## 2 Results and Discussion
### Optimization of HS Collection for VOC Profiling
#### 2.2.3 Optimization of Incubation Conditions  
We used a 100-\(\upmu\)m PDMS fiber and evaluated collection efficiencies by changing incubation conditions, times and temperatures. The PCA was applied to visualize relationships between incubation time or temperature and collection efficiency of volatiles (Figure 4). We first evaluated incubation time to optimize the collection efficiency of volatiles. We fixed the incubation temperature at 60 \({}^{\circ}\)C and then performed HS-SPME-GC-TOF-MS analysis by setting the incubation times at 10, 20, 30, 40, 50, 60, 70, 80 or 90 min (Figure 4a). Most of the samples were clustered together on the PCA scatter plot; however, samples incubated at 60 and 90 min showed relatively low reproducibility (Figure 4a, left). The PCA loading scatter plot demonstrated that longer incubation times resulted in more peaks than shorter incubation times did (Figure 4a, right). As metabolomics requires \begin{table}
\begin{tabular}{|c c c c c c c|} \hline \hline
**Compound annotation** & **log\({}_{2}\)-FC** & **FDR** & **log\({}_{2}\)-FC** & **FDR** & **log\({}_{2}\)-FC** & **FDR** \\  & **(C/P 75 \(\upmu\)s. P 100)** & & **(P 7 \(\upmu\)s. P 100)** & & **(C-PEG \(\upmu\)s. P 100)** \\ \hline \hline \multirow{2}{*}{Heptane, 5-ethyl-2-methyl-Dodecane, 4,6-dimethyl-PDMS and C-PEG fibers, unlike that observed in the case of 100-\(\upmu\)m PDMS fiber.} & \multirow{2}{*}{3.8} & \multirow{2}{*}{0.0000} & \multirow{2}{*}{n.s.} & \multirow{2}{*}{n.s.} \\  & & & & & \\ \hline
**45514** & n.s. & & 4.6 & 0.0139 & n.s. \\
1H-Indole, 1-methoxy- & n.s. & & n.s. & 4.5 & 0.0090 \\
2-Undecanone & n.s. & & n.s. & 4.4 & 0.0000 \\ Undecanal & n.s. & & n.s. & 3.7 & 0.0001 \\ Biphenyl & n.s. & & n.s. & 6.6 & 0.0000 \\ Dodecanal & n.s. & n.s. & & 3.9 & 0.0000 \\ \(n\)-Tridecan-1-ol & n.s. & & n.s. & 5.4 & 0.0010 \\ \hline \hline \end{tabular}  
* Significant, false discovery rate (FDR) of \(<\)0.05. Biological replicates, \(n\) = 4 per fiber. Abbreviations: log\({}_{2}\)-FC, log\({}_{2}\)-fold change; C/P 75, a 75-\(\upmu\)m C/P fiber; P 7, a 7-\(\upmu\)m PDMS fiber; C-PEG, a C-PEG fiber; P 100, a 100-\(\upmu\)m PDMS fiber; n.s., not significant.  
\end{table} 
Table 1: Log\({}_{2}\)-fold changes in fiber-specific peaks collected using the 75-\(\upmu\)m C/P, 7-\(\upmu\)m PDMS and C-PEG fibers, unlike that observed in the case of 100-\(\upmu\)m PDMS fiber. high-throughput analysis with high reproducibility, we chose 20 min as the optimum time for the incubation of the SPME vials. Next, we evaluated incubation temperature by setting it at 30 \({}^{\circ}\)C, 40 \({}^{\circ}\)C, 50 \({}^{\circ}\)C, 60 \({}^{\circ}\)C or 70 \({}^{\circ}\)C. The HS-VOCs were collected for 20 min by SPME with a 100-\(\upmu\)m PDMS fiber. The PCA indicated that the largest source of variation was the difference in incubation temperature and, in particular, the difference between incubation at 30 \({}^{\circ}\)C and 70 \({}^{\circ}\)C (Figure 4b, left, PC1). Peaks were extracted at low and high incubation temperatures (Figure 4b, right), but samples incubated at 30 \({}^{\circ}\)C showed low reproducibility. On the other hand, the second source of variation could be explained by the differences between the samples incubated at 50 \({}^{\circ}\)C-70 \({}^{\circ}\)C, and those at 30 \({}^{\circ}\)C (Figure 4b, left, PC2) include aliphatic alcohols, aliphatic/aromatic aldehydes, esters, ethers, hydrocarbons, including alkanes, ketones and terpen [39, 50, 53]. _Arabidopsis_ and _Brassicaceae_ species can emit isothiances and nitriles [58]. These VOCs have high vapor pressures and low-to-medium water solubility. Since some terpen are unstable at high temperature [59, 60], incubations must be performed under mild conditions. Therefore, the incubation temperature was set at 60 \({}^{\circ}\)C.  
Figure 4: Evaluation of incubation time (a) and temperature (b) to optimize collection efficiency of volatiles in the HS vials containing _Arabidopsis_ seedlings grown in water and MS conditions. Left, PCA score scatter plot.