Science of the Total Environment 691 (2019) 827–834

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Science of the Total Environment

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Dynamics of Selenium uptake, speciation, and antioxidant response in

rice at different panicle initiation stages
Zhihua Dai a,b, Muhammad Imtiaz c, Muhammad Rizwan a, Yuan Yuan a, Hengliang Huang a, Shuxin Tu a,b,⁎
a
College of Resources and Environment, Huazhong Agricultural University, Wuhan 430070, China
b
Microelement Research Center, Huazhong Agricultural University, Wuhan 430070, China
c
Soil and Environmental Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad 38000, Pakistan

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• Low levels of Se (b5 mg kg−1) promoted

the growth and yield of rice.
• SeMet was the major Se-species in
young spikes and subcellular fractions
of rice leaves.
• SeCys was the main Se-species in brown
rice.
• The percentage of organic Se decreased,
but the percentage of inorganic Se in-
creased with elevated levels of Se appli-
cation.
• High levels of Se (≥5 mg kg−1) reduced
the antioxidant capacity of rice.

a r t i c l e i n f o a b s t r a c t

Article history: Selenium (Se) is an essential element in animals and humans, and its deficiency may cause conditions such as
Received 1 June 2019 cardiac disease. The production of Se-enriched rice is one of the most important ways to supply Se in the
Received in revised form 12 July 2019 human body, and thus, understanding of the mechanisms of Se-enriched rice is of great significance. A pot exper-
Accepted 12 July 2019
iment was conducted to study the effects of Se addition on the growth, antioxidation, Se uptake and distribution,
Available online 13 July 2019
and Se speciation in three different stages of panicle initiation stage (i.e., pistil and stamen formation stage, pollen
Editor: Xinbin Feng mother cell formation stage, pollen mother cell meiosis stage) and the maturity stage. The results showed that
soil Se application significantly increased Se uptake in rice. Low rates of Se (b5 mg kg−1) application enhanced
Keywords: the plant growth and rice yield. Se speciation assays showed that SeCys and SeMet were the two main forms
Selenium found in rice, of which SeMet accounted for 65.5%–100% in the ears and leaves, while SeCys accounted for
Rice growth stages 61.4%–75.6% in brown rice. SeMet was also the main Se-species found in different subcellular parts at the panicle
Biofortification initiation stage. However, inorganic Se was present in brown rice, mainly as Se(VI), when the soil Se addition
Species exceeded 5 mg kg−1. Lower rates of Se (b5 mg kg−1) promoted the antioxidant capacity, while high levels of
Subcellular distribution
Se (≥5 mg kg−1) reduced the antioxidant capacity of rice. The results indicate that Se effects are dose dependent,
and the suitable amount of soil Se application for Se-enriched rice production would be b5 mg kg−1.
© 2019 Elsevier B.V. All rights reserved

⁎ Corresponding author at: College of Resources and Environment, Huazhong Agricultural University, Wuhan 430070, China.
E-mail addresses: [email protected] (Z. Dai), [email protected] (M. Imtiaz), [email protected] (M. Rizwan),
[email protected] (Y. Yuan), [email protected] (H. Huang), [email protected] (S. Tu).

https://doi.org/10.1016/j.scitotenv.2019.07.186
0048-9697/© 2019 Elsevier B.V. All rights reserved
828 Z. Dai et al. / Science of the Total Environment 691 (2019) 827–834

