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

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Science of the Total Environment

journal homepage: www.elsevier.com/locate/scitotenv

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: daizhihua@webmail.hzau.edu.cn (Z. Dai), m.imtiazpk92@hotmail.com (M. Imtiaz), muhammadrizwan@webmail.hzau.edu.cn (M. Rizwan),
yuany@webmail.hzau.edu.cn (Y. Yuan), hhl@webmail.hzau.edu.cn (H. Huang), stu@mail.hzau.edu.cn (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
Selenium (Se) is an essential micronutrient element for humans and
animals, and it is reported as a component of 25 proteins, including glu-
tathione peroxidase (GSH-Px), the antioxidant capacity of which is N200
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-
ducing the risk of cancer, cardiovascular disease, and diabetes, etc.
(Benstoem et al., 2015; Cai et al., 2016; Wei et al., 2015). In recent de-
cades, hidden hunger caused by Se deficiency is commonly found world-
wide (D'Amato et al., 2018; H.H. Gao et al., 2018; Kumar et al., 2016),
becoming an important health issue as daily intake of Se is below the rec-
ommended allowance (50–200 μg), especially in rice-dependent Asian
countries (D'Amato et al., 2018; EFSA, 2014, H.H. Gao et al., 2018). How-
ever, some soils and mineral deposits are naturally rich in Se (Sun, 2017),
and high Se areas can be found in many different countries, such as
Australia, China, Ireland, India and the USA (Dhillon and Dhillon, 2003),
due to anthropogenic activities such as mining, agriculture, and oil pro-
duction, resulting in excess contents of Se in agriculture products (Both
et al., 2018; Cui et al., 2018; Eiche et al., 2015; Wang et al., 2012) and
harming human and animal health (Song et al., 2018).
Rice fulfils more than half of the food requirement of the world's
population, and in China, N60% of the 1.4 billion people consume rice
on a daily basis (Tang and Cheng, 2019). Hence, the development of
Se-enriched rice is the most popular and effective way to meet human
nutritional needs for Se. However, a daily intake of Se above the recom-
mended range may potentially causes Se toxicity in humans and in-
duces diseases such as hypothyroidism and cardiac disease (Hatfield
et al., 2014). For adults, daily Se intake of 800 μg is considered toxic
(H. Zhang et al., 2014).
The plants exhibited different forms of Se mainly Se(IV), Se(VI),
selenocysteine (SeCys) and selenomethionine (SeMet). Plants take up
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
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-
ficial (Longchamp et al., 2015; Marschall et al., 2016; Rayman, 2008).
Thus, it is important to analyze both the total Se concentrations and dif-
ferent types of Se speciation in edible plant parts to avoid toxicity.
In practical production, soil addition and foliar application of inor-
ganic Se are the best ways to produce Se-enriched agriculture products
(Deng et al., 2017; Ros et al., 2016). Previous studies focused on the Se
accumulation in the edible parts of rice only, not at different growth
stages: the seedling, tillering, heading and maturity stages which re-
spond differently regarding Se accumulation (Gong et al., 2018; Hu
et al., 2018). To the best of our knowledge, there is no information
about the role of Se on different growth stages, especially the spike dif-
ferentiation stages (at which Se starts to transport into brown rice), as
well as the accumulation of Se species in rice.
The objectives of this research were to (1) investigate the uptake,
transformation and distribution of Se in rice plants under different Se
levels, (2) reveal the relationship between Se species and the antioxi-
dant capacity of rice plants, and (3) determine the optimal dose of Se
in soil applications for rice growth. The obtained results will be support-
ive to understand the dynamic behaviors of Se at different rice growth
stages and the mechanisms of Se accumulation as well as its speciation
in rice plants.
2. Materials and methods
2.1. Experimental materials
A pot experiment was conducted under greenhouse conditions at
Huazhong Agricultural University (N 31°28′26″, E 114° 20′15″). Brown-
red paddy soil was used and sampled from the top 20 cm of field soil of
the school farm. The basic soil physical and chemical properties were as
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-
sium 87.16 mg kg−1, and total soil Se 0.27 mg kg−1.
