1 LWMOAs content and Pearson correlation coefficients (r) between content of LWMOAs and Cd in A. aegerita.
Formic
Malonic
Lactic
Acetic
Total
The subcellular distribution of Cd in A. aegerita is showed in Fig. 3. The concentrations of Cd in all subcellular fractions of pileus and stipe increased with the increase of Cd concentrations in soil. In general, most of Cd (53–75%) was stored in the soluble fraction, followed by the cell wall fraction (19–42%), and the part of organelle fraction accounted for only 5–6% of the total. The proportion of Cd in the cell wall fraction at pileus and stipe increased with the increase of Cd added to soil, and the proportion of Cd in the cell wall fraction at pileus was higher than that at stipe. However, the proportions of the soluble fraction at stipe (75% and 67% in T1 and T2, respectively) were higher than those at pileus (61% and 53% in T1 and T2, respectively). In this study, the concentration of Cd in pileus was higher than in stipe. High concentrations of Cd would severely damage the A. aegerita. Previous researches have shown that the regionalization of Cd in the cell wall and soluble fraction is a measure to alleviate the toxicity of Cd (Fu et al., 2011; Lu et al., 2017; Zhou et al., 2017). The result of this study that most of Cd was compartmentalized in the cell wall and soluble fractions, was similar to previous researches. The cell wall plays a protective role under Cd stress, because it is the first barrier for cell to reduce the damage of Cd. The carboxyl, amino, sulfhydryl, phosphoryl and hydroxyl groups of polysaccharide and protein at the cell wall can interact with Cd2+ making Cd2+ adsorb on the cell wall to restrict Cd2+ transportation across cytoplasm (Das and Guha, 2007). In this study, the proportion of cell wall-bound Cd in pileus was higher than in stipe. The proportion of Cd in the cell wall fraction increased with an increase in the concentration of Cd. This result suggested that fixation Cd to cell wall was a strategy to reduce the poisonousness of Cd in A. aegerita. Moreover, the compartmentation of Cd in the vacuole could also be helpful to alleviate the toxicity of Cd (Xu et al., 2018; Yang et al., 2018). Vacuole contains significant amounts of sulfur-rich peptides and organic acids which can chelate and compartmentalize Cd to decrease the amount of Cd interfering with the organelles in organisms (Xu et al., 2018; Yang et al., 2018). In this study, most of Cd (53–75%) was compartmentalized in soluble fraction which mostly consists of vacuoles (Wang et al., 2016; Zhou et al., 2017). Similarly, previous research revealed that a large proportion of Cd (54–68%) in pokeweed compartmentalized with organo-ligands and sequestrated in soluble fraction (Fu et al., 2011). Correspondingly, the A. aegerita contained plenty of thiol groups and organic acids (Fig. 2d and Table 1). Thiol groups and organic acids in vacuoles of A. aegerita play an essential role in the tolerance and accumulation of Cd (Wang et al., 2008). In this research, the concentration of Cd in pileus was higher than in stipe, as well as the soluble fraction in stipe was higher than in pileus, indicating that chelation with organo-ligands may enhance the transport of Cd from stipe to pileus in A. aegerita.
14.21 ± 0.47 B 19.65 ± 5.81 B 31.95 ± 5.37 A 0.818* 12.14 ± 0.31 b 17.53 ± 3.46 ab 26.66 ± 4.44 a 0.799
3.3. Subcellular distribution of Cd
Values are means ± standard deviation (n = 3). Different letters in the same organs means significant differences (P < 0.05) among different treatments. CK: without the addition of Cd in the soil; T1 and T2: Cd concentrations of 10 mg/kg and 20 mg/kg respectively in soil. DW and r represent dry weight and pearson correlation coefficients, respectively. * Significant at p < 0.05 ** significant at p < 0.01.
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Fig. 4. Metal speciation in A. aegerita grown in Cd-polluted soil. F1, F2, F3, F4, F5, and F6 denoted ethanol-extractable, water-extractable, NaCl-extractable, HAcextractable, HCl-extractable and residual Cd, respectively. The values are represented as the means ± standard. The letters in the same organs means significant differences (P < 0.05) among different treatments and tissues. T1 and T2: Cd concentrations of 10 mg/kg and 20 mg/kg respectively in soil.
and to enhance the accumulation of Cd (Goliński et al., 2015). LMWOAs, based on the internal complexation and compartmentation in the vacuole rather than on extracellular mechanism, take part in the detoxification of Cd in maize (Chaffai et al., 2006). Previous research showed that T. caerulescens synthesize more LMWOAs in Cd stress and accumulate more Cd in the shoots (Pence et al., 2000). Cadmium elevated LMWOAs in leaves of maize with positive effect (Dresler et al., 2014). In this study, A. aegerita produced more LMWOAs (total acids have increased 1.49–2.08 times and 1.35–2.03 times in pileus and stipe, respectively) in the Cd supplied soil compared to CK. This result suggested that LMWOAs played a crucial part in reducing the damage of Cd and improving the accumulation of Cd in A. aegerita. When Cd enters into cell, LMWOAs can compartmentalize Cd into vacuole based on metal-ligand complexation (Dresler et al., 2014; Mleczek et al., 2016). Vacuole is rich in LMWOAs which can chelate and compartmentalize Cd (Xu et al., 2018; Yang et al., 2018). The chelation with LMWOAs is one reason that the proportion of soluble fraction was higher than other fractions. Carboxylic acids, particularly citric acid, treated as metal-complexing agents, can be combined with divalent cations strongly and form stable complexes (Nigam et al., 2001). The mobile organicallybound Cd was created by the interaction of Cd with citric acid (Nigam et al., 2001). For example, citric acid can interact with Cd and form Cd (CH2)2COH(COO)3- and Cd(CH2)2COH(COO)3H (Rauser, 1999). Previous research showed that citric acid is the ligand for Cd moving from roots to leaves (Rauser, 1999). This study revealed that the content of citric acid had a positive relation to the level of Cd in A. aegerita (r = 0.915 in stipe and r = 0.939 in pileus). This finding suggested that the chelate of citric acid with Cd participated in the transport of Cd from stipe to pileus. The toxicity and bioavailability of Cd depend in part on solubility and chemical reactivity. Cadmium ions reacted with oxalic acid forming the insoluble chelated complexes to reduce the toxicity of Cd in Pleurotus ostreatus (Li et al., 2017). Previous research has revealed that oxalic acid secreted by white-rot fungi was induced by exogenous Cd, and oxalic acid played an important role in Cd detoxification with a negative correlation between oxalic acid concentration and Cd-induced growth inhibition ratios (Xu et al., 2015). The Cd oxalate, extracted by 0.6 M HCl, has relatively low toxicity (Lu et al., 2017). In this research, the proportions of HCl-extractable Cd in pileus were higher than in stipe (1.62% in pileus and 1.16% in stipe for T1, and 3.28% in pileus and 1.61% in stipe for T2, P < 0.05), corresponding to the higher concentrations of oxalic acid in pileus compared to stipe. Therefore,
extractable fractions (water-soluble Cd in inorganic form and organic form) is higher than other forms (Xu et al., 2018), and the toxicity of these forms to organisms is higher than other forms, correspondingly. In this study, the proportions of ethanol- and H2O-extractable Cd in stipe were higher than those in pileus. The higher water-soluble Cd in inorganic and organic form with higher migration capacity in stipe can explain why the translocation factor of A. aegerita was greater than 1. Higher concentration and proportion of Cd in water-soluble inorganic form and organic form (extracted by 80% ethanol and deionized water) with higher migration capacity, implied that Cd in A. aegerita was transported mainly in water soluble forms. The Cd integrated with pectates and protein ligands (extracted by 1 M NaCl) with less toxicity in pileus were higher than those in stipe. When the concentrations of Cd in pileus were higher than those in stipe, the proportions of pectate and protein-bound form Cd in pileus were higher than those in stipe, suggesting that converting Cd into undissolved pectate and protein-bound form Cd can reduce the toxicity of Cd to A. aegerita and increase the accumulation of Cd in the fruiting body. The proportions of Cd in HAc-, HCl- and residual-fractions were low, which may be owing to the influence of the thiol groups and LMWOAs (Xiao et al., 2016).
