Ecotoxicology and Environmental Safety 120 (2015) 7–12
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Effect of arsenite-oxidizing bacterium B. laterosporus on arsenite toxicity and arsenic translocation in rice seedlings Gui-Di Yang a,n, Wan-Ying Xie b, Xi Zhu a, Yi Huang a, Xiao-Jun Yang a, Zong-Qing Qiu a, Zhen-Mao Lv a, Wen-Na Wang a, Wen-Xiong Lin a,n a Fujian Provincial Key Laboratory of Agroecological Processing and Safety Monitoring, College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China b Key Laboratory of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen 361021, China
art ic l e i nf o
a b s t r a c t
Article history: Received 1 December 2014 Received in revised form 14 May 2015 Accepted 14 May 2015
Arsenite [As (III)] oxidation can be accelerated by bacterial catalysis, but the effects of the accelerated oxidation on arsenic toxicity and translocation in rice plants are poorly understood. Herein we investigated how an arsenite-oxidizing bacterium, namely Brevibacillus laterosporus, influences As (III) toxicity and translocation in rice plants. Rice seedlings of four cultivars, namely Guangyou Ming 118 (GM), Teyou Hang II (TH), Shanyou 63 (SY) and Minghui 63 (MH), inoculated with or without the bacterium were grown hydroponically with As (III) to investigate its effects on arsenic toxicity and translocation in the plants. Percentages of As (III) oxidation in the solutions with the bacterium (100%) were all significantly higher than those without (30–72%). The addition of the bacterium significantly decreased As (III) concentrations in SY root, GM root and shoot, while increased the As (III) concentrations in the shoot of SY, MH and TH and in the root of MH. Furthermore, the As (III) concentrations in the root and shoot of SY were both the lowest among the treatments with the bacterium. On the other hand, its addition significantly alleviated the As (III) toxicity on four rice cultivars. Among the treatments amended with B. laterosporus, the bacterium showed the best remediation on SY seedlings, with respect to the subdued As (III) toxicity and decreased As (III) concentration in its roots. These results indicated that As (III) oxidation accelerated by B. laterosporus could be an effective method to alleviate As (III) toxicity on rice seedlings. & 2015 Published by Elsevier Inc.
Keywords: Rice (Oryza sativa L.) Arsenite-oxidizing bacteria Arsenic speciation B. laterosporus CE-ICP-MS Remediation
1. Introduction With the development of industry and agriculture, large amounts of arsenic-containing waste water were discharged into the environment and increased arsenic (As) pollution in paddy soil and underground water (Sierra-Alvarez et al., 2006; Garelick et al., 2008). There are four main As species in soil solution of rice rhizosphere, namely arsenite [As (III)], arsenate [As (V)], dimethylarsinate [DMA (V)] and methylarsonate [MMA (V)], with As (III) as the predominant form under flooded conditions (Abedin et al., 2002). Arsenic accumulation in rice seedlings inhibits the photosynthesis and the activities of the defending enzymes in the plants (Abedin and Meharg, 2002). On the other hand, As accumulation in rice grains will pose serious risk on human health (Zhu et al., 2008,b). As (III) is the most toxic species among the above four species for the growth of rice seedlings (Abedin and Meharg, 2002). Therefore, it is vital to alleviate the As (III) toxicity on the n
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http://dx.doi.org/10.1016/j.ecoenv.2015.05.023 0147-6513/& 2015 Published by Elsevier Inc.
growth of rice seedling and decrease As translocation from roots to shoots to improve rice yield and quality. Several strategies have potential to alleviate As toxicity for rice growth and to decrease As accumulation in rice plants, including screening for rice cultivars with high As resistance and/or low As accumulation ability, and the management of the cultivation environment. Screening for rice cultivars with high As resistance and/or low As accumulation ability is a good technology to alleviate As toxicity for rice growth. However, it is a time- and costconsuming process. In contrast, the management of the cultivated environments is a simpler and easier pathway to alleviate As toxicity and accumulation in rice. Many attentions have been paid to the interactions between the uptakes of As and some nutrient elements, such as Si, S, P, and Fe (Hu et al., 2007; Zhang et al., 2011), especially to the competitive uptake between P and As (Abedin et al., 2002; Hu et al., 2005; Liu et al., 2006; Hossain et al., 2009). However, over addition of nutrient elements could easily lead to secondary pollution in soil and water. Bioremediation is another potential technique to alleviate As toxicity. Microbial remediation has gained close attention because
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of its affordability and compatibility with soil medium. Microbe in the soil could detoxify As toxicity by efficiently oxidizing As (III) to As (V) (Tamaki and Frankenberger 1992; Kashyap et al., 2006). Several arsenite-oxidizing bacteria, such as Agrobacterium, Herminiimonas arsenicoxydans and Alcaligenes faecalis, have been isolated from As polluted soils since the first arsenite-oxidizing bacterium was found in 1918 (Green, 1918). Genes responsible for the oxidation have also been identified from these bacteria (Oremland and Stolz, 2003; Silver and Phung, 2005). So far, most of the studies on arsenite-oxidizing bacteria focused on the screening processes, physiological and biochemical characteristics of the bacteria and the mechanisms of arsenite-oxidizing. Though microbes play critical roles in As (III) oxidation, little attention has been paid to the effects of accelerated As (III) oxidation by the microbes on As toxicity and translocation in rice plants. In our previous research, an arsenite-oxidizing bacterium, B. laterosporus, was isolated from paddy soil (Yang et al., 2014). It could oxidize 200 mgL 1 of As (III) into As (V) within 24 h (5% inoculation, v/v). However, the effect of this bacterium on the As toxicity and translocation in rice seedlings is still unknown. Therefore, the objective of this study is to evaluate the effects of B. laterosporus on the As (III) oxidation, As toxicity and translocation in rice seedlings.
was 23.00, 7.00 and 21.00 mg L 1, respectively.). Prior to the transplantation, seedlings with similar status in each cultivar were selected and washed with 18.2 MΩ DDI water to remove the transparent sticky secretions adhered to their roots. All treatments were conducted for 6 d and 18.2 MΩ DDI water was daily added into the plates to keep stable solution volumes. Three of the six replicates were used to determine the pH value, the residual concentration of NH4 þ , K þ , HPO42 and NO3 , and As (III) oxidation percentage in the solutions, the concentrations of N, P, K, and As species in the roots and shoots, the chlorophyll SPAD (Soil and Plant Analyzer Development) value in the leaves, length of the roots, height of the shoots, and dry weights of the roots and shoots. The other three replicates were used to determine the defending enzyme activities in the leaves as described in Section 2.5.
2. Materials and methods
2.4. Determination of pH, nutrient concentrations and the As (III) oxidation in the solutions
2.3. Arsenate reduction by the rice roots To investigate the reduction of As (V) to As (III) by the rice roots, the rice seedlings of four cultivars were hydroponically cultivated in the nutrient solutions with 3 mg L 1 As (V) as described in Section 2.2. After 6 days of incubation, the seedlings were harvested and the concentrations of As (III) and As (V) in the roots of four cultivars were determined as described in Section 2.5.
