Effect of Endogenous Selenium on Arsenic Uptake and Antioxidative Enzymes in As-exposed Rice Seedlings

Effect of Endogenous Selenium on Arsenic Uptake and Antioxidative Enzymes in As-exposed Rice Seedlings

Abstract

Arsenic (As) and selenium (Se) are two metalloids found in the environment. As poses the most significant threat to human health and plant growth due to its prevalence and toxicity, whereas Se is a required micronutrient for human health. In this study, hydroponic experiments were performed to investigate whether Se can mitigate As toxicity in rice (Oryza sativa L.) seedlings. We found that As uptake by rice roots was increased by pretreatment for 7 days with Se in the form of selenite [Se(IV)] or selenate [Se(VI)]; however, co-application of arsenite [As(III)] or arsenate [As(V)] with Se(VI) markedly reduced As uptake by roots. Co- or pretreatment with Se in 1 µM As(III) or 5 µM As(V) solution decreased As content in the shoot. Conversely, Se pretreatment prior to addition of 5 µM As(III) or 1 µM As(V) resulted in As accumulation in the shoot as compared to co-application of Se and As. The translocation of As in the shoot was lower whereas the transfer factor was higher upon simultaneous application of Se and As as compared to Se pretreatment. Se supplementation of As(III) or pretreatment increased the activities of antioxidant enzymes [peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT)] in the root and shoot, but decreased glutathione (GSH) and malondialdehyde (MDA) contents in the shoot. Plants under As(V) treatment showed the same trend except that CAT content was decreased in the root and shoot while MDA content was increased in the shoot. These results suggest that cultivating rice in the presence of Se can reduce accumulation of toxic As in seedlings, ensuring the safety of this important crop for human consumption.

Keywords: As, Se, Rice seedling, Uptake, Translocation, Antioxidant.

1. Introduction

Arsenic (As) and selenium (Se) are among the inorganic environmental contaminants causing the greatest concern due to their influence on humans and animals. The presence of As and Se in the environment is regulated by environmental and public health authorities (Goh et al., 2004). As and Se are also toxic to plants, acting either directly or indirectly through accumulation in plant tissues, which can in turn lead to their entry into the animal and human food chains. Rice cultivation is a process that requires an abundance of water; as such, accumulation of inorganic As in form of As(III) is higher in rice as compared to other crops (Kumar et al., 2013). At high concentrations, As can reduce crop growth and productivity and alter mineral uptake (Kumar et al., 2016). As(V) uptake can be reduced by iron (Fe) through formation of Fe plaques in the root (Tripathi et al., 2007). The As content of lowland or paddy rice grain is generally much higher than that of upland cereal crops (Williams et al., 2007). As a result of environmental contamination, tens of millions of people are exposed to high levels of As through drinking water and diets.

Arsenite [As(III)] and arsenate [As(V)] are inorganic, phytoavailable forms of As that are highly toxic to plants (Mkandawire et al., 2004). In natural waters, As predominantly exists as As(V), which is taken up by and is toxic to plants (Zhang et al., 2008). As(III) and As(V) concentrations of 25 and 250 µM, respectively, were toxic to Hydrilla verticillata growth (Srivastava et al., 2007), and application of 20–50 µg/L As(III) or As(V) is sufficient for Lemna gibba growth inhibition (Mkandawire et al., 2005).

Se is not an essential element for plants, but is an important micronutrient for both humans and animals. Selenium is an essential trace element in mammals, with the majority specifically encoded as seleno-L-cysteine into a range of selenoproteins. Many of these proteins play a key role in modulating oxidative stress, via either direct detoxification of biological oxidants, or repair of oxidised residues (Carroll et al., 2015). Se supplementation was found to reduce As(III) in rice by increasing antioxidant defence (Kumar et al., 2016) as well as the thiol and cadmium (Cd) stress responses and lead (Pb) stress response in Vicia faba L. (Nadgórska-Socha et al., 2013). The biochemical functions of Se in plants have been widely studied (El-Sharaky et al., 2007; Terry et al., 2000). At high concentrations, Se can act as a pro-oxidant and cause damage to plants (Feng et al., 2012); however, at low concentrations, Se exerts positive effects such as growth enhancement, increased antioxidative capacity, a reduction of reactive oxygen species and lipid peroxidation, and increased accumulation of starch (Turakainen et al., 2004; Xue et al., 2001). Se in selenoproteins protects against oxidative stress and promotes immune and thyroid functions (Lazarus et al., 2011), and can counteract the detrimental effects of environmental stresses through various mechanisms (Kumar et al., 2013). A recent study reported that interaction between inorganic As and Se species alters their uptake and accumulation in rice plants grown under hydroponic conditions (Hu et al., 2014), although the detailed mechanism remains unclear. There is also limited information on the effects of endogenous Se on the uptake, translocation, and oxidative stress caused by As exposure in plants.