3.5. Concentration of LMWOAs Nine types of LMWOAs were all detected in the pileus and stipe of A. aegerita (Table 1). The citric acid was the dominant organic acid in pileus (30–53%) and stipe (60–73%) of A. aegerita. The tartaric acid concentration in the fruiting body of A. aegerita was lowest among the nine types of LMWOAs. According to the result, the concentrations of citric and tartaric acids in A. aegerita showed highly positive correlation with the accumulation of Cd in A. aegerita. The content of citric acid in stipe was higher than in pileus (P < 0.05). Nevertheless, the contents of oxalic and lactic acids in pileus were higher than these in stipe (P < 0.05). In general, the concentrations of oxalic, citric, lactic and acetic acids were elevated in pileus of A. aegerita exposed to Cd compared to CK (P < 0.05), whereas the levels of succinic, malic, tartaric, malonic and formic acids in pileus were no significant difference between different treatments (P > 0.05). The concentrations of oxalic, citric, succinic, tartaric, lactic and acetic acids in stipe of Cd-exposed A. aegerita increased compared to CK (P < 0.05). LMWOAs take part in many metabolic pathways in organisms, such as cation transportation, respiration, metal chelating and detoxification (Dresler et al., 2014). The increasing of LMWOAs in the presence of Cd can chelate and compartmentalize the Cd to reduce the toxicity of Cd 72
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there is one strategy used by A. aegerita to alleviate the toxicity of Cd by complexation with oxalic acid to change the chemical forms of Cd. 4. Conclusions The accumulation and detoxification of Cd as well as stress responses of A. aegerita were analyzed in this work for the first time. A. aegerita could tolerate high concentrations of Cd (up to 20 mg/kg) and could utilize various strategies to detoxify and survive. Antioxidant enzyme system, GSH and T-SH played an important role in the detoxification and accumulation of Cd. Nine types of LMWOAs were detected in A. aegerita and these acids were closely related to accumulation and detoxification of Cd in this study. The concentration of Cd in pileus was higher than that in stipe. Its subcellular distribution showed that vast majority of Cd (94–95%) was in the cell wall and soluble fractions. Furthermore, the proportions of NaCl- and ethanol-extractable Cd were the dominant forms in pileus and stipe, respectively. The overall study clearly suggested the ability of A. aegerita to remediate Cd contaminated soils and systematically provided new insights into the cellular mechanisms of Cd tolerance and detoxification of mushrooms. Acknowledgements This study was financially supported by the Key Research and Development Program of Sichuan Province (2017SZ0181) and the Agricultural Science and Technology Achievements Transformation Program of Sichuan Province (2017NZZJ008). The authors also wish to thank Professor Guanglei Cheng and Dong Yu from Sichuan University for the technical assistance. Conflict of interest There was no conflict of interest. References Aravind, P., Prasad, M.N., 2005. Modulation of cadmium-induced oxidative stress in Ceratophyllum demersum by zinc involves ascorbate-glutathione cycle and glutathione metabolism. Plant Physiol. Biochem. 43, 107–116. Amor, N.B., Hamed, K.B., Debez, A., Grignon, C., Abdelly, C., 2005. Physiological and antioxidant responses of the perennial halophyte Crithmum maritimum to salinity. Plant Sci. 168, 889–899. Barros, L., Pereira, C., Ferreira, I.R., 2013. Optimized analysis of organic acids in edible mushrooms from Portugal by ultra fast liquid chromatography and photodiode array detection. Food Anal. Methods 6, 309–316. Beers, R.F., Sizer, I., 1952. A spectrophotometric method for measuring the breakdown of hydrogen by catalase. J. Biochem. 195, 133–140. Cen, F., Hu, Y.J., Xu, H., 2012. Responses of antioxidant defenses in Coprinus comatus exposed to cadmium and mercury toxicity. Asian J. Chem. 24, 4679–4685. Chaffai, R., Ali, T., Ferjani, E.E., 2006. A comparative study on the organic acid content and exudation in maize (Zea mays L.) seedlings under conditions of copper and cadmium stress. Asian J. Plant Sci. 21, 228–232. Chen, N.C., Zhang, Y.J., He, X.F., Li, X.F., Zhang, X.X., 2017. Analysis of the report on the national general survey of soil contamination. J. Agro-Environ. Sci. 36, 1689–1692. D'Alessandro, A., Taamalli, M., Gevi, F., Timperio, A.M., Zolla, L., Ghnaya, T., 2013. Cadmium stress responses in Brassica juncea: hints from proteomics and metabolomics. J. Proteome Res. 12, 4979. Damodaran, D., Shetty, K.V., Mohan, B.R., 2014. Uptake of certain heavy metals from contaminated soil by mushroom–Galerina vittiformis. Ecotoxicol. Environ. Saf. 104, 414–422. Das, S.K., Guha, A.K., 2007. Biosorption of chromium by termitomyces clypeatus. Colloids Surf. B Biointerfaces 60, 46–54. Dresler, S., Hanaka, A., Bednarek, W., Maksymiec, W., 2014. Accumulation of low-molecular-weight organic acids in roots and leaf segments of Zea mays plants treated with cadmium and copper. Acta Physiol. Plant. 36, 1565–1575. Ebbs, S.D., Kochian, L., 1997. Toxicity of zinc and copper to Brassica species: implications for phytoremediation. J. Environ. Qual. 26, 776–781. Fu, X.P., Dou, C.M., Chen, Y.X., Chen, X.C., Shi, J.Y., Yu, M., Xu, J., 2011. Subcellular distribution and chemical forms of cadmium in Phytolacca americana L. J. Hazard. Mater. 186, 103. Fujita, T., Komemushi, S., Yamagata, K., 2010. Analysis of organic acids in fruit-bodies of Tricholoma giganteum by high performance liquid chromatography. Lett. Appl. Microbiol. 11, 27–29. Goliński, P., Mleczek, M., Magdziak, Z., Gąsecka, M., Borowiak, K., Dąbrowski, J., Kaczmarek, Z., Rutkowski, P., 2015. Efficiency of Zn phytoextraction, biomass yield
