AGRONOMIC BIOFORTIFICATION WITH IRON AND ZINC ON YIELD AND QUALITY OF OAT GRAINS FOR THE VALIDATION OF A POTENTIAL RESOURCE FOR NUTRITIONAL SECURITY

Purpose: The objective of this study was to develop a management proposal for biofortification of oat crops with iron and zinc by foliar application and validate the technology by analyzing indicators of yield and industrial and chemical quality of grains. Method/design/approach: The experiment was carried out in Augusto Pestana, RS, in 2020 and 2021. A randomized block experimental design with four replications was used, in a 3×5 factorial arrangement. The treatments consisted of applications of zinc sulfate, iron sulfate, and zinc sulfate + iron sulfate at rates of 0, 500, 1000, 2000, and 4000 g ha-1. Indicators of yield and industrial and chemical quality of oat grains were analyzed. Results and conclusion: The agronomic biofortification with iron and zinc by foliar application at the grain filling stage does not affect yield and industrial and chemical quality of oat grains. Zinc biofortification with sulfate sources promotes increases in zinc contents in oat caryopses, however, with a decrease in iron contents. Iron biofortification with sulfate sources increases iron contents in the oat caryopsis, however, with a decrease in zinc contents.


INTRODUCTION
Oat (Avena sativa L.) is a winter cereal crop recognized worldwide that stands out for its use for soil protection and quality and as human and animal food (Mantai et al., 2021a;Scremin et al., 2023). Although large part of the oat produced is used for animal feed, human consumption has increased in recent years due to its nutritional and functional properties, which result in several health benefits Savicki et al., 2023). Oat composition includes a high concentration of dietary fibers, proteins, flavonoids, and antioxidant compounds (Chudan et al., 2023;Dornelles et al., 2023), including soluble fibers such as β-glucans, which stands out for their bioactive compounds that lower LDL cholesterol and promote glucose control, as well as insoluble fibers, which improve intestinal microflora (Rasane et al., 2015;Dornelles et al., 2021). Therefore, this food has a high biological quality for direct consumption and for the development of several products (Marolli et al., 2021;Loro et al., 2022).
A proper diet with nutrient balance is essential to meet human needs. Iron and zinc are among nutrients that have important functions (Dobermann et al., 2022;Zhang et al., 2022). Iron assists in enzyme activation, enhances metabolic activity, cellular respiration, and synthesis of nucleic acids, and facilitates the movement of oxygen by red blood cells to tissues (Kumari & Chauhan 2022;Rohr, Brandenburg & Rocca, 2023). Zinc assists in enzyme activities, promotes protein synthesis, has antioxidant functions, and is part of important enzymes for the nervous system, assisting in the learning process (Dussiot et al., 2022;Duan et al., 2023). Iron deficiency causes fatigue, attention deficits, reduced defense mechanisms, and anemia. Zinc deficiency impairs growth, reduces immunity, causes skin diseases and hair loss, and delays wound healing (Ho, Wong & King, 2022;Cappellini et al., 2022).
Iron and zinc deficiency has been currently observed in populations of developing countries due to limited access to foods rich in these nutrients, mainly red meats and fish, which often have a high cost (Mantadakis, Chatzimichael & Zikidou, 2020;Von Braun et al., 2023). In this scenario, the increased consumption of highly processed foods (fast foods), which are quickly made and easy to consume, has also promoted health problems due to an inadequate nutritional balance (Rasane et al., 2015;Güven & Öncü, 2022).
The recovery of iron and zinc from the soil by most plants is generally very low, which raises the need to enrich agricultural species with these elements and make them more available to the population. In this scenario, agronomic biofortification is a method that assists in the accumulation of nutrients and important compounds in plants, based on agronomic practices and focused on the enrichment of edible structures (Ramzan et al., 2020;Zhang et al., 2022). Although genetic biofortification is a possibility, increasing nutrients by crossing and selection is difficulty, may result in an inverse relationship between yield and mineral concentration, takes a long time to reach high concentrations in the cultivars and make them available for planting, and is interconnected with the availability of these nutrients in the soil (Kumar et al., 2019;Krishna, Maharajan & Ceasar, 2023).
