Annals of Botany 114: 271– 278, 2014 doi:10.1093/aob/mcu117, available online at www.aob.oxfordjournals.org
Selenium addition alters mercury uptake, bioavailability in the rhizosphere and root anatomy of rice (Oryza sativa) Xun Wang1, Nora Fung-Yee Tam2,3, Shi Fu4, Aray Ametkhan1, Yun Ouyang1 and Zhihong Ye1,* 1
State Key Laboratory of Biocontrol and Guangdong Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat-sen University, Guangzhou 510006, PR China, 2Department of Biology and Chemistry, City University of Hong Kong, Hong Kong SAR, PR China, 3State Key Laboratory in Marine Pollution, City University of Hong Kong, Hong Kong SAR, PR China and 4Fine Arts College, Sichuan Normal University, Chengdu 610110, PR China * For correspondence. E-mail [email protected] Received: 11 March 2014 Returned for revision: 25 April 2014 Accepted: 7 May 2014 Published electronically: 19 June 2014
† Background and Aims Mercury (Hg) is an extremely toxic pollutant, especially in the form of methylmercury (MeHg), whereas selenium (Se) is an essential trace element in the human diet. This study aimed to ascertain whether addition of Se can produce rice with enriched Se and lowered Hg content when growing in Hg-contaminated paddy fields and, if so, to determine the possible mechanisms behind these effects. † Methods Two cultivars of rice (Oryza sativa, japonica and indica) were grown in either hydroponic solutions or soil rhizobags with different Se and Hg treatments. Concentrations of total Hg, MeHg and Se were determined in the roots, shoots and brown rice, together with Hg uptake kinetics and Hg bioavailability in the soil. Root anatonmy was also studied. † Key Results The high Se treatment (5 mg g – 1) significantly increased brown rice yield by 48 % and total Se content by 2.8-fold, and decreased total Hg and MeHg by 47 and 55 %, respectively, compared with the control treatments. The high Se treatment also markedly reduced ‘water-soluble’ Hg and MeHg concentrations in the rhizosphere soil, decreased the uptake capacity of Hg by roots and enhanced the development of apoplastic barriers in the root endodermis. † Conclusions Addition of Se to Hg-contaminated soil can help produce brown rice that is simultaneously enriched in Se and contains less total Hg and MeHg. The lowered accumulation of total Hg and MeHg appears to be the result of reduced bioavailability of Hg and production of MeHg in the rhizosphere, suppression of uptake of Hg into the root cells and an enhancement of the development of apoplastic barriers in the endodermis of the roots. Key words: Apoplastic barriers, mercury pollution, Hg bioavailability, methylmercury, Oryza sativa, rice, root endodermis, selenium, Se addition, uptake process.
IN T RO DU C T IO N Mercury (Hg) is now a global and extremely toxic contaminant (Jiang et al., 2006), which has received considerable attention due to its great ability to be methylated, accumulated and biomagnified along the food chain. Methylmercury (MeHg), the most common form of organic Hg, is of greatest concern because of the high toxicity and potentially severe effects on humans (Mergler et al., 2007). Consumption of fish, fish products and marine mammals is generally considered the main pathway of human exposure to MeHg, posing a worldwide human health threat (Clarkson, 1993). However, recent studies have shown that rice (Oryza sativa) is the primary source of MeHg for those living in Hg mining areas and also in certain inland areas in South-western China (Feng et al., 2008; Zhang et al., 2010). Selenium (Se) is an essential trace element as it functions through seleno-proteins in humans and other animals (Goldhaber, 2003; Thomson, 2004). For most populations, the dominant food source of Se is cereals, such as rice and wheat (Duan et al., 2013). However, it has been reported that Se levels in major rice-producing and rice-consuming countries
such as China, Thailand and Egypt are low (Williams et al., 2009), and about 15 % of the world’s population is estimated to be Se deficient (Zhu et al., 2009). Protective effects of Se against Hg toxicity have been demonstrated in a range of organisms (Hansen et al., 1981; Chen et al., 2001; Belzile et al., 2006), suggesting that a universal Se – Hg antagonism may exist. However, there have been few studies investigating this putative Se – Hg antagonism in plants, most of which have been focused on the detoxifying effects of Se on inorganic Hg (IHg) but not MeHg (Shanker et al., 1996a; Mounicou et al., 2006). From these limited reports, it appears that an increased Se supply could effectively reduce the accumulation of Hg in plants, which may be primarily due to the formation of a high molecular weight Hg – Se-containing complex in plant roots that is difficult to be metabolized or translocated to aerial parts of plants (Afton and Caruso, 2009; McNear et al., 2012). Rice is the staple food for 3 billion people (Stone, 2008). However, the effects of Se on the accumulation of total Hg (THg), MeHg and total Se (TSe) in rice plants and the reasons why Se has such effects all remain unclear. It is generally accepted that plant uptake of Hg involves the same processes (symplastic and apoplastic pathways) as for the
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Wang et al. — Se addition and mercury uptake in rice
essential micronutrient ions (Patra and Sharma, 2000). In the symplastic pathway, Hg uptake is via one or various essential element membrane transporters for other metals such as manganese (Mn) and copper (Cu) in plant roots (Esteban et al., 2013; Regier et al., 2013). A low affinity transporter has also been suggested to take part in Hg uptake by roots of white lupin (Lupinus albus; Esteban et al., 2008). Esteban et al. (2013) further observed that Hg uptake was significantly decreased with increasing Mn concentrations in oilseed rape roots, indicating that Mn can compete with the Hg transporter in this plant. However, whether or not Se can affect the process of Hg uptake via membrane transporters in root tissues of rice plants has never been reported. From the perspective of the apoplastic pathway, the entry of heavy metals such as cadmium (Cd) and zinc (Zn) into the xylem can be delayed by the development of endodermis, exodermis and other extracellular barriers (Martinka and Lux, 2004; Cheng et al., 2010). Some essential elements such as iron (Fe), sulphur (S) and silicon (Si) can induce changes in root anatomy that may delay the uptake of heavy metals and contribute to a higher tolerance (Cheng et al., 2012; Vaculik et al., 2012). Cheng et al. (2012) demonstrated that Fe2+or S2 – , or both, induced a lignified exodermis that restricted the entry of Zn2+ into roots of a mangrove species, Bruguiera gymnorrhiza. Vaculik et al. (2012) found that suberization of individual endodermal cells commenced closer to the root apex in maize roots treated with Cd and Si than those treated with Cd only. However, whether Se can bring about similar changes in root anatomy that may affect the Hg uptake by roots of rice plants is unclear. There is a need to understand further the process involved in the Se – Hg antagonism in root tissues of rice plants. In the rhizosphere, the bioavailability of Hg and MeHg has been recognized to be one of the most important factors affecting their accumulation in rice plants (Meng et al., 2011; Peng et al., 2012; Wang et al., 2014). Local conditions in the rhizosphere can influence the bioavailability of Hg/MeHg (Hurley et al., 1995) and the various microbially facilitated processes that may affect Hg speciation (Marvin-DiPasquale et al., 2003; Wang et al., 2014). However, the specific effects of Se on the bioavailability of Hg (and MeHg) in the rhizosphere of rice plants are not fully understood. The present study therefore aims to investigate the effects of Se treatments on: (1) the accumulation of THg, MeHg and TSe in rice plants; (2) the uptake process for Hg into root tissues; (3) the anatomy of root tissues; and (4) the bioavailability of Hg and the concentration of MeHg in the rhizosphere. MAT E RI ALS A ND METH O DS Pre-culture of rice seedlings
