The Role of Metabolites in Abiotic and Biotic Stress Tolerance in Legumes

Abstract

Population growth in the world has made the production of food to feed this population a major challenge. The Food and Agriculture Organization (FAO) estimates that to meet human food needs by 2050, crop productivity must double. Legumes family plays an important role in food security, poverty alleviation, and sustainability. It is determined that plant development and stress responses, as well as processes such as growth, the integrity of cells, energy storing, cellular signaling, formation of membrane and scaffolding, cellular replenishing, and whole-plant resource assignment, are managed by plant metabolites. One of the important parts of early stress responses concerns changes in plant metabolism, which includes the accumulation of antioxidants for the protection of cellular components from oxidative damage and the accumulation of compatible solutes that retain water in the cell. Other components, such as GABA and amino acids, including threonine, leucine, methionine, lysine, valine, and isoleucine, were usually induced during environmental stress conditions. In general, it was determined that plants containing various metabolites alter their physiology to adapt to various situations, such as stress. Important metabolites that play a role in tolerance to stress in legumes can help breeding programs in developing stress-tolerant cultivars to increase food security in the world.

1. Introduction

Concerning the increasing population of the world, the production of food to feed this population is a major challenge. The Food and Agriculture Organization (FAO) estimates that to meet human food needs by 2050, crop productivity must increase. However, because of climate change, the increase in production is not promising. In many countries, it is expected that the productivity of many crops will decrease by roughly 50% by 2080 [1, 2]. Changing global climatic conditions has a negative impact on food security and human health in two ways: 1- It directly affects the amount of food and indirectly affects the spread of disease and pests, water availability, and environmental pollution and 2- It changes CO₂ concentration that causes a change in plant biomass [1]. The third largest flowering family is the legume family, which is classified into five subfamilies, including Detarioideae, Cercidoideae, Dialioideae, Papilionoideae, Caesalpinioideae, and Duparquetioideae [2]. Due to their symbiotic relationship with nitrogen-fixing bacteria, the legume family is able to fix nitrogen, so they provide nitrogen for subsequent crops and reduce future nitrogen fertilizer usage [3, 4]. On the other hand, legumes are a family with a rich source of plant proteins, fibers, carbohydrates, dietary fibers, vitamins, and minerals, and they are low in fat [3, 4, 5, 6]. Hence, seeds of the legume family have an important role in the human diet as they are rich in proteins, minerals, vitamins, and bioactive compounds [7]. Moreover, the legumes family plays an important role in food security, poverty alleviation, and sustainability [8]. Legumes provide 27% of global primary crop production and 33% of protein requirements and take the second place in food production after cereals [9]. In addition to primary metabolites, legumes are rich in secondary metabolites such as isoflavones, triterpenoid saponins, and inositol phosphates [5, 10, 11]. It is documented that stress tolerance in the legume family can be induced through the production of secondary metabolites such as phenolic, flavonoids, various alkaloids, and carotenoids [12].

Therefore, investigating the metabolites affecting the tolerance of biotic and abiotic stress in legumes helps researchers in breeding legumes to produce more yield under different stresses. In this part, with the aim of identifying important metabolites that play a role in tolerance to stresses in legumes, we reviewed metabolomics studies that focused on important abiotic stressors of legumes.

2. Drought stress

It is determined that water stress modifies the transcriptomic and metabolic profiles in plants. During drought, processes, such as the accumulation of compatible solutes and the production of hormones and antioxidants, occur for the regulation of the physiological processes. Also, the plant’s ability to tolerate water stress is associated with soluble compounds such as polyamines, sucrose, trehalose, amino acids, polyhydric alcohols, mannose, and oligosaccharides. Therefore, the regulation of these compounds can be used for the improvement of drought tolerance in legume plants [13, 14, 15]. Several studies reported that changes in some key primary and secondary metabolites, such as sugars, amino acids, polyamines, phytohormones, secondary metabolites, antioxidants, and TCA cycle metabolites, are the response and adaptation mechanism of legumes to water-deficient conditions [16]. In a study by Goufo and colleagues, when cowpea (Vigna unguiculata L. Walp.) was exposed to water deficiency, 41 primary metabolites such as 5 sugars, 4 polyols, 24 amino acids, and 8 organic acids were detected. In addition, secondary metabolites such as Quercetin 3-O-6″-malonylglycoside, kaempferol 3-O-diglycoside, quercetin, and galactinol were identified [14]. Drought stress increased soybean plants’ leucine, isoleucine, glycine, and proline. It seems that leucine, isoleucine, proline, and glycine are indicative of the potential for an advanced tolerance reaction under drought stress [17]. Cowpea plants responded to drought with an increase in quercetin 3-O-6′′-malonylglycoside and quercetin as secondary metabolites, as well as phenylalanine and ornithine as primary metabolites in leaves. However, compounds such as glutamine, glycerate, γ-aminobutyrate, and kaempferol derivatives decreased [14]. Metabolism profile in cowpea plants during drought stress indicated that compounds, such as kaempferol, amino acid derivatives, glycine, glutamine, glutamate, aspartate, asparagine, serine, alanine, threonine, sucrose, glucose, and fructose, had decreased but raffinose, catechin derivatives, unidentified phenolics, myricetin, trehalose, phenolic acids, polyols, and quercetin were induced [14].

