# NMR and LC-MS-Based Metabolomics to Study the Effect of Surfactin on the Metabolome of Flax
## 3 Results and Discussion
### Metabolite Changes in Aerial Parts  
The metabolic changes were studied in the aerial organs. These implicated the same biochemical families as those observed in roots, such as amino acids, carbohydrates, organic acids, and cyanogenic compounds, as well as phenolic compounds. The OPLS-DA score plot (Figure 6) also showed a good separation between the two groups. Symbols in green represent the control plant samples, which are grouped on the left side, whereas symbols in blue represent the surfactin-treated plant samples, which are grouped on the right side. Stems:  
The values for PC1 were 34% and 36% for the NMR dataset and the LC-MS dataset, respectively, suggesting the similar contribution of more and less accumulated metabolites in the separation between the control and surfactin-treated samples. The values of PC2 were 3% for the NMR dataset and 17% for the LC-MS dataset. This component can explain the kinetic effect. 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. Equivalent clustering on the OPLS-DA score plot corresponding to the LC-MS dataset was observed, but most of the 56 h samples were in the lower part, whereas most of the 10 D samples were in the upper part. Leaves:  
The values for PC1 were 33% and 44% for the NMR dataset and the LC-MS dataset, respectively, suggesting the highest contribution of less accumulated metabolites in the separation between the control and surfactin-treated samples. The values of PC2 were 23% for the NMR dataset and 10% for the LC-MS dataset. This component can explain the kinetic effect. The clustering of samples from a kinetic point of view was also observed for both the NMR and LC-MS datasets, with most of the 56 h samples in the upper part, while most of the 10 D samples were in the lower part. Figure 5: Heatmap of the discriminant metabolites differently accumulated in flax root extracts between surfactin-treated and control conditions. The scale bar (from red to blue) on the right represents the ratio of metabolite content in surfactin-treated flax roots to that in control flax roots. The column on the left indicates the kinetic point at 56 h and the column on the right indicates the kinetic point at 10 D. The analytical methods used to determine discriminant metabolites are indicated on the left of the graph; the red color corresponds to the combination of NMR and LC-MS, the blue color corresponds to LC-MS, and the purple color corresponds to NMR. Among the annotated variables in the VIP list corresponding to all the NMR and LC-MS data from the leaf and stem extracts, the discriminating metabolites belonged to the same biochemical families as the ones of root extracts (Table S1). Amino acids and organic acids:  
In the stem and leaf extract, the discriminant amino acids due to surfactin treatment were the same as those observed in the root extract, with the observation of threonine for stems and leaves and without the observation of proline in leaves (Figure 7). In the stems, the accumulation of all amino acids decreased at 56 h, except for threonine, and increased at 10 D, except for glutamic acid and GABA. The decrease in amino acid content observed at 56 h could be due to the remobilization of the amino acids from stems to roots. At 10 D, the plants could activate the synthesis of these primary metabolites to correct the lack of them at 56 h. Glutamic acid was still less accumulated. The same hypothesis regarding a deviation occurring mainly through proline and less through GABA was established as previously for the roots. Malic acid, as for the roots, was less accumulated at 56 h and more at 10 D, whereas formic acid was more accumulated at the two kinetic points and fumaric acid content decreased at 56 h and at 10 D. The organic acids represent a way to store fixed carbon and can be allocated to form amino acids or carbohydrates [38].