We previously demonstrated that the addition of Se(IV) significantly increased the rice root As content, but decreased the rice shoot As content (Camara et al., 2018). We speculated that Se might influence the As translocation to the shoot, but not the uptake process. To test this hypothesis, in this study we used endogenous Se to investigate the potential role of Se in translocation of As and influence of antioxidant enzyme activities in rice seedlings pretreated with Se.

2. Materials and Methods
   1. Rice culture conditions

Rice (Oryza sativa L., Fengyuanyou 299) seeds were surface sterilised using 30% hydrogen peroxide (v/v) for 15 min, rinsed with distilled water, and soaked in a saturated CaSO₄ solution overnight at 25℃ ± 2℃ in the dark. Seeds were germinated in a moist, pre-sterilised plastic net floating sheet in deionised water at 25℃. After 7 days, the seedlings of uniform size were selected from germinated seeds and transferred to 2.5-L plastic pots (four plants per pot) containing nutrient solution for 35 days. The composition of half-strength Kimuar nutrient solution was (mM): KNO₃ (0.091), Ca(NO₃)₂∙4H₂O (0.183), MgSO₄∙7H₂O (0.274), KH₂PO₄ (0.1), (NH₄)₂SO₄ (0.183), MnSO₄∙H₂O (1 × 10⁻³), H₃BO₃ (3 × 10⁻³), (NH₄)₆Mo₇O₂₄∙4H₂O (1 × 10⁻³), ZnSO₄∙7H₂O (1 × 10⁻³), CuSO₄∙5H₂O (2 × 10⁻⁴), and Fe(III)-EDTA (6 × 10⁻²). The pH was adjusted to 5.5 using KOH or HCl and each treatment was carried out in triplicate (in three pots). The nutrient solution was renewed twice a week. Plants were grown in a controlled environment greenhouse at 25 ± 4℃/20 ± 2℃ day/night temperatures, 14-h photoperiod, light intensity of 240–350 µmol·(m²·s)⁻¹, and relative humidity of 60%–70%.

2. As and Se treatments

In all treatments, arsenite, arsenate, selenite and selenate were abbreviated as As(III) for NaAsO₂, As(V) for Na₂HAsO₄, Se(IV) for Na₂SeO₃, and Se(VI) for Na₂SeO₄, respectively.

Experiment 1. Effect of Se pretreatment on As(III) uptake

The effect of endogenous Se on As(III) uptake and translocation was investigated. After 42 days of seedling growth, those of uniform size were transferred to pots (two plants per pot) containing 2.5-L nutrient solution to which Se (5 µM Na₂SeO₃ or Na₂SeO₄), or As (1 or 5 µM NaAsO₂) was added for a total of 10 treatments: (1) 1As(III); (2) 5As(III); (3) 1As(III) + Se(IV); (4) 1As(III) + Se(VI); (5) 5As(III) + Se(IV); (6) 5As(III) + Se(VI); (7) pre-Se(IV) + 1As(III); (8) pre-Se(VI) + 1As(III); (9) pre-Se(IV) + 5As(III); and (10) pre-Se(VI) + 5As(III). Plants were pretreated with Se for 2 days before As application. The composition of the other nutrients was the same as that in normal nutrient solution (pH 5.5). Plants were collected after 2 and 7 days.

Experiment 2. Effect of Se pretreatment on As(V) uptake

The effect of endogenous Se on As(V) uptake and translocation was investigated. After 42 days of seedling growth, those of uniform size were transferred to pots (two plants per pot) containing 2.5-L nutrient solution to which Se (5 µM Na₂SeO₃ or Na₂SeO₄) or As (1 or 5 µM Na₂HAsO₄) was added for a total of 10 treatments: (1) 1As(V); (2) 5As(V); (3) 1As(V) + Se(IV); (4) 1As(V) + Se(VI); (5) 5As(V) + Se(IV); (6) 5As(V) + Se(VI); (7) pre-Se(IV) + 1As(V); (8) pre-Se(VI) + 1As(V); (9) pre-Se(IV) + 5As(V); and (10) pre-Se(VI) + 5As(V). Plants were pretreated with Se for 2 days before As application. The composition of the other nutrients was the same as that in normal nutrient solution (pH 5.5). Plants were collected after 2 and 7 days.

Experiment 3. Effect of Se on enzymatic activities in plants exposed to As(III) or As(V)

The effect of Se on enzymatic activity in rice seedlings exposed to As(III) or As(V) was investigated. After 42 days of seedling growth, those of uniform size were transferred to pots (two plants per pot) containing 2.5-L nutrient solution to which Se (5 µM Na₂SeO₃ or Na₂SeO₄) or As (5 µM NaAsO₂ or Na₂HAsO₄) was added for a total of 11 treatments: (1) As(III); (2) As(V); (3) As(III) + Se(IV); (4) As(III) + Se(VI); (5) As(V) + Se(IV); (6) As(V) + Se(VI); (7) pre-Se(IV) + As(III); (8) pre-Se(VI) + As(III); (9) pre-Se(IV) + As(V); (10) pre-Se(VI) + As(V); (11) CK (without As and Se addition). Plants were pretreated with Se for 2 days before As application. The composition of the other nutrients was the same as that in normal nutrient solution (pH 5.5). Plants were collected after 7 days.

