Journal of Soil Science and Plant Nutrition (2023) 23:1397–1407

https://doi.org/10.1007/s42729-023-01131-8

ORIGINAL PAPER

Selenium Increases Photosynthetic Pigments, Flavonoid Biosynthesis,

Nodulation, and Growth of Soybean Plants (Glycine max L.)
Matheus Luís Oliveira Cunha1 · Lara Caroline Alves de Oliveira1 · Nandhara Angélica Carvalho Mendes1 ·
Vinicius Martins Silva1 · Eduardo Festozo Vicente2 · André Rodrigues dos Reis2

Received: 10 November 2022 / Accepted: 5 January 2023 / Published online: 20 January 2023
© The Author(s) under exclusive licence to Sociedad Chilena de la Ciencia del Suelo 2023

Abstract
This study aimed to investigate the physiological roles of selenium (Se) in the photosynthetic pigment, antioxidant metabo-
lism, flavonoid biosynthesis, nodulation, and its relationship with soybean production. The treatments consisted of five doses
of Se (0 μg ­kg−1, 7.5 μg k­ g−1, 15 μg k­ g−1, 30 μg k­ g−1, and 45 μg k­ g−1) applied via soil and in the form of sodium selenate at
the ­V2 phenological stage of soybean. The concentration of chlorophyll b and carotenoids, the activity of antioxidant enzymes
(superoxide dismutase, catalase, ascorbate peroxidase), and the concentration of total sugars in soybean leaves increased in
response to Se fertilization. In addition, Se increased ureide concentration in leaves and activity of nitrate reductase, result-
ing in greater bioavailability of amino acids and proteins in the leaves, generating greater stimulation of plant growth and
root dry weight, but did not increase yield. The number of nodules and the synthesis of rutin and genistein in the roots were
significantly increased by the Se application. Se also stimulated the translocation of amino acids from leaves to the nodules
and of ureides from nodules to leaves, increasing efficiency in biological nitrogen fixation. This study presents fundamental
new insights regarding Se effect on nitrogen metabolism, flavonoid synthesis, antioxidant metabolism, and nodulation of
soybean that can be helpful in increasing the crop tolerance to abiotic stress.

Keywords  Ascorbate peroxidase · Catalase · Chlorophyll · Daidzein · Genistein · Superoxide dismutase · Total sugar ·
Yield

1 Introduction 2021). Application of Se at low doses can increase pho-

tosynthetic pigments, sugars, nitrate reductase, and anti-
Selenium (Se) is an essential element for humans and oxidant enzymes and decrease the production of reactive
animals and a beneficial element for plants; however, the oxygen species (ROS) (Lanza and dos Reis 2021; Cunha
average Se content in most soils is low (0.01–2 mg ­kg−1) et al. 2022). Plants fertilized with Se show greater tolerance
(Fordyce 2013). The low availability of Se in arable soils to abiotic and biotic stress (Silva et al. 2018, 2020; Cunha
can result in the production of foods with low nutritional Se et al. 2022). On the other hand, Se application at high con-
content (Silva et al. 2020; Lanza and dos Reis 2021). centration can cause phytotoxicity (Lanza et al. 2021). The
A good strategy to increase Se concentration in the use of Se in agriculture has become a trend in annual crops;
edible parts of plants is through agronomic biofortification however, information on the dose-response behavior of Se
by applying fertilizers containing Se (Lanza and dos Reis is scarce. Therefore, further studies are needed to establish
optimal doses and sources of Se to avoid detrimental effects
in legume plants.
* André Rodrigues dos Reis Soybean (Glycine max L.) is an important crop for human
[email protected]
food, so biofortifying soybeans with Se is a good strategy
1
Faculty of Agricultural and Veterinary Sciences, São to improve human health. Therefore, new developments are
Paulo State University, Via de Acesso Prof. Paulo Donato important to improve agronomic practices, yield, and soy-
Castellane, 14884‑900, Jaboticabal, São Paulo, Brazil bean quality especially in the face of contemporary climate
2
School of Science and Engineering, São Paulo State and environmental challenges. In addition, soybean plants
University, Rua Domingos da Costa Lopes 780, perform biological nitrogen fixation with bacteria of the
Tupã 17602‑496, Brazil