A study of metabolomics under water stress time in the leaves and roots of cowpeas showed that leaves are more suitable than roots for recording metabolism changes [14]. It was necessary to identify if these metabolomics compounds were controlled by genetics through a distinct pathway that could help improve drought tolerance. In the leaves of both cultivars, most pathways are regulated consistently, but this is not the case in the roots [14]. Other studies show that raffinose family oligosaccharides (RFOs) [18, 19, 20] and myoinositol [21] have an important role in drought resistance. This can probably be attributed to an increase in branched-chain amino acids such as valine, leucine, and isoleucine connecting with the substrates storage for critical metabolic pathways. It is documented that drought tolerance in plants is enhanced by the overaccumulation of phenolic and other secondary metabolites [22, 23]. A study of altered metabolomics in model and forage legumes in response to drought stress by Sanchez et al. [13] showed a significant increase in organic acids, sugars, and polyols. Organic acids, including TCA cycle intermediate succinic and malic acid, along with fructose, glucose-galactose, maltose, arabitol, ononitol, and galactitol as sugars and polyols, are accumulated in response to water deficit, while glutamic, aspartic, and phosphoric acid dropped during drought [13]. The change patterns in amino acids were different. Some amino acids such as proline, leucine, and isoleucine increased, while serine, glycine, and threonine decreased. Other amino acids, such as asparagine, lysine, and valine, showed no significant changes [13]. Previous studies indicated that the accumulation of small molecules, such as compatible solutes, played an important role in drought tolerance. So, drought tolerance was enhanced in transgenic plants with a high potential for compatible solutes [24, 25, 26]. In drought stress, the observed metabolic responses may reflect the basic metabolic adaptation. Thus, conserved metabolic responses indicate not only an osmotic adjustment but also a wide range of processes including protection of membranes and proteins, radical scavenging, signaling, or buildup of reusable nitrogen and carbon repositories [27, 28].

Among the global metabolic rearrangements found in primary metabolism under water stress and after recovery from dehydration, proline increases in lotus genotypes in response to drought, reflecting the critical physiological role of proline in drought stress [13, 29]. The metabolism response of two cultivars of cowpea (Pinhel and Fradel) to drought is different. In response to drought, first primary and secondary metabolites accumulated in the root of the Pinhel cultivar, but as water stress persisted, primary metabolites decreased to roughly the control level whereas some secondary metabolites increased. In the meantime, primary and secondary metabolites initially decreased in Fradel cultivar roots, but subsequently, primary metabolites increased [14]. Using cowpea as a model species, researchers found that during drought stress plants produce organic solutes, which act as a compatible osmolyte and store them in the cytosol for the maintenance of turgor and osmotic adjustment. It is proposed that metabolite-based markers could improve drought tolerance [30]. Cowpea is a drought-tolerant legume that, during drought stress, limits water deficiency through drought avoidance strategies including stomatal closure, paraheliotropism, and moisture conservation through osmolyte accumulation [14].