Two rice (Oryza sativa) cultivars, japonica, ‘Zixiang’ (‘ZX’) and indica, ‘Nanfeng’ (‘NF’), were selected as they are both grown widely in China. Seeds were surface sterilized with 30 % (v/v) hydrogen peroxide (H2O2) for 30 min and washed thoroughly with deionized water. The seeds were then germinated in acid-washed quartz sand for 10 d. Afterwards, the seedlings were transferred to plastic containers (12 L) and grown in half-strength Hoagland’s nutrient solution for 3 d. The seedlings were then cultivated for another 25 d in quarter-strength solution with the
following nutrient composition (mmol L – 1): NH4NO3 500, K2SO4 200, CaCl2 400, MgSO4.7H2O 1500, KH2PO4 1.3, Fe-EDTA 50, H3BO3 10, ZnSO4.7H2O 1.0, CuSO4.5H2O 1.0, MnSO4.5H2O 5.0, Na2MoO4.2H2O 0.5 and CoSO4.7H2O 0.25. The nutrient solution was adjusted to pH 6.0 with NaOH or HCl and changed every 3 d. Plant cultivation under hydroponic conditions
At the end of 28 d growth, the rice seedlings grown under the above hydroponic conditions were divided into two batches. The first batch was used for the comparison of Hg uptake kinetics in root tissues and the second one was used for the determination of Hg concentrations in rice plants and the observations of root anatomy. No Hg was supplied to the first batch during cultivation, but Hg was added to the excised roots as described in the following section. Hg as HgCl2 (5 mM) was supplied to the second batch. In each batch, three treatments of Se were prepared: (1) high Se (10 mM Na2SeO3); (2) low Se (1.0 mM Na2SeO3); and (3) no Se addition (control) and Se was substituted by 10 mM KCl. There were four replicates with 16 seedlings per treatment. All nutrient solutions were quarter-strength, adjusted to pH 6 and renewed twice a week. The seedlings were grown for 2 weeks in a growth cabinet with the following conditions: day/night temperatures were 25/20 8C, day/night relatively humidity was 60/ 80 % and 16 h of light with . 350 mmol m – 2 s – 1 photon flux density. Kinetics of Hg uptake
The rice seedlings obtained from the three treatments (control, low and high) as described above were washed in deionized water and excised at the basal node. The excised roots (four replicates per treatment) were incubated in an aerated solution containing 0.5 mM CaCl2, 2 mM MES and 0, 0.5, 2.5, 5.0, 12.5 and 25 mM Hg at pH 6.0 for 20 min at room temperature (25 8C). After incubation, roots were rinsed in a fresh ice-cold solution containing 5 mM CaCl2 and 5 mM MES at pH 6.0 for 2 min and incubated in a fresh ice-cold nutrient solution of the same composition for 15 min to remove Hg adsorbed onto the root surface and from root free space, following the method described by Esteban et al. (2008). Fresh roots were weighed, freeze-dried at – 50 8C and ground to a powder for the determination of total Hg concentration, as described below. Histochemical studies to detect apoplastic barriers in the roots
After 2 weeks of cultivation, the rice plants obtained from three treatments in the second batch were divided into roots and shoots, weighed, freeze-dried at – 50 8C, ground to a powder and stored at 4 8C until further analysis. The concentration of Hg in each plant component was measured, with four replicates per treatment, as described below. Healthy adventitious roots of rice plants grown in the three treatments were crosssectioned at a distance of 1.0 and 5.0 cm from the root tip. To detect the development of Casparian bands (CBs) in the root endodermis and exodermis, sections were stained with 0.1 % (w/v) berberine hemisulphate for 1 h and with 0.5 % (w/v) aniline blue (Kotula et al., 2009) for another hour, and then viewed under a fluorescence microscope (Zeiss, Germany). To
Wang et al. — Se addition and mercury uptake in rice
Plant cultivation under soil conditions
Soil was collected from the plough layer (0 – 20 cm) of a paddy field, located at Wanshan in Guizhou Province, South-west China. The soil, pH 7.0, contained 2.34 % total C, 0.14 % total N, 0.7 mg g – 1 total Se, 46 mg g – 1 total Hg and 3.7 ng g – 1 MeHg. After air-drying, sieving to ,10 mesh and homogenization, the soil was supplemented with basal fertilizers [125 mg N kg – 1 soil as (NH2)2CO, 80 mg P kg – 1 and 125 mg K kg – 1 soil as KH2PO4 and K2SO4]. As with the hydroponic culture, three Se treatments were applied to the soil: (1) high Se (5 mg g – 1 Na2SeO3); (2) low Se (1 mg g – 1 Na2SeO3); and (3) no Se addition (control). Soils were mixed thoroughly and equilibrated for a month. For each treatment, a cylindrical nylon rhizobag (30 mm nylon mesh, 12 cm diameter and 15 cm high) was designed to separate the rhizosphere from the non-rhizosphere. The rhizobag filled with 0.75 kg of soil was placed in the centre of a PVC pot (20 cm diameter and 18 cm high) containing 1.0 kg of soil. Rice seedlings were carefully transplanted into the rhizobags with two plants per bag. There were four replicates per treatment. Soil in each pot was kept submerged using deionized water, and the growth conditions were the same as for the hydroponic culture. Rice plants were harvested at the maturity stage (about 90 d after transplanting) and separated into roots, straw, husk and brown rice. All the plant samples were freeze-dried at – 50 8C, ground to a fine powder and stored at 4 8C prior to further analysis of THg, MeHg and TSe. The soil samples were freeze-dried at – 50 8C, and crushed to pass a 150-mesh sieve for the analysis of THg and MeHg (Meng et al., 2011).
R E S ULT S Growth and accumulations of TSe, THg and MeHg in rice plants
The growth of rice plants was significantly improved by the addition of Se under both hydroponic and soil pot conditions (see Supplementary Data Tables S1 and S2). Under hydroponic conditions, the high Se treatment (10 mM Se) increased the biomass of roots and shoots by 32 and 14 %, respectively. Under soil conditions, the yields of brown rice in the high Se treatment (5 mg g – 1 Se) were 29– 66 % higher than those in the control treatment. Whether under hydroponic or soil conditions, different Se treatments produced a dramatic effect on THg, MeHg and TSe accumulation in rice plants, with the lowest THg and MeHg concentrations in the plants subject to the high Se treatment (Figs 1 and 2; Supplementary Data Fig. S1). Under hydroponic conditions, the high Se treatment decreased the concentrations of THg in roots and shoots by 22 and 29 %, respectively, compared with the control treatment. Under soil conditions, THg and MeHg concentrations in brown rice in the high Se treatment were 47 and 55 % lower than those in the control treatment, whereas TSe
70 Shoot THg concentration (mg g–1)
detect the suberin lamellae in the root, sections were stained with Sudan red 7B (Brundrett et al., 1991) and viewed under the same microscope but under bright light.
Chemical analysis
Control Low Se High Se
A
60
a a
50
a
b b
40 b 30
20 3000 Root THg concentration (mg g–1)
For THg and TSe analysis, soil and plant samples were microwave digested in concentrated HNO3 (16 mol L – 1) and measured by atomic fluorescence spectrometry (AFS; Beijing Titan Instrument Co., Ltd). For MeHg analysis, soil and plant samples were extracted by KBr – CuSO4/solvent and KOH – methanol/solvent, respectively (Liang et al., 1996), and determined with a MERX Automatic Methylmercury System (Brooks Rand Laboratories, Seattle, WA, USA), following method 1630 of USEPA (2001). For the analysis of Hg bioavailability, a sequential extraction technique for soil and sediment was employed, and only the water-soluble fraction among the five fractions represented the ‘bioavailable inorganic Hg’ (Bloom et al., 2003). Information on the quality assurance/ quality control (QA/QC) of our analytical data is available in the Supplementary Data.
273
B a
2700 a 2400
2100
b
b b
b
1800
Statistical analysis
Data were analysed using the statistical software package SPSS 17.0 and summarized by means + standard errors (s.e.). Means were compared by least significant difference (LSD) at the 5 % level. Coefficients of determination (R 2) and significance probabilities (P) were computed for linear regression fits.
‘NF’
‘ZX’
F I G . 1. Concentrations of THg in shoots (A) and roots (B) of two rice cultivars (‘NF’, ‘Nanfeng’; and ‘ZX’, ‘Zixiang’) grown in 5 mM Hg solution without Se (control), and with low Se and high Se amendments for 2 weeks. Data are means + s.e. (n ¼ 4). Control, no Se supplied; Low Se, 1.0 mM Se supplied; High Se, 10 mM Se supplied.
