We discovered 23 metabolic targets that were highly induced by biostimulant inoculations in both maize and sugarcane seedlings (Table 1). We would like to highlight the significantly greater concentrations of these core metabolites in leaf extracts, independent of plant species as being indicative of their usefulness as markers of the positive biostimulation-associated phenotype. The repression or inhibition of cell metabolites, leading to lower concentrations, is equally as, or more, important than high concentrations when discriminating the physiological effects. However, it is much easier to detect one compound by simple GC–MS or HPLC in an extract if this compound is present at a greater concentration. Simplistic solutions to complex problems can lead to ill-advised choices; however, here, we studied the metabolites present in greater concentrations to determine the correlations between their levels and the phenotype/agronomic trait. This strategy has been used previously, including in recent studies of metabolic responses under drought-stress conditions [19].
Among the metabolite targets found in both plants’ extracts, those from the tricarboxylic acid (TCA) cycle and one amino acid indicated that the biostimulant application increased the plants’ metabolic levels. This was reflected in the accumulations of citric, isocitric, aconitic, malic, maleic and fumaric acids (Table 1). The intensification of the TCA flux must be fed by the accumulation of substrates required for enzymatic reaction that will lead to the next step. The main function of the TCA cycle is the generation of ATP, which the cell consumes for energy. The pivotal roles of TCA-related compounds found in greater concentrations include photosynthesis, photorespiration, nitrogen metabolism, reductant transport and the maintenance of photosynthetic redox balance [20]. In addition, the TCA cycle is responsible for the production of various biosynthetic precursors, such as ascorbate, vitamin co-factors, fatty acids and amino acids. We found a number of amino acids at greater concentrations in only one of the plants, such as glycine in maize and proline, phenylalanine, homoserine and glutamine in sugarcane, but only aspartic acid was found in significant amounts compared with the controls in both extracts. Aspartate is the precursor of the essential amino acids lysine, threonine, methionine and isoleucine. It is formed by the transamination of oxaloacetate and may be derived from the TCA cycle [21]. Oxaloacetate is the intermediate compound between malic and citric acids, and the observed accumulations of both concurs with aspartic acid production and oxaloacetate consumption. The use of compounds from the TCA cycle and aspartic acid as markers of biostimulant application is facilitated by these compounds being well described and the availability of kits for their identification by HPLC. Enzymes linked to the TCA cycle were induced by humic substances in maize seedlings [22] as were amino acid, including aspartic acid, synthesis and accumulation [23]. Increased carbohydrate consumption and nitrogen assimilation enzyme activities has been observed in maize treated with an HA plus PGPB biostimulant [6], which was in agreement with the promotion of the primary metabolism observed in this study.
Lipids have essential structural functions in plant cell membranes, are a highly energetic carbon source for cells and can act as cell signal messengers [24]. These compounds are chemically defined by their low-aqueous solubility, and this broad definition includes molecules from primary and secondary plant metabolisms. We identified three fatty acids, linoleic, 2-monopalmitin and myristic, present at greater concentrations in extracts of both treated plant species in comparison with untreated plants. Linoleic acid has a long unsaturated chain (18:2) and is a predominant fatty acid constituent of storage lipids. It has previously been found to preferentially accumulate at high osmotic potentials [25]. 2-Monopalmitin is a glyceride with a fatty acid chain (16:0) covalently bound to a glycerol molecule through an ester linkage, and it can be transported and utilized for energy production or metabolic pathways. Myristic acid (tetradecanoic acid) is another fatty acid (14:0) found at relatively high concentrations in the leaf extracts of treated plants, while tocopherol (vitamin E) is a lipid-soluble molecule that’s biosynthesis is strongly conditioned by the availability of phytyl pyrophosphate, its aromatic biosynthetic precursor [26]. The stimulative effect on the TCA pathway indicates quantitative and qualitative changes in amino acids and lipid accumulation. Future studies can be performed to evaluate these lipid accumulations as markers for biostimulative responses in plants treated with endophytic diazotrophic bacteria and humic substances.