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1398 Journal of Soil Science and Plant Nutrition (2023) 23:1397–1407

genus Rhizobium. The isoflavonoids genistein and daidzein 1.4, Mg ­mmolc ­dm−3 = 4.0, pH ­CaCl2 = 4.6, P mg ­dm−3 =
play a crucial role in the expression of nod genes in bacte- 4.0, Ca m­ molc ­dm−3 = 4.0, H+Al ­mmolc ­dm−3 = 18.0, Al
ria, which leads to the synthesis and secretion of nodula- ­mmolc ­dm−3 = 3.0, organic matter mg ­dm−3 = 13.0, base sat-
tion factors that promote a series of physiological changes uration ­mmolc ­dm−3 = 9.4, cation exchange capacity ­mmolc
in plant cells that initiate the formation of nodules in plant ­dm−3 = 27.4, Se = 3.6 μg ­kg−1, and V % = 34.0.
roots (Bosse et al. 2021). Inside the nodules, ammonia is The experiment was conducted in pots filled with 5 kg of
converted into ureides (allantoin and allantoic acid) which soil. Liming was carried out about 30 days before sowing
are transported to the shoot by the xylem sap. Once in the and consisted of applying 2.9 g of lime per 5 kg of soil. Sub-
cytosol of cells, ureides are transformed into ammonium, sequently, each pot received 0.25 g of potassium chloride,
amino acids, and proteins that can be used for chlorophyll 0.124 g of simple superphosphate, 0.058 g of boric acid,
production, photosynthetic processes, and plant growth 0.083 g of copper, and 0.25 g of zinc.
(Baral et al. 2016). The soybean cultivar BRS 1003 PRO was used due to
Fertilization of plants with Se can affect nodulation, its economic importance and its wide cultivation in Brazil.
biological nitrogen fixation, and antioxidant metabolism of The seeds were inoculated with Bradyrhizobium japonicum
soybean plants. However, this phenomenon has still not been SEMIA 5079. Sowing was performed manually using 6
deeply investigated. This study hypothesizes that Se affects seeds per pot. After seedling emergence, thinning was per-
flavonoid metabolism by increasing nodulation, ureide con- formed, leaving only 2 seedlings per pot. Weed control was
centration, and soybean plant yield. This study aimed to performed manually, and when necessary, pyrethroid-based
investigate the physiological roles of Se on photosynthetic insecticide was used to control pests.
pigment, antioxidant metabolism, flavonoid biosynthesis,
nodulation, and its relationship with soybean production. 2.4 Determination of Enzymatic Antioxidant
Metabolism and Photosynthetic Pigments

2 Material and Methods During flowering period in both experiments, diagno-

sis leaves of the soybean plants were collected into liquid
2.1 Site Description nitrogen and stored in ultra-freezer until analysis. Hydro-
gen peroxide (­ H2O2) was evaluated according to Alexieva
To test our hypothesis, we performed an experiment con- et al. (2001), and malondialdehyde (MDA) concentration
ducted in a greenhouse at the São Paulo State University was evaluated according to Heath and Packer (1968). For
(UNESP), Campus de Tupã, located in the city of Tupã, antioxidant metabolism analysis, leaves were extracted using
Brazil, during the year 2021. 100 mM potassium phosphate buffer (pH 7.5). The plant
extracts were homogenized and centrifuged at 10,000 × g
2.2 Experimental Design and Treatment for 30 min at 4 °C. After centrifugation, the supernatant was
Application used to measure total proteins (Bradford 1976), superoxide
dismutase (SOD) activity (Giannapolitis and Ries 1977),
The treatments consisted of the application of five doses of catalase (CAT) activity (Azevedo et al. 1998), and ascorbate
Se (0 μg ­kg−1, 7.5 μg ­kg−1, 15 μg ­kg−1, 30 μg ­kg−1, and 45 μg peroxidase (APX) activity (Moldes et al. 2008).
­kg−1) applied via soil. The application of Se was carried out For the quantification of photosynthetic pigments (chloro-
when the plants were in the phenological stage ­(V2) (Fehr phyll a, chlorophyll b, total chlorophyll, carotenoids, pheo-
and Caviness 1976), and the source of Se used was sodium phytin a, and total pheophytin), the leaves were collected at
selenate (Sigma-Aldrich). A stock solution of ­Na2SeO4 was flowering and the analyses were performed according to the
prepared and distributed around the plants according to the methodology of Lichtenthaler (1987).
doses of each treatment. Se doses were established according
to the results of Cunha et al. (2022). The experiment had 8 2.5 Quantification of N Compounds in Leaves
replications: 4 were used for destructive analysis at flower- and Nodules
ing, and 4 were carried out until the full maturation of the
grains to determine the yield. The concentration of total sugars and N compounds (nitrate,
ammonia, amino acids, and ureides) was determined in the
2.3 Cultivation leaves and nodules. To determine the total sugars and N
compounds, 1 g of fresh leaves and nodules was macerated
Before the installation of the experiment, the soil was col- and added to falcon tubes containing 10 mL of MCW solu-
lected in the 0–20-cm layer to determine the chemical analy- tion (60% v/v methanol, 25% v/v chloroform, and 15% v/v
sis that presented the following results: K m­ molc ­dm−3 = ­H2O). After 48 h, 4 mL of the supernatant was collected and