Wang et al. — Se addition and mercury uptake in rice
Brown rice THg concentration (ng g–1)
40
A
Control Low Se High Se
35 30
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a
a 25 20
b
15 b
b
10
3·5
Hg influx (μmol g–1 f. wt h –1)
274
2·8
2·1
1·4
0·7
0
B
3·2
28
a
24 20 16
a
12
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8
Hg influx (μmol g–1 f. wt h –1)
32
B
2·4
1·6
0·8
4 0
0
0
3·0 Brown rice TSe concentration (mg g–1)
Control Low Se High Se
5 0
Brown rice MeHg concentration (ng g–1)
A
C
5
10
15
20
25
Hg concentration in solution (μM)
2·5
a a
2·0
1·5
b b c
1·0
F I G . 3. Hg influx in excised roots of two rice cultivars, ‘Nanfeng’ (A) and ‘Zixiang’ (B). The excised roots were incubated in an aerated solution containing 0, 0.5, 2.5, 5.0, 12.5 or 25 mM Hg without Se (control, filled circles), and with low Se (open circles) and high Se (triangles) amendments at pH 6.0 for 20 min at room temperature (25 8C). Data are means + s.e. (n ¼ 4). Control, no Se supplied; Low Se, 1.0 mM Se supplied; High Se, 10 mM Se supplied.
grown under contaminated soil conditions was below the National Guidance Limit for crops ( , 20 ng g – 1) recommended by the Chinese National Standard Agency (Fig. 2A).
c
Kinetics of Hg uptake
0·5
0
’ ‘NF’
‘ZX’
F I G . 2. Concentrations of THg (A), MeHg (B) and TSe (C) in brown rice of two rice cultivars (‘NF’, ‘Nanfeng’; and ‘ZX’, ‘Zixiang’) grown in a Hg-contaminated paddy soil without Se (control), and with low Se and high Se amendments. Data are means + s.e. (n ¼ 4). Control, no Se amendment; Low Se, 1 mg Se g – 1 amended; High Se, 5 mg Se g – 1 amended. The dashed line in (A) refers to the National Guidance Limit for crops recommended by the Chinese National Standard Agency.
concentrations in brown rice in the high Se treatment were 2.8-fold higher than those in the control treatment. It should be noted that the THg concentration in the high Se-treated rice
For both rice cultivars, Hg uptake could be described satisfactorily by Michaelis – Menten kinetics (Fig. 3). The kinetic parameters shown in Table 1 suggested that the uptake kinetics for Hg influx were significantly affected by different Se treatments. The Vmax values in the control treatment were 3.59 + 0.75 and 3.37 + 0.58 mmol Hg g – 1 f. wt h – 1 for ‘NF’ and ‘ZX’, respectively, whereas the respective values declined to 2.19 + 0.67 and 2.56 + 0.47 mmol Hg g – 1 f. wt h – 1 in the high Se treatment. Root anatomy: development of Casparian bands and suberin lamellae
At 5.0 cm from the root tip, no CBs were observed in the endodermis of either cultivar grown in the control treatment (Fig. 4A,
Wang et al. — Se addition and mercury uptake in rice TA B L E 1. Kinetic parameters for Hg influx into excised roots of cultivars ‘Nanfeng’ (‘NF’) and ‘Zixiang’ (‘ZX’) Rice cultivar ‘NF’
Se treatment
Vmax (mmol Hg g f. wt h – 1)
‘ZX’
Km (mM)
R2
6.40 + 2.37 7.15 + 3.02 4.66 + 3.63 7.37 + 2.47 8.70 + 2.24 7.84 + 1.73
0.948 0.961 0.905 0.980 0.983 0.978
The excised roots were incubated in an aerated solution containing 0, 0.5, 2.5, 5.0, 12.5 or 25 mM Hg without Se (control), and with low Se (1.0 mM Se) and high Se (10 mM Se) amendments at pH 6.0 for 20 min at room temperature (25 8C). Kinetic parameters were calculated from mean Hg influx (n ¼ 4) using a Michaelis–Menten function model.
Control
Control
Low Se
High Se
A
B
C
D
E
F
–1
3.59 + 0.75 3.19 + 0.59 2.19 + 0.67 3.37 + 0.58 2.95 + 0.47 2.56 + 0.47
Control Low Se High Se Control Low Se High Se
275
Low Se
‘NF’
‘ZX’
F I G . 5. Appearance of Casparian bands (A– F) in rice roots. Cross-sections were made at 1.0 cm from the root tip in cultivars ‘Nanfeng’ (‘NF’) and ‘Zixiang’ (‘ZX’) grown with the control (without Se) (A, D), low Se (1.0 mM Se supplied) (B, E) and high Se (10 mM Se supplied) (C, F) treatments under hydroponic conditions. The presence of Casparian bands was indicated by green-yellow fluorescence (see arrows in B, C, F). Scale bars ¼ 50 mm.
High Se
A
B
C
D
E
F
G
H
I
‘NF’
‘ZX’
‘NF’
in both cultivars, although they were more prominent in ‘NF’ (Fig. 4C, F). The suberin lamellae were detected as red stains in the cells walls after staining with Sudan red. In the control treatment, only a few cells in the endodermis of both cultivars had suberin lamellae at 5.0 cm from the root tip (Fig. 4G, J), whereas a single complete ring was observed in the low Se treatment (Fig. 4H, K). Suberin lamellae also appeared as 2 – 3 complete rings with thickened cell walls in the roots of the two cultivars grown in the high Se treatment (Fig. 4I, L). At 1.0 cm from the root tip of ‘NF’, dot-like CBs had begun to be observable in the endodermis in the low Se treatment (Fig. 5B), and well-developed CBs could be detected in the high Se treatment (Fig. 5C). However, for ‘ZX’, dot-like CBs were only observed in the endodermis in the high Se treatment (Fig. 5F). THg, MeHg concentration and Hg bioavailability in rhizosphere soil
J
K
L
‘ZX’
F I G . 4. Appearance of Casparian bands (A–F) and suberin lamellae (G–L) in the endodermis of rice roots. Cross-sections were made at 5.0 cm from the root tip in cultivars ‘Nanfeng’ (‘NF’) and ‘Zixiang’ (‘ZX’) grown with the control (without Se) (A, D, G, J), low Se (1.0 mM Se supplied) (B, E, H, K) and high Se (10 mM Se supplied) (C, F, I, L) treatments under hydroponic conditions. The presence of Casparian bands was indicated by green-yellow fluorescence (see arrows in B, C, F). Cross-sections were stained with the lipophilic dye Sudan red 7B; only a few cells were observed with lamellae in ‘Nanfeng’ (G) and ‘Zixaing’ (J) grown in the control treatment, but a complete ring of lamellae with red stain appeared in the low Se treatment (H, K). Two to three completes rings of lamellae with thickened cell walls were detected in the high Se treatment (I, L). Scale bars ¼ 50 mm.
