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Soil and foliar application of selenium in rice biofortification
Journal of Food Composition and Analysis 31 (2013) 238–244
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Original Research Article
Soil and foliar application of selenium in rice biofortification Paulo Fernandes Boldrin a, Valdemar Faquin a, Sı´lvio Ju´nio Ramos a, Karina Volpi Furtini Boldrin b, Fabrı´cio William A´vila a, Luiz Roberto Guimara˜es Guilherme a,* a b
Department of Soil Science, Federal University of Lavras, Zip Code 3037, 37200-000 Lavras, MG, Brazil Department of Agriculture, Federal University of Lavras, Zip Code 3037, 37200-000 Lavras, MG, Brazil
A R T I C L E I N F O
A B S T R A C T
Article history: Received 12 June 2012 Received in revised form 30 May 2013 Accepted 20 June 2013
Selenium (Se) is essential for humans and animals because of its antioxidant properties, which form part of a series of chemical reactions. The aim of this study was to evaluate the effect of different Se application forms and sources on rice growth, grain yield, and rice Se concentration and accumulation, as well the content of N, P, Mg, S, B, Cu, Fe, Mn, and Zn in rice grains. The experiment was carried out in a greenhouse with 4-dm3 pots containing a sandy clay loam Red-Yellow Latosol. The experimental design was a completely randomized 2 2 2 factorial scheme (two Se doses two forms of Se application, soil or foliar two Se sources, selenate or selenite), with five replicates. Selenium in rice plants was analyzed by total reflection X-ray fluorescence spectrometry (TXRF). The results shows that soil selenate application was more effective for shoot dry matter production and grain Se accumulation than selenite. Foliar application of both selenate and selenite increased grain yield. This study provides useful information concerning agronomic biofortification of rice, showing that both soil and foliar Se application could be used for increasing Se content in edible parts, which could result in health benefits. ß 2013 Elsevier Inc. All rights reserved.
Keywords: Oryza sativa Rice Biofortification Selenate Selenite Nutrient contents Horticultural practices and nutrition Food analysis Food composition
1. Introduction Selenium (Se) is essential for humans and animals because of its antioxidant properties, which form part of a series of chemical reactions (Fairweather-Tait et al., 2011). In fact, Se is considered as one of the most important trace elements for human and animal health (Chaudhary et al., 2010). The deficiency of this element has been associated with the occurrence of serious diseases like heart disease and many types of cancers (Rayman, 2002). Unlike the case for humans and animals, for plants Se is not considered essential (Pilon-Smits and Quinn, 2010). Due to its chemical properties similar to sulfur (S), Se is absorbed and metabolized by plants in the same assimilation pathway as S, which is present in sulfur amino acids such as selenomethionine and selenocysteine (Sors et al., 2005). Depending on the Se concentration used, it may exert a double effect on plant growth. In
* Corresponding author at: Department of Soil Science, Federal University of Lavras, CP 3037, Campus UFLA, 37200-000 Lavras (MG), Brazil. Tel.: +55 35 3829 1259; fax: +55 35 3829 1251. E-mail addresses: [email protected] (P.F. Boldrin), [email protected]fla.br (V. Faquin), [email protected] (S.J. Ramos), [email protected] (K.V.F. Boldrin), [email protected] (F.W. A´vila), [email protected]fla.br (L.R.G. Guilherme). 0889-1575/$ – see front matter ß 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jfca.2013.06.002
this sense, Hartikainen et al. (2000) reported that, at low concentrations, Se acts as an antioxidant and can stimulate plant growth and production, whereas at high concentrations, it favours cell oxidation, leading to a reduction in yield. The low Se intake by the Brazilian population is linked with the consumption of foods with low contents of this element, a result of its low concentration in most Brazilian agricultural soils (Ferreira et al., 2002; Sillanpa¨a¨ and Jansson, 1992). According to Combs (2001), approximately 1 billion people worldwide have Se deficiency, considered the fourth most serious deficiency of minerals after iron (Fe), the most severe, followed by zinc (Zn) and iodine (I) (White and Broadley, 2009). Due to the importance of Se in human health and the strong deficiency of this element worldwide, it is necessary to find strategies to increase its content in foods. Accordingly, Chen et al. (2002), for rice, Hawkesford and Zhao (2007), Lyons et al. (2004), for wheat varieties, and, recently, Ramos et al. (2010) for lettuce, have showed that the Se content in these cultures increased with the introduction of this element through fertilization, which enables increased Se intake by the population. The choice of crops highly consumed by the population allows greater success in biofortification programmes. Rice is particularly interesting in this context, because is a staple food in at least 33 countries and provides about 80% of the daily caloric intake to 3
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billion people (Lucca et al., 2006; Meng et al., 2005). Rice is widely cultivated in Brazil–both under lowland and upland conditions– and is ranked 4th place in cultivated area and 6th in terms of fertilizer consumption. Thus, increasing the Se concentration in rice grains via fertilization could be beneficial to human health. However, for programmes.involving biofortification with this element, the application form and sources of Se must be taken into account, since Se in the form of selenite can undergo specific adsorption into Fe and Al oxides in the clay fraction, which compromises the availability of Se to plants (Mouta et al., 2008). Because of the importance of rice and Se for human nutrition and health, this study aimed to evaluate the effect of different application forms and sources of Se on rice growth, grain yield, and grain Se content and accumulation, as well as the content of N, P, Mg, S, B, Cu, Fe, Mn, and Zn in rice grains. 