Foliar Application of Nitrogen Affects Metabolism and Productivity of Soybean

Soybean is one of the world’s most economically important crops and several factors can affect the productivity of this culture. Among these factors is the supply of needed nitrogen, especially in the reproductive stage, as it acts in photosynthetic activity and in grain filling. In view of this, the objective of our work was to evaluate the effect of foliar application of nitrogen in different reproductive stages in soybean culture. Two sources of nitrogen were used: conventional urea and urea-formaldehyde/triazone, both applied in reproductive stages R2, R3, R4, or R5, as well as a control treatment without foliar application of nitrogen. Plants submitted to foliar application of urea-formaldehyde/triazone showed an increase in nitrogen metabolism (percentage of nitrogen derived from the atmosphere [Ndfa] and nitrate reductase activity [NR]), an increase in peroxidase (POD), and the consequent reduction in hydrogen peroxide (H2O2) in all stages of application of this treatment. When urea-formaldehyde/triazone was applied in R4, it resulted in a 7% increase in yield. The application of conventional urea in reproductive stages R4 and R5 increased nitrogen metabolism and resulted in an increase in yield by 4%. However, conventional urea reduced yield when applied in stages R2 and R3. The use of low doses of foliar nitrogen in stages R4 and R5, increased nitrogen metabolism in soybean plants. The timing of the application has a direct impact on the results with the slow-release nitrogen (urea formaldehyde /triazone) showing better results when applied in stage R4 and better results for conventional urea in R5.

development of chlorosis due to the reduction of chlorophyll (Taiz et al., 2017). In soybean culture, N deficiency in the vegetative phase is related to chlorosis and the reduction in the size of leaflets (Borket et al., 1994;Prado et al., 2010). Several studies have been carried out with the application of different N sources in soybean crops (Hungary et al., 2006;Mendes et al., 2008;Marcon et al., 2017;Moreno et al., 2018;Pierozan Junior et al., 2020). However, there are still few studies related to N fertilizers based on urea-formaldehyde/triazone in soybeans, especially at different stages of plant development. Urea-formaldehyde/triazone fertilizers, classified as chemically modified because they have properties of gradual release of nitrogen (Clapp & Parham, 1991;Widders, 1991;Clapp, 2001;Bondada et al., 2001), provide greater efficiency in the use of applied N and are the most promising in an attempt to maximize crop yields. Cassim et al. (2020) demonstrated that the foliar application of nitrogen in stage R 3 is effective in parameters such as productivity in soybean crop. However, we believe that different responses can be obtained from the application of N at different phenological stages, especially related to the reproductive period, a period with high nitrogen demand in the soybean crop.
Thus, the objective of this study was to evaluate the influence of foliar application of nitrogen based on urea-formaldehyde/triazone and conventional urea on physiological, biochemical, and phenological variables, as well as, grain productivity in soybean culture.