D). As indicated by green-yellow fluorescence, dot-like CBs occurred in the radial walls of the endodermis of ‘NF’ in the low Se treatment, whereas in ‘ZX’ there were no CBs apparent (Fig. 4B, E). In the high Se treatment, well-developed CBs were observed
The THg concentration was 46 + 3.4 mg g – 1 in rhizosphere soil, and showed no significant difference between the Se treatments (data not shown). The MeHg concentration in rhizosphere soil ranged from 2.6 to 5.9 ng g – 1, and was significantly reduced by the addition of Se (Table 2). The low and high Se treatments decreased MeHg concentrations by 13 and 44 %, respectively, compared with the control treatment. The ‘water-soluble Hg’ fraction accounted for 0.34–0.45 % of the THg content of the soil, and was significantly affected by the addition of Se (Table 3). The low and high Se treatments decreased the ‘water-soluble Hg’ concentrations by 11 and 16 %, respectively, compared with the control treatment. A significant positive correlation was also found between the ‘watersoluble Hg’ concentrations in the rhizosphere soils and the THg concentrations in the brown rice (R 2 ¼ 0.87, P , 0.01) (Fig. 6). DISCUSSION Effects of Se on growth and accumulation of THg, MeHg and TSe in rice plants
The present study clearly demonstrates that the bioaccumulations of both THg and MeHg in rice tissues (brown rice, straw
Wang et al. — Se addition and mercury uptake in rice
TA B L E 2. MeHg concentrations in rhizosphere soils of two rice cultivars (‘NF’, ‘Nanfeng’; and ‘ZX’, ‘Zixiang’) grown in a Hg-contaminated paddy soil without Se (control), and with low Se and high Se amendments in a pot trial MeHg concentration (ng g – 1) Se treatment Control Low Se High Se
‘NF’
‘ZX’
4.8 + 0.48a 3.9 + 0.73b 2.6 + 0.74c
5.9 + 0.60a 5.4 + 0.97a 3.1 + 0.39b
Data are means + s.e. (n ¼ 4). Different letters within the same column indicate a significant difference between the Se treatments at the level of P , 0.05. Control, no Se amendment; Low Se, 1 mg Se g – 1 amended; High Se, 5 mg Se g – 1 amended.
TA B L E 3. Water-soluble Hg concentrations in rhizosphere soils of two rice cultivars (‘NF’, ‘Nanfeng’; and ‘ZX’, ‘Zixiang’) grown in a Hg-contaminated paddy soil without Se (control), and with low Se and high Se amendments in a pot trial Water-soluble Hg concentration (ng g – 1) Se treatment Control Low Se High Se
‘NF’
‘ZX’
194 + 28a 170 + 20a 158 + 17b
205 + 11a 187 + 37b 179 + 10b
Data are means + SE (n ¼ 4) Different letters within the same column indicate a significant difference between the Se treatments at the level of P , 0.05. Control, no Se amendment; Low Se, 1 mg Se g – 1 amended; High Se, 5 mg Se g – 1 amended.
and roots) were significantly reduced by the addition of Se, whereas total Se accumulations in brown rice and yields significantly increased in Se treatments under soil conditions (Fig. 2; Supplementary Data Fig. S1, Table S2). A similar response has also been observed in tomato (Solanum lycopersicum), radish (Raphanus sativus), soybean (Glycine max) and other plants (Shanker et al., 1996a, b; Yathavakilla and Caruso, 2007). Furthermore, THg concentrations in brown rice grown in Hg-contaminated soil amended with 5 mg Se g – 1 declined below the National Guidance Limit for crops ( , 20 ng g – 1) (Fig. 2A). This result suggests that the addition of Se not only produced high yielding and Se-enriched brown rice but also lowered its Hg and MeHg contents when plants were grown in Hg-contaminated soils.
Effects of Se on the Hg uptake process
The determination of the kinetics of Hg uptake is a necessary preliminary step for modelling soil – plant transfer in Hg-contaminated environments (Nowack et al., 2006). However, only a few studies have reported the kinetic parameters of Hg uptake in higher plants (Esteban et al., 2008, 2013). Our study suggests that Hg uptake by roots of rice
30 Brown rice THg concentration (ng g–1)
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R 2 = 0·87, P < 0·01
27 24 21 18 Control Low Se High Se
15 12 9 0·15
0·16
0·17
0·18
0·19
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Water-soluble Hg concentration (mg
0·21
g–1)
F I G . 6. Relationship between water-soluble Hg concentrations in rhizosphere soils and THg concentrations in brown rice from plants grown in a Hg-contaminated paddy soil without Se (control, filled squares), and with low Se (1 mg Se g – 1, open circles) and high Se (5 mg Se g – 1, solid triangles) amendments in a pot trial.
plants is an active process (Esteban et al., 2008), which needs selective binding sites and an energy source as the driving force. Furthermore, it is worth noting that Vmax values for both cultivars in the high Se treatment were significantly lower than those in the control treatment (Table 1), suggesting that the uptake of Hg into root cells through the symplastic pathway was restricted by the addition of Se via lowering the activity of membrane transporters. To the best of our knowledge, the effects of Se on the kinetic parameters of Hg uptake in roots of rice plants have never been reported. The only study by Feng et al. (2013) showed that Se could compete with Hg for specific binding sites, such as the thiol groups of cysteine in membrane proteins, to defend higher plants against abiotic stresses. Effects of Se on root anatomy
Uptake of elements through the apoplastic pathway is controlled by apoplastic barriers in the endodermis and exodermis (Schreiber, 2010). The development of these barriers varies and often differs between plant species and with environmental conditions (Meyer et al., 2009; Redjala et al., 2011). Our study suggests that the addition of Se could induce and enhance the development of apoplastic barriers in the endodermis, which may help to restrict the uptake and translocation of Hg into shoots. Previous studies also showed that some essential elements (e.g. Fe, S and Si) could induce the changes in root anatomy that may delay the uptake of heavy metals (e.g. Zn and Cd) and contribute to a higher metal resistance by plants (Cheng et al., 2012; Vaculik et al., 2012). However, the development of CBs and suberin lamellae was not so obvious in the exodermis of rice plants and showed no differences among the three Se treatments. This may be explained by the fact that the exodermis usually develops later and is less sensitive to environmental conditions, compared with the endodermis (Ma and Peterson, 2003; Vaculik et al., 2012). Furthermore, it is worth noting that the development of apoplastic barriers in the endodermis of ‘NF’ was more
Wang et al. — Se addition and mercury uptake in rice obvious than in ‘ZX’ (Figs 4 and 5), irrespective of the level of Se treatments. This suggests that the effects of Se on the development of apoplastic barriers in the endodermis might vary among cultivars, which may have different impacts on the uptake and translocation of Hg into shoots.
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ACK NOW LED GE MENTS We thank Professor A. J. M. Baker (The Universities of Melbourne and Queensland, Australia) for help in the initial preparation and improvement of this paper. This work was supported by the National Natural Science Foundation of China (30770417, 31070450) and the National ‘863’ project of China (2012AA061510, 2013AA062609).
Effects of Se on Hg bioavailability and MeHg content in the rhizosphere
The bioavailability of Hg plays an important role in affecting its accumulation in rice plants (Peng et al., 2012; Wang et al., 2014). In our study, the bioavailable Hg was represented by the ‘water-soluble’ Hg fraction, which is the most mobile and bioavailable fraction and can be easily transported by natural processes and absorbed by plants (Issaro et al., 2009). Furthermore, this fraction is the major source of inorganic Hg accumulated in rice plants (Meng et al., 2010) and MeHg in paddy soil (Meng et al., 2014). Our results suggest that Se amendment enhanced the immobilization of Hg in the soil, leading to lower Hg bioavailability and restricting the production of MeHg (Table 2), which could finally lead to a reduction in the uptake and accumulation of THg and MeHg in rice plants.
Conclusions
This study systematically explored the effects of Se on the uptake process of Hg, root anatomy, an aspect of the Hg bioavailability in the rhizosphere and their relationships with Hg/MeHg accumulation in rice plants. Selenium addition not only significantly increased yields and TSe but also decreased THg and MeHg contents in brown rice in plants grown under Hgcontaminated soil conditions. Reducing the bioavailability of Hg and production of MeHg in the rhizosphere, suppressing the uptake process of Hg into root cells and enhancing the development of apoplastic barriers in the endodermis of roots may be major reasons for the lowered accumulation of THg and MeHg in rice plants. This study enhances our understanding of the factors involved in Se – Hg antagonism in rice plants and so provides a potentially useful method to produce simultaneously safe and enriched Se brown rice for plants grown under Hg-contaminated paddy field conditions.