2. Material and methods 2.1. Experimental design and rice cultivation The experiment was conducted under greenhouse conditions at the Department of Soil Science, Federal University of Lavras, using 4dm3 pots filled with samples from the 0–20 cm layer of a sandy clay loam Red-Yellow Latosol. Soil physical and chemical analyses followed the methods described by EMBRAPA (1997), and mineralogical analyses were performed according to Souza (2005), with results as follows: sand = 600 g kg1; silt = 170 g kg1; 1; clay = 230 g kg1; pH H2O = 5.4; soil organic matter = 12 g kg1; P (Mehlich 1) = 0.9 mg kg1; K = 23.4 mg kg1; Ca = 0.3 cmolc kg1; Mg = 0.1 cmolc kg1; Al = 0.1 cmolc kg1; H + Al = 0.9 cmolc kg1; Fe2O3 = 37.8 g kg1; and Al2O3 = 168.1 g kg1. The total soil Se content was 0.064 mg kg1, according to the methodology described by Ramos et al. (2010). The experimental design was a completely randomized 2 2 2 factorial scheme as follows: with and without application of Se, two forms of Se application (soil application at a dose of 0.75 mg kg1 and foliar application at a concentration of 50 mmol L1), and two sources of Se (selenate – Na2SeO4 and selenite – Na2SeO3, both purchased from Sigma–Aldrich, Saint Louis, USA), with five replications, totalling 40 plots. Each experimental unit consisted of two plants per pot. For soil Se application, the dose was chosen in order not to compromise the plant development in our preliminary studies (Boldrin et al., 2012). The dose for foliar application was chosen within the range of concentrations suggested by Kapolna et al. (2009). Based on soil chemical analysis, soil samples were limed to increase base saturation to 50%, using quick lime with 32% CaO, 15% MgO, and an effective neutralizing value of 94.5%. After 30 days of incubation with a moisture content near 60% of the total pore volume (TPV), the sources of Se were applied in the soil along with a basic fertilization, which consisted of 80 mg of N, 250 mg of P, 70 mg of K, and 60 mg of S per dm3 of soil, using the following sources: ammonium nitrate - NH4NO3, monoammonium phosphate – NH4H2PO4, and potassium sulfate – KSO4. Basic fertilization with micronutrients consisted in the application of 3.6 mg of Mn, 1.5 mg of Cu, 5 mg of Zn, 0.5 mg of B, and 0.15 mg of Mo per dm3 of soil, as manganese chloride – MnCl2.4H2O, copper sulfate – CuSO45H2O, zinc sulfate – ZnSO47H2O, boric acid – H3BO3, and ammonium molybdate – (NH4)6Mo7O244H2O. Ten rice seeds were sown (Oryza sativa L. cv BRSMG Relaˆmpago) per pot, with two seedlings left in each pot one week after emergence. In the present study, we selected an early upland rice cultivar with high grain quality that is extensively cultivated in Brazil (Soares et al., 2010). During the growing period, rice plants received top-dressed N (450 mg dm3) and K (350 mg dm3), split into eight applications, using ammonium nitrate – NH4NO3 and
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potassium nitrate – KNO3. During the experiment, soil moisture was rigorously controlled by daily weighing of the soil-plant-pot set, replacing the volume lost through evapotranspiration with deionized water. Foliar application of the different Se sources was performed during flowering with three pulverizations applied in the morning (from 8:00 to 9:00) with an interval of three days between each application. A compression sprayer (Guarany Ind., Itu, Brazil) of 1.25 L was used, distributing the Se solution uniformly in all leaves of each plant separately, to avoid contamination of neighbouring plants. Rice plants were harvested at the end of the cycle. 2.2. Analytical procedures Leaves, stem, and grains were washed under deionized running water and oven-dried at 65–70 8C prior to digestions and analyses. For P, Mg, S, Cu, Fe, Mn, and Zn analyses, dried tissues (500 mg) were weighed and acid-digested with 4.0 mL of concentrated HNO3 + 2.0 mL of concentrated HClO4 (Sigma–Aldrich, Saint Louis, MO, USA) at 120 8C for 1 h and then at 220 8C until HClO4 fumes were observed. Total Mg, S, Cu, Fe, Mn, and Zn contents in the samples were determined using an atomic absorption spectrophotometer (PerkinElmer Inc., San Jose, CA, USA), and total P contents in the sample was determined using a spectrophotometer to measure a phospho-molybdenum complex colorimetrically at 680 nm (Malavolta et al., 1997). Total nitrogen content was determined by the Kjeldahl method, after sulfuric acid digestion. Boron was analyzed after digestion of the samples in a muffle-oven at 550 8C, being determined colorimetrically at 540 nm, after the reaction of boric acid with curcumin producing the compound rosocyanine (Malavolta et al., 1997). Selenium concentration in grains was determined according to a method described by Ramos et al. (2010). Briefly, dried tissues (approximately 500 mg) were weighed and acid-digested in 5 mL of concentrated HNO3 (Sigma–Aldrich, Saint Louis, MO, USA) in Teflon1 PTFE flasks (CEM Corporation, Matthews, NC, USA) and submitted to 0.76 MPa for 10 min in a CEM microwave oven (Mars model 5, CEM Corporation, Matthews, NC, USA). After cooling to room temperature, the extracts were filtered (Whatman No. 40 filter) and diluted by adding 5 mL of bi-distilled water. A certified reference material (White Clover-BCR402, Institute for Reference Materials and Measurements (IRMM), Geel, Belgium) was included for quality control. Blank and certified reference samples were analyzed along with every batch of digestion. Selenium content in the extracts was determined by total reflection X-ray fluorescence spectrometry (TXRF), using a PicofoxTM S2 instrument (Bruker, Karlsruhe, Germany), as described by Stosnach (2005). For TXRF analysis, gallium was used for internal standardization (final gallium concentration of 10; 100 and 1000 mg L1). After homogenization, an aliquot of 5 mL was transferred onto a quartz glass sample carrier and dried before TXRF analysis. The instrument analytical detection limit (LD) for Se pffiffiffiffiffiffiffiffi in plant extracts was calculated as: LD ¼ ð3 Ci N BG Þ=Ni; where Ci is the concentration of the element, NBG is the background area subjacent to the fluorescence peak, and Ni is area of the fluorescence peak in counts. The calculated detection limit for the method (after considering the dilution of plant extracts) was 50 mg kg1. 2.3. Statistical analysis All results were analyzed using analysis of variance (ANOVA), and significantly different means between treatments were separated with the Scoot-Knott’s test at the 0.05 significance level
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Fig. 1. Shoot dry matter production (A), grain yield (B) and selenium content in rice grains (C), in rice plants, depending on Se application forms and sources. Same letters, comparing Se sources within each application form do not differ (p 0.05). Asterisks (*), comparing Se application forms within each Se source, differ between each other (p 0.05).
