Journal of Cereal Science 79 (2018) 508e515

Contents lists available at ScienceDirect

Journal of Cereal Science

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

Agronomic biofortification of upland rice with selenium and nitrogen

and its relation to grain quality
Heitor Pontes Gestal Reis a, Je
ssica Pigatto de Queiroz Barcelos a, Enes Furlani Junior a,
Elcio Ferreira Santos b, Vinícius Martins Silva a, Milton Ferreira Moraes c,
Fernando Ferrari Putti d, Andre
Rodrigues dos Reis a, d, *
a ~o Paulo State University - UNESP, 15385-000, Ilha Solteira, SP, Brazil
Sa
b ~o Paulo - USP, 13416-000, Piracicaba, SP, Brazil
University of Sa
c
Federal University of Mato Grosso e UFMT, 78600-000, Barra do Garças, MT, Brazil
d ~ ~, SP, Brazil
Sao Paulo State University - UNESP, School of Sciences and Engineering, 17602-496, Tupa

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

Article history: The objective of the present study was to evaluate agronomic biofortification with nitrogen (N) and
Available online 16 January 2018 selenium (Se) with the aim of increasing the daily Se intake by the population and the nutritional quality
of the grain. A randomized block experimental design with a 5
2 factorial scheme was used; the factors
Keywords: were the five levels of N (0, 20, 40, 80, and 120 kg ha1), applied as topdressing fertilization, and the two
Reserve proteins levels of Se (0 and 25 g ha1). The reserve protein fractions in the seeds, glutelin, and globulin increased
Physiological responses
significantly when Se application was combined with N fertilization. Grain Se content varied from 0.03 to
Human health
0.35 mg kg1, which was within the food safety limit of 0.3 mg kg1 established by the Codex Ali-
Sodium selenate
mentarius. The estimated daily Se intake originating from Se-biofortified rice varied between 2.05 and
24.7 mg per day, representing an increase from 3.72% to 44.9% of the daily Se requirement. Because the
recommended Se daily intake for adults is 55 mg per day, the present study presents relevant information
about agronomic biofortification to increase Se concentrations in edible plant parts, with possible
benefits to human health.
© 2018 Elsevier Ltd. All rights reserved.

1. Introduction hypothyroidism, cardiovascular diseases, fragile immune system,

Food is the main nutrient source, and its demand increases with
the increase in population. The current world's population is 7.2
billion inhabitants and is estimated to reach 12.3 billion in 2100
(Gerland et al., 2014). Cereal production has been growing at the
same rate as the population. Ray et al. (2013) estimated that food
production should increase between 60 and 110% until 2050 to
meet the growing global demand. In turn, malnutrition has been
increasing, affecting half of the world's population, especially
pregnant women, adolescents, and children (Graham et al., 2007).
This includes selenium (Se) deficiency in humans, which is
increasingly more frequent (Reis et al., 2017).
Combs (2011) estimated that approximately 1 billion people are
likely deficient in Se. In humans, Se deficiency is associated with
* Corresponding author. Sa ~o Paulo State University - UNESP, 17602-496, Tupa
~, SP,
Brazil.
E-mail address: andrereis@tupa.unesp.br (A.R. Reis).
male infertility, and an incidence of several types of cancer
(Fordyce, 2013). The recommended Se intake is 55 mg per day for
adults (White, 2016). Considering that dietary Se is basically
derived from food, Se deficiency in humans is related to the con-
sumption of foods with low Se content in the edible parts (Reis
et al., 2017). This is due to the low soil Se available for plant up-
take (White, 2016).
Se may increase growth and improve the nutritional status of
vascular plants (Graham et al., 2007). The role of Se in grain for-
mation is still little understood, but Seregina et al. (2001) showed
an interaction effect between Se and N on increased wheat pro-
ductivity. The authors also observed stronger positive effects of Se
on wheat grain formation when combined with higher N levels. In
addition to its nutritional role, Se also plays an important part in
plant antioxidant protection. Depending on the dose, Se may acti-
vate enzymes such as superoxide dismutase (SOD) and catalase
(CAT). These enzymes are activated in the presence of Se,
decreasing the lipid peroxidation and hydrogen peroxide formation

