## 2 Results
### Screening of Candidate Genes Related to Terpenoid Biosynthetic Pathways  
The biosynthesis of terpenoids in plants is mainly from acetyl-CoA via the MVA pathway and glyceraldehyde phosphate and pyruvate via the MEP pathway. In such pathways, IPP and different terpenoids are synthesized through a series of catalytic reactions. By integrating homology-based BLAST and phylogenetic analysis, isoforms involved in the terpenoid metabolic pathway were screened from the transcriptome database of _P. lucliflora_ cv. Zifengyu (Figure 5A). To note, two acetyl-CoA acetyltransferaseases (AACTs), two hydroxymethylglutaryl-CoA synthases (HMGSs), two hydroxymethylglutaryl-CoA reductase (HMGRs), mevalonate kinase (MVK), phosphomevalonate kinase (PMK), two mevalonate diphosphate decarboxylases (MVDs), isopentenyl-diphosphate \(\delta\)-isomerase (IDI), and two farnesyl diphosphate synthases (FPPSs) were annotated into the upstream of MVA pathway. The 1-deoxy-D-xylulose-5-phosphate synthase (DXS), 1-deoxyxylulose-5-phosphate reducto isomerase (DXR), 2-C-methyl-D-erythnitol 4-phosphate cytidylyl-transferase (MCT), 4-diphosphocytidyl-2-C-methyl-D-erythnitol kinase (CMK), 2-C-methylerythnitol-2,4-diphosphate synthase (MDS), 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (HDS), hydroxymethylbutenyl diphosphate reductase (HDR), and geranyl diphosphate synthase (GPPS) were annotated into the upstream of the MEP pathway. ACCT1, HMGS1, HMGR1/2, and IDI were highly expressed across the three flowering stages, signifying their input to sesquiterpene backbone biosynthesis in herbaceous peonies. Similarly, DXS, DXR, MDS, HDS, and HDR were highly expressed across the three flowering stages, indicating their contribution to monoterpene backbone biosynthesis in peonies. Notably, MEP and MVA pathway genes exhibited comparable expression levels. However, the expression level of GPPS was relatively higher than FPPS, which may be associated with a higher production of monoterpenes than sesquiterpenes. Generally, these findings provided insights into specific pathway enzymes that are associated with the intricate mechanisms underpinning terpenoid synthesis in herbaceous peony at different flowering stages.  
A total of eight _TPS_ genes were obtained from the transcriptome database of _P. lucliflora_ cv. Zifengyu, among which _TPS_2 and _TPS_3 have been identified before [41; 42]. The phylogenetic analysis of PITPSs with proteins from other plants (Table S3) showed that TPS1, TPS2, TPS4, TPS6, TPS7, and TPS8 fall into angiosperm-specific TPS-a while TPS3 and TPS5 into TPS-b clades (Figure 5B). Amino acid sequence alignment displayed salient TPS features such as the presence of R(R,P,K)(X)\({}_{8}\)W, DDX(D/E), and (N,D)DXX(S,T,G) XXXE (NSE/DTE) motifs involved in catalysis (Figure S2). Even so, they are solely required for their catalytic features, and the diversity of TPS enzyme-mediated products in plants cannot be entirely accounted for by these conserved motifs. Transcriptome analysis showed that _TPS_2, _TPS_3, and _TPS_8 had higher expression levels than their homologs, suggesting their more dominant roles in catalyzing terpenoid formation (Figure 5C). Subcellular localization prediction analysis revealed that all eight TPSs were supposed to localize into chloroplasts, suggesting their possible roles in converting GPP into monoterpenes (Figure 5D). Nevertheless, comprehensive functional characterizations of these TPSs are still required to completely elucidate their roles in terpenoid biosynthesis. Overall, these findings offer details on the diversity, evolution, and expression patterns of terpenoid metabolic genes in _Paeonia_ genus, enabling us to investigate their functions in terpene biosynthesis in the future.