of probability, using Sisvar 4.6 software (Build 6.1) (Ferreira, 2011) and the graphs were made in the Sigma Plot Programme (version 12.0, Systat Software Inc., Chicago, IL, USA). 3. Results and discussion The mean Se concentrations of the repeated analysis (n = 5) of the standard reference materials are presented in Table 1. The Se content recoveries in the certified samples show a reliable analytical data accuracy for Se analysis. The shoot dry matter production (SDMP), grain yield (GY) and Se content in grains were significantly affected by the application forms and sources of Se, as well as by the interaction between these factors (Fig. 1). Foliar application of Se did not change SDMP. However, when the sources of Se were applied to the soil, selenate provided an increase of about 6% in SDMP (Fig. 1A). For GY, foliar application of both sources of Se provided increases as follows: 22% for selenate and 17% for selenite (Fig. 1B). However, neither source of Se changed the GY. The increase in SDMP when Se was applied as selenate in the soil and in grain yield, for both Se sources applied on leaves, can be related to the beneficial effects of Se at low doses. Our results are consistent with those found by Djanaguiraman et al. (2005) for soybean, Rios et al. (2008) for lettuce, and Boldrin et al. (2012) for rice, which showed increased production at low Se concentration. Moreover, earlier studies indicate beneficial effects of Se, because it increases the antioxidant activity in plants, leading to better plant yield (Hartikainen et al., 2000; Lyons et al., 2009). Soil application of selenate provided higher Se content in grains, being 450% higher than the foliar application of this Se source
(Fig. 1C). This result may be related to the longer plant-Se contact time promoted by the soil application. Since foliar application was performed at the flowering stage, possibly the time required for Se transport via phloem from the leaves to the grains was not sufficient to increase the content of this element in the grain. It is noteworthy that, among the Se sources applied to soil, the highest concentrations of this element were provided by selenate. This fact is probably due to the specific adsorption that occurs with selenite on Fe and Al oxide surfaces, which are abundant in tropical soils (Rovira et al., 2008), decreasing Se in the soil solution, and thus its absorption by plants. Furthermore, it is known that selenite is less translocated in plants (Cartes et al., 2005; Ramos et al., 2010; Rios et al., 2008; Terry et al., 2000) when compared with selenate. Combs (2001) reports that the adequate Se level in a population is highly correlated with the Se content in agricultural crops. Thus, due to the fact that rice is the staple food of over half the world’s population, in at least 33 countries (Lucca et al., 2006), and that Se deficiency reaches nearly 1 billion people worldwide (White and Broadley, 2009), this crop has enormous potential to increase the Se consumption by the world population. According to the Food and Agriculture Organization of the United Nations - FAO (2011), the average rice consumption per person in the world is 180 g day1, and the recommended minimum daily Se intake for adults is 70 mg day1, with a maximum tolerable level of 400 mg day1 (USDA, 2003). Considering the data of our study, we estimate that the Se content in rice grains could contribute to the dietary Se intake with 210 and 48 mg day1, via foliar application, and 1150 and 75 mg day1 via soil application, by using selenate and selenite respectively. Thus, further studies
Table 1 Certified value, determined concentration and Se recovery on certified material. Certified material
Certified value (mg kg1)
Determined concentrationa (mg kg1)
Mean recovery (%)
BCR-402
6.7
5.2 0.3
78 4.5
a
Average of 5 measurements of standard reference material samples.
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Fig. 2. Se accumulation (A) and percentual distribution (B) in parts of the rice plant according to Se application forms and sources. Different letters indicate significant differences between means among Se sources within each application form (p < 0.05): (A–C) compare total Se accumulation; (a–c) compare rice grain Se accumulation; (d–f) compare shoots Se accumulation; (g–i) compare roots Se accumulation. Asterisks (*), comparing Se application forms within each Se source, differ from each other (p 0.05).
should be carried out assessing agronomic rice biofortification with Se in order to adjust the doses to be applied in soils with different characteristics, and under different application times when applying Se on leaves for assuring food security. Selenite and selenate absorption and mobility have been clearly differentiated in plants (Sors et al., 2005; Terry et al., 2000). Fig. 2 shows the total Se accumulation in rice roots, shoots and grains and the percent distribution of this accumulation among these plant parts. Since there was little variation in shoot dry matter production and grain yield due to the use of different sources of Se in both forms of application (Fig. 1A and B), the Se accumulation was dependent on the content of this element in the plant parts. Thus, as seen in Fig. 1C, for the Se content in grains, soil application
of both selenite and selenate resulted in greater total Se accumulation in all parts of the plant and selenate yielded higher Se accumulation when compared with selenite in both forms of application (Fig. 2A). Although selenate in the soil has enabled high accumulation of Se in the plant, most of the Se (70%) was accumulated in the vegetative part of the plant (shoots), and lower amounts in roots and grains. For selenite applied to the soil, besides shoots, roots accumulated high amounts of Se at the expense of grains (Fig. 2B). Foliar applications of the different sources of Se resulted in a greater proportion of Se in grains when selenate was applied (Fig. 2B). This fact allows inferring that selenate is more easily transported through the phloem when applied to leaves, as occurs
Fig. 3. N (A), P (B), Mg (C) and S (D) contents in rice grains according to the Se application form and sources. Same letters, comparing Se sources within each application form, do not differ from each other (p 0.05). Asterisks (*), comparing Se application forms within each Se source, differ from each other (p 0.05).