2.1. Plant materials and cultivation conditions Four rice cultivars, namely Guangyou Ming 118 (GM), Teyou Hang II (TH), Shanyou 63 (SY) and Minghui 63 (MH), were used in the present study. They were all indica rice, from the Rice Research Institute, Academy of Agriculture Sciences, Fujian, China. The seeds were germinated separately in 250 mL beakers with tap water till the shoot length reached 3–4 cm in a temperature-controlled culture chamber at 31 °C. The rice seedlings of each plant were transferred into a plate with holes (4 4 cm2), and affixed in the holes with Styrofoam. The Styrofoam plate with seedlings was allowed to float in a pot (17 10 6 cm3) filled with 500 mL full nutrient solution with the following compositions per liter: 14.9 mg K2SO4, 48.2 mg (NH4)2SO4, 24.8 mg KH2PO4, 18.5 mg KNO3, 86.3 mg Ca(NO3)2 4H2O, 135.0 mg MgSO4 7H2O, 200.0 mg Na2SiO3 9H2O, 27.8 mg FeSO4 7H2O, 37.2 mg Na2EDTA, 14.3 mg H3BO3, 0.4 mg CuSO4 5H2O, 1.1 mg ZnSO4 7H2O, 9.1 mg MnCl2 4H2O, 0.5 mg Na2MoO4 2H2O. The pH value of the full nutrient solution was adjusted to 5.5–6.0 with 0.1 mol L 1 HCl. 2.2. Arsenic treatment and B. laterosporus amendment The four cultivars of rice seedlings (at the 2-leaf stage) were transferred into new Styrofoam plates (5 seedlings each) as described in Section 2.1 and grown hydroponically within three treatments (6 replicates each), respectively. Treatment 1 (CT), control seedlings treated without As (III) or B. laterosporus; Treatment 2 (AT), seedlings treated with 3 mg L 1 As (III); Treatment 3 (BLT), seedlings treated with 3 mg L 1 As (III) and 0.25% (v/v) rejuvenated B. laterosporus. Control seedlings treated with 0.25% (v/v) B. laterosporus was not cultivated because their physiological and biochemical properties had no significant difference (p 40.01) from CT within the regulation period of our study. The rejuvenation of B. laterosporus could be found in the supplementary material. B. laterosporus was inoculated into the incubation solutions just before the transplantation. The incubation solutions were prepared with NH4NO3, K2SO4 and NaH2PO4 2H2O in 18.2 MΩ doubly deionized (DDI) water (the pH values were adjusted to 5.5–6.0 with 0.1 mol L 1 HCl; the concentration of N, P, K
After 6 days of incubation, the pH values in the nutrient solutions were determined by a pHS-3C meter. Nutrient solutions (around 5.0 mL) were filtered through 0.22 μm polypropylene membranes, kept in 20 °C until further determination of nutrient concentrations by CIC-260 Ion Chromatography (IC) and As species by Capillary Electrophoresis-Inductively Coupled PlasmaMass Spectrometry (CE-ICP-MS) as described in Section 2.5. 2.5. Extraction and determination of N, P, K and As in rice roots and shoots After 6 days of incubation, the seedlings were harvested. The fresh roots were washed three times with 18.2 MΩ DDI water to remove the adsorbed As on the surface. The roots and shoots were separated with stainless steel scissors, dried at 40 °C in an oven till the weights were constant, and cut into pieces by stainless steel scissors. The crushed roots or shoots were used to extract N, P, K and As by microwave assisted extraction (Yuan et al., 2005). Around 0.3000 g (with the precision of 0.0001 g) crushed roots or shoots were weighed into separate PFA jars and added with 10.0 mL HPLC grade CH3OH–H2O (1:1, v/v). The jars were sealed in PEEK cans and extracted for 15 min at 120 °C in a MDS-6 microwave extraction apparatus (Sineo Microwave Chemistry Technology Co. Ltd., China). After the extraction, the extracts were filtered through 0.22 μm polypropylene membranes into polyethylene centrifuge tubes and diluted to the appropriate volume (according to N, P, K and As concentration in the sample) with 18.2 MΩ DDI water, and the final solution was used for the determination of N, P, K, total As and As species. In speciation analysis of arsenic, it is important to know if all arsenics are completely extracted during the extraction process. The shoots of SY in BLT treatment were chosen as the materials to evaluate the extraction efficiency of arsenic species by CH3OH–H2O (1:1, v/v). The digestion solution of HNO3–H2O2 (5:1, v/v) was used to completely decompose the rice shoots according to the extraction procedure described above. The extraction efficiency (%, n ¼3) was 99.63 72.32, namely the percent between the total As concentration extracted by the mixture of CH3OH–H2O
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and the total As concentration digested by the mixture of HNO3–H2O2, the results indicated that all arsenics had been extracted by CH3OH–H2O (1:1, v/v) in the above procedure. In speciation analysis of arsenic, it is also important to know if each arsenical species remains constant during the extraction and analytical process. The shoots of SY in BLT treatment were chosen as the materials, spiked by As (III), MMA (V), DMA (V) and As (V), and also analyzed in the same manner, the average recoveries (%, n ¼3) was 93.13 74.89, 98.62 73.67, 105.4 73.3 and 107.1 74.5, respectively, for the above targets at the spiked concentration of 2.00, 2.00, 2.00 and 10.00 mg kg 1. The approximate 100% recovery for As (III), DMA (V), MMA (V) and As (V) indicated that no arsenical species changed during extraction and analysis. The total contents of N, P and K in the roots and shoots of rice seedling were determined by a KDN-08C azotometer (Shanghai new instrument co., LTD., China), a UV-9100 ultraviolet–visible spectrophotometer (Shanghai precision scientific instrument co., LTD., China), and a FP640 flame spectrophotometer (Shanghai precision scientific instrument co., LTD., China), respectively. Concentrations of the total As in the shoots of SY (BLT treatment) were determined by NexION 300X ICP-MS (PerkinElmer, USA) (Llorente-Mirandes et al., 2011). Germanium (Ge) was used as an internal standard to calculate the matrix effect of rice plants on As signal. Total As concentration in Table 1 was calculated according to the sum of As species. The concentrations of As species in the extracts and in the nutrient solutions were determined by CE-ICP-MS according to Yang et al. (2008, 2009). Briefly, A 57.0 cm CE capillary was used to separate the arsenic species and coupled with a NexION 300X ICP-MS by an interface designed by our group (Yang et al., 2009). The CE capillary was conditioned daily by purging with 18.2 MΩ DDI water for 10 min, 0.1 mol L 1 NaOH solution for 10 min, 18.2 MΩ DDI water for 10 min and running buffer solution (50 mmol L 1 H3BO4–12.5 mmol L 1 Na2B4O7, pH 9.1) for 10 min. Between each run, the CE capillary was flushed with 18.2 MΩ DDI water and running buffer solution for 2 min, respectively. The sample was injected into the CE capillary for determination by electro-migration injection (15 kV, 10 s). The nebulizer type was PFA (optimum flow 10–20 μL min 1). Mixed standard solution of As (III), As (V), DMA (V) and MMA (V) with Table 1 Concentration of As (III), As (V) and total As in roots and shoots in the treatments amended with or without B. laterosporus. Arsenic concentration (mg kg 1DW) Cultivar
Organ
Treatment
As (III)
As (V)
Total As
TH
Roots
AT BLT AT BLT
2577 13A 2727 17A 177 1 B 72 72A
194 7 9A 91 7 4 B 38 7 2A 17 7 1 B
452 716A 363 713 B 55 72 B 88 74A
AT BLT AT BLT
467 2 B 567 2A 67 1 B 267 1A
39 7 2 B 91 7 3A 17 7 1 B 30 7 2A
85 710 B 147 716A 23 71 B 56 73A
AT BLT AT BLT
13 71A n n.d. B 67 1 B 97 1A
170 7 5 B 240 7 8A 64 7 3A 43 7 2 B
183 712 B 240 713A 71 74A 53 73 B
AT BLT AT BLT
7447 22A 6117 15 B 27 71A 257 0 B
152 7 7 B 238 7 10A 21 7 2A 12 7 1 B
896 723A 848 720 B 48 73A 37 72 B
Shoots
MH
Roots Shoots
SY
Roots Shoots
GM
Roots Shoots
n
n.d. not detected
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10.0–100.0 μg L 1 was used to calibrate the concentration of As species in the roots and shoots of rice seedling, the linear correlation coefficient for above four As standard solutions from 10.0 to 100.0 μg L 1 was higher than 0.995, the relative standard deviation (RSD) for 100.0 μg L 1 mixed As standard solution was lower than 5.0% within 5 replicates. Qualitative confirmation of As species was based on their retention time and spiked tests. Quantitative determination of As species was based on peak areas. The limit of detection (LOD) of the analytical method for above four As species in the roots and shoots of dried rice seedling was about 0.1 mg kg 1 As according to the extraction, dilution and CE-ICPMS determination. 2.6. Determination of the physiological and biochemical properties of the rice seedlings The values of chlorophyll SPAD in the fresh leaves were determined by a SPAD-502 chlorophyll meter with the precision of 0.1. The length of the roots and the height of the shoots were determined by a ruler with the precision of 0.01 cm. The dry weights of the roots and shoots were weighed by a balance with the precision of 0.0001 g. The defending enzyme activities, such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and malondialdehyde (MDA) in the shoots were determined as described below. Fresh shoots (about 0.5000 g) were homogenized with 5.0 mL extraction buffer (pH7.80, 50.0 mmol L 1 phosphorus (P), 10.0 mg mL 1 polyvinylpyrrolidone) in a pre-chilled mortar and pestle on ice bath. The homogenate was transferred into a 15.0 mL Polyethylene centrifuge tube, diluted with extraction buffer to 10.0 mL, and centrifuged at 12,000 rpm for 3 min at 4 °C. The supernatant with the enzyme extract was used for the determination of SOD, POD and CAT. For the extraction of MDA, fresh shoots (about 0.9000 g) was homogenized with 9.0 mL of 5% (m/v) trichloroacetic acid (TCA) in the same manner as described before, and the supernatant was collected from the mixture without dilution by centrifuging at 3,000 rpm for 10 min at 4 °C. During the determination process, the supernatant was kept in 4 °C refrigerator until the assays were completed. SOD activity, POD activity, CAT activity and MDA concentration were measured according to nitro blue tetrazolium (NBT) method (Beyer and Fridovich, 1987), guaiacol oxidation method, spectrophotometric method at 240 nm and thiobarbituric acid (TBA) method (Wang, 2006), respectively. 2.7. Statistics analysis The average values of the three replicates were used for statistical analysis. Software DPS 7.5 was used to conduct the significance test by least significant difference (LSD) among the three treatments within the same rice cultivar.