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Journal of Soil Science and Plant Nutrition (2023) 23:1397–1407 1399

Fig. 1  Selenium effect on chlorophyll a (a), chlorophyll b (b), total lowercase letters indicate significance between Se rates according to
chlorophyll (c), carotenoids (d), pheophytin a (e), and total pheophy- the Tukey test (P ≤ 0.05). The bars indicate the standard deviation of
tin (f) concentrations in the leaf tissue of soybean plants. Different the mean (n = 4)

transferred to another falcon tube and 1 mL of chloroform results were determined from a standard solution containing
and 1.5 mL of deionized water were added. naringenin, rutin, genistein, daidzein, and kaempferol.
To quantify N compounds and total sugar concentration,
the following analyses were performed: nitrate, ammonia, 2.7 Determination of Yield, Development,
total amino acid, total sugar, and ureide (allantoin and allan- and Nodulation of Soybean Plants
toic acid) concentration in leaves and nodules. The com-
pound was determined, respectively, according to Cataldo To determine the dry weight of roots and shoots, during full
et  al. (1975), McCullough (1967), Yemm et  al. (1955), grain maturation, the plants were collected and roots and
Dubois et al. (1956), and Vogels and Van Der Drift (1970), shoots were separated and then dried in a forced-air circula-
and the results were expressed in μmol ­g−1 fresh weight tion oven until constant dressing. The number of seeds per
(FW). pot was determined by counting the seeds of the two plants
The activity of the nitrate reductase enzyme was per- contained in the pot. The weight of 100 seeds was deter-
formed according to the methodology described by Reis mined by weighing 100 seeds and expressed in g per pot. To
et al. (2009). All measured analyses described above were determine the yield, the seeds of the plants were collected
performed using a spectrophotometer (SP-220, Bioespec- and weighed and the results were expressed in g per pot. The
tro™, Brazil). number and weight of nodules on the roots were determined
during plant anthesis due the highest metabolic activity.
2.6 Quantification of Flavonoids in Soybean Root
2.8 Statistical Analysis
The quantification of the concentration of isoflavonoids
(genistein, rutin, kaempferol, and daidzein) in soybean roots Data were submitted to analysis of variance (ANOVA), and
was performed according to the methodology described by results were submitted to the Tukey test (P < 0.05). The
Cunha et al. (2022). Flavonoid readings were performed on graphics were made using the SigmaPlot software, ver-
high-performance liquid chromatography (HPLC), and the sion 12.5. In order to analyze the relationship between the

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Fig. 2  Selenium effect on lipid peroxidation (MDA) (a) and hydrogen bean plants. Different lowercase letters indicate significance between
peroxide ­(H2O2) concentrations (b); superoxide dismutase (SOD) (c), Se rates according to the Tukey test (P ≤ 0.05). The bars indicate the
catalase (CAT) (d), and ascorbate peroxidase (APX) (e) activities; standard deviation of the mean (n = 4)
and total soluble protein (f) concentration in the leaf tissue of soy-