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Subcellular distribution, chemical forms and physiological responses involved in cadmium tolerance and detoxification in Agrocybe Aegerita ⁎
Xuedan Lia, Hang Maa, LingLing Lia, Yufeng Gaoa, Yunzhen Lib, , Heng Xua, a b
T
⁎
Key Laboratory of Bio-Resource and Eco-Environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu 610065, Sichuan, PR China Institute of Soil and Groundwater Pollution Control of Sichuan Academy of Environmental Sciences, Chengdu 610065, Sichuan, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Subcellular distribution Chemical forms Cadmium Accumulation Detoxification Physiological responses
A pot experiment was conducted to investigate the detoxification mechanism of Agrocybe aegerita (A. aegerita). The physiological responses, subcellular distribution and chemical forms of cadmium (Cd) in A. aegerita grown in Cd stress were analyzed. The results showed that the biomass was decreased under Cd stress, while the production of malonaldehyde, thiols, and low-molecular-weight organic acids (LWMOAs) as well as the antioxidant enzymes in A. aegerita was increased compared with control group. The HPLC results showed that nine LWMOAs were found in A. aegerita with critic acid as the dominant and they played important role in the detoxification and accumulation of Cd in A. aegerita. More Cd was accumulated in pileus than in stipe. Differential centrifugation technique showed that the majority of Cd was compartmentalized in the soluble fraction (53–75%) and bound to the cell wall (19–42%). The proportion of Cd in the cell wall increased with the increase of the accumulation of Cd in the fruiting body, but in the soluble fraction showed an opposite trend. Furthermore, most of the Cd in A. aegerita was mainly in the forms of NaCl- (29–49%) and ethanol-extractable Cd (20–40%). The ethanol- and water-extractable Cd in stipe (60–66%) was higher than in pileus (43–49%). Thus intracellular detoxification mechanisms of Cd in A. aegerita is related to subcellular partitioning and chemical forms of Cd and well-coordinated physiological responses.
1. Introduction With the development of industrialization and the agricultural activity of human beings, soil pollution by heavy metals has become a serious problem to human beings. Over 80% of contaminated soils are polluted by excess metals, among which 7% was accounted by cadmium (Cd) in China (Chen et al., 2017). Cadmium, which has low mobility and degradability, is non-essential element for human beings, but it often enters humans body via food chain (Volpe et al., 2009). Due to the high toxicity of Cd, it poses a serious threat to human's health. Because of the ability of inhibiting apoptosis and DNA repair, Cd can cause prostate, breast, and lung cancer (Luevano and Damodaran, 2014). Many methods have been used to remedy the Cd-contaminated soil, such as chemical immobilization, soil washing and phytoremediation. However, chemical immobilization and soil washing are easy to cause secondary pollution for soil remediation (Murtaza et al., 2014). Phytoremediation is affected not only by the climate conditions but also by the hyper-accumulator plant species (Ebbs and Kochian, 1997; Wang et al., 2008). Fortunately, it has been reported that macrofungi have an enormous potential for bioaccumulation of Cd from soil
⁎
(Damodaran et al., 2014). Previous research has shown that the accumulation of Cd in Agrocybe aegerita (A. aegerita) was 1.43–3.16 mg/kg when A. aegerita was planted in Cd-polluted soils (Li et al., 2016b). The mycelia of mushroom can spread over the available area and effectively accumulate heavy metals in mycelia and fruiting body. Mycoremediation is an important and promising repair method to remove heavy metals from contaminated soil, due to its low cost, rapid growth, and high biomass. However, the tolerance and detoxification mechanism of Cd within mushroom is still unclear. The subcellular distribution and chemical forms of Cd in organisms can influence the Cd accumulation and tolerance of organisms (Lu et al., 2017; Shi et al., 2016). When Cd is absorbed by organisms, organisms may produce a variety of responses to the harmful effect of Cd, such as enhancing their antioxidant abilities (Cen et al., 2012), binding with the Cd and compartmentalization of Cd (Lu et al., 2017). Previous researches have revealed that most of Cd was deposited in the cell wall and vacuole, such as Oryza sativa, Kandelia obovate, Phytolacca americana, and Lactuca sativa (Fu et al., 2011; Li et al., 2016a; Ramos et al., 2002; Weng et al., 2012). The toxicity of Cd in the cell wall and vacuole is lower than in the organelle fraction (Yang et al., 2018). Therefore,
Corresponding author. E-mail address: [email protected] (H. Xu).
https://doi.org/10.1016/j.ecoenv.2018.12.063 Received 28 June 2018; Received in revised form 19 December 2018; Accepted 21 December 2018 0147-6513/ © 2018 Elsevier Inc. All rights reserved.
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holding capacity, 11.98 ± 0.68%; Alkeline-N (ALN), 46 ± 1.70 mg/ kg; Olsen-P (OSP), 32 ± 1.20 mg/g; Olsen-K (OSK), 45.40 ± 2.30 mg/ kg and pH, 8.26 ± 0.08. The CdCl2·2.5H2O was added to the unpolluted soil to obtain 10 mg/kg (T1) and 20 mg/kg (T2) Cd-polluted soil. The unpolluted soil was marked as CK. Each treatment included three replicates. The spiked soil samples were equilibrated for three months prior to the experiment. Each pot contained 2.0 kg of the as-prepared soil and 100 g of the A. aegerita mycelia. The experiments were conducted in a green-house to keep a constant temperature at 19 ± 3 ℃ and avoid direct sunshine. All pots received unified water to maintain 60% moisture of its water holding capacity till harvesting. About 20 days later, the matured fruiting bodies at the growth time of 3 days after emergence of fruiting bodies were successively collected.