3. Results 3.1. Effect of B. laterosporus on the pH value, nutrient uptake and As (III) oxidation in the solutions The addition of As (III) in AT treatment significantly (po 0.01) increased the pH value in the solutions compared to the control (Fig.1). While when B. laterosporus was added (BLT treatment), the pH values were maintained at the same level as those in the control except MH. The addition of B. laterosporus significantly (po0.01) promoted the uptake of NH4 þ , NO3 , HPO42 and K þ in the solutions with the four cultivars except for the NH4 þ by MH compared to AT treatments (Fig. 2). B. Laterosporus promoted the nutrient uptake by SY most
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Fig. 1. The pH value in the solutions of the three treatments. Results are presented as mean 7standard deviation; n¼3; TH, Teyou Hang II; MH, Minghui 63; SY, Shanyou 63; GM, Guangyou Ming 118; CT, control seedlings treated without As (III) or B. laterosporus; AT, seedlings treated with 3 mg L 1 As (III); BLT, seedlings treated with 3 mg L 1 As (III) and 0.25% (v/v) rejuvenated B. laterosporus. Different letters mean significant differences among the treatments within the same cultivar (po 0.01).
significantly among the BLT treatments with the four cultivars. The addition of B. laterosporus significantly (p o0.01) increased the As (III) oxidation in the solution (Fig. 3). Rice cultivars alone were able to oxidize just 30–72% of the As (III) they were exposed
Fig. 3. As (III) oxidation percentage in the solutions of the three treatments. Results are presented as mean 7 standard deviation; n¼3; TH, Teyou Hang II; MH, Minghui 63; SY, Shanyou 63; GM, Guangyou Ming 118; AT, seedlings treated with 3 mg L 1 As (III); BLT, seedlings treated with 3 mg L 1 As (III) and 0.25% (v/v) rejuvenated B. laterosporus. Different letters mean significant differences among the treatments within the same cultivar (p o 0.01).
to, whereas for rice cultivars amended with B. laterosporus the oxidation was 100%.
3.2. Effect of B. laterosporus on the accumulation and translocation
Fig. 2. Uptake concentrations of NH4 þ , NO3 , HPO42 and K þ in the solutions of the three treatments. c(N-NH4 þ ), uptake concentration of N in NH4 þ ; c(N-NO3 ), uptake concentration of N in NO3 ; c(P-HPO42 ), uptake concentration of P in HPO42 ; c(K-K þ ), uptake concentration of K in K þ . Results are presented as mean 7 standard deviation; n¼ 3; TH, Teyou Hang II; MH, Minghui 63; SY, Shanyou 63; GM, Guangyou Ming 118; CT, control seedlings treated without As (III) or B. laterosporus; AT, seedlings treated with 3 mg L 1 As (III); BLT, seedlings treated with 3 mg L 1 As (III) and 0.25% (v/v) rejuvenated B. laterosporus. Different letters mean significant differences among the treatments within the same cultivar (p o0.01). Uptake concentration of different elements was calculated according to the difference value between their initial concentration and their residual concentration in the nutrient solutions after 6 days of incubation.
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of As species in rice seedlings There was not any detectable MMA (V) or DMA (V) in the roots and shoots of 4 rice seedlings, only As (III) and As (V) were determined in 6 days of AT or BLT treatments with 4 rice seedlings, the total As concentration in Table 1 was calculated according to the sum of As (III) and As (V) concentration. 3.2.1. Effect of B. laterosporus on the total As concentration in rice seedlings Compared with AT treatments (Table 1), Addition of B. laterosporus significantly (p o0.01) decreased the total As concentrations in GM roots, GM shoots, TH roots and SY shoots. However, the addition increased (p o0.01) the total As concentrations in MH roots, MH shoots, SY roots and TH shoots. B. laterosporus decreased the translocation ratio of total As from roots to shoots in GM and SY. However, B. laterosporus increased the transportation ratio of total As from roots to shoots in MH and TH. The concentrations of the total As among the root and shoot of the four cultivars all showed significant differences in the AT and BLT treatments (p o0.01). However, the difference among the shoots of the four cultivars was much lower than that among their roots (Table 1). Furthermore, the concentrations of total As in the root of SY and MH were much lower than those in the root of GM and TH, the concentration of total As in the shoot of SY and GM were lower than those in the shoot of TH and MH among the BLT treatments of four cultivars. 3.2.2. Effect of B. laterosporus on the As (III) concentration in rice seedlings Compared with AT treatments (Table 1), the addition of B. laterosporus significantly decreased the As (III) concentrations in GM roots, GM shoots and SY roots. However, the addition of B. laterosporus increased As (III) concentrations in MH roots, MH shoots and TH shoots. The As (III) concentrations in the roots and shoots of SY were both the lowest in the BLT treatments of four cultivars, with no As (III) detected in SY roots due to the 100% of As (III) oxidation in the nutrient solution by B. Laterosporus and the weakest As (V) reduction capacity by SY roots (Fig. 3 and Fig. 4), only 9.48 mg kg 1 As (III) detected in SY shoots.
Fig. 4. As (III) percentage in the roots of four cultivars with 3 mg L 1 As (V) in the solutions after 6 days of incubation. Results are presented as mean 7standard deviation; n¼ 3; TH, Teyou Hang II; MH, Minghui 63; SY, Shanyou 63; GM, Guangyou Ming 118. Different letters mean significant differences among four cultivars (po 0.01).
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3.2.3. Effect of B. laterosporus on As (V) concentration in rice seedlings Compared with AT treatments (Table 1), the addition of B. laterosporus significantly decreased the As (V) concentrations in TH roots, TH shoots, SY shoots and GM shoots. However, the addition of B. laterosporus increased As (V) concentrations in MH roots, MH shoots, SY roots and GM roots. 3.3. Effect of B. laterosporus on the physiological and biochemical properties of the rice seedlings Compared with AT treatments, the physiological and biochemical properties such as chlorophyll SPAD value, root and shoot lengths, dry weights of rice roots and shoots, and the defending enzyme activities in the four cultivars were all dramatically restored by B. laterosporus amendment. The repairing effect was the most significant in SY, with respect to the chlorophyll SPAD value (Fig. A.1), root lengths (Fig. A.2) and MDA concentration (Fig. A.3), the repairing effect was the most significant in TH, with respect to shoot lengths and shoot dry weight (Fig. A.2) and the activities of SOD and CAT (Fig. A.3). B. laterosporus had the best repair effect on As (III) toxicity for roots dry weight of MH (Fig. A.2) and POD activities of GM (Fig. A.3). However, among the BLT treatments of the four cultivars, GM had the highest values for the chlorophyll SPAD value (Fig. A.1), roots dry weight, shoot lengths and shoot dry weight (Fig. A.2), all the second were SY. Furthermore, SY still had the highest values for root lengths (Fig. A.2) and SOD activity (Fig. A.3). Therefore, B. laterosporus had better repair effect on the toxicity of As (III) for SY and GM than for MH and TH. On the other hand, B. laterosporus significantly (po 0.01) promoted the uptakes of N, P and K in the roots and shoots of the four cultivars except for MH shoots (Fig. A.4), the most significant promotion was observed in the treatment with SY.