parameters, the Pearson correlation test was performed and The highest activity of SOD was observed in plants that
represented as a heatmap graphic using the R Core Team were exposed to 15 μg k­ g−1 of Se, being an increase of 17%
software, version 4.0.3. To better verify the effects of Se in relation to the control (Fig. 2c). Plants that received Se,
on the physiological, biochemical, and productive traits of regardless of the dose, presented increased CAT activity
soybean, cluster analysis was performed using the hierarchi- compared to the control group, and this increase is more
cal method unweighted pair-group method using arithmetic evident in the dose of 15 μg ­kg−1 (Fig. 2d). Se applica-
averages (UPGMA) using the Euclidean distance and the tion at doses of 15 μg k­ g−1, 30 μg ­kg−1, and 45 μg k­ g−1
results were expressed in the form of heatmap graph. (Fig. 2e) increased APX activity in soybean leaves. Higher
soluble proteins were detected in plants that were exposed
to Se regardless of the dose, except for the dose of 15 μg
3 Results ­kg−1 which presented a lower result compared the control
(Fig. 2f).
3.1 Se Enhances Photosynthetic Pigments
and Antioxidant Metabolism 3.2 Se Enhances Sugar, N Compounds,
and Biometric Parameters of Soybean Plants
The soybean leaves at the flowering stage exposed to Se at
a dose of 7.5 μg ­kg−1 presented higher chlorophyll b and Nitrate reductase activity was increased in plants that
carotenoids (Fig. 1b, d), with no influence of Se on the received Se at doses of 15 μg ­kg −1, 30 μg ­kg −1, and 45
concentration of chlorophyll a and pheophytins. The high- μg ­k g −1 , representing an increment of 78%, 79%, and
est concentration of MDA in leaves was observed under 91% compared to the control, respectively (Fig. S1a). Se
45 μg k­ g−1, which was 200% higher than control (Fig. 2a). application at doses of 15 μg ­kg−1, 30 μg ­kg−1, and 45 μg
Furthermore, Se at doses of 15 μg k­ g−1 and 30 μg k­ g−1 ­kg −1 increased total soluble sugar (Fig. S1b). Curiously,
significantly increased H­ 2O2 concentration compared to there was no influence of Se on nitrate concentration
control (Fig. 2b). compared to control groups (Fig. 3a). Se decreased the

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Journal of Soil Science and Plant Nutrition (2023) 23:1397–1407 1401

Fig. 3  Selenium effect on nitrate (a), ammonia (b), amino acids (c), significance between Se rates according to the Tukey test (P ≤ 0.05).
allantoin (d), allantoic acid (e), and total ureide (f) concentrations in The bars indicate the standard deviation of the mean (n = 4)
the leaf tissue of soybean plants. Different lowercase letters indicate

ammonia concentration at doses of 15 μg k­ g −1, 30 μg 3.3 Se Increases Nodulation, N Compounds

­kg −1, and 45 μg ­k g −1 (Fig. 3b). On the other hand, the in Nodules, Isoflavonoid in Soybean Root,
content of the amino acids has increased, regardless of and Yield
the dose of Se applied and this increase is more evident
in the dose of 45 μg k­ g −1 which increased the content Se application also influenced isoflavonoid concentration in
of amino acids in the leaves by 268% in relation to the soybean roots. Se at the dose of 7.5 μg ­kg−1 increased the
control (Fig. 3c). concentration of rutin in the roots (Fig. S2a). Genistein and
Application of Se at doses of 7.5 μg k­ g −1 and 15 μg naringenin production was induced at all Se doses applied
−1
­kg significantly increased allantoin concentration in (Fig. S2c, d). However, there was no influence of Se on the
leaves by 10% and 17% compared to control (Fig. 3d). concentration of daidzein in roots. A higher number of nod-
Allantoic acid was also increased when Se was applied at ules were recorded in the roots exposed either to 7.5 μg ­kg−1,
doses of 15 μg k­ g−1 (Fig. 3e). The highest concentration 15 μg ­kg−1, or 45 μg ­kg−1; however, Se did not influence the
of ureides was observed in plants under Se application fresh weight of nodules (Fig. 5).
of 15 μg ­kg−1, being an increase of 20% compared to the The lowest concentration of nitrate was observed at
control (Fig. 3f). the dose of 45 μg ­k g −1 (Fig. S3a). On the other hand,
The highest root dry weight was observed under 45 μg Se fertilization at 15 μg ­kg−1 significantly increased the
­kg−1, which was 44% higher than control. On the other concentration of ammonia and amino acids in the nodules
hand, there was no influence of Se application on shoot dry by 18% and 16% compared to the control, respectively
weight (Fig. 4b), but increased plant height, regardless of (Fig. S3b, c). There was no influence of Se on allantoin
the dose applied (Fig. 4c). concentration compared to control plants. However, Se

