## 2 Results
### Insect Attack-Uregulated CsAOS2 Functions in JA Synthesis in Tea Flowers
#### 2.1.1 Phylogenetic Analysis of CsAOSs and Their Subcellular Localization
The first method involved prodding 12 holes in each leaf using a needle. The second method involved cutting the leaves into eight parts. Different treatments led to different levels of JA increase in the _CsAOS2_ overexpression group. This is because the intensity of the mechanical damage can affect the degree of expression changes observed for several genes in the JA synthetic pathway; these variation ranges may be higher than the overexpression level of _CsAOS2_. Figure 3: Comparison of JA contents in control and overexpression of CsAOS2 in _N. benthamiana_. (**A**) The treated leaves were harvested at the indicate time points 0 h, 1 h, and 12 h (\(n=6\)), after mechanical damage; (**B**) Mechanical damage method 1 was damage by making 12 holes on one leaf using needle. Mechanical damage method 2 was damage by cutting one leaf into eight pieces. The treated leaves were harvested at 1 h, after mechanical damage. Data represent the mean value \(\pm\) standard deviation. Figure 2: Subcellular localizations of CsAOS1 and CsAOS2. The CsAOS1-GFP and CsAOS2-GFP fusion proteins were transiently expressed in _Nicotiana benthamiana_. Green fluorescence indicated CsAOS1-GFP and CsAOS2-GFP fusion protein. Red fluorescence showed the chloroplast auto-fluorescence. In Contrast to Tea Leaves, the Metabolite Profiles of Tea Flowers Were Not Significantly Affected by JA Treatment  
It is well-known that JA can affect metabolite profiles in plants, especially vegetative parts, for example, the secondary metabolites in _Nicotiana attenuata_ leaves [32]. To investigate whether JA affect the metabolite profiles in tea flowers, tea flowers were treated with JA (2.5 mM) for 10 h (from flower half-open stage to fully-open stage) and metabolite profiles were subsequently analyzed by gas chromatography-mass spectrometry (GC-MS, for volatile metabolites) and ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS, for nonvolatile metabolites). Emitted volatile and internal volatile profiles were not significantly changed in JA-treated tea flowers (Figure 4A). In the present study, UPLC-QTOF-MS was equipped with either an ACQUITY UPLC BEH Amide column (suitable for determination of high polarity metabolites) or an ACQUITY UPLC HSS T3 C18 column (suitable for determination of low polarity metabolites). In analyses with separations using both columns and positive or negative MS modes, nonvolatile metabolite profiles were also not significantly changed in JA-treated tea flowers (Figure 4B). In contrast, we also investigated the effects of the same JA treatment on metabolite profiles in tea leaves. Unlike tea flowers, volatile metabolite profiles and partial nonvolatile metabolite profiles of tea leaves were changed (Figure 5). These results suggest that the responses of metabolites to JA in tea flowers was weaker than in tea leaves. Figure 4: Effect of JA treatment on metabolite profiles of tea flowers. (**A**) JA treatment: 2.5 mM JA dissolved in 0.5% ethanol; control: distilled water dissolved in 0.5% ethanol. Principal component analysis (PCA) of emitted volatiles and internal volatiles, which were analyzed by GC-MS (\(n=5\)). (**B**) PCA analysis of nonvolatile metabolites, which were analyzed by UPLC-QTOF-MS (\(n=5\)).