2.2. Experimental design
Se with seven different levels, 0 (control), 0.5, 1.25, 2.5, 5, 10 and
20 mg kg−1, were applied as sodium selenite (Na2SeO3). Each treatment
was replicated 8 times. Each pot (30 cm × 30 cm) was filled with 5 kg
air-dried soil, and fertilizers were applied as follows: N fertilizer
(urea) 0.36 g kg−1, P fertilizer (SSP) 0.087 g kg−1, and K fertilizer
(KCl) 0.50 g kg−1 of soil, with the addition of a mixture of micronutrient
Hoagland solution (Supplementary Table 1) at a rate of 1 ml kg−1 soil.
To generate a good mixture of soil and chemicals, all the fertilizers and
Na2SeO3 were thoroughly mixed 10 days before of rice planting.
The rice (Oryza sativa L. cv. Yangliangyou 6) seeds were germinated
and cultured in an incubator under constant temperature (30 °C) and
light (14 h/d). After 2 weeks of germination, 10 seedlings with a uni-
form size were transplanted into each pot, and Se-free water was used
for irrigation throughout the experiment.
The rice samples were collected at the pistil and stamen formation
stage, pollen mother cell formation stage, pollen mother cell meiosis
stage, and maturity stage, related to plant growth of 108 d, 112 d,
115 d and 134 d, respectively.
The collected samples were rinsed with tap water and then washed
with deionized water and dried with clean tissue paper. The samples
were separated into two parts: one part was stored in an ultra-low tem-
perature freezer for antioxidant enzyme and Se-speciation analyses,
while the remaining part was dried in an oven first at 105 °C for
30 min and then at 60 °C for 2 d to determine the contents of Se. The
Se-speciation was determined from the samples that were collected at
different stages.
2.3. Chemical analysis methods
2.3.1. Determination of Se contents
For total Se content determination, 1 g pulverized rice samples were
weighted and placed in a 50-ml beaker. Then, 10 ml of mixed acid
[HNO3 and HClO4 (9:1)] was added, followed by overnight digestion
in the room temperature. The next day, the samples were digested at
180 °C until the liquid become clear and transparent in approximately
2 ml, followed by the addition of 5 ml of 6 mol L−1 HCl after cooling.
Then, the solutions were again digested on a hot plate with a sand
bath for reduction Se6+ to Se4+, until the solutions became clear and
transparent after white smoke. After cooling, the solutions were trans-
ferred to a 25-ml volumetric flask, followed by an increase in the vol-
ume up to the mark with deionized water. Finally, the 10 ml of
digested solution was collected from each sample into a tube with
2 ml HCl and 1 ml of 100 g L−1 K3[Fe(CN)6]. The Se contents were deter-
mined using an atomic fluorescence spectrometer (AFS-8220). The ac-
curacy of the Se analysis was checked by standard reference material
GBW07602 (GSV-1) (branches and leaves of shrubs certified at 0.184
± 0.013 μg Se g−1) purchased from the center for the standard refer-
ence of China. The standard reference material was run in parallel
with the samples, and the obtained value was found to be in good agree-
ment with the certified value.
2.3.2. Se speciation
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
nitrogen and then transferred to a new 50-ml centrifuge tube. Then,
10 ml of ultrasonic extraction solution was added to the ground samples
and centrifuged at 5000 ×g for 15 min. Subsequently, the supernatant
was collected, and methanol was removed from the samples using ro-
tary evaporator to a volume up to 10 ml. The samples were then filtered
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
Fig. 1. The standard HPLC curve for SeCys, SeMet, Se(IV), and Se(VI) (the oxidizing agent
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
(pH 6.0, 1.0 ml min−1). The AFS conditions were adjusted as follows: Se hollow cathode
lamp current, 50 mA; negative high voltage of photomultiplier tube, 270 V; flow rate of
carrier gas, 400 ml min−1; flow rate of makeup gas, 600 ml min−1).
The atomic fluorescence speciation analyzer (SA-10), atomic fluo-
rescence spectrometer (AFS-8220) of Beijing Titan Instruments Co.
Ltd. and Shimadzu HPLC were used to determine Se-speciation. The
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
(pH 6.0, 1.0 ml min−1). The working conditions for AFS conditions
were as follows: Se hollow cathode lamp current (general research in-
stitute for nonferrous metals, Beijing, China), 50 mA; negative high volt-
age of the photomultiplier tube, 270 V; flow rate of the carrier gas,
400 ml min−1; flow rate of makeup gas, 600 ml min−1.