the vacuolar compartmentalization of Cd and the affinity of cell wall for Cd synchronously take part in the detoxification of Cd. The subcellular distribution of Cd depends on the species, according to previous researches that the greatest amount of Cd was in the cell wall for Koelreuteria paniculata (Yang et al., 2018) and the highest concentration of Cd in Phytolacca americana L. was in the soluble fraction (Fu et al., 2011). The subcellular distribution of Cd in mushroom is unclear, which is necessary to research. Chemical forms of Cd indicate distinct bioavailability and toxicity, relating to different biological functions (Wei et al., 2014). Some researches have suggested that the bioavailability, migration, and toxicity of water-soluble Cd in the inorganic and organic form were higher than pectate and protein integrated Cd, nevertheless the insoluble Cd-phosphate complexes and Cd-oxalate with little toxicity (Fu et al., 2011; Qiu et al., 2011). The proportion of pectate and protein integrated Cd and inorganic Cd was highest in peanut cultivars, Haihua 1 (Shi et al., 2016), whereas, the highest amount of Cd for Siegesbeckia orientalis was bound to oxalate, pectate and protein (Xu et al., 2018). In the Koelreuteria paniculata, protein and pectates integrated Cd and undissolved Cd phosphate are the predominant forms (Yang et al., 2018). Given the above, the major chemical form of Cd in organisms is species dependent. The dominant chemical forms of Cd accumulated in mushroom are largely unknown. Therefore, detailed investigation is needed to elucidate the chemical forms of Cd in mushroom. The low-molecular-weight organic acids (LMWOAs) take part in the metal chelation, transportation, sequestration, and accumulation in organism (Kutrowska and Szelag, 2014). When plants grown in Cdpolluted soil, organic acids secreted by plants can bond to Cd adsorbed by the soil particles, consequently promoting the absorption of Cd into the plant (D'Alessandro et al., 2013). In addition, LMWOAs can be involved in the intracellular detoxification via Cd-ligand complexation and compartmentation of the complexation into the vacuole when Cd enters into the cell (Dresler et al., 2014). To our knowledge, macrofungi contain a lot of organic acids (Barros et al., 2013; Fujita et al., 2010), but the relation between cadmium and LMWOAs in mushrooms has not be elucidated. Agrocybe aegerita (A. aegerita) is a very common mushroom (an edible mushroom and has extraordinary nutritional value) with the most effective components of cyclooxygenase inhibitory and antioxidant compounds, which is able to accumulate Cd from Cd-polluted soil (Li et al., 2016b). However, the mechanism of Cd tolerance, detoxification, and accumulation within A. aegerita is absent. The aims of this study were to research the subcellular distribution and chemical forms of Cd in A. aegerita and to analyze their influence on the Cd tolerance and detoxification, as well as to evaluate the relationship between Cd accumulation and LMWOA content in A. aegerita. The physiological changes of A. aegerita under Cd stress were also analyzed.
2.3. Superoxide dismutase (SOD), guaiacol peroxidase (POD), and catalase (CAT) activities analysis The fresh tissue samples (1 g) were ground on the ice with 0.05 M phosphate buffers (pH 7.8) containing 1% PVP (5 mL) and then the homogenates were centrifuged (4 °C, 25 min, 15,000 rpm) to collect the supernatants. The supernatants were used immediately for measuring the activities of antioxidant enzymes. The activity of SOD (EC 1.15.1.1) was determined by measuring its ability to inhibit the photochemical reduction of nitroblue tetrazolium (NBT) at 560 nm (METASH, UV5500PC) (Xu et al., 2010; Li et al., 2017). The reaction medium consisted of 1.5 mL of 50 mM phosphate buffer (pH 7.8), 300 µL of methionine (130 mM), 300 µL of NBT (750 µM), 300 µL riboflavin (20 µM), 300 µL EDTA-Na2 (100 µM), and 50 µL of enzyme extract. One unit of SOD was defined as the amount of enzyme which causes 50% inhibition of the initial reduction of NBT under light. The activity of POD (EC 1.11.1.7) was spectrophotometrically following the increase in absorbance at 470 nm due to the oxidation of guaiacol (METASH, UV5500PC) (Zhu et al., 2004). The reaction mixture in a total volume of 50 mL 100 mM phosphate buffer (pH 6.0) containing 19 µL H2O2 (30%), 28 µL guaiacol was prepared immediately before use. Then 0.1 mL enzyme extract with 0.9 mL phosphate buffer was added to 3 mL reaction mixture. One unit of POD activity was defined as an absorbance change of 0.01 units/min. Catalase activity (EC1.11.1.6) was determined as described by Beers and Sizer (1952). Phosphate buffer (2.5 mL, pH 7.5) and 1% H2O2 (0.1 mL) were mixed in an ice bath and enzyme extract (0.1 mL) was added immediately. The absorption was determined spectrophotometrically at a wavelength of 240 nm (METASH, UV-5500PC). One unit of CAT activity was defined as an absorbance change of 0.01 units/min. 2.4. Measurement of total thiol content (T-SH), reduced glutathione (GSH) and malondialdehyde (MDA)
2. Materials and methods The tissue sample was homogenized in 0.02 M EDTA on ice and the total thiol content was measured by the spectrophotometer at 412 nm using Ellman's reagent, 5,5′-dithio-bis-(2-nitrobenzoicacid) (DTNB), according Aravind and Prasad (Aravind and Prasad, 2005). The content of GSH was measured using a kit which purchased from Nanjing Jian Cheng Bioengineering Institute (China), according to the instruction of the kit. The concentration of MDA was determined spectrophotometrically by reaction with thiobarbituric acid (TBA) and was calculated using 155 per mM per cm as extinction coefficient (Amor et al., 2005).
2.1. Materials The A. aegerita was purchased from Sichuan Academy of Agricultural Sciences (China). The LMWOAs standards (oxalic, citric, malic, tartaric, formic, succinic, malonic, lactic and acetic acids) with certified standard grade and the chromatographic-grade reagents (H3PO4, KH2PO4 and methanol) were purchased from Solarbio (China) and Aladdin (China), respectively. The rest of the chemicals in our experiment were analytical reagents bought from Chengdu Kelong Corp (China).
2.5. Extraction of Cd subcellular distribution 2.2. Soil preparation and pot experiment The fresh sample (1 g) was homogenized in 50 mL precooled extraction solution (250 mM sucrose, 20 mM Tris-HCl pH 7.5, 1 mM DLdithioerythritol) on ice. Then the homogenate was centrifuged at 300 g for 1 min and the solid residue was defined as cell wall fraction. The
Unpolluted soil without Cd was collected from Sichuan University, China, at a depth of 5–20 cm. The properties of soil are as follows: CEC, 10.41 ± 0.92 cmol/kg; organic carbon, 11.70 ± 0.27 g/kg; water 67
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Fig. 1. Biomass (a), MDA contents (b), Cd concentration (c) and Cd accumulation (d) of A. aegerita. The values are represented as the means ± standard. The letters in same pattern column indicate significant differences among different treatments (P < 0.05). CK: without the addition of Cd in the soil; T1 and T2: Cd concentrations of 10 mg/kg and 20 mg/kg respectively in soil.
digested using HNO3-HClO4 (3:1, v/v).
supernatant was centrifuged at 15,000 g for 45 min. The precipitate and the supernatant were designated as organelle fraction and a soluble fraction, respectively (Su et al., 2014). All centrifugations were done at 4 °C. The three fractions were evaporated and digested using HNO3HClO4 (3:1, v/v).