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Fig. 4. B (a), Cu (b), Fe (c), Mn (d) and Zn (e) contents in rice grains according to the Se application form and sources. Same letters, comparing Se sources within each application form, do not differ from each other (p 0.05). Asterisks (*), comparing Se application forms within each source, differ from each other (p 0.05).
in the xylem when applied via roots. These results are similar to those observed by Poggi et al. (2000), who applied selenite and selenate on leaves of potato and also observed greater mobility in the phloem when using selenate, which increased the Se content in this culture. It is noteworthy that plants that had not received Se application (control treatment) grew in soil with low natural selenium content (0.064 mg dm3), which was not sufficient to promote rice biofortification. Based on this result and on other studies reporting low Se concentrations in Brazilian soils and food (Ferreira et al., 2002; Moraes et al., 2009; Sillanpa¨a¨ and Jansson, 1992) and elsewhere (Antoine et al., 2012; Sirichakwal et al., 2005), it could be inferred that to increase the Se concentration in agricultural crops in soils poor in Se, it is necessary to apply Se in fertilization programmes. Neither the application form nor the Se source affected the N content in grains (Fig. 3a). Rı´os et al. (2010) assessed the effect of sources and concentrations of Se on N use efficiency in lettuce plants and found no variation in the concentration of N-NO2 in plants. For the P in grains (Fig. 3b), soil application resulted in higher P contents when compared with foliar applications of Se. In foliar application, regardless of the source of Se, there was a reduction in the P content in grains. Although the competitive
inhibition of selenite in P absorption has been documented (Hopper and Parker, 1999), which affects the P content in plants (Li et al., 2008), in the present study, no significant difference in the P content was found when selenite was applied to the soil. It should also be considered that selenite undergoes specific adsorption onto Fe and Al oxides in the soil, reducing its availability and, as shown in Fig. 2a, its absorption by plants. Thus, there would be less competition with phosphate in the absorption process. Foliar application of different sources of Se did not affect the Mg content in rice grains (Fig. 3c). Nevertheless, an increase in Mg content was observed when these sources have been applied to the soil. For S in grains, the highest contents were observed when the Se sources were applied through the leaves (Fig. 3d). Comparing the sources of Se within the foliar application, selenite provided increased S concentration when compared to selenate. Due to the chemical similarity between S and Se, plants absorb and metabolize Se via the S absorption and assimilation pathway (Pilon-Smits and Quinn, 2010), and several studies have indicated synergistic effect of Se by increasing the S content in plants (Barack and Goldman, 1997; Mikkelsen and Wan, 1990). There are some interactions concerning the responses of plants exposed to different concentrations of Se on micronutrients levels, like antagonistic or synergistic effects (Pazurkiewicz-Kocot et al.,
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2008; Feng et al., 2009). In the present study, we found that the different sources and application forms of Se promoted different behaviour in terms of micronutrient contents (Fig. 4). The B content reduced in both application forms and Se sources used (Fig. 4a). For the Cu content, no significant difference was observed when the Se sources were applied in the soil (Fig. 4b). However, for this variable, the foliar application of selenate increased the Cu content, while selenite promoted reduction. Pazurkiewicz-Kocot et al. (2008) also found a decrease in Cu contents when selenite was added to the corn crop. An increase in Fe content was verified when selenate was applied to leaves (Fig. 4c). The same behaviour was observed for the Mn content when this source of Se was applied to the soil (Fig. 4d). However, significant differences between the application forms were observed only for the Mn content (Fig. 4d). Fang et al. (2008) also observed increase in Fe content with the foliar application of Se. Regarding the Zn content, only selenate differed between the application forms of Se, with the highest Zn values being observed when this source of Se was applied to the soil. Studies carried out by Zembala et al. (2010) with rape plant (Brassica napus) and wheat (Triticum spp.) have shown that the application of Se increased Zn concentrations whereas decreased Fe concentrations in the plant. Our results showed that selenate application increased the contents of Cu, Zn, and Mn in rice grains, indicating a possible synergistic effect. 4. Conclusions Soil application of Se as selenate increased rice dry matter production by 6%, and foliar application of both sources of Se – selenate and selenite – increased grain yield. The application of selenium, both foliar (applied during flowering) and soil, promotes an increase in Se contents in rice grains, and soil application was the most effective procedure. In both application forms, selenate resulted in higher contents of Se in rice grains, with soil application yielding nearly 450% higher Se contents than foliar application. When compared with selenate, soil application of selenite resulted in a higher percentage of Se in roots than in shoots and grains. The application forms and sources of Se affected the macro and micronutrient contents in rice grains. Our results showed that it is possible to promote biofortification with Se in rice, both via soil and foliar application. Further research is needed in order to define adequate application rates for both Se forms, either via soil or foliar sprays. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgments The authors are grateful to CNPq, CAPES and FAPEMIG for financial support and scholarships. References Antoine, J.M.R., Hoo Fung, L.A., Grant, C.N., Dennis, H.T., Lalor, G.C., 2012. Dietary intake of minerals and trace elements in rice on the Jamaican market. Journal of Food Composition and Analysis 26, 111–121. Barack, P., Goldman, I.L., 1997. Antagonistic relationship between selenate and sulfate uptake in onion (Allium cepa): implications for the production of organosulfur and organoselenium compounds in plants. Journal of Agricultural and Food Chemistry 45, 1290–1294. Boldrin, P.F., Faquin, V., Ramos, S.J., Guilherme, L.R.G., Bastos, C.E.A., Carvalho, G.S., Costa, E.T.d.S., 2012. Selenato e selenito na produc¸a˜o e biofortificac¸a˜o agronoˆmica com seleˆnio em arroz. Pesquisa Agropecua´ria Brasileira 47, 831–837. Cartes, P., Gianfreda, L., Mora, M.L., 2005. Uptake of selenium and its antioxidant activity in ryegrass when applied as selenate and selenite forms. Plant and Soil 276, 359–367.
