The score plot of OPLS-DA in Fig. 1 shows that for the two cultures C1 and C2, the bio stimulant-treated samples were grouped away from the control samples. These observations highlighted the different metabolic profiles in the leaves, stems, and roots of the bio stimulant-treated and control samples, demonstrating that the flax plants were responding to the bio stimulant treatment. In the C2 culture, the effect of the bio stimulant was investigated after treatment with two doses (1 kg/ha and 2 kg/ha) on D21.
The OPLS-DA analysis revealed no distinct differences between samples treated with 1 kg/ha and 2 kg/ha in the different plant parts. In addition, a Wilcoxon Rank Sum test was performed on the datasets of the control and treated samples, and no significant differences were found in the metabolite content of the samples treated with the 2 doses of AGRO-K® after 21 days (data not shown). Thus, the samples treated with different doses of bio stimulant could be considered as a group. These observations provide the information necessary for farmers to select 1 kg/ha as an effective dose to obtain a response to the bio stimulant with lower cost in flax culture. Figure 2 represents the corresponding loading plots of the OPLS-DA obtained from NMR and LC-MS data showing the distribution of metabolites in the control and treated samples in the different plant parts for the two cultures C1 and C2.
The identification of discriminant metabolites detected by NMR was based on the matching of the 1D and 2D NMR spectra with the spectra of standard references available in the database developed in our laboratory.
For the discriminant metabolites detected by LC-MS, some features were identified by matching the exact masses, retention times and MS/MS spectra to those of an analytical reference standard. Others were identified by comparison with accurate masses and MS/MS spectra of molecules available in an accessible mass spectral library. Identification was also confirmed by comparing identical molecules in previous publications (Tchoumtchoua et al., 2019) (Elboutachfaiti et al., 2022) (Chantreau et al., 2014) (Morreel et al., 2010) (Tais et al., 2021) (Hanhineva et al., 2012) (Moheb et al., 2011) (Li et al., 2018). All information on the identification of discriminant metabolites are indicated in Table 1.
Table 1. List of discriminant metabolites identified with 1H-NMR and LC-MS.
In the roots, stems, and leaves of the two cultures, a significant increase in the amount of HCAAs was observed in the treated samples compared to that of the control samples at different harvest time points. For both cultures, the level of N-feruloyl putrescine in the AGRO-K®-treated plants increased significantly compared to that in the control plants, with T/C ratios (calculated by the formula: ratio T/C = content in treated plant /content in control plant) of 5.67 in the roots of the C1 culture at D19 and 3.11 in the leaves of the C2 culture at D21. A similar increase in the levels of N-feruloyl-spermine and N-coumaroyl-N-feruloyl-spermine were also observed in the different plant parts.
Furthermore, a significant decrease in the level of the amino acid arginine was observed in the stems, leaves, and roots of flax after treatment with the bio stimulant, with a T/C ratio ≤ 0.4 over the harvest period in the two cultures C1 and C2. This amino acid is a direct precursor of the synthesis of polyamines such as putrescine, spermidine, and spermine through the action of arginine decarboxylase (ADC), spermidine synthase, and spermine synthase (Gill and Tuteja, 2010). HCAAs result from the conjugation of polyamines with hydroxycinnamic acids, such as p-coumaric, ferulic, and caffeic acids, together with some glycosylated forms (Liu et al., 2022). Therefore, the observed accumulation in the content of N-feruloyl-putrescine, N-feruloyl-spermine, and N-coumaroyl-N-feruloyl-spermine in the treated samples provides an explanation for the simultaneous bio stimulant-induced decrease in arginine levels, as arginine is converted into putrescine and spermine, essential for production of the above-mentioned HCAAs.
A significant increase in the content of phenylpropanoids such as coniferyl alcohol and oligolignols such as G(8-O-4)S(8–8)G was observed in the flax stems and roots after application of the bio stimulant compared to that of the control plants. The largest increase in coniferyl alcohol was observed in the stems, when compared to the roots, with T/C ratios of 2.91 and 2.03 for the C1 culture at D10 and D19, respectively, and 2.09 and 2.97 for the C2 culture at D12 and D21, respectively. The increase in the amount of G(8-O-4)S(8–8)G followed the same trend, with T/C ratios of 2.42 and 2.95 in the stems of the C1 culture at D10 and D19, respectively, and 2.09 and 2.97 in the stems of the C2 culture at D12 and D21, respectively.
In addition, an increase in the content of flavonoids, such as triticuside-A, vitexin, schaftoside, vicenin-2, and lucenin-2, was observed in the stems and leaves of treated samples compared to those in the control in the two cultures.
Concurrent with the accumulation of HCAAs, phenylpropanoids, and flavonoids, the level of phenylalanine in the stems, roots, and leaves decreased after application of the bio stimulant in the two flax cultures. Phenylalanine ammonia lyase (PAL) is a key gateway enzyme that links primary and secondary metabolism mainly through the phenylpropanoid pathway, which branches into a network of other pathways. This enzyme catalyzes the deamination of phenylalanine giving rise to cinnamic acid. The latter then serves as precursor leading to the generation of various phenolic compounds such as HCAAs, flavonoids, and lignins (Kong, 2015). Thus, the decrease in the amount of phenylalanine observed in the treated flax samples could be explained by the redirection of the plant to induce deamination of this compound in order to yield cinnamic acid, which is involved in the biosynthesis of HCAAs, phenylpropanoids, and flavonoids.
