The tomato (Solanum lycopersicum L.) is one of the most-consumed and demanded crops worldwide due to its great nutritional and antioxidant properties. This high demand makes it one of the most important crops at the social and economic levels. In agreement with the statistics from the Food and Agriculture Organization [FAO] (2020), more than 180 million tons of tomato have been grown in the past year worldwide, of which 24 million come from Europe. Spain is the second-most important producer in Europe, with an approximate production of 5 million tons per year.

Due to the increase in the world’s population since the industrial revolution, the performance of the crops has had to increase fast and exponentially to provide and answer to the demand for food; however, in the past few years, the production and quality of the harvest of various crops, among which we find tomatoes, have been greatly affected as a result of climate change. The high temperatures and periods of drought, which have become more intense and prolonged, have affected crop production (Wallace et al., 2003; Parry, 2019; Mutale-Joan et al., 2020). Aside from the challenge faced by agriculture for increasing production in an era of climate change, we also find another important challenge, such as the practice of sustainable agriculture that affects the environment the least. Thus, at present, new agronomic strategies are being designed and evaluated, which could help take on these challenges, starting with the acquisition of basic scientific knowledge about the physiological and metabolic processes of crops. As an alternative to conventional agronomic strategies, such as the application of synthetic inorganic fertilizers, which could have damaging effects on the environment, other more ecological alternatives are being adopted (Foley et al., 2011; Tilman et al., 2002; Mutale-Joan et al., 2020). Among these new agronomic strategies, we find the use of biostimulants (Lucini et al., 2015). It has been observed that the use of these products significantly improves the performance of crops, as they have beneficial effects on the physiological processes of plants, such as the absorption of water and nutrients, among others (Mutale-Joan et al., 2020).

Biostimulants are composed of bioactive compounds such as humic acids, hydrolyzed proteins, seaweed and microorganism extracts, among others (Rouphael et al., 2018). The hydrolyzed proteins and seaweed extracts are a rich source of amino acids, and these compounds tend to be added freely to enrich the biostimulant products, as they have very beneficial effects on the crops due to their role in the synthesis of proteins, vitamins, nucleotides and alkaloids, aside from having effects as elicitors (Khan et al., 2019). Some of the most important effects described in the past few years are: (i) they palliate the negative impact of certain environmental stresses such as drought (L-methionine in bitter gourd, Momordica charantia L.; Akram et al., 2020), salinity (mixture of amino acids in tomato plants; Tantawy et al., 2009), Cd toxicity (Glutamate in rice; Jiang et al., 2020), (ii) they induce hormonal responses and improve the absorption of nitrogen (mixture of amino acids and polypeptides in tomato, corn and peas; Colla et al., 2014), (iii) they regulate the antioxidant metabolism (Glutamate, Cysteine, Phenylalanine, Glycine in soy Teixeira et al., 2017).

Despite the fact that as of today no doubts exist about the positive effects of the application of amino acids, as mentioned above, little is known about the role played by each of these amino acids on the physiological and metabolic processes of plants. On many occasions, the biostimulant products are formulated from the chemical hydrolysis of raw materials rich in amino acids (residues from the agro-food industry), but there is a lack of knowledge on their amino acid profile or what specific function they play in crops. Also, the application of biostimulant products rich in amino acids can vary depending on the species, cultivar, climate, dose used, origin and application time (Parađiković et al., 2019). Therefore, to create highly efficient biostimulant products, it is indispensable to understand what role the different amino acids play on the physiological and metabolic processes of crops, and the possible antagonistic, neutral and synergistic effects between them.