S U P P L E M E N TARY D ATA Supplementary data are available online at www.aob.oxford journals.org and consist of the following. Details of quality assurance/quality control (QA/QC) for THg, TSe and MeHg analysis in the samples. Figure S1: concentrations of THg in roots and straw of rice grown without Se, and with low Se and high Se amendments in a pot trial. Figure S2: relationships between TSe concentrations in brown rice, straw, root tissues and THg and MeHg concentrations in brown rice tissues of rice plants. Table S1: biomass of root and shoot of rice grown in 5 mM Hg solution without Se, and with low Se and high Se amendments for 2 weeks. Table S2: biomass of roots, straw, husk and brown rice tissues of rice grown in a Hg-contaminated paddy soil without Se, and with low Se and high Se amendments in a pot trial.
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Selenium addition alters mercury uptake, bioavailability in the rhizosphere and root anatomy of rice (Oryza sativa) Xun Wang1, Nora Fung-Yee Tam2,3, Shi Fu4, Aray Ametkhan1, Yun Ouyang1 and Zhihong Ye1,* 1
State Key Laboratory of Biocontrol and Guangdong Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat-sen University, Guangzhou 510006, PR China, 2Department of Biology and Chemistry, City University of Hong Kong, Hong Kong SAR, PR China, 3State Key Laboratory in Marine Pollution, City University of Hong Kong, Hong Kong SAR, PR China and 4Fine Arts College, Sichuan Normal University, Chengdu 610110, PR China * For correspondence. E-mail [email protected] Received: 11 March 2014 Returned for revision: 25 April 2014 Accepted: 7 May 2014 Published electronically: 19 June 2014
† Background and Aims Mercury (Hg) is an extremely toxic pollutant, especially in the form of methylmercury (MeHg), whereas selenium (Se) is an essential trace element in the human diet. This study aimed to ascertain whether addition of Se can produce rice with enriched Se and lowered Hg content when growing in Hg-contaminated paddy fields and, if so, to determine the possible mechanisms behind these effects. † Methods Two cultivars of rice (Oryza sativa, japonica and indica) were grown in either hydroponic solutions or soil rhizobags with different Se and Hg treatments. Concentrations of total Hg, MeHg and Se were determined in the roots, shoots and brown rice, together with Hg uptake kinetics and Hg bioavailability in the soil. Root anatonmy was also studied. † Key Results The high Se treatment (5 mg g – 1) significantly increased brown rice yield by 48 % and total Se content by 2.8-fold, and decreased total Hg and MeHg by 47 and 55 %, respectively, compared with the control treatments. The high Se treatment also markedly reduced ‘water-soluble’ Hg and MeHg concentrations in the rhizosphere soil, decreased the uptake capacity of Hg by roots and enhanced the development of apoplastic barriers in the root endodermis. † Conclusions Addition of Se to Hg-contaminated soil can help produce brown rice that is simultaneously enriched in Se and contains less total Hg and MeHg. The lowered accumulation of total Hg and MeHg appears to be the result of reduced bioavailability of Hg and production of MeHg in the rhizosphere, suppression of uptake of Hg into the root cells and an enhancement of the development of apoplastic barriers in the endodermis of the roots. Key words: Apoplastic barriers, mercury pollution, Hg bioavailability, methylmercury, Oryza sativa, rice, root endodermis, selenium, Se addition, uptake process.
IN T RO DU C T IO N Mercury (Hg) is now a global and extremely toxic contaminant (Jiang et al., 2006), which has received considerable attention due to its great ability to be methylated, accumulated and biomagnified along the food chain. Methylmercury (MeHg), the most common form of organic Hg, is of greatest concern because of the high toxicity and potentially severe effects on humans (Mergler et al., 2007). Consumption of fish, fish products and marine mammals is generally considered the main pathway of human exposure to MeHg, posing a worldwide human health threat (Clarkson, 1993). However, recent studies have shown that rice (Oryza sativa) is the primary source of MeHg for those living in Hg mining areas and also in certain inland areas in South-western China (Feng et al., 2008; Zhang et al., 2010). Selenium (Se) is an essential trace element as it functions through seleno-proteins in humans and other animals (Goldhaber, 2003; Thomson, 2004). For most populations, the dominant food source of Se is cereals, such as rice and wheat (Duan et al., 2013). However, it has been reported that Se levels in major rice-producing and rice-consuming countries
such as China, Thailand and Egypt are low (Williams et al., 2009), and about 15 % of the world’s population is estimated to be Se deficient (Zhu et al., 2009). Protective effects of Se against Hg toxicity have been demonstrated in a range of organisms (Hansen et al., 1981; Chen et al., 2001; Belzile et al., 2006), suggesting that a universal Se – Hg antagonism may exist. However, there have been few studies investigating this putative Se – Hg antagonism in plants, most of which have been focused on the detoxifying effects of Se on inorganic Hg (IHg) but not MeHg (Shanker et al., 1996a; Mounicou et al., 2006). From these limited reports, it appears that an increased Se supply could effectively reduce the accumulation of Hg in plants, which may be primarily due to the formation of a high molecular weight Hg – Se-containing complex in plant roots that is difficult to be metabolized or translocated to aerial parts of plants (Afton and Caruso, 2009; McNear et al., 2012). Rice is the staple food for 3 billion people (Stone, 2008). However, the effects of Se on the accumulation of total Hg (THg), MeHg and total Se (TSe) in rice plants and the reasons why Se has such effects all remain unclear. It is generally accepted that plant uptake of Hg involves the same processes (symplastic and apoplastic pathways) as for the
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essential micronutrient ions (Patra and Sharma, 2000). In the symplastic pathway, Hg uptake is via one or various essential element membrane transporters for other metals such as manganese (Mn) and copper (Cu) in plant roots (Esteban et al., 2013; Regier et al., 2013). A low affinity transporter has also been suggested to take part in Hg uptake by roots of white lupin (Lupinus albus; Esteban et al., 2008). Esteban et al. (2013) further observed that Hg uptake was significantly decreased with increasing Mn concentrations in oilseed rape roots, indicating that Mn can compete with the Hg transporter in this plant. However, whether or not Se can affect the process of Hg uptake via membrane transporters in root tissues of rice plants has never been reported. From the perspective of the apoplastic pathway, the entry of heavy metals such as cadmium (Cd) and zinc (Zn) into the xylem can be delayed by the development of endodermis, exodermis and other extracellular barriers (Martinka and Lux, 2004; Cheng et al., 2010). Some essential elements such as iron (Fe), sulphur (S) and silicon (Si) can induce changes in root anatomy that may delay the uptake of heavy metals and contribute to a higher tolerance (Cheng et al., 2012; Vaculik et al., 2012). Cheng et al. (2012) demonstrated that Fe2+or S2 – , or both, induced a lignified exodermis that restricted the entry of Zn2+ into roots of a mangrove species, Bruguiera gymnorrhiza. Vaculik et al. (2012) found that suberization of individual endodermal cells commenced closer to the root apex in maize roots treated with Cd and Si than those treated with Cd only. However, whether Se can bring about similar changes in root anatomy that may affect the Hg uptake by roots of rice plants is unclear. There is a need to understand further the process involved in the Se – Hg antagonism in root tissues of rice plants. In the rhizosphere, the bioavailability of Hg and MeHg has been recognized to be one of the most important factors affecting their accumulation in rice plants (Meng et al., 2011; Peng et al., 2012; Wang et al., 2014). Local conditions in the rhizosphere can influence the bioavailability of Hg/MeHg (Hurley et al., 1995) and the various microbially facilitated processes that may affect Hg speciation (Marvin-DiPasquale et al., 2003; Wang et al., 2014). However, the specific effects of Se on the bioavailability of Hg (and MeHg) in the rhizosphere of rice plants are not fully understood. The present study therefore aims to investigate the effects of Se treatments on: (1) the accumulation of THg, MeHg and TSe in rice plants; (2) the uptake process for Hg into root tissues; (3) the anatomy of root tissues; and (4) the bioavailability of Hg and the concentration of MeHg in the rhizosphere. MAT E RI ALS A ND METH O DS Pre-culture of rice seedlings
