The relationship of root porosity and radial oxygen loss on arsenic tolerance and uptake in rice grains and straw

Environmental Pollution 157 (2009) 2550–2557

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The relationship of root porosity and radial oxygen loss on arsenic tolerance and uptake in rice grains and straw X.Q. Mei a, Z.H. Ye a, *, M.H. Wong b, ** a b

State Key Laboratory for Bio-control, and School of Life Sciences, Sun Yat-sen (Zhongshan) University, 135 Xin Gang West Road, Guangzhou 510275, PR China Croucher Institute for Environmental Sciences, and Department of Biology, Hong Kong Baptist University, Kowloon Tong, Hong Kong SAR, PR China

Rice with high radial oxygen loss and porosity of root accumulates low As in grains.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 18 September 2008 Received in revised form 24 February 2009 Accepted 28 February 2009

The correlations among arsenic (As) accumulation in grains and straw, rates of radial oxygen loss (ROL), and porosity of roots using 25 rice cultivars were investigated based on two pot experiments: (1) soil with addition of 100 mg As kg1 for analysis of As in grains and straw, and (2) deoxygenated solution for analyzing rates of ROL and porosity of roots. The results showed that there were great differences in grain As (0.71–1.72 mg kg1) and straw As (15.6–31.7 mg kg1), rates of ROL (7.40–13.24 mmol O2 kg1 root d.w. h1), and porosity (20.91–33.08%) among the cultivars. There were significant negative correlations between As in grains or straw and ROL and porosity, and significant positive correlations between rates of ROL and porosities, respectively. Rice cultivars with high porosities tended to possess higher rates of ROL, and had higher capacities for limiting the transfer of As to aboveground tissues. Ó 2009 Elsevier Ltd. All rights reserved.

Keywords: Oryza sativa L. Accumulation Aerenchyma Arsenic Radial oxygen loss (ROL) Root porosity

1. Introduction Arsenic (As) has imposed adverse environmental problems that have received worldwide attention, especially As contamination of paddy soils throughout southeast Asia (Meharg, 2004; Juhasz et al., 2006; Stone, 2008). Paddy rice (Oryza sativa L.) grown on Ascontaminated soils usually possesses high As levels in shoot (including grains) (Xie and Huang, 1998; Abedin et al., 2002; Zhu et al., 2008a). Rice grains collected in As-contaminated districts of Bangladesh had concentrations that were 10-fold higher than the ‘normal’ level of about 0.2 mg g1 As (Meharg and Rahman, 2003). Besides As contamination, grain yield is also decreased when rice is grown in paddy soils contaminated by As (Akter et al., 2005). Since it is not cost-effective to remediate these paddy soils, and rice is particularly susceptible to As accumulation compared to other cereals as it is generally grown under flooded conditions where As mobility is high (Zhu et al., 2008b), the only option is to select and breed rice cultivars with low accumulation and high tolerance of As (Meharg, 2004), in order to ensure safe crop production, even under on As-contaminated sites. * Corresponding author. Tel.: þ86 20 39332858. ** Corresponding author. Tel.: þ86 852 34117746; fax: þ86 852 34117743. E-mail addresses: [email protected] (Z.H. Ye), [email protected] (M.H. Wong). 0269-7491/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.envpol.2009.02.037

Some rice genotypes accumulated significantly lower As levels in their grains than others although they grew in the same Ascontaminated paddy soil (Williams et al., 2005; Liu et al., 2006). Currently, the mechanisms involved in As accumulation by rice are still poorly understood, henceforth it is crucial to understand the internal/external factors of rice that may play important roles in As uptake and tolerance in order to select/breed the rice varieties with high tolerance and low accumulation of As in grains. Wetland plants commonly possess aerenchyma tissues containing enlarged gas spaces (Evans, 2003). Porosity (% gas space per unit tissue volume) in plant tissues results from the intercellular gas-filled spaces which form a constitutive part of the development. It has been reported that the porosity of root differs markedly among rice varieties, and can be further enhanced by the formation of aerenchyma, flooding or deoxygenated nutrient solution (Colmer, 2003a; Colmer et al., 2006). In roots, O2 is required for respiration to provide sufficient energy for growth, maintenance and nutrient uptake. However, significant amounts of O2 supplied via aerenchyma to roots in anaerobic substrates may diffuse into the rhizosphere, a process termed radial oxygen loss (ROL) (Armstrong, 1979). ROL from root to the rhizosphere is considered to be essential for detoxification of phytotoxins such as Fe2þ, Mn2þ, H2S, S2, HS and organic acids by direct oxidation or by oxidizing aerobic microorganisms maintained in the rhizosphere region (Armstrong and