Method
The experiment was conducted in the experimental field of the Cooperativa Agropecuária do Alto Paranaíba (COOPADAP), located in the state of Minas Gerais, in the Alto Paranaíba region, municipality of Rio Paranaíba. This location, according to Köppen classification, has a tropical climate of altitude (Cwa), with average annual precipitation around 1540 mm and an average annual temperature of approximately 20 ºC (Peel, 2007).
Soil analysis was carried out before sowing and, based on this analysis, fertilization was calculated. According to the soil analysis (Table 1), it was not necessary to apply limestone to correct the soil. The maintenance fertilizer used was composed of 31.2 kg ha -1 of N, 117 kg ha -1 of P, 39 kg ha -1 of K, 12.7 kg ha -1 of Ca, 16.6 kg ha -1 of S, 0.8 kg ha -1 of B, and 0.2 kg ha -1 of Zn.    Note. P-Mel and K + : Mehich 1 extractor; Ca + , Mg + and Al + : KCl 1 mol L -1 extractor; P-rem: Remaining phosphorus-phosphorus concentration of the equilibrium solution after stirring 1 hour with 10 mmol L -1 CaCl 2 solution containing 60g of P (1:10).
Sowing was carried out in October 2017 using the cultivar TEC 7849 IPRO with a spacing of 0.60 m between rows to maintain a population density of 240,000 plants ha -1 . Each plot was composed of four rows, seven meters in length, with 0.45 m between rows, totaling an area of 12.6 m 2 . The area of each plot was constituted by the central lines, discarding 0.5 m at each end of the plot.
For weed control, the herbicide glyphosate was applied in stage V 3 (three nodes on the main stem) [650 grams of active ingredient (gai) at a rate of 2.1 kg of commercial product (cp) ha -1 ]. Insect control was performed with teflubenzuron [150 gai L -1 at a rate of 0.30 kg cp ha -1 ] in stage V 5 (five nodes on the main stem); chlorfenapyr [240 gai L -1 at the rate of 0.8 L cp ha -1 ] at stage R 1/2 (beginning of flowering), and imidacloprid and bifenthrin [250 gai L -1 , 50 gai L -1 , respectively, at the rate of 0.4 L cp ha -1 ] applied in R 3 (start of pod development) and R 5.1 (start of grain formation and filling).
The experiment was designed with two N sources and four time of application with an additional treatment (control-without N), with eight replications (Table 2). Note. * Applied Dose: 2.5 kg Nitrogen per hectare.
Foliar applications were carried out with a pack sprayer pressurized with CO 2 at 2 bar. For all foliar applications, the spray volume of 200 L ha -1 was used.
The urea-formaldehyde/triazone fertilizer (N Hexion) was a 28% N fertilizer composed of urea, intermediate-release methylene-urea compounds, and triazone; the latter two responsible for the gradual release of N. Slow-release compounds accounted for 70% of the composition with a solids content of 73%. In comparison, the conventional urea used had 45% N in its composition.

Biochemical Analyzes
The antioxidant metabolism (lipid peroxidation, hydrogen peroxide, catalase enzymes, superoxide dismutase, and peroxidase) was evaluated. Additionally, nitrogen metabolism was assessed using biological fixation and the enzyme nitrate reductase. For all these analyses, completely expanded leaves, collected from the middle third of five plants, were used for each repetition.
(1) Antioxidant Metabolism The determination of the activity of antioxidant enzymes was carried out in the R 5.5 stage (pods with 76% to 100% of formed grains), at 109 days after sowing. The extraction of plant material was carried out according to the method of Kar and Mishra (1976). The determination of enzymatic activity was performed based on the protein content in the leaf with the protein being quantified using the method proposed by Bradford (1976). From this analysis, the activity of the enzyme superoxide dismutase (Beauchamp & Fridovich, 1971), catalase (Peixoto et al., 1999) and peroxidase (Teisseire & Guy, 2000) was determined. To further aid in the verification of oxidative stress in plants, the content of proline (Bates et al. 1973), hydrogen peroxide (Alexieva et al., 2001), and lipid peroxidation (Heath & Packer, 1968) was quantified.
(2) Nitrogen Metabolism The analysis of the enzyme nitrate reductase (NR) was carried out in R 5.5 stage. The method was performed according to the method proposed by Mulder et al. (1959). The biological fixation was determined based on the levels of ureides, amino acids, and nitrate present in the stem of the plants in stages R 3 , R 4 (pods fully developed) and R 5.5 . The material was dried in an oven with forced air circulation at a temperature of 65 °C and then crushed with the aid of an IKA mill. For the determination of nitrate (NO 3 -), total amino acids (Aa), and ureides, 200 mg of plant material was transferred to a Falcon tube and combined with 10mL of Milli-Q water. After this procedure, the material was incubated in a water bath at 45 °C for one hour and then centrifuged at 10,000 rpm for 15 minutes. The quantification of NO 3 was performed by the method proposed by Cataldo et al. (1975). For the determination of total amino acids, the protocol of Yemm and Cocking (1955) with adaptations described in Herridge (1984) was used. The ureides were determined based on the method proposed by Young and Conway jas.ccsenet.org Journal of Agricultural Science Vol. 13, No. 10;(1942. From the nitrate, amino acid, and ureide evaluations, the relative abundance of ureides (Rur) and the nitrogen derived from the atmosphere (Ndfa) was estimated according to equations proposed by Herridge and Peoples (1990).