There is a hypothesis that better grain yield and survival during drought and the subsequent recovery depend on organs, developmental stage, and cultivar-specific similarities in metabolic reaction. During water stress in cowpeas, nearly 88 metabolites were identified. Modifications in the metabolome profile were more intense during long periods of stress, regardless of developmental stage [14]. The most important reactions to water stress, including combinations such as quercetin 3O-6′′-malonylglycoside, kaempferol 3-O-diglycoside, quercetin, galactinol, and proline, were identified in response to water stress [14]. A study of metabolomics in the roots and leaves of cowpeas during water stress indicated that, compared to roots, leaves were more suitable for recording metabolism changes [14]. Cowpea plants respond to water stress after 12 days by stimulating the arginine/proline pathway, which may be associated with the greater damage sustained under previous drought stresses. Results showed two other pathways that have important roles: the glycine/serine/threonine pathway and the alanine/aspartate/glutamate pathway. Researchers found that the content of the glycine/serine/threonine had decreased, and using chlorophyll fluorescence data [14] faster consumption of these metabolites through enhanced photorespiration [31] is suggested. It is shown that when cowpea plants are exposed to water deficiency, they modify their metabolism to resist this condition. This mechanism occurred via the interplay between the shikimate and arginine/proline pathways, which induced three drought-responsive metabolites, namely proline, galactinol, and quercetin 3-O-6′′-malonylglycoside [14]. Proline, L-arginine, L-histidine, L isoleucine, allantoin, tyrosine, and tryptophan showed higher levels of accumulation in the leaves of drought-stressed plants, whereas GABA, adenosine, alanine, alpha-ketoglutaric acid, phenylalanine, choline, glucosamine, guanine, and aspartic acid decreased under drought stress [30]. Accumulation of aromatic amino acids in chickpeas during drought stress acts as a different source of energy, leading to stress tolerance in chickpeas [30].

It is found that during drought stress in the leaves of chickpeas, compounds, such as tryptophan, proline, adenosine, alanine, choline, and histidine, were highly increased, and hence, they can be defined as responses to drought conditions [30]. Metabolites, such as guanosine, guanine, phenylalanine, aspartic acid, adenosine, GABA, and choline, constantly reduced during water stress, but glutathione disulfide was induced after water stress imposition but decreased because of longer periods of water stress [30]. During drought stress, increments of amino acids, such as histidine, isoleucine, tyrosine, proline, and tryptophan at both two-time points in chickpea, occur. Previous studies showed that amino acid content increased during drought stress in soybeans [32] and Phaseolus vulgaris [33]. Increased levels of amino acids influenced different physiological mechanisms, including regulation of osmotic changes, disintoxication of reactive oxygen species, and adjusting the pH range in intracellular, along with enhanced stress tolerance in plants [34]. D1 protein is necessary for damage repair in photosystem II and the photosynthetic machinery generally produces various ROS. During stress conditions, the production of ROS can be induced, which can prevent D1 protein production [35, 36] and can oxidase various proteins such as D1 protein [37].

Under drought stress in chickpeas, the amount of tryptophan increased, which is involved in the ROS scavenging mechanism, leading to decreased damage to the photosystem. When chickpeas were exposed to drought stress, tyrosin and histidine as amino acids increased [30], and it is reported that tyrosin plays a role in biotic and abiotic tolerance [38, 39, 40, 41, 42]. Different reports indicate that arginine is an important amino acid that produces a mechanism for the transmission of nitrogen in plants during high-stress conditions [43, 44]. In chickpeas, during drought conditions, arginine content was induced [30]. In chickpeas and soybeans, during water stress, the majority of the soluble sugar and sugar derivatives were downregulated [30, 32]. It was found that the content of choline and glutathione disulfide gamma-aminobutyric in the chickpea was reduced. GABA has critical roles in plant physiology and results in tolerance under abiotic stresses via maintenance of cell pH, osmoregulation, and metabolism [20, 32, 45]. During water stress in chickpeas, the synthesis of some amino acids, including isoleucine, tryptophan, tyrosine, phenylalanine, and arginine, was raised [30]. The three main molecules in plant metabolism are phenylalanine, tryptophane, and tyrosine [46]. Chorismate is the precursor of these molecules that originate from the shikimate pathway. This pathway serves a basic function in the plant reproductive system, development, insect defense, and abiotic stresses [47].