2.7. Analysis of low-molecular-weight organic acids (LMWOAs) The mushroom sample was dried at 50 °C for 96 h and powered. Then 8 mL 1% H3PO4 was added to suspend the power (1 g) and then the homogenate subjected to ultrasound action (400 W, 30 min, 25 °C) to extract LMWOAs. The solution was centrifuged at 2500g for 10 min, and the supernatant was filtered through a 0.45 µm cellulose membrane for analysis (Mleczek et al., 2016). The content of LMWOAs (oxalic, citric, malic, tartaric, formic, succinic, malonic, lactic and acetic acids) ware analyzed by high-performance liquid chromatography (HPLC, Shimadzu CBM-20A, Japan) with a SWELL Chromplus C18 column (250 mm × 4.6 mm × 5 µm). HPLC conditions were as follows: mobile phase, 20 mM KH2PO4 (pH 2.2) and methanol (98:2, v/v); flow rate, 1 mL/min; temperature, 25 ℃; UV detection wavelength, 210 nm (Mleczek et al., 2016).
2.6. Extraction of Cd in chemical forms The chemical forms of Cd were analyzed using successively extracted by designated solutions in the following order: (1) 80% ethanol, extracting inorganic Cd; (2) deionized water, extracting water-soluble organic Cd; (3) 1 M NaCl, extracting pectate and protein-integrated Cd; (4) 2% acetic acid, extracting insoluble Cd-phosphate complexes; (5) 0.6 M HCl, extracting Cd-oxalate and (6) the residual Cd. The fresh sample (4 g) was homogenized in 80 mL extraction solution, and shaken for 18 h at 25 °C, and then the solution was centrifuged at 4000g for 10 min, and the first supernatant was collected in a conical beaker. The precipitate was suspended twice in the extraction solution, shaken for 2 h at 25 °C, and centrifuged. The supernatants of the three centrifugations were pooled and the solid residue was subjected to the next extraction in the indicated order with the same steps mentioned above (Lu et al., 2017). The five extraction solutions and the residue were evaporated on an electroplated plate at 70 °C to a constant weight and
2.8. Cd concentration analysis Dried and powered mushroom samples were digested with a mixture of HNO3 and H2O2 (5:2, v/v) using a microwave oven (SINEO, MDS-6) following the procedure of Tuzen et al. (2007). The total 68
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Fig. 2. Activities of SOD (a), CAT (b) and POD (c), and concentration of GSH and T-SH (d) of A. aegerita. The values are represented as the means ± standard. The letters in same pattern column indicate significant differences among different treatments (P < 0.05). CK: without the addition of Cd in the soil; T1 and T2: Cd concentrations of 10 mg/kg and 20 mg/kg respectively in soil.
concentration of Cd in mushroom samples was determined by flame atomic adsorption spectrometry (FAAS, VARIAN, SpectrAA-220Fs). The conditions were as follows: acetylene flow rate, 2.0 L/min; the air flow rate, 13.5 L/min; wave length, 228.8 nm; Cd lamp current, 4.0 nm; slit width, 0.5 nm. The Cd concentrations in different fractions (subcellular distribution and chemical forms) were determined by inductively coupled plasma mass spectrometry (ICP-MS, aurora M90). The isotopes selected for measurement were 113Cd with internal standard 103Rh. The conditions were as follows: cooling air flow rate, 18 L/min; auxiliary gas flow rate, 1.8 L/min; atomizing gas flow rate, 1 L/min; sample depth, 6.5 mm; radio frequency (RF) power, 1400 W.
following formula:
TF = Concentration of Cd in pileus / Concentration of Cd in stipe 3. Results and discussions 3.1. Biomass, MDA, Cd concentration and accumulation The biomass of A. aegerita decreased with an increase in the concentration of Cd (Fig. 1a). The fresh weights (FW) of pileus and stipe of A. aegerita in T1 had decreased by 16% and 20% respectively compared to CK. Furthermore, the biomass of pileus and stipe in T2 were 73% and 71% of CK, respectively. According to the tolerance index, the A. aegerita belonged to high tolerance (Tolerance index > 70%) in the polluted soil containing 20 mg/kg Cd (Yang et al., 2018). Biomass is a significant indicator of stress response for mushroom. It is reported that Cd can inhibit metabolic enzyme activities and influence auxin homeostasis, as well as affect the synthesis of protein (Luevano and Damodaran, 2014; Thomet et al., 1999; Yu et al., 2015), which may explain why accumulation of Cd in A. aegerita caused the decrease of biomass. The addition of Cd greatly influenced the MDA concentrations in pileus and stipe of A. aegerita (Fig. 1b). The contents of MDA in pileus and stipe of T1 were 41% and 36% higher than the control,
2.9. Statistical analysis All results were tested by one-way ANOVA using the SPSS statistics 17.0 package. All data were the means ± SD of three independent replicates and the significance of the difference between different treatments were compared using least significant difference (LSD) calculated at a significance level of P < 0.05. All figures were performed using OriginPro 8. The tolerance index (TI), which was defined as the ratio of biomass exposed to Cd to the control, was calculated to determine the tolerance of A. aegerita. The translocation factor (TF) was used to describe the transport of Cd in fruiting body of A. aegerita, calculated by the 69
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through secreting antioxidant enzymes (Zhang et al., 2012). SOD is the first line of defense against the ROS, converting the superoxide radical to molecular oxygen and H2O2 (Irfan et al., 2014). CAT participates in the decrease of H2O2 concentration in the cell, which breaks H2O2 to H2O and oxygen (Irfan et al., 2014). The H2O2 can also be scavenged by SOD using chlorogenic acid as a substrate (Takahama and Oniki, 1997). A large amounts of active oxygen were produced when fungi were exposed in Cd stress (Li et al., 2017). The generation of active oxygen can result in high toxicity to organisms. Organisms have an effective system to counter this toxicity. Previous research showed that Cd stress can induce the response of SOD, CAT and POD in fungi (Li et al., 2017). In this study, the activities of SOD, CAT and POD were increased under Cd stress. These results suggested that antioxidant enzymes, such as SOD, CAT and POD, take part in the detoxification of Cd. The effect of Cd on glutathione (GSH) and total thiol content (T-SH) in A. aegerita are showed in Fig. 2d. The concentrations of GSH in pileus and stipe were significantly increased compared to those in CK and reached their maximum level of 60.82 and 55.25 μmol/g FW respectively at T2. The concentrations of GSH in pileus and stipe of T1 were improved by 129% and 117% over CK levels, respectively. Furthermore, the contents of T-SH in pileus and stipe were also dramatically increased compared to those in CK. The maximum concentrations of TSH in pileus and stipe at T2 were 1.51 and 1.38 times higher than those of CK, respectively. In general, the contents of GSH and T-SH in pileus were higher than those in stipe, but the difference between pileus and stipe was not significant (P > 0.05). The complexation of Cd with thiol compounds is another intracellular detoxification of Cd (Weng et al., 2012). GSH is the main non-protein thiols in the cells and can to relieve Cd stress using various mechanisms, such as the activation of antioxidation mechanism and chelation of cadmium ions (Xu et al., 2011; Zhuo et al., 2017). On the one hand, GSH is an important antioxidation molecule, which can take part in the removal of active oxygen induced by Cd stress (Xu et al., 2018). On the other hand, GSH is the precursors of phytochelatins (PCs) which can bind to Cd2+ forming Cd-PC complexes that facilitate the sequestration of Cd in the vacuoles (Rahman et al., 2017). The thiol groups of T-SH can bind to Cd leading to the sequestration of Cd in the vacuole and reducing the damage of Cd to A. aegerita. Therefore, the increase of GSH and T-SH contents in A. aegerita under Cd stress revealed that they play an important role in reducing the toxic effect of Cd on mushrooms.