X.Q. Mei et al. / Environmental Pollution 157 (2009) 2550–2557

Armstrong, 2005). A number of wetland plants, including rice, are known to form iron (Fe) plaque on their roots by oxidizing Fe2þ to Fe3þ, resulting from the oxidizing activity of plant roots and associated microorganisms (Crowder and St-Cyr, 1991; Ye et al., 1997). Recently, it has been discovered that Fe plaque formed on root surface has an important effect on As accumulation by rice (Chen et al., 2005). The oxidizing capacity and ROL of plant root are considered the most important biotic factor controlling Fe plaque formation (Mendellsohn et al., 1995). It was reported that flood tolerance was positively related with ROL or aerenchyma and porosity of plants grown in waterlogged soil (Armstrong, 1979; Chabbi et al., 2000). However, so far the effects of ROL on As accumulation and tolerance by rice are uncertain. The objectives of this study were therefore to investigate the relationships between the rate of ROL, porosity of roots, levels of As accumulation in aboveground parts (grains and straw), and As tolerance of rice plant among different cultivars. 2. Materials and methods 2.1. Pot trial under waterlogged condition Seeds of 25 rice (O. sativa L.) cultivars [Basmati 370, C039, Fengaizhan, Fenghuazhan, Ganxiangnuo, Guangluai, IR 56, Jingxian 89, Khazar, Molixinzhan, Qiguizao, Qihuangzhan, Qishanzhan, Sanerai, Sanhuangzhan, Sanluzhan 7, Shengtai 1, Suyunuo, Texianzhan 13, Zhenguiai, Zhong 4188, Zhonghua 11, Zhongerruanzhan, Yuefengzhan, Yuexiangzhan] were obtained from the Rice Research Institute in Guangdong Province and Professor Chuanguo Li (Principal Breeder), and Professor Guiquan Zhang, Academy of Agriculture, South China Agricultural University. All cultivars belonged to Indica, except for Khazar, Suyunuo and Zhonghua 11 which belonged to Japonica. The control soil (average As concentration: 9.10 mg kg1) collected from a paddy field (0–20 cm depth) at the campus of South China Agricultural University was amended with As (0 and 100 mg As kg1 as Na2HAsO4 $ 7H2O, As(V)), thoroughly mixed and allowed to equilibrate for 3 weeks, and then placed in plastic pots (7.5 cm diameter and 14 cm high, 1 kg soil pot1). The soil used for the pot experiment contained 11.2% organic matter, 1.30 g kg1 total N and 1.14 g kg1 total P, respectively, with a pH value of 5.98. The pots were painted black and had no drainage holes. Seeds were disinfected in 30% H2O2 (w/v) solution for 15 min, followed by thorough washing with deionized water, and then germinated in moist perlites. After 3 weeks, uniform seedlings were selected and transplanted to PVC pots (same size as described above, one plant per pot). There were four replicates for each treatment/cultivar. The plant pots were placed in a greenhouse and arranged in a randomized completed block design during the rice-growing season (from earlyMarch to mid-July). To ensure normal growth and development of rice plants, potassium chloride (KCl) solution (in distilled water) was applied after transplantation of the seedlings to give 28.6 mg K kg1 soil, and nitrogen was supplied as a solution of urea [CO(NH2)2] (in distilled water) in four equal splits to give a total of 76.3 mg N kg1 soil during the growth period. The soil was maintained under flooded conditions (with 2 cm of water above soil surface) during the whole growth period of 95–135 days, which varied among different cultivars. After measuring plant height (panicle top to level of soil in the pot) and tiller numbers, the aboveground portion of each rice plant was harvested at maturity by cutting at 4 cm above the soil, and separated into straw and grain. The samples were washed thoroughly with tap water and deionized water, and then oven-dried at 55  C to constant weight. Oven-dried straw and grain samples were ground separately using a Retschgrinder (Type: 2 mm, Made in Germany), and digested with HNO3/H2O2 below 110  C (Cai et al., 2000). Arsenic concentrations both in straw and grain digests were determined using inductively coupled plasma mass spectrometry (ICP-MS). Blanks and tea standard material (GBW-08303) (China Standard Materials Research Center, Beijing, PR China) were used for quality control. The As recovery rates were 90  10%.

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decreased O2, increased ethylene) better than other methods used to impose rootzone O2 deficiency in solution culture. The pots were placed in a greenhouse and arranged in a completely randomized design, and the solutions were renewed once every 6 days. After 30 days, the plants were used for measurements of ROL and rate of ROL, and root porosity. 2.2.2. Preparation of titanium(III) citrate solutions Stock solutions of the reduced titanium(III) citrate buffer were prepared using commercially available titanium(III) chloride in HCl solution (Aldrich Chemical Co., Milwaukee, WI, USA), as described by Zehnder and Wuhrmann (1976) and Kludze et al. (1993). Deoxygenated water (300 ml) was added to 17.65 g of sodium citrate to give 0.2 M sodium citrate solution. Titanium chloride (1.16 M, 30 ml) was then added to the sodium citrate solution, with pH adjusted to 5.6 by adding saturated sodium carbonate. All preparations were done under N2 atmosphere. 2.2.3. Measurement of radial oxygen loss (ROL) ROL of rice seedlings was determined according to the method described by Zehnder and Wuhrmann (1976) and Kludze et al. (1993). Nutrient solution (40 ml) was poured into each 50 ml test tube and the solution was purged with Ar gas for 1200 s to remove dissolved O2. Rice seedlings, previously carefully washed of any foreign matter, with the base of the plants coated with paraffin, were inserted into the tubes (one per test tube). The roots were completely immersed into the nutrient solution. A 5 ml aliquot of Ti3þ-citrate solution was then injected into each test tube with a plastic syringe, followed immediately by layering the solution surface with 20 mm of paraffin oil to inhibit contamination by atmospheric O2. Control treatments did not contain any plants. All the test tubes were kept at 25  C. After 6 h, the test tubes were gently shaken and solution samples were collected with a syringe through a rubber tubing that had been introduced into the solution alongside the roots. Absorbance of the partly oxidized Ti3þ-citrate solution was measured at 527 nm using a Perkin–Elmer 3 UV/VIS spectrophotometer. Released O2 on the whole-plant root system was determined by extrapolation of the measured absorbance to a standard curve. Net rate of ROL was calculated (Kludze et al., 1993) as: ROL ¼ cðy  zÞ where ROL ¼ radial oxygen loss, mmol O2 plant1 day1; c ¼ initial volume of Ti3þcitrate added to each test tube, L; y ¼ concentration of Ti3þ in solution control (without plant), mmol Ti3þ L1; and z ¼ concentration of Ti3þ in solution after 6 h treatment with plants, mmol Ti3þ in solution plant1 L1. Rate of ROL ¼