1. Introduction follows: pH 6.32, organic matter 14.33 g kg−1, alkali-hydrolysable nitro-

gen 125.31 mg kg−1, available phosphorus 7.63 mg kg−1, available potas-
Selenium (Se) is an essential micronutrient element for humans and sium 87.16 mg kg−1, and total soil Se 0.27 mg kg−1.
animals, and it is reported as a component of 25 proteins, including glu-
tathione peroxidase (GSH-Px), the antioxidant capacity of which is N200 2.2. Experimental design
times that of vitamin E (Taneja, 2016). In addition, Se plays a very impor-
tant role in maintaining the immune system for human health and re- Se with seven different levels, 0 (control), 0.5, 1.25, 2.5, 5, 10 and
ducing the risk of cancer, cardiovascular disease, and diabetes, etc. 20 mg kg−1, were applied as sodium selenite (Na2SeO3). Each treatment
(Benstoem et al., 2015; Cai et al., 2016; Wei et al., 2015). In recent de- was replicated 8 times. Each pot (30 cm × 30 cm) was filled with 5 kg
cades, hidden hunger caused by Se deficiency is commonly found world- air-dried soil, and fertilizers were applied as follows: N fertilizer
wide (D'Amato et al., 2018; H.H. Gao et al., 2018; Kumar et al., 2016), (urea) 0.36 g kg−1, P fertilizer (SSP) 0.087 g kg−1, and K fertilizer
becoming an important health issue as daily intake of Se is below the rec- (KCl) 0.50 g kg−1 of soil, with the addition of a mixture of micronutrient
ommended allowance (50–200 μg), especially in rice-dependent Asian Hoagland solution (Supplementary Table 1) at a rate of 1 ml kg−1 soil.
countries (D'Amato et al., 2018; EFSA, 2014, H.H. Gao et al., 2018). How- To generate a good mixture of soil and chemicals, all the fertilizers and
ever, some soils and mineral deposits are naturally rich in Se (Sun, 2017), Na2SeO3 were thoroughly mixed 10 days before of rice planting.
and high Se areas can be found in many different countries, such as The rice (Oryza sativa L. cv. Yangliangyou 6) seeds were germinated
Australia, China, Ireland, India and the USA (Dhillon and Dhillon, 2003), and cultured in an incubator under constant temperature (30 °C) and
due to anthropogenic activities such as mining, agriculture, and oil pro- light (14 h/d). After 2 weeks of germination, 10 seedlings with a uni-
duction, resulting in excess contents of Se in agriculture products (Both form size were transplanted into each pot, and Se-free water was used
et al., 2018; Cui et al., 2018; Eiche et al., 2015; Wang et al., 2012) and for irrigation throughout the experiment.
harming human and animal health (Song et al., 2018). The rice samples were collected at the pistil and stamen formation
Rice fulfils more than half of the food requirement of the world's stage, pollen mother cell formation stage, pollen mother cell meiosis
population, and in China, N60% of the 1.4 billion people consume rice stage, and maturity stage, related to plant growth of 108 d, 112 d,
on a daily basis (Tang and Cheng, 2019). Hence, the development of 115 d and 134 d, respectively.
Se-enriched rice is the most popular and effective way to meet human The collected samples were rinsed with tap water and then washed
nutritional needs for Se. However, a daily intake of Se above the recom- with deionized water and dried with clean tissue paper. The samples
mended range may potentially causes Se toxicity in humans and in- were separated into two parts: one part was stored in an ultra-low tem-
duces diseases such as hypothyroidism and cardiac disease (Hatfield perature freezer for antioxidant enzyme and Se-speciation analyses,
et al., 2014). For adults, daily Se intake of 800 μg is considered toxic while the remaining part was dried in an oven first at 105 °C for
(H. Zhang et al., 2014). 30 min and then at 60 °C for 2 d to determine the contents of Se. The
The plants exhibited different forms of Se mainly Se(IV), Se(VI), Se-speciation was determined from the samples that were collected at
selenocysteine (SeCys) and selenomethionine (SeMet). Plants take up different stages.
Se(IV) or Se(VI), most of which is converted into SeCys and SeMet (Zhu
et al., 2009). Generally, inorganic Se(IV) and Se(VI) are the most toxic 2.3. Chemical analysis methods
to human health (Hatfield et al., 2014; Pilon-Smits et al., 2009; Yin
et al., 2019), whereas organic SeMet and SeCys are safer and more bene- 2.3.1. Determination of Se contents
ficial (Longchamp et al., 2015; Marschall et al., 2016; Rayman, 2008). For total Se content determination, 1 g pulverized rice samples were
Thus, it is important to analyze both the total Se concentrations and dif- weighted and placed in a 50-ml beaker. Then, 10 ml of mixed acid
ferent types of Se speciation in edible plant parts to avoid toxicity. [HNO3 and HClO4 (9:1)] was added, followed by overnight digestion
In practical production, soil addition and foliar application of inor- in the room temperature. The next day, the samples were digested at
ganic Se are the best ways to produce Se-enriched agriculture products 180 °C until the liquid become clear and transparent in approximately
(Deng et al., 2017; Ros et al., 2016). Previous studies focused on the Se 2 ml, followed by the addition of 5 ml of 6 mol L−1 HCl after cooling.
accumulation in the edible parts of rice only, not at different growth Then, the solutions were again digested on a hot plate with a sand
stages: the seedling, tillering, heading and maturity stages which re- bath for reduction Se6+ to Se4+, until the solutions became clear and
spond differently regarding Se accumulation (Gong et al., 2018; Hu transparent after white smoke. After cooling, the solutions were trans-
et al., 2018). To the best of our knowledge, there is no information ferred to a 25-ml volumetric flask, followed by an increase in the vol-
about the role of Se on different growth stages, especially the spike dif- ume up to the mark with deionized water. Finally, the 10 ml of
ferentiation stages (at which Se starts to transport into brown rice), as digested solution was collected from each sample into a tube with
well as the accumulation of Se species in rice. 2 ml HCl and 1 ml of 100 g L−1 K3[Fe(CN)6]. The Se contents were deter-
The objectives of this research were to (1) investigate the uptake, mined using an atomic fluorescence spectrometer (AFS-8220). The ac-
transformation and distribution of Se in rice plants under different Se curacy of the Se analysis was checked by standard reference material
levels, (2) reveal the relationship between Se species and the antioxi- GBW07602 (GSV-1) (branches and leaves of shrubs certified at 0.184
dant capacity of rice plants, and (3) determine the optimal dose of Se ± 0.013 μg Se g−1) purchased from the center for the standard refer-
in soil applications for rice growth. The obtained results will be support- ence of China. The standard reference material was run in parallel
ive to understand the dynamic behaviors of Se at different rice growth with the samples, and the obtained value was found to be in good agree-
stages and the mechanisms of Se accumulation as well as its speciation ment with the certified value.
in rice plants.
2.3.2. Se speciation
2. Materials and methods The assay of Se speciation in plants was conducted as described by
Han et al. (2013). Briefly, 1 g fresh plant samples was ground in liquid
2.1. Experimental materials nitrogen and then transferred to a new 50-ml centrifuge tube. Then,
10 ml of ultrasonic extraction solution was added to the ground samples
A pot experiment was conducted under greenhouse conditions at and centrifuged at 5000 ×g for 15 min. Subsequently, the supernatant
Huazhong Agricultural University (N 31°28′26″, E 114° 20′15″). Brown- was collected, and methanol was removed from the samples using ro-
red paddy soil was used and sampled from the top 20 cm of field soil of tary evaporator to a volume up to 10 ml. The samples were then filtered
the school farm. The basic soil physical and chemical properties were as with a 0.22-μm membrane and stored at 4 °C for the Se speciation assay.
 Z. Dai et al. / Science of the Total Environment 691 (2019) 827–834 829

0.5 g fresh leaves of each sample were homogenized in 15 ml solution

containing 0.25 mol L−1 sucrose, 50 mmol L−1 pH 7.8 Tris-maleate
buffer, 1 mmol L−1 MgCl2 and 10 mmol L−1 cysteine. After homogeniza-
tion, the homogenate was centrifuged at 2200 ×g for 30 s at 4 °C, and the
resulting precipitate was considered the cell wall. The supernatant was
further centrifuged at 11,000 ×g for 45 min at 4 °C, and the residue was
collected as organelles and the supernatant as the soluble fraction. Se
speciation was determined using an atomic fluorescence speciation an-
alyzer (SA-10) and atomic fluorescence spectrometer (AFS-8220).