Foliar application of nutrients for agronomic biofortification can be a more practical and cheaper method, considering that the nutrients cross cell membranes and follow routes that promote absorption, transport, storage, and redistribution in the plant. Many transporters in plants are involved in carrying iron and zinc to the endosperm of seeds (Szeremen et al., 2022;Huertas et al., 2023). Agronomic biofortification of high-biological-value foods, such as oats, with iron and zinc can increase nutritional quality, bringing benefits to human health, which is consistent with the interest of several countries that seek quality products and nutritional safety. Therefore, the objective of this study was to develop a management proposal for agronomic biofortification of oat crops with iron and zinc by foliar application and validate the technology by analyzing indicators of yield and industrial and chemical quality of grains.

METHODOLOGY
The study was carried out in 2021, in Augusto Pestana, RS, Brazil (28°26'30''S and54°00'58''W). The soil of the experimental area was classified as Typic Hapludox (Latossolo Vermelho Distroferrico tipico), with a deep and well-drained profile and a dark red color. The climate of the region is Cfa, humid subtropical, according to the Köppen classification. The study for validating the biofortification technology was carried out using the oat cultivar URS Corona, which is recommended for crops in southern Brazil. Oat seeds were mechanically sown on June 15 at a density of 450 seeds per square meter, distributed in five 5meter rows spaced 0.20 m apart; the experimental unit was 5 m 2 . P2O5 and K2O fertilizers were applied at sowing, using 60 and 50 kg ha -1 , respectively, based on the soil P and K contents. Nitrogen was applied at sowing, using 10 kg ha -1 , and as topdressing at the fourth expanded leaf stage, using 40 kg ha -1 ; the expected grain yield was 3,000 kg ha -1 . Tebuconazole (Folicur® CE) fungicide was applied at a rate of 0.75 L ha -1 . Weed control was carried out using metsulfuron-methyl herbicide (Ally®) at a rate of 2.4 g ha -1 and additional weeding whenever necessary.
The experiment was carried out in a soybean-oat rotation system. A randomized block experimental design with four replications was used, in a 3×5 factorial arrangement. The treatments consisted of applications of zinc sulfate, iron sulfate, and zinc sulfate + iron sulfate, at the rates of 0, 500, 1000, 2000, and 4000 g ha -1 . The iron sulfate + zinc sulfate combination was applied maintaining the same rates for each source. The biofortification treatments were applied at the beginning of grain filling stage. Zinc and iron were applied using the compounds ZnSO4.5H2O and FeSO4.7H2O, respectively, through an electric knapsack sprayer with cone jet nozzles at a constant pressure of 30 lb pol -2 . Iron rates were defined considering the study carried out by Gupta (1991), using foliar application of iron sulfate heptahydrate (FeSO4.7H2O) at rates ranging from 500 to 1,000 g ha -1 , in a volume of 500 L ha -1 of water. Zinc rates were defined based on the study carried out by Pascoalino (2014), using zinc sulfate pentahydrate (ZnSO4.5H2O) at the rate of 910 g ha -1 in a volume of 200 L ha -1 of water. Zhang et al. (2010) combined ferric citrate and zinc sulfate, using a volume of 1,000 L ha -1 of water. Based on these studies, an intermediate water volume of 500 L ha -1 was used for the application of the biofortification treatments. Tests carried out before field application were used to determine the volume and application time for each experimental unit, setting the time at 21 seconds for the specified volume and working pressure.
