## 2 Results
### Screening of Candidate Genes Related to 2-PE Biosynthetic Pathway  
Previously, enzymes associated with the 2-PE biosynthetic pathway were characterized (Figure 6A). To further investigate the 2-PE pathway in peony and elucidate the genetic limitations underlying 2-PE biosynthesis in _P. lactiflora_ cv. Zifengyu, the homology-based BLAST and phylogenetic analysis approach were employed to mine 2-PE pathway genes from the transcriptome database of _P. lactiflora_ cv. Zifengyu. Consequently, four candidate AADCs, one candidate CYP79D, and nine candidate PARs or closely related GERs (geranial reductases) were identified (Figure 6B). Their transcripts during flower developmental stages were assessed using FPKM values from the transcriptome database, suggesting that the homologs may function differently in _P. lactiflora_ cv. Zifengyu (Figure 6C). As illustrated, there are at least four pathways for PALd biosynthesis in plants, whereas PAR is the only characterized enzyme responsible for PALd conversion into 2-PE (Figure 6A). To investigate the genetic mechanism underlying 2-PE deficiency in _P. lactiflora_ cv. Zifengyu, the highly expressed _PAR_ and _GER_ genes, subsequently designated as _PIPAR1_, _PIPAR2_, and _PIGER1_,  
Figure 5: Screening of candidate genes related to terpenoid biosynthetic pathways. **(A)** The expression profiles of putative genes encoding enzymes involved in terpene backbone biosynthesis. Full names of the MVA and MEP pathway enzymes appear in the main text. **(B)** Phylogenetic relationship between TPS proteins from _Pconia_ and other plants. Bootstrap values are shown as a percentage of 1000 replicates. The PITPS proteins in this study are highlighted (red). The TPS-clades are highlighted in different color patterns. Accession numbers are given in Table S3. **(C)** Relative expression level of _PITPS_ at different flowering stages. Data were obtained from FPKM values of RNA-seq. **(D)** Subcellular localization. The PITPS proteins were predicted to be localized in the chloroplasts. were cloned and functionally characterized via in vitro enzymatic assays. Consequently, both PIPAR1 and PIGER1 efficiently converted PALd into 2-PE, albeit with traces of PALd, while no 2-PE was detected in reactions catalyzed by PIPAR2 (Figure 6D). The results suggest that the absence of PALd, rather than the inactivation of enzymes catalyzing PALd transformation, blocks 2-PE biosynthesis in _P. lactisiflora_ cv. Zifengyu. To further validate this hypothesis, we tentatively cloned the highly expressed _PIAADCI_ candidate from _P. lactisiflora_