3. Sample preparation and analysis of As content

After exposure to As for different times, plants were harvested and rinsed with deionised water, and the roots were dipped in an ice-cold desorption solution composed of 0.5 mM Ca(NO₃)₂, 5 mM MES (pH 5.5), and 1 mM K₂HPO₄ (Hu et al., 2014) for 15 min to remove adhered As. The roots and shoots were separated, dried, weighed, and powdered. To determine the As contents of roots and shoots, approximately 0.2500 g of fine plant material powder was digested with 8 mL concentrated nitric acid. Digestion tubes were allowed to stand overnight at room temperature and the following day, the sample was heated in the microwave oven (MARSS; CEM Corp., Matthews, NC, USA). The supernatant solution was cooled, diluted in 50 mL deionised water, and passed through a 0.45-µm filter before analysis with an atomic fluorescence spectrometer (AFS-920; Beijing Jitian Instruments Co., Beijing, China). For quality control, the standard reference material GBW10049 (GBS-27) and blanks were included in the digestion procedure. The recovery of the reference material was 85%–110%.

4. Sample preparation and analysis of antioxidant enzyme activities and MDA content

Fresh plant samples were frozen in liquid nitrogen, pulverised, and stored at −25℃. To assess antioxidant enzyme levels, shoot and root samples exposed to As for 7 days were ground in phosphate buffer (pH 7.3) and the extract was centrifuged at 4000 rpm for 10 min by using kits (Jiancheng Bioengineering Institute, Nanjing, China). The extract for glutathione (GSH) analysis was centrifuged at 10000 rpm for 15 min. The extraction and the centrifugation were performed under 4℃, to determine antioxidative enzyme (POD, SOD, and CAT) activities   and antioxidative non enzyme (GSH and MDA) levels.

For POD activity, the reaction mixture contained 1 mL extraction buffer was added with 0.3 mL 20 mM guaiacol and 0.2 mL 40 mM H₂O₂. The absorbance at 470 nm of the mixture was performed after 30 min of reaction at 37℃ in water for enzyme calculation. For SOD, the reaction mixture was performed by adding the enzyme extract to 0.3 mL of reaction that obtained 10 mm L-methionine, 50 µM NBT, and 0.005% riboflavin (w/v). The absorbance was determined at 550 nm then illuminated after 40 min at 37℃ water-bath. CAT activity was obtained by analyzing the amount of H₂O₂. 1 mL of supernatant was mixed with 1 mL reaction mixture solution containing 163 mM H₂O₂ and 100 mM (NH₄)₆Mo₇O₂₄. The CAT was tested by monitoring the decrease of the absorbance at 405 nm for 1 min at 37℃ in water.

The GSH concentration was determined based on the reduction of DTNB. A 1 mL aliquot of supernatant was mixed in a 3 mL of reaction solution containing 0.5 mM EDTA, 3 mM NADPH. The absorbance at 412 nm of reaction mixture was expressed after 1 min of water-bath at 37℃. The MDA content was calculated based on the MDA’s extinction coefficient of 155 mM^–1 cm^–1.

5. Data analysis

As contents in roots and shoots (C_Root−As, C_Shoot−As) were calculated based on dry weight. Total As (T_As), the proportion of As distributed to shoots (Shoot−As%), and transfer factor (TF) were calculated with the following equations:

T_Root−As

=

C_Root−As

×

Root_{Dry biomass}      (1)

T_Shoot−As

=

C_Shoot−As

×

Shoot_{Dry biomass}      (2)

T_As

=

T_Root−As + T_Shoot−As       (3)

As uptake

=

T_As/Root_{Dry biomass}      (4)

Shoot–As%

=

(T_Shoot−As/T_As)

×

100%      (5)

TF

=

C_Shoot−As/C_Root−As       (6)

 

6. Statistical analysis

Statistical analysis were performed using SPSS v.20.0 software for Windows and Microsoft Excel 2010. One-way analysis of variance with multiple comparisons using Turkey’s test was used to compare means among different treatments and to evaluate significant effects at P < 0.05.

3. Results
   1. Effect of Se pretreatment on As(III) uptake and translocation in rice seedlings

Se pretreatment for 2 days did not affect As uptake by rice roots (Fig. 1). As content was increased by 29.6% (P < 0.05) by co-application of 1 µM As(III) and Se(IV), but was decreased by 28.0% when Se(VI) was added (Fig. 1A). Meanwhile, co-treatment of 5 µM As(III) with Se(IV) increased As uptake by 25.5% after 2 days (Fig. 1B). After 7 days, the highest As uptake by roots relative to treatment with As(III) alone was observed by co-application of As(III) and Se(IV). As uptake was increased by 27.1% upon co-treatment with 1 µM As(III) and Se(IV) and was decreased by 31.0% when Se(VI) was used instead (Fig. 1A). On the other hand, uptake was increased by 35.7% in the presence of 5 µM As combined with Se(IV) and was decreased by 26.8% with Se(VI) supplementation (P < 0.05) (Fig. 1B). In contrast to co-application of the two compounds, for lower concentrations of As (1 µM) Se pretreatment for 2 days—i.e. pre-Se(IV) + As(III) or pre-Se(VI) + As(III)—decreased As uptake by 41.8% and 14.9%, respectively, as compared to As(III) single treatment for longer times (Fig. 1A). However, at higher As concentration, Se(IV) or Se(VI) pretreatment increased As uptake by 15.2% and 15.7%, respectively, as compared to As alone after 7 days (Fig. 1B).