The Se standard material of SeMet (Sigma, USA), SeCys (Sigma,
USA), selenite radical (GBW10032, 42.9 μg g−1 Se, National Institute of
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.
We tested the prepared Se standard solutions, and the chromatogram
analysis clearly showed four kinds of Se species (Fig. 1). To validate the
method and test conditions, we added four Se species standard solutions
accurately to the Se-enriched samples of rice and performed an extrac-
tion with methanol + water (1:2) to determine their recovery rates.
The recovery rates of the four Se species were 94%–103%, indicating
that the method was reasonable. In addition, reagent blanks and internal
standards were used to ensure the accuracy and precision in the Se spe-
ciation analysis.
2.3.3. Plant subcellular fractionation and Se analysis
The Se contents in subcellular fractions of rice leaves were deter-
mined according to the method described by Xiao et al. (2006). Briefly,
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)
Control 122.0 ± 4.6cd 10.9 ± 1.1e
0.5 129.3 ± 3.5c 14.6 ± 0.8d
1.25 132.3 ± 3.8b 17.8 ± 1.2bc
2.5 138.7 ± 4.0b 20.2 ± 1.0a
5 146.3 ± 2.5a 21.0 ± 1.5a
10 149.7 ± 4.2a 17.7 ± 0.4b
20 132.3 ± 3.1b 16.0 ± 0.7cd
Values are the mean ± SD (n = 3). Different letters indicate a significant difference in the same column at p b 0.05.
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
method of Huang et al. (2013) and expressed as U/mg protein, where
1 U was defined as 0.001 ΔOD470 min−1. Glutathione peroxidase
(GSH-Px) activity was determined according to the method of Flohe
and Gunzler (1984), and the absorbance was recorded at 412 nm, and
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-
termined according to the method used by Cui et al. (2011). Lipid perox-
idation was estimated by measuring the amount of malondialdehyde
(MDA) as previously described (Jin et al., 2013).
2.3.5. Measurement of rice growth parameters and yield
The plant height at maturity stage was recorded by the meter scale.
All plant samples were separated into the straw and rice grain and
dried first at 105 °C for 30 min and then at 60 °C until a constant weight
was attained. The dry biomass, 1000-grain weight, and rice yield were
thus recorded.
2.4. Statistical analysis
The experimental data were analyzed by one-way ANOVA using SAS
8.1 software. Different letters indicated significant differences between
treatments at p b 0.05. All data represent means ± standard deviations
(SD) of three replicates for each treatment.
3. Results
3.1. Effect of Se addition on rice growth and yield
To examine the effect of Se on the growth and development of rice
plants, we recorded the plant height, dry biomass, 1000-grain weight,
and yield at maturity stage (Table 1). Among 7 treatments, Se levels at
2.5–5 mg kg−1 achieved the best parameters. For instances, plant height,
dry biomass, 1000-grain weight, and rice yield increased by 19.91%,
1000-grain weight (g) Rice yield (g plant−1)
28.9 ± 1.3bc 30.9 ± 2.0d
28.9 ± 0.7c 32.8 ± 1.1cd
29.0 ± 0.8bc 33.2 ± 2.8bcd
30.0 ± 0.3ab 38.6 ± 2.9ab
29.9 ± 0.5ab 40.3 ± 1.2a
28.7 ± 0.4c 34.3 ± 0.7c
29.6 ± 0.6bc 31.0 ± 0.5d
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
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
that high Se was not beneficial to plant growth.
3.2. Effect of the Se supply on Se concentrations in rice
3.3. Effect of Se application on Se speciation in rice
Se species were divided into Se(IV), Se(VI), SeCys and SeMet. Sam-
ples collected from different parts of rice at different growth and devel-
opmental stages were analyzed to understand the dynamic changes of
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
Se concentrations in rice shoots (stem + leaves) at different growth
stages ranked as follows: heading stage (16.17 mg kg−1) N maturity
stage (13.72 mg kg−1) N tillering stage (9.20 mg kg−1). As the soil Se
levels increased from 0.5 to 20 mg kg−1, the Se concentrations in the
rice shoots correspondingly increased. The average Se concentrations
in different rice organs at the maturity stage ranked as follows: shoots
(13.72 mg kg−1) N brown rice (2.73 mg kg−1) N husk (1.18 mg kg−1).
