## 2 Results  
### Insect Attack-Uregulated CsAOS2 Functions in JA Synthesis in Tea Flowers  
#### 2.1.1 Phylogenetic Analysis of CsAOSs and Their Subcellular Localization  
In our previous study, we investigated the effects of _T. hawaiiensis_ treatments on the expression levels of JA synthesis-related genes in tea flowers. Only _CsAOS2_ expression was upregulated by _T. hawaiiensis_ treatment, while other JA synthesis genes were not significantly affected [16]. To further validate the function of CsAOS2 in JA synthesis in tea flowers, in the present study, we first compared the homologous protein sequences of AOSs among different plant species, including GmAOS1 (_Glycine max_), GmAOS2, HvAOS1 (_Hordeum vulgare_), HvAOS2, SIAOS1 (_Solanum lycopersicum_), SIAOS2, SIAOS3, SIAOS1 (_Solanum tuberosum_), StAOS2, StaAOS3, NaAOS (_Nicotiana attenuata_),CsAOS1 (_C. sinensis_), CsAOS2, LuAOS (_Linnum isitatissimum_), AtAOS (_Arabidopsis thaliana_), OsAOS (_Oryza sativa_), and ZmAOS1 (_Zea mays_) [22; 23; 24; 25] (Figure 1). The phylogenetic analysis indicated that CsAOS1 had lower similarity with CsAOS2, which only showed 55.01% (Figure 1). It was reported that wounding causes the release of linoleic acid (LA) from chloroplast lipids, catalyzed by lipoxygenenses (LOX), AOS, and allene oxide cyclase (AOC) to 12-oxo-phytodienoic acid. 12-oxo-Phytodienoic acid produced in the chloroplast is transported to the peroxisome where it is used to synthesize JA [26; 27; 28; 29]. In the present study, we investigated the subcellular localization of CsAOS1 and CsAOS2 by GFP-fusion protein pCAMBIA3300. CsAOS1 was located in the plasma membrane and CsAOS2 was detected in the chloroplast membrane (Figure 2). 1.2 Transient Expression Analysis in _N. benthamiana_ Plants Confirmed That CsAOS2 Functioned in JA Synthesis  
To further confirm whether CsAOS2 functions in JA synthesis, transient expression analysis in _N. benthamiana_ plants was employed (Figure 3). As the JA synthetic pathway derived from LA involves several enzymes/genes, the overexpression of individual genes may be insufficient to increase JA content. Therefore, mechanical damage was used to activate the genes in the JA synthetic pathway. In the present study, there were two groups of _N. benthamiana_ plants including control (vector) and _CsAOS2_ overexpression plants. These two groups of plants were treated by mechanical damage. As shown in Figure 3A, JA content in the control increased at 1 h after mechanical damage (damage by making 12 holes on one leaf using needle) because of upregulation of the genes in the JA synthetic pathway. Furthermore, the _CsAOS2_ overexpression group had higher JA content than the control  
Figure 1: Phylogenetic analyses of plant allene oxidase synthases (AOSs) related to the function of jasmonic acid (JA) synthesis. Phylogenetic tree was performed by software MEGA 5.1 with neighbour joining computation. SI: _Solanum lycopersicum_; St: _Solanum tuberosum_; Na: _Nicotiana attenuata_; Cs: _Camellia sinensis_; Gm: _glycine max_; Lu: _Linum usitatissimum_; At: _Arabidopsis thaliana_; Os: _Oryza sativa_; Zm: _zea mays_; Hv: _Hordeum vulgare_. The protein sequences of AOS were SIAOS2 (NP_001274707.1), StAOS2 (CAD29736.2), NaAOS (CAC82911.1), SIAOS1 (CAB88032.1), StaOS1 (CAD29735.1), CsAOS2 (AHY03308.1), GmAOS2 (NP_001236445.1), GmAOS1 (NP_001236432.1), LuAOS (P48417.1), AtAOS (CAA63266.1), OsAOS (AAL17675.1), ZmAOS1 (NP_001105244.2), HvAOS1 (CAB86383.1), HvAOS2 (CAB86384.1), CsAOS1 (BAU24784.1), SIAOS3 (NP_001265949.1), and StAOS3 (CA130876.1). group at 1 h after the mechanical damage, indicating that CsAOS2 functions in JA synthesis. At 12 h after mechanical damage, JA content in both groups decreased; this was reasonable given that the JA response is relatively quick when plants are exposed to stresses [30; 31]. We also investigated the effect of mechanical damage treatments of different intensities on functional characterization of _CsAOS2_ at 1 h after mechanical damage. Figure 3B shows the two mechanical damage methods.