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1402 Journal of Soil Science and Plant Nutrition (2023) 23:1397–1407

Fig. 4  Selenium effect on root dry weight (a), shoot dry weight (b), according to the Tukey test (P ≤ 0.05). The bars indicate the standard
plant height (c), and visual growth development of soybean plants deviation of the mean (n = 4)
(d). Different lowercase letters indicate significance between Se rates

decreased allantoic acid, regardless of the applied dose, the formation of ROS that can destroy the cell membrane
and ureides at doses of 7.5 μg k­ g−1 and 15 μg k­ g−1 com- causing cell death (Sachdev et al. 2021). Lanza et al.
pared to control (Fig. S3d–f). (2021) and De Brito Mateus et al. (2020) reported an
There was no influence of Se on the number of seeds, increase in the concentration of chlorophyll, carotenoids,
weight of 100 seeds, and soybean yield (Fig. 6). According and pheophytin in cowpea and coffee plants treated with
to the results obtained in the heatmap cluster, the fertiliza- Se, respectively.
tion of plants with Se is promising to increase the physio- The concentration of MDA and ­H2O2 was higher at higher
logical and biochemical traits and to stimulate the growth of doses of Se. However, there were no significant inhibitions
the shoot and accumulation of biomass in the roots (Fig. 7). of any trait at the plant level in this study, indicating that
applied doses did not exceed the toxicological threshold for
causing toxicity at the whole plant level.
4 Discussion In this study, plants that received an intermediate
dose of Se showed higher SOD, CAT, and APX activi-
Plants fertilized with Se (7.5 μg ­k g −1 ) showed higher ties. These enzymes act in the enzymatic antioxidant
concentrations of chlorophyll a, chlorophyll b, and metabolism of plants and are responsible for mitigating
carotenoids. Photosynthetic pigments contribute to non- the formation of ROS, which, in high concentrations,
enzymatic antioxidant metabolism and play an important can cause cell wall destruction and cell death (Silva
role in increasing plant tolerance against environmental et al. 2020). This information can be used as a tool in
stresses (Agathokleous et al. 2020; Agathokleous 2021). agriculture to increase the tolerance of plants to abi-
Chlorophyll b and pheophytins are responsible for dis- otic stresses, since these enzymes act in the reduction
sipating excess sunlight energy (Kitajima and Hogan of oxidative stress and confer greater ability of plants
2003; Klimov 2003) while chlorophyll a plays a role in against stresses. Similar results were obtained by Silva
photosynthesis (Stirbet et al. 2018). Carotenoids protect et al. (2020) and Lanza et al. (2021) in cowpea plants.
plants from excessive light (Silva et al. 2020) and prevent In quinoa plants, Khalofah et al. (2021) reported that

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Journal of Soil Science and Plant Nutrition (2023) 23:1397–1407 1403

Fig. 5  Selenium effect on the number of nodules per pot (a), the nod- between Se rates according to the Tukey test (P ≤ 0.05). The bars
ule fresh weight (b), and the visual number of nodules per pot (c) indicate the standard deviation of the mean (n = 4)
of soybean plants. Different lowercase letters indicate significance

