## 2 Results
### Phylogenetic Tree and Multiple Sequence Alignment Analysis of PsTPS1  
Phylogenetic tree construction by downloading 32 representative TPS protein sequences of 9 other species from the TPS-a, TPS-b, TPS-c, TPS-d, TPS-e/f, and TPS-g subfamilies, respectively, showed that PsTPS1 clustered in the TPS-a subfamily (Figure 5A). And PsTPS1 also had a shorter branch length in the phylogenetic tree (Figure 5A), indicating that it differentiated later than other plants, the result that was the same as phylogenetic tree of PsHMGR1 in Figure 3A. In addition, PsTPS1 showed the closest homology to PdTPS (TPS of _P. delavayi_), which also belonged to the genus _Paeonia_, in agreement with the plant  
Figure 4: The electrophoresis map of the _PsTPS1_ gene and PsTPS1 protein characterization. (**A**) Electrophoresis map of _PsTPS1_ gene. M, marker. (**B**) Secondary structure prediction, (**C**) 3D model building, (**D**) transmembrane helices prediction, (**E**) hydrophobicity or hydrophilicity analysis, (**F**) signal peptide analysis, and (**G**) conserved domains analysis of PsTPS1 protein. taxonomy, as well as clustering on the same branch with PITPS of _P. lactisflora_, PdTPS2 of _P. delawayi_, and LfTPS01 of _Liquidambar formosana_ (Figure 5A). Based on the results of the tree, three TPSs were selected for multiple sequence alignment analysis. The results of multiple sequence alignment indicated that PsTPS1 had the conserved R(R,P,Q)(X)\({}_{8}\)W (motif I) at the N-termini that could catalyze the cyclization of substrates, as well as two typical DDXXD (motif II) and NSE/DTE (motif III) motifs at the C-termini that can be involved in the binding of divalent metal cations Mg\({}^{2+}\) or Mn\({}^{2+}\) (Figure 5B). Compared to the amino acid sequences of other species, the PsTPS1 protein had the highest identity to the TPS protein of _P. delawayi_ (PdTPS) (99.10%), followed by the PITPS (95.15%) and LfTPS01 (70.18%) (Figure 5B). Figure 5: Phylogenetic tree and multiple sequence alignment of TPS proteins. (**A**) Phylogenetic tree of PsTPS1 and TPS proteins from other 12 plant species using the NJ method by MEGA X software V10.2.6. Bootstrap values are shown as a percentage of 1000 replicates. PsTPS1 is marked with a green dot. Clades of TPS-a, TPS-b, TPS-c, TPS-d, TPS-e/f, and TPS-g are highlighted with different color lines. At, _Arabidopsis thaliana_; SI, _Solanum lycopersicum_; Mt, _Medicago truncatula_; Ag, _Abies grandis_; _Lf. Liquidambar formosana_; Ma, _Melia aezdarach_; Pa, _Populus alba_; Pd, _Paoenia delawayi_; Pl, _Paoenia lackiflora_; Vr, _Vitis riparia_; Vv, _Vitis vinifera_; Rh, _Rosa hybrida_. (**B**) Alignment and analysis of PsTPS1 with TPS protein sequences of _P. delawayi_, _P. lactiflora_, and _L. formosana_. Motif I, R(R,P,Q)(X)\({}_{8}\)W_; motif II, DDXXD; motif III, (N,D)DX(S,T,G)XXCE (NSE/DTE). Accession information of TPS proteins of other 12 plant species is detailed in Supplemental Table S2. PsHMGR1 and PsTPS1 Genes Show Different Expression Patterns in Flower Development Stages and Different Tissues  
The expression patterns of _PsHMGR1_ and _PsTPS1_ were analyzed by qRT-PCR in _P. suffrruticossion '_Oukan'. The results indicated that both _PsHMGR1_ and _PsTPS1_ were expressed at every flower developmental stage and every organ and tissue (Figure 6). _PsHMGR1_ expression levels increased from Stage 1 to Stage 3 and then decreased to Stage 4 (Figure 6A). The expression level of _PsTPS1_ also showed a trend of increasing and then decreasing, with the highest expression level at Stage 2 (Figure 6B). The expression levels of the _PsHMGR1_ and _PsTPS1_ at different flower development stages were consistent with the release amount of floral fragrance volatile from 'Oukan' [42], demonstrating that they may play an important role in floral volatile biosynthesis.