Amino acids such as Glutamic acid, Aspartic acid and Alanine are three of the amino acids used in the formulation of biostimulant products (Colla et al., 2015). These are added in their free form or as part of the raw materials from hydrolyzed proteins. Glutamic acid is important in the metabolism of nitrogen, as it intervenes in the assimilation of nitrogen in plants and in the reactions of amino transferases (Lea and Ireland, 1999; Cao et al., 2010). This amino acid, aside from its intrinsic value as an amino acid itself, is the precursor of other amino acids such as Aspartic acid, Serine, Alanine, Lysine and Proline, among others. Alanine is synthesized from Glutamic acid, and its transamination with oxoglutarate produces glutamate and pyruvate, a reversible reaction, granting this amino acid a dual function between carbon and nitrogen metabolism (Kendziorek et al., 2012). Lastly, this amino acid is linked with the synthesis of chlorophylls and photosynthetic activity (Sánchez-Pale, 2017). Aspartic acid is obtained from a transamination reaction between glutamate and oxaloacetate in plants, and is metabolized to produce the amino acids lysine, threonine, methionine, and isoleucine, in a series of reactions known as the Aspartic acid metabolic pathway. The application of these three amino acids to plants helps them to endure adverse environmental conditions such as drought, salinity and heavy metal toxicity (Rai, 2002). However, up to the present, the effects of the individual application of these amino acids on the physiology and the primary metabolism of plants, or the antagonistic or synergistic effects that could result from the mixture of amino acids, are unknown. This knowledge could be essential when designing biostimulants that contain amino acids, or even when selecting the raw materials for which the profile of amino acids is better adapted to the requirements of a specific crop. For this, the objectives of the study were to understand the effects of the application of the amino acids Glutamic acid, Aspartic acid and Alanine, applied individually or as a mixture, on the physiological and metabolic processes, and the nutritional state of tomato plants of the ‘Optima’ variety; and to discover if these changes stimulate the vegetative growth of the plants. To characterize the metabolic state of the plants, the ¹H-NMR omics technique was utilized, as it allows analyzing a great quantity of primary metabolites in a fast and simple manner.

Amino acids in plants are involved in primary and secondary metabolism, and participate in a wide range of cellular enzymatic reactions as constituents of different enzymes such as aminotransferases, dehydrogenases, lyases and decarboxylases. Therefore, they can have an influence on diverse phenological and physiological processes such as vegetative development of the plants, seed germination, fruit maturation, signaling and activation of defense systems against abiotic and biotic stresses, osmotic adjustment, deactivation of reactive oxygen species, and as a reserve source of nitrogen, among others (Teixeira et al., 2017). These important functions of the AAs in plants lead to the application of biostimulant products containing AAs, either added in their free form or added as botanical and seaweed extracts and hydrolyzed proteins, becoming a common practice in agriculture (Khan et al., 2019). Nevertheless, in order for the biostimulant products to become more efficient in the improvement of the agronomic performance of the crops, and so that they can be specifically formulated depending on the crop varieties, it necessary to understand the function of each amino acid in each crop, to identify antagonistic, synergistic or neutral effects among the amino acid mixtures, and to determine the doses and the best time of application.

In our experiment, it was observed that the foliar application of AAs can alter the vegetative growth of the plants. However, this effect was dependent on the AAs utilized for the preparation of the products applied. According to the shoot dry weight data (Figure 1), the best treatment found from all those applied was the Asp + Glu mixture. And, taking account that the application of these two AAs individually did not affect this parameter or even decreased it, as in the Glu treatment, relative to the control plants, it was concluded that there was a synergistic effect these two AAs. The opposite behavior was found for the plants to which L-Ala was applied individually or as part of a mixture. In this case, it became apparent that L-Ala was harmful to tomato plants when applied at a concentration of 15 mM. In addition, when mixed with Asp + Glu, its negative effect was enhanced, as observed in the decrease in dry weight of the shoot as well as the appearance of damage to the leaves (Figure 1). Some synergic effects between AAs has been previously reported. In an experiment conducted with this same tomato variety under saline conditions (Alfosea-Simón et al., 2020), it was observed that the Pro + Glu and Met + Trp mixtures were more efficient in palliating the effects of salinity as compared to the individual application of these AAs. Despite the positive effects of the application of Glutamic or Aspartic acids being observed on several occasions, such as in Kimchi plants grown in low temperatures (Lee et al., 2017) or rice under an excess of Cd (Rizwan et al., 2017), the positive effects of these two AAs applied simultaneously had never been previously described. On the other hand, plants from Asp + Glu + Ala applications received three times more amino acid content compared to those from individual Asp, Glu and Ala spraying, and 1.8 time more relative to Asp + Glu treatment. Thus, the excess of aminoacid content in the spraying Asp + Glu + Ala solution could have damaged the plants. In several cases, for instance, in Arabidopsis plants, it has been shown that amino acids as low as 100 μM applied via root caused damage in the root growth (Ravelo-Ortega et al., 2021). Thus, low concentrations of amino acids in some plant species can have un-expected effects.