3. Cold stress

Cold stress causes dehydration, resulting in plasma membrane damage. According to some studies, this might occur since the flexibility of the membrane under such conditions might be low, and it may lose its function [48, 49]. The growth and productivity of economically important crops are affected by low-temperature stress as a main abiotic stress. Osmoprotectants, such as glycine betaine, sugars (trehalose and fructans), and polyamines, were accumulated to tolerate cold stress [49]. Plants adapt to low (nonfreezing) temperatures with an accumulation of soluble sugars, among which sucrose is the most abundant [50]. Sucrose is an important signaling molecule that regulates plant metabolism under stress conditions [51, 52]. In addition, it has an important role in various processes related to the growth and development of plants [52]. Protection of cell membranes against freeze dehydration is a major factor in freezing tolerance, which is related to the accumulation of solutes such as sugars, proline, and betaines in the cytosol and alteration in membrane lipid composition [53, 54, 55]. When chickpeas were subjected to cold stress, higher levels of components such as putrescine (Put) (322%), spermidine (Spd) (45%), spermine (Spm) (69%), and the highest ratio of Put/(Spd + Spm) were observed in the tolerant genotype after the sixth day. This time, gamma-aminobutyric acid (GABA) was accumulated up to 74% more in the tolerant genotype [56]. According to reports on the effect of cold stress on the legume family, it was evident that cold stress can increase metabolites with changes in gene expression. These metabolites play a role in stress protection. Using metabolite information and genetic engineering, new cold stress-tolerant varieties of the legume crop could be developed [57].

4. Salinity stress

Salt stress removes water from the cytoplasm, causing osmotic stress. Plants respond to salt stress by the accumulation or decrease of specific secondary metabolites [58]. Polyphenol content is increased in response to biotic or abiotic stresses [59, 60]. Parida and Das showed that under salinity stress, the polyphenol content in different tissues in some plants is increased [61]. In another study, El-Shintinawy and El-Shourbagy reported that in response to salinity stress, amino acids, including cysteine, arginine, and methionine decrease, while proline concentration increases [62]. Glycine betaine is one of the nontoxic cellular osmolytes that increases osmolarity during stress periods and then helps with stress mitigation through ROS reduction, protein stabilization, and osmotic modification [63, 64, 65]. The metabolic profile in lotus legumes subjected to drought stress and salt stress indicated that their metabolic acclimation had standard features. Also, Sanchez and colleagues showed that one third to half of the metabolic changes within all lotus species were conserved [13]. Similarly, research shows that the metabolic pathways of response to environmental stress within these species are relatively conserved [66]. With increased salinity in the soil, the accumulation of ions occurs. This is one of the main abiotic stresses that negatively affect the productivity of cultivated plants. A study by Sarri and colleagues revealed that in the roots of M. arborea exposed to salt stress, the number of saponins, flavonoids, and triterpenic acids was reduced, while benzyl tetrahydrofurans, lignans, and phenols increased [67]. Also, research shows that the metabolic response of vicia faba to salt stress includes increased leaf proline and decreased fumaric and malic acid [68].

5. Heat stress

Drought and heat stresses regulate various metabolic pathways in different ways. These metabolic pathways include the metabolism of carbohydrates, amino acids, peptides, secondary metabolites, and the biosynthesis of purine and pyrimidine [17]. Heat stress in soybean decreased primary metabolic, including citrate, alpha-ketoglutarate, malate, oxaloacetate, glucose, dihydroxy acetone phosphate, succinate, pyruvate, and mannitol but drought-stress-induced carbohydrates such as glucose, fructose, sucrose, raffinose, ribose, deoxyribose, gluconate, xylose, and xylitol [17]. In a study, Liu and colleagues reported that metabolites have a critical role in response to heat stress in legume seed setting [69]. In soybean plants, the amino acid biosynthesis pathway shikimate and total aspartate family-derived amino acids are reduced in response to heat stress. So heat stress results in the reduction of tryptophan, tyrosine, phenylalanine, lysine, alanine, methionine, and isoleucine, but drought stress increases leucine, isoleucine, glycine, and proline in soybean plants. Leucine, isoleucine, proline, and glycine indicate the potential for an improved tolerance reaction under drought stress [17]. Soybean plants react to heat stress with a reduction in thymine, cytosine, and uracil as major building blocks of pyrimidine and thymidine, deoxycytidine, orotate, 2′,3′-cyclic UMP, and dUMP as a pyrimidine biosynthetic pathway, while drought stress induces the number of total metabolites in purine biosynthesis pathway [17]. Using metabolic profiling, Das and colleagues showed that upon drought stress 73% of observed flavonoids and phenylpropanoids are upregulated, and 84% of the detected secondary metabolites increase at a lower content relative to the control in reaction to heat stress, which signifies the activation of the defense mechanism for relieving stress [17]. Also, A study by Jansen, Jürgens, and Ordon shows that high temperature in Lupinus angustifolius increased alkaloid accumulation [70].