Tocopherol has presented antioxidative properties that protect against the oxygen toxicity of scavenging lipid peroxyl radicals, thereby preventing the lipid peroxidation of membranes [27]. Alpha-tocopherol levels can change in response to environmental cues depending on the magnitude of the stress and species’ sensitivity to stress, and it is generally assumed that increases in tocopherol contribute to plant stress tolerance [28].
Another compound found in greater concentrations in both treated seedlings and often linked to plant stress was trehalose, a disaccharide formed by two molecules of glucose. Trehalose accumulation has been observed in symbiosis and plant–pathogen interactions, as well as during abiotic stress, but its role in plant defense remains unclear [29]. Trehalose is highly soluble but chemically unreactive owing to its non-reducing nature, making it compatible with cellular metabolism even at high concentrations in response to abiotic stress [30]. In addition, trehalose production is a feature of many beneficial microbes, such as rhizobial symbionts [31], and the inoculation of grapevine with a plant growth-promoting rhizobacterium Burkholderia phytofirmans led to the up-regulation of trehalose metabolism and improved chilling-stress tolerance in the plant [32].
Three compounds linked to ascorbate metabolism–catabolism (vitamin C), threonic, isothreonic and oxalic acids, were also observed in greater concentrations in both treated species. The pathways by which ascorbate is catabolized to form oxalic, threonic and isothreonic acids have been previously reported, as well as its roles in many aspects of redox control and anti-oxidant activities in plant cells [33]. Reactive oxygen species (including OH, O−2, H2O2, HO˙2, RO˙, ROO˙ and 1O2) are cytotoxic to plants and induced by various environmental disturbances [34]. Reactive oxygen species are scavenged by various antioxidative defense systems, including hydrophilic and hydrophobic redox buffers, namely ascorbate and tocopherol, respectively. According to Foyer and colleagues [35], tocopherol is an effective scavenger of singlet oxygen species and, in this case, the reduced scavenging form is regenerated by ascorbate. Thus, the greater tocopherol concentration may be linked to the accumulations of ascorbate-degradative metabolites.
Another pivotal compound in secondary plant metabolism found in relatively greater concentrations in both maize and sugarcane was shikimic acid, a precursor of aromatic amino acids, indole compounds and their derivatives and alkaloids. Higher plants possess a mechanism to convert quinic acid to shikimic acid, phenylalanine and tyrosine [36]. Quinic acid, a cyclic polyol, was also found in greater concentrations, which indicates coherence among the metabolite pathways induced by plant inoculations. In addition, 4-hydroxybenzoic acid and 3,4-dihydroxycinnamic acid, two precursors of salicylic acid, which is another compound known to accumulate in response to different stresses, were found at greater concentrations. Salicylic acid is an important plant hormone that regulates many aspects of plant growth and development, as well as resistance to biotic and abiotic stresses [37]. Humic substances can promote phenylpropanoid metabolism, inducing plant accumulations of diverse phenolic compounds [38], and that bacteria used in the biostimulant are able to produce indole derivatives [39]. Thus, it was not surprising to find greater concentrations of shikimic acid and its derivatives in both leaf extracts.
Erythritol is an important nutrient for several α-Proteobacteria, including N2-fixing plant endosymbionts, and is used to feed the pentose phosphate pathway [40]. Here, it was also found at greater concentrations (Table 1). Galactaric acid is also known as mucic acid and is a product of galactose oxidation. Positive correlations between metabolite levels and drought-tolerance traits were identified for galactaric acid and other metabolites (allantoin, gluconic acid, glucose, and a salicylic acid glucopyranoside) in rice genotypes [41]. The concentration of mucic acid also increases in response to heat stress [42] as do other compounds found in this study, like shikimic acid, malonic acid, threonic acid and citric acid, indicating that these metabolites are promising candidate biostimulant markers.