The Se concentrations in brown rice and husk followed trends similar
to those in rice shoots (Fig. 2).
3.3.1. Dynamic Se speciation in young spikes at three panicle initiation
stages
The Se speciation at three panicle initiation stages (i.e. pistil and sta-
men formation stage, pollen mother cell formation stage, and pollen
mother cell meiosis stage) showed that organic SeMet was the main
forms of Se accumulated in young spikes of rice plants (Fig. 3). The per-
centages of SeMet ranged from 65.5 to 100%. While, SeCys (organic
form), Se(IV), and Se(VI) (inorganic form) were accumulated about
8.4–24.2%, 5.3–15.4%, and 0–10.7%, respectively. It was interesting to
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
Fig. 4. Selenium speciation in brown rice with soil application of Se 0–20 mg kg−1.
percentage of organic SeMet and SeCys but increased the percentages of
inorganic Se(IV) and Se(VI) .
Further analysis of the Se species at different panicle initiation
stages revealed that the SeMet content ranked as follows: pistil and
stamen formation stage (16.52 mg kg−1) N pollen mother cell forma-
tion stage (10.96 mg kg−1) N pollen mother cell meiosis stage
(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
cell formation stage (1.48 mg kg−1) N pollen mother cell meiosis
stage (0.65 mg kg−1). The average Se contents of the pistil and sta-
men formation stage, pollen mother cell formation stage, and pollen
mother cell meiosis stages were 1.76, 1.81 and 0.32 mg kg−1, respec-
tively, while Se(VI) was only detected following the 20 mg kg−1 Se
treatment, indicating that higher levels of Se could not be good for
the young spike of rice.
3.3.2. Se speciation in brown rice
Unlike the results obtained for young spikes of rice (Fig. 4), SeCys
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
(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
soil.
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.
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
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.
Overall, as compared with the control, the POD activity increased by
161% to 176%. The response of both CAT and GSH-Px activity against
Se levels was a second-degree parabola, and the highest activity was ob-
served at the 1.25–2.50 mg kg−1 Se level. Compared with the control,
the activity of CAT and GSH-Px following 1.25–2.50 mg kg−1 Se treat-
ment was 104–151% and 104–121%, respectively.
3.4.2. GSH, AsA and MDA contents
The contents of GSH in rice leaves increased by 6.2% to 66.5% when
exposed to 0.5 to 20 mg kg−1 of Se treatments. However, the highest
content of AsA recorded at the Se levels of 5 mg kg−1, with increases
by 62.9% compared to the control (Fig. 7).
The MDA contents in the cell membrane were not increased under
soil Se (0.5–1.25 mg kg−1); however, a significant reduction of MDA
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).
4. Discussion
4.1. Effects of Se treatments on rice growth and yield
The soil application Se at lower levels (i.e. 0.5–5 mg kg−1) signifi-
cantly increased rice growth and yield (Table 1). This finding was
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
et al., 2017) and tobacco (Han et al., 2013), indicating that lower Se has
positive effects on plants, although Se is not an essential element. For in-
stance, previously studies have reported that appropriate application of
Se could promote the photosynthesis in rice (M. Gao et al., 2018; M.
Zhang et al., 2014), wheat (Kaur and Sharma, 2018) and Brassica juncea
(Handa et al., 2019). The appropriate Se application could also promote
the synthesis of amino acids and protein in rice (Hu et al., 2018; Singh
et al., 2018) and soybean (Zhao et al., 2019). Se applications could alle-
viate the detrimental effects of rice leaf senescence (Duan et al., 2019).
Additionally, Se could increase the contents of pectin, hemicelluloses
and lignin in cell walls to resist heavy metal absorption (Liu et al., 2019).
4.2. Changes in Se species in rice with soil Se levels
Assay of Se species found that four main Se-species [i.e. Se(IV), Se
(VI), SeMet and SeCys] were present in rice plants, and the SeMet was
the major species found in rice shoots (Fig. 3). A similar trend was ob-
served in previous studies, which confirmed that Se mainly exists as
SeMet in rice (Zhang et al., 2012). Earlier research has shown that the
inorganic Se transforms into organic Se after being absorbed by plants,
and first into SeCys and then into other forms, such as SeMet, due to
the activation of a variety of enzymes (Dinh et al., 2019). However,
our study showed that SeCys was the major species in brown rice, po-
tentially due to the rapid and active transformation of SeMet during
the process of being converted into rice grain.