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Original Research Article
Soil and foliar application of selenium in rice biofortification Paulo Fernandes Boldrin a, Valdemar Faquin a, Sı´lvio Ju´nio Ramos a, Karina Volpi Furtini Boldrin b, Fabrı´cio William A´vila a, Luiz Roberto Guimara˜es Guilherme a,* a b
Department of Soil Science, Federal University of Lavras, Zip Code 3037, 37200-000 Lavras, MG, Brazil Department of Agriculture, Federal University of Lavras, Zip Code 3037, 37200-000 Lavras, MG, Brazil
A R T I C L E I N F O
A B S T R A C T
Article history: Received 12 June 2012 Received in revised form 30 May 2013 Accepted 20 June 2013
Selenium (Se) is essential for humans and animals because of its antioxidant properties, which form part of a series of chemical reactions. The aim of this study was to evaluate the effect of different Se application forms and sources on rice growth, grain yield, and rice Se concentration and accumulation, as well the content of N, P, Mg, S, B, Cu, Fe, Mn, and Zn in rice grains. The experiment was carried out in a greenhouse with 4-dm3 pots containing a sandy clay loam Red-Yellow Latosol. The experimental design was a completely randomized 2 2 2 factorial scheme (two Se doses two forms of Se application, soil or foliar two Se sources, selenate or selenite), with five replicates. Selenium in rice plants was analyzed by total reflection X-ray fluorescence spectrometry (TXRF). The results shows that soil selenate application was more effective for shoot dry matter production and grain Se accumulation than selenite. Foliar application of both selenate and selenite increased grain yield. This study provides useful information concerning agronomic biofortification of rice, showing that both soil and foliar Se application could be used for increasing Se content in edible parts, which could result in health benefits. ß 2013 Elsevier Inc. All rights reserved.
Keywords: Oryza sativa Rice Biofortification Selenate Selenite Nutrient contents Horticultural practices and nutrition Food analysis Food composition
1. Introduction Selenium (Se) is essential for humans and animals because of its antioxidant properties, which form part of a series of chemical reactions (Fairweather-Tait et al., 2011). In fact, Se is considered as one of the most important trace elements for human and animal health (Chaudhary et al., 2010). The deficiency of this element has been associated with the occurrence of serious diseases like heart disease and many types of cancers (Rayman, 2002). Unlike the case for humans and animals, for plants Se is not considered essential (Pilon-Smits and Quinn, 2010). Due to its chemical properties similar to sulfur (S), Se is absorbed and metabolized by plants in the same assimilation pathway as S, which is present in sulfur amino acids such as selenomethionine and selenocysteine (Sors et al., 2005). Depending on the Se concentration used, it may exert a double effect on plant growth. In
* Corresponding author at: Department of Soil Science, Federal University of Lavras, CP 3037, Campus UFLA, 37200-000 Lavras (MG), Brazil. Tel.: +55 35 3829 1259; fax: +55 35 3829 1251. E-mail addresses: [email protected] (P.F. Boldrin), [email protected]fla.br (V. Faquin), [email protected] (S.J. Ramos), [email protected] (K.V.F. Boldrin), [email protected] (F.W. A´vila), [email protected]fla.br (L.R.G. Guilherme). 0889-1575/$ – see front matter ß 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jfca.2013.06.002
this sense, Hartikainen et al. (2000) reported that, at low concentrations, Se acts as an antioxidant and can stimulate plant growth and production, whereas at high concentrations, it favours cell oxidation, leading to a reduction in yield. The low Se intake by the Brazilian population is linked with the consumption of foods with low contents of this element, a result of its low concentration in most Brazilian agricultural soils (Ferreira et al., 2002; Sillanpa¨a¨ and Jansson, 1992). According to Combs (2001), approximately 1 billion people worldwide have Se deficiency, considered the fourth most serious deficiency of minerals after iron (Fe), the most severe, followed by zinc (Zn) and iodine (I) (White and Broadley, 2009). Due to the importance of Se in human health and the strong deficiency of this element worldwide, it is necessary to find strategies to increase its content in foods. Accordingly, Chen et al. (2002), for rice, Hawkesford and Zhao (2007), Lyons et al. (2004), for wheat varieties, and, recently, Ramos et al. (2010) for lettuce, have showed that the Se content in these cultures increased with the introduction of this element through fertilization, which enables increased Se intake by the population. The choice of crops highly consumed by the population allows greater success in biofortification programmes. Rice is particularly interesting in this context, because is a staple food in at least 33 countries and provides about 80% of the daily caloric intake to 3
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billion people (Lucca et al., 2006; Meng et al., 2005). Rice is widely cultivated in Brazil–both under lowland and upland conditions– and is ranked 4th place in cultivated area and 6th in terms of fertilizer consumption. Thus, increasing the Se concentration in rice grains via fertilization could be beneficial to human health. However, for programmes.involving biofortification with this element, the application form and sources of Se must be taken into account, since Se in the form of selenite can undergo specific adsorption into Fe and Al oxides in the clay fraction, which compromises the availability of Se to plants (Mouta et al., 2008). Because of the importance of rice and Se for human nutrition and health, this study aimed to evaluate the effect of different application forms and sources of Se on rice growth, grain yield, and grain Se content and accumulation, as well as the content of N, P, Mg, S, B, Cu, Fe, Mn, and Zn in rice grains. 