Figure 1. Effect of Se pretreatment and co-application on As uptake by rice seedlings exposed to low (A) and high (B) As(III) levels. Data represent mean + SE (n = 3). Different letters indicate statistically significant differences among treatments (P < 0.05).

The accumulation of As in the rice shoot showed different trends. There was little difference among treatment groups after 2 days of exposure, except that 1 µM As(III) co-applied with Se(IV) decreased As distribution in the shoot to 12.7% (Fig. 2A). However, after 7 days, Se co- and pretreatments significantly affected the distribution of As between roots and shoots (Fig. 2A): 68.9% of As taken up in the As-only treatment group was distributed in the shoots as compared to just 18.0% and 20.0% in plants co-treated with As plus Se(IV) or Se(VI), respectively, and 37.1% and 39.3% in plants pretreated with Se(IV) or Se(VI) for 2 days, respectively (Fig. 2A). However, at an As concentration of 5 µM, addition of Se significantly affected As uptake at short exposure times (Fig. 2B). Compared to application of As alone, As(III) and Se(VI) co-treatment increased the As content in the shoot by 30%, whereas the other treatments decreased As distribution between the roots and shoots by 26.5% to 68.6% after 2 days. At an exposure time of 7 days, Se pretreatment had no effect on the As accumulation in rice shoots. However, co-application of Se and As(III) decreased the proportion of As in the shoots by 78.4% with Se(IV) and by 49.2% with Se(VI) (Fig. 2B) relative to As single treatment.

Figure 2. Effect of Se pretreatments and co-application on As distribution in the shoot of rice seedlings exposed to a low (A) and high (B) concentration of As(III). Data represent mean + SE (n = 3). Different letters indicate statistically significant differences among treatments (P < 0.05).

The shoot-to-root TF value for As was higher in plants pretreated with Se than in those co-treated with the As and Se (Table 1). The TF value was higher for plants exposed to a lower as compared to a higher As concentration. For 1 µM As, the average shoot-to-root TF values in Se-pretreated plants were 0.37 and 0.34 for exposure times of 2 and 7 days, respectively, which were higher than the values in co-application groups (0.26 and 0.10, respectively). TF values were lower at an As(III) concentration of 5 µM: the average TF with Se(IV) or Se(VI) pretreatment was 0.05 and 0.08 at 2 and 7 days, respectively. However, upon co-application of As and Se, TF values were 0.1 and 0.02, respectively, whereas for As(III) alone, the values were 0.1 and 0.09, respectively (Table 1).

Table 1. Effect of Se pretreatments co-application on the transfer factor of As in rice seedlings exposed to As(III)

Treatment  （1 µM As）	Exposure time (days)			Treatment  （5 µM As）	Exposure time (days)
	2	7			2	7
1AsIII	0.54 ± 0.91 a	0.93 ± 0.05 a		5AsIII	0.10 ± 0.01 a	0.09 ± 0.01 a
Pre-SeIV+AsIII	0.42 ± 0.08 a	0.28 ± 0.03 b		Pre-SeIV+AsIII	0.03 ± 0.01 a	0.09 ± 0.01 a
Pre-SeVI+AsIII	0.33 ± 0.05 a	0.40 ± 0.08 b		Pre-SeVI+AsIII	0.07 ± 0.01 a	0.07 ± 0.00 a
1AsIII+5SeIV	0.05 ± 0.01 b	0.10 ± 0.01 c		5AsIII+5SeIV	0.05 ± 0.01 a	0.02 ± 0.00 a
1AsIII+5SeVI	0.47 ± 0.01 a	0.10 ± 0.01 c		5AsIII+5SeVI	0.15 ± 0.02 a	0.03 ± 0.00 a

Data represent mean ± SE (n = 3). Different letters in the same column indicate statistically significant differences among treatments (P < 0.05).

2. Effect of Se pretreatment on As(V) uptake and translocation in rice seedlings

Co-application of Se(IV) or Se(VI) with As(V) or Se pretreatment (2 days) did not significantly alter As uptake after 2 days as compare to As(V) treatment alone (Fig. 3), except in the case of simultaneous treatment with 5 µM As and Se(IV), which increased As uptake by 47.1% as compared to treatment with As only (Fig. 3B). Increasing the exposure time to 7 days increased As uptake by roots; addition of Se(IV) increased As uptake by 54.3% and 57.5% at As concentrations of 1 and 5 µM, respectively (P < 0.05) (Fig. 3A, B) as compared to As treated alone. In contrast to Se(IV), Se(VI) application did not affect the As uptake by the roots. Moreover, unlike simultaneous exposure to As and Se, pretreatment with Se increased As accumulation in roots; however, this increase was only significant (P < 0.05) in pre-Se(IV) with a lower As concentration (Fig. 3A), which increased As accumulation by 37.0%.