In this research, the inorganic Se-species increased in higher levels of
soil Se (Figs. 3 and 4). Similar results were reported by Han et al. (2013)
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
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-
tuce and chard leaves, while SeMet N Se(VI) N Se(IV) in parsley leaves
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
SeCys, Se(IV), and Se(VI) are available for the synthesis of selenoproteins,
whereas SeMet can substitute for methionine in proteins where it can act
as a Se store (Nemeth and Dernovics, 2015).
In the present study, Se was mainly deposited in the cell wall fraction
in the form of SeMet (Fig. 5). Thus, certain substances or structures of
the rice cell wall might have absorbed the SeMet. The obtained results
revealed that different species were observed in the soluble fraction
and cell organelles. With increasing soil Se levels, the percentage of in-
organic Se (especially Se(VI)) increased in the soluble fraction, while
SeCys decreased and SeMet increased in cell organelles. The subcellular
distribution of Se species was closely related to the pathway of Se me-
tabolisms and detoxicity in plant. However, few reports have examined
the subcellular distribution of Se speciation in plants. Plants may accu-
mulate Se species in different subcellular parts. For instance, Ding
et al. (2015) reported that Se was primarily concentrated in the cell
wall fraction and cytosol fractions in paddy rice. In contrast, Sabbioni
et al. (2015) reported the presence of Se in all subcellular fractions, es-
pecially in the cytosol of microalgae, as well as in membranes and or-
ganelles of spring tea leaves (Du et al., 2010).
4.3. Effects of Se treatments on antioxidant system and nonenzymatic
antioxidants
Se is an active center of GSH-Px and plays an important role in the
plant antioxidant defense system (Ahsan et al., 2014). All the SOD,
POD, CAT and GSH-Px enzymes can protect the structure and func-
tion of the cell membrane from oxidation and damage by removing
free radicals, such as peroxide and H2O2. In this study, soil applica-
tion of Se significantly influenced the activities of GSH-Px, SOD,
POD and CAT, as well as the contents of GSH and AsA (Figs. 6 and
7). The activities of SOD, POD, CAT and GSH-Px exhibited basically
a quadratic parabolic curve, which indicated that the activities of an-
tioxidant enzymes were increased in rice at lower Se levels, while an
opposite trend was observed at higher levels of soil Se, especially for
CAT and GSH-Px. Earlier studies have also reported that the activities
of SOD, POD, CAT and GSH-Px increased with the increasing Se levels
at low levels. For instance, SOD and CAT activities were significantly
increased in Ulva sp. when exposed to sodium selenate (Schiavon
et al., 2012). Similarly, POD and CAT activities were also higher
than that in the control when Se b5 mg L−1 was applied (Feng and
Wei, 2012). In the present study, with continuously increasing Se
levels, SOD, POD and CAT activities were increased. When Se treat-
ment ranged from 5 to 20 mg kg−1, Se(VI) was detected in brown
rice, and SOD activity was increased and CAT activity decreased com-
pared with the control. The underlying mechanism for this phenom-
enon might be explained the production of hydrogen peroxide and
superoxide radicals (O− 2 ) in rice, which enhanced SOD activity. It
has been suggested that high Se toxicity mainly derives from its
role as an oxidizing agent, resulting in the production of peroxides,
which are poisonous for crop plants (Feng and Wei, 2012).
5. Conclusions
Soil Se application at low Se levels (b5 mg kg−1) promoted plant
growth and rice yield. Organic SeCys and SeMet were the main species
in rice, and the relative content decreased with the Se levels. The inor-
ganic Se(IV) and Se(VI) increased in brown rice at high Se levels (10
and 20 mg kg−1), indicating a potential threat to human health. Thus,
based on the present findings, 0.5–5 mg kg−1 could be a suitable and
safe dose for Se-enriched rice production.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.scitotenv.2019.07.186.
Declaration of Competing Interest
The authors declare no conflict of interests.
833
Acknowledgments
This research was supported in part by the Guangxi Major Special
Project of Science and Technique (Guike AA17202026-3), Hubei Special
Project for Technique Innovation (2017ABA154), and Special Fund for
Agro-scientific Research in the Public Interest (201303106).
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