2.3.4. Assay of enzymatic and nonenzymatic antioxidants

Superoxide dismutase (SOD) activity was determined according to
the method of Ge et al. (2018). SOD activity is expressed as U/mg pro-
tein, where U is defined as the inhibition of 50% of the photochemical re-
duction of nitroblue tetrazolium (NBT). Catalase (CAT) activity was
assayed following the method of Ren et al. (2017), and the unit CAT ac-
tivity was defined as the amount required to decompose 1 μmol H2O2 in
1 min. Peroxidase (POD) activity was determined according to the
Fig. 1. The standard HPLC curve for SeCys, SeMet, Se(IV), and Se(VI) (the oxidizing agent method of Huang et al. (2013) and expressed as U/mg protein, where
was 0.3% KI (m/v), carrier solution was 10% HCl (v/v), reducing agents were 2.0% KBH4
(m/v) + 0.5% KOH (m/v), the mobile phase for HPLC was 60 mmol L−1 (NH4)2HPO4
1 U was defined as 0.001 ΔOD470 min−1. Glutathione peroxidase
(pH 6.0, 1.0 ml min−1). The AFS conditions were adjusted as follows: Se hollow cathode (GSH-Px) activity was determined according to the method of Flohe
lamp current, 50 mA; negative high voltage of photomultiplier tube, 270 V; flow rate of and Gunzler (1984), and the absorbance was recorded at 412 nm, and
carrier gas, 400 ml min−1; flow rate of makeup gas, 600 ml min−1). the unit 1 U defined as 1 μmol GSH mg−1 protein min−1.
The ascorbate (AsA) content was determined by titration with 2,6-
dichloroindophenol as described by Gao et al. (2016) and expressed as
μg AsA/g fresh weight (FW). The glutathione (GSH) contents were de-
The atomic fluorescence speciation analyzer (SA-10), atomic fluo-
termined according to the method used by Cui et al. (2011). Lipid perox-
rescence spectrometer (AFS-8220) of Beijing Titan Instruments Co.
idation was estimated by measuring the amount of malondialdehyde
Ltd. and Shimadzu HPLC were used to determine Se-speciation. The
(MDA) as previously described (Jin et al., 2013).
working conditions were as follows: oxidizing agent, 0.3% KI (m/v); car-
rier solution, 10% HCl (v/v); reducing agents, 2.0% KBH4 (m/v) + 0.5%
KOH (m/v); mobile phase for HPLC, 60 mmol L−1 (NH4)2HPO4 2.3.5. Measurement of rice growth parameters and yield
(pH 6.0, 1.0 ml min−1). The working conditions for AFS conditions The plant height at maturity stage was recorded by the meter scale.
were as follows: Se hollow cathode lamp current (general research in- All plant samples were separated into the straw and rice grain and
stitute for nonferrous metals, Beijing, China), 50 mA; negative high volt- dried first at 105 °C for 30 min and then at 60 °C until a constant weight
age of the photomultiplier tube, 270 V; flow rate of the carrier gas, was attained. The dry biomass, 1000-grain weight, and rice yield were
400 ml min−1; flow rate of makeup gas, 600 ml min−1. thus recorded.
The Se standard material of SeMet (Sigma, USA), SeCys (Sigma,
USA), selenite radical (GBW10032, 42.9 μg g−1 Se, National Institute of 2.4. Statistical analysis
Metrology P.R. China) and selenate radical (GBW10033, 41.5 μg g−1
Se, P.R. China) were used to prepare the Se species standard solutions. The experimental data were analyzed by one-way ANOVA using SAS
We tested the prepared Se standard solutions, and the chromatogram 8.1 software. Different letters indicated significant differences between
analysis clearly showed four kinds of Se species (Fig. 1). To validate the treatments at p b 0.05. All data represent means ± standard deviations
method and test conditions, we added four Se species standard solutions (SD) of three replicates for each treatment.
accurately to the Se-enriched samples of rice and performed an extrac-
tion with methanol + water (1:2) to determine their recovery rates. 3. Results
The recovery rates of the four Se species were 94%–103%, indicating
that the method was reasonable. In addition, reagent blanks and internal 3.1. Effect of Se addition on rice growth and yield
standards were used to ensure the accuracy and precision in the Se spe-
ciation analysis. To examine the effect of Se on the growth and development of rice
plants, we recorded the plant height, dry biomass, 1000-grain weight,
2.3.3. Plant subcellular fractionation and Se analysis and yield at maturity stage (Table 1). Among 7 treatments, Se levels at
The Se contents in subcellular fractions of rice leaves were deter- 2.5–5 mg kg−1 achieved the best parameters. For instances, plant height,
mined according to the method described by Xiao et al. (2006). Briefly, dry biomass, 1000-grain weight, and rice yield increased by 19.91%,

Table 1
Effect of soil Se addition on growth and yield characters of rice.

Soil Se (mg kg−1) Plant height (cm) Dry biomass (g plant−1) 1000-grain weight (g) Rice yield (g plant−1)

Control 122.0 ± 4.6cd 10.9 ± 1.1e 28.9 ± 1.3bc 30.9 ± 2.0d

0.5 129.3 ± 3.5c 14.6 ± 0.8d 28.9 ± 0.7c 32.8 ± 1.1cd
1.25 132.3 ± 3.8b 17.8 ± 1.2bc 29.0 ± 0.8bc 33.2 ± 2.8bcd
2.5 138.7 ± 4.0b 20.2 ± 1.0a 30.0 ± 0.3ab 38.6 ± 2.9ab
5 146.3 ± 2.5a 21.0 ± 1.5a 29.9 ± 0.5ab 40.3 ± 1.2a
10 149.7 ± 4.2a 17.7 ± 0.4b 28.7 ± 0.4c 34.3 ± 0.7c
20 132.3 ± 3.1b 16.0 ± 0.7cd 29.6 ± 0.6bc 31.0 ± 0.5d

Values are the mean ± SD (n = 3). Different letters indicate a significant difference in the same column at p b 0.05.
830 Z. Dai et al. / Science of the Total Environment 691 (2019) 827–834

Fig. 2. The Se contents in shoots (steam + leaves) at different stages (tillering, heading and maturity stage), husk and brown rice in the presence of different levels of Se. Data are means ±
SD (n = 3). Different letters indicate a significant difference between treatments at the p b 0.05 level.

