Effects of silicon (Si) on arsenic (As) accumulation and speciation in rice (Oryza sativa L.) genotypes with different radial oxygen loss (ROL)

Chemosphere 138 (2015) 447–453

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Effects of silicon (Si) on arsenic (As) accumulation and speciation in rice (Oryza sativa L.) genotypes with different radial oxygen loss (ROL) Chuan Wu a, Qi Zou a, Shengguo Xue a,⇑, Jingyu Mo a, Weisong Pan b, Laiqing Lou c, Ming Hung Wong d a

School of Metallurgy and Environment, Central South University, Changsha 410083, PR China College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha 410128, PR China c College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, PR China d Consortium on Health, Environment, Education and Research (CHEER), Hong Kong Institute of Education, Tai Po, Hong Kong Special Administrative Region b

h i g h l i g h t s  ROL were higher in hybrid rice genotypes than that in conventional genotypes.  Si addition significantly increased straw biomass.  Si addition significantly reduced shoot total As and inorganic As concentrations.  Si addition decreased more As in lower ROL genotype than higher ROL genotype.  Si fertilization increased DMA concentrations.

a r t i c l e

i n f o

Article history: Received 27 January 2015 Received in revised form 15 June 2015 Accepted 25 June 2015 Available online 11 July 2015 Keywords: Arsenic Radial oxygen loss (ROL) Rice Silicon fertilization

a b s t r a c t Arsenic (As) contamination of paddy soils has adversely affected the health of millions of people those consuming rice for staple food. The present study was aimed at investigating the effects of silicon (Si) fertilization on As uptake, speciation in rice plants with different radial oxygen loss (ROL). Six genotypes were planted in pot soils under greenhouse conditions until late tillering state. The results showed that the rates of ROL were higher in hybrid rice genotypes varying from 19.76 to 27 lmol O2 g1 root dry weight h1 than that in conventional indica rice genotypes varying from 9.55 to 15.41 lmol O2 g1 root dry weight h1. Si addition significantly increased straw biomass (p < 0.005), but with no significant effects on root biomass. Si fertilization significantly reduced shoot and root total As concentrations (p < 0.001) in six genotypes grown in 40 mg As/kg soil. Si addition decreased the inorganic As in shoots of ‘Xiangfengyou-9’ with lower ROL and ‘Xiangwanxian-12’ with higher ROL by 31% and 25% respectively and had the tendency to increase DMA concentrations. It is potential to reduce As contamination of rice efficiently by combining Si fertilization and selecting genotypes with high radial oxygen loss. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Arsenic (As) has been documented as a human carcinogen (Zhu et al., 2008a). As-contaminated ground water has been used as drinking water in some parts of the world, such as Bangladesh and India, resulting in adverse health effects (Meharg, 2004; Geen et al., 2006; Williams et al., 2006). In addition, irrigation with As-contaminated water gave rise to high levels of As in agricultural soils, especially paddy soils (Ullah, 1998; Alam and Satter, 2000), affecting different crops, such as vegetables, wheat and rice (Roychowdhury et al., 2002; Meharg, 2004; Norra et al., 2005). ⇑ Corresponding author. E-mail address: [email protected] (S. Xue). http://dx.doi.org/10.1016/j.chemosphere.2015.06.081 0045-6535/Ó 2015 Elsevier Ltd. All rights reserved.

Rice is a staple food of about 3 billion people all over the world, predominantly in Asia (Stone, 2008). Grown in As-contaminated paddy soils, rice will assimilate As from soils and accumulate As in grains, contributing to a major As exposure pathway to human beings (Meharg, 2004; Stone, 2008; Zhu et al., 2008b). Understanding the processes involved in As tolerance and uptake in rice is of importance for mitigating the risks caused by As contamination in rice grains. Arsenic compounds exist in rice dominantly as arsenate (As(V)), arsenite (As(III)), monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) (Aurillo et al., 1994; Williams et al., 2005; Zhu et al., 2008b). Inorganic As including As(V) and As(III) are generally considered to be more toxic than organic arsenic, and the toxic order of As species is as follows: As(III) > As(V) > MMA > DMA