Agronomic indicators, industrial quality and organic chemical composition of grains, and iron and zinc concentrations in oat grains and caryopses were evaluated, focused on validating the technology. The agronomic indicators evaluated were: i) grain yield (GY, kg ha -1 ): the three central rows in each plot were mechanically harvested, with a grain moisture content of approximately 18%, then, the grains were sent to a laboratory for adjusting grain moisture to 13%, threshing, and weighing to obtain the yield in grams, which was converted into kg ha-1; ii) one thousand grain weight (g) was obtained by weighing 250 grains on a precision scale and multiplying the result by four to convert into 1000-grain weight; iii) hectoliter weight (kg hl -1 ) was obtained by the grain weight from a cube of 250 cm³, which was converted into kg hl -1 . The industrial quality indicators evaluated were: i) number of grains larger than 2 mm (NG>2 mm, n), determined by placing one hundred grains on a 2 mm mesh sieve and counting those with higher sizes; ii) weight of grains larger than 2 mm (g), determined by weighing 50 grains larger than 2 mm on a precision scale; iii) caryopsis weight of grains larger than 2 mm, obtained by weighing 50 grains larger than 2 mm and without husk on a precision scale; iv) husking index (HI, g g -1 ), determined by the ratio between the caryopsis weight of 50 grains larger than 2 mm and its grain weight; v) industrial grain yield (IGY, kg ha -1 ), obtained by multiplying the grain yield by the number of grains larger than 2 mm and by the husking index (IGY = GY × NG>2 mm × HI).
The organic chemical composition indicators evaluated were: crude protein (g kg -1 ), crude fiber (g kg -1 ), neutral detergent fiber (g kg -1 ), and starch (g kg -1 ), evaluated in 40-gram samples through near infrared spectrophotometry (Perten DA7200 Diode Array). Iron (g ha -1 ) and zinc (g ha -1 ) concentrations were obtained by weighing 20-gram samples of grains with and without caryopsis on a precision scale. The samples were taken to an oven at temperature between 70 and 80 °C for drying and weight correction and then ground in a mill for approximately 90 seconds. These samples were then sieved in a 270-mesh sieve to obtain a particle size of 53 microns, resulting in weights of 5 to 6 grams. The samples were identified and analyzed for zinc and iron concentrations in the oat grains and caryopses. The procedures and materials used were based on Tedesco et al. (1995).
Air temperature (°C) and rainfall depth (mm) information during the crop seasons were obtained from an automatic weather station installed 500 meters from the experimental area. The data were subjected to analysis of variance to assess main and interaction effects, and the means were tested using the Scott & Knott test at 5% error probability level. Regression analysis was used to determine the dynamics and define the adjusted iron and zinc rates in oat grains and caryopsis. The statistical analyses were carried out using the free software GENES (Cruz, 2013).  Table 1 shows the data of temperature and rainfall depths. The lowest minimum temperatures in July and August were found in 2020, with values below 0 °C. The highest mean temperature in 2020 denotes a trend of high variation. Rainfalls were concentrated in the months of June and July, which correspond to the establishment and tillering stages, with a period of plant elongation and grain filling, denoting a significant water restriction, mainly from the time the biofortification treatments were applied until the grain harvest (Table 1; Figure 1). These results explain the lower mean yield obtained (1192 kg ha -1 ) compared to the expected yield (3000 kg ha -1 ), showing that 2020 was an unfavorable year for oat crop (Table 1). In 2021 (Table 1), June and July had minimum temperatures below 0 °C, with a mean air temperature lower than that in the 2021 crop cycle, showing a smaller range of variation. A dry spell was observed in July, at the oat establishment, tillering, and elongation stages. Therefore, the nitrogen management for the expected yield was compromised. Regular rainfall was observed after 75 days after emergence; this condition ensured a more favorable soil moisture condition during the biofortification period (Table 1; Figure 1). The results found for 2021 also showed that it was an unfavorable year for oat crop, presenting a mean yield of 1,516 kg ha -1 (Table 1). Rainfall has a significant effect on the efficiency of nitrogen, which is the macronutrient that most affects grain yield (Kraisig et al., 2020a;Mantai et al., 2021b). Nitrogen management is focused on applications under adequate soil moisture conditions without high rainfall volumes or intensity after application to avoid leaching (Silva et al., 2020;Pereira et al., 2023). Temperature is a catalyst for biological processes, and plants require specific minimum and maximum temperatures for normal physiological activities (Scremin et al., 2023;Berlezi et al., 2023). Milder temperatures and high-quality radiation favor tillering and grain filling in cereal crops, including oat and wheat, with direct effects on grain yield (Trautmann et al., 2019;Henrichsen et al., 2023). Low temperatures from the germination to grain filling stages and high daytime temperatures during the maturation stage are favorable for obtaining high grain yield and quality (Marolli et al., 2018;Savicki et al., 2023). Milder temperatures and low rainfall volumes with adequate distribution throughout the crop cycle favor oat crops Scremin et al., 2017). Grain yield and quality increase as weather conditions and agricultural technologies improve (Henrichsen et al., 2022;Reginatto et al., 2022).