92.66%, 3.46%, and 30.42%, respectively, with Se of 5 mg kg−1 compared 3.3. Effect of Se application on Se speciation in rice
to the control (Table 1). When the amount of Se (N5 mg kg−1) continued
to increase, the growth effects did not increase but decreased, indicating Se species were divided into Se(IV), Se(VI), SeCys and SeMet. Sam-
that high Se was not beneficial to plant growth. ples collected from different parts of rice at different growth and devel-
opmental stages were analyzed to understand the dynamic changes of
3.2. Effect of the Se supply on Se concentrations in rice the Se forms.

With greater additions of Se to the soil, more Se accumulated in rice

plants, regardless of the growth stage or plant part (Fig. 2). The average 3.3.1. Dynamic Se speciation in young spikes at three panicle initiation
Se concentrations in rice shoots (stem + leaves) at different growth stages
stages ranked as follows: heading stage (16.17 mg kg−1) N maturity The Se speciation at three panicle initiation stages (i.e. pistil and sta-
stage (13.72 mg kg−1) N tillering stage (9.20 mg kg−1). As the soil Se men formation stage, pollen mother cell formation stage, and pollen
levels increased from 0.5 to 20 mg kg−1, the Se concentrations in the mother cell meiosis stage) showed that organic SeMet was the main
rice shoots correspondingly increased. The average Se concentrations forms of Se accumulated in young spikes of rice plants (Fig. 3). The per-
in different rice organs at the maturity stage ranked as follows: shoots centages of SeMet ranged from 65.5 to 100%. While, SeCys (organic
(13.72 mg kg−1) N brown rice (2.73 mg kg−1) N husk (1.18 mg kg−1). form), Se(IV), and Se(VI) (inorganic form) were accumulated about
The Se concentrations in brown rice and husk followed trends similar 8.4–24.2%, 5.3–15.4%, and 0–10.7%, respectively. It was interesting to
to those in rice shoots (Fig. 2). note that increasing the amount of Se addition decreased the

Fig. 3. Selenium speciation in young spikes of rice treated with Se 0–20 mg kg−1 at three panicle initiation stages (A: pistil and stamen formation stage, B: pollen mother cell formation
stage, C: pollen mother cell meiosis stage).
 Z. Dai et al. / Science of the Total Environment 691 (2019) 827–834 831

3.3.3. Se speciation in subcellular fractions of rice leaves

The cell tissues of rice leaves and stems at the pollen mother cell
meiosis stage were separated into the cell wall, cell organelles and sol-
uble fraction for Se species determination. It was found that organic Se
was the main species but was unevenly distributed in different parts
(Fig. 5). In cell walls, no Se species were detected at the soil Se level of
0.5 mg kg−1, and SeMet, SeCys and Se(IV) were observed only at the
soil Se level of 20 mg kg−1, with percentages of 74.0%, 14.1%, and
11.9%, respectively (Fig. 5). All the Se species (SeMet, SeCys, Se(IV)
and Se(VI)) were present in cell organelles. With increasing soil Se
levels, SeMet increased up to 100% at the soil Se level of 20 mg kg−1.
Se species in cell soluble parts were similar to cell organelles. It is note-
worthy that Se(VI) appeared only at soil Se levels N10 mg kg−1, with the
percentages reaching 13.1% at the 20 mg kg−1 soil Se level.

3.4. Effect of Se on the rice antioxidant enzyme system

3.4.1. SOD, POD, CAT and GSH-Px

Se at higher levels (≥5 mg kg−1) significantly increased the SOD ac-
tivity in rice leaves (Fig. 6). The SOD activities following 5 mg kg−1,
10 mg kg−1 and 20 mg kg−1 Se treatments were 2.28 times, 2.71
Fig. 4. Selenium speciation in brown rice with soil application of Se 0–20 mg kg−1. times and 2.29 times greater than the control, respectively. Changes in
POD activity with Se levels followed the same trends observed for
SOD, and the maximum activity was recorded at Se levels ≥5 mg kg−1.
percentage of organic SeMet and SeCys but increased the percentages of Overall, as compared with the control, the POD activity increased by
inorganic Se(IV) and Se(VI) . 161% to 176%. The response of both CAT and GSH-Px activity against
Further analysis of the Se species at different panicle initiation Se levels was a second-degree parabola, and the highest activity was ob-
stages revealed that the SeMet content ranked as follows: pistil and served at the 1.25–2.50 mg kg−1 Se level. Compared with the control,
stamen formation stage (16.52 mg kg−1) N pollen mother cell forma- the activity of CAT and GSH-Px following 1.25–2.50 mg kg−1 Se treat-
tion stage (10.96 mg kg−1) N pollen mother cell meiosis stage ment was 104–151% and 104–121%, respectively.
(2.99 mg kg−1). While the ranking of SeCys content were different:
pistil and stamen formation stage (2.39 mg kg−1) N pollen mother 3.4.2. GSH, AsA and MDA contents
cell formation stage (1.48 mg kg−1) N pollen mother cell meiosis The contents of GSH in rice leaves increased by 6.2% to 66.5% when
stage (0.65 mg kg−1). The average Se contents of the pistil and sta- exposed to 0.5 to 20 mg kg−1 of Se treatments. However, the highest
men formation stage, pollen mother cell formation stage, and pollen content of AsA recorded at the Se levels of 5 mg kg−1, with increases
mother cell meiosis stages were 1.76, 1.81 and 0.32 mg kg−1, respec- by 62.9% compared to the control (Fig. 7).
tively, while Se(VI) was only detected following the 20 mg kg−1 Se The MDA contents in the cell membrane were not increased under
treatment, indicating that higher levels of Se could not be good for soil Se (0.5–1.25 mg kg−1); however, a significant reduction of MDA
the young spike of rice. contents was noted in response to a soil Se of 2.5–20 mg kg−1 compared
with the control, with the lowest content in response to a soil Se of
5–10 mg kg−1 (Fig. 7).
3.3.2. Se speciation in brown rice
Unlike the results obtained for young spikes of rice (Fig. 4), SeCys 4. Discussion
was the main form in brown rice, with percentages ranging from 61.4
to 75.6%, followed by SeMet (9.9–18.4%), Se(IV) (12.6–17.9%) and Se 4.1. Effects of Se treatments on rice growth and yield
(VI) (0–10.3%). Likewise, SeCys was decreased, while inorganic Se (es-
pecially the Se(VI)) was increased with the increase in Se levels in the The soil application Se at lower levels (i.e. 0.5–5 mg kg−1) signifi-
soil. cantly increased rice growth and yield (Table 1). This finding was

