## 2 Results and Discussion
### Optimization of HS Collection for VOC Profiling
#### 2.2.2 SPME Fiber Selection  
Many types of SPME fibers are available, which is one of the advantages of choosing the SPME method to collect the HS. According to the molecular weight and polarity of the analytes, an appropriate fiber should be chosen. We tested nine fibers to collect the HS from seedlings grown under the two conditions (Figure 2). Figure 3a presents representative TICs of HS-VOCs collected by using a 7-\(\upmu\)m-thick layer of polydimethylsiloxane (PDMS), 100-\(\upmu\)m PDMS, 75-\(\upmu\)m carboxen/PDMS (C/P) and carboxax-polyethylene glycol (C-PEG) fibers. Many peaks were eluted later in the TIC when we used a 7-\(\upmu\)m PDMS fiber, as well as a C-PEG fiber (Figure 2a, top and bottom). This is because a 7-\(\upmu\)m PDMS fiber favors non-polar and high-molecular-weight compounds or semi-volatile compounds, while a PEG fiber prefers to collect alcohols and polar compounds. On the other hand, a 75-\(\upmu\)m C/P fiber can absorb gases and low-molecular-weight compounds, which are eluted early in the TIC (Figure 3a). The VOC profiles obtained using a 100-\(\upmu\)m PDMS fiber were highly scattered in the TIC (Figure 3a). The HS-SPME-GC-TOF-MS analysis showed 437 peaks and their mass spectra. A total of 99 peaks were annotated as known VOCs or peaks with original IDs provided by VocBinBase [36] (Supplementary Table S1, S2). Principal component analysis (PCA) was applied to create a projection of multivariate data onto a space of lower dimensionality for visualization purposes (Figure 3b). The PCA score scatter plot showed clear separation in accordance with each fiber property. Often, SPME fibers coated with either 100-\(\upmu\)m PDMS [42,53,56] or 65-\(\upmu\)m-thick PDMS/divinylbenzene (DVB) [39,57] are used as a first choice, because the former prefers to absorb non-polar metabolites,  
Figure 3: Collection efficiency of HS-VOCs by using various solid phase microextraction (SPME) fibers. **(a)** Total ion chromatograms (TICs) of VOC profiles collected by using a 7-\(\upmu\)m polydimethylsiloxane (PDMS) fiber (P 7), a 100-\(\upmu\)m PDMS fiber (P 100), a 75-\(\upmu\)m carboxen/PDMS fiber (C/P 75) and a carboxax-polyethylene glycol fiber (C-PEG). X-axis represents retention time, while the Y-axis shows peak intensity. **(b)** Principal component analysis (PCA) score scatter plot of VOC profiles using nine SPME fibers (observation: samples, \(n=36\); variables: annotated peaks, \(n=99\)). Four independent samples were analyzed using each SMPE fiber. HS-VOCs were collected from _Arabidopsis_ seedlings grown in water and MS conditions (\(n=2\) each). **(c)** Venn diagram of the annotated peaks that showed significant changes in the profiles of (i) the 75-\(\upmu\)m C/P (blue), (ii) the 7-\(\upmu\)m PDMS (yellow) and (iii) the C-PEG fiber collection (green) when compared to those collected by the 100-\(\upmu\)m PDMS fiber. The number in the blue circle represents how many annotated peaks showed significant changes in the profiles from the HS-GC-TOF-MS analysis by using the 75-\(\upmu\)m C/P fiber. The number in the yellow circle represents the number of significant peaks in the profile from the 7-\(\upmu\)m PDMS analysis. The number in the green circle represents the number of significant peaks in the HS profile from the C-PEG fiber collection. Control, the HS profile collected by the 100-\(\upmu\)m PDMS fiber (see Table 1). Abbreviation: PC, principal component. while the latter can extract short-chain polar compounds, e.g., alcohols and amines. The PCA score scatter plot illustrates that samples obtained by using a 100-\(\upmu\)m PDMS fiber and those obtained by using a 65-\(\upmu\)m PDMS/DVB fiber were clustered very closely (Figure 3b). This suggests that the collection efficiency of these two fibers is probably very similar.