cðy  zÞ G

where Rate of ROL ¼ rate of radial oxygen loss, mmol O2 kg1 root d.w. h1; G ¼ root dry weight, kg. 2.2.4. Measurement of root porosity Root porosity (% gas volume/root volume, POR) was measured for adventitious roots of rice by pycnometer method (Jensen et al., 1969; Kludze et al., 1993). About 0.6 g of fresh roots (2–2.5 cm from the root apex) were used for porosity determination. Porosity was calculated using the following formula: Porosity ¼

ðp and vrÞ  ðp and rÞ  100 ðp þ rÞ  ðp and rÞ

r ¼ mass of fresh roots, in g; p ¼ mass of water filled pycnometer, in g; p and r ¼ mass of pycnometer with fresh roots and water, in g; and p and vr ¼ mass of pycnometer with vacuumed roots and water, in g. 2.3. Statistical analysis All results are presented as arithmetic means with standard errors. A statistical comparison of means of plant data was examined with one-way ANOVA followed by the Tukey-HSD test as available in the SPSS statistical package. Correlation coefficient analyses were conducted using Origin 6.0.

3. Results

2.2. Pot trial under deoxygenated nutrient condition

3.1. Growth and biomass of rice

2.2.1. Rice cultivation The same seeds used in the soil pot trials were used in the deoxygenated nutrient experiment. Five days after germination, seedlings were subsequently exposed to 0.25 and 0.5 strength Hoagland nutrient solutions (Hoagland and Amon, 1950) for 7 days and 14 days, respectively, and then uniform seedlings were selected and transplanted to PVC pots (7.5 cm diameter and 14 cm high, one plant per pot) filled with deoxygenated nutrient solution containing 0.1% (w/v) agar. Wiengweera et al. (1997) showed that dilute agar prevents convective movements in the solution so that it may mimic the changes in gas composition found in waterlogged soil (e.g.

A number of visible toxic symptoms, including growth retardation, reduction in tiller, and grain or straw dry weight, associated with As treatment were observed, especially in some varieties (such as: C039, Sanerai, Shengtai 1). Grain and straw biomass (% of control) of 25 rice cultivars grown in soil with the addition of 100 mg As kg1 (As(V)) soil are presented in Fig. 1. Compared with rice grown in control soil, grain and straw biomass of all tested rice

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X.Q. Mei et al. / Environmental Pollution 157 (2009) 2550–2557

Grain biomass (% of control)

100 95 90 85 80 75 70 65 60 55 50 45 Shengtai 1

Sanerai

Sanhuangzhan

Zhong 4188

Yuefengzhan

Texianzhan 13

Ganxiangnuo

C 039

Molixinzhan

Zhenguiai

Guangluai

Fengaizhan

Qishanzhan

Qihuangzhan

Qiguizao

Jingxian 89

Zhongerruanzhan

Khazar

Fenghuazhan

Sanluzhan 7

Suyunuo

Zhonghua 11

Basmati 370

IR 56

Yuexiangzhan

40

Straw biomass (% of control)

100 95 90 85 80

Indica

75

Japonica

70 65 60 55 50 45 Shengtai 1

C 039

Sanerai

Guangluai

Sanhuangzhan

Yuefengzhan

Jingxian 89

Ganxiangnuo

Sanluzhan 7

Fenghuazhan

Texianzhan 13

Zhenguiai

Yuexiangzhan

IR 56

Qiguizao

Zhong 4188

Molixinzhan

Qishanzhan

Fengaizhan

Basmati 370

Zhonghua 11

Khazar

Qihuangzhan

Suyunuo

Zhongerruanzhan

40

Cultivars Fig. 1. Grain and straw biomass (% of control) of rice grown in soil with addition of 100 mg As kg1 (as Na2HAsO4 $ 7H2O) (mean  SE, n ¼ 4).

cultivars were reduced under As treatment, and the reduction (% of control) was significantly different (P < 0.01) among the 25 rice cultivars, ranging from 12.6 (IR 56) to 55.5% (Shengtai 1) for grains, and from 13.8 (Suyunuo) to 56.0% (Shengtai 1) for straw biomass. There were significant (P < 0.01) correlations between grain As dry weight (% of control) and straw As dry weight (% of control) of the rice varieties tested (r ¼ 0.68, n ¼ 25, data not shown). 3.2. Total arsenic in straw and grain The total As concentrations in husked grain and straw of all rice cultivars under both the control soil and soil with addition of 100 mg As kg1 (As(V)) conditions are presented in Table 1. Under the control soil condition, husked grain As concentrations ranged from 0.08 to 0.22 mg kg1, with an average of 0.15 mg kg1. Straw As concentration ranged from 0.59 to 2.52 mg kg1, with an average of 1.53 mg kg1. While under 100 mg kg1 As exposure, husked grain As concentrations ranged from 0.71 to 2.72 mg kg1, with an average of 1.72 mg kg1. Straw As concentration ranged from 15.60 to 31.70 mg kg1, with an average of 22.27 mg kg1.