Fig. 5. Percentages of Se species in subcellular fractions (A: cell wall, B: cell organelles, C: soluble fraction) of rice leaves at the pistil and stamen formation stage.
832 Z. Dai et al. / Science of the Total Environment 691 (2019) 827–834

Fig. 6. Antioxidant enzyme activity in rice leaves treated with Se of 0–20 mg kg−1. Different letters represent different significant between treatments at the p b 0.05 level.

similar to the previous studies on rice (Zhang et al., 2019), maize (Jiang the major species found in rice shoots (Fig. 3). A similar trend was ob-
et al., 2017) and tobacco (Han et al., 2013), indicating that lower Se has served in previous studies, which confirmed that Se mainly exists as
positive effects on plants, although Se is not an essential element. For in- SeMet in rice (Zhang et al., 2012). Earlier research has shown that the
stance, previously studies have reported that appropriate application of inorganic Se transforms into organic Se after being absorbed by plants,
Se could promote the photosynthesis in rice (M. Gao et al., 2018; M. and first into SeCys and then into other forms, such as SeMet, due to
Zhang et al., 2014), wheat (Kaur and Sharma, 2018) and Brassica juncea the activation of a variety of enzymes (Dinh et al., 2019). However,
(Handa et al., 2019). The appropriate Se application could also promote our study showed that SeCys was the major species in brown rice, po-
the synthesis of amino acids and protein in rice (Hu et al., 2018; Singh tentially due to the rapid and active transformation of SeMet during
et al., 2018) and soybean (Zhao et al., 2019). Se applications could alle- the process of being converted into rice grain.
viate the detrimental effects of rice leaf senescence (Duan et al., 2019). In this research, the inorganic Se-species increased in higher levels of
Additionally, Se could increase the contents of pectin, hemicelluloses soil Se (Figs. 3 and 4). Similar results were reported by Han et al. (2013)
and lignin in cell walls to resist heavy metal absorption (Liu et al., 2019). who found that inorganic Se was the major Se species in the leaves of
flue-cured tobacco in high soil Se. However, various results have also
4.2. Changes in Se species in rice with soil Se levels been observed in the edible portion of the plant. For instance, the accu-
mulation of Se-species ranked as Se(VI) N SeMet N Se(IV) in cabbage, let-
Assay of Se species found that four main Se-species [i.e. Se(IV), Se tuce and chard leaves, while SeMet N Se(VI) N Se(IV) in parsley leaves
(VI), SeMet and SeCys] were present in rice plants, and the SeMet was when treated with Se (Funes-Collado et al., 2013). Upon absorption,

Fig. 7. Effects of Se treatments on the content of GSH, AsA and MDA in rice leaves. Different letters represent significant differences between treatments at the p b 0.05 level.
 Z. Dai et al. / Science of the Total Environment 691 (2019) 827–834 833

