## 2 Results
### Nootkatone Does Not Induce the Biosynthesis of Sesquiterpenes in A. thaliana  
In order to additionally investigate whether the nootkatone presence has an effect on the plant's secondary metabolism of terpenoids, the volatile profile of _A. thaliana_ was investigated using headspace solid-phase microextraction (HS-SPME)-gas chromatography (GC)\(\times\)GC-time-of-flight mass spectrometer (TOF-MS). A total of 16 sesquiterpene hydrocarbons were found in the headspace of 30-day-old plants growing on both nootkatone-containing medium and the DMSO control (Table 1). The contour plot of the two-dimensional separation is shown in Figure 7 and the corresponding structural formulas of the sesquiterpenes in Figure S2. No differences in the sesquiterpene profiles were detected between the treatments. None of the compounds listed in Table 1 could be detected in blank tests with media-containing nootkatone or DMSO, except the sesquiterpene hydrocarbon nootkatene (7), which was already found in the medium containing nootkatone. The GC measurements performed indicated that nootkatone did not induce the formation of additional sesquiterpenes in _A. thaliana_.  
Figure 5: _Arabidopsis_ leaf disc reactive oxygen species (ROS) production in response to nootkatone exposure. _A. thaliana_ leaves were exposed to different concentrations of nootkatone, the bacterial elicitor peptide flg22 as a positive control or water and 0.5% DMSO as negative controls. The ROS presence was measured in relative light units (RLU) using a luminol-based assay during a 75-min-long incubation. Data show the mean value of 3 biological replicates, \(n\) = 12.  
\begin{table}
\begin{tabular}{c c c c c} \hline
**No.** & **Compound a** & **\(I\) b** & **\(I\) (lit.) c** & **Identification d** \\ \hline
12 & \(\alpha\)-cedrene & 1586 & 1582 [21] & ms (936, 942), ri, std \\
15 & \(\alpha\)-barbatene & 1596 & 1572 [21] & ms (844, 864), ri \\
5 & \(\beta\)-elemene & 1600 & 1605 [22] & ms (876, 876), ri, std \\
13 & \(\beta\)-cedrene & 1607 & 1599 [23] & ms (820, 834), ri \\
6 & (\(E\))-\(\beta\)-caryophyllene & 1613 & 1619 [22] & ms (909, 913), ri, std \\
14 & _cis_-thuiopsene & 1640 & 1620 [24] & ms (924, 927), ri \\
2 & (\(E\))-\(\beta\)-farnesene & 1666 & 1666 [25] & ms (913, 923), ri \\
16 & \(\beta\)-barbatene & 1669 & n.a. e & ms (874, 876) \\
10 & 4-epi-\(\alpha\)-acoradiene & 1692 & n.a. e & ms (885, 895) \\
9 & \(\beta\)-chamigrene & 1722 & 1740 [26] & ms (888, 905), ri \\
3 & \(\beta\)-bisabolene * & 1734 & 1729 [21] & ms (889, 893), ri \\
1 & (\(E\))-\(\alpha\)-farnesene * & 1750 & 1747 [27] & ms (902, 922), ri \\
8 & \(\alpha\)-cannigrene & 1755 & 1753 [21] & ms (845, 894), ri \\
4 & \(\alpha\)-curcumene & 1780 & 1780 [22] & ms (876, 892), ri \\
7 & Nootkatene & 1829 & 1815 [24] & ms (872, 887), ri \\
11 & Cuparene & 1838 & 1825 [24] & ms (906, 915), ri \\ \hline \end{tabular} a
[FOOTNOTE:a]Footnote a: footnotetext: Unidentified compounds are not listed. b  
NDFOOTNOTE]  
\end{table}
Table 1: Sesquietrpene hydrocarbons identified in the headspace of 30-day-old _Arabidopsis thaliana_.  
Figure 6: Relative mRNA levels of _Arabidopsis thaliana_ defense-related genes upon nootkatone exposure quantified by using qRT-PCR. Fold change values were calculated and represent changes in mRNA levels in _Arabidopsis_ plants cultivated on a medium mixed with 5-ppm nootkatone relative to that of _Arabidopsis_ cultivated on a medium mixed with 0.5% DMSO as the control. Data are averages \(\pm\) SE of three biologically independent experiments, each consisting of three technical replicates. _A. thaliana_\(\beta\) tubulin transcript was used as an internal control to normalize the data. The asterisk marks indicate significant differences based on a \(t\)-test (\(p<0.05\)). SA: salicylic acid, JA: jasmonic acid and ET: ethylene.