3.3. Radial oxygen loss and porosity of roots of rice grown in 0.1% agar solution ROL and porosity of the same 25 rice cultivars were measured after being grown in 0.1% agar nutrient solutions for 30 days (Table 1). The results showed that both the rate of ROL and the root porosity of rice were varied due to genetic differences among the cultivars. Rates of oxygen loss ranged from 7.40 to 13.24 mmol O2 kg1 root d.w. h1 (average 9.75 mmol O2 kg1 root d.w. h1), and porosities of rice roots ranged from 20.91 to 33.08% (average 26.73%). 3.4. Correlation between ROL, porosity, biomass (% of control) and As concentration in grain and straw There were positive significant relationships between grain or straw biomass (% of control) and ROL and porosity of roots (Fig. 2). Furthermore, significant negative correlations were found between porosity of root and grain total As concentration (P < 0.01) or straw total As concentration (P < 0.01). The negative correlations

X.Q. Mei et al. / Environmental Pollution 157 (2009) 2550–2557

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Table 1 Total As concentrations (mg kg1 d.w.) in grain and straw in 25 rice cultivars grown in the control soils (contained 9.10 mg As kg1, CK) and soil with addition of 100 mg As kg1 (As(V)), and root porosity (POR) (%) and rate of ROL (mmo1 O2 kg1 root d.w. h1) of the rice cultivars exposed to 0.1% agar with 1/2 strength Hoagland solution for 30 days (mean  SE, range, n ¼ 4). Cultivara

Basmati 3 C039 Fengaizh Fenghuaz Ganxiang Guanglua IR 56 Jingxian Molixinz Qiguizao Qihuangz Qishanzh Sanerai Sanhuang Sanluzha Shengtai Texianzh Zhenguia Zhong 418 Zhongerr Yuefengz Yuexiang Khazarb Suyunuob Zhonghuab a b

Total grain As

Total straw As

CK

Added As

CK

Added As

0.16  0.008 (0.15–0.17) 0.17  0.002 (0.17–0.18) 0.16  0.004 (0.16–0.17) 0.19  0.009 (0.18–0.20) 0.17  0.002 (0.17–0.18) 0.17  0.006 (0.15–0.19) 0.14  0.008 (0.13–0.15) 0.15  0.005 (0.14–0.15) 0.15  0.008 (0.14–0.16) 0.15  0.015 (0.13–0.17) 0.13  0.008 (0.13–0.14) 0.13  0.007 (0.12–0.14) 0.21  0.011 (0.20–0.22) 0.19  0.008 (0.18–0.19) 0.14  0.008 (0.13–0.14) 0.17  0.004 (0.16–0.17) 0.21  0.003 (0.21–0.21) 0.20  0.001 (0.19–0.20) 0.16  0.002 (0.16–0.16) 0.11  0.010 (0.10–0.12) 0.19  0.006 (0.18–0.19) 0.13  0.001 (0.12–0.13) 0.08  0.002 (0.08–0.08) 0.12  0.009 (0.11–0.13) 0.09  0.002 (0.09–0.10)

1.33  0.02 (1.31–1.35) 2.72  0.22 (2.50–2.94) 2.33  0.09 (2.24–2.42) 1.78  0.12 (1.66–1.90) 2.05  0.06 (1.99–2.11) 2.28  0.02 (2.26–2.30) 1.05  0.07 (0.98–1.12) 2.22  0.15 (2.07–2.37) 0.71  0.02 (0.69–0.73) 1.93  0.03 (1.90–1.96) 1.87  0.15 (1.72–2.02) 1.34  0.06 (1.28–1.40) 1.52  0.11 (1.41–1.63) 2.65  0.11 (2.54–2.76) 1.47  0.17 (1.30–1.64) 2.14  0.14 (2.00–2.28) 2.24  0.08 (2.16–2.32) 2.26  0.06 (2.20–2.32) 1.85  0.09 (1.76–1.94) 1.07  0.07 (1.00–1.14) 1.41  0.02 (1.39–1.43) 0.96  0.02 (0.94–0.98) 1.73  0.23 (1.50–1.96) 0.92  0.06 (0.90–0.94) 1.09  0.18 (0.91–1.27)

1.25  0.09 (1.16–1.34) 1.66  0.01 (1.65–1.67) 1.89  0.06 (1.84–1.95) 1.97  0.09 (1.88–2.06) 1.82  0.09 (1.73–1.90) 1.44  0.10 (1.34–1.53) 0.78  0.06 (0.72–0.84) 1.35  0.11 (1.24–1.47) 1.54  0.05 (1.49–1.60) 1.09  0.12 (0.97–1.21) 1.96  0.13 (1.82–2.09) 2.10  0.12 (1.98–2.22) 1.60  0.15 (1.45–1.75) 2.42  0.10 (2.33–2.52) 0.72  0.04 (0.67–0.76) 1.26  0.07 (1.19–1.33) 1.74  0.08 (1.66–1.82) 1.94  0.09 (1.85–2.04) 1.88  0.11 (1.78–1.99) 1.53  0.08 (1.46–1.61) 2.32  0.07 (2.24–2.39) 1.67  0.03 (1.64–1.70) 0.64  0.04 (0.59–0.68) 0.79  0.03 (0.76–0.82) 0.86  0.04 (0.82–0.90)