4. Discussion Arsenic speciation is key to its toxicity for rice growth (Bissen and Frimmel, 2003). It was reported when exposed to As, the activities of key enzymes and the corresponding gene expression closely related to photosynthesis were significantly decreased in rice seedlings (Shri et al., 2009). Activities of the defending enzymes, such as SOD, POD and CAT, were significantly decreased, while the concentration of MDA was significantly increased (Ma et al., 2008). In this study, after incubation in AT solutions for 6 d, the physiological and biochemical properties except the increased MDA concentration in the four cultivars of rice seedlings were all severely inhibited by As (III). As (III) was much more toxic than As (V) for the growth of rice seedling (Abedin and Meharg, 2002). The inoculation of B. laterosporus significantly increased the percentages of As (III) oxidation in the solutions with four cultivars, thus greatly decreased As (III) toxicity for the physiological and biochemical characteristics of four cultivars (Figs. A.1– A.3). The pH value in the medium is affected by Nitrogen assimilation of rice roots. It was reported that plants tend to acidify the rhizosphere because the net uptake of NH4 þ by rice roots was faster than that of NO3 when both forms of nitrogen were present (Colmer and Bloom, 1998). The NH4 þ uptake in AT treatments with four cultivars was 9.67–39.28% of that in CT treatments, the NO3 uptake in AT treatments with four cultivars was 26.24– 60.83% of that in CT treatments (Fig. 2). Therefore, pH value in AT treatments with four cultivars was significantly higher than that in CT treatments (Fig. 1). The inoculation of B. laterosporus significantly increased the NH4 þ uptake in the solutions of BLT treatments with four cultivars, 1.27–9.61 times of that in AT treatments (Fig. 2), the pH value in BLT treatments with four
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cultivars was maintained at the same level as that in the control except MH (Fig. 1). The increase of nutrient uptakes by B. laterosporus alleviated As (III) toxicity for the growth of rice seedlings. Furthermore, B. laterosporus showed the better repair effect on the As (III) toxicity for SY and GM than for MH and TH, due to the higher nutrient uptake level in the CT treatments with SY and GM (Figs. A.1–A.4). On the other hand, The pH value in the medium will affect the stability of As (III) speciation (Meharg and HartleyWhitaker, 2002). It was reported that the increase of pH value in the solutions would inhibit the oxidation of As (III) to As (V) by the bacteria (Cullen and Reimer, 1989). Compared with AT treatments, B. laterosporus significantly (p o0.01) decreased the pH value in the nutrient solutions of BLT treatments with four cultivars (Fig. 1). Therefore, B. laterosporus accelerated the oxidation of As (III). Arsenic translocation and accumulation in rice seedlings are greatly affected by its speciation in the environmental medium. The inoculation of B. laterosporus in the solution accelerated the oxidation of As (III) to As (V), As (III) concentration in the roots of SY and GM were significantly decreased (Table 1). However, As (V) in rice root might be further reduced into As (III) and then followed by As (III) efflux into the medium (Duan et al., 2007; Xu et al., 2007). Furthermore, the As (III) discharged out of the root may once more be oxidized into As (V) under the B. laterosporus catalysis and generate a dynamic cycle of As redox processes. In this study, the increase of As (III) concentration in the roots of TH and MH might result from their very strong reduction capacity for As (V) (Fig. 4). Phosphate as an important environmental factor could influence As translocation and accumulation. As (V) is taken up by phosphate transporters and is strongly suppressed by P nutrient in the medium (Abedin et al., 2002; Verbruggen et al., 2009). Compared with AT treatments, the addition of B. laterosporus significantly (p o0.01) promoted the uptake of HPO42 in the nutrient solutions with the four cultivars (Fig. 2), the residual P concentration in the BLT solution with the four cultivars was significantly decreased due to the higher uptake of P into their roots, which in turn enhanced the uptake of As (V) (Table 1). Therefore, the As (V) concentration in their roots except TH was increased after the addition of B. laterosporus (Table 1). In a word, the inoculation of B. laterosporus significantly inhibited the increase of pH values in the solutions, promoted As (III) oxidation in the solutions and increased the uptakes of N, P and K by rice seedlings, the physiological and biochemical properties of four cultivars were all dramatically restored. As a whole, B. laterosporus had the best repair effect on SY, with respect to the subdued As (III) toxicity, the decreased As (III) concentrations in its roots and the decreased total As translocation from its roots to shoots. The results demonstrated that B. laterosporus amendment could be an effective method to alleviate As (III) toxicity for rice seedling.
Acknowledgments This work was financially supported by National Natural Science Foundation of China (31271670), Fujian Provincial Department of Science and Technology (2013Y0005), Program for New Century Excellent Talents in Fujian Province University, China (JA10093) and Key Subject of Ecology, Fujian Province, China (6112C0600). We thank Professor Yong-Guan Zhu in the Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, for his kindly proofreading the manuscript. Appendix A. Supplementary material Supplementary data associated with this article can be found in
the online version at http://dx.doi.org/10.1016/j.ecoenv.2015.05. 023.
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Sulfur (S)-induced enhancement of iron plaque formation in the rhizosphere reduces arsenic accumulation in rice (Oryza sativa L.) seedlings. Environ. Pollut. 147, 387–393. Kashyap, D.R., Botero, L.M., Franck, W.L., Hassett, D.J., McDermott, T.R., 2006. Complex regulation of arsenite oxidation in Agrobacterium tumefaciens. J. Bacteriol. 188, 1081–1088. Liu, W.J., Zhu, Y.G., Hu, Y., Williams, P., Gault, A., Meharg, A.A., Charnock, J., Smith, F.A., 2006. Arsenic sequestration in iron plaque, its accumulation and speciation in mature rice plants (Oryza sativa L.). Environ. Sci. Technol. 40, 5730–5736. Llorente-Mirandes, T., Ruiz-Chancho, M.J., Barbero, M., Rubio, R., López-Sánchez, J.F., 2011. Determination of water-soluble arsenic compounds in commercial edible seaweed by LC-ICPMS. J. Agric. Food Chem. 59, 12963–12968. Ma, J.F., Yamaji, N., Mitani, N., Xu, X.-Y., Su, Y.-H., McGrath, S.P., Zhao, F.-J., 2008. Transporters of arsenite in rice and their role in arsenic accumulation in rice grain. Proc. Natl. Acad. Sci. 105, 9931–9935. Meharg, A.A., Hartley-Whitaker, J., 2002. Arsenic uptake and metabolism in arsenic resistant and nonresistant plant species. New Phytol. 154, 29–43. Oremland, R.S., Stolz, J.F., 2003. The ecology of arsenic. Science 300, 939–944. Shri, M., Kumar, S., Chakrabarty, D., Trivedi, P.K., Mallick, S., Misra, P., Shukla, D., Mishra, S., Srivastava, S., Tripathi, R.D., Tuli, R., 2009. Effect of arsenic on growth, oxidative stress, and antioxidant system in rice seedlings. Ecotoxicol. Environ. Saf. 72, 1102–1110. Sierra-Alvarez, R., Yenal, U., Field, J.A., Kopplin, M., Gandolfi, A.J., Garbarino, J.R., 2006. Anaerobic biotransformation of organoarsenical pesticides monomethylarsonic acid and dimethylarsinic acid. J. Agric. Food Chem. 54, 3959–3966. Silver, S., Phung, L.T., 2005. Genes and enzymes involved in bacterial oxidation and reduction of inorganic arsenic. Appl. Environ. Microbiol. 71, 599–608. Tamaki, S., Frankenberger Jr, W.T., 1992. Environmental biochemistry of arsenic. Rev. Environ. Contam. Toxicol. 124, 79–110. Verbruggen, N., Christian, H., Henk, S., 2009. Mechanisms to cope with arsenic or cadmium excess in plants. Curr. Opin. Plant Biol. 12, 364–372. Wang, X.K., 2006. Principle and Technology of Plant Physiological and Biochemical Experiments in Chinese. Higher Education Press, Beijing, China. Xu, X.Y., McGrath, S.P., Zhao, F.J., 2007. Rapid reduction of arsenate in the medium mediated by plant roots. New Phytol. 