RESULTS AND DISCUSSIONS
The analysis of variance in 2020 and 2021 showed that grain yield and other industrial quality indicators of interest were not affected by the sources and rates of the compounds with iron, zinc, and their combination (Table 2). These results are significant by showing that biofortification does not change variables of interest to farmers and industries, especially the number of grains larger than 2 mm and husking index, which are components that define industrial yield. 20 Note: CV = Coefficient of variation; DF = degrees of freedom; GY = grain yield; 1000GW = one thousand grain weight; MH = hectoliter weight; NG>2 mm = number of grains larger than 2 mm, HI = husking index; IGY = industrial yield. Source: Prepared by the authors (2023).
The single and combined sources and rates of zinc and iron did not change the components of the grain organic composition, regardless of the evaluated agricultural year, ensuring that biofortification does not compromise the variables of interest for food consumption (Table 3). However, the sources and concentrations of the compounds with zinc and iron changed the iron and zinc contents in oat grains and caryopses, regardless of the evaluated year. Table 3. Analysis of variance for organic contents and zinc and iron contents in oat grains and caryopses due to biofortification with sources and rates of iron and zinc sulfates.

Source of Variation DF
Mean Square CP CF NDF ST ZnG ZnC FeG FeC (g kg -1 ) (g kg -1 ) (g kg -1 ) (g kg -1 ) (mg kg -1 ) (mg kg -1 ) (mg kg -1 ) (mg kg -1 ) 2020 The zinc sulfate rates did not change zinc contents in oat grains in 2020 (Figure 2.A); however, increased zinc concentrations in the caryopsis (Figure 2.B). Zinc concentrations in caryopses increased up to a limit, confirming a quadratic response to the biofortification treatments. The estimated optimal rate of the compound was 2330 g ha-1, resulting in an estimated zinc content of 44.3 mg kg -1 in the caryopsis, representing an increase of more than 50% compared to the treatment with no biofortification (27.9 mg kg -1 ). The increase in the rates of the compound with zinc decreased iron contents in the oat grains (Figure 2.C) and caryopses (Figure 2.D). The use of the optimal rate of the compound decreased iron contents from 85 to 34 mg kg -1 in grains and from 64 to 32.7 mg kg -1 in caryopses.
The zinc sulfate rates did not change zinc contents in oat grains in 2021 (Figure 2.A); however, increased zinc concentrations on caryopses (Figure 2.B). Zinc concentrations in caryopses presented a linear response to the biofortification treatments. Zinc in oat caryopses increased 0.0039 mg kg -1 for every 1 g ha -1 of the compound (Figure 2.B). The actual zinc concentration for the compound rate of 2,000 and 4000 g ha -1 on the regression line showed similar nutrient expression in the caryopsis (Figure 2.B). Therefore, the rate of 2000 g ha -1 of zinc sulfate is more viable.