2. Material and methods 2.1. Experimental design and rice cultivation The experiment was conducted under greenhouse conditions at the Department of Soil Science, Federal University of Lavras, using 4dm3 pots filled with samples from the 0–20 cm layer of a sandy clay loam Red-Yellow Latosol. Soil physical and chemical analyses followed the methods described by EMBRAPA (1997), and mineralogical analyses were performed according to Souza (2005), with results as follows: sand = 600 g kg1; silt = 170 g kg1; 1; clay = 230 g kg1; pH H2O = 5.4; soil organic matter = 12 g kg1; P (Mehlich 1) = 0.9 mg kg1; K = 23.4 mg kg1; Ca = 0.3 cmolc kg1; Mg = 0.1 cmolc kg1; Al = 0.1 cmolc kg1; H + Al = 0.9 cmolc kg1; Fe2O3 = 37.8 g kg1; and Al2O3 = 168.1 g kg1. The total soil Se content was 0.064 mg kg1, according to the methodology described by Ramos et al. (2010). The experimental design was a completely randomized 2 2 2 factorial scheme as follows: with and without application of Se, two forms of Se application (soil application at a dose of 0.75 mg kg1 and foliar application at a concentration of 50 mmol L1), and two sources of Se (selenate – Na2SeO4 and selenite – Na2SeO3, both purchased from Sigma–Aldrich, Saint Louis, USA), with five replications, totalling 40 plots. Each experimental unit consisted of two plants per pot. For soil Se application, the dose was chosen in order not to compromise the plant development in our preliminary studies (Boldrin et al., 2012). The dose for foliar application was chosen within the range of concentrations suggested by Kapolna et al. (2009). Based on soil chemical analysis, soil samples were limed to increase base saturation to 50%, using quick lime with 32% CaO, 15% MgO, and an effective neutralizing value of 94.5%. After 30 days of incubation with a moisture content near 60% of the total pore volume (TPV), the sources of Se were applied in the soil along with a basic fertilization, which consisted of 80 mg of N, 250 mg of P, 70 mg of K, and 60 mg of S per dm3 of soil, using the following sources: ammonium nitrate - NH4NO3, monoammonium phosphate – NH4H2PO4, and potassium sulfate – KSO4. Basic fertilization with micronutrients consisted in the application of 3.6 mg of Mn, 1.5 mg of Cu, 5 mg of Zn, 0.5 mg of B, and 0.15 mg of Mo per dm3 of soil, as manganese chloride – MnCl2.4H2O, copper sulfate – CuSO45H2O, zinc sulfate – ZnSO47H2O, boric acid – H3BO3, and ammonium molybdate – (NH4)6Mo7O244H2O. Ten rice seeds were sown (Oryza sativa L. cv BRSMG Relaˆmpago) per pot, with two seedlings left in each pot one week after emergence. In the present study, we selected an early upland rice cultivar with high grain quality that is extensively cultivated in Brazil (Soares et al., 2010). During the growing period, rice plants received top-dressed N (450 mg dm3) and K (350 mg dm3), split into eight applications, using ammonium nitrate – NH4NO3 and
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potassium nitrate – KNO3. During the experiment, soil moisture was rigorously controlled by daily weighing of the soil-plant-pot set, replacing the volume lost through evapotranspiration with deionized water. Foliar application of the different Se sources was performed during flowering with three pulverizations applied in the morning (from 8:00 to 9:00) with an interval of three days between each application. A compression sprayer (Guarany Ind., Itu, Brazil) of 1.25 L was used, distributing the Se solution uniformly in all leaves of each plant separately, to avoid contamination of neighbouring plants. Rice plants were harvested at the end of the cycle. 2.2. Analytical procedures Leaves, stem, and grains were washed under deionized running water and oven-dried at 65–70 8C prior to digestions and analyses. For P, Mg, S, Cu, Fe, Mn, and Zn analyses, dried tissues (500 mg) were weighed and acid-digested with 4.0 mL of concentrated HNO3 + 2.0 mL of concentrated HClO4 (Sigma–Aldrich, Saint Louis, MO, USA) at 120 8C for 1 h and then at 220 8C until HClO4 fumes were observed. Total Mg, S, Cu, Fe, Mn, and Zn contents in the samples were determined using an atomic absorption spectrophotometer (PerkinElmer Inc., San Jose, CA, USA), and total P contents in the sample was determined using a spectrophotometer to measure a phospho-molybdenum complex colorimetrically at 680 nm (Malavolta et al., 1997). Total nitrogen content was determined by the Kjeldahl method, after sulfuric acid digestion. Boron was analyzed after digestion of the samples in a muffle-oven at 550 8C, being determined colorimetrically at 540 nm, after the reaction of boric acid with curcumin producing the compound rosocyanine (Malavolta et al., 1997). Selenium concentration in grains was determined according to a method described by Ramos et al. (2010). Briefly, dried tissues (approximately 500 mg) were weighed and acid-digested in 5 mL of concentrated HNO3 (Sigma–Aldrich, Saint Louis, MO, USA) in Teflon1 PTFE flasks (CEM Corporation, Matthews, NC, USA) and submitted to 0.76 MPa for 10 min in a CEM microwave oven (Mars model 5, CEM Corporation, Matthews, NC, USA). After cooling to room temperature, the extracts were filtered (Whatman No. 40 filter) and diluted by adding 5 mL of bi-distilled water. A certified reference material (White Clover-BCR402, Institute for Reference Materials and Measurements (IRMM), Geel, Belgium) was included for quality control. Blank and certified reference samples were analyzed along with every batch of digestion. Selenium content in the extracts was determined by total reflection X-ray fluorescence spectrometry (TXRF), using a PicofoxTM S2 instrument (Bruker, Karlsruhe, Germany), as described by Stosnach (2005). For TXRF analysis, gallium was used for internal standardization (final gallium concentration of 10; 100 and 1000 mg L1). After homogenization, an aliquot of 5 mL was transferred onto a quartz glass sample carrier and dried before TXRF analysis. The instrument analytical detection limit (LD) for Se pffiffiffiffiffiffiffiffi in plant extracts was calculated as: LD ¼ ð3 Ci N BG Þ=Ni; where Ci is the concentration of the element, NBG is the background area subjacent to the fluorescence peak, and Ni is area of the fluorescence peak in counts. The calculated detection limit for the method (after considering the dilution of plant extracts) was 50 mg kg1. 2.3. Statistical analysis All results were analyzed using analysis of variance (ANOVA), and significantly different means between treatments were separated with the Scoot-Knott’s test at the 0.05 significance level
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Fig. 1. Shoot dry matter production (A), grain yield (B) and selenium content in rice grains (C), in rice plants, depending on Se application forms and sources. Same letters, comparing Se sources within each application form do not differ (p 0.05). Asterisks (*), comparing Se application forms within each Se source, differ between each other (p 0.05).
