# NMR and LC-MS-Based Metabolomics to Study the Effect of Surfactin on the Metabolome of Flax
## 3 Results and Discussion
### Metabolite Changes in Root  
The OPLS-DA score plot (Figure 4) showed a good separation between the two groups. Symbols in green represent the control plant samples, which are grouped in the left part, whereas symbols in blue represent the surfactin-treated plant samples, which are grouped in the right part. The values for PC1 were 44% and 30% for the NMR dataset and LC-MS dataset, respectively, suggesting the highest contribution of more accumulated metabolites in the separation between the control and surfactin-treated samples because NMR analysis is less sensitive than LC-MS analysis. The values of PC2 were 9% for the NMR dataset and 17% for the LC-MS dataset. This component can explain the effect of kinetics on the separation. For the OPLS-DA score plot corresponding to the NMR dataset, most of the 56 h samples were in the upper part, whereas most of the 10 D samples were in the lower part, especially for the surfactin-treated samples. Among the annotated variables from the corresponding VIP list, the discriminant metabolites belong to several biochemical families such as amino acids, carbohydrates, organic acids, or secondary metabolites (Table S1). \begin{table}
\begin{tabular}{c c c} \hline
**Surfactin Type** & & \\
**Molecular Formula** & **Chemical Structure** & **ID Reference** \\
**Monoisotopic Mass** & & **ChemSpider** \\ \hline Surfactin C & & \\ C\({}_{53}\)H\({}_{93}\)N\({}_{7}\)O\({}_{13}\) & & \\
1035.6831 & & \\ \hline \end{tabular}
\end{table} 
Table 1: _Cont._Figure 3: Signals of surfactin detected (**A**) by NMR analysis; doublet (\(\delta\) 0.85 ppm, J = 6.7 Hz), present in standard (red) and 56 h surfactin-treated flax root extract (blue) or absent in 56 h control flax root extract (green); (**B**) by LC-MS analysis; peaks at 6.27, 6.35, and 6.43 min present in 56 h surfactin-treated flax root extract (blue) or absent in 56 h control flax root extract (green), (**C**) with the corresponding \(m/z\) of [M-H]\({}^{-}\) below. Amino acids and organic acids:  
Seven discriminant amino acids were annotated in the NMR dataset from root extracts, namely GABA, alanine, proline, valine, leucine, isoleucine, and glutamic acid. As can be seen in Figure 5, most of these amino acids were more accumulated in surfactin-treated flax roots compared to control flax roots, while in surfactin-treated flax, only one amino acid presented a decrease in its content for both kinetic points, namely glutamic acid. This amino acid is a precursor of proline and GABA, which were both accumulated at 56 h and 10 D in surfactin-treated conditions. The importance of this branchpoint has been recently described in plant defense and, in this work, could be associated with surfactin treatment [34]. In this review, they also mentioned the implication of malic acid in this biosynthesis regulation; changes in malic acid content were also observed in our study, with a decrease at 56 h and an increase at 10 D in the surfactin-treated flax roots. The different accumulation of amino acids has already been described in metabolomic studies on flax samples subjected to osmotic stress, suggesting that treatment with surfactin induces metabolic changes to prepare the plant for potential stress [16]. Two other amino acids, valine and isoleucine, were differentially accumulated in flax roots between surfactin treatment and control conditions. Their content highly increased at 10 D, similar to the content of cyanogenic compounds because valine is a precursor of linamarin biosynthesis, and isoleucine is a precursor of lotaustralin. Cyanogenic compounds:  
Both NMR and LC-MS datasets showed increases in linamarin and lotaustralin in surfactin-treated flax roots (Figure 5). These increases were more important at 56 h than at 10 D, whereas the two precursors were less accumulated at 56 h than at 10 D, suggesting that an increase in cyanogenic compounds led to precursor consumption at the beginning of surfactin treatment and that the flax plant responded by increasing the pool of precursors later after the beginning of surfactin treatment.