Changes in Root Architecture After Amino Acid Application in a Soybean Crop

Improving the root system favors better plant growth, since it promotes water and nutrient absorption, resulting in higher plant yield. In this respect, the use of products for this purpose has become promising. Applying amino acids has benefitted the root system of Arabidopsis and in some vegetables; however, little is known about their influence on soybean plants. As such, the aim of this study was to assess the effect of applying amino acids to seeds and leaves on the root architecture of soybean plants. Effects of amino acids such as glutamate, cysteine, glycine and phenylalanine on the main root length (MRL), total root length (TRL), projected area (PA), root volume (RV), number of secondary roots (NSR), secondary root length (SRL) and number of tertiary roots (NTR) were evaluated. All the amino acids studied improved root architecture. Seed-applied cysteine increased TRL by 55%, in relation to control. When applied on leaves, it raised TRL by 27% and MRL by 69%, compared to control. Applying glycine to seeds increased MRL by 54%, PA by 69%, RV by 96% and NTR by 119%, all in relation to control. Thus, amino acids enhanced the architecture of soybean roots. However, glutamate, glycine and phenylalanine produced better responses when applied to seeds, and cysteine, when applied to leaves. All of these changes may help roots absorb more water and nutrients, thereby raising crop yield.


Introduction
Plant growth depends on water and the nutrients absorbed from the soil, substrate or nutrient solution.However, they are commonly grown in areas at risk of water deficit, due to unstable rainfall (FAO, 2011), and in environments with low levels of some nutrients, including nitrogen, phosphorous, zinc and boron (IPNI, 2016;Prochnow et al., 2018).Thus, the characteristics of the root system and its architecture are important in determining crop development.
Root system architecture is defined as the spatial arrangement of its individual parts (Shahzad and Amtmann, 2017).As such, it is determined from a set of traits, particularly morphology, topology and root distribution (Lynch, 1995), which establish how efficiently plants use the resources of the crop environment (Shahzad & Amtmann, 2017).
In Brazil, Paula Neto (2013) demonstrated that the roots of coffee cultivars most efficient at absorbing phosphorous had a larger surface area, length, volume and tissue density.In China, Mi et al. (2010) observed that corn plants with deeper roots and vigorous lateral roots were more efficient at absorbing nitrogen in an intensive cropping system.Moreover, in a review, Li et al. (2016) found that a rise in efficient nutrient use by plants is related to better root system architecture.
architecture.The use of amino acids to treat seeds or in foliar applications is one of the techniques employed to improve root development.
Glutamate is one of the amino acids that causes the largest number of changes in root system architecture.In this case, the effects are related to its role as signaler, via GLR receptors, as demonstrated by Forde's review (2014).Moreover, glycine and cysteine have also exerted an effect on root system architecture; however, the mechanisms that control these effects are even more obscure in relation to glutamate (Teixeira, 2016).
Although amino acids cause changes in root growth, a large number of studies have been conducted with products containing a mixture of amino acids, making it difficult to understand the effect that each one has when applied separately.Furthermore, to the best of our knowledge, this is the first study that assesses the effect of amino acids applied separately in seed treatments or to the leaves of a soybean crop.
As such, the aim of the present study was to evaluate the effect of applying glutamate, cysteine, glycine and phenylalanine in seed or leaf treatments on the modulation of soybean root architecture.

Method
The experiment was carried out in the greenhouse of the Department of Plant Production of the "Luiz de Queiroz" Superior Agricultural School (Esalq/USP), in Piracicaba, São Paulo state (22º41′ S, 47º38′ W and 546 m of altitude).The study was conducted in 11 dm 3 vases, containing washed sand as substrate, using the NS 7901 RR cultivar (Glycine max L. Merrill).Ten seeds per vase were planted and thinned after emergence, leaving only three plants per vase.
The experiment, performed using a random block design, consisted of applying amino acids to seeds or leaves, using eight blocks per treatment (Table 1).The dose used of each amino acid was determined from previous experiments that tested several doses of amino acids and were carried out by the research group itself.Before treatment application, all the seeds were treated with fungicide and insecticide [fipronil (250 g L -1 ) + methyl thiophanate (225 g L -1 ) + pyraclostrobin (25 g L -1 )] at a dose of 1 mL kg -1 of seeds.Note. 1 The sources used correspond to the pure amino acids, with optical isomerism levogyrous (L-amino acid).
During the experiment, the vases were watered daily according to water needs (400 mL per vase).Nutrient solution was applied weekly, as proposed by Johnson et al. (1957).

Assessments
This analysis was carried out in stages V 4 and V 6 (25 and 45 DAS), where two plants from each repetition were sampled for computational analysis of roots using Winrhizo ® 4.1 software, and an Epson XL 10000 scanner.Analysis followed the procedures proposed by Bouma et al. (2000).A resolution of 600 dpi was used to obtain the digital images.The roots were placed (not overlapped) on an acrylic cube containing 1 dm 3 of water.
Analysis was conducted based on the grey tones of each of the pixels that make up the image.The program automatically establishes a grey tone value, from which each plant tissue can be identified.These data were used to obtain main root length (MRL, cm plant -1 ), total root length (TRL, cm plant -1 ), projected area (PA, cm 2 plant -1 ), root volume (RV, cm 3 plant -1 ), number of secondary roots (NSR), secondary root length (SRL, cm plant -1 ) and number of tertiary roots (NTR).