Two rice (Oryza sativa) cultivars, japonica, ‘Zixiang’ (‘ZX’) and indica, ‘Nanfeng’ (‘NF’), were selected as they are both grown widely in China. Seeds were surface sterilized with 30 % (v/v) hydrogen peroxide (H2O2) for 30 min and washed thoroughly with deionized water. The seeds were then germinated in acid-washed quartz sand for 10 d. Afterwards, the seedlings were transferred to plastic containers (12 L) and grown in half-strength Hoagland’s nutrient solution for 3 d. The seedlings were then cultivated for another 25 d in quarter-strength solution with the
following nutrient composition (mmol L – 1): NH4NO3 500, K2SO4 200, CaCl2 400, MgSO4.7H2O 1500, KH2PO4 1.3, Fe-EDTA 50, H3BO3 10, ZnSO4.7H2O 1.0, CuSO4.5H2O 1.0, MnSO4.5H2O 5.0, Na2MoO4.2H2O 0.5 and CoSO4.7H2O 0.25. The nutrient solution was adjusted to pH 6.0 with NaOH or HCl and changed every 3 d. Plant cultivation under hydroponic conditions
At the end of 28 d growth, the rice seedlings grown under the above hydroponic conditions were divided into two batches. The first batch was used for the comparison of Hg uptake kinetics in root tissues and the second one was used for the determination of Hg concentrations in rice plants and the observations of root anatomy. No Hg was supplied to the first batch during cultivation, but Hg was added to the excised roots as described in the following section. Hg as HgCl2 (5 mM) was supplied to the second batch. In each batch, three treatments of Se were prepared: (1) high Se (10 mM Na2SeO3); (2) low Se (1.0 mM Na2SeO3); and (3) no Se addition (control) and Se was substituted by 10 mM KCl. There were four replicates with 16 seedlings per treatment. All nutrient solutions were quarter-strength, adjusted to pH 6 and renewed twice a week. The seedlings were grown for 2 weeks in a growth cabinet with the following conditions: day/night temperatures were 25/20 8C, day/night relatively humidity was 60/ 80 % and 16 h of light with . 350 mmol m – 2 s – 1 photon flux density. Kinetics of Hg uptake
The rice seedlings obtained from the three treatments (control, low and high) as described above were washed in deionized water and excised at the basal node. The excised roots (four replicates per treatment) were incubated in an aerated solution containing 0.5 mM CaCl2, 2 mM MES and 0, 0.5, 2.5, 5.0, 12.5 and 25 mM Hg at pH 6.0 for 20 min at room temperature (25 8C). After incubation, roots were rinsed in a fresh ice-cold solution containing 5 mM CaCl2 and 5 mM MES at pH 6.0 for 2 min and incubated in a fresh ice-cold nutrient solution of the same composition for 15 min to remove Hg adsorbed onto the root surface and from root free space, following the method described by Esteban et al. (2008). Fresh roots were weighed, freeze-dried at – 50 8C and ground to a powder for the determination of total Hg concentration, as described below. Histochemical studies to detect apoplastic barriers in the roots
After 2 weeks of cultivation, the rice plants obtained from three treatments in the second batch were divided into roots and shoots, weighed, freeze-dried at – 50 8C, ground to a powder and stored at 4 8C until further analysis. The concentration of Hg in each plant component was measured, with four replicates per treatment, as described below. Healthy adventitious roots of rice plants grown in the three treatments were crosssectioned at a distance of 1.0 and 5.0 cm from the root tip. To detect the development of Casparian bands (CBs) in the root endodermis and exodermis, sections were stained with 0.1 % (w/v) berberine hemisulphate for 1 h and with 0.5 % (w/v) aniline blue (Kotula et al., 2009) for another hour, and then viewed under a fluorescence microscope (Zeiss, Germany). To
Wang et al. — Se addition and mercury uptake in rice
Plant cultivation under soil conditions
Soil was collected from the plough layer (0 – 20 cm) of a paddy field, located at Wanshan in Guizhou Province, South-west China. The soil, pH 7.0, contained 2.34 % total C, 0.14 % total N, 0.7 mg g – 1 total Se, 46 mg g – 1 total Hg and 3.7 ng g – 1 MeHg. After air-drying, sieving to ,10 mesh and homogenization, the soil was supplemented with basal fertilizers [125 mg N kg – 1 soil as (NH2)2CO, 80 mg P kg – 1 and 125 mg K kg – 1 soil as KH2PO4 and K2SO4]. As with the hydroponic culture, three Se treatments were applied to the soil: (1) high Se (5 mg g – 1 Na2SeO3); (2) low Se (1 mg g – 1 Na2SeO3); and (3) no Se addition (control). Soils were mixed thoroughly and equilibrated for a month. For each treatment, a cylindrical nylon rhizobag (30 mm nylon mesh, 12 cm diameter and 15 cm high) was designed to separate the rhizosphere from the non-rhizosphere. The rhizobag filled with 0.75 kg of soil was placed in the centre of a PVC pot (20 cm diameter and 18 cm high) containing 1.0 kg of soil. Rice seedlings were carefully transplanted into the rhizobags with two plants per bag. There were four replicates per treatment. Soil in each pot was kept submerged using deionized water, and the growth conditions were the same as for the hydroponic culture. Rice plants were harvested at the maturity stage (about 90 d after transplanting) and separated into roots, straw, husk and brown rice. All the plant samples were freeze-dried at – 50 8C, ground to a fine powder and stored at 4 8C prior to further analysis of THg, MeHg and TSe. The soil samples were freeze-dried at – 50 8C, and crushed to pass a 150-mesh sieve for the analysis of THg and MeHg (Meng et al., 2011).
R E S ULT S Growth and accumulations of TSe, THg and MeHg in rice plants
The growth of rice plants was significantly improved by the addition of Se under both hydroponic and soil pot conditions (see Supplementary Data Tables S1 and S2). Under hydroponic conditions, the high Se treatment (10 mM Se) increased the biomass of roots and shoots by 32 and 14 %, respectively. Under soil conditions, the yields of brown rice in the high Se treatment (5 mg g – 1 Se) were 29– 66 % higher than those in the control treatment. Whether under hydroponic or soil conditions, different Se treatments produced a dramatic effect on THg, MeHg and TSe accumulation in rice plants, with the lowest THg and MeHg concentrations in the plants subject to the high Se treatment (Figs 1 and 2; Supplementary Data Fig. S1). Under hydroponic conditions, the high Se treatment decreased the concentrations of THg in roots and shoots by 22 and 29 %, respectively, compared with the control treatment. Under soil conditions, THg and MeHg concentrations in brown rice in the high Se treatment were 47 and 55 % lower than those in the control treatment, whereas TSe
70 Shoot THg concentration (mg g–1)
detect the suberin lamellae in the root, sections were stained with Sudan red 7B (Brundrett et al., 1991) and viewed under the same microscope but under bright light.
Chemical analysis
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For THg and TSe analysis, soil and plant samples were microwave digested in concentrated HNO3 (16 mol L – 1) and measured by atomic fluorescence spectrometry (AFS; Beijing Titan Instrument Co., Ltd). For MeHg analysis, soil and plant samples were extracted by KBr – CuSO4/solvent and KOH – methanol/solvent, respectively (Liang et al., 1996), and determined with a MERX Automatic Methylmercury System (Brooks Rand Laboratories, Seattle, WA, USA), following method 1630 of USEPA (2001). For the analysis of Hg bioavailability, a sequential extraction technique for soil and sediment was employed, and only the water-soluble fraction among the five fractions represented the ‘bioavailable inorganic Hg’ (Bloom et al., 2003). Information on the quality assurance/ quality control (QA/QC) of our analytical data is available in the Supplementary Data.
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Statistical analysis
Data were analysed using the statistical software package SPSS 17.0 and summarized by means + standard errors (s.e.). Means were compared by least significant difference (LSD) at the 5 % level. Coefficients of determination (R 2) and significance probabilities (P) were computed for linear regression fits.