176, 590–599. Yang, G.D., Xu, J.H., Zheng, J.P., Xu, X.Q., Wang, W., Xu, L.J., Chen, G.N., Fu, F.F., 2009. Speciation analysis of arsenic in Mya arenaria Linnaeus and Shrimp with capillary electrophoresis-inductively coupled plasma mass spectrometry. Talanta 78, 471–476. Yang, G.D., Xu, X.Q., Wang, W., Xu, L.J., Chen, G.N., Fu, F.F., 2008. A new interface used to couple capillary electrophoresis with an inductively coupled plasma mass spectrometry for speciation analysis. Electrophoresis 29, 2862–2868. Yang, X.J., Huang, Y., Qiu, Z.Q., Bai, Y.F., Lin, Z.H., He, H.B., Yang, G.D., 2014. Isolation and characterization of Brevibacillus laterosporus from the paddy soil and its renovation for paddy rice seedling stressed by arsenite. J. Fujian Agric. For. Univ. Chin.: Nat. Sci. Ed. 43, 172–177. Yuan, C., Jiang, G., He, B., 2005. Evaluation of the extraction methods for arsenic speciation in rice straw, Oryza sativa L., and analysis by HPLC-HG-AFS. J. Anal. At. Spectrom. 20, 103–110. Zhang, J., Zhao, Q.-Z., Duan, G.-L., Huang, Y.-C., 2011. Influence of sulphur on arsenic accumulation and metabolism in rice seedlings. Environ. Exp. Bot. 72, 34–40. Zhu, Y.G., Sun, G.X., Lei, M., Teng, M., Liu, Y.X., Chen, N.C., Wang, L.H., Carey, A.M., Deacon, C., Raab, A., 2008a. High percentage inorganic arsenic content of mining impacted and nonimpacted Chinese rice. Environ. Sci. Technol. 42, 5008–5013. Zhu, Y.G., Williams, P.N., Meharg, A.A., 2008b. Exposure to inorganic arsenic from rice: a global health issue? Environ. Pollut. 154, 169–171.
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Effect of arsenite-oxidizing bacterium B. laterosporus on arsenite toxicity and arsenic translocation in rice seedlings Gui-Di Yang a,n, Wan-Ying Xie b, Xi Zhu a, Yi Huang a, Xiao-Jun Yang a, Zong-Qing Qiu a, Zhen-Mao Lv a, Wen-Na Wang a, Wen-Xiong Lin a,n a Fujian Provincial Key Laboratory of Agroecological Processing and Safety Monitoring, College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China b Key Laboratory of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen 361021, China
art ic l e i nf o
a b s t r a c t
Article history: Received 1 December 2014 Received in revised form 14 May 2015 Accepted 14 May 2015
Arsenite [As (III)] oxidation can be accelerated by bacterial catalysis, but the effects of the accelerated oxidation on arsenic toxicity and translocation in rice plants are poorly understood. Herein we investigated how an arsenite-oxidizing bacterium, namely Brevibacillus laterosporus, influences As (III) toxicity and translocation in rice plants. Rice seedlings of four cultivars, namely Guangyou Ming 118 (GM), Teyou Hang II (TH), Shanyou 63 (SY) and Minghui 63 (MH), inoculated with or without the bacterium were grown hydroponically with As (III) to investigate its effects on arsenic toxicity and translocation in the plants. Percentages of As (III) oxidation in the solutions with the bacterium (100%) were all significantly higher than those without (30–72%). The addition of the bacterium significantly decreased As (III) concentrations in SY root, GM root and shoot, while increased the As (III) concentrations in the shoot of SY, MH and TH and in the root of MH. Furthermore, the As (III) concentrations in the root and shoot of SY were both the lowest among the treatments with the bacterium. On the other hand, its addition significantly alleviated the As (III) toxicity on four rice cultivars. Among the treatments amended with B. laterosporus, the bacterium showed the best remediation on SY seedlings, with respect to the subdued As (III) toxicity and decreased As (III) concentration in its roots. These results indicated that As (III) oxidation accelerated by B. laterosporus could be an effective method to alleviate As (III) toxicity on rice seedlings. & 2015 Published by Elsevier Inc.
Keywords: Rice (Oryza sativa L.) Arsenite-oxidizing bacteria Arsenic speciation B. laterosporus CE-ICP-MS Remediation
1. Introduction With the development of industry and agriculture, large amounts of arsenic-containing waste water were discharged into the environment and increased arsenic (As) pollution in paddy soil and underground water (Sierra-Alvarez et al., 2006; Garelick et al., 2008). There are four main As species in soil solution of rice rhizosphere, namely arsenite [As (III)], arsenate [As (V)], dimethylarsinate [DMA (V)] and methylarsonate [MMA (V)], with As (III) as the predominant form under flooded conditions (Abedin et al., 2002). Arsenic accumulation in rice seedlings inhibits the photosynthesis and the activities of the defending enzymes in the plants (Abedin and Meharg, 2002). On the other hand, As accumulation in rice grains will pose serious risk on human health (Zhu et al., 2008,b). As (III) is the most toxic species among the above four species for the growth of rice seedlings (Abedin and Meharg, 2002). Therefore, it is vital to alleviate the As (III) toxicity on the n
Corresponding authors. Fax: þ 86 59183789352
http://dx.doi.org/10.1016/j.ecoenv.2015.05.023 0147-6513/& 2015 Published by Elsevier Inc.
growth of rice seedling and decrease As translocation from roots to shoots to improve rice yield and quality. Several strategies have potential to alleviate As toxicity for rice growth and to decrease As accumulation in rice plants, including screening for rice cultivars with high As resistance and/or low As accumulation ability, and the management of the cultivation environment. Screening for rice cultivars with high As resistance and/or low As accumulation ability is a good technology to alleviate As toxicity for rice growth. However, it is a time- and costconsuming process. In contrast, the management of the cultivated environments is a simpler and easier pathway to alleviate As toxicity and accumulation in rice. Many attentions have been paid to the interactions between the uptakes of As and some nutrient elements, such as Si, S, P, and Fe (Hu et al., 2007; Zhang et al., 2011), especially to the competitive uptake between P and As (Abedin et al., 2002; Hu et al., 2005; Liu et al., 2006; Hossain et al., 2009). However, over addition of nutrient elements could easily lead to secondary pollution in soil and water. Bioremediation is another potential technique to alleviate As toxicity. Microbial remediation has gained close attention because
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of its affordability and compatibility with soil medium. Microbe in the soil could detoxify As toxicity by efficiently oxidizing As (III) to As (V) (Tamaki and Frankenberger 1992; Kashyap et al., 2006). Several arsenite-oxidizing bacteria, such as Agrobacterium, Herminiimonas arsenicoxydans and Alcaligenes faecalis, have been isolated from As polluted soils since the first arsenite-oxidizing bacterium was found in 1918 (Green, 1918). Genes responsible for the oxidation have also been identified from these bacteria (Oremland and Stolz, 2003; Silver and Phung, 2005). So far, most of the studies on arsenite-oxidizing bacteria focused on the screening processes, physiological and biochemical characteristics of the bacteria and the mechanisms of arsenite-oxidizing. Though microbes play critical roles in As (III) oxidation, little attention has been paid to the effects of accelerated As (III) oxidation by the microbes on As toxicity and translocation in rice plants. In our previous research, an arsenite-oxidizing bacterium, B. laterosporus, was isolated from paddy soil (Yang et al., 2014). It could oxidize 200 mgL 1 of As (III) into As (V) within 24 h (5% inoculation, v/v). However, the effect of this bacterium on the As toxicity and translocation in rice seedlings is still unknown. Therefore, the objective of this study is to evaluate the effects of B. laterosporus on the As (III) oxidation, As toxicity and translocation in rice seedlings.
was 23.00, 7.00 and 21.00 mg L 1, respectively.). Prior to the transplantation, seedlings with similar status in each cultivar were selected and washed with 18.2 MΩ DDI water to remove the transparent sticky secretions adhered to their roots. All treatments were conducted for 6 d and 18.2 MΩ DDI water was daily added into the plates to keep stable solution volumes. Three of the six replicates were used to determine the pH value, the residual concentration of NH4 þ , K þ , HPO42 and NO3 , and As (III) oxidation percentage in the solutions, the concentrations of N, P, K, and As species in the roots and shoots, the chlorophyll SPAD (Soil and Plant Analyzer Development) value in the leaves, length of the roots, height of the shoots, and dry weights of the roots and shoots. The other three replicates were used to determine the defending enzyme activities in the leaves as described in Section 2.5.
