## 2 Results  
### Identification of \(\alpha\)-Farnesene Synthases in Tea Plants  
To identify the \(\alpha\)-farnesene synthase (CsAFS) in tea plants, we compared homologous sequences of monoterpene and sesquiterpene synthases from the TPS family. Accordingly, we selected the syntheses of sesquiterpene, farnesene, \(\alpha\)-farnesene, (\(E\))-\(\beta\)-farnesene, (\(E\),\(E\))-\(\alpha\)-farnesene, and (\(E\))-\(\beta\)-ocimene in different plants belonging to the TPS-a, TPS-b, TPS-g, and TPS-d1 subfamilies (Figure 2A). Phylogenetic analysis indicated that CsAFS had a high similarity (66%) to MdAFS and farnesene synthase in _Pyrus communis_ (pear) (Figure 2A), while only MdAFS in apple was related to (\(E\),\(E\))-\(\alpha\)-farnesene formation. These results suggested that the _CsAFS_ probably encoded \(\alpha\)-farnesene synthase in tea plants. To validate the enzyme function of CsAFS in \(\alpha\)-farnesene synthesis in tea leaves, cDNA of CsAFS was cloned into _Escherichia coli_ (\(E\). _coli_) expression vector pET32a, and the obtained recombinant protein was assayed for CsAFS function (Figure 2B). We used solid-phase microextraction (SPME) to collect products and utilized gas chromatography-mass spectrometry (GC-MS) to perform analysis. The results showed that recombinant protein CsAFS converted substrate famesyl diphosphate (FPP) into \(\alpha\)-farnesene as the final product. Furthermore, with geranyl diphosphate (GPP) as substrate, the CsAFS enzyme produced only \(\beta\)-ocimene, with no \(\alpha\)-farnesene detected. No terpene products were found in the control, which carried the empty pET32a vector (Figure 3A,B). Our analysis of the recombinant CsAFS protein showed that the enzyme can produce \(\alpha\)-farnesene or \(\beta\)-ocimene _in vitro_ depending on the substrate. CsAFS enzyme activity produced \(\alpha\)-farnesene as the only product from FPP as substrate. Figure 2: Phylogenetic analysis and SDS-PAGE analysis of \(\alpha\)-farnesene synthase CsAFS. **(A)** The phylogenetic tree of \(\alpha\)-farnesene synthase in different plant species, which was obtained by using MEGA-7 software with the neighbor-joining computation. Am, _Antirrhinum majus_; At, _Arabidopsis thaliana_; Cs, _Cucumis sativus_; Eo, _Eleais oleifera_; Md, _Malus domestica_; Mp, _Mentha x piperia_; Pa, _Picea abies_; Pc, _Pyrus communis_; Pm, _Pseudotsuga menziesii_; Pt, _Pinus taeda_; CsAFS, \(\alpha\)-farnesene synthase in _Camellia sinensis_. **(B)** SDS-PAGE analysis of recombinant CsAFS expressed in _Escherichia coli_ (\(E\). _coli_). Black arrows indicate target proteins. To further confirm the CsAFS function _in vivo_, we investigated the subcellular localization of CsAFS using green fluorescent protein (GFP)-fusion protein pCAMBIA3300. GFP analysis of epidermis cells from several independent tobacco plants transformed with CsAFS-GFP construct showed green fluorescence located in the cytoplasm, which clearly indicated that the CsAFS protein was targeted to the cytoplasm (Figure 4). GC-MS analysis of the products in transformed _Nicotiana benthamiana_ showed that the CsAFS enzyme produced \(\alpha\)-farnesene as a single product _in vivo_ (Figure 3C). These findings supported that CsAFS probably used FPP as substrate to form \(\alpha\)-farnesene in tea plants. Figure 3: GC-MS analysis of the products formed by recombinant CsAFS enzyme _in vitro_ and _in vivo_. (**A**) Functional identification of CsAFS _in vitro_ with farnesyl diphosphate (FPP) as substrates; (**B**) Functional identification of CsAFS _in vitro_ with GPP as substrates. CK 1, empty pET32a vector; T 1, recombinant CsAFS-PET32a protein. (**C**) Functional identification of CsAFS _in vivo_. The CsAFS-green fluorescent protein (GFP) fusion protein was transiently expressed in _N. benthamiana_. CK 2, empty GFP vector; T 2, recombinant CsAFS-GFP protein.