Figure 3. Effect of Se pretreatments and co-application on As uptake by rice seedlings exposed to low (A) and high (B) As(V) concentrations. Data represent mean + SE (n = 3). Different letters indicate statistically significant differences among treatments (P < 0.05).

A greater percentage of As was transported to shoots in plants pretreated with Se as compared to those that exposed to As and Se simultaneously (Fig. 4). After 2 days at a low As(V) concentration [pre-Se(IV)+As(V)], 70.1% of As was distributed in rice shoot, whereas only 48.4% of As was transported from the root to shoot in the pre-Se(VI)+As(V) group. Co-treatment groups showed less As accumulation in the shoot at 31.2% and 39% in the presence of Se(IV) and Se(VI), respectively (Fig. 4A). However, at 5 µM As there was no difference between As-treated and pre-Se(VI)+As(V) groups (47.2% vs. 49.2%). Pre-Se(IV)+As(V) and co-application with Se(IV) or Se(VI) decreased the fraction of As in the shoot by 20.9%, 13.3%, and 22.2% respectively (Fig. 4B).

In addition to As uptake, As distribution in the shoot of rice seedlings was also affected by longer exposure times and As concentration. At 1 μM, 48.9%, 46.6%, and 31.1% of As was taken up by the shoot in the As single treatment and pre-Se(IV) and -Se(VI) groups, respectively. In contrast, only 10.1% and 28.3% of As was transported to the shoot upon co-application of Se(IV) or Se(VI) (Fig. 4A). At a concentration of 5 μM, 30.4% of As was distributed in the shoot in the absence of Se. However, pre-Se(IV) or -Se(VI) decreased As distribution in the shoot by 56.8% and 33.5%, respectively (P < 0.05) relative to As(V) alone, as compared to 81.1% and 31.7%, respectively, by co-treatment with Se(IV) or Se(VI) (Fig. 4B).

Figure 4. Effect of Se pretreatments and co-application on As distribution in the shoot of rice seedlings exposed to low (A) and high (B) As(V) concentrations. Data represent mean + SE (n = 3). Different letters indicate statistically significant differences among treatments (P < 0.05).

Se pretreatment and co-application decreased the TF values in rice, except that Pre-Se(VI)+1 µM As(V) treatment increased by 50.9%, compared with As(V) treatment alone. And among the Se addition treatments, Pre-Se(VI)+As(V) treatments showed higher TF values (Table 2). At 5 µM As, co-application with Se decreased TF values by 82.6% and 85.7% with Se(IV) and 69.6% and 57.1% with Se(VI) at 2 and 7 days, respectively, relative to treatment with As(V) alone. Similarly, Se pretreatment also decreased the TF value by 73.9% and 71.4% for Pre-Se(IV) and 8.7% and 50% for Pre-Se(VI) at 2 and 7 days, respectively, relative to the value for As(V) treatment alone (Table 2).

Table 2. Effect of Se pretreatment and co-application on the transfer factor of As in rice seedlings exposed to As(V)

Treatment  （1 µM As）	Exposure time (days)			Treatment  （5 µM As）	Exposure time (days)
	2	7			2	7
1AsV	0.26 ± 0.03 b	0.27 ± 0.02 a		5AsV	0.23 ± 0.02 a	0.14 ± 0.02 a
Pre-SeIV+AsV	0.24 ± 0.01 b	0.25 ± 0.01 a		Pre-SeIV+AsV	0.06 ± 0.01 b	0.04 ± 0.01 b
Pre-SeVI+AsV	0.53 ± 0.02 a	0.13 ± 0.02 b		Pre-SeVI+AsV	0.21 ± 0.01 a	0.07 ± 0.00 b
1AsV+5SeIV	0.11 ± 0.02 c	0.02 ± 0.00 c		5AsV+5SeIV	0.04 ± 0.00 b	0.02 ± 0.00 b
1AsV+5SeVI	0.17 ± 0.03 c	0.11 ± 0.01 b		5AsV+5SeVI	0.07 ± 0.00 b	0.06 ± 0.01 b

Data represent mean ± SE (n = 3). Different letters in the same column indicate statistically significant differences among treatments (P < 0.05).