2. Materials and methods
2.4. Determination of pH, nutrient concentrations and the As (III) oxidation in the solutions
2.3. Arsenate reduction by the rice roots To investigate the reduction of As (V) to As (III) by the rice roots, the rice seedlings of four cultivars were hydroponically cultivated in the nutrient solutions with 3 mg L 1 As (V) as described in Section 2.2. After 6 days of incubation, the seedlings were harvested and the concentrations of As (III) and As (V) in the roots of four cultivars were determined as described in Section 2.5.
2.1. Plant materials and cultivation conditions Four rice cultivars, namely Guangyou Ming 118 (GM), Teyou Hang II (TH), Shanyou 63 (SY) and Minghui 63 (MH), were used in the present study. They were all indica rice, from the Rice Research Institute, Academy of Agriculture Sciences, Fujian, China. The seeds were germinated separately in 250 mL beakers with tap water till the shoot length reached 3–4 cm in a temperature-controlled culture chamber at 31 °C. The rice seedlings of each plant were transferred into a plate with holes (4 4 cm2), and affixed in the holes with Styrofoam. The Styrofoam plate with seedlings was allowed to float in a pot (17 10 6 cm3) filled with 500 mL full nutrient solution with the following compositions per liter: 14.9 mg K2SO4, 48.2 mg (NH4)2SO4, 24.8 mg KH2PO4, 18.5 mg KNO3, 86.3 mg Ca(NO3)2 4H2O, 135.0 mg MgSO4 7H2O, 200.0 mg Na2SiO3 9H2O, 27.8 mg FeSO4 7H2O, 37.2 mg Na2EDTA, 14.3 mg H3BO3, 0.4 mg CuSO4 5H2O, 1.1 mg ZnSO4 7H2O, 9.1 mg MnCl2 4H2O, 0.5 mg Na2MoO4 2H2O. The pH value of the full nutrient solution was adjusted to 5.5–6.0 with 0.1 mol L 1 HCl. 2.2. Arsenic treatment and B. laterosporus amendment The four cultivars of rice seedlings (at the 2-leaf stage) were transferred into new Styrofoam plates (5 seedlings each) as described in Section 2.1 and grown hydroponically within three treatments (6 replicates each), respectively. Treatment 1 (CT), control seedlings treated without As (III) or B. laterosporus; Treatment 2 (AT), seedlings treated with 3 mg L 1 As (III); Treatment 3 (BLT), seedlings treated with 3 mg L 1 As (III) and 0.25% (v/v) rejuvenated B. laterosporus. Control seedlings treated with 0.25% (v/v) B. laterosporus was not cultivated because their physiological and biochemical properties had no significant difference (p 40.01) from CT within the regulation period of our study. The rejuvenation of B. laterosporus could be found in the supplementary material. B. laterosporus was inoculated into the incubation solutions just before the transplantation. The incubation solutions were prepared with NH4NO3, K2SO4 and NaH2PO4 2H2O in 18.2 MΩ doubly deionized (DDI) water (the pH values were adjusted to 5.5–6.0 with 0.1 mol L 1 HCl; the concentration of N, P, K
After 6 days of incubation, the pH values in the nutrient solutions were determined by a pHS-3C meter. Nutrient solutions (around 5.0 mL) were filtered through 0.22 μm polypropylene membranes, kept in 20 °C until further determination of nutrient concentrations by CIC-260 Ion Chromatography (IC) and As species by Capillary Electrophoresis-Inductively Coupled PlasmaMass Spectrometry (CE-ICP-MS) as described in Section 2.5. 2.5. Extraction and determination of N, P, K and As in rice roots and shoots After 6 days of incubation, the seedlings were harvested. The fresh roots were washed three times with 18.2 MΩ DDI water to remove the adsorbed As on the surface. The roots and shoots were separated with stainless steel scissors, dried at 40 °C in an oven till the weights were constant, and cut into pieces by stainless steel scissors. The crushed roots or shoots were used to extract N, P, K and As by microwave assisted extraction (Yuan et al., 2005). Around 0.3000 g (with the precision of 0.0001 g) crushed roots or shoots were weighed into separate PFA jars and added with 10.0 mL HPLC grade CH3OH–H2O (1:1, v/v). The jars were sealed in PEEK cans and extracted for 15 min at 120 °C in a MDS-6 microwave extraction apparatus (Sineo Microwave Chemistry Technology Co. Ltd., China). After the extraction, the extracts were filtered through 0.22 μm polypropylene membranes into polyethylene centrifuge tubes and diluted to the appropriate volume (according to N, P, K and As concentration in the sample) with 18.2 MΩ DDI water, and the final solution was used for the determination of N, P, K, total As and As species. In speciation analysis of arsenic, it is important to know if all arsenics are completely extracted during the extraction process. The shoots of SY in BLT treatment were chosen as the materials to evaluate the extraction efficiency of arsenic species by CH3OH–H2O (1:1, v/v). The digestion solution of HNO3–H2O2 (5:1, v/v) was used to completely decompose the rice shoots according to the extraction procedure described above. The extraction efficiency (%, n ¼3) was 99.63 72.32, namely the percent between the total As concentration extracted by the mixture of CH3OH–H2O
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and the total As concentration digested by the mixture of HNO3–H2O2, the results indicated that all arsenics had been extracted by CH3OH–H2O (1:1, v/v) in the above procedure. In speciation analysis of arsenic, it is also important to know if each arsenical species remains constant during the extraction and analytical process. The shoots of SY in BLT treatment were chosen as the materials, spiked by As (III), MMA (V), DMA (V) and As (V), and also analyzed in the same manner, the average recoveries (%, n ¼3) was 93.13 74.89, 98.62 73.67, 105.4 73.3 and 107.1 74.5, respectively, for the above targets at the spiked concentration of 2.00, 2.00, 2.00 and 10.00 mg kg 1. The approximate 100% recovery for As (III), DMA (V), MMA (V) and As (V) indicated that no arsenical species changed during extraction and analysis. The total contents of N, P and K in the roots and shoots of rice seedling were determined by a KDN-08C azotometer (Shanghai new instrument co., LTD., China), a UV-9100 ultraviolet–visible spectrophotometer (Shanghai precision scientific instrument co., LTD., China), and a FP640 flame spectrophotometer (Shanghai precision scientific instrument co., LTD., China), respectively. Concentrations of the total As in the shoots of SY (BLT treatment) were determined by NexION 300X ICP-MS (PerkinElmer, USA) (Llorente-Mirandes et al., 2011). Germanium (Ge) was used as an internal standard to calculate the matrix effect of rice plants on As signal. Total As concentration in Table 1 was calculated according to the sum of As species. The concentrations of As species in the extracts and in the nutrient solutions were determined by CE-ICP-MS according to Yang et al. (2008, 2009). Briefly, A 57.0 cm CE capillary was used to separate the arsenic species and coupled with a NexION 300X ICP-MS by an interface designed by our group (Yang et al., 2009). The CE capillary was conditioned daily by purging with 18.2 MΩ DDI water for 10 min, 0.1 mol L 1 NaOH solution for 10 min, 18.2 MΩ DDI water for 10 min and running buffer solution (50 mmol L 1 H3BO4–12.5 mmol L 1 Na2B4O7, pH 9.1) for 10 min. Between each run, the CE capillary was flushed with 18.2 MΩ DDI water and running buffer solution for 2 min, respectively. The sample was injected into the CE capillary for determination by electro-migration injection (15 kV, 10 s). The nebulizer type was PFA (optimum flow 10–20 μL min 1). Mixed standard solution of As (III), As (V), DMA (V) and MMA (V) with Table 1 Concentration of As (III), As (V) and total As in roots and shoots in the treatments amended with or without B. laterosporus. Arsenic concentration (mg kg 1DW) Cultivar