SeCys, Se(IV), and Se(VI) are available for the synthesis of selenoproteins, Acknowledgments
whereas SeMet can substitute for methionine in proteins where it can act
as a Se store (Nemeth and Dernovics, 2015). This research was supported in part by the Guangxi Major Special
In the present study, Se was mainly deposited in the cell wall fraction Project of Science and Technique (Guike AA17202026-3), Hubei Special
in the form of SeMet (Fig. 5). Thus, certain substances or structures of Project for Technique Innovation (2017ABA154), and Special Fund for
the rice cell wall might have absorbed the SeMet. The obtained results Agro-scientific Research in the Public Interest (201303106).
revealed that different species were observed in the soluble fraction
and cell organelles. With increasing soil Se levels, the percentage of in- References
organic Se (especially Se(VI)) increased in the soluble fraction, while
Ahsan, U., Kamran, Z., Raza, I., Ahmad, S., Babar, W., Riaz, M.H., et al., 2014. Role of sele-
SeCys decreased and SeMet increased in cell organelles. The subcellular nium in male reproduction-a review. Anim. Reprod. Sci. 146 (1–2), 55–62.
distribution of Se species was closely related to the pathway of Se me- Benstoem, C., Goetzenich, A., Kraemer, S., Borosch, S., Manzanares, W., Hardy, G., et al.,
tabolisms and detoxicity in plant. However, few reports have examined 2015. Selenium and its supplementation in cardiovascular disease-what do we
know? Nutrients 7 (5), 3094–3118.
the subcellular distribution of Se speciation in plants. Plants may accu- Both, E.B., Shao, S.X., Xiang, J.Q., Jokai, Z., Yin, H.Q., Liu, Y.F., et al., 2018. Selenolanthionine
mulate Se species in different subcellular parts. For instance, Ding is the major water-soluble selenium compound in the selenium tolerant plant
et al. (2015) reported that Se was primarily concentrated in the cell Cardamine violifolia. BBA-Gen Subjects 1862 (11), 2354–2362.
Cai, X.L., Wang, C., Yu, W.Q., Fan, W.J., Wang, S., Shen, N., et al., 2016. Selenium exposure
wall fraction and cytosol fractions in paddy rice. In contrast, Sabbioni and cancer risk: an updated meta-analysis and meta-regression. Sci Rep-UK 6, 1–18.
et al. (2015) reported the presence of Se in all subcellular fractions, es- Cui, W.T., Fu, G.Q., Wu, H.H., Shen, W.B., 2011. Cadmium-induced heme oxygenase-1 gene
pecially in the cytosol of microalgae, as well as in membranes and or- expression is associated with the depletion of glutathione in the roots of Medicago
sativa. Biometals 24 (1), 93–103.
ganelles of spring tea leaves (Du et al., 2010).
Cui, L.W., Zhao, J.T., Chen, J.T., Zhang, W., Gao, Y.X., Li, B., et al., 2018. Translocation and
transformation of selenium in hyperaccumulator plant Cardamine enshiensis from
4.3. Effects of Se treatments on antioxidant system and nonenzymatic Enshi, Hubei, China. Plant Soil 425 (1–2), 577–588.
antioxidants D'Amato, R., Fontanella, M.C., Falcinelli, B., Beone, G.M., Bravi, E., Marconi, O., et al., 2018.
Selenium biofortification in rice (Oryza sativa L.) sprouting: effects on se yield and
nutritional traits with focus on phenolic acid profile. J Agr Food Chem 66 (16),
Se is an active center of GSH-Px and plays an important role in the 4082–4090.
plant antioxidant defense system (Ahsan et al., 2014). All the SOD, Deng, X.F., Liu, K.Z., Li, M.F., Zhang, W., Zhao, X.H., Zhao, Z.Q., et al., 2017. Difference of se-
lenium uptake and distribution in the plant and selenium form in the grains of rice
POD, CAT and GSH-Px enzymes can protect the structure and func- with foliar spray of selenite or selenate at different stages. Field Crop Res. 211,
tion of the cell membrane from oxidation and damage by removing 165–171.
free radicals, such as peroxide and H2O2. In this study, soil applica- Dhillon, K.S., Dhillon, S.K., 2003. Distribution and management of seleniferous soils. Adv.
Agron. 79, 119–184.
tion of Se significantly influenced the activities of GSH-Px, SOD, Ding, Y.Z., Wang, R.G., Guo, J.K., Wu, F.C., Xu, Y.M., Feng, R.W., 2015. The effect of selenium
POD and CAT, as well as the contents of GSH and AsA (Figs. 6 and on the subcellular distribution of antimony to regulate the toxicity of antimony in
7). The activities of SOD, POD, CAT and GSH-Px exhibited basically paddy rice. Environ Sci Pollut R 22 (7), 5111–5123.
Dinh, Q.T., Wang, M.K., Tran, T.A.T., Zhou, F., Wang, D., Zhai, H., et al., 2019. Bioavailability
a quadratic parabolic curve, which indicated that the activities of an- of selenium in soil-plant system and a regulatory approach. Crit Rev Env Sci Tec 49
tioxidant enzymes were increased in rice at lower Se levels, while an (6), 443–517.
opposite trend was observed at higher levels of soil Se, especially for Du, Q., Wang, C., Li, B., Li, H., Liu, Y., 2010. Effects of Se, Zn and their interaction on the sub-