3.3 Rice root and shoot POD, SOD, and CAT activities and GSH and MDA contents

Simultaneous application of Se and As or Se pretreatment increased root GSH content relative to the control treatment (P < 0.05). GSH content in the roots was decreased when plants were challenged with As(V) in the presence of Se if compared to As(V) single treatment (Fig. 6A). The percentage decrease in GSH content in the roots compared to the As(V) alone was 14.8% with pre-Se(IV) and from 37.0% and 25.9% with Se(IV) and Se(VI) respectively in their simultaneous treatment (Fig. 6A). Pre-Se treatment and Se(VI) co-application did not affect GSH content in root if compared to As(III) alone, except Se(IV) co-exposure with As(III) where GSH content in the root was decreased by 40.9% (Fig. 6A) compare to As(III) alone. However, Se co-application  had no effect on GSH content in the shoot, but increased by 20% and 25% with pre-Se(IV) and pre-Se(VI) respectively as compared to As(III) alone (Fig. 6B). Addition of pre-Se(IV) or pre-Se(VI) before As(V) increased GSH content in shoot by 25% and 14.3% and increased by 34% – 29.8% with Se(IV) and Se(VI) respectively if compare to As(V) alone (Fig. 6B). POD activity was unchanged by treatment with As alone or in combination with Se as compared to that in control seedlings (Fig. 5A, B). However, POD activity was slightly increased in roots treated with As(V) (Fig. 5B) as compared to As(V) alone.

No much difference was observed in SOD activity upon exposure pre-Se treatment if compare to As(III) alone. However, simultaneous application with Se(IV) decrease SOD activity by 29.9%, but increased (P < 0.05) by 29.7% as compared to As(III) single treatment (Fig. 5C). In the root, SOD activity increased from 17.6 to 35.7 U/g fresh weights. Similarly, As(V) in conjunction with Se(VI) significantly increase (P < 0.05) SOD activity in the roots by 44.2% compare to As(V) alone. However, Se(IV) or se(VI) pretreatment decreased SOD activity in the root by 47.9% and 59.6%, respectively, as compared to As(V) alone (Fig. 5C). The activity was decreased in the shoots relative to the As(III) or As(V) in all treatment groups (Fig. 5D), except in the case of pre-Se(IV)+As(V) and As(V)+Se(IV) which showed a significant increase in SOD by 24.3% and 29.1% as compare to A(V) alone (Fig. 5D).

CAT activity in rice roots was increased relative to control plants As(III) groups (P < 0.05) but not As(V) (Fig. 5E). In seedlings pretreated with Se(VI), exposure to As(III) stress increased CAT activity by 26.4% in the root as compared to As(III) single treatment (Fig. 5E). However, if compare to As(V) alone, CAT activity in root significantly increase (P <0.05) and was ranged from 28% to 46.1% with pre-Se(IV) and pre-Se(VI) and by 24.3% to 37.1% with Se(IV) or Se(VI) respectively (Fig. 5E). The activity in the shoots significantly increase by 31.9%, 21.2% and 21.5% upon pre-Se(IV), pre-Se(VI) and Se(IV) co-exposure with As(III) as compare to As(III) alone addition (Fig. 5F). On the other hand, CAT activity was increased significantly by 39.3% and 35.2% respectively with pre-Se(IV) and pre-Se(VI) in the shoot while co-exposure of As(V) with Se(IV) or Se(VI) significantly increase CAT activity by 72.0% and 57.7% respectively as compare to As(V) single treatment (Fig. 5F).

Since the MDA content of rice seedling roots was below the limit of detection, only the shoots were analysed (Fig. 6C). Compared to As(V) alone treated plants, MDA content in the shoot was increased significantly by 54.3% with Se(IV) pretreatment for 2 days before As(V) application (Fig. 6C). The MDA content of the shoot was higher in the presence of As(V) as compared to As(III), although there was no significant difference between the two groups except in the case of pre-Se(IV)+As(V) plants (Fig. 6C).

Figure 5. Effect of Se pretreatments and co-application on antioxidative enzyme levels in rice roots (A, C, E) and shoots (B, D, F) exposed to As (As: 5 µM; Se: 5 µM). Data represent mean + SE (n = 3). Dashed lines indicate the reference lines on the control. Different letters indicate statistically significant differences among treatments (P < 0.05).

Figure 6. Effect of Se pretreatments and co-application on antioxidative non enzyme levels in rice roots (A) and shoots (B, C) exposed to As (As: 5 µM; Se: 5 µM). Data represent mean + SE (n = 3). Dashed lines indicate the reference lines of the control. Different letters indicate statistically significant differences among treatments (P < 0.05).

4. Discussion
   1. As uptake by rice seedlings

Se is known as essential trace element for humans and animals (Allan et al., 1999); its deficiency can lead to Keshan and Kashin–Beck diseases, which have been reported in regions characterised by extremely low Se content in the soil and crops (Fordyce et al., 2000); cereals and cereal products are the main dietary source of Se for these populations (Rayman, 2008). Se may have a beneficial role in plants under As stress (Hartikainen and Xue, 1999). The present study investigated the effects of Se and As co-exposure and Se pretreatment (2 days before addition of As) on As uptake and translocation from root to shoot as well as enzymes activities in rice seedlings.

