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

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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).

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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%.

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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

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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%.

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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:

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T_Root−As

=

C_Root−As

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Root_{Dry biomass}      (1)

T_Shoot−As

=

C_Shoot−As

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Shoot_{Dry biomass}      (2)

T_As

=

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T_Root−As + T_Shoot−As       (3)

As uptake

=

T_As/Root_{Dry biomass}      (4)

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Shoot–As%

=

(T_Shoot−As/T_As)

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100%      (5)

TF

=

C_Shoot−As/C_Root−As       (6)

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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).

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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)