cellular distribution of selenium in spring tea leaves. Acta Horticulturae Sinica 37 (5),
CAT and GSH-Px. Earlier studies have also reported that the activities
794–800.
of SOD, POD, CAT and GSH-Px increased with the increasing Se levels Duan, M.Y., Cheng, S.R., Lu, R.H., Lai, R.F., Zheng, A.X., Ashraf, U., et al., 2019. Effect of foliar
at low levels. For instance, SOD and CAT activities were significantly sodium selenate on leaf senescence of fragrant rice in South China. Appl. Ecol. Envi-
increased in Ulva sp. when exposed to sodium selenate (Schiavon ron. Res. 17 (2), 3343–3351.
EFSA, 2014. Scientific opinion on the safety and efficacy of DL-selenomethionine as a feed
et al., 2012). Similarly, POD and CAT activities were also higher additive for all animal species. EFSA J. 12 (2), 3567.
than that in the control when Se b5 mg L−1 was applied (Feng and Eiche, E., Bardelli, F., Nothstein, A.K., Charlet, L., Gottlicher, J., Steininger, R., et al., 2015. Se-
Wei, 2012). In the present study, with continuously increasing Se lenium distribution and speciation in plant parts of wheat (Triticum aestivum) and
Indian mustard (Brassica juncea) from a seleniferous area of Punjab, India. Sci.
levels, SOD, POD and CAT activities were increased. When Se treat- Total Environ. 505, 952–961.
ment ranged from 5 to 20 mg kg−1, Se(VI) was detected in brown Feng, R.W., Wei, C.Y., 2012. Antioxidative mechanisms on selenium accumulation in Pteris
rice, and SOD activity was increased and CAT activity decreased com- vittata L., a potential selenium phytoremediation plant. Plant Soil Environ. 58 (3),
105–110.
pared with the control. The underlying mechanism for this phenom- Flohe, L., Gunzler, W.A., 1984. Assays of glutathione-peroxidase. Method Enzymol 105,
enon might be explained the production of hydrogen peroxide and 114–121.
superoxide radicals (O− 2 ) in rice, which enhanced SOD activity. It
Funes-Collado, V., Morell-Garcia, A., Rubio, R., Lopez-Sanchez, J.F., 2013. Selenium uptake
by edible plants from enriched peat. Sci Hortic-Amsterdam 164, 428–433.
has been suggested that high Se toxicity mainly derives from its
Gao, H., Zhang, Z.K., Chai, H.K., Cheng, N., Yang, Y., Wang, D.N., et al., 2016. Melatonin
role as an oxidizing agent, resulting in the production of peroxides, treatment delays postharvest senescence and regulates reactive oxygen species me-
which are poisonous for crop plants (Feng and Wei, 2012). tabolism in peach fruit. Postharvest Bio Tec 118, 103–110.
Gao, H.H., Chen, M.X., Hu, X.Q., Chai, S.S., Qin, M.L., Cao, Z.Y., 2018. Separation of selenium
species and their sensitive determination in rice samples by ion-pairing reversed-
5. Conclusions phase liquid chromatography with inductively coupled plasma tandem mass spec-
trometry. J. Sep. Sci. 41 (2), 432–439.
Soil Se application at low Se levels (b5 mg kg−1) promoted plant Gao, M., Zhou, J., Liu, H.L., Zhang, W.T., Hu, Y.M., Liang, J.N., et al., 2018. Foliar spraying
with silicon and selenium reduces cadmium uptake and mitigates cadmium toxicity
growth and rice yield. Organic SeCys and SeMet were the main species in rice. Sci. Total Environ. 631-632, 1100–1108.
in rice, and the relative content decreased with the Se levels. The inor- Ge, Y.H., Duan, B., Li, C.Y., Tang, Q., Li, X., Wei, M.L., et al., 2018. Gamma-aminobutyric acid
ganic Se(IV) and Se(VI) increased in brown rice at high Se levels (10 delays senescence of blueberry fruit by regulation of reactive oxygen species metab-
olism and phenylpropanoid pathway. Sci Hortic-Amsterdam 240, 303–309.
and 20 mg kg−1), indicating a potential threat to human health. Thus, Gong, R.Y., Ai, C.Y., Zhang, B.J., Cheng, X.L., 2018. Effect of selenite on organic selenium
based on the present findings, 0.5–5 mg kg−1 could be a suitable and speciation and selenium bioaccessibility in rice grains of two Se-enriched rice culti-
safe dose for Se-enriched rice production. vars. Food Chem. 264, 443–448.
Han, D., Li, X.H., Xiong, S.L., Tu, S.X., Chen, Z.G., Li, J.P., et al., 2013. Selenium uptake, spe-
Supplementary data to this article can be found online at https://doi. ciation and stressed response of Nicotiana tabacum L. Environ. Exp. Bot. 95, 6–14.
org/10.1016/j.scitotenv.2019.07.186. Handa, N., Kohli, S.K., Sharma, A., Thukral, A.K., Bhardwaj, R., Abd Allah, E.F., et al., 2019.
Selenium modulates dynamics of antioxidative defence expression, photosynthetic
attributes and secondary metabolites to mitigate chromium toxicity in Brassica
Declaration of Competing Interest
juncea L. plants. Environ. Exp. Bot. 161, 180–192.
Hatfield, D.L., Tsuji, P.A., Carlson, B.A., Gladyshev, V.N., 2014. Selenium and selenocysteine:
The authors declare no conflict of interests. roles in cancer, health, and development. Trends Biochem. Sci. 39 (3), 112–120.
834 Z. Dai et al. / Science of the Total Environment 691 (2019) 827–834