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(Schoof et al., 1999; Abedin et al., 2002a; Meharg and Hartley-Whitaker, 2002). There are two main mechanisms reported of inorganic As entry into rice roots, which are related to As speciation. It has been reported that As(III), a silicic acid analog, is taken up by roots via silicic acid transport systems (Ma et al., 2008; Chen et al., 2012), and that As(V), a phosphate analog, is assimilated by phosphate transport proteins (Abedin et al., 2002b). Moreover, MMA is considered to be mediated by the silicic acid transporter Lsi1 (Li et al., 2009). The transport pathway of DMA needs to be further investigated. Due to the fact that As shares the same transport systems with Silicon (Si), there exists a competition relationship between As and Si (Bogdan and Manfred, 2008). A hydroponic culture experiment showed that the increase of external Si concentration decreased root and shoot As concentrations and total As uptake by rice seedlings (Guo et al., 2005). Guo et al. (2007) reported that both external and internal Si inhibited the uptake of As in rice. Moreover, a greenhouse study showed that the application of Si to soil decreased total As concentration of rice straw and grains by 78% and 16% respectively, and increased organic As in grains (Li et al., 2009). It is reported that increasing Si concentration in soil pore-water increased Si concentrations in rice straws and husks and decreased As accumulation in grains (Seyfferth and Fendorf, 2012). The addition of Si ameliorated As induced oxidative stress in rice seedlings by lowering As accumulation and improving antioxidant and thiolic system (Preeti et al., 2013). Adaptation of plants in waterlogged soils is the production of numerous adventitious roots (Smirnoff and Crawford, 1983; Blom and Voesenek, 1996; Visser et al., 1996). Adventitious roots of wetland plants including rice (Oryza sativa L.) have acclimated to oxygen deficient environment by developing plentiful aerenchyma and induction of a barrier to radial oxygen loss (ROL), defined as the transfer of oxygen from aerenchyma to the rhizosphere (Armstrong, 1971, 1979; Colmer, 2003a; Deng et al., 2009). ROL are essential for the detoxification of phytotoxins, including Fe2+, Mn2+, and H2S, S2, HS, and organic acids (McDonald et al., 2001; Armstrong and Armstrong, 2005). It is reported that ROL of roots was relevant to As tolerance and accumulation in rice (Mei et al., 2009; Deng et al., 2010; Wu et al., 2011). Our previous study showed that rice genotypes with higher rates of ROL inclined to accumulate less As concentrations in rice grains (Wu et al., 2011). Previous investigations mainly focused on the effects of ROL on As tolerance and uptake (Mei et al., 2009; Wu et al., 2011) and the effects of Si on As uptake by rice plants (Li et al., 2009; Seyfferth and Fendorf, 2012; Preeti et al., 2013), but there is a lack of information concerning the effects of Si on As accumulation and speciation of rice genotypes with different ROL. Does silicon (Si) affect As uptake and speciation in rice genotypes with different ROL? The present study aims to investigate the effects of Si on: (i) root and straw biomass of rice plants treated with As; (ii) As accumulation in rice plants with different ROL; and (iii) As speciation in rice plants with different ROL. 2. Materials and methods

major elements including N, P, K, Mg, Ca and trace elements such as Fe, Mn, Cu, Zn, Mo, and B. The solution pH was adjusted to 5.6 with KOH and HCl. The nutrient solution was refreshed every 5 days. After 15 days, agar (0.1%, w/v) was added in the nutrient solution to imitate the stagnant condition in waterlogged soil, as dilute agar could prevent convection between atmosphere and solution (Wiengweera et al., 1997). All seedlings were cultured for 30 d before measuring radial oxygen loss (ROL) of the entire root. Vessels were randomly arranged in a greenhouse at a temperature of 25/20 °C day/night, with natural light and a day/night photoperiod of 12/12 h, and with relative humidity of 70%. 2.2. Measurement of ROL of entire rice root The radial oxygen loss of entire rice roots was quantified colorimetrically using a titanium(III) citrate buffer method (Kludze et al., 1994), which has been described in detail in our previous studies (Mei et al., 2009; Wu et al., 2011). The released O2 was determined by extrapolation of the absorbance to a standard curve, obtained by previous Ti3+-citrate solution. The ROL rates were calculated with the following formula (Kludze et al., 1994).