https://doi.org/10.1016/j.jcs.2018.01.004
0733-5210/© 2018 Elsevier Ltd. All rights reserved.
 H.P.G. Reis et al. / Journal of Cereal Science 79 (2018) 508e515
rates in plant cells and resulting in reduced senescence (Saidi et al.,
2014).
According to the results of decades-long monitoring of bio-
fortification programs in Northern European countries, such as
Finland, currently, the addition of Se to the NPK fertilizers used in
agricultural areas seems to be the most effective and safest way to
avoid Se deficiency in humans and animals (Haug et al., 2007). The
authors also state that after these programs were established,
mortality indexes greatly decreased, especially those related to
heart diseases, cancer, and incidence of vitamin E deficiency.
Attempts to increase the Se content in edible plant parts may
change the crop metabolism and nutritional status because Se in-
terferes with the sulfur metabolism, and such an increase probably
affects the whole nitrogen (N) metabolism and grain yield (Ríos
et al., 2010; White, 2016). Se addition via fertilizers is involved in
the quality improvement of agricultural products. Agronomic bio-
fortification with Se increases the selenocysteine and selenome-
thionine contents, which are essential for human and animal health
(Rayman et al., 2012). Studies are therefore needed to evaluate the
interactions between the N and Se levels and facilitate a better
understanding of Se accumulation in rice grains, as rice is one of the
planet's main food sources. The objective of the present study was
to evaluate the potential of agronomic biofortification with Se, used
in combination with N topdressing fertilization of upland rice,
under field conditions, with the aim of increasing daily Se intake by
the Brazilian population, which may result in improved human
health.
2. Materials and methods
2.1. Description of the experimental site
The study was performed between January and May 2015 in
Selvíria, Mato Grosso do Sul, Brazil (20 220 S and 51220 W, 335 m
altitude). The experimental site belongs to the Cerrado biome, has
been cultivated for more than 25 years, and has been under no-
tillage for the last 10 years. According to the Ko€ ppen climate clas-
sification, the region's climate is Aw, tropical humid, with a rainy
season in the summer and dry season in the winter. The mean
annual rainfall is 1232 mm, and the mean annual temperature is
24.5  C. During the experiment, the mean daily temperature varied
between 27.2  C and 15.3  C, the mean rainfall was 3.0 mm, and the
mean relative humidity was 86%.
The soil in the area was classified as Oxisol. The soil analysis
revealed the following chemical characteristics: phosphorus (resin)
29 mg dm3; organic mater 21 g dm3; pH calcium chloride (CaCl2)
5.3; potassium 3.5 mmolc dm3; calcium 38 mmolc dm3; mag-
nesium 22 mmolc dm3; Hþ þ Al mmolc dm3; aluminum 0 mmolc
dm3; Se 62 mg kg1; cation exchange capacity 92.5 mmolc dm3;
and base saturation (V%) - 69%.
A randomized block experimental design was used, with a 5
2
factorial scheme, with four replicates, totaling 40 plots. The factors
were five levels of N (0, 20, 40, 80, and 120 kg ha1), applied as urea,
and there were two levels of Se (0 and 25 g ha1), applied as sodium
selenate. A Se stock solution was prepared, diluted in 500-mL
bottles, and distributed over the planting furrow. The Se dose
applied (25 g ha1) was selected based on preliminary results (Reis,
2015).
2.2. Experimental setup
The experiment was performed under a no-tillage system. Soil
acidity was not corrected because the base saturation was adequate
for upland rice. Twenty days before sowing, the area was desiccated
with glyphosate (4.0 L ha1), carfentrazone-ethyl (200 mL ha1),
509
and 0.5% mineral oil.
The upland rice cultivar used was ANa 5015, sown at a density of
approximately 70 kg ha1. The experimental plots consisted of four
6-m-long rows, spaced 0.35 m apart. The useful area consisted of
the two central rows, excluding 0.5 m of the row ends. Concur-
rently, seeds were treated with fipronil at 2 mL c.p. (commercial
product) kg1 seeds. Fertilization was performed at sowing, with
the application of 250 kg ha1 08-28-16 NPK. Irrigation was per-
formed as needed, using a center-pivot sprinkler irrigation system,
with a mean water depth of 14 mm, and 72-h irrigation frequency.
Plant health management during the crop cycle was performed
with the application of metsulfuron-methyl (3.3 g ha1), chloran-
traniliprole (50 mL ha1), flubendiamide (60 mL ha1), and
imidacloprid þ beta-cyfluthrin (0.8 L ha1).
2.3. Chlorophyll index
Fourteen days after the treatment application, indirect chloro-
phyll measurements were performed in five plants from each plot,
on three different points of the flag leaf, using a portable chloro-
phyll meter (SPAD-502, MINOLTA).
2.4. Antioxidant system
Twenty days after the treatment application, the flag leaves
from 15 plants from each plot were collected, placed in liquid ni-
trogen, and used for quantification of the hydrogen peroxide con-
tent, lipid peroxidation, soluble protein content, and SOD and CAT
activity. The plant material was ground in liquid N and stored
at 80  C until analyzed.
2.4.1. Hydrogen peroxide content
Hydrogen peroxide (H2O2) content was determined after reac-
tion of the plant material with potassium iodide (KI), according to
Alexieva et al. (2001). Leaf hydrogen peroxide contents were
calculated based on a standard curve, and the results were
expressed in nmol g1 FW (fresh weight).
2.4.2. Lipid peroxidation
Lipid peroxidation was determined by the production of 2-
thiobarbituric acid (TBA)-reactive substances, especially malon-