Se increased photosynthetic pigments and SOD, CAT, nitrogen such as nitrate reductase resulting in higher plant
and APX activities and decreased MDA and H ­ 2O 2. De biomass and reduced N concentration in leaves (Rios et al.
Brito Mateus et al. (2020), Silva et al. (2018), and Cunha 2010; Ríos et al. 2010).
et al. (2022) showed an increase in the activity of anti- Fertilization of plants with Se increased foliar levels
oxidant enzymes in coffee, cowpea, and peanut plants, of allantoin (7.5 μg ­kg−1 and 15 μg ­kg−1), allantoic acid
respectively. (7.5 μg ­kg −1), and ureides (15 μg ­kg −1). In leguminous
Nitrate reductase is the first enzyme to act in the pro- plants, ureides can represent more than 90% of all trans-
cess of N assimilation in plants, being responsible for the ported by the xylem (Raso et al. 2007) and the increase
reduction of nitrate to nitrite (Karwat et al. 2019). The in the ureide production is important to enhance the N
higher concentration of amino acids in leaves observed in transport to the shoot and can be used for the production
plants that were fertilized with Se at a dose of 45 μg k­ g−1 of photosynthetic pigments since nitrogen is part of the
may be related to the increase in the activity of the nitrate chlorophyll molecule (Tibiletti et al. 2016).
reductase enzyme at the same dose, since that nitrate Soybean plants that received Se at doses of 15 μg
reductase activity had a direct and significant correla- ­kg −1, 30 μg ­kg −1, and 45 μg ­kg−1 showed a higher con-
tion with the concentration of amino acids in the leaves centration of total sugars in the leaves. The increase in
(Fig. S4). Thus, increasing the activity of this enzyme can osmolyte compounds such as total soluble sugars may
increase the concentration of reduced N in leaves (Dos demonstrate a defense mechanism for the osmotic read-
Santos et al. 2017; Cunha et al. 2022). In a study regarding justment (Khalofah et al. 2021) of plants helping to miti-
Se application in lettuce plants, Se increased the efficiency gate stress, because these substances can keep cells tur-
of N metabolism by increasing the activity of enzymes gid under water stress conditions, maintaining cell turgor,
related to the process of assimilation and reduction of resulting in greater tolerance to water deficit. An increase

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The highest dose of Se applied significantly increased

the soybean root dry weight, and this may have occurred
due to the increase in the concentration of sugars, amino
acids, and proteins also caused by Se at that same dose,
which were important to stimulate the plant height and
root dry weight. An increase in the root system of plants
can help in water and nutrient uptake and increasing tol-
erance to water deficit. Ikram et al. (2020) and Cunha
et al. (2022) also found that Se increased root dry weight.
Se stimulated genistein production in soybean roots.
These isoflavonoids are crucial molecules for nodula-
tion to occur in leguminous plants, being inducers for
the expression of nod genes, which are responsible for
the production and secretion of compounds known as
nodulation factors (Nod factors) by symbiotic bacteria,
which induce a series of modifications or physiological
changes in plant cells for the formation of the nodule
(Bosse et al. 2021).
The stimulation in the synthesis of genistein at all
doses of Se applied in the experiment carried out in the
soil influenced the nodulation of the plants; however, the
only dose that promoted an increase in the concentration
of total ureides was 15 μg k­ g−1. Thus, the increase in the
concentration of ureides in the leaves at the dose of 15 μg
­kg−1 could probably have occurred due to the increase in
the translocation of amino acids to the nodules observed
at the same dose, which may have contributed to the
improvement of efficiency in the biological fixation of N
and the viability of the nodules. In addition, a significant
reduction in the concentration of ureides in the nodules
was observed at a dose of 15 μg ­kg−1 and this may indi-
cate that Se stimulated the translocation of ureides to the
shoot where they are catabolized into amino acids and
proteins (Baral et al. 2016). In our recent study carried
out with the peanut crop, we found that Se increased the
synthesis of daidzein and genistein in the roots, resulting
in a greater number of nodules and production and trans-
location of ureides to the leaves, generating greater plant
growth and yield (Cunha et al. 2022).
In this study, Se did not influence soybean yield. Simi-
lar results were obtained in rice (Reis et al. 2018) and
cowpea (Silva et al. 2019). Although Se can increase crop
Fig. 6  Selenium effect on the number of seeds per pot (a), the weight yield, this effect is more commonly reported in studies in
of 100 seeds (b), and the yield (c) of soybean plants. Different low- which plants are submitted to stressful conditions: under
ercase letters indicate significance between Se rates according to the salt stress conditions, Se can enhance the yield of cow-
Tukey test (P ≤ 0.05). The bars indicate the standard deviation of the
mean (n = 4) pea (Manaf 2018) and mung bean (Kaur and Nair 2015),
while wheat under ultraviolet stress presented increased
yield under Se application (Yao et al. 2013). Those ben-
in total sugar concentration as a function of Se fertiliza- eficial Se effects on yield are more common in plants
tion has been reported in quinoa (Khalofaf et al. 2021), under stress most likely due to Se well-known enhance-
coffee (De Brito Mateus et al. 2020), rice (Gouveia et al. ment in oxidative stress mitigation and ROS scavenging
2020), cowpea (Silva et al. 2018), and peanut (Cunha systems (Silva et al. 2020; Lanza and dos Reis 2021).
et al. 2022). It is also noteworthy that Se might impair plant yield