Dry Mass of Stem, Leaves and Pods
Four plants were collected per repetition for evaluation in stage R 5.5 . Subsequently, these were placed in individualized paper bags, identified, and dried at 65°C until constant mass was achieved to determine the dry matter mass. Weighing was performed using a digital scale with an accuracy of 0.01 gram.

Productivity
The plants were collected from the two central rows of each plot. The water content of the grains was determined and the yield (mass per unit area) was calculated with the water content corrected to 13%. To measure the mass of the samples, a digital scale with an accuracy of 0.01 gram was used.

Statistical Analysis
The results obtained were analyzed for normality and homogeneity of variances and then the analyzed with One-Way Analysis of Variance with means being compared using Tukey's multiple comparisons test at a level of 5% significance.
A multivariate analysis was performed through Principal Component Analysis. All analyses were performed using the statistical software SAS 9.3 (SAS Institute, 2011).

Evaluations in R 3 Stage
Application of two sources of N in R 3 stage increased the total amino acids (Aa) in plants ( Figure 1A), in comparison to application in R 2 stage; however, there was no difference in comparison to the control. The application of Conventional Urea in the R 2 and R 3 stages reduced the ureides ( Figure 1B) in comparison to the control.
When observing the percentage of Rur and Ndfa, it is noted that urea-formaldehyde/triazone was more efficient compared to conventional urea when applied in the R 2 stage ( Figures 1D and 1E), providing an increase of 20% and 18% for the variables Rur and Ndfa, respectively.   % ( Figure 4E) igure 4F) comp hyde/triazone i ontent ( Figure   4D), POD (F vity ( Figure 4H