dialdehyde, according to Heath and Packer (1968).
2.4.3. Protein and antioxidant enzyme extraction
The plant material stored at 80  C was ground in liquid ni-
trogen with a mortar and pestle. For protein extraction, approxi-
mately 0.3 g processed plant material was placed into 15-mL Falcon
tubes and extracted with 5 mL of 100 mM potassium phosphate
buffer, pH 6.8, with 1 mM ethylenediaminetetraacetic acid (EDTA),
3 mM dithiothreitol (DTT), and 4% polyvinylpolypyrrolidone (PVPP)
(w/v). The supernatant was transferred to Eppendorf tubes and
used for protein and antioxidant enzyme activity quantification.
2.4.4. Reserve protein determination
For extraction of the reserve proteins, 0.25 g of dry and ground
grain was weighed and subjected to sequential extraction with
5 mL of deionized water (for albumin), 5 mL of 5% NaCl (for glob-
ulin), 2.5 mL of 60% ethanol (for prolamin), and 5 mL of 0.4% NaOH
(for glutelin). Protein concentration was determined according to
Bradford (1976), using BSA as the standard.
2.4.5. Superoxide dismutase (SOD, EC 1.15.1.1)
SOD activity was determined according to Giannopolitis and
Ries (1977).
510 H.P.G. Reis et al. / Journal of Cereal Science 79 (2018) 508e515
2.4.6. Catalase (CAT, 1.11.1.6)
CAT activity was determined by monitoring the decomposition
of H2O2 according to Azevedo et al. (1998).
2.5. Growth and yield evaluation
At the end of the experiment, panicles were collected and
weighed. For determination of the total crop yield, a 100-g sample
of upland rice was collected from each plot and processed in a rice
huller for 1 min. The percent filled grain was determined as the
weight of the polished grain. The polished grains were then placed
in a trieur, and the grains were separated for 30 s. The grains that
remained in the trieur were weighed, and the percent undamaged
grains and percent broken grains were determined.
2.6. Nutrient content analysis
The flag leaves from 20 plants from each plot were collected 15
days after treatment application, dried, and used for quantification
of the leaf macro- and micronutrient concentrations. Grain
collected at the end of the crop cycle was also analyzed. N con-
centrations were determined using the semi-micro Kjeldahl
method, following sulfuric acid digestion. For quantification of the
remaining nutrients, the material was subjected to nitric acid-
hydrogen peroxide digestion according to Chilimba et al. (2011).
The ground grain was digested in a microwave oven for 45 min
under controlled pressure (20 bar), in 3 mL of 70% nitric acid
(Merck), 2 mL of hydrogen peroxide (Merck) and 3 mL of milli-Q
water. Se and nutrient analysis was performed by inductively
coupled plasma mass spectroscopy (ICP-MS). The final Se and
micronutrient contents are expressed in mg kg1 dry weight.
2.7. Statistical analysis
For all data sets, normality was tested using the Anderson-
Darling test and homoscedasticity using Levene's test for the
equality of variances. The data were subjected to analysis of vari-
ance using the F test, at p  .05. When significant differences were
found, the Tukey test was used to test for differences between
means, at p  .05, using the R statistical software. Graphs were
plotted using the SigmaPlot software.
3. Results and discussion
Se application in combination with N topdressing fertilization
did not affect the Soil-Plant Analyses Development (SPAD) index,
plant height, percent filled grains, percent undamaged grains,
percent broken grains, or total crop yield.
In the present study, increasing the N doses resulted in an
increasing SPAD index, which varied from 33 to 45 (Fig. 1A).
Increasing the N doses applied to rice crops has been observed to
increase the values of the readings performed on a portable SPAD
chlorophyll meter (Chen et al., 2015; Yuan et al., 2016).
N is one of the main components of chlorophyll and other pig-
ments that absorb solar energy and trigger photosynthesis.
Therefore, greater N accumulation is suggested to result in higher
production of photoassimilates. Because the SPAD index is an in-
direct measure of the leaf chlorophyll concentration, higher N
doses are expected to result in a higher SPAD index.
Increasing the N fertilization resulted in increasing plant height.
N fertilization at levels lower than 40 kg ha1 did not result in the
adequate growth of upland rice (Fig. 1B). By contrast, high N levels
promote tillering and leaf formation, causing self-shading, which
increases competition and lowers productivity (Chen et al., 2015).
In the present study, the percent filled grain was lower without
N fertilization (Fig. 1C). The treatment that received N fertilization
performed better in this evaluation, and the highest percent filled
grains value was observed with 80 kg N ha1. This finding agrees
with Cardoso et al. (2015). The effect of N on the grain yield has
been studied by several authors, and the results indicate that the
effects of N application on the percent of undamaged grains varies
and likely depends on the cultivar and environmental factors (Chen
et al., 2015; Yuan et al., 2016).
N fertilization also had a direct effect on the percent undamaged
grains and percent broken grains (Fig. 1D and E). The percent un-
damaged grains increased, and the percent broken grains
decreased with increasing N fertilization. Cardoso et al. (2015)
observed that N fertilization affected the undamaged grain yield,
with 65% undamaged grains observed for the maximum economic
dose.
The total yield of the cultivar ANa 5015 increased with
increasing N fertilization. This increase was slight for treatment
80 kg N ha1, indicating that this dose was the most economically
