## 2 Results and Discussion
### VOC Profiling by Using a Developed Workflow - A Case Example  
We hypothesized that VOC profiles may differ when grown under the two nutrient conditions, because the biomass of the _Arabidopsis_ seedlings grown in the MS condition was dramatically higher than that in water (Figure 1a,b). This implies that samples in water may be exposed to severe stress compared to those in MS. Using the optimized analytical conditions, we performed VOC profiling of _Arabidopsis_ seedlings grown under the two conditions. We obtained 263 peaks and their mass spectra by performing HS-SPME-GC-TOF-MS analysis. A total of 69 peaks were annotated as known VOCs or peaks with VocBinBase IDs (Supplementary Table S1, S3). To find the VOCs that contributed to differences between the VOC profiles of samples harvested from MS and those from water, bidirectional orthogonal projection onto latent structure-discriminant analysis (O2 PLS-DA) were performed [61; 62]. In this case, O2 PLS-DA can extract as much of the class-separating (MS _vs._ water) variation as possible. The O2 PLS-DA score scatter plot of samples grown under the two conditions showed a clear separation (Figure 5a). For example, we could detect the peak annotated as 1-isothiocyanato-heptane (Figure 5b), which is produced from aliphatic glucosinolates [58; 63]. When considering the biosynthesis of isothiocyanates in _Arabidopsis_, the isothioxane would contain a branched chain rather than an odd-numbered chain [64]. GC-high-resolution (HR)-TOF-MS analysis has great potential in more reliably estimating the structure of such a peak. The 39 VOCs were predicted to be discriminative metabolites by the O2 PLS-DA loadings in the first Y-predictive component. Of these, 37 peaks tended to increase in water (e.g., calamenen-10-one), while the levels of two peaks tended to increase when the samples were grown in MS (e.g., 3-Buten-2-one, 4-(2,6,6-trimethyl-1-cyclohexen-1-yl)-, (E)-). Figure 5: O2 PLS-DA score scatter plot (_left_) and loading plot (_right_) of VOC profiles elicited from _Arabidopsis_ seedlings grown under the two different conditions. Each symbol represents an independent sample in the score scatter plot and an independent annotated peak in the loading plot. The levels of VOCs annotated as V126, V174 and V273 emitted from the samples grown in water (biological replicates, \(n\) = 10) showed significant changes, when compared with those grown in MS (biological replicates, \(n\) = 10) (see Table 2). The metabolites were extracted according to the first-weight vector (w* 1) in addition to the 95% confidence intervals. A _p_-value (calculated by cross-validated predictive residuals, CV-ANOVA; \(p\) cv) of \(<\)0.01 for the model was defined as significant. Abbreviations: pred comp, predictive component; ortho comp orthogonal component; HepICT, 1-isothiocyanato-heptane. Subsequently, a _t_-test was applied to compare the two groups, MS _vs._ water (Table 2). The nine volatiles showed significant changes (false discovery rate (FDR) of \(<\)0.05). A significant decrease was observed in the level of the VOC, called V126, when compared to that from samples grown in MS. The peak V126 annotated as \(\beta\)-ionone is known to be a flavor VOC emitted from various plant tissues [65, 66]. \(\beta\)-ionone is produced via the enzymatic cleavage of carotenoid by dioxygenase in tomatoes [65]. The significant decrease in the level of \(\beta\)-ionone grown in water may be linked to carotenoid breakdown, since only the existence of water is very severe for the survival of _Arabidopsis_ seedlings (Figure 1a,b). For instance, carotenoid content decreases during aging in tobacco leaves [67], and it also decreases when _Arabidopsis_ leaves are shifted from a nutrient-sufficient medium to water [68]. The levels of eight volatiles, including unknown peaks with a VocBinBase ID, showed significant increases when compared to those from samples in MS.