15.60  0.15 (15.45–15.75) 25.77  0.34 (25.43–26.11) 23.37  0.49 (22.88–23.86) 29.20  0.38 (28.82–29.58) 23.80  0.78 (23.02–24.58) 25.13  0.91 (24.22–26.04) 16.20  0.22 (15.98–16.42) 21.60  0.99 (20.61–22.59) 18.80  0.39 (18.41–19.19) 21.40  0.69 (20.71–22.09) 24.17  0.53 (23.64–24.70) 18.90  0.82 (18.08–19.72) 22.10  0.87 (21.23–22.97) 25.40  0.85 (24.55–26.25) 16.90  0.49 (16.41–17.39) 24.03  0.94 (23.09–24.97) 31.70  0.89 (30.81–32.59) 28.30  0.84 (27.46–29.14) 19.77  0.54 (19.23–20.31) 23.35  0.66 (22.69–24.01) 29.80  0.12 (29.68–29.92) 21.07  0.75 (20.32–21.82) 15.97  0.16 (15.81–16.13) 17.73  0.59 (17.14–18.32) 16.77  0.42 (16.35–17.19)

POR (%)

Rate of ROL

28.97  0.43 (28.54–29.40) 26.40  0.64 (25.76–27.04) 25.77  0.29 (25.48–26.06) 23.33  0.91 (22.42–24.24) 25.75  1.09 (24.66–26.84) 25.20  0.99 (24.21–26.19) 25.50  0.53 (24.97–26.03) 25.39  0.78 (24.61–26.17) 29.15  0.69 (28.46–29.84) 31.08  0.45 (30.63–31.53) 24.30  0.16 (24.14–24.46) 29.00  0.88 (28.12–29.88) 27.40  0.75 (26.65–28.15) 21.43  0.57 (20.86–22.00) 28.53  0.76 (27.77–29.29) 21.63  0.78 (20.85–22.41) 20.91  0.13 (20.78–21.04) 24.85  0.46 (24.39–25.31) 21.81  0.51 (21.30–22.32) 29.48  1.06 (28.42–30.54) 26.80  1.02 (25.78–27.82) 29.92  0.87 (29.05–30.79) 30.40  0.52 (29.88–30.92) 33.08  0.39 (32.69–33.47) 32.14  0.27 (31.87–32.41)

11.64  0.36 (11.28–12.00) 8.23  0.24 (7.99–8.47) 8.05  0.26 (7.79–8.31) 8.05  0.63 (7.42–8.68) 7.59  0.11 (7.48–7.70) 9.03  0.33 (8.7–9.36) 10.39  0.59 (9.80–10.98) 9.52  0.24 (9.28–9.76) 10.00  0.10 (9.90–10.10) 11.36  0.55 (10.81–11.91) 9.31  0.18 (9.13–9.49) 12.44  0.37 (12.07–12.81) 8.05  0.14 (7.91–8.19) 8.05  0.25 (7.80–8.30) 11.58  0.35 (11.23–11.93) 7.40  0.16 (7.24–7.56) 8.82  0.15 (8.67–8.97) 8.23  0.17 (8.06–8.40) 8.21  0.09 (8.12–8.30) 11.57  0.37 (11.20–11.94) 8.18  0.31 (7.87–8.49) 11.99  0.27 (11.72–12.26) 10.47  0.13 (10.34–10.60) 13.24  0.24 (13.00–13.48) 12.36  0.40 (11.96–12.76)

Only first eight letters will be used to name cultivars. The cultivars are Japonica, and the others are Indica.

100 R SD N P ------------------------------0.76 8.22 25 < 0.001

90

Straw biomass (% of control)

Grain biomass (% of control)

100

80 70 60 50

80 70 60 50 40

40 7

8

9

10

11

Rate of ROL (mmol O2

kg-1

12

13

root d.w.

7

14

8

9

10

11

12

13

14

Rate of ROL (mmol O2 kg-1 root d.w. h-1)

h-1) 100

100 R SD N P -------------------------------90 0.62 9.87 25 < 0.001

Straw biomass (% of control)

Grain biomass (% of control)

R SD N P ------------------------------0.66 8.93 25 < 0.01

90

80 70 60 50

90

R SD N P -----------------------------0.55 9.96 25 < 0.01

80 70 60 50 40

40 20

22

24

26

28

POR (%)

30

32

34

20

22

24

26

28

30

32

34

POR (%)

Fig. 2. Correlation between the grain or straw biomass (% of control) of rice grown in soils added with 100 mg As kg1 and the rates of radial oxygen loss (ROL) and root porosity (POR) of rice grown in 0.1% agar solution.

X.Q. Mei et al. / Environmental Pollution 157 (2009) 2550–2557

0.25

Straw total As conc. in CK (mg kg-1)

Grain total As conc. in CK (mg kg-1)

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R SD N P ----------------------------------0.74 0.023 25 < 0.001

0.20

0.15

0.10

0.05 7

8

9

10

11

12

13

2.50

2.00

1.50

1.00

0.50 7

14

0.25 R SD N P ----------------------------------0.67 0.025 25 < 0.001

0.15

0.10

0.05 20

22

24

26

28

30

32

34

POR (%)

8

9

10

11

12

13

14

Rate of ROL (mmol O2 kg-1 root d.w. h-1) Straw total As conc. in CK ( mg kg-1)

Grain total As conc. in CK (mg kg-1)

Rate of ROL (mmol O2 kg-1 root d.w. h-1)