ROL ¼ cðy  zÞ where ROL = radial oxygen loss, in lmol O2 plant1 d1; c = initial volume of Ti3+-citrate added to each test tube, in L; y = concentration of Ti3+-citrate solution of control (without plant), in lmol Ti3+/L; z = concentration of Ti3+-citrate solution after 6 h with plants, in lmol Ti3+/L. The rate of ROL (lmol O2 g1 dry weight d1) was calculated as (Wu et al., 2013):

Rate of ROL ¼ cðy  zÞ=G where the rate of ROL is rate of radial oxygen loss, lmol O2 g1 dry weight d1; G is the root dry weight, in g. 2.3. Pot experiment Seeds of six rice genotypes were germinated and cultured in nutrient solution for 30 d (Yoshida et al., 1976). The soils used in this pot trial were the surface (1–20 cm depth) soils of a paddy field located at the Central South University. It is a sandy soil which had a pH of 6.6 and contained 9.4 mg/kg total As. The soils were then air-dried, milled and sieved through a 2-mm sieve. Mineral nutrients (P as CaH2PO4H2O at 0.15 g/kg P2O5, K as KCl at 0.2 g/kg K2O and N as CO(NH2)2 at 0.2 g/kg N) (Wu et al., 2011) were mixed with the soils for rice seedling growth. Arsenate was added as Na2HAsO47H2O at 40 mg As /kg soils and Si was added as SiO2 gel at 20 g SiO2/kg soil (Li et al., 2009). Six rice genotypes were grown in four treatments (SiAs, +SiAs, Si+As and +Si+As). After the soils had been equilibrated for 2 weeks, 5 kg of the soils were placed in a polyethylene pot (18 cm diameter, 24 cm height) for plant growth. Three seedlings were planted per pot with three replicates per treatment. All plants were randomly arranged in the same greenhouse and harvested until tillering stage (90 days after rice seedling germination).

2.1. Hydroponic experiment 2.4. Analysis of total As and As speciation Six genotypes of rice (O. sativa L.) were selected in this experiment: Shengyou 9586 (‘SY-9586’), Fengyuanyou 299 (‘FYY-299’), Xiangfengyou 9 (‘XFY-9’), T-you207 (‘TY-207’) Xiangwanxian 17 (‘XWX-17’) and Xiangwanxian 12 (‘XWX-12’). Seeds were obtained from Hunan Agricultural University. All seeds were sterilized with 30% H2O2 for 15 min and washed thoroughly with deionized water. They were then germinated in Petri dishes, each containing a piece of moist filter paper. After two days, the germinated seeds were transplanted to nutrient solution (Yoshida et al., 1976), containing

Until late tillering state, plant samples were harvested and washed with deionized water. Roots and shoots were separated, oven-dried at 70 °C until reaching a constant weight, then ground for total As analysis. Shoot and root samples were freeze-dried for As speciation analysis. Sample treatments followed those described by Zhu et al. (2008b). Milled samples (0.5 g) were weighed into conical flasks and digested with 5 mL of concentrated nitric acid (HNO3). The