The application of zinc sulfate, at some rates of the compound, decreased iron contents in oat grains and caryopses in 2021. It showed a cubic degree trend in iron expression, generating decreases, mainly at the rate of 1000 g ha -1 of the compound (Figure 2.C). Furthermore, considering the decreases in iron in the caryopsis, stability reached 2000 and 4000 g ha -1 , denoting a quadratic response (Figure 2.D). The use of the recommended zinc sulfate rate resulted in decreases in iron from 281.4 to 201.4 mg kg -1 in grains and from 111.8 to 37.8 mg kg -1 in the caryopsis. These results show that biofortification using zinc sulfate should be carried out with caution, based on the interest in the iron content in the raw material intended for food consumption.   Biofortification with iron sulfate showed a cubic trend in iron contents in oat grains (Figure 3.A) and caryopses (Figure 3.B) in 2020. Therefore, the regression line at 2000 and 4000 g ha -1 of the compound showed similar iron contents in grains on the ordinate axis ( Figure  3.A). This similarity is also found in the actual iron contents in caryopses at 1000 and 4000 g ha -1 on the regression line (Figure 3.B).
Considering that caryopsis is the edible raw material for humans and constitutes the largest volume in the grain composition, the rate of 1000 g ha -1 of the compound is recommended for biofortification. This rate promoted increases from 37.8 to 51.8 mg kg -1 , representing a 37% increase in iron. In 2020, there was an opposite effect due to increases in iron rates, with a trend of decreases in zinc until stability in the oat caryopsis structure, presenting a quadratic response. The use of the recommended iron sulfate rate resulted in a decrease from 51.2 to 35.2 mg kg -1 in the oat caryopsis (Figure 3.D).
In 2021, biofortification with iron sulfate also showed a cubic trend in the expression of iron content in grains (Figure 3.A), however, expressing a quadratic response (Figure 3.B). This quadratic regression showed an optimal iron sulfate rate of 2230 g ha -1 , with an estimated iron concentration of 63.6 mg kg -1 in the caryopsis, representing an increase of 57%.
In 2021, zinc contents in oat grains and caryopses decreased as the iron sulfate rate was increased ( Figure 3). The use of the recommended iron sulfate rate for iron biofortification of oat caryopsis (2230 g ha -1 ) decreased zinc contents from 37 to 27.8 mg kg -1 in grains and from 36.5 to 33.6 mg kg -1 in the oat caryopsis. These results denote a competition between these nutrients; thus, further studies are necessary to enhance the combination of these elements and to understand the dynamics of the transporters of these nutrients in plant tissues.
In 2020, the combined biofortification with zinc and iron sulfates showed a cubic trend in zinc expression in oat grains (Figure 4.A). Zinc contents increased up to the 1000 g ha -1 , followed by a decrease at 2000 g ha -1 , and an increase at the highest rate when using the combination of sulfates. However, no changes in zinc concentrations were found in the edible structure (caryopsis) (Figure 4.B).
The combined application of zinc sulfate and iron sulfate showed a trend of decrease and stability in iron concentrations in oat grains, presenting a quadratic response (Figure 4.C). However, it was found a lack of effectiveness of biofortification with combination of sulfates in iron concentrations in oat caryopses (Figure 4.D). In 2021, the combined use of compounds with iron and zinc showed no changes in zinc contents in oat grains (Figure 4.A) and caryopses (Figure 4.B). Despite some differences in iron contents in grains and caryopses, the combined biofortification with iron sulfate and zinc sulfate decreased their contents in oat grains (   Studies have sought for strategies to improve the nutritional quality of foods, and regression models in experiments with quantitative treatments are efficient in describing the relationship between independent variables and a dependent one, enabling behavior analysis and simulations (Kraisig et al., 2020b;Trautmann et a., 2022;Rosa et al., 2022). In this context, oats have raised the attention of physicians, nutritionists, and consumers due to being one of the most complete foods, with high contents of proteins and quality fibers (Hawerroth et al., 2014;Pradebon et al., 2023). In this sense, studies focused on grain yield and nutritional quality have been developed due to a high demand for this cereal Loro et al., 2021). The growth of the world's population has been followed by changes in dietary habits, with approximately 66% of the diets lacking some micronutrients. In this sense, studies have reported that agronomic biofortification is a technique that can be accessible and efficient in increasing minerals and vitamins and is a sustainable way to provide access to more nutritious and healthy foods for the population (Zhang et al., 2010).