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Journal of Soil Science and Plant Nutrition (2023) 23:1397–1407 1405

Fig. 7  Cluster heatmap between the physiological, biochemical, and development parameters evaluated in response to rates of Se applied to soy-
bean. MDA, malondialdehyde; ­H2O2, hydrogen peroxide; SOD, superoxide dismutase; CAT, catalase; APX, ascorbate peroxidase

due to phytotoxicity (Silva et al. 2018); thus, the lack nodulation and synthesis of flavonoids in the soybean
of yield response in the present study suggests that the crop.
doses used are not detrimental to soybean in the observed
conditions.
The heatmap cluster revealed the hormetic effect of Se 5 Conclusions
in soybean plants, at both the molecular and whole-plant
levels, in agreement with other studies that also showed The concentration of chlorophyll b and carotenoids, the
a hormetic effect of plants exposed to chemical ele- activity of antioxidant enzymes (superoxide dismutase,
ments (Ferrari et al. 2021a, b, 2022; Marsala et al. 2022). catalase, ascorbate peroxidase), and the concentration of
However, dose responses were not identical among the total sugars in soybean leaves increased in response to
studied traits. This is because hormetic responses are selenium fertilization. In addition, selenium increased the
endpoint-specific (Marchica et  al. 2021). Despite the ureide concentration in leaves and the activity of nitrate
appearance of several biphasic responses, the number reductase, resulting in greater bioavailability of amino
of nodules, nitrate reductase activity, plant height, and acids and proteins in the leaves, generating greater stimu-
total sugar concentration significantly increased at all lation of plant growth and root dry weight, but did not
doses of Se applied. These results show trait-specific increase the yield of soybean.
improvement, but no adverse effects were observed at the The number of nodules and the synthesis of rutin and gen-
applied doses. Although no trend of possible toxicity was istein in the roots were significantly increased by the applica-
detected at doses greater than 45 μg ­kg−1, this should be tion of selenium. Selenium also stimulated the translocation
tested experimentally in the future. of amino acids from leaves to the nodules and ureides from
This study contributes to providing important knowl- nodules to leaves, increasing efficiency in biological nitrogen
edge about the role of flavonoids, ureides, and N metabo- fixation. This study presents fundamental new insights regard-
lism at the whole plant level, an aspect little explored in ing selenium effect on nitrogen metabolism, flavonoid synthe-
the literature on plant nutrition and physiology. Further- sis, antioxidant metabolism, and nodulation of soybean that
more, this study is the first report on the role of Se in the can be helpful in increasing the crop tolerance to abiotic stress.

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1406 Journal of Soil Science and Plant Nutrition (2023) 23:1397–1407

Supplementary Information  The online version contains supplemen- Dubois M, Gilles KA, Hamilton JK, Rebers PT, Smith F (1956) Colori-
tary material available at https://d​ oi.o​ rg/1​ 0.1​ 007/s​ 42729-0​ 23-0​ 1131-8. metric method for determination of sugars and related substances.
Anal Chem 28:350–356
Funding  This research was supported by FAPESP (2020/10053-8). Ferrari S, Marsala L, Luis Oliveira Cunha M (2021b) Effects
of low doses of glyphosate on agronomic traits of upland
Data Availability  The datasets are available from the corresponding rice. Gesun Pf lan 73:533–539. https:// ​ d oi. ​ o rg/ ​ 1 0. ​ 1 007/​
author upon request. s10343-​021-​00573-3
Ferrari S, Marsala L, Oliveira Cunha ML, dos Santos Cordeiro LF, Tro-
Declarations  paldi L, de Mattos Barretto VC, Alves de Oliveira LC (2021a) Can
the application of low doses of glyphosate induce the hormesis
Competing Interests  The authors declare no competing interests. effect in upland rice? J Environ Sci Health B 56:814–820. https://​
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Ferrari S, do Valle Polycarpo G, Vargas PF, Fernandes AM, Luís
Oliveira Cunha M, Pagliari P (2022) Mix of trinexapac-ethyl
and nitrogen application to reduce upland rice plant height and
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