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digestion solution was stood overnight at laboratory temperature (about 25 °C) and then heated using a hot block at 120 °C until the extracts became clear. The digested extracts were made up to a volume of 20 mL with ultrapure water. The standard reference material of bush branches and leaves (GBW07603) was used to verify the accuracy of the analyses with recoveries of 80.4–89.5%. The extracts were analyzed for total arsenic by hydride generation atomic fluorescence spectrometry (HG-AFS, AFS-8230, Beijing Jitian Instruments Co., China). The method of measuring As speciation has been described by Zhu et al. (2008b) and Shi et al. (2013). Milled samples (1.0 g) were weighed into 50 mL centrifuge tubes and extracted with 20 mL 1% nitric acid (HNO3) at 95 °C for 1.5 h. After extraction, samples were cooled to room temperature and centrifuged at 5000 r/min for 10 min. The supernatant was filtered through 0.22 lm membrane filter. As speciation was determined by high performance liquid chromatography – hydride generation atomic fluorescence spectrometry (HPLC-HG-AFS) (Shimadzu LC-15C Suzhou Instruments Co., China and HG-AFS, AFS-8230, Beijing Jitian Instruments Co., China). 2.5. Statistical analysis Data were analyzed using the statistical package SPSS 19.0 and Excel 2007 for Windows. Figures were drawn with the software of Origin 8.0. 3. Results and discussion 3.1. Effects of Si on the growth of rice genotypes with different ROL Table 1 shows the results of statistical analysis of ROL with six genotypes. The total ROL of six rice genotypes used in this investigation was significantly different (p < 0.01) in genotypes and types, which was ranked in the order of: XFY-9 < SY-9586 < FYY-299 < TY-207 < XWX-17 < XWX-12. The ROL of indica genotypes varied from 19.76 to 27 while hybrid genotypes from 9.55 to 15.41 lmol O2 g1 root dry weight h1 (Table 1). The results of shoot and root biomass of six genotypes in different treatments are shown in Table 2. There were significant differences in biomass of shoot (p < 0.001) and root (p < 0.05) among genotypes (Table 2). In +SiAs treatment, shoot biomass was significantly higher (p < 0.005) than that in SiAs treatment, but +Si treatment had no significant effects on root biomass (Table 2). As addition (+As) did not impose significant effects on shoot and root biomass, compared with As treatment, regardless of Si treatment. There exist several factors influencing ROL of rice roots, such as stagnant and aerated conditions (Colmer et al., 1998) and different genotypes (Wu et al., 2011). In the present study, conventional indica rice possessed higher rates of ROL than that of hybrid rice Table 1 Characteristics of genotypes used in this investigation with ROL (lmol O2 g1 DW h1) of six rice genotypes subjected to stagnant nutrient solution for 30 d (data are means ± SD, n = 3). Values followed by different letters on the same column are significantly different (p < 0.05; least significant difference (LSD) test). Genotype

Type

Origin

ROL

SY-9586 FYY-299 XFY-9 TY-207 XWX-17 XWX-12 Genotypes (G) Subspecies (S)

Hybrid Hybrid Hybrid Hybrid Indica Indica

Hunan, China Hunan, China Jiangxi, China Hubei, China Hunan, China Hunan, China

10.83 ± 0.73a 15.31 ± 1.15b 9.55 ± 0.85a 15.41 ± 1.33b 19.76 ± 1.74c 27.00 ± 1.29d p < 0.01 p < 0.01

Table 2 Biomass (g/plant, dry weight) of rice roots and shoots of six genotypes subjected to SiAs, +SiAs, Si+As and +Si+As treatments for 60 days (data = mean ± SD, n = 3). Genotype

SY-9586 FYY-299 XFY-9 TY-207 XWX-17 XWX-12

Treatment

Si +Si Si +Si Si +Si Si +Si Si +Si Si +Si

Analysis of variance Genotype (G) As Si G  As G  Si As  Si G  As  Si

Shoot

Root

As

+As

As

+As

1.62 ± 0.04 1.90 ± 0.41 2.24 ± 0.40 3.30 ± 0.15 2.82 ± 0.85 3.25 ± 0.84 1.68 ± 0.47 2.97 ± 0.65 1.54 ± 0.41 2.24 ± 0.83 2.48 ± 0.41 2.07 ± 0.07

1.95 ± 0.29 2.22 ± 0.42 3.01 ± 0.28 2.83 ± 0.33 2.77 ± 0.34 2.73 ± 0.45 2.56 ± 0.23 2.67 ± 0.65 1.98 ± 0.45 1.92 ± 0.22 1.58 ± 0.12 2.35 ± 0.75

0.46 ± 0.15 0.68 ± 0.25 0.32 ± 0.07 0.55 ± 0.11 0.51 ± 0.12 0.47 ± 0.09 0.38 ± 0.07 0.63 ± 0.15 0.48 ± 0.11 0.61 ± 0.25 0.69 ± 0.03 0.61 ± 0.09