viable (Fig. 1F).
Lipid peroxidation was significantly lower with than without Se
application for the treatments with 0, 20, and 40 kg N ha1 (Fig. 2A).
Without Se application, the malondialdehyde (MDA) contents were
higher for the treatments with 80 and 120 kg N ha1, indicating a
high peroxidation rate of the lipid bilayer of leaf cell membranes.
Ríos et al. (2008) applied Se to lettuce and observed that MDA
contents tended to decrease in the presence of Se. Similar results
were reported by Xue et al. (2001). In the present study, the N and
Se application had no significant effects on the H2O2 contents, but
the MDA contents clearly indicated a beneficial effect of Se, which
decreased the peroxidation rate of the lipid bilayer in rice plants.
The application of low sodium selenate concentrations may
promote the induction of the antioxidant system in plants, there-
fore increasing the resistance to oxidative stress (Rios et al., 2010),
as observed in the present study. Plant treatment with sodium
selenate induces significant increases in the activity of enzymes
such as CAT and peroxidase, which detoxify hydrogen peroxide
(Rios et al., 2010).
In the treatments with Se application, the total leaf protein
content increased with increasing N fertilization up to 80 kg N ha1,
followed by stabilization at 120 kg N ha1. Without Se application,
the leaf total protein content was highest, with 120 kg N ha1
(Fig. 2B).
During oxidative stress, excess production of reactive oxygen
species (ROS) causes damage to cell membranes, eventually leading
to cell death. Plants possess antioxidant enzymes, such as SOD and
CAT, and a wide variety of non-enzymatic antioxidant compounds
for protection against ROS (White, 2016). Increased activity of the
antioxidant enzymes CAT and SOD was observed in rice leaves.
CAT is mainly found in peroxisomes and converts H2O2 into
water; however, its affinity to the substrate is weaker than that of
other antioxidant enzymes (Feng et al., 2013). In the present study,
CAT activity was highest for the treatment with 120 kg N ha1 and
25 g Se ha1 (Fig. 2C). Significant differences in the CAT activity
between the treatments with and without Se were only observed
for treatments with 0 and 120 kg N ha1, with CAT activity being
higher without Se for 0 kg N ha1 and higher with Se for 120 kg N
ha1. Rios et al. (2010) studied the effect of Se application on the
antioxidant system in lettuce and observed no effect of Se appli-
cation on CAT activity. The authors concluded that CAT is not one of
the main enzymes for H2O2 detoxification.
SOD is the enzyme responsible for eliminating superoxide rad-
icals and converting them into H2O2 because it is the first enzyme
acting in the antioxidant system (Saidi et al., 2014). Increasing the N
fertilization resulted in no significant differences in SOD activity in
upland rice (Fig. 2D). Significant differences were only observed
 H.P.G. Reis et al. / Journal of Cereal Science 79 (2018) 508e515
Fig. 1. SPAD index, plant height, percent filled grains, percent undamaged grains, percent broken grains, and total crop yield of the upland rice cultivar ANa 5015 in response to
different N fertilization levels.
between the treatments with and without Se because the Se
application resulted in increased SOD activity. Rios et al. (2010) also
observed higher SOD activity in plants treated with Se.
The contents of the reserve proteins albumin, prolamin, and
glutelin in the rice grains increased with increasing N fertilization
(Fig. 3). This result was expected because these proteins are the
main N reserve in the rice grains, and their accumulation increases
with increasing plant N uptake (Poluboyarinov and Golubkina,
2015). However, albumin, prolamin, and glutelin contents were
higher for plants with Se application for all N treatments except 0 N.
This result indicates a beneficial effect of Se on N metabolism,
inducing N redistribution to improve grain formation in rice plants.
Prolamin concentrations were not affected by the N or Se
applications.
Regarding grain nutrient concentrations, Se application com-
bined with N fertilization resulted in higher grain concentrations of
N, P, K, Ca, Se, B, Cu, Zn, and Mo (Table 1). Se, Mg, Mn, and Fe
contents were not affected by Se. Grain N contents were higher
with Se application.
511
Differences in the P content between the treatments with and
without Se were observed starting at 40 kg N ha1 (Table 1). This
finding agrees with Wu and Huang (1992), who reported increased
P contents with selenate application, which provided low Se levels.
Se contents were higher with 40 kg N ha1. Se application resulted
in increased S concentration in the treatment with 20 kg N ha1
and decreased with 40 kg N ha1 (Table 1). White et al. (2004)
suggested that selenate promotes the S uptake at 4 mM S in the
rhizosphere. By contrast, Rios et al. (2013) reported that high Se
levels have a negative effect on S uptake in lettuce, resulting in
decreased S content in plants. For K contents, significant in-
teractions between the N fertilization level and Se application were
only observed starting at 40 kg N ha1. Se application increased the
grain K content for treatments of 40 and 120 kg N ha1 (Table 1).
Grain Se contents were directly affected by sodium selenate
application. Se application resulted in increased grain Se contents
independently of N fertilization. N fertilization helped plants up-
take Se, since grain Se content was 0.22 mg kg1 with 0 kg N ha1
and reached a mean of 0.34 mg kg1 with N fertilization (Table 1).
512 H.P.G. Reis et al. / Journal of Cereal Science 79 (2018) 508e515