One of the reasons why the plants treated with Asp + Glu had a greater growth can be found in the results from the physiological study. In this study (Figure 8), it was observed that one of the common responses of the plants when treated with any AAs treatment was the increase in stomatal conductance (g_s). However, this generalized response was not observed in the A_CO2. The only plants that had an increase in the CO₂ assimilation rate, in parallel to the opening of stomata, were those from the Asp + Glu treatment. Thus, the simultaneous effect of Asp + Glu could have enhanced the biochemical reactions of photosynthesis. This has already been reported by Lee et al. (2017) with the foliar application of Glutamic acid, with positive effects observed on the photosynthetic rate and stomatal conductance. In an experiment carried out in rice plants under conditions of cadmium stress, it was determined that the foliar application of Asp also improved the photosynthetic rate and stomatal conductance (Rizwan et al., 2017). In our experiment in tomato plants, the simultaneous application of Asp + Glu was necessary to enhance A_CO2. In addition, in these plants, A_CO2 and g_s had similar increases (as a percentage), relative to control plants. Thus, the WUE was maintained similar to the control plants, resulting in the maintenance of a good balance between the CO₂ captured and water lost by the plants. On the other hand, the application of L-Ala seems to have altered the non-stomatal factors of photosynthesis (biochemical factors), as indicated by the decrease in the assimilation rate of CO₂ in plants treated with L-Ala and Asp + Glu + Ala in parallel to the decrease in the Ci/Ca ratio (Farquhar and Sharkey, 1982). In plant cell culture studies, evidence has been shown about the harmful effects of L-Alanine. Chen et al. (2006) in cell cultures of V. labrusca and V. vinifera observed that the application of L-Ala to the growth media caused the death of cells due to the activation of Phenylalanine ammonium lyase, 4-hydroxylase cinnamic acid, the expression of the stilbene synthase gene, and the accumulation of stilbenes. Nevertheless, the applications of L-Ala to crops has barely been studied, except in the Plumbago indica crop, where the application of Alanine (5mM) combined with chitosan (150 mg L^–1) improved its production (Jaisi and Panichayupakaranant, 2020).

The ionomics study indicated that the AAs applied to the tomato plants of the variety ‘Optima’ did not induce changes in the absorption of nutrients relative to control plants, as the concentrations of macronutrients and micronutrients in plants from any of the treatments were within the normal range for this crop (Watanabe et al., 2016). However, some foliar treatments with amino acids are able to increase or decrease the absorption of diverse macronutrients and micronutrients as indicated by Sainju et al. (2003). The data from our experiment showed that the plants treated with L-Ala (L-Ala and Asp + Glu + Ala) had an altered nutritional status as in these plants it was increased the K, P and N concentration. This could be due to a concentration effect, as the dry weight of the shoot decreased. However, as this effect was not observed in the concentration of Ca and Mg, it should not be discarded that the L-Ala amino acid could have influenced the absorption and transport of the nutrients in plants. Nevertheless, the increase in K, P and N with respect to Ca and Mg could have created nutritional imbalances which may have caused a decrease in the growth of the shoots. Another possible reason for the highest leaf N concentration observed in the Asp + Glu + Ala treated plants could be due to the high N concentration (0.75 mg N L^–1) of the solution sprayed relative to the rest of treatments.