Se can alleviate the toxicity of heavy metals in plants by inhibiting the uptake of As, Hg, and Pb (Ebbs et al., 2001; Yathavakilla et al., 2007). In this study, rice plants accumulated high levels of As particularly in the roots; this was blocked by Se in a dose-dependent manner. This is consistent with previous findings in mung bean (Malik et al., 2012). Meanwhile, all plants in the As(III)+Se(IV) group had higher As concentrations in the roots than in the shoots as compared to treatment with As alone (Fig. 1A, B); a similar result was obtained for the As(V)+Se(IV) group (Fig. 3A, B). It was previously reported that simultaneous application of Se(IV) and PO₄³⁻ at lower concentrations reduced As(III) uptake (Kumar et al., 2013). However, we found that there was less As accumulation when plants were pretreated as opposed to co-treated with Se. It has been suggested that there is competitive inhibition among Se(IV), As(III), and PO₄³⁻ or As(V), Se(IV), and PO₄³⁻(Kumar et al., 2016). However, the opposite was reported by another study showing that Cd concentrations in the shoots and roots were higher for Cd+Se than for pre-Se+Cd (Lin et al., 2012). Se was found to lower the Cd content in both shoots and roots in rape seedlings (Filek et al., 2008). Thus, in rice as in rape seedlings, phosphate can inhibit heavy metal uptake; the rate of uptake is expected to be similar in the two species (Li et al., 2008; Meharg et al., 1991; Zhang et al., 2014). Competition for specific binding sites in proteins can partly explain the reduced heavy metal uptake and protective effect of Se against heavy metal toxicity (Kumar et al., 2013). Additionally, Se has been shown to mitigate stress in plants induced by other abiotic factors via several different mechanisms (Feng et al., 2013).

Co-application of Se(IV) and As(III) resulted in greater accumulation of As in the roots than co-application of Se(VI) and As(III) (Fig. 1A, B). The opposite was observed upon Se pretreatment, where total As uptake in the root was higher in the pre-Se(IV)+As(III) than in the pre-Se(IV)+As(III) group. As accumulation was lower under 1 µM As(III) than at a concentration of 5 µM (Fig. 1A, B); moreover, As(III) was taken up more rapidly than As(V) (Fig. 3A, B). These findings suggest that uptake of Se(IV) can suppress that of both As(III) and As(V) and that accumulation of the two compounds occurs via two distinct transport pathways likely involving silicon and phosphate, respectively (Kumar et al., 2016). Co-treatment with Se and As(V) increased As uptake by Thunbergia alata (Bluemlein et al., 2009). Stimulation of the heavy metal uptake by Se has also been reported in Salix alba (Cd and Cu) (Fargašová et al., 2006), wheat, and pea (Cd and Cu) (Landberg and Greger, 1994). These findings suggest a mutual antagonism between Se and As or other heavy metals in plants, although no such relationship was observed between As(V) and Se(IV) in plants or animals (Kumar et al., 2016). Se/As antagonism in the soil environment may occur via several mechanisms. For instance, it was reported that the two species form an insoluble complex such as orpiment (As₂Se₃), thereby decreasing their bioavailability (Afton et al., 2009). Moreover, abiotically or microbially produced sulphide can chemically reduce As, resulting in the formation of As₂Se₃ (Stolz and Oremland, 1999).

2. As translocation from root to shoot in rice seedlings

Plants have several protective mechanisms against As toxicity, including compartmentalisation and translocation (Tu and Ma, 2003). When these mechanisms are inadequate, biochemical processes are induced for detoxification (Srivastava et al., 2005). In this study, the amount of As translocated from the root to shoot of rice seedling was lower in the presence of low as compared to high As(III) concentration (Fig. 2A, B). A greater decrease in As translocation in the shoot of rice plants was observed upon Se co-treatment as compared to pretreatment, indicating that endogenous Se does not affect this process, possibly due to the presence of As adsorption on surfaces or in the apoplastic pathway in the root (Liu et al., 2005) that affect the movement of As from roots to shoots via the xylem, which is driven by transpiration from the leaves (Salt et al., 1995). Similar observations on As translocation from root to shoot were made when rice seedlings were exposed to As(V) (Fig. 4A, B). This may be explained by the deficiency in sulphur hydride-rich compounds in the roots of plants, which restricts As translocation to the shoot through binding and eventual sequestration into root vacuoles (Tripathi et al., 2007). The decrease in root to shoot As translocation was most evident upon co-treatment with Se(IV) and As(V) (Fig. 4A, B). Similar results have been reported in T. alata (Bluemlein et al., 2009). In addition, the improvement of heavy metal-induced membrane disturbance by Se may be associated with enhancement of fatty acid unsaturation (Kumar et al., 2013).