Organ
Treatment
As (III)
As (V)
Total As
TH
Roots
AT BLT AT BLT
2577 13A 2727 17A 177 1 B 72 72A
194 7 9A 91 7 4 B 38 7 2A 17 7 1 B
452 716A 363 713 B 55 72 B 88 74A
AT BLT AT BLT
467 2 B 567 2A 67 1 B 267 1A
39 7 2 B 91 7 3A 17 7 1 B 30 7 2A
85 710 B 147 716A 23 71 B 56 73A
AT BLT AT BLT
13 71A n n.d. B 67 1 B 97 1A
170 7 5 B 240 7 8A 64 7 3A 43 7 2 B
183 712 B 240 713A 71 74A 53 73 B
AT BLT AT BLT
7447 22A 6117 15 B 27 71A 257 0 B
152 7 7 B 238 7 10A 21 7 2A 12 7 1 B
896 723A 848 720 B 48 73A 37 72 B
Shoots
MH
Roots Shoots
SY
Roots Shoots
GM
Roots Shoots
n
n.d. not detected
9
10.0–100.0 μg L 1 was used to calibrate the concentration of As species in the roots and shoots of rice seedling, the linear correlation coefficient for above four As standard solutions from 10.0 to 100.0 μg L 1 was higher than 0.995, the relative standard deviation (RSD) for 100.0 μg L 1 mixed As standard solution was lower than 5.0% within 5 replicates. Qualitative confirmation of As species was based on their retention time and spiked tests. Quantitative determination of As species was based on peak areas. The limit of detection (LOD) of the analytical method for above four As species in the roots and shoots of dried rice seedling was about 0.1 mg kg 1 As according to the extraction, dilution and CE-ICPMS determination. 2.6. Determination of the physiological and biochemical properties of the rice seedlings The values of chlorophyll SPAD in the fresh leaves were determined by a SPAD-502 chlorophyll meter with the precision of 0.1. The length of the roots and the height of the shoots were determined by a ruler with the precision of 0.01 cm. The dry weights of the roots and shoots were weighed by a balance with the precision of 0.0001 g. The defending enzyme activities, such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and malondialdehyde (MDA) in the shoots were determined as described below. Fresh shoots (about 0.5000 g) were homogenized with 5.0 mL extraction buffer (pH7.80, 50.0 mmol L 1 phosphorus (P), 10.0 mg mL 1 polyvinylpyrrolidone) in a pre-chilled mortar and pestle on ice bath. The homogenate was transferred into a 15.0 mL Polyethylene centrifuge tube, diluted with extraction buffer to 10.0 mL, and centrifuged at 12,000 rpm for 3 min at 4 °C. The supernatant with the enzyme extract was used for the determination of SOD, POD and CAT. For the extraction of MDA, fresh shoots (about 0.9000 g) was homogenized with 9.0 mL of 5% (m/v) trichloroacetic acid (TCA) in the same manner as described before, and the supernatant was collected from the mixture without dilution by centrifuging at 3,000 rpm for 10 min at 4 °C. During the determination process, the supernatant was kept in 4 °C refrigerator until the assays were completed. SOD activity, POD activity, CAT activity and MDA concentration were measured according to nitro blue tetrazolium (NBT) method (Beyer and Fridovich, 1987), guaiacol oxidation method, spectrophotometric method at 240 nm and thiobarbituric acid (TBA) method (Wang, 2006), respectively. 2.7. Statistics analysis The average values of the three replicates were used for statistical analysis. Software DPS 7.5 was used to conduct the significance test by least significant difference (LSD) among the three treatments within the same rice cultivar.
3. Results 3.1. Effect of B. laterosporus on the pH value, nutrient uptake and As (III) oxidation in the solutions The addition of As (III) in AT treatment significantly (po 0.01) increased the pH value in the solutions compared to the control (Fig.1). While when B. laterosporus was added (BLT treatment), the pH values were maintained at the same level as those in the control except MH. The addition of B. laterosporus significantly (po0.01) promoted the uptake of NH4 þ , NO3 , HPO42 and K þ in the solutions with the four cultivars except for the NH4 þ by MH compared to AT treatments (Fig. 2). B. Laterosporus promoted the nutrient uptake by SY most
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Fig. 1. The pH value in the solutions of the three treatments. Results are presented as mean 7standard deviation; n¼3; TH, Teyou Hang II; MH, Minghui 63; SY, Shanyou 63; GM, Guangyou Ming 118; CT, control seedlings treated without As (III) or B. laterosporus; AT, seedlings treated with 3 mg L 1 As (III); BLT, seedlings treated with 3 mg L 1 As (III) and 0.25% (v/v) rejuvenated B. laterosporus. Different letters mean significant differences among the treatments within the same cultivar (po 0.01).
significantly among the BLT treatments with the four cultivars. The addition of B. laterosporus significantly (p o0.01) increased the As (III) oxidation in the solution (Fig. 3). Rice cultivars alone were able to oxidize just 30–72% of the As (III) they were exposed
Fig. 3. As (III) oxidation percentage in the solutions of the three treatments. Results are presented as mean 7 standard deviation; n¼3; TH, Teyou Hang II; MH, Minghui 63; SY, Shanyou 63; GM, Guangyou Ming 118; AT, seedlings treated with 3 mg L 1 As (III); BLT, seedlings treated with 3 mg L 1 As (III) and 0.25% (v/v) rejuvenated B. laterosporus. Different letters mean significant differences among the treatments within the same cultivar (p o 0.01).
to, whereas for rice cultivars amended with B. laterosporus the oxidation was 100%.
3.2. Effect of B. laterosporus on the accumulation and translocation
Fig. 2. Uptake concentrations of NH4 þ , NO3 , HPO42 and K þ in the solutions of the three treatments. c(N-NH4 þ ), uptake concentration of N in NH4 þ ; c(N-NO3 ), uptake concentration of N in NO3 ; c(P-HPO42 ), uptake concentration of P in HPO42 ; c(K-K þ ), uptake concentration of K in K þ . Results are presented as mean 7 standard deviation; n¼ 3; TH, Teyou Hang II; MH, Minghui 63; SY, Shanyou 63; GM, Guangyou Ming 118; CT, control seedlings treated without As (III) or B. laterosporus; AT, seedlings treated with 3 mg L 1 As (III); BLT, seedlings treated with 3 mg L 1 As (III) and 0.25% (v/v) rejuvenated B. laterosporus. Different letters mean significant differences among the treatments within the same cultivar (p o0.01). Uptake concentration of different elements was calculated according to the difference value between their initial concentration and their residual concentration in the nutrient solutions after 6 days of incubation.