0.20

R SD N P -------------------------------------0.56 0.43 25 < 0.01

2.50 R SD N P ----------------------------------0.54 0.43 25 < 0.01

2.00

1.50

1.00

0.50 20

22

24

26

28

30

32

34

POR (%)

Fig. 3. Correlation between total As concentrations in grain or straw of rice grown in control soil (contained 9.10 mg As kg1) and rates of radial oxygen loss (ROL) and root porosity (POR) of rice grown in 0.1% agar solution.

between rate of ROL and grain total As concentration (P < 0.01) or straw total As concentration (P < 0.01) were also significant for the 25 rice cultivars examined under both the control soil and soil with addition of 100 mg As kg1 conditions (Figs. 3 and 4). 4. Discussion 4.1. Biomass (yield) and As levels in straw and husked grain Rice growth and yield were usually depressed when grown in soils containing high levels of As under both greenhouse and field conditions (Akter et al., 2005; Williams et al., 2005). Results from the present pot trial study clearly showed that the grain and straw biomass of all rice cultivars were reduced when grown in soil with the addition of 100 mg As kg1 compared to the control (Fig. 1). Similar results have been reported by Williams et al. (2005) when rice was grown in soil with the addition of 100 mg As kg1. Akter et al. (2005) showed that As in soil damaged the roots of rice, resulting in inhibition of nutrient uptake; while Rahman et al. (2007) revealed that the reduction of growth and yield of rice were due to reduced chlorophyll content in rice leaf under As toxicity. The present results also showed that toxic effects of As on rice biomass varied considerably among the rice cultivars studied. These indicated that there are great variations in As tolerance among different rice cultivars. Accumulative evidence from pot trials and field samples clearly showed that rice grown on As-contaminated land results in elevated As levels in rice straw and grains (Abedin et al., 2002;

Williams et al., 2005, 2007; Xu et al., 2008). Concentrations of As in rice tissues usually follow the order of root > straw > grain husk > grain (Abedin et al., 2002; Xu et al., 2008). Our results showed that As concentrations in husked grains and straws ranged from 0.08 to 0.22 mg kg1 and from 0.59 to 2.52 mg kg1 under the control soil (contained 9.10 mg kg1 As) condition, and from 0.71 to 2.72 mg kg1 and from 15.6 to 31.7 mg kg1 under the soil with addition of 100 mg kg1 (As(V)) condition, respectively (Table 1), which were within the range reported by others (Abedin et al., 2002; Meharg and Rahman, 2003; Xu et al., 2008; Zhu et al., 2008a). Under greenhouse conditions, Xu et al. (2008) reported that up to 2.5, 6.0 and 30 mg kg1 As were accumulated in rice grain (unpolished), rice husk and straw, respectively, when rice was grown on soils with the addition of 10 mg As kg1. Another pot experiment conducted by Abedin et al. (2002) showed that straw As concentration reached 91.8 mg kg1 when rice grown on soil was continuously irrigated with As-contaminated water. Under field conditions, some rice cultivated in areas irrigated with Ascontaminated water in Bangladesh contained 1.23–2.05 mg kg1 total As in grain (Meharg and Rahman, 2003; Williams et al., 2005). Zhu et al. (2008a) reported 0.07–0.62 mg kg1 total As in rice grain samples from mining-impacted areas in Hunan, China. It has been noted that the uptake of As to consumable parts of rice varied greatly with cultivars within a species (Williams et al., 2005; Liu et al., 2006). Our results indicated that As levels in both straw and husked grain varied considerably among different cultivars under both treatment conditions. Under the control soil treatment, grain total As concentration of Sanerai and Texianzhan

X.Q. Mei et al. / Environmental Pollution 157 (2009) 2550–2557

32 R SD N P --------------------------------------0.71 0.43 25 < 0.001

2.50

Straw total As conc. (mg kg-1)

Grain total As conc. (mg kg-1)

3.00

2.00

1.50

1.00

R SD N P -------------------------------------0.67 3.39 25 < 0.001

30 28 26 24 22 20 18 16 14

0.50 7

8

9

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7

Rate of ROL (mmol O2 kg-1 root d.w. h-1)

8

9

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Rate of ROL (mmol O2 kg-1 root d.w. h-1)

3.00

32 R SD N P ------------------------------------0.66 0.44 25 < 0.001

2.50

2.00

1.50

1.00

Straw total As conc. (mg kg-1)

Grain total As conc. (mg kg-1)

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R SD N P -------------------------------------0.62 3.67 25 < 0001 .

30 28 26 24 22 20 18 16 14

0.50 20

22

24

26

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34

POR ( % )

20

22

24

26

28

30

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34

POR ( % )

Fig. 4. Correlation between total As concentrations in grain or straw of rice grown in soils added with 100 mg As kg1 and rates of radial oxygen loss (ROL) and root porosity (POR) of rice grown in 0.1% agar solution.

was 2.6-fold higher than that of Khazar, while straw total As concentration of Sanhuangzhan was 3.8-fold high than that of Khazar. Similar results were also found when the cultivars grown in the soil with addition of 100 mg As kg1 (Table 1). Williams et al. (2005) found that in all As-treated rice, shoot As increased compared to the control, but the magnitude of this increase varied considerably among cultivars, with shoot As increasing up to 50% for some cultivars, and 400% for others. Similar results were found in the present study (Table 1). These results showed that different genotypes have different capacities for assimilating/accumulating As within the rice plant. It was subsequently observed that large variation existed in the shoot transport of As to rice grain, with the grain/shoot transfer factor (Grain/Shoot TF) ranging from 0.002 to 0.36 (Williams et al., 2007). The variation of Grain/Shoot TF obtained in our study was smaller than the results of Williams et al. (2007), ranging from 0.038 in Molixinzhan to 0.11 in Khazar when exposed to 100 mg As kg1 soil. The present data also showed that the grain total As concentrations were significant positively correlated with straw total As concentrations under both the control soil and the As treatment soil conditions (P < 0.01) (Fig. 5), indicating that more As was accumulated in straw, and eventually transported to grains.