The difference in As distribution between shoots and roots suggest variations in As translocation mechanisms in different parts of the plant. The lower shoot to root As ratio in rice suggested that less As accumulated in the shoots, possibly facilitating an intracellular detoxification mechanism that may be at least partly responsible for the reduced As sensitivity of rice seedlings (Lou-Hing et al., 2011). When the seedlings were exposed to 1 µM As(III) along with Se(IV) or Se(VI), the same TF value of 0.10 was observed after 7 days, which was lower than that of the pre-Se(IV) and -Se(VI) groups (0.28 and 0.40, respectively) (Table 1). The root-to-shoot ratios of 0.02 and 0.03 upon co-treatment with 5 µM As(III) and Se(IV) or Se(VI), respectively, were also lower than those observed for pre-Se(IV) or -Se(VI) (0.09 and 0.07, respectively). Similar results were obtained in seedlings exposed to As(V), which showed TF values that were higher than those of Se pretreated plants (Table 2). These observations are consistent with previous findings (Hu et al., 2014a). In contrast, in plants exposed to Cd, Se did not affect the root-to-shoot TF value (Hu et al., 2014b).

3. Enzymatic activities in rice seedlings

Clarifying the biochemical and molecular responses of plants to drought stress is essential for a broad understanding of resistance mechanisms. Low concentrations of Se can protect plants from several types of abiotic stress by enhancing their antioxidant capacity (Djanaguiraman et al., 2010; Hasanuzzaman et al., 2011). In the present study, we investigated the relationship between Se levels and POD, SOD, and CAT activities and GSH and MDA contents in rice seedlings. GSH is a particularly important soluble antioxidant that protects cellular components from oxidative stress. We found here that root GSH content was increased in the presence of As, although this was non-significant relative to the control group (Fig. 6A). However, a significant increase in GSH content relative to the untreated control was observed in the shoot under all treatment conditions, which was greater in plants pretreated as opposed to co-treated with Se for both As(III) and As(V) (Fig. 6B). The increased GSH content may be due to enhanced glutathione reductase (GR) and glutathione-dependent dehydroascorbate reductase activities as well as higher biosynthesis rates (Mittova et al., 2003). An increase in GSH content under drought stress has been reported by others (Hasanuzzaman and Fujita, 2011; Kadioglu et al., 2011; Sharma and Dubey, 2005). It is possible that application of Se increased GR activity, resulting in an increase in GSH and decrease in oxidised GSH content in the plant (Hasanuzzaman and Fujita, 2011). The competition between As and Se to integrate GSH may also contribute to the formation of organic Se (Feng et al., 2013), since addition of Se increased GSH content in the shoot (Han et al., 2013; Srivastava et al., 2009).

Co-exposure of rice seedlings to Se and As or pre-treatment with Se increased POD activity both in the root and shoot (Fig. 5A, B). The fact that there was no difference in POD activity against As and Se indicates that Se does not influence key antioxidative enzymes. Se increased POD activity in tobacco (Han et al., 2013) and sunflower leaves (Saidi et al., 2014), whereas a previous study found no changes in POD activity in the rice shoots in the presence of As(V) or As(III) (Shri et al., 2009).

SOD activity was increased in the root (Fig. 5C) but slightly reduced in the shoot (Fig. 5D) upon treatment with Se as compared to As(III) treatment alone or Se and As co-treatment. The opposite trend was observed upon As(V) and Se co-treatment (Fig. 5C, D). For both As(III) and As(V), Se pretreatment reduced SOD activity to a greater extent than co-treatment. Simultaneous exposure to As and Se was shown to increase SOD activity relative to treatment with As only (Malik et al., 2012). CAT activity was higher in As(III)-treated as compared to As(V)-treated plants (Fig. 5E, F). The increased activities of SOD and CAT against As can be attributed to an enhanced antioxidative response to As(V) or As(III) (Kumar et al., 2013; Lou-Hing et al., 2011; Mishra et al., 2011; Kumar et al., 2016). An increase in SOD activity and GSH content in presence of As or phenanthrene typically indicates greater stress in plants (Sun et al., 2011).

MDA is an indicator of membrane lipid peroxidation whose increase reflects oxidative damage in cellular defences under stress (Tripathi et al., 2012). We found here that MDA level was elevated in the shoot of plants pretreated with Se as compared to those that were co-treated or exposed only to As (Fig. 6C). However, MDA content was reduced in plants exposed to As(III) as compared to As(V). Previous studies have reported decreases in MDA content in the presence of As resulting from alterations in lipoxygenase activity (Kumar et al., 2014; Mishra et al., 2011). An increase in the levels of cysteine, proline, and MDA in plant tissue can alleviate As-induced stress (Ahmad et al., 2013; Tripathi et al., 2012).

5. Conclusion

The results of this study indicate that simultaneous exposure of rice seedlings to As and Se [particularly Se(IV)] leads to As accumulation in roots and inhibits its translocation from root to shoot. Our study has shown that Se pretreatment in As(III) groups can be taken up by rice roots, at higher rate than that of As(V) groups. But clearly demonstrated that endogenous Se in As(V) group appeared to have a higher susceptibility to reduce As translocation from root to shoot than As(III) group.  Se pretreatment prior to As addition was less effective in alleviating As stress than As single application, although addition of Se before or during As exposure increased the activities of antioxidant enzymes along with GSH and MDA contents, thereby protecting against oxidative stress after 7 days exposure. Taken together, our results suggest that modulating the Se content of rice paddies can prevent the accumulation of As in rice plants and thus ensure their safety for human consumption.

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