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of As species in rice seedlings There was not any detectable MMA (V) or DMA (V) in the roots and shoots of 4 rice seedlings, only As (III) and As (V) were determined in 6 days of AT or BLT treatments with 4 rice seedlings, the total As concentration in Table 1 was calculated according to the sum of As (III) and As (V) concentration. 3.2.1. Effect of B. laterosporus on the total As concentration in rice seedlings Compared with AT treatments (Table 1), Addition of B. laterosporus significantly (p o0.01) decreased the total As concentrations in GM roots, GM shoots, TH roots and SY shoots. However, the addition increased (p o0.01) the total As concentrations in MH roots, MH shoots, SY roots and TH shoots. B. laterosporus decreased the translocation ratio of total As from roots to shoots in GM and SY. However, B. laterosporus increased the transportation ratio of total As from roots to shoots in MH and TH. The concentrations of the total As among the root and shoot of the four cultivars all showed significant differences in the AT and BLT treatments (p o0.01). However, the difference among the shoots of the four cultivars was much lower than that among their roots (Table 1). Furthermore, the concentrations of total As in the root of SY and MH were much lower than those in the root of GM and TH, the concentration of total As in the shoot of SY and GM were lower than those in the shoot of TH and MH among the BLT treatments of four cultivars. 3.2.2. Effect of B. laterosporus on the As (III) concentration in rice seedlings Compared with AT treatments (Table 1), the addition of B. laterosporus significantly decreased the As (III) concentrations in GM roots, GM shoots and SY roots. However, the addition of B. laterosporus increased As (III) concentrations in MH roots, MH shoots and TH shoots. The As (III) concentrations in the roots and shoots of SY were both the lowest in the BLT treatments of four cultivars, with no As (III) detected in SY roots due to the 100% of As (III) oxidation in the nutrient solution by B. Laterosporus and the weakest As (V) reduction capacity by SY roots (Fig. 3 and Fig. 4), only 9.48 mg kg 1 As (III) detected in SY shoots.
Fig. 4. As (III) percentage in the roots of four cultivars with 3 mg L 1 As (V) in the solutions after 6 days of incubation. Results are presented as mean 7standard deviation; n¼ 3; TH, Teyou Hang II; MH, Minghui 63; SY, Shanyou 63; GM, Guangyou Ming 118. Different letters mean significant differences among four cultivars (po 0.01).
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3.2.3. Effect of B. laterosporus on As (V) concentration in rice seedlings Compared with AT treatments (Table 1), the addition of B. laterosporus significantly decreased the As (V) concentrations in TH roots, TH shoots, SY shoots and GM shoots. However, the addition of B. laterosporus increased As (V) concentrations in MH roots, MH shoots, SY roots and GM roots. 3.3. Effect of B. laterosporus on the physiological and biochemical properties of the rice seedlings Compared with AT treatments, the physiological and biochemical properties such as chlorophyll SPAD value, root and shoot lengths, dry weights of rice roots and shoots, and the defending enzyme activities in the four cultivars were all dramatically restored by B. laterosporus amendment. The repairing effect was the most significant in SY, with respect to the chlorophyll SPAD value (Fig. A.1), root lengths (Fig. A.2) and MDA concentration (Fig. A.3), the repairing effect was the most significant in TH, with respect to shoot lengths and shoot dry weight (Fig. A.2) and the activities of SOD and CAT (Fig. A.3). B. laterosporus had the best repair effect on As (III) toxicity for roots dry weight of MH (Fig. A.2) and POD activities of GM (Fig. A.3). However, among the BLT treatments of the four cultivars, GM had the highest values for the chlorophyll SPAD value (Fig. A.1), roots dry weight, shoot lengths and shoot dry weight (Fig. A.2), all the second were SY. Furthermore, SY still had the highest values for root lengths (Fig. A.2) and SOD activity (Fig. A.3). Therefore, B. laterosporus had better repair effect on the toxicity of As (III) for SY and GM than for MH and TH. On the other hand, B. laterosporus significantly (po 0.01) promoted the uptakes of N, P and K in the roots and shoots of the four cultivars except for MH shoots (Fig. A.4), the most significant promotion was observed in the treatment with SY.
4. Discussion Arsenic speciation is key to its toxicity for rice growth (Bissen and Frimmel, 2003). It was reported when exposed to As, the activities of key enzymes and the corresponding gene expression closely related to photosynthesis were significantly decreased in rice seedlings (Shri et al., 2009). Activities of the defending enzymes, such as SOD, POD and CAT, were significantly decreased, while the concentration of MDA was significantly increased (Ma et al., 2008). In this study, after incubation in AT solutions for 6 d, the physiological and biochemical properties except the increased MDA concentration in the four cultivars of rice seedlings were all severely inhibited by As (III). As (III) was much more toxic than As (V) for the growth of rice seedling (Abedin and Meharg, 2002). The inoculation of B. laterosporus significantly increased the percentages of As (III) oxidation in the solutions with four cultivars, thus greatly decreased As (III) toxicity for the physiological and biochemical characteristics of four cultivars (Figs. A.1– A.3). The pH value in the medium is affected by Nitrogen assimilation of rice roots. It was reported that plants tend to acidify the rhizosphere because the net uptake of NH4 þ by rice roots was faster than that of NO3 when both forms of nitrogen were present (Colmer and Bloom, 1998). The NH4 þ uptake in AT treatments with four cultivars was 9.67–39.28% of that in CT treatments, the NO3 uptake in AT treatments with four cultivars was 26.24– 60.83% of that in CT treatments (Fig. 2). Therefore, pH value in AT treatments with four cultivars was significantly higher than that in CT treatments (Fig. 1). The inoculation of B. laterosporus significantly increased the NH4 þ uptake in the solutions of BLT treatments with four cultivars, 1.27–9.61 times of that in AT treatments (Fig. 2), the pH value in BLT treatments with four
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cultivars was maintained at the same level as that in the control except MH (Fig. 1). The increase of nutrient uptakes by B. laterosporus alleviated As (III) toxicity for the growth of rice seedlings. Furthermore, B. laterosporus showed the better repair effect on the As (III) toxicity for SY and GM than for MH and TH, due to the higher nutrient uptake level in the CT treatments with SY and GM (Figs. A.1–A.4). On the other hand, The pH value in the medium will affect the stability of As (III) speciation (Meharg and HartleyWhitaker, 2002). It was reported that the increase of pH value in the solutions would inhibit the oxidation of As (III) to As (V) by the bacteria (Cullen and Reimer, 1989). Compared with AT treatments, B. laterosporus significantly (p o0.01) decreased the pH value in the nutrient solutions of BLT treatments with four cultivars (Fig. 1). Therefore, B. laterosporus accelerated the oxidation of As (III). Arsenic translocation and accumulation in rice seedlings are greatly affected by its speciation in the environmental medium. The inoculation of B. laterosporus in the solution accelerated the oxidation of As (III) to As (V), As (III) concentration in the roots of SY and GM were significantly decreased (Table 1). However, As (V) in rice root might be further reduced into As (III) and then followed by As (III) efflux into the medium (Duan et al., 2007; Xu et al., 2007). Furthermore, the As (III) discharged out of the root may once more be oxidized into As (V) under the B. laterosporus catalysis and generate a dynamic cycle of As redox processes. In this study, the increase of As (III) concentration in the roots of TH and MH might result from their very strong reduction capacity for As (V) (Fig. 4). Phosphate as an important environmental factor could influence As translocation and accumulation. As (V) is taken up by phosphate transporters and is strongly suppressed by P nutrient in the medium (Abedin et al., 2002; Verbruggen et al., 2009). Compared with AT treatments, the addition of B. laterosporus significantly (p o0.01) promoted the uptake of HPO42 in the nutrient solutions with the four cultivars (Fig. 2), the residual P concentration in the BLT solution with the four cultivars was significantly decreased due to the higher uptake of P into their roots, which in turn enhanced the uptake of As (V) (Table 1). Therefore, the As (V) concentration in their roots except TH was increased after the addition of B. laterosporus (Table 1). In a word, the inoculation of B. laterosporus significantly inhibited the increase of pH values in the solutions, promoted As (III) oxidation in the solutions and increased the uptakes of N, P and K by rice seedlings, the physiological and biochemical properties of four cultivars were all dramatically restored. As a whole, B. laterosporus had the best repair effect on SY, with respect to the subdued As (III) toxicity, the decreased As (III) concentrations in its roots and the decreased total As translocation from its roots to shoots. The results demonstrated that B. laterosporus amendment could be an effective method to alleviate As (III) toxicity for rice seedling.
Acknowledgments This work was financially supported by National Natural Science Foundation of China (31271670), Fujian Provincial Department of Science and Technology (2013Y0005), Program for New Century Excellent Talents in Fujian Province University, China (JA10093) and Key Subject of Ecology, Fujian Province, China (6112C0600). We thank Professor Yong-Guan Zhu in the Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, for his kindly proofreading the manuscript. Appendix A. Supplementary material Supplementary data associated with this article can be found in
the online version at http://dx.doi.org/10.1016/j.ecoenv.2015.05. 023.
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