constitutive porosity of their adventitious root when grown in aerated solutions (lowest was 16%, highest was 30%), and in stagnant condition between 28 and 38% (Colmer et al., 1998). The porosities of adventitious roots in 25 selected rice genotypes investigated in the present study varied from 20.9 (Texianzhan 13) to 33.1% (Suyunuo) when grown under stagnant conditions, which further confirmed the great variation in porosity among rice genotypes. Similar to porosity of roots, substantial differences on rates of ROL of roots among wetland species (Chabbi et al., 2000; Jensen et al., 2005; Van Bodegom et al., 2005) and rice genotypes have been commonly observed (Kludze et al., 1993). Sorrell (1999) reported that the rates of ROL of Juncus inflexu and Juncus effuse were 8.1 and 14.57 mmol O2 kg1 root d.w. h1, respectively. Chabbi et al. (2000) showed that Typha domingensis (1.8 mmol O2 kg1 root d.w. h1) and Cladium jamaicense (1.6 mmol O2 kg1 root d.w. h1) differed in rates of ROL under flooded conditions. The present results also showed significant differences in rates of ROL among different rice cultivars, from the lowest 7.40 mmol O2 kg1 root d.w. h1 for Shengtai 1 to the highest 13.24 mmol O2 kg1 root d.w. h1 for Suyunuo.

4.2. Porosity of root and rate of ROL

4.3. Correlations between porosity or ROL and As tolerance and accumulation

Porosity resulting from constitutive intercellular gas spaces can differ markedly among different species and genotypes, e.g. ranging from 2 to 22% in selected non-wetland species and from 15 to 52% in selected wetland species under O2-deficient conditions (Colmer, 2003b). Rice genotypes also differed significantly in the

Aerenchyma and/or porosity has long been regarded as an essential feature for the survival of emergent macrophytes, such as rice, in anaerobic (flooded) soils, and ROL from root to the rhizosphere is essential for the detoxification of phytotoxins (e.g. Fe2þ, Mn2þ, and H2S) (Armstrong and Armstrong, 2005). Previous studies

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A

0.25 R SD N P --------------------------------0.60 0.027 25 < 0.01

Rate of ROL (mmol O2 kg-1 root d.w. h-1)

Grain total As conc. in CK(mg kg-1)

A

X.Q. Mei et al. / Environmental Pollution 157 (2009) 2550–2557

0.20

0.15

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0.05 0.50

0.75

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14 13 12 11 10 9 8 7

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R SD N P ---------------------------------0.57 0.48 25 < 0.01

14

22

POR (%)

Rate of ROL (mmol O2 kg-1 root d.w. h-1)

Grain total As conc. (mg kg-1)

B

High grain total As conc. in CK (0.17-0.21 mg kg-1) Neutral grain total As conc. in CK (0.14-0.16 mg kg-1) Low grain total As conc. in CK (0.08-0.13 mg kg-1)

24

26

28

Straw total As conc. (mg

30

32

34

kg-1)

High grain total As conc. (2.05-2.72 mg kg-1) Neutral grain total As conc. (1.33-1.93 mg kg-1) Low grain total As conc. (0.71-1.09 mg kg-1)

14 13 12 11 10 9 8 7 20

22

24

26

28

30

POR (%) Fig. 5. Correlation between grain total As concentration and straw total As concentration grown in the control soil (A) and in soils added with 100 mg As kg1 (B).

showed that flood tolerance (Youssef and Saenger, 1996; Moog and Bruggemann, 1998; Grimoldi et al., 2005; Mano et al., 2006) and salinity tolerance (Rogers et al., 2008) of plants were correlated to their degrees of aerenchyma/porosity and oxidizing ability. However, little information is available about the relationship between As tolerance or accumulation and degree of porosity and/ or ROL of rice, respectively. The positive (significant) correlations between grain or straw biomass (% of control) and porosity and ROL of rice (Fig. 2), and negative (significant) correlations between grain As or straw As concentrations and porosity and ROL of rice, respectively (Figs. 3 and 4). These results revealed that rice cultivars with higher porosity and higher ROL tended to have higher As tolerance, and the ability to inhibit As accumulation in grains and straw under both the control and As-contaminated soil conditions (Fig. 6). The results in Fig. 6 also showed that the correlation between rate of ROL and porosity was positively significant (P < 0.01). These results suggest that the rice cultivars with higher porosity had higher rates of ROL, and also confirmed that higher root porosity will facilitate more oxygen loss from roots to rhizosphere soil (Van Bodegom et al., 2005). Fig. 1 and Table 1 also showed that three Japonica cultivars of the 25 selected cultivars possessed higher porosities and rates of ROL,

Fig. 6. Correlation between grain total As concentration and rate of radial oxygen loss (ROL) and root porosity (POR) of rice grown in the control soil (A) and in soils added with 100 mg As kg1 (B). Symbols indicate Ward’s analysis clusters for grain As concentrations.