Hu, Z.Y., Cheng, Y.X., Suzuki, N., Guo, X.P., Xiong, H., Ogra, Y., 2018. Speciation of selenium Singh, R., Upadhyay, A.K., Singh, D.P., 2018. Regulation of oxidative stress and mineral nu-
in brown rice fertilized with selenite and effects of selenium fertilization on rice pro- trient status by selenium in arsenic treated crop plant Oryza sativa. Ecotox Environ
teins. Int. J. Mol. Sci. 19 (11), 1–15. Safe 148, 105–113.
Huang, W.S., Bi, X.F., Zhang, X., Liao, X.J., Hu, X.S., Wu, J.H., 2013. Comparative study of en- Song, T.J., Su, X.S., He, J., Liang, Y.K., Zhou, T., Liu, C., 2018. Selenium (Se) uptake and dy-
zymes, phenolics, carotenoids and color of apricot nectars treated by high hydrostatic namic changes of Se content in soil-plant systems. Environ Sci Pollut R 25 (34),
pressure and high temperature short time. Innov Food Sci Emerg 18, 74–82. 34343–34350.
Jiang, C.Q., Zu, C.L., Lu, D.J., Zheng, Q.S., Shen, J., Wang, H.Y., et al., 2017. Effect of exogenous Sun, H.B., 2017. Associations of spatial disparities of Alzheimer's disease mortality rates
selenium supply on photosynthesis, Na+ accumulation and antioxidative capacity of with soil selenium and sulfur concentrations and four common risk factors in the
maize (Zea mays L.) under salinity stress. Sci Rep-UK 7, 1–14. United States. J. Alzheimers Dis. 58 (3), 897–907.
Jin, Q.J., Zhu, K.K., Xie, Y.J., Shen, W.B., 2013. Heme oxygenase-1 is involved in ascorbic Taneja, S.S., 2016. Re: difference in association of obesity with prostate cancer risk be-
acid-induced alleviation of cadmium toxicity in root tissues of Medicago sativa. tween US African American and non-Hispanic white men in the Selenium and Vita-
Plant Soil 366 (1–2), 605–616. min E cancer Prevention Trial (SELECT). J Urology 195 (3), 627–628.
Kaur, M., Sharma, S., 2018. Influence of selenite and selenate on growth, leaf physiology Tang, D., Cheng, Z., 2019. From basic research to molecular breeding - Chinese scientists
and antioxidant defense system in wheat (Triticum aestivum L.). J Sci Food Agr 98 play a central role in boosting world rice production. Genom Proteom Bioinf 16 (6),
(15), 5700–5710. 389–392.
Kumar, J., Sen Gupta, D., Kumar, S., Gupta, S., Singh, N.P., 2016. Current knowledge on ge- Wang, S.S., Liang, D.L., Wang, D., Wei, W., Fu, D.D., Lin, Z.Q., 2012. Selenium fractionation
netic biofortification in lentil. J Agr Food Chem. 64 (33), 6383–6396. and speciation in agriculture soils and accumulation in corn (Zea mays L.) under field
Liu, Y., Lv, H., Yang, N., Li, Y., Liu, B., Rensing, C., et al., 2019. Roles of root cell wall compo- conditions in Shaanxi Province. China. Sci Total Environ. 427, 159–164.
nents and root plaques in regulating elemental uptake in rice subjected to selenite Wei, J., Zeng, C., Gong, Q.Y., Yang, H.B., Li, X.X., Lei, G.H., Yang, T.B., 2015. The association
and different speciation of antimony. Environ. Exp. Bot. 163, 36–44. between dietary selenium intake and diabetes: a cross-sectional study among
Longchamp, M., Castrec-Rouelle, M., Biron, P., Bariac, T., 2015. Variations in the accumula- middle-aged and older adults. Nutr. J. 14, 1–6.
tion, localization and rate of metabolization of selenium in mature Zea mays plants Xiao, X., Liao, X., Chen, T., Yan, X., Xie, H., Zhai, L., et al., 2006. Subcellular distributions of
supplied with selenite or selenate. Food Chem. 182, 128–135. phosphorus and calcium in arsenic hyperaccumulator Pteris vittata L. and its toler-
Marschall, T.A., Bornhorst, J., Kuehnelt, D., Schwerdtle, T., 2016. Differing cytotoxicity and ance to phytotoxicity of arsenic. Acta Scien. Circum. 26 (6), 954–961.
bioavailability of selenite, methylselenocysteine, selenomethionine, selenosugar 1 Yin, H.Q., Qi, Z.Y., Li, M.Q., Ahammed, M., Chu, X.Y., Zhou, J., 2019. Selenium forms and
and trimethylselenonium ion and their underlying metabolic transformations in methods of application differentially modulate plant growth, photosynthesis, stress
human cells. Mol. Nutr. Food Res. 60 (12), 2622–2632. tolerance, selenium content and speciation in Oryza sativa L. Ecotox Environ Safe
Nemeth, A., Dernovics, M., 2015. Effective selenium detoxification in the seed proteins of 169, 911–917.
a hyperaccumulator plant: the analysis of selenium-containing proteins of Zhang, L., Li, Q., Yang, X.D., Xia, Z.L., 2012. Effects of sodium selenite and germination on
monkeypot nut (Lecythis minor) seeds. J. Biol. Inorg. Chem. 20 (1), 23–33. the sprouting of chickpeas (Cicer arietinum L.) and its content of selenium,
Pilon-Smits, E.A.H., Quinn, C.F., Tapken, W., Malagoli, M., Schiavon, M., 2009. Physiological formononetin and biochanin a in the sprouts. Biol. Trace Elem. Res. 146 (3), 376–380.
functions of beneficial elements. Curr. Opin. Plant Biol. 12 (3), 267–274. Zhang, H., Feng, X.B., Chan, H.M., Larssen, T., 2014. New insights into traditional health
Rayman, M.P., 2008. Food-chain selenium and human health: emphasis on intake. Brit J risk assessments of mercury exposure: implications of selenium. Environ Sci Technol
Nutr 100 (2), 254–268. 48 (2), 1206–1212.
Ren, Y.F., He, J.Y., Liu, H.Y., Liu, G.Q., Ren, X.L., 2017. Nitric oxide alleviates deterioration Zhang, M., Tang, S.H., Huang, X., Zhang, F.B., Pang, Y.W., Huang, Q.Y., et al., 2014. Selenium
and preserves antioxidant properties in 'Tainong' mango fruit during ripening. Hortic uptake, dynamic changes in selenium content and its influence on photosynthesis
Environ Biote 58 (1), 27–37. and chlorophyll fluorescence in rice (Oryza sativa L.). Environ. Exp. Bot. 107, 39–45.
Ros, G.H., van Rotterdam, A.M.D., Bussink, D.W., Bindraban, P.S., 2016. Selenium fertiliza- Zhang, M., Xing, G.F., Tang, S.H., Pang, Y.W., Yi, Q., Huang, Q.Y., et al., 2019. Improving soil
tion strategies for bio-fortification of food: an agro-ecosystem approach. Plant Soil selenium availability as a strategy to promote selenium uptake by high-Se rice culti-
404 (1–2), 99–112. var. Environ. Exp. Bot. 163, 45–54.
Sabbioni, E., Polettini, A.E., Fortaner, S., Farina, M., Groppi, F., Manenti, S., et al., 2015. Up- Zhao, X., Zhao, Q., Chen, H., Xiong, H., 2019. Distribution and effects of natural selenium in
take from water, internal distribution and bioaccumulation of selenium in soybean proteins and its protective role in soybean beta-conglycinin (7S globulins)
Scenedesmus obliquus, Unio mancus and Rattus norvegicus: part B. B Environ under AAPH-induced oxidative stress. Food Chem. 272, 201–209.
Contam Tox 94 (1), 90–95. Zhu, Y.G., Pilon-Smits, E.A.H., Zhao, F.J., Williams, P.N., Meharg, A.A., 2009. Selenium in
Schiavon, M., Moro, I., Pilon-Smits, E.A.H., Matozzo, V., Malagoli, M., Dalla Vecchia, F., higher plants: understanding mechanisms for biofortification and phytoremediation.
2012. Accumulation of selenium in Ulva sp and effects on morphology, ultrastructure Trends Plant Sci. 14 (8), 436–442.
and antioxidant enzymes and metabolites. Aquat. Toxicol. 122, 222–231.