In soybean plants, the amino acid biosynthesis pathway shikimate and all aspartate family-derived amino acids are reduced in response to heat stress. So, heat stress results in the reduction of tryptophan, tyrosine, phenylalanine, lysine, alanine, methionine, and isoleucine [17]. Moreover, a significant reduction in phytochemicals, such as genistin, daidzin, formononetin, glycitin, syringic acid, genistein, and daidzein, is reported as the soybean plants’ reaction to heat stress, but these compounds are increased in soybean plants exposed to drought stress [17]. Gill and Tuteja argued that plants alleviate stress by activating their tolerance mechanisms and antioxidant activity [71]. When soybean plants were briefly exposed to heat stress, several metabolites were downregulated. Nonetheless, under water stress, the majority of the metabolites were considerably induced or were similar to the control plants [17].

6. Flooding stress

Planting areas of Soybean, as the most important oleaginous seeds in the world, are affected by flooding. In a study, flooding stress reduced plant stand growth and grain yield and underscored the importance of genetic diversity for detecting flooding tolerance in various cultivars [72]. To distinguish the traits that mediate flooding tolerance among different soybean cultivars, Coutinho and colleagues evaluated the metabolites that play a role in flooding stress response [73]. Fumarate, succinate, pinitol, citrate, alanine, malate, and phenolic compounds are the metabolites that were detected in the leaves of soybeans upon flooding stress. Soybeans that were exposed to flooding stress had more isoflavones accumulated in the roots compared to their leaves [73]. A large percentage of the metabolites in the roots and leaves of soybeans changed by flooding stress. Browne and colleagues reported that during flooding stress, the amount of several metabolites in the leaves decreased, whereas most of the compounds in the roots increased [38]. Similarly, an important change in the primary metabolism of nitrogen and carbon was observed during flooding stress. Metabolites, such as succinate, citrate, sucrose, acetate, GABA, and alanine, accumulated in the roots, while most of these metabolites were reduced in the leaves under flooding stress [38]. Soybean plants respond to flooding stress by changing compounds that are involved in the primary metabolism of carbon and nitrogen such as carbohydrates (glucose, fructose, and sucrose), organic acids of the TCA cycle, amino acids, and metabolite profiling of isoflavone glycosides [38]. In the leaves of plants during flooding stress, the amount of fructose as a carbohydrate was decreased, but the increase in sucrose and glucose is time-dependent. This change is different in roots, where a significant increase in carbohydrates, mainly sucrose, is detected for all stress periods [38]. Previous studies showed that the amount of succinate was induced compared to the other compound of the TCA cycle [74, 75], which is in agreement with succinate accumulation and lower fumarate and malate contents in soybean plants during low oxygen conditions [73]. Soybean plants respond to flooding stress by changing compounds involved in the primary metabolism of carbon and nitrogen, such as carbohydrates (glucose, fructose, and sucrose), organic acids of the TCA cycle, amino acids, and metabolite profiling of isoflavone glycosides [73]. During flooding stress, in the leaves of plants, the amount of fructose as a carbohydrate was decreased, but the accumulation of glucose and sucrose was time-dependent. This change is different in roots, where a remarkable accumulation of carbohydrates, especially sucrose, was detected for all stress periods [73].

Previous studies showed that, compared to other compounds of the TCA cycle, the amount of succinate was induced [74, 75]. This is in agreement with succinate accumulation and lower fumarate and malate contents in soybean plants during low oxygen conditions [73]. Previous studies indicated that alanine and GABA had an important role under low oxygen conditions [76, 77]. The precursor of glycine betaine (GlyBet) is an essential osmoprotectant in plants. Research shows that GlyBet changes tolerance to drought, salinity, and other stresses [78]. During flooding stress, choline content was induced in soybean roots, while the content of this metabolite decreased in leaves. Since flooding may induce stomatal resistance and inhibit water uptake because of internally limited water [79], leaf cells need an osmoprotectant for the cellular structures. This means that the higher amount of GlyBet is part of the osmoprotectant mechanism needed in this tissue. Thus, a lower amount of choline in the leaves may show a higher demand for converting this metabolite into GlyBet [73]. It is suggested that primary and secondary metabolisms in soybean plants are strongly affected by flooding stress. Most of the changed metabolites were active in carbon and nitrogen metabolism and also in the phenylpropanoid pathway. Various responses were observed in the roots and leaves and also within flood-tolerant and flood-sensitive cultivars [73]. When Medicago truncatula was exposed to flooding stress, root components such as γ-aminobutyrate and alanine, accumulated raffinose, sucrose, and hexoses, while pentoses decreased. Leaves showed a significant increase in starch, sugars, sugar derivatives, and phenolics (tyrosine, tryptophan, phenylalanine, benzoate, ferulate). As a result, there was an accumulation of sugar and a reduction in organic acids in phloem sap exudates during flooding stress [73].