Statistical Analysis
The data obtained were assessed for normality and homogeneity using the Shapiro-Wilk and Levene tests, respectively, both at a 5% significance level.Analysis of variance was carried out and, when significant, the Duncan's multiple-range test test was applied at a 5% significance level.All the analyses were performed using SAS 9.3 software (SAS Institute, 2011).

Results
The use of glutamate, cysteine, phenylalanine and glycine promoted an increase in all the parameters related to root growth in stage V 4 (Figures 1 and 2).
The plant seeds treated with glutamate, cysteine, phenylalanine and glycine exhibited greater main root length (13, 15, 19 and 17%, respectively) compared to controls (Figure 1A).These treatments also raised root volume and number of tertiary roots to 100%, when compared to controls (Figures 1D and 1G).
Cysteine application stood out most in terms of root growth parameters.This treatment increased root length by 55% in relation to controls (Figure 1B).The use of cysteine in seed treatment also raised the number and length of secondary roots by 52% (Figures 1E and 1F).Increases of 29 and 39% were observed in the projected root area after cysteine and glutamate application, respectively, when compared to controls (Figure 1C).Moreover, glycine is also involved in one of the production routes of glycine betaine, a compound that helps minimize possible saline or temperature stress (Sakamoto & Murata, 2002;Ashraf & Foolad, 2007;Taiz et al., 2017).Glycine betaine can stabilize protein and enzyme structures and protect cell membrane integrity (Sakamoto & Murata, 2002), characteristics that can also benefit root development.
Cysteine and glycine have also been related to stress attenuation in plants (Azarakhsh et al., 2015;Teixeira et al., 2017).Glycine can help in the production of glyoxylate, an H 2 O 2 -reducing compound, thereby decreasing lipid peroxidation.Furthermore, glyoxylate produces NADPH and ATP, energy molecules used in a number of metabolic processes (Azarakhsh et al., 2015).These authors observed that the use of cysteine in seeds and leaves may increase the activity of antioxidant enzymes such as catalase and phenylalanine ammonia lyase and reduce lipid peroxidation.
In an experiment with soybean, the use of cysteine and glycine in seeds increased catalase activity and when applied to the leaves, these amino acids helped decrease lipid peroxidation (Teixeira et al., 2017).
Glutamate produced better results when applied in the seed treatment, with improvements in the projected area (Figure 1C) and root volume (Figure 3D).According to the literature, this amino acid is a signaler (Forde & Roberts, 2014;Weiland et al., 2015).In roots, glutamate inhibits main root growth and increases lateral root development, which may augment root volume and area.Similar characteristics were observed in Arabidopsis after exogenous application of glutamate.In this case, at concentrations between 1 and 50 µM, main root growth declined when compared to controls (Walch-Liu & Forde, 2007).This decrease is due to cell division inhibition of the apical meristem of the main root, and since the other regions of the root are not sensitive to glutamate, secondary root growth increases (Forde, 2014).The authors reported that these effects are only observed in L-glutamate (the form used here) and not in the isomer D-glutamate (Walch-Liu & Forde, 2007).All these responses occurred because plants have glutamate receptors (GLRs) that can activate a series of physiological processes.These receptors can also be activated by other amino acids such as glycine and cysteine (Vincill et al., 2012;Forde & Roberts, 2014).
The use of phenylalanine was also significant in seed treatment, with a rise in the projected area and volume of roots (Figures 3C and 3D).The effect of this amino acid seems to be more connected to pathways of secondary metabolism, such as flavonoid and lignin production (Taiz et al., 2017).
In addition to favoring nutrient absorption, the increased absorption area of roots caused by the amino acids seems to promote a rise in contact area in order for plant nodulation and greater nutrient absorption to occur (Li, Zeng & Liao, 2016).This characteristic is extremely favorable, since 50-60% of the nitrogen required by the soybean crop is obtained via biological fixation (Salvagiotti et al., 2008).Amino acids can also increase transcription of genes involved in nitrate, ammonium, phosphorous, magnesium and iron transport (Santi et al., 2017).Teixeira et al. (2018) demonstrated that applying glycine, cysteine and glutamate to seeds or leaves raises nitrate and amino acid accumulation in leaves.These characteristics are essential to plants, since they guarantee better growth and increase photosynthetic area, which leads to higher production of photoassimilates that can be used during the grain-filling phase, resulting in greater yield (Board & Modale, 2005;Soares et al., 2016;Teixeira et al., 2018).

Conclusions
Applying amino acids to seeds or leaves changes the architecture of soybean roots, thereby influencing important parameters such as root volume, projected area and number of secondary and tertiary roots.Glutamate, glycine and phenylalanine induced better responses when applied to seeds, and cysteine when applied to leaves.All of these changes may help increase water and nutrient absorption, as well as crop yields.

Table 1 .
Concentration of different amino acids applied only on seed (ST) and foliar application (FA) at V 4 stage