of probability, using Sisvar 4.6 software (Build 6.1) (Ferreira, 2011) and the graphs were made in the Sigma Plot Programme (version 12.0, Systat Software Inc., Chicago, IL, USA). 3. Results and discussion The mean Se concentrations of the repeated analysis (n = 5) of the standard reference materials are presented in Table 1. The Se content recoveries in the certified samples show a reliable analytical data accuracy for Se analysis. The shoot dry matter production (SDMP), grain yield (GY) and Se content in grains were significantly affected by the application forms and sources of Se, as well as by the interaction between these factors (Fig. 1). Foliar application of Se did not change SDMP. However, when the sources of Se were applied to the soil, selenate provided an increase of about 6% in SDMP (Fig. 1A). For GY, foliar application of both sources of Se provided increases as follows: 22% for selenate and 17% for selenite (Fig. 1B). However, neither source of Se changed the GY. The increase in SDMP when Se was applied as selenate in the soil and in grain yield, for both Se sources applied on leaves, can be related to the beneficial effects of Se at low doses. Our results are consistent with those found by Djanaguiraman et al. (2005) for soybean, Rios et al. (2008) for lettuce, and Boldrin et al. (2012) for rice, which showed increased production at low Se concentration. Moreover, earlier studies indicate beneficial effects of Se, because it increases the antioxidant activity in plants, leading to better plant yield (Hartikainen et al., 2000; Lyons et al., 2009). Soil application of selenate provided higher Se content in grains, being 450% higher than the foliar application of this Se source
(Fig. 1C). This result may be related to the longer plant-Se contact time promoted by the soil application. Since foliar application was performed at the flowering stage, possibly the time required for Se transport via phloem from the leaves to the grains was not sufficient to increase the content of this element in the grain. It is noteworthy that, among the Se sources applied to soil, the highest concentrations of this element were provided by selenate. This fact is probably due to the specific adsorption that occurs with selenite on Fe and Al oxide surfaces, which are abundant in tropical soils (Rovira et al., 2008), decreasing Se in the soil solution, and thus its absorption by plants. Furthermore, it is known that selenite is less translocated in plants (Cartes et al., 2005; Ramos et al., 2010; Rios et al., 2008; Terry et al., 2000) when compared with selenate. Combs (2001) reports that the adequate Se level in a population is highly correlated with the Se content in agricultural crops. Thus, due to the fact that rice is the staple food of over half the world’s population, in at least 33 countries (Lucca et al., 2006), and that Se deficiency reaches nearly 1 billion people worldwide (White and Broadley, 2009), this crop has enormous potential to increase the Se consumption by the world population. According to the Food and Agriculture Organization of the United Nations - FAO (2011), the average rice consumption per person in the world is 180 g day1, and the recommended minimum daily Se intake for adults is 70 mg day1, with a maximum tolerable level of 400 mg day1 (USDA, 2003). Considering the data of our study, we estimate that the Se content in rice grains could contribute to the dietary Se intake with 210 and 48 mg day1, via foliar application, and 1150 and 75 mg day1 via soil application, by using selenate and selenite respectively. Thus, further studies
Table 1 Certified value, determined concentration and Se recovery on certified material. Certified material
Certified value (mg kg1)
Determined concentrationa (mg kg1)
Mean recovery (%)
BCR-402
6.7
5.2 0.3
78 4.5
a
Average of 5 measurements of standard reference material samples.
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Fig. 2. Se accumulation (A) and percentual distribution (B) in parts of the rice plant according to Se application forms and sources. Different letters indicate significant differences between means among Se sources within each application form (p < 0.05): (A–C) compare total Se accumulation; (a–c) compare rice grain Se accumulation; (d–f) compare shoots Se accumulation; (g–i) compare roots Se accumulation. Asterisks (*), comparing Se application forms within each Se source, differ from each other (p 0.05).
should be carried out assessing agronomic rice biofortification with Se in order to adjust the doses to be applied in soils with different characteristics, and under different application times when applying Se on leaves for assuring food security. Selenite and selenate absorption and mobility have been clearly differentiated in plants (Sors et al., 2005; Terry et al., 2000). Fig. 2 shows the total Se accumulation in rice roots, shoots and grains and the percent distribution of this accumulation among these plant parts. Since there was little variation in shoot dry matter production and grain yield due to the use of different sources of Se in both forms of application (Fig. 1A and B), the Se accumulation was dependent on the content of this element in the plant parts. Thus, as seen in Fig. 1C, for the Se content in grains, soil application
of both selenite and selenate resulted in greater total Se accumulation in all parts of the plant and selenate yielded higher Se accumulation when compared with selenite in both forms of application (Fig. 2A). Although selenate in the soil has enabled high accumulation of Se in the plant, most of the Se (70%) was accumulated in the vegetative part of the plant (shoots), and lower amounts in roots and grains. For selenite applied to the soil, besides shoots, roots accumulated high amounts of Se at the expense of grains (Fig. 2B). Foliar applications of the different sources of Se resulted in a greater proportion of Se in grains when selenate was applied (Fig. 2B). This fact allows inferring that selenate is more easily transported through the phloem when applied to leaves, as occurs
Fig. 3. N (A), P (B), Mg (C) and S (D) contents in rice grains according to the Se application form and sources. Same letters, comparing Se sources within each application form, do not differ from each other (p 0.05). Asterisks (*), comparing Se application forms within each Se source, differ from each other (p 0.05).