respectively. The MDA contents in pileus and stipe at T2 were increased by 1.52 and 1.92 times compared to CK, respectively. MDA is an indicator of lipid peroxidation to reflect the peroxidation degree, indicating the toxic effects of Cd to mushroom (Li et al., 2017; Zhao et al., 2012). In this research, the result indicated that the membrane lipids of A. aegerita were oxidized by Cd stress. Cadmium concentration in pileus and stipe increased with the increase of Cd level in the soils (Fig. 1c). On the whole, the contents of Cd in pileus were higher than in stipe, with a TF of 1.38–1.90. Similarly, the accumulation of Cd in pileus was higher than in stipe (Fig. 1d). Cadmium accumulation in pileus was 85–88% of the total. Although the ability of accumulating Cd for A. aegerita is limited, the A. aegerita possesses abilities of rapid growth and high biomass, and can be cultivated from February to November, which is a eurythermic species. Therefore, A. aegerita can be used as an effective accumulator for bioremediation of the Cd-polluted soil. Despite the biomass of A. aegerita decreased with an increase in the concentration of Cd, the accumulation of Cd in A. aegerita had improved. A. aegerita may employ some measures to detoxify Cd ions within the cell and to enhance the capacity of Cd tolerance and accumulation. 3.2. Effect of Cd on the activities of SOD, POD and CAT as well as on the production of GSH and T-SH In this research, the effect of Cd on the activities of antioxidant enzymes showed an increasing tendency (Fig. 2a, b, c). The activities of SOD in T1 of pileus and stipe increased by 60% and 45% compared to those in CK, respectively. The SOD activities in T2 reached the maximum amount of 368–369 U/g FW. The activity of CAT increased in a Cd concentration dependent manner, and the maximum value of 254 U/g FW was appeared in stipe at T2. CAT activity in pileus of T2 was 208 U/g FW, but there was no significant difference in pileus and stipe at T2 according to statistical analysis (P > 0.05). The activities of CAT in T1 of pileus and stipe were 1.57 and 1.31 times higher than those of CK, respectively. POD activities under Cd stress were also higher compared with the CK. The soil spiked with 20 mg/kg Cd led to the maximal values of 230 and 240 U/g FW for POD in pileus and stipe, respectively. Cadmium can induce the production of reactive oxygen species (ROS) in organisms directly or indirectly, thereby disturbing the balance of the cellular redox status and seriously impairing the function of cells (Zhang et al., 2016). To resist the damage from ROS such as superoxide (O2-) and hydrogen peroxide (H2O2), organisms activate cellular immune system for the removal of cellular ROS, for example
Fig. 3. Subcellular distribution of Cd in A. aegerita grown in Cd polluted soil. The values are represented as the means ± standard. The letters in the same fraction means significant differences (P < 0.05) among different treatments and tissues. T1 and T2: Cd concentrations of 10 mg/kg and 20 mg/kg respectively in soil. 70
71
244.45 ± 2.84 C 364.71 ± 24.73 B 509.04 ± 24.55 A 0.908* 232.25 ± 9.27 c 313.01 ± 38.45 b 470.54 ± 17.52 a 0.846* 14.15 ± 2.88 B 42.14 ± 4.22 A 27.84 ± 6.20 AB 0.741 14.21 ± 0.47 b 19.65 ± 5.81 ab 31.95 ± 5.37 a 0.772 13.84 ± 1.67 C 62.52 ± 8.35 B 164.53 ± 1.95 A 0.811 6.79 ± 1.58 c 17.60 ± 1.27 b 83.38 ± 4.98 a 0.725 22.18 ± 5.82 A 28.60 ± 4.83 A 30.30 ± 7.44 A 0.634 9.79 ± 1.32 a 11.73 ± 1.81 a 13.46 ± 7.79 a 0.411 6.65 ± 2.09 A 9.96 ± 1.26 A 10.05 ± 1.06 A 0.83* 4.21 ± 1.11 a 5.17 ± 0.33 a 6.92 ± 0.45 a 0.79 1.52 ± 0.01 A 2.00 ± 0.26 A 2.78 ± 0.31 A 0.848* 1.04 ± 0.02 b 1.66 ± 0.67 b 1.80 ± 0.09 a 0.987** 23.42 ± 5.30 A 23.17 ± 2.37 A 27.53 ± 12.88 A 0.174 12.15 ± 4.90 a 12.36 ± 0.66 a 17.93 ± 0.419 a 0.525 8.95 ± 0.27 A 10.02 ± 2.43 A 11.27 ± 1.13 A 0.632 4.21 ± 0.58 b 2.95 ± 0.93 b 7.12 ± 0.99 a 0.36 129.70 ± 5.24 B 155.43 ± 3.85 A 154.52 ± 6.44 A 0.939** 167.71 ± 1.01 c 224.36 ± 23.64 b 281.31 ± 14.59 a 0.915*
Tartaric Malic Succinic Citric
Stipe
CK T1 T2 r CK T1 T2 r Pileus
Oxalic
Low-molecular-weight organic acid content (mg/g DW)
To understand the mechanisms of Cd detoxification, transportation and accumulation in A. aegerita, the chemical forms of Cd in A. aegerita were analyzed. Overall, the Cd concentrations in different chemical forms increased with the increase of Cd concentrations in the soil (Fig. 4a). The proportion of Cd in different chemical forms is shown in Fig. 4b. The ethanol- (40% and 34% in T1 and T2, respectively) and NaCl-extractable Cd (29% and 35% in T1 and T2, respectively) were predominant in stipe, followed by H2O-extractable Cd. Whereas, NaClextractable Cd occupied 46% and 49% of the total concentrations of Cd at pileus in T1 and T2, respectively, followed by ethanol- (26% and 20% in T1 and T2, respectively) and H2O-extractable Cd (24% and 22% in T1 and T2, respectively). The chemical forms of Cd have a great influence on the toxicity and migration capability of Cd in organisms, and Cd exists in organisms in different chemical forms to perform distinctive biological function (Yang et al., 2018). The migration ability of Cd in ethanol- and H2O-
Treatments
3.4. Chemical forms of Cd
Tissues
Table 1 LWMOAs content and Pearson correlation coefficients (r) between content of LWMOAs and Cd in A. aegerita.