higher grain and straw biomass (% of control) and lower As concentrations in grain and straw than other Indica cultivars when grown in both the control and As-contaminated soils. Further study on this is needed. Oxidizing capacity or ROL is considered the most important biotic factor controlling Fe plaque formation (Mendellsohn et al., 1995). Liu et al. (2006) reported that the amount (degree) of iron plaque formed on the root surface of rice was significantly different among the genotypes tested. The difference in iron plaque formation could reflect, at least partly, the genotypic difference in the oxidation capacity of root during the growth period. Due to the high adsorption capacity of iron (hydr)oxides, Fe plaque provides a reactive substrate for As sequestration (Blute et al., 2004; Liu et al., 2006). The formation of Fe plaque on root surface and in rhizosphere resulted in an effective fixation and detoxification of As (Doyle and Otte, 1997). As(III) is predominant species of As in anaerobic soils (Zhao et al., 2009). The oxidative rhizosphere in paddy soil is also likely to alter the speciation of As associated with root surface. It is reported that most (70–80%) of the As in the Fe plaque on the root of Phalaris arundinacea, Typha latifolia and rice

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was As(V), with the remaining (20–30%) As being As(III) (Hansel et al., 2002; Blute et al., 2004; Liu et al., 2006). The presence of Fe plaque enhances As(III) and decreases As(V) uptake by rice, but the reasons are not clear (Chen et al., 2005). These all suggest that there may be a strong relationship among porosity and ROL, degree of iron plaque (formation) on root surface and in rhizosphere, and As tolerance, accumulation in aboveground tissues of rice. 5. Conclusion This is the first study revealing that rice cultivars with higher porosity in their roots tend to result in more ROL from their roots, leading to higher As tolerance and lower As levels in their grain and straw. Porosity of root is considered a constitutive attribute of rice. The present results are significant in screening rice cultivars with high As tolerance and low As accumulation. The effects of ROL on the changes of concentration and/or speciation of As, Fe and P, and Fe plaque formation on roots and in rhizosphere soil, and the correlations among the rates of ROL, degrees of Fe plaque, and As levels and speciation in grains need further investigation. Acknowledgements We sincerely thank Guangdong Rice Research Institute and South China Agricultural University for provision of rice seeds; and Dr. H. Deng (South East Normal University) for technical assistance in ROL measurement. We are also very grateful to National Natural Science Foundation of China (30770417, 30570345), NSFC–Guangdong United Foundation (U0833004), Guangdong Natural Science Group Foundation (06202438), and the Research Grant Council, Hong Kong (HKBU 261407) for financial support. References Abedin, J., Cresser, M., Meharg, A.A., Feldmann, J., Cotter-Howells, J., 2002. Arsenic accumulation and metabolism in rice (Oryza sativa L.). Environmental Science and Technology 36, 962–968. Akter, K.F., Owens, G., Davey, D.E., Naidu, R., 2005. Arsenic speciation and toxicity in biological systems. Reviews Environmental Contamination and Toxicology 184, 97–149. Armstrong, W., 1979. Aeration in higher plants. In: Woolhouse, H.W. (Ed.), Advances in Botanical Research, vol. 7. Academic Press, London, pp. 225–332. Armstrong, J., Armstrong, W., 2005. Rice: sulfide-induced barriers to root radial oxygen loss, Fe2þ and water uptake, and lateral root emergence. Annals of Botany 96, 625–638. Blute, N.K., Drabander, D.J., Hemond, H.F., Sutton, S.R., Newville, M.G., Rivers, M.L., 2004. Arsenic sequestration by ferric iron plaque on cattail roots. Environmental Science and Technology 38, 6074–6077. Cai, Y., Georgiadis, M., Fourqurean, J.W., 2000. Determination of arsenic in seagrass using inductively coupled plasma mass spectrometry. Spectrochimica Acta Part B 55, 1411–1422. Chabbi, A., McKee, K.L., Mendelssohn, I.A., 2000. Fate of oxygen losses from Typha domingensis (Typhaceae) and Cladium jamaicense (Cyperaceae) and consequences for root metabolism. American Journal of Botany 87, 1081–1090. Chen, Z., Zhu, Y.G., Liu, W.J., Meharg, A.A., 2005. Direct evidence showing the effect of root surface iron plaque on arsenite and arsenate uptake into rice (Oryza sativa) roots. New Phytologist 165, 91–97. Colmer, T.D., 2003a. Aerenchyma and an inducible barrier to radial oxygen loss facilitate root aeration in upland, paddy and deep-water rice (Oryza sativa L.). Annals of Botany 91, 301–309. Colmer, T.D., 2003b. Long-distance transport of gases in plants: a perspective on internal aeration and radial oxygen loss from roots. Plant, Cell and Environment 26, 17–36. Colmer, T.D., Cox, M.C.H., Voesenek, L.A.C.J., 2006. Root aeration in rice (Oryza sativa L.): evaluation of oxygen, carbon dioxide, and ethylene as possible regulators of root acclimatizations. New Phytologist 170, 767–777. Colmer, T.D., Gibbered, M.R., Wiengweera, A., Tinh, T.K., 1998. The barrier to radial oxygen loss from roots of rice (Oryza sativa L.) is induced by growth in stagnant solution. Journal of Experimental Botany 49, 1431–1436. Crowder, A.A., St-Cyr, L., 1991. Iron oxide plaque on wetland roots. Trends in Soil Science 1, 315–329.

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