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Fig. 4. B (a), Cu (b), Fe (c), Mn (d) and Zn (e) contents in rice grains according to the Se application form and sources. Same letters, comparing Se sources within each application form, do not differ from each other (p 0.05). Asterisks (*), comparing Se application forms within each source, differ from each other (p 0.05).
in the xylem when applied via roots. These results are similar to those observed by Poggi et al. (2000), who applied selenite and selenate on leaves of potato and also observed greater mobility in the phloem when using selenate, which increased the Se content in this culture. It is noteworthy that plants that had not received Se application (control treatment) grew in soil with low natural selenium content (0.064 mg dm3), which was not sufficient to promote rice biofortification. Based on this result and on other studies reporting low Se concentrations in Brazilian soils and food (Ferreira et al., 2002; Moraes et al., 2009; Sillanpa¨a¨ and Jansson, 1992) and elsewhere (Antoine et al., 2012; Sirichakwal et al., 2005), it could be inferred that to increase the Se concentration in agricultural crops in soils poor in Se, it is necessary to apply Se in fertilization programmes. Neither the application form nor the Se source affected the N content in grains (Fig. 3a). Rı´os et al. (2010) assessed the effect of sources and concentrations of Se on N use efficiency in lettuce plants and found no variation in the concentration of N-NO2 in plants. For the P in grains (Fig. 3b), soil application resulted in higher P contents when compared with foliar applications of Se. In foliar application, regardless of the source of Se, there was a reduction in the P content in grains. Although the competitive
inhibition of selenite in P absorption has been documented (Hopper and Parker, 1999), which affects the P content in plants (Li et al., 2008), in the present study, no significant difference in the P content was found when selenite was applied to the soil. It should also be considered that selenite undergoes specific adsorption onto Fe and Al oxides in the soil, reducing its availability and, as shown in Fig. 2a, its absorption by plants. Thus, there would be less competition with phosphate in the absorption process. Foliar application of different sources of Se did not affect the Mg content in rice grains (Fig. 3c). Nevertheless, an increase in Mg content was observed when these sources have been applied to the soil. For S in grains, the highest contents were observed when the Se sources were applied through the leaves (Fig. 3d). Comparing the sources of Se within the foliar application, selenite provided increased S concentration when compared to selenate. Due to the chemical similarity between S and Se, plants absorb and metabolize Se via the S absorption and assimilation pathway (Pilon-Smits and Quinn, 2010), and several studies have indicated synergistic effect of Se by increasing the S content in plants (Barack and Goldman, 1997; Mikkelsen and Wan, 1990). There are some interactions concerning the responses of plants exposed to different concentrations of Se on micronutrients levels, like antagonistic or synergistic effects (Pazurkiewicz-Kocot et al.,
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2008; Feng et al., 2009). In the present study, we found that the different sources and application forms of Se promoted different behaviour in terms of micronutrient contents (Fig. 4). The B content reduced in both application forms and Se sources used (Fig. 4a). For the Cu content, no significant difference was observed when the Se sources were applied in the soil (Fig. 4b). However, for this variable, the foliar application of selenate increased the Cu content, while selenite promoted reduction. Pazurkiewicz-Kocot et al. (2008) also found a decrease in Cu contents when selenite was added to the corn crop. An increase in Fe content was verified when selenate was applied to leaves (Fig. 4c). The same behaviour was observed for the Mn content when this source of Se was applied to the soil (Fig. 4d). However, significant differences between the application forms were observed only for the Mn content (Fig. 4d). Fang et al. (2008) also observed increase in Fe content with the foliar application of Se. Regarding the Zn content, only selenate differed between the application forms of Se, with the highest Zn values being observed when this source of Se was applied to the soil. Studies carried out by Zembala et al. (2010) with rape plant (Brassica napus) and wheat (Triticum spp.) have shown that the application of Se increased Zn concentrations whereas decreased Fe concentrations in the plant. Our results showed that selenate application increased the contents of Cu, Zn, and Mn in rice grains, indicating a possible synergistic effect. 4. Conclusions Soil application of Se as selenate increased rice dry matter production by 6%, and foliar application of both sources of Se – selenate and selenite – increased grain yield. The application of selenium, both foliar (applied during flowering) and soil, promotes an increase in Se contents in rice grains, and soil application was the most effective procedure. In both application forms, selenate resulted in higher contents of Se in rice grains, with soil application yielding nearly 450% higher Se contents than foliar application. When compared with selenate, soil application of selenite resulted in a higher percentage of Se in roots than in shoots and grains. The application forms and sources of Se affected the macro and micronutrient contents in rice grains. Our results showed that it is possible to promote biofortification with Se in rice, both via soil and foliar application. Further research is needed in order to define adequate application rates for both Se forms, either via soil or foliar sprays. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgments The authors are grateful to CNPq, CAPES and FAPEMIG for financial support and scholarships. References Antoine, J.M.R., Hoo Fung, L.A., Grant, C.N., Dennis, H.T., Lalor, G.C., 2012. Dietary intake of minerals and trace elements in rice on the Jamaican market. Journal of Food Composition and Analysis 26, 111–121. Barack, P., Goldman, I.L., 1997. Antagonistic relationship between selenate and sulfate uptake in onion (Allium cepa): implications for the production of organosulfur and organoselenium compounds in plants. Journal of Agricultural and Food Chemistry 45, 1290–1294. Boldrin, P.F., Faquin, V., Ramos, S.J., Guilherme, L.R.G., Bastos, C.E.A., Carvalho, G.S., Costa, E.T.d.S., 2012. Selenato e selenito na produc¸a˜o e biofortificac¸a˜o agronoˆmica com seleˆnio em arroz. Pesquisa Agropecua´ria Brasileira 47, 831–837. Cartes, P., Gianfreda, L., Mora, M.L., 2005. Uptake of selenium and its antioxidant activity in ryegrass when applied as selenate and selenite forms. Plant and Soil 276, 359–367.
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