Formic
Malonic
Lactic
Acetic
Total
The subcellular distribution of Cd in A. aegerita is showed in Fig. 3. The concentrations of Cd in all subcellular fractions of pileus and stipe increased with the increase of Cd concentrations in soil. In general, most of Cd (53–75%) was stored in the soluble fraction, followed by the cell wall fraction (19–42%), and the part of organelle fraction accounted for only 5–6% of the total. The proportion of Cd in the cell wall fraction at pileus and stipe increased with the increase of Cd added to soil, and the proportion of Cd in the cell wall fraction at pileus was higher than that at stipe. However, the proportions of the soluble fraction at stipe (75% and 67% in T1 and T2, respectively) were higher than those at pileus (61% and 53% in T1 and T2, respectively). In this study, the concentration of Cd in pileus was higher than in stipe. High concentrations of Cd would severely damage the A. aegerita. Previous researches have shown that the regionalization of Cd in the cell wall and soluble fraction is a measure to alleviate the toxicity of Cd (Fu et al., 2011; Lu et al., 2017; Zhou et al., 2017). The result of this study that most of Cd was compartmentalized in the cell wall and soluble fractions, was similar to previous researches. The cell wall plays a protective role under Cd stress, because it is the first barrier for cell to reduce the damage of Cd. The carboxyl, amino, sulfhydryl, phosphoryl and hydroxyl groups of polysaccharide and protein at the cell wall can interact with Cd2+ making Cd2+ adsorb on the cell wall to restrict Cd2+ transportation across cytoplasm (Das and Guha, 2007). In this study, the proportion of cell wall-bound Cd in pileus was higher than in stipe. The proportion of Cd in the cell wall fraction increased with an increase in the concentration of Cd. This result suggested that fixation Cd to cell wall was a strategy to reduce the poisonousness of Cd in A. aegerita. Moreover, the compartmentation of Cd in the vacuole could also be helpful to alleviate the toxicity of Cd (Xu et al., 2018; Yang et al., 2018). Vacuole contains significant amounts of sulfur-rich peptides and organic acids which can chelate and compartmentalize Cd to decrease the amount of Cd interfering with the organelles in organisms (Xu et al., 2018; Yang et al., 2018). In this study, most of Cd (53–75%) was compartmentalized in soluble fraction which mostly consists of vacuoles (Wang et al., 2016; Zhou et al., 2017). Similarly, previous research revealed that a large proportion of Cd (54–68%) in pokeweed compartmentalized with organo-ligands and sequestrated in soluble fraction (Fu et al., 2011). Correspondingly, the A. aegerita contained plenty of thiol groups and organic acids (Fig. 2d and Table 1). Thiol groups and organic acids in vacuoles of A. aegerita play an essential role in the tolerance and accumulation of Cd (Wang et al., 2008). In this research, the concentration of Cd in pileus was higher than in stipe, as well as the soluble fraction in stipe was higher than in pileus, indicating that chelation with organo-ligands may enhance the transport of Cd from stipe to pileus in A. aegerita.
14.21 ± 0.47 B 19.65 ± 5.81 B 31.95 ± 5.37 A 0.818* 12.14 ± 0.31 b 17.53 ± 3.46 ab 26.66 ± 4.44 a 0.799
3.3. Subcellular distribution of Cd
Values are means ± standard deviation (n = 3). Different letters in the same organs means significant differences (P < 0.05) among different treatments. CK: without the addition of Cd in the soil; T1 and T2: Cd concentrations of 10 mg/kg and 20 mg/kg respectively in soil. DW and r represent dry weight and pearson correlation coefficients, respectively. * Significant at p < 0.05 ** significant at p < 0.01.
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Fig. 4. Metal speciation in A. aegerita grown in Cd-polluted soil. F1, F2, F3, F4, F5, and F6 denoted ethanol-extractable, water-extractable, NaCl-extractable, HAcextractable, HCl-extractable and residual Cd, respectively. The values are represented as the means ± standard. The letters in the same organs means significant differences (P < 0.05) among different treatments and tissues. T1 and T2: Cd concentrations of 10 mg/kg and 20 mg/kg respectively in soil.
and to enhance the accumulation of Cd (Goliński et al., 2015). LMWOAs, based on the internal complexation and compartmentation in the vacuole rather than on extracellular mechanism, take part in the detoxification of Cd in maize (Chaffai et al., 2006). Previous research showed that T. caerulescens synthesize more LMWOAs in Cd stress and accumulate more Cd in the shoots (Pence et al., 2000). Cadmium elevated LMWOAs in leaves of maize with positive effect (Dresler et al., 2014). In this study, A. aegerita produced more LMWOAs (total acids have increased 1.49–2.08 times and 1.35–2.03 times in pileus and stipe, respectively) in the Cd supplied soil compared to CK. This result suggested that LMWOAs played a crucial part in reducing the damage of Cd and improving the accumulation of Cd in A. aegerita. When Cd enters into cell, LMWOAs can compartmentalize Cd into vacuole based on metal-ligand complexation (Dresler et al., 2014; Mleczek et al., 2016). Vacuole is rich in LMWOAs which can chelate and compartmentalize Cd (Xu et al., 2018; Yang et al., 2018). The chelation with LMWOAs is one reason that the proportion of soluble fraction was higher than other fractions. Carboxylic acids, particularly citric acid, treated as metal-complexing agents, can be combined with divalent cations strongly and form stable complexes (Nigam et al., 2001). The mobile organicallybound Cd was created by the interaction of Cd with citric acid (Nigam et al., 2001). For example, citric acid can interact with Cd and form Cd (CH2)2COH(COO)3- and Cd(CH2)2COH(COO)3H (Rauser, 1999). Previous research showed that citric acid is the ligand for Cd moving from roots to leaves (Rauser, 1999). This study revealed that the content of citric acid had a positive relation to the level of Cd in A. aegerita (r = 0.915 in stipe and r = 0.939 in pileus). This finding suggested that the chelate of citric acid with Cd participated in the transport of Cd from stipe to pileus. The toxicity and bioavailability of Cd depend in part on solubility and chemical reactivity. Cadmium ions reacted with oxalic acid forming the insoluble chelated complexes to reduce the toxicity of Cd in Pleurotus ostreatus (Li et al., 2017). Previous research has revealed that oxalic acid secreted by white-rot fungi was induced by exogenous Cd, and oxalic acid played an important role in Cd detoxification with a negative correlation between oxalic acid concentration and Cd-induced growth inhibition ratios (Xu et al., 2015). The Cd oxalate, extracted by 0.6 M HCl, has relatively low toxicity (Lu et al., 2017). In this research, the proportions of HCl-extractable Cd in pileus were higher than in stipe (1.62% in pileus and 1.16% in stipe for T1, and 3.28% in pileus and 1.61% in stipe for T2, P < 0.05), corresponding to the higher concentrations of oxalic acid in pileus compared to stipe. Therefore,
extractable fractions (water-soluble Cd in inorganic form and organic form) is higher than other forms (Xu et al., 2018), and the toxicity of these forms to organisms is higher than other forms, correspondingly. In this study, the proportions of ethanol- and H2O-extractable Cd in stipe were higher than those in pileus. The higher water-soluble Cd in inorganic and organic form with higher migration capacity in stipe can explain why the translocation factor of A. aegerita was greater than 1. Higher concentration and proportion of Cd in water-soluble inorganic form and organic form (extracted by 80% ethanol and deionized water) with higher migration capacity, implied that Cd in A. aegerita was transported mainly in water soluble forms. The Cd integrated with pectates and protein ligands (extracted by 1 M NaCl) with less toxicity in pileus were higher than those in stipe. When the concentrations of Cd in pileus were higher than those in stipe, the proportions of pectate and protein-bound form Cd in pileus were higher than those in stipe, suggesting that converting Cd into undissolved pectate and protein-bound form Cd can reduce the toxicity of Cd to A. aegerita and increase the accumulation of Cd in the fruiting body. The proportions of Cd in HAc-, HCl- and residual-fractions were low, which may be owing to the influence of the thiol groups and LMWOAs (Xiao et al., 2016).