0.59 ± 0.20 0.41 ± 0.04 0.52 ± 0.12 0.40 ± 0.03 0.60 ± 0.23 0.41 ± 0.06 0.50 ± 0.15 0.43 ± 0.12 0.43 ± 0.10 0.46 ± 0.07 0.60 ± 0.07 0.61 ± 0.09

p < 0.001 NS p < 0.005 p < 0.01 NS NS p < 0.05

p < 0.05 NS NS NS NS p < 0.005 NS

when grown in the same hydroponic condition (Table 1). It has been reported that the rates of ROL differed among rice genotypes (Mei et al., 2009; Wu et al., 2011; Li et al., 2013). Wu et al. (2011) reported that there were significant differences in ROL among different genotypes, but there were neither significant difference between indica and japonica subspecies, nor between upland and paddy rice. However, our results found that there were significant differences in ROL between conventional and hybrid genotypes (p < 0.01). This may be due to the variation of aerenchyma or the barrier to ROL in rice roots. Aerenchyma in rice provides a low resistance internal pathway for movement of O2 within the roots (Colmer, 2003b). Effective longitudinal diffusion of O2 in roots increases as the volume of aerenchyma becomes large, especially if roots are relatively thick (Armstrong, 1979). In addition to extensive aerenchyma, the roots of wetland species including rice contain a barrier to ROL in the basal zones (Visser et al., 2000; McDonald et al., 2002). These traits act synergistically to enhance O2 diffusion to root tip and thus enable an aerobic rhizosphere around root tip (Armstrong, 1979). Silicon had not been considered an essential element, but it has been increasingly regarded as a ‘‘quasi-essential element’’ for plant growth (Epstein, 1994; Epstein and Bloom, 2005). It was found that Si treatment significantly increased shoot biomass in the pot experiment (Table 2). It has been shown in the previous studies that Si addition not only increased shoot dry weight significantly in the hydroponic experiment (Guo et al., 2005) but increased grain and straw yield significantly in the pot experiment (Li et al., 2009). Our results also showed that Si addition increased shoot biomass of six genotypes compared with the Si treatment, which is consistent with the findings of Guo et al. (2005) and Li et al. (2009). 3.2. Effects of Si on total As concentrations in rice genotypes with different ROL The concentrations of As were under detection limits in rice plants (including the parts of shoot and root) grown in the As treatment (results are not shown). Rice genotypes had significant influences (p < 0.001) on shoot and root total As concentrations (Table 3). The shoot As concentration of six genotypes grown in Si+As treatment ranged from 2.13 to 3.82 mg/kg with the ranking of XFY-9 > SY-9586 > FYY-299 > TY-207 > XWX-12 > XWX-17,

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Table 3 Arsenic concentrations (mg/kg, dry weight) in roots and shoots of six rice genotypes subjected to different treatments for 60 days (data = mean ± SD, n = 3). Genotype

Treatment

As in shoot

As in root

SY-9586

Si,+As +Si,+As Si,+As +Si,+As Si,+As +Si,+As Si,+As +Si,+As Si,+As +Si,+As Si,+As +Si,+As

2.90 ± 0.18 2.40 ± 0.13 2.34 ± 0.13 2.06 ± 0.09 3.82 ± 0.27 2.23 ± 0.30 2.33 ± 0.26 2.02 ± 0.23 2.13 ± 0.18 2.01 ± 0.10 2.20 ± 0.24 1.66 ± 0.15

90.40 ± 8.91 32.98 ± 10.8 111.6 ± 23.3 104.9 ± 21.9 86.83 ± 7.27 50.60 ± 11.5 85.14 ± 10.0 57.54 ± 10.5 147.8 ± 32.3 86.39 ± 18.9 129.2 ± 28.6 49.20 ± 17.7