In the metabolomics study, it was observed that the most significant change observed after the application of the AAs Aspartic acid, Glutamic acid and their Asp + Glu mixture, occurred in the concentration of Proline. The Asp and Glu treatments, and their Asp + Glu mixture increased the concentration of this amino acid, and this increase was greater with the Asp + Glu mixture. Proline is an AA with multiple functions. It has the ability to act as an osmoprotector, and plays a role in osmotic adjustment, deactivation of free oxygen radicals, regulation of the absorption of nutrients, and can also induce the CO₂ assimilation rate (Pervaiz et al., 2019). These functions are especially important when the plants suffer adverse environmental conditions. In the Mediterranean area, the typical greenhouse conditions can make temperature be a limiting factor in tomato cultivation. Therefore, the increase of proline in these plants could have exerted a protective role that favored the increase in the CO₂ assimilation rate and the plant’s growth. In addition, glutamate, aside from being a precursor of proline, is also related to the production of glutathione and arginine. These two compounds are involved in oxidative metabolism, directly as glutathione or indirectly as arginine, as they activate PAL activity (Ben-Rejeb et al., 2014; Zheng et al., 2011). On the contrary, the application of L-Ala and Asp + Glu + Ala decreased the concentration of proline, and this response coincided with a decrease of A_CO2 and decreased shoot growth. These results suggest that Asp + Glu improved the physiological and morphological parameters of the plants through the synthesis of proline, while Ala altered the metabolism of the plants by reducing the concentration of proline, and therefore reducing their growth.

In plants, the AAs Glutamate and Aspartate are precursors of up to eleven AAs. The reaction of Glutamate + Oxaloacetate produces Aspartate + α-ketoglutarate. This reaction is reversible, so that in the biosynthesis pathways, the AAs from glutamate (such as Alanine, Valine, Leucine, Glutamine, Proline and Arginine) are connected with those from Aspartate (such as Asparagine, Methionine, Threonine, Isoleucine and Lysine) (Forde and Lea, 2007). The application of Glutamate and Aspartate and their mixture did not increase the foliar concentration of these AAs, but were metabolized to increase the concentration of proline. Instead, the application of L-Ala individually or in a mixture increased the concentration of L-Ala in leaves. This AA was not metabolized, so that the increase in concentration could have caused damages to the cells, as observed in Chen et al. (2006) in cell cultures. Another of the alterations produced by the application of L-Ala was the decrease in Tyrosine. This AA is the precursor of secondary metabolites derived from the p-Coumaroyl-CoA pathway, which produces compounds such as coumarins, flavonoids, isoflavonoids, stilbenes, aurones, cutin, suberin, sporopollenin, catechins, proanthocyanidins, lignans, lignins, phenylpropenes, acylated polyamines and many other alkaloid derivatives. These compounds are related with the antioxidant protection capacity of the cells and with the mechanisms of protection of plants against plagues and diseases.

In the metabolomics study it was also observed that some compounds from the pentose phosphate pathway, such as fructose, sucrose, glucose and formate, as well as the tricarboxylic acid pathway (citrate, malate and fumarate), were affected. For the individual application of Asp, Glu and L-Ala and their mixture Asp + Glu, different responses were observed in the pentose phosphate pathway and the tricarboxylic acid one, which demonstrate that the mix of amino acids has a different effect than the AA applied individually. The mixtures that contained L-Ala produced a decrease in the concentration of fructose, sucrose and glucose, malate and citrate. This could suggest that the L-Ala applied at a concentration of 15mM produces alterations in the metabolism of C such as in A_CO2, which could have caused a decrease in the growth of the shoot. Therefore, the causes of L-Ala toxicity in tomato plants should be studied further, using as the report from Chen et al. (2006) a reference, where it was observed that AAs can activate the enzymes Phenylalanine ammonium lyase, 4-hydroxylase cinnamic acid, the expression of the stilbene synthase gene, and the accumulation of stilbenes. In the case of Asp, Glu, and their mixture, it was observed that while the mixture maintained the concentration of glucose at similar values as those of the control, the individual application decreased its concentration. This could indicate that the simultaneous application of Asp + Glu improved the capture and assimilation of CO₂ in the plants, improving the growth of these plants, where proline could have played an important role in increasing the performance of the photosynthetic system. For the tricarboxylic acid pathway, it was observed that the application of this Asp + Glu treatment induced a reduction in the concentration of citrate, and an increase in the concentration of fumarate, which could have modulated the A_CO2 and g_s regulation in plants under this treatment. Some studies have revealed a negative correlation between the concentrations of fumarate and gas exchange through the opening of stomata (Nunes-Nesi et al., 2007; Araújo et al., 2012).