3.5. Concentration of LMWOAs Nine types of LMWOAs were all detected in the pileus and stipe of A. aegerita (Table 1). The citric acid was the dominant organic acid in pileus (30–53%) and stipe (60–73%) of A. aegerita. The tartaric acid concentration in the fruiting body of A. aegerita was lowest among the nine types of LMWOAs. According to the result, the concentrations of citric and tartaric acids in A. aegerita showed highly positive correlation with the accumulation of Cd in A. aegerita. The content of citric acid in stipe was higher than in pileus (P < 0.05). Nevertheless, the contents of oxalic and lactic acids in pileus were higher than these in stipe (P < 0.05). In general, the concentrations of oxalic, citric, lactic and acetic acids were elevated in pileus of A. aegerita exposed to Cd compared to CK (P < 0.05), whereas the levels of succinic, malic, tartaric, malonic and formic acids in pileus were no significant difference between different treatments (P > 0.05). The concentrations of oxalic, citric, succinic, tartaric, lactic and acetic acids in stipe of Cd-exposed A. aegerita increased compared to CK (P < 0.05). LMWOAs take part in many metabolic pathways in organisms, such as cation transportation, respiration, metal chelating and detoxification (Dresler et al., 2014). The increasing of LMWOAs in the presence of Cd can chelate and compartmentalize the Cd to reduce the toxicity of Cd 72
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there is one strategy used by A. aegerita to alleviate the toxicity of Cd by complexation with oxalic acid to change the chemical forms of Cd. 4. Conclusions The accumulation and detoxification of Cd as well as stress responses of A. aegerita were analyzed in this work for the first time. A. aegerita could tolerate high concentrations of Cd (up to 20 mg/kg) and could utilize various strategies to detoxify and survive. Antioxidant enzyme system, GSH and T-SH played an important role in the detoxification and accumulation of Cd. Nine types of LMWOAs were detected in A. aegerita and these acids were closely related to accumulation and detoxification of Cd in this study. The concentration of Cd in pileus was higher than that in stipe. Its subcellular distribution showed that vast majority of Cd (94–95%) was in the cell wall and soluble fractions. Furthermore, the proportions of NaCl- and ethanol-extractable Cd were the dominant forms in pileus and stipe, respectively. The overall study clearly suggested the ability of A. aegerita to remediate Cd contaminated soils and systematically provided new insights into the cellular mechanisms of Cd tolerance and detoxification of mushrooms. Acknowledgements This study was financially supported by the Key Research and Development Program of Sichuan Province (2017SZ0181) and the Agricultural Science and Technology Achievements Transformation Program of Sichuan Province (2017NZZJ008). The authors also wish to thank Professor Guanglei Cheng and Dong Yu from Sichuan University for the technical assistance. Conflict of interest There was no conflict of interest. References Aravind, P., Prasad, M.N., 2005. Modulation of cadmium-induced oxidative stress in Ceratophyllum demersum by zinc involves ascorbate-glutathione cycle and glutathione metabolism. Plant Physiol. Biochem. 43, 107–116. Amor, N.B., Hamed, K.B., Debez, A., Grignon, C., Abdelly, C., 2005. Physiological and antioxidant responses of the perennial halophyte Crithmum maritimum to salinity. Plant Sci. 168, 889–899. Barros, L., Pereira, C., Ferreira, I.R., 2013. Optimized analysis of organic acids in edible mushrooms from Portugal by ultra fast liquid chromatography and photodiode array detection. Food Anal. Methods 6, 309–316. Beers, R.F., Sizer, I., 1952. A spectrophotometric method for measuring the breakdown of hydrogen by catalase. J. Biochem. 195, 133–140. Cen, F., Hu, Y.J., Xu, H., 2012. Responses of antioxidant defenses in Coprinus comatus exposed to cadmium and mercury toxicity. Asian J. Chem. 24, 4679–4685. Chaffai, R., Ali, T., Ferjani, E.E., 2006. A comparative study on the organic acid content and exudation in maize (Zea mays L.) seedlings under conditions of copper and cadmium stress. Asian J. Plant Sci. 21, 228–232. Chen, N.C., Zhang, Y.J., He, X.F., Li, X.F., Zhang, X.X., 2017. Analysis of the report on the national general survey of soil contamination. J. Agro-Environ. Sci. 36, 1689–1692. D'Alessandro, A., Taamalli, M., Gevi, F., Timperio, A.M., Zolla, L., Ghnaya, T., 2013. Cadmium stress responses in Brassica juncea: hints from proteomics and metabolomics. J. Proteome Res. 12, 4979. Damodaran, D., Shetty, K.V., Mohan, B.R., 2014. Uptake of certain heavy metals from contaminated soil by mushroom–Galerina vittiformis. Ecotoxicol. Environ. Saf. 104, 414–422. Das, S.K., Guha, A.K., 2007. Biosorption of chromium by termitomyces clypeatus. Colloids Surf. B Biointerfaces 60, 46–54. Dresler, S., Hanaka, A., Bednarek, W., Maksymiec, W., 2014. Accumulation of low-molecular-weight organic acids in roots and leaf segments of Zea mays plants treated with cadmium and copper. Acta Physiol. Plant. 36, 1565–1575. Ebbs, S.D., Kochian, L., 1997. Toxicity of zinc and copper to Brassica species: implications for phytoremediation. J. Environ. Qual. 26, 776–781. Fu, X.P., Dou, C.M., Chen, Y.X., Chen, X.C., Shi, J.Y., Yu, M., Xu, J., 2011. Subcellular distribution and chemical forms of cadmium in Phytolacca americana L. J. Hazard. Mater. 186, 103. Fujita, T., Komemushi, S., Yamagata, K., 2010. Analysis of organic acids in fruit-bodies of Tricholoma giganteum by high performance liquid chromatography. Lett. Appl. Microbiol. 11, 27–29. Goliński, P., Mleczek, M., Magdziak, Z., Gąsecka, M., Borowiak, K., Dąbrowski, J., Kaczmarek, Z., Rutkowski, P., 2015. Efficiency of Zn phytoextraction, biomass yield
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