‘NF’
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F I G . 1. Concentrations of THg in shoots (A) and roots (B) of two rice cultivars (‘NF’, ‘Nanfeng’; and ‘ZX’, ‘Zixiang’) grown in 5 mM Hg solution without Se (control), and with low Se and high Se amendments for 2 weeks. Data are means + s.e. (n ¼ 4). Control, no Se supplied; Low Se, 1.0 mM Se supplied; High Se, 10 mM Se supplied.
Wang et al. — Se addition and mercury uptake in rice
Brown rice THg concentration (ng g–1)
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F I G . 3. Hg influx in excised roots of two rice cultivars, ‘Nanfeng’ (A) and ‘Zixiang’ (B). The excised roots were incubated in an aerated solution containing 0, 0.5, 2.5, 5.0, 12.5 or 25 mM Hg without Se (control, filled circles), and with low Se (open circles) and high Se (triangles) amendments at pH 6.0 for 20 min at room temperature (25 8C). Data are means + s.e. (n ¼ 4). Control, no Se supplied; Low Se, 1.0 mM Se supplied; High Se, 10 mM Se supplied.
grown under contaminated soil conditions was below the National Guidance Limit for crops ( , 20 ng g – 1) recommended by the Chinese National Standard Agency (Fig. 2A).
c
Kinetics of Hg uptake
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F I G . 2. Concentrations of THg (A), MeHg (B) and TSe (C) in brown rice of two rice cultivars (‘NF’, ‘Nanfeng’; and ‘ZX’, ‘Zixiang’) grown in a Hg-contaminated paddy soil without Se (control), and with low Se and high Se amendments. Data are means + s.e. (n ¼ 4). Control, no Se amendment; Low Se, 1 mg Se g – 1 amended; High Se, 5 mg Se g – 1 amended. The dashed line in (A) refers to the National Guidance Limit for crops recommended by the Chinese National Standard Agency.
concentrations in brown rice in the high Se treatment were 2.8-fold higher than those in the control treatment. It should be noted that the THg concentration in the high Se-treated rice
For both rice cultivars, Hg uptake could be described satisfactorily by Michaelis – Menten kinetics (Fig. 3). The kinetic parameters shown in Table 1 suggested that the uptake kinetics for Hg influx were significantly affected by different Se treatments. The Vmax values in the control treatment were 3.59 + 0.75 and 3.37 + 0.58 mmol Hg g – 1 f. wt h – 1 for ‘NF’ and ‘ZX’, respectively, whereas the respective values declined to 2.19 + 0.67 and 2.56 + 0.47 mmol Hg g – 1 f. wt h – 1 in the high Se treatment. Root anatomy: development of Casparian bands and suberin lamellae
At 5.0 cm from the root tip, no CBs were observed in the endodermis of either cultivar grown in the control treatment (Fig. 4A,
Wang et al. — Se addition and mercury uptake in rice TA B L E 1. Kinetic parameters for Hg influx into excised roots of cultivars ‘Nanfeng’ (‘NF’) and ‘Zixiang’ (‘ZX’) Rice cultivar ‘NF’
Se treatment
Vmax (mmol Hg g f. wt h – 1)
‘ZX’
Km (mM)
R2
6.40 + 2.37 7.15 + 3.02 4.66 + 3.63 7.37 + 2.47 8.70 + 2.24 7.84 + 1.73
0.948 0.961 0.905 0.980 0.983 0.978
The excised roots were incubated in an aerated solution containing 0, 0.5, 2.5, 5.0, 12.5 or 25 mM Hg without Se (control), and with low Se (1.0 mM Se) and high Se (10 mM Se) amendments at pH 6.0 for 20 min at room temperature (25 8C). Kinetic parameters were calculated from mean Hg influx (n ¼ 4) using a Michaelis–Menten function model.
Control
Control
Low Se
High Se
A
B
C
D
E
F
–1
3.59 + 0.75 3.19 + 0.59 2.19 + 0.67 3.37 + 0.58 2.95 + 0.47 2.56 + 0.47
Control Low Se High Se Control Low Se High Se
275
Low Se
‘NF’
‘ZX’
F I G . 5. Appearance of Casparian bands (A– F) in rice roots. Cross-sections were made at 1.0 cm from the root tip in cultivars ‘Nanfeng’ (‘NF’) and ‘Zixiang’ (‘ZX’) grown with the control (without Se) (A, D), low Se (1.0 mM Se supplied) (B, E) and high Se (10 mM Se supplied) (C, F) treatments under hydroponic conditions. The presence of Casparian bands was indicated by green-yellow fluorescence (see arrows in B, C, F). Scale bars ¼ 50 mm.
High Se
A
B
C
D
E
F
G
H
I
‘NF’
‘ZX’
‘NF’
in both cultivars, although they were more prominent in ‘NF’ (Fig. 4C, F). The suberin lamellae were detected as red stains in the cells walls after staining with Sudan red. In the control treatment, only a few cells in the endodermis of both cultivars had suberin lamellae at 5.0 cm from the root tip (Fig. 4G, J), whereas a single complete ring was observed in the low Se treatment (Fig. 4H, K). Suberin lamellae also appeared as 2 – 3 complete rings with thickened cell walls in the roots of the two cultivars grown in the high Se treatment (Fig. 4I, L). At 1.0 cm from the root tip of ‘NF’, dot-like CBs had begun to be observable in the endodermis in the low Se treatment (Fig. 5B), and well-developed CBs could be detected in the high Se treatment (Fig. 5C). However, for ‘ZX’, dot-like CBs were only observed in the endodermis in the high Se treatment (Fig. 5F). THg, MeHg concentration and Hg bioavailability in rhizosphere soil
J
K
L
‘ZX’
F I G . 4. Appearance of Casparian bands (A–F) and suberin lamellae (G–L) in the endodermis of rice roots. Cross-sections were made at 5.0 cm from the root tip in cultivars ‘Nanfeng’ (‘NF’) and ‘Zixiang’ (‘ZX’) grown with the control (without Se) (A, D, G, J), low Se (1.0 mM Se supplied) (B, E, H, K) and high Se (10 mM Se supplied) (C, F, I, L) treatments under hydroponic conditions. The presence of Casparian bands was indicated by green-yellow fluorescence (see arrows in B, C, F). Cross-sections were stained with the lipophilic dye Sudan red 7B; only a few cells were observed with lamellae in ‘Nanfeng’ (G) and ‘Zixaing’ (J) grown in the control treatment, but a complete ring of lamellae with red stain appeared in the low Se treatment (H, K). Two to three completes rings of lamellae with thickened cell walls were detected in the high Se treatment (I, L). Scale bars ¼ 50 mm.
D). As indicated by green-yellow fluorescence, dot-like CBs occurred in the radial walls of the endodermis of ‘NF’ in the low Se treatment, whereas in ‘ZX’ there were no CBs apparent (Fig. 4B, E). In the high Se treatment, well-developed CBs were observed
The THg concentration was 46 + 3.4 mg g – 1 in rhizosphere soil, and showed no significant difference between the Se treatments (data not shown). The MeHg concentration in rhizosphere soil ranged from 2.6 to 5.9 ng g – 1, and was significantly reduced by the addition of Se (Table 2). The low and high Se treatments decreased MeHg concentrations by 13 and 44 %, respectively, compared with the control treatment. The ‘water-soluble Hg’ fraction accounted for 0.34–0.45 % of the THg content of the soil, and was significantly affected by the addition of Se (Table 3). The low and high Se treatments decreased the ‘water-soluble Hg’ concentrations by 11 and 16 %, respectively, compared with the control treatment. A significant positive correlation was also found between the ‘watersoluble Hg’ concentrations in the rhizosphere soils and the THg concentrations in the brown rice (R 2 ¼ 0.87, P , 0.01) (Fig. 6). DISCUSSION Effects of Se on growth and accumulation of THg, MeHg and TSe in rice plants
The present study clearly demonstrates that the bioaccumulations of both THg and MeHg in rice tissues (brown rice, straw
Wang et al. — Se addition and mercury uptake in rice
TA B L E 2. MeHg concentrations in rhizosphere soils of two rice cultivars (‘NF’, ‘Nanfeng’; and ‘ZX’, ‘Zixiang’) grown in a Hg-contaminated paddy soil without Se (control), and with low Se and high Se amendments in a pot trial MeHg concentration (ng g – 1) Se treatment Control Low Se High Se
‘NF’
‘ZX’
4.8 + 0.48a 3.9 + 0.73b 2.6 + 0.74c
5.9 + 0.60a 5.4 + 0.97a 3.1 + 0.39b
Data are means + s.e. (n ¼ 4). Different letters within the same column indicate a significant difference between the Se treatments at the level of P , 0.05. Control, no Se amendment; Low Se, 1 mg Se g – 1 amended; High Se, 5 mg Se g – 1 amended.
