## 2 Results
### Identification of the Bifunctional Terpene Synthes RcTPS23  
TPS functions are often related to their subcellular localization (plastid or cytosol), and some TPSs can react with both GPP and FPP when expressed in vitro. The most common of these potentially bifunctional TPSs, whose products are monoterpene linalool and sesquiterpene nerolidol, have been reported in _A. thaliana_, strawberry, tomato, cotton, and other species (Figure 6A) [16; 43; 44; 45; 46]. BLAST (version 2.13.0) analysis of the protein sequences in our data revealed that most of these TPSs belong to the TPS-g subgroup and synnet Cluster126. We constructed a syntenic network of AtTPS14, FaNESI, GhITS12, and SlyTPS39 and found that most nodes were in subgroup TPS-g and belong to various species (Figure 6B). Two rose TPSs were present in this syntenic network. We cloned these genes and named them _RcTPS23_ (_RchiOBHmChr5g000471_) and _RcTPS10_ (_RchiOBHmChr2g0160421_). The sequence of _RcTPS10_ contained a frameshift. _RcTPS23_ reached its highest expression levels in stage FB_CP petals but was expressed at very low levels in stamens, stems, roots, and OF_PP stage petals (Figure 6C). We investigated the subcellular localization of RcTPS23 by expressing RcTPS23-eGFP fusion protein in _A. thaliana_ protoplasts (Figure 6D). Confocal laser scanning microscopy revealed that RcTPS23-eGFP localized to the cytosol, suggesting that _RcTPS23_ might function in sesquiterpene biosynthesis. Figure 6: Syntenic localization, spatiotemporal expression, and subcellular localization of RcTPS23. (**A**) Catalog of TPS enzymes capable of catalyzing the substrates GPP and FPP to produce linalool or  
neroidold [16; 43; 44; 45; 46]. (**B**) Syntenic analysis of key TPSs, including AtTPS14, FaNES1, GhTPS12, and SlyTPS39 (highlighted with red asterisks). Each node is annotated with species abbreviations and color-coded: nodes in the outer loop represent different plant groups--super-asterids (light blue), super-rosids (light orange), basal eudicots (light purple), monocots (light green), _L. chitense_ and _A. trichopoda_ (light gray); nodes in the inner loop represent TPS subgroups, coded as TPS-a (orange), TPS-b (yellow), TPS-c (purple), TPS-e/f (green), and TPS-g (blue). (**C**) Expression analysis of _RcTPS23_ across four developmental stages of rose petals and various tissues, conducted via qRT-PCR: FB_GP (green petals in a flower bud), FB_CP (petals changing color in a flower bud), FB_PP (pink petals in a flower bud), and OF_PP (pink petals in an open flower). Expression data are expressed as mean \(\pm\) standard deviation from three biological replicates. (**D**) Subcellular localization of 355::RcTPS23-eGFP fusion protein (green) in _A. thaliana_ protoplasts. Using 355::GFP as the control. Red fluorescence corresponds to chloroplast autofluorescence detected under 488 nm excitation. Scale bars = 10 \(\upmu\)m.  
Finally, to perform functional analysis, we heterologous expressed _RcTPS23_ in _Escherichia coli_ strain BL21 (DE3) and analyzed the in vitro chemical products of the resulting recombinant proteins using different substrates by GC-MS. The product produced by RcTPS23 and the substrate FPP was (_E_)-neroidol (Figure 7A,B). However, linalool was produced when GPP was used as the substrate (Figure 7C,D). In these assays, linalool and nerolidol were identified by comparing mass spectra. Each reaction had a blank control and three biological replications. In summary, we used the syntenic relationships of TPSs to identify the TPS-g subgroup member _RcTPS23_ and analyzed its gene expression pattern in rose. We determined that RcTPS23 was a bifunctional terpene synthase that produces nerolidol and linalool in vitro and might possess nerolidol biosynthetic activity in vivo due to its localization in the cytoplasm. Figure 7: Dual catalytic activity of RcTPS23 in producing (_E_)-neroidol and linalool in vitro.