FYY-299 XFY-9 TY-207 XWX-17 XWX-12

Analysis of variance Genotypes (G) Si G  Si

p < 0.001 p < 0.001 p < 0.001

p < 0.001 p < 0.001 p < 0.05

while the root As concentration ranged from 85.14 to 147.8 mg/kg with the ranking of TY-207 < XFY-9 < SY-9586 < FYY-299 < XWX-12 < XWX-17 (Table 3). It is worthwhile mentioning that the shoot total As concentration in XFY-9 (3.82 mg/kg) with the lowest rate of ROL was higher than that in XWX-12 (2.2 mg/kg) with the highest ROL, while the root total As concentration in XFY-9 (86.83 mg/kg) was 42.4 mg/kg lower than that of XWX-12 (129.23 mg/kg) in Si+As treatment (Table 3). It is showed in Table 3 that Si addition (+Si+As) significantly reduced shoot and root total As concentrations (p < 0.001) when compared to Si+As treatment. The shoot total As concentrations of six genotypes ranged from 1.66 to 2.4 mg/kg with the treatment of +Si+As and from 2.13 to 3.82 mg/kg with the treatment of Si+As (Table 3). The root total As concentrations of six genotypes ranged from 32.98 to 104.92 mg/kg in the treatment of +Si+As and from 85.14 to 147.8 mg/kg in the treatment of Si+As (Table 3). Si addition significantly decreased As concentrations in shoots of four genotypes including XFY-9, SY-9586, FYY-1 299 and XWX-12 (p < 0.01), but decreased As concentration in shoots of TY-207 and XWX-17 insignificantly (Fig. 1). The shoot total As concentrations of XFY-9, SY-9586, FYY-299 and XWX-12 in the treatment of +Si+As were decreased by 42%, 17%, 12% and 25% respectively, compared to the treatment of Si+As (Fig. 1a). It is reported that wetland plants with higher rates of ROL possessed higher concentrations of As on root surfaces (Li et al., 2011), and rice genotypes with higher ROL had a strong ability to reduce shoot As accumulation (Wu et al., 2011; Mei et al., 2012). The present results showed that genotypes with higher ROL accumulated less As contents in shoot than genotypes with lower ROL. However, the results showed that rice plants with higher ROL contained higher As concentrations in root compared with the genotypes with lower ROL (Fig. 1b). Due to the formation of Fe plaque on root surfaces, arsenic has been thought to be absorbed on Fe plaque, which is enhanced by genotypes with higher ROL (Wu et al., 2012). Therefore, our results could be explained by the fact that without the extraction of iron plaque on the surface of rice roots in this investigation, higher total As concentrations of roots were observed on genotypes with higher ROL rather than genotypes with lower ROL. Bogdan and Manfred (2008) suggested that soils with a high level of plant available Si resulted in low As concentrations in rice plant and that application of Si to soils may decrease the As concentrations in rice. Furthermore, Li et al. (2009) reported that the total As in rice straw were found to be decreased by 78% when adding Si fertilization to soils. Our results showed that in soils with the

Fig. 1. Total arsenic (As) concentrations in shoots (a) and roots (b) of six rice (Oryza sativa) genotypes (SY-9586, FYY-299, XFY-9, TY-207, XWX-17 and XWX-12) in silicon (+Si) treatment and no silicon (Si) treatment, exposed to 40 mg As/kg soils (data = mean ± SD; n = 3).

+Si+As treatment, the total As concentrations in rice shoot of XFY-9, SY-9586, FYY-299, TY-207, XWX-17 and XWX-12 were decreased compared to the Si+As treatment. This is also consistent with the results of Li et al. (2009) and Seyfferth and Fendorf (2012). Because of a shortage of O2, As(III) is more likely the predominant As species in waterlogged soil (Fitz and Wenzel, 2002; Pan et al., 2014). There may exist a competing relationship between As(III) and Si for the uptake into rice roots (Ma et al., 2008). Si addition affected the uptake of As(III) into rice roots and as a result decreased the total As in shoot and root. The present study also found that the shoot As concentration of genotype with lower ROL (‘XFY-9’) was decreased by 42% while that of higher ROL genotype (‘XWX-12’) was decreased by 25% with the addition of 40 mg Si/kg (Fig. 1a). This suggested that Si addition decreased more As concentrations in shoots of lower ROL genotype (‘XFY-9’) than that of higher ROL genotype (‘XWX-12’). The reason may be due to the fact that Si addition increased the barrier against ROL in rice roots by attributing to a suberized exodermis and lignified sclerenchyma cells which may inhibit the process of ROL (Kotula and Steudle, 2008; Fleck et al., 2011). Rice roots develop a barrier (Armstrong, 1979). This barrier against ROL is present in the basal parts of the root (Colmer, 2003b). In general, densely packed cells, suberin deposition, and lignification in the outer cell