Another of the responses that differentiated the treatments that contained L-Ala with those that did not have this AA, was observed in the concentrations of GABA and Trigonelline. The GABA increased while the Trigonelline decreased with respect to the control, and this response was greater when L-Ala was applied with the Asp + Glu mixture. GABA is a non-protein amino acid with multiple functions in plants, playing an important role in metabolic processes such as signaling, interconnection between Carbon and Nitrogen metabolisms and the tolerance to different stresses such as low light intensity, salinity, and lack of nitrogen, drought or temperature (Ramos-Ruiz et al., 2019). The increase in GABA observed in plants treated with L-Ala could be the result of some type of stress produced by this amino acid, unchaining a protection response related with GABA. In these plants, this response was not enough to counter the stress created, and therefore, led to a decrease in growth. The concentration of Trigonelline also increases in the plant as a response to different stresses, mainly salinity, drought or UV light (Cho et al., 1999; Cho et al., 2003; Willmon et al., 2017). However, the application of L-Ala in this experiment decreased its concentration. It is important to point out that the increased in GABA and the decrease of Trigonelline are found in plants suffering some abiotic stress, so that these metabolites could be used as biomarkers to detect the possible negative effects of these AAs in tomato plants. However, more research is needed to verify this hypothesis.

The different changes observed in the metabolic processes of the plants utilized in the present research could be due that the AAs, aside from having physiological functions, may also be involved in the metabolism of plants in signaling processes (Häusler et al., 2014). It has been described that plants have glutamate receptors (GLRs) that could be activated not only by glutamate, but also by other AAs such as Serine, Alanine, Methionine, Tryptophan, Phenylalanine, Leucine, Asparagine, Threonine, Cysteine, Glycine, Tyrosine, and Peptides (Vincill et al., 2012; Forde and Roberts, 2014). The activation of these receptors could unchain a series of signaling mechanisms in processes related to the absorption of nitrogen by the roots (Miller et al., 2008), growth and architecture of the roots (Weiland et al., 2016), antioxidant metabolism (Hildebrandt et al., 2015; Weiland et al., 2016), regulation of transcription factors (Santi et al., 2017), regulation of stomas and photosynthesis, and plant defense (Weiland et al., 2016), as well as balances between C and N metabolism (Kang and Turano, 2003; Price et al., 2012).

In this experiment, it was observed that the application of AA to tomato plants could be beneficial for increasing their vegetative development, but this application should be first optimized. To increase the efficiency of biostimulant products in agriculture, much research is needed that may reveal: i) the effects of the AAs on physiological and metabolic processes of plants, either applied individually or as a mixture; in this experiment it was observed that the combined application of the AAs (in this case Asp and Glu) had better effects that their individual application, and ii) what dose needs to be applied; in this experiment, it was observed that the application of 15 mM of Alanine is toxic to tomato plants, and it is not reversed by the simultaneous application of the Asp + Glu + Ala mixture. The application of AAs to plants resulted in changes in their ionomic, physiological and metabolomic profiles; however, as these compounds have many different functions in plants, a systems biology approach that integrates metabolomics, proteomic, genomic and hormonal studies to delve into these changes, is necessary.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

FG-S and EZ-G conceived and designed the research. MA-S, SS-G, IS, and VL conducted the experiments and collected the data in the field and in the lab. JC-Z and JM-N analyzed the data and did tables and figures. FG-S and MA-S wrote the manuscript. SS-G and EZ-G commented and revised the manuscript. All authors contributed to the article and approved the submitted version.

EZ-G was employed by the company Atlantica Agricola. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.