Discussion
It was observed that the application of urea-formaldehyde/triazone increased nitrogen metabolism in all stages of application (R 2 , R 3 , R 4 and R 5 ). As a result of increased nitrogen metabolism, higher leaf protein content was observed in stages R 2 and R 4 (Figures 4 and 6). These characteristics demonstrate the relationship between foliar application of low-dose nitrogen and signalling in plants. The concentration of nitrogen in the form of NH 4 + in plant tissue triggers the signalling role primarily because it induces the production of glutamate and glutamine (Coruzzi & Bush, 2001) which can signal various physiological processes in plants (Forde & Roberts, 2014;Weiland et al., 2015).
The increase in glutamate production in response to NH 4 + can play an important role in signalling in plants, as it is linked to physiological processes such as activation of permeate amino acids, calcium transporters, genes that are resistant to diseases and defend against stress, in addition to being involved in the activation of metabolism, transport, and growth genes (Kan et al., 2017). Glutamate also acts on the concentration of calcium that is propagated through phloem and intracellular channels (Toyota et al., 2018). According to Kang and Turano (2003), these signals can also alter the metabolism of carbon and nitrogen in plants. Therefore, the increase in nitrogen metabolism observed in our experiment may be a result of the indirect effect of the role of NH 4 + in plant signalling.
The maintenance of nitrogen and carbon metabolism must be strongly coordinated in plants in order to sustain plant growth, development, and productivity (Zheng, 2009). Plants with adequate nitrogen levels increase carbon metabolism, which can have an impact on increased productivity. The benefits of applying urea-formaldehyde/triazone were especially evident in the R 4 stageresulting in an increase in the protein content in leaves and a 7% increase in yield in comparison to the control ( Figures 4H and 5). There is a positive relationship between the concentration of protein in the leaves and yield ( Figure 5). Plants with a higher protein concentration may show higher yield (Salvagiotti et al., 2008). These characteristics appear to be related to the gradual release of nitrogen combined with the period that precedes one of the stages of higher nitrogen demand in soybean plants. Triazone is responsible for the slow release of nitrogen (Widders, 1991) allowing the signalling process to occur gradually. This provides an increase in nitrogen metabolism and delay in leaf senescence, causing the plant to have greater nutritional support for fruit. Additionally, the availability of N in the leaves is essential for extending the grain filling period. Furthermore, there is a linear relationship between leaf N concentration in R 5 and soybean productivity (Munier-Jolain et al., 1996;Shibles & Sundberg, 1998).
Understanding the periods of higher nitrogen demand in soybean plants is essential to ensure a positive effect of foliar fertilization of this nutrient. According to Câmara (2014), soybean has two biological fixation peaks; the first at R 1/2 at full bloom and the second at R 5.1 and R 5.2 in grain filling. According to the author, the biological fixation of nitrogen peaks occurs as a result of photosynthetic peaks that provide energy for functioning of the nodules.
The application of urea-formaldehyde/triazone was effective in increasing the antioxidant metabolism, with an increase in the activity of peroxidase enzyme and in protein content, and the consequent reduction in H 2 O 2 content (Figure 4), which can also assist in increasing yield due to reduced plant stress. This effect can also be related to the induction of glutamate production from NH 4 + signalling. This amino acid increases antioxidant metabolism in plants (Mittler 2002;Gill & Tuteja, 2010;Rejeb et al., 2014). The increase in the activity of antioxidant enzymes from glutamate was also observed by Teixeira et al. (2017Teixeira et al. ( , 2019. The application of conventional urea provided positive results only in R 4 and R 5 , in which it increased the metabolism of nitrogen and proteins, in addition to helping increase the antioxidant metabolism. This was reflected in the increase in yield by 4% compared to the control ( Figure 4H). The positive effect of urea in these two application phases can also be related to the signalling role provided by NH 4 + , which modulates nitrogen metabolism (Kang & Turano, 2003) and antioxidant metabolism leading to increased productivity. Conventional urea is rapidly absorbed by the leaves, so its application was only effective in periods close to the higher nitrogen demand in the soybean crop.
One of the first works that aimed to evaluate the effects of the application of conventional urea via leaves was carried out by Ashour and Thalooth (1983). These authors observed that the application of 1% urea in the R 6 stage of soybean had an impact on the increase of fruiting, oil and protein content, and on pod mass.
It was shown that application of conventional urea reduced productivity by 6% and 4% when applied in stages R 2 and R 3 , respectively, compared to control plants ( Figure H). These two stages do not correspond to higher nitrogen demand by plants and; therefore, this fact associated with the rapid absorption of conventional urea may have led to the toxic and negative effect of NH 4 + in plants. Due to the fact that these stages do not correspond to periods of higher nitrogen demand in plants (Câmara, 2014), the application may have led to a change in the carbon and nitrogen balance and the signalling process (Kang and Turano (2003), which leads to changes in the entire process of cellular metabolism, resulting in a negative impact on yield.
The use of low doses of nitrogen via foliar application in stages R 4 and R 5 , increases nitrogen metabolism in soybean plants. The timing of the application directly affects the results of the application. Slow-release Nitrogen (urea-formaldehyde/triazone) presents better results when applied in stage R 4 . On average, the use of urea-formaldehyde/triazone increased soybean yield by approximately 1,44% and 1,58% compared with control and conventional urea, respectively. The late application of urea-formaldehyde/triazone (R 5 ) did not differ statistically from conventional urea on soybean yield, showing no benefits from the gradual release of N in that phenological stage. However, in other N application stages (R 2 , R 3 and R 4 ), the urea-formaldehyde/triazone promotes a greater increase in productivity when compared to conventional urea, which allows greater flexibility in foliar application of N, via urea-formaldehyde/triazone, when compared to conventional urea. The results suggest that further studies should be carried out at the genetic level, aiming to evaluate the role of foliar application of NH 4 + , from a conventional source and gradual release. In this way, it will be possible to understand more clearly how this nutrient can signal changes in plants.