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C. Wu et al. / Chemosphere 138 (2015) 447–453 Table 4 Proportions of arsenic species in shoots for 3 genotypes using HPLC-AFS-HG measurement. Genotype

Treatment

Total As (mg/kg)

Inorganic As (mg/kg)

DMA (mg/kg)

MMA (mg/kg)

Inorganic Asa (%)

Recoveryb (%)

Fengyuanyou299

Si +Si Si +Si Si +Si

2.34 ± 0.13 2.06 ± 0.09 3.82 ± 0.27 2.23 ± 0.30 2.20 ± 0.24 1.66 ± 0.15

1.85 ± 0.25 1.41 ± 0.13 2.96 ± 0.11 2.05 ± 0.08 2.06 ± 0.06 1.54 ± 0.13

nd 0.06 ± 0.02 0.08 ± 0.01 0.10 ± 0.01 0.08 ± 0.007 0.13 ± 0.04

0.07 ± 0.03 0.03 ± 0.008 0.05 ± 0.01 0.07 ± 0.02 0.05 ± 0.01 0.06 ± 0.006

96 94 96 92 94 89

82 73 81 97 99 104

Xiangfengyou9 Xiangwanxian12 a b

Inorganic arsenic (%) = [(inorganic As]/(species sum)]  100. Recovery (%) = [(species sum]/(total As)]  100.

Fig. 2. Concentrations of arsenic speciation [As(III), As(V), MMA and DMA] in shoots of FYY-299, XFY-9 and XWX-12 in soils added with 40 mg As/kg with Si (+Si) or without Si (Si) addition.

layers are thought to serve as barrier formation (Sorrell, 1994; Armstrong et al., 2000). In rice roots, the barrier against ROL is attributed to a suberized exodermis with casparian bands and lignified sclerenchyma cells (Kotula and Steudle, 2008). Fleck et al. (2011) reported that silicic acid supply clearly enhanced the formation of casparian bands in the exodermis and endodermis. Hence, Si addition may weak ROL and the degree of effects of ROL on As uptake in rice, and higher ROL genotypes may be weakened more. As a result, As concentrations in shoots of lower ROL genotype (‘XFY-9’) was decreased more than that of higher ROL genotype (‘XWX-12’) after Si was added. 3.3. Effects of Si on As speciation in rice genotypes with different ROL Three genotypes including XFY-9 (the lowest ROL), XWX-12 (the highest ROL) and FYY-299 (mediate ROL) were selected for As speciation analysis. In our study, inorganic As was the predominant species in shoot from 89% to 96% (Table 4) and As(III) concentrations was generally higher than As(V) concentrations in above-ground parts of rice (Fig. 2). The shoot inorganic As concentration of three genotypes ranged from 1.85 to 2.96 mg/kg in the treatment of Si+As, and from 1.41 to 2.05 mg kg1 in the treatment of +Si+As (Table 4). The result showed that the shoot DMA contents of XFY-9, FYY-299 and XWX-12 ranged from 0 to 0.08 mg/kg in Si+As treatment and from 0.06 to 0.13 mg kg1 in +Si+As treatment, and that the shoot MMA contents of XFY-9, FYY-299 and XWX-12 ranged from 0.05 to 0.07 mg/kg in Si+As treatment and ranged from 0.03 to 0.07 mg/kg in +Si+As treatment (Table 4).