In summary, the quantitative pathway analysis of the differentially abundant metabolites in the roots, stems, and leaves of flax, a dicotyledonous plant, suggested that the application of AGRO-K® induces changes in the metabolic pathways of HCAAs, flavonoids, lignin, and phenylpropanoids. A similar postulated framework describing AGRO-K®-induced events was observed in our previous metabolomic study conducted in wheat, a monocotyledonous plant. Studies in several plant species have shown a positive correlation between HCAA content and root growth rate and elongation (BIONDI et al., 1993) (Tang and Newton, 2005) (Couée et al., 2004) (Tarenghi and Martin-Tanguy, 1995) (Hummel et al., 2002) (Ebeed et al., 2017). In addition, HCAAs were found to increase cell wall thickness (Zeiss et al., 2021) (Gunnaiah et al., 2012) (Graça, 2010)(Macoy et al., 2022) (Macoy et al., 2015).
Lignin content in the stems and roots of plants is significantly related to lodging resistance (Li et al., 2021). Lignin is mainly present in secondary-thickened cell walls; it provides structural support, giving rigidity and strength to stems to stand upright (Yi Chou et al., 2018).
Flavonoids have long been known to be key regulators of physiological processes, including root gravitropism and branching (Buer et al., 2006) ; (Buer and Muday, 2004) ; (Brown et al., 2001) ; (Buer and Djordjevic, 2009) (Peer et al., 2004).
An enhancement in similar pathways with common discriminant metabolites has been observed in different plant species treated with AGRO-K®. The pathways which seem to be reproducibly impacted are associated with root development, soil anchorage, and cell wall stiffening and lignification. The findings herein, therefore, contribute towards the generation of a fundamental knowledge base describing the molecular mechanisms underlying the effects of this bio stimulant on enhancing plant vigor and improving resistance to lodging and provide precise information necessary for industries and farmers to confidently use this bio stimulant in different agricultural practices.
Moreover, metabolic changes in amino acids and tricarboxylic acid (TCA) intermediates were observed only in flax (not wheat), and especially in the roots, after treatment with the bio stimulant. A significant increase in the concentration of amino acids such as proline, glutamate, aspartate, and glycine and of TCAs such a succinic, fumaric, and malic acid, was observed only in the roots of flax. A study conducted by Biancucci et al. showed that proline can promote root elongation in Arabidopsis plants via the modulation of the size of the root meristematic zone likely by controlling cell division and, in turn, by modulating the ratio between cell division and cell differentiation (Biancucci et al., 2015). Recent studies have shown that glutamate usually exerts a signaling role by its receptors (GLRs), which are also capable of binding to other amino acids. These receptors, when activated by amino acids, can trigger a series of physiological processes such as the stimulation of lateral root growth and development (Kong et al., 2015). Another study showed that, in Brassica napus, aspartate induces cell division and promotes root development and elongation through the modulation of ethylene and/or the ethylene signaling pathway (Lemaire et al., 2013). In Habanero pepper (Capsicum chinense), glycine promotes root hair growth that allows access to a greater surface area and volume of soil, which can favor the acquisition of water and nutrients (Domínguez-May et al., 2013).
Furthermore, amino acids generally play several roles in plants, from serving as basic building blocks of proteins to signaling metabolites that are involved in numerous metabolic pathways which stimulate plant growth (Pratelli and Pilot, 2014). Following degradation, the amino acids’ carbon skeletons are generally converted into precursor intermediates of the TCA cycle that produce energy in the form of ATP (Hildebrandt et al., 2015).This energy can fuel a wide range of energy-demanding biochemical processes that contribute to plant growth and development such as gene expression, mobility and metabolism (De Col et al., 2017).
Herein, the simultaneous increase in the amount of amino acids and TCA intermediates in the roots of flax treated with the bio stimulant led to the hypothesis that treatment with the bio stimulant induces ATP production through amino acid accumulation in order to enhance root growth and elongation.
Major changes in the primary and secondary metabolic pathways of roots, stems, and leaves of flax after AGRO-K® treatment, as well as the proposed relations between these metabolic pathways are presented in Fig. 4.
Agro-K® is composed of a mixture of potassium oxide (50%), phosphorus oxide (32%), amino acids (10%), as well as galacturonic acid (5%) extracted from the Nopal cactus. Galacturonic acid is regarded as an efficient elicitor used for activating plant defense responses (Wan et al., 2021). This compound has been reported to be a damage-associated molecular pattern (DAMP), that can be recognized by pattern recognition receptors (PRRs) responsible for the stimulation of the immune system (Ferrari et al., 2013). Several studies on various plant models showed that treatment with galacturonic acid, as an elicitor, accumulates transcripts such as PAL, which constitutes the point of connection between the primary metabolism of shikimate, leading to aromatic amino acids, and the secondary metabolism of phenylpropanoids, leading to flavonoid and lignin production, and stimulates changes in defense genes, including salicylic acid, which is known to be involved in strengthening the cell wall by increasing polyamine, lignin, and callose biosynthesis (Butselaar and Ackerveken, 2020) (Ochoa-Meza et al., 2021) (Canales et al., 2019) Thus, the mechanism of action of the bio stimulant could be attributed to the elicitation effect of galacturonic acid.