TA B L E 3. Water-soluble Hg concentrations in rhizosphere soils of two rice cultivars (‘NF’, ‘Nanfeng’; and ‘ZX’, ‘Zixiang’) grown in a Hg-contaminated paddy soil without Se (control), and with low Se and high Se amendments in a pot trial Water-soluble Hg concentration (ng g – 1) Se treatment Control Low Se High Se
‘NF’
‘ZX’
194 + 28a 170 + 20a 158 + 17b
205 + 11a 187 + 37b 179 + 10b
Data are means + SE (n ¼ 4) Different letters within the same column indicate a significant difference between the Se treatments at the level of P , 0.05. Control, no Se amendment; Low Se, 1 mg Se g – 1 amended; High Se, 5 mg Se g – 1 amended.
and roots) were significantly reduced by the addition of Se, whereas total Se accumulations in brown rice and yields significantly increased in Se treatments under soil conditions (Fig. 2; Supplementary Data Fig. S1, Table S2). A similar response has also been observed in tomato (Solanum lycopersicum), radish (Raphanus sativus), soybean (Glycine max) and other plants (Shanker et al., 1996a, b; Yathavakilla and Caruso, 2007). Furthermore, THg concentrations in brown rice grown in Hg-contaminated soil amended with 5 mg Se g – 1 declined below the National Guidance Limit for crops ( , 20 ng g – 1) (Fig. 2A). This result suggests that the addition of Se not only produced high yielding and Se-enriched brown rice but also lowered its Hg and MeHg contents when plants were grown in Hg-contaminated soils.
Effects of Se on the Hg uptake process
The determination of the kinetics of Hg uptake is a necessary preliminary step for modelling soil – plant transfer in Hg-contaminated environments (Nowack et al., 2006). However, only a few studies have reported the kinetic parameters of Hg uptake in higher plants (Esteban et al., 2008, 2013). Our study suggests that Hg uptake by roots of rice
30 Brown rice THg concentration (ng g–1)
276
R 2 = 0·87, P < 0·01
27 24 21 18 Control Low Se High Se
15 12 9 0·15
0·16
0·17
0·18
0·19
0·20
Water-soluble Hg concentration (mg
0·21
g–1)
F I G . 6. Relationship between water-soluble Hg concentrations in rhizosphere soils and THg concentrations in brown rice from plants grown in a Hg-contaminated paddy soil without Se (control, filled squares), and with low Se (1 mg Se g – 1, open circles) and high Se (5 mg Se g – 1, solid triangles) amendments in a pot trial.
plants is an active process (Esteban et al., 2008), which needs selective binding sites and an energy source as the driving force. Furthermore, it is worth noting that Vmax values for both cultivars in the high Se treatment were significantly lower than those in the control treatment (Table 1), suggesting that the uptake of Hg into root cells through the symplastic pathway was restricted by the addition of Se via lowering the activity of membrane transporters. To the best of our knowledge, the effects of Se on the kinetic parameters of Hg uptake in roots of rice plants have never been reported. The only study by Feng et al. (2013) showed that Se could compete with Hg for specific binding sites, such as the thiol groups of cysteine in membrane proteins, to defend higher plants against abiotic stresses. Effects of Se on root anatomy
Uptake of elements through the apoplastic pathway is controlled by apoplastic barriers in the endodermis and exodermis (Schreiber, 2010). The development of these barriers varies and often differs between plant species and with environmental conditions (Meyer et al., 2009; Redjala et al., 2011). Our study suggests that the addition of Se could induce and enhance the development of apoplastic barriers in the endodermis, which may help to restrict the uptake and translocation of Hg into shoots. Previous studies also showed that some essential elements (e.g. Fe, S and Si) could induce the changes in root anatomy that may delay the uptake of heavy metals (e.g. Zn and Cd) and contribute to a higher metal resistance by plants (Cheng et al., 2012; Vaculik et al., 2012). However, the development of CBs and suberin lamellae was not so obvious in the exodermis of rice plants and showed no differences among the three Se treatments. This may be explained by the fact that the exodermis usually develops later and is less sensitive to environmental conditions, compared with the endodermis (Ma and Peterson, 2003; Vaculik et al., 2012). Furthermore, it is worth noting that the development of apoplastic barriers in the endodermis of ‘NF’ was more
Wang et al. — Se addition and mercury uptake in rice obvious than in ‘ZX’ (Figs 4 and 5), irrespective of the level of Se treatments. This suggests that the effects of Se on the development of apoplastic barriers in the endodermis might vary among cultivars, which may have different impacts on the uptake and translocation of Hg into shoots.
277
ACK NOW LED GE MENTS We thank Professor A. J. M. Baker (The Universities of Melbourne and Queensland, Australia) for help in the initial preparation and improvement of this paper. This work was supported by the National Natural Science Foundation of China (30770417, 31070450) and the National ‘863’ project of China (2012AA061510, 2013AA062609).
Effects of Se on Hg bioavailability and MeHg content in the rhizosphere
The bioavailability of Hg plays an important role in affecting its accumulation in rice plants (Peng et al., 2012; Wang et al., 2014). In our study, the bioavailable Hg was represented by the ‘water-soluble’ Hg fraction, which is the most mobile and bioavailable fraction and can be easily transported by natural processes and absorbed by plants (Issaro et al., 2009). Furthermore, this fraction is the major source of inorganic Hg accumulated in rice plants (Meng et al., 2010) and MeHg in paddy soil (Meng et al., 2014). Our results suggest that Se amendment enhanced the immobilization of Hg in the soil, leading to lower Hg bioavailability and restricting the production of MeHg (Table 2), which could finally lead to a reduction in the uptake and accumulation of THg and MeHg in rice plants.
Conclusions
This study systematically explored the effects of Se on the uptake process of Hg, root anatomy, an aspect of the Hg bioavailability in the rhizosphere and their relationships with Hg/MeHg accumulation in rice plants. Selenium addition not only significantly increased yields and TSe but also decreased THg and MeHg contents in brown rice in plants grown under Hgcontaminated soil conditions. Reducing the bioavailability of Hg and production of MeHg in the rhizosphere, suppressing the uptake process of Hg into root cells and enhancing the development of apoplastic barriers in the endodermis of roots may be major reasons for the lowered accumulation of THg and MeHg in rice plants. This study enhances our understanding of the factors involved in Se – Hg antagonism in rice plants and so provides a potentially useful method to produce simultaneously safe and enriched Se brown rice for plants grown under Hg-contaminated paddy field conditions.
S U P P L E M E N TARY D ATA Supplementary data are available online at www.aob.oxford journals.org and consist of the following. Details of quality assurance/quality control (QA/QC) for THg, TSe and MeHg analysis in the samples. Figure S1: concentrations of THg in roots and straw of rice grown without Se, and with low Se and high Se amendments in a pot trial. Figure S2: relationships between TSe concentrations in brown rice, straw, root tissues and THg and MeHg concentrations in brown rice tissues of rice plants. Table S1: biomass of root and shoot of rice grown in 5 mM Hg solution without Se, and with low Se and high Se amendments for 2 weeks. Table S2: biomass of roots, straw, husk and brown rice tissues of rice grown in a Hg-contaminated paddy soil without Se, and with low Se and high Se amendments in a pot trial.
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