The shoot inorganic As concentrations in the rice genotype with lower ROL (‘XFY-9’ 2.96 mg/kg in Si+As and 2.05 mg/kg in +Si+As treatment) were higher than those in the genotype with higher ROL (‘XWX-12’ 2.06 mg/kg in Si+As and 1.54 mg/kg in +Si+As treatment), while there were no significant differences in DMA concentration between ‘XFY-9’ (0.08 mg/kg in Si+As and 0.1 mg/kg in +Si+As treatment) and ‘XWX-12’ (0.08 mg/kg in S+As and 0.13 mg/kg in Si+As treatment) (Table 4). The shoot inorganic As concentrations of XFY-9 and XWX-12 in +Si+As treatment were decreased by 31% and 25% respectively, when compared to the treatment of Si+As (Fig. 2). Si accumulated in plants helps to resist biotic and abiotic stresses (Ma, 2004; Ma and Yamaji, 2006). A previous investigation reported that Si decreased inorganic As accumulation in rice grain (Li et al., 2009). It has been reported that As(III) shares the same pathway with silicic acid into rice roots (Ma et al., 2008). The present study showed that the shoot inorganic As concentrations of three genotypes in +Si+As treatment were decreased from 24% to 31% compared with Si+As treatment (Table 4 and Fig. 2). The results also showed that As(III) concentration was higher than As(V) in shoot (Table 4). It may be the fact that As(III) is the main As species in the rhizosphere of waterlogged soils (Fitz and Wenzel, 2002; Pan et al., 2014). Furthermore, compared with the treatment of Si+As, the shoot inorganic As concentrations of lower ROL rice genotype (‘XFY-9’) and higher ROL genotype (‘XWX-12’) in +Si+As treatment were decreased by 31% and 25% respectively. This trend is consistence with the results that Si addition decreased total As concentrations in shoots of XFY-9 and XWX-12 by 42% and 25% respectively (Figs. 1a and 2). Because of sharing silicic acid transport systems, Si addition inhibited the uptake of As(III) in rice (Ma et al., 2008; Chen et al., 2012). Consequently, the effects of Si on total As uptake in rice genotypes with different ROL could be explained by the effects of Si on inorganic As uptake, especially As(III), which is the predominant As species in rice rhizosphere soils (Fitz and Wenzel, 2002; Pan et al., 2014). As no evidence has been reported that rice plants are able to methylate As, methylated As species including DMA and MMA in rice plant are more likely derived from soils and rice rhizosphere in which microbial methylation occurs (Lomax et al., 2011; Zhao et al., 2013; Jia et al., 2013). With the lack of methylated ability in rice plants, DMA and MMA must have been taken up from rice rhizosphere in soils. Si probably may increase the uptake of DMA and MMA into rice roots or facilitate the methylation process in rhizosphere soils. It has been reported that MMA is mediated by the silicic acid transporter Lsi1 (Li et al., 2009), but the mechanism of DMA uptake in rice is a complicate process and needs to be further investigated.

4. Conclusion Six rice genotypes were selected to investigate the effects of Si on As accumulation and speciation in rice plants with different radial oxygen loss (ROL) under greenhouse conditions. The results

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revealed that indica rice genotypes (19.76–27 lmol O2 g1 root dry weight h1) had higher rates of ROL than that of hybrid rice genotypes (from 9.55 to 15.41 lmol O2 g1 root dry weight h1). Regardless of As treatment, +Si treatment significantly increased shoot biomass (p < 0.005) compared with Si treatment. The shoot and root total As concentrations in +Si+As treatment were significantly lower than that in Si+As treatment (p < 0.001). The shoot total As in rice genotypes with higher ROL (‘XWX-17’ 2.13 mg/kg; ‘XWX-12’ 2.2 mg/kg) were lower than that in rice genotypes with lower ROL (‘SY-9586’ 2.9 mg/kg; ‘XFY-9’ 3.82 mg/kg). Moreover, As concentrations in the rice genotype with low ROL (‘XFY-9’ 42%) grown in +Si+As were decreased more substantially than that of the genotype with high ROL (‘XWX-12’ 25%), compared with Si+As treatment. Furthermore, the shoot inorganic As concentrations of ‘XFY-9’ and ‘XWX-12’ were decreased by 31% and 25% respectively due to the addition of Si, which demonstrated the effects of Si on the shoot total As. It is worthwhile to mention that Si addition decreased inorganic As concentrations including As(III) and As(V) and increased the concentration of organic As especially DMA.

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