7. Biotic stress

Infesting alfalfa with thrips increased flavonoids and amino acids [2]. Chen and colleagues that in common beans infested with Fusarium pathogens, isoflavonoids, proline, amino acids, purines, and flavonoids were induced [80]. In addition to glycoside, terpenes, phenols, flavonoids, lipids, carbohydrates, peptides, and amino acids content were accumulated in common bean plants infected with the pathogen R. solani [81]. In soybean plants exposed to Melodegyne pinodes, and Heterodera glycines pressure, the number of components such as tropane, alkaloid, cysteine methionine, and phenylpropanoids increased, which could be related to the resistance properties of the crop to nematodes [30]. The same results were recorded for common beans infected with F. solani [80] T. velutinum, and R. solani [81]. The content of primary metabolites, including alkaloids, alcohols, and amino acids, was accumulated and Wood and colleagues found that precursor molecules of these metabolites play a role in the defense and energy supply of the plant [82]. Legumes, such as red clover, pea, and alfalfa, respond to aphid infestation by increasing triterpene, flavonoid, and saponin [83] and in alfalfa plants, infected by thrips the content of flavonoid, and amino acids were induced [84]. Another study found that soybean responds to Melodegyne pinodes, and Heterodera glycines pressure by increasing phenylpropanoids, cysteine, methionine, alkaloid, and tropane, which results in a crop resistant to nematodes [85].

8. Conclusion

It is determined that plant development and stress responses, as well as processes, such as growth, the integrity of cells, energy storing, cellular signaling, formation of membrane and scaffolding, cellular replenishing, and whole-plant resource assignment, are managed by plant metabolites [86]. There are nearly 200,000 to 1,000,000 metabolites in plants [87, 88]. In plants that produce various metabolites, changes in metabolism alter plants’ physiology to adapt to various situations, such as stresses [31, 86, 89]. One of the important parts of early stress responses is changes in plant metabolism, which includes the accumulation of antioxidants to protect cellular components from oxidative damage and the accumulation of compatible solutes that retain water in the cell [15]. Phenolic compounds, such as phenolic acids, hydroxybenzoic acids, hydroxycinnamic acids, flavonoids, anthocyanins, flavonols and flavones, flavanones, and tannins, are detected in the legume family [90]. Moreover, negative and positive correlations were detected between certain metabolites and grain yield [21]. So metabolomics profiles in plants could be used as a strong selection tool to provide the correlation between phenotype and genotype and, hence, to improve plant responses to stress [30].

Polyphenol components play an important role in protecting against oxidative stresses in plants and can also indirectly protect them through the activation of endogenous defense systems and modulation of cellular signaling processes [91]. It is reported that Medicago truncatula responds to biotic and abiotic elicitors with changes in metabolite profiling, particularly amino acid and carbohydrate metabolism [92]. It is found that proline has an important role in stress tolerance in plants. It acts as a signaling molecule that inhibits oxidative damage [93, 94, 95]. In most stress conditions, the amount of sucrose, as a major transport sugar, was increased [96]. In addition, compatible solutes are induced during various environmental stresses. These compounds that are highly soluble in water are nontoxic. They play a role in sustaining the ordered vicinal water around proteins [26, 97]. Compatible solutes include betaines and related compounds; polyols and sugars, such as mannitol, sorbitol, and trehalose; and amino acids, such as proline [26, 98]. Previous studies indicate that compatible solutes protect plants from osmotic stress and various stress factors [26, 72, 99, 100]. Raffinose is a sugar that acts as an osmoprotectant that protects plant cells during most stressful situations, especially at the subsequent stages of stress treatment [21], and protects plants from oxidative damage [101]. GABA is another metabolite that plays a role in biotic and abiotic stress responses [40, 102, 103]. This compound protects plants against different stress situations such as protection against oxidative stress, regulation of cytosolic pH, and functions of GABA as a signaling and osmoregulation molecule [40]. Obata and Farnie showed that amino acids, including threonine, leucine, methionine, lysine, valine, and isoleucine, were usually induced during environmental stress conditions [21].