Studies on iron biofortification through foliar application at the beginning of soybean flowering stage showed that concentrations of 0, 0.5, 1.0, and 1.5 of iron sulfate (FeSO4.7H2O) increased iron contents in grains by 56.7, 64.2, 65.9, and 67.29 mg kg -1 , respectively, reaching an increase of 18% (Makdoh et al., 2023). A study on wheat crops reported increase from 27 to 43 mg kg -1 in iron contents in grains when using foliar application of iron sulfate at a rate of 0.25% at the beginning of grain filling. The biofortification treatments did not affect wheat grain yield, with a 37% increase in iron concentration (Jalal et al., 2020). Chickpea (Cicer arietinum L.) plants biofortified with iron sulfate through foliar application showed better performance at the rate of 1875 g ha -1 . Iron contents in grains were 55 mg kg -1 in the treatment without iron sulfate application, and increased 69.97 mg kg -1 (25%) when iron sulfate was applied at the flowering stage. Additionally, sequential iron sulfate application at the flowering and pod formation stages can increase iron contents in grains by 35% (75.34 mg kg −1 ) (Pal, Singh & Dhaliwal, 2019).
A study on foliar and soil applications of zinc sulfate (ZnSO4.7H2O) for biofortification of corn carried out 25 days after emergence showed increases in zinc contents in corn grains from 22.3 to 41.9 mg kg −1 , representing an increase of 53% (Imran & Rehim, 2017). A study on foliar application of 0.5% zinc sulfate for biofortification showed increases in zinc contents in wheat, triticale, and durum wheat grains, ranging from 31.0 to 63.0, 29.3 to 61.8, and 30.2 to 62.4 mg kg -1 , respectively . The biofortification of rice with zinc sulfate by foliar and soil applications showed no changes in grain yield; however, zinc concentrations in brown rice increased approximately 30% when using foliar applications, and only 2.4% when using soil application . Studies on biofortification of arugula evaluating foliar application of zinc sulfate at rates of 0, 500, 1,000, and 1500 g ha -1 at 15, 20, 25, 15, 20, and 25 days after emergence showed no effect on plant height, leaf area, and shoot weight; the application of the rate of 1500 g ha -1 at 25 days after emergence resulted in the highest increase (279%) in zinc contents in arugula leaves: from 65 to 246 mg kg -1 (Rugeles-reyes et al., 2019).
A study showed that increases in zinc sulfate rates through foliar application for biofortification of wheat grains caused decreases in iron contents in wheat grains from 47.8 to 40.8 mg kg -1 for the zinc sulfate rates of 0 and 0.3%, respectively, representing a decrease of 17%; increases in iron contents by application of iron sulfate rates of 0 and 2% decreased zinc concentrations in wheat grains from 38.3 to 35.2 mg kg -1 , respectively, representing a decrease of 8%. Thus, increasing one nutrient decreases the other, denoting a competition between these elements (Jalal et al., 2020).
The analyzed studies on different agricultural species did not show results that validate biofortification with zinc and iron through foliar application involving characteristics of agronomic, industrial, and food consumption interests, such as those evaluated in oat crops in the present study. Therefore, these valuable results show the potential of this technology for food enrichment, the possibility of practical use in field productions, and the direct perspective of improving the health of populations due to the consumption of more nutritious foods.

CONCLUSION
Agronomic biofortification with iron and zinc through foliar application at the grain filling stage does not affect yield indicators, industrial quality, and organic chemistry of oat grains.
Foliar application of zinc sulfate for biofortification increases zinc contents in oat caryopses, however, decreases iron contents. Foliar application of iron sulfate for biofortification increases iron contents in oat caryopses, however, decreases zinc contents.
The combined application of iron and zinc does not promote enrichment of oat grains through biofortification, indicating that this technology is feasible when using single sources.