Fig. 2. Malondialdehyde (MDA) and total soluble protein contents and catalase (CAT) and superoxide dismutase (SOD) activity in the leaves of the upland rice cultivar ANa 5015 in
response to different N fertilization levels and Se application. Different lowercase letters indicate significant differences between treatments with and without Se application,
according to the t-test (p  .05). Error bars represent the standard error of the mean (n ¼ 4).

Fig. 3. Grain albumin, globulin, prolamin, and glutelin contents in the upland rice cultivar ANa 5015 in response to different N fertilization levels and Se application. Different
lowercase letters indicate significant differences among treatments with and without Se application, according to the t-test (p  .05). Error bars represent the standard error of the
mean (n ¼ 4). Abbreviation: ns e non-significant.
 H.P.G. Reis et al. / Journal of Cereal Science 79 (2018) 508e515 513

Table 1
Grain nutrient contents in response to different N fertilization levels and Se application in the upland rice cultivar ANa 5015.

N P S K Ca Mg Se B Mn Fe Cu Zn Mo

g kg1 mg kg1

Se (g ha1) 0 18.48b 3.87b 1.32 2.84b 0.07 1.69 0.03b 0.40b 22.35 13.28 5.59b 27.25b 0.04b
25 19.18a 4.04a 1.32 2.91a 0.08 1.74 0.32a 0.71a 23.15 13.45 5.75a 29.09a 0.09a
N Lin. ns p  .05 p  .05 Ns ns ns p  .05 Ns p  .05 ns p  .05 Ns ns
Quad ns p  .05 p  .05 p  .05 ns ns p  .05 p  .05 p  .05 ns p  .05 Ns p  .05
0 (kg ha1) 0 18.39 4.31A 1.22B 2.99A 0.07 1.56Bb 0.03b 0.35Bb 23.39Bb 11.22Cb 5.19B 29.17A 0.04b
25 18.69 4.16A 1.18C 3.01A 0.08 1.77a 0.22Ca 0.95Aa 26.47Aa 15.31Aa 5.26C 29.16AB 0.11Ba
20 (kg ha1) 0 17.96 4.24A 1.22Bb 2.84AB 0.07 1.80A 0.03b 0.37Bb 26.21Aa 16.41Aa 5.03Bb 27.66AB 0.05a
25 18.80 4.18A 1.33ABa 2.97A 0.07 1.76 0.34ABa 0.61Ca 23.74Bb 10.16Cb 5.52BCa 28.49B 0.02Db
40 (kg ha1) 0 18.78 3.45Bb 1.39Aa 2.81ABb 0.07 1.68AB 0.04b 0.33Bb 21.60BC 12.03BC 6.22Aa 24.43Cb 0.04b
25 19.27 4.11Aa 1.30Bb 3.06Aa 0.08 1.79 0.31Ba 0.70Ba 21.54B 14.98Aa 5.66Bb 31.74Aa 0.10Ba
80 (kg ha1) 0 18.74 3.69B 1.41A 2.84ABa 0.07 1.75A 0.02b 0.47Ab 20.76BC 12.72BC 6.19Ab 29.01A 0.04b
25 19.16 3.81AB 1.38A 2.58Bb 0.07 1.63 0.35Ab 0.66BCa 21.82B 14.23AB 6.48Aa 27.38B 0.17Aa
120 (kg ha1) 0 18.55 3.66Bb 1.35A 2.75Bb 0.07 1.63AB 0.02b 0.48Ab 19.81Cb 14.00B 5.32Bb 25.95BC 0.045b
25 19.99 3.94Ba 1.39A 2.91Aa 0.08 1.75 0.35Aa 0.64BCa 22.18Ba 12.55B 5.81Ba 28.68B 0.06C
CV 5.28 3.74 2.83 3.42 3.95 5.54 9.23 5.81 5.70 8.31 2.52 4.69 20.42
Se p  .05 p  .05 ns p  .05 p  .05 ns p  .05 p  .05 p  .05 ns p  .05 Ns p  .05
N ns p  .05 p  .05 p  .05 ns ns p  .05 p  .05 Ns ns p  .05 p  .05 p  .05
Se x N ns p  .05 p  .05 p  .05 ns p  .05 p  .05 p  .05 p  .05 p  .05 p  .05 p  .05 p  .05

Abbreviation: NS: Not significant, ** significant at p  .05; * significant at p  .01; Means followed by the same lowercase letters are not significantly different for the same N
level and followed by the same case letter are not significantly different between treatments with and without Se application, according to the t-test (p  .05); MSD: Minimum
significant difference; C.V.: coefficient of variation; Lin.: linear regression; Quad.: quadratic regression.

Grain Fe contents were higher with than without Se application
for treatments 0 and 40 kg N ha1, increasing from 11.22 to
15.31 mg kg1 and from 12.03 to 14.98 mg kg1, respectively. By
contrast, for the 20 kg N ha1 treatment, the Fe content with Se
application decreased from 16.41 mg kg1 to 10.16 mg kg1.
Se application only affected the Zn content in the treatment
with 40 kg N ha1, which presented the highest Zn concentration.
Grain Mo contents were not affected by the N fertilization but were
affected by the Se application. For treatments with 0, 20, and
80 kg N ha1, the Se application considerably increased the Mo
content. The highest grain Mo contents were observed with ap-
plications of 80 kg N ha1 in combination with Se.
The leaf nutrient contents in response to the N and Se applica-
tion are summarized in Table 2. Leaf N, Cu, and Se contents were
higher with Se application. Se application had different effects on
the leaf P contents depending on the N fertilization level.
Leaf S contents were not affected by the N level and were only
affected by the Se application in the treatment with 0 kg N ha1,
where it resulted in decreased leaf S contents. This was because
both the S and the Se, when applied as selenate, use sulfate
transporters (SULTR), i.e., S transporter proteins, in plants (White,
2016). Therefore, the higher the soil Se content is, the lower the
plant S uptake and transport. Increased leaf K contents were
observed for treatments with 20, 80, and 120 kg N ha1 combined
with Se application. The highest leaf K contents were observed for
the highest N levels (80 and 120 kg ha1) combined with Se. Leaf Ca
contents were lower with the Se application combined with the N
fertilization. The highest Ca contents were observed for the treat-
ments of 80 and 120 kg N ha1 without Se application (8.20 and
8.14 mg kg1, respectively).
Leaf Se contents were directly affected by Se application. Leaf Se
contents were higher with the application of 25 g Se ha1 for all

Table 2
Leaf nutrient contents in response to different N fertilization levels and Se application in the upland rice cultivar ANa 5015.

N P S K Ca Mg Se Cu Zn
1 1
g kg mg kg

Se (g ha1) 0 17.47b 1.97 2.89a 10.61a 7.82a 2.73 0.02b 6.32b 9.18a
25 18.44a 1.98 2.72b 11.43b 7.21b 2.74 0.38a 6.73a 8.46b
N Linear p  .05 p  .05 p  .05 ns ns p  .05 ns p  .05 p  .05
Quadratic ns p  .05 p  .05 ns p  .05 Ns p  .05 ns p  .05
0 (kg ha1) 0 16.14 2.90Aa 3.41Aa 11.57Ba 7.25BC 2.58Db 0.02b 5.67Bb 10.61Aa
25 17.87 2.51Ab 2.76b 10.33ABb 7.54AB 2.75ABa 0.23Ea 6.75Aa 8.64Ab
20 (kg ha1) 0 16.08 2.19Bb 2.82B 10.61Bb 7.67ABa 2.71BC 0.02b 6.13ABb 9.13B
25 18.87 2.41ABa 2.92 11.50Ca 6.98Ab 2.69B 0.37Ca 7.04Aa 8.79A
40 (kg ha1) 0 17.76 1.76Ca 2.75B 11.37C 7.83Aa 2.64CDb 0.01b 6.46A 9.17Ba
25 17.71 1.39Cb 2.81 11.31BC 7.33ABb 2.76ABa 0.54Aa 6.04B 7.46Bb
80 (kg ha1) 0 18.74 1.69Ca 2.65B 9.75Bb 8.20C 3.01Aa 0.01b 6.72A 8.40C
25 18.81 1.32Cb 2.47 12.69Aa 7.76B 2.72ABb 0.44Ba 7.04A 8.67A
120 (kg ha1) 0 18.62 1.33Db 2.82C 9.74Ab 8.14ABa 2.73A 0.01b 6.60A 8.61BC
25 18.94 2.28Ba 2.63 11.33Aa 6.43Cb 2.78B 0.32Da 6.76A 8.72A
C.V. 7.94 5.42 4.06 3.53 4.33 1.34 8.19 4.64 8.81
Se p  .05 ns p  .05 p  .05 p  .05 p  .05 p  .05 p  .05 p  .05
N Ns p  .05 p  .05 p  .05 p  .05 Ns p  .05 p  .05 p  .05
Se x N Ns p  .05 p  .05 p  .05 p  .05 p  .05 p  .05 p  .05 p  .05

Legend: NS: Not significant, ** significant at p  .05; * significant at p  .01; Means followed by the same lowercase letters are not significantly different for the same N level
and followed by the same uppercase letter are not significantly different between treatments with and without Se application, according to the t-test (p  .05); C.V.: coefficient
of variation.
514 H.P.G. Reis et al. / Journal of Cereal Science 79 (2018) 508e515
treatments, independently of the N level (Table 2). Se is not an
essential nutrient for plants but is considered a beneficial element
because it stimulates growth and confers tolerance to oxidative
stress and resistance to pathogens and herbivores (El Mehdawi and
Pilon-Smits, 2012; Feng et al., 2013; Reis et al., 2017). The interac-
tion between N and Se is still not well understood, but Rios et al.
(2013) also observed higher leaf Se contents in lettuce grown
with high N concentrations.
Leaf Se contents varied from 0.01 to 0.54 mg kg1 (Table 2). Se
translocation from leaves to grains was observed in the present
study, with grain Se contents varying from 0.03 to 0.35 mg kg1
(Table 1). According to the Codex Alimentarius, Se-biofortified foods
should not have Se contents higher than 0.3 mg kg1 (Reis et al.,
2017).
The application of 25 g Se ha1 resulted in a maximum mean
grain Se content of 0.35 mg kg1 (Table 1). The average rice con-
sumption by the Brazilian population is 25 kg year1, i.e., 68.5 g
day1 per capita (Reis, 2015). To calculate the daily Se intake via
biofortified rice, the daily rice consumption should be multiplied by
the Se content in the sample, according to equation 1. The observed
daily Se intake from biofortified rice (24.7 mg day1) is thus still
under the recommended level of 55 mg day1 (Fig. 4).
68:5 g day1  x 0:35 mg kg1 ¼ 24:7 mg of  Se day1 (1)
The calculated daily Se intake varied from 2.05 for non-
biofortified rice to 24.7 mg day1 for Se-biofortified rice (Fig. 4),
representing an increase from 3.72% to 44.9% of the daily Se
requirement. Considering that the recommended daily Se intake for
adults is 55 mg day1, the present study presents relevant infor-
mation about agronomic biofortification to increase the Se content
in edible plant parts, which may result in benefits to human health.
This work is one of the first studies of Se agronomic bio-
fortification of upland rice in the Brazilian Cerrado under field
conditions. Further studies are therefore needed to determine the
best Se dose to be applied. Future experiments using different
combinations of Se doses and sources, as well as different rice ge-
notypes, should be performed to better understand Se uptake and
accumulation in edible plant parts.
4. Conclusions
Interactions between the Se and N did not increase the upland
Fig. 4. Daily Se intake (mg day1) from the grains of the upland rice cultivar ANa 5015
grown at different N fertilization levels and with or without Se application.
rice yield, but the Se contents in biofortified rice grains increased
from 0.03 to 0.35 mg kg1. The estimated Se daily intake from
biofortified rice, based on the average daily consumption of the
Brazilian population, varied from 2.05 to 24.7 mg day1, represent-
ing an increase from 3.72% to 44.9% of the Se daily requirements.
Because the recommended Se daily intake for adults is 55 mg
day1, the present study presents relevant information about
agronomic biofortification to increase the Se contents in plant
edible parts, which may result in benefits to human health.
Declaration of interest
The authors have no conflicts of interest to declare.
Role of the funding source
The authors wish to thank the Conselho Nacional de Desen-
volvimento Científico e Tecnolo
gico (CNPq Process Number:
448783/2014-2) for financial support to conduct the research.
Acknowledgments
The authors wish to thank the S~
ao Paulo State Research Support
Foundation (Fundaça ~o de Amparo a  Pesquisa do Estado de Sa ~o
Paulo - FAPESP) (Process FAPESP: 2015/11690-3) for the master's
scholarship granted to the first author and Dr. Martin Broadley and
Dr. Scott Young from the University of Nottingham, England, for the
total Se content analyses.
References
Alexieva, V., Sergiev, I., Mapelli, S., Karanov, E., 2001. The effect of drought and
ultraviolet radiation on growth and stress markers in pea and wheat. Plant Cell
Environ. 24, 1337e1344.
Azevedo, R.A., Alas, R.M., Smith, R.J., Lea, P.J., 1998. Response of antioxidant enzymes
to transfer from elevated carbon dioxide to air and ozone fumigation, in the
leaves and roots of wild-type and a catalase-deficient mutant of barley. Physiol.
Plantarum 104, 280e292.
Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of micro-
gram quantities of protein utilizing the principle of protein-dye binding. Anal.
Biochem. 72, 248e254.
Cardoso, E.A., De Souza, M.A., Fontes, P.C.R., 2015. Fertilizaç~ ao nitrogenada na
absorça ~o de nutrientes e rendimento de gr~ aos em arroz irrigado. Agri. Environ.
Sci. 1, 39e49.
Chen, Y., Peng, J., Wang, J., Fu, P., Hou, Y., Zhang, C., Fahad, S., Peng, S., Cui, K., Nie, L.,
Huang, J., 2015. Crop management based on multi-split topdressing enhances
grain yield and nitrogen use efficiency in irrigated rice in China. Field Crop. Res.
184, 50e57.
Chilimba, A.D.C., Young, S.D., Black, C.R., Rogerson, K.B., Ander, E.L., Watts, M.J.,
Lammel, J., Broadley, M.R., 2011. Maize grain and soil surveys reveal suboptimal
dietary selenium intake is widespread in Malawi. Sci. Rep. 1, 72. https://doi.org/
10.1038/srep00072.
Combs, G.F., 2011. Selenium in global food systems. Br. J. Nutr. 85, 517e547.
El Mehdawi, A.F., Pilon-Smits, E.A.H., 2012. Ecological aspects of plant selenium
hyperaccumulation. Plant Biol. 14, 1e10. https://doi.org/10.1111/j.1438-
8677.2011.00535.x.
Feng, R., Wei, C., Tu, S., 2013. The roles of selenium in protecting plants against
abiotic stresses. Environ. Exp. Bot. 87, 58e68. https://doi.org/10.1016/
j.envexpbot.2012.09.002.
Fordyce, F.M., 2013. Selenium deficiency and toxicity in the environment. In:
Selinus, O., Alloway, B., Centeno, J.A. (Eds.), Essentials of Medical Geology,
Revised Ed. Springer, Dordrecht, pp. 375e416.
Gerland, P., Raftery, A.E., Sev cíkova
, H., Li, N., Gu, D., Spoorenberg, T., Alkema, L.,
Fosdick, B.K., Chunn, J., Lalic, N., Bay, G., Buettner, T., Heilig, G.K., Wilmoth, J.,
2014. World population stabilization unlikely this century. Science 346,
234e237.
Giannopolitis, C.N., Ries, S.K., 1977. Superoxidedismutases: I. Occurrence in high-
erplants. Plant Physiol. 59, 309e314.
Graham, R.D., Welch, R.M., Saunders, D.A., Ortiz-Monasterio, I., Bouis, H.E.,
Bonierbale, M., de Haan, S., Burgos, G., Thiele, G., Liria, R., Meisner, C.A.,
Beebe, S.E., Potts, M.J., Kadian, M., Hobbs, P.R., Gupta, R.K., Twomlow, S., 2007.
Nutritious subsistence food systems. Adv. Agron. 92, 1e74.
Haug, A., Graham, R.D., Christophersen, O.A., Lyons, G.H., 2007. How to use the
world's scarce selenium resources efficiently to increase the selenium con-
centration in food. Microb. Ecol. Health Dis. 19, 209e228.
 H.P.G. Reis et al. / Journal of Cereal Science 79 (2018) 508e515
Heath, R.L., Packer, L., 1968. Photoperoxidation in isolated chloroplasts. I. Kinetics
and stoichiometry of fatty acid peroxidation. Arch. Biochem. Biophys. 125,
189e198.
Poluboyarinov, P.A., Golubkina, N.A., 2015. Investigation of biochemical function of
selenium and its influence on the content of protein fractions and peroxidase
activity in maize seedlings. Russ. J. Plant Physiol. 62, 367e374.
Ray, D.K., Mueller, N.D., West, P.C., Foley, J.A., 2013. Yield trends are insufficient to
double global crop production by 2050. PLoS One 8, e66428.
Rayman, M.P., Blundell-Pound, G., Pastor-Barriuso, R., Guallar, E., Steinbrenner, H.,
Stranges, S., 2012. A randomized trial of selenium supplementation and risk of
type-2 diabetes, as assessed by plasma adiponectin. PLoS One 7, e45269.
https://doi.org/10.1371/journal.pone.0045269.
Reis, A., El-Ramady, H., Santos, E.F., Grata~o, P.L., Schomburg, L., 2017. Overview of
selenium deficiency and toxicity worldwide: affected areas, selenium-related
health issues, and case studies. In: De Kok, L.J., Hakesford, M.J. (Eds.), Plant
Ecophysiology, vol. 11. Springer International Publishing, pp. 209e230.
Reis, A.R., 2015. Selenium status in Brazilian soils and crops: agronomic bio-
fortification as a strategy to improve food quality. In: Banuelos, G.S., Lin, Z.A.,
Moraes, M.F., Guilherme, L.R.G., Reis, A.R. (Eds.), Proceedings of the 4th Inter-
national Selenium Conference in the Environment and Human Health. CRC
Press Taylor & Francis Group, Londres, pp. 179e180.
Rios, J.J., Blasco, B., Leyva, R., Sanchez-Rodriguez, E., Rubio-Wilhelmi, M.M.,
Romero, L., Ruiz, J.M., 2013. Nutritional balance changes in lettuce plant grown
under different doses and forms of selenium. J. Plant Nutr. 36, 1344e1354.
Rios, J.J., Blasco, B., Rosales, M.A., Sanchez-Rodriguez, E., Leyva, R., Cervilla, L.M.,
515
Romero, L., Ruiz, J.M., 2010. Response of nitrogen metabolism in lettuce plants
subjected to different doses and forms of selenium. J. Sci. Food Agric. 90,
1914e1919.
Ríos, J.J., Rosales, M.A., Blasco, B., Cervilla, L.M., Romero, L., Ruiz, J.M., 2008. Bio-
fortification of Se and induction of the antioxidant capacity in lettuce plants.
Sci. Hortic. 116, 248e255.
Saidi, I., Chtourou, Y., Djebali, W., 2014. Selenium alleviates cadmium toxicity by
preventing oxidative stress in sunflower (Helianthus annuus) seedlings. J. Plant
Physiol. 171, 85e91.
Seregina, I.I., Niloskaya, N.T., Ostapenko, N.O., 2001. The role of selenium in the
formation of the grain yield in spring wheat. Agrokhimiya 1, 44e50.
White, P.J., 2016. Selenium accumulation by plants. Ann. Bot. 117, 213e235.
White, P.J., Bowen, H.C., Parmaguru, P., Fritz, M., Spracklen, W.P., Spiby, R.E.,
Meacham, M.C., Mead, A., Harriman, M., Trueman, L.J., Smith, B.M., Thomas, B.,
Broadley, M.R., 2004. Interactions between selenium and sulfur nutrition in
Arabidopsis thaliana. J. Exp. Bot. 55, 1927e1937.
Wu, L., Huang, Z.Z., 1992. Selenium assimilation and nutrient element uptake in
white clover and tall fescue under influence of sulfate concentration and se-
lenium tolerance of the plants. J. Exp. Bot. 43, 549e555.
Xue, T., Hartikainen, H., Piironen, V., 2001. Antioxidative and grown-promoting
effect of selenium on senescing lettuce. Plant Soil 237, 55e61.
Yuan, Z., Ata-Ul-Karim, S.T., Cao, Q., Lu, Z., Cao, W., Zhu, Y., Liu, X., 2016. Indicators
for diagnosing nitrogen status of rice based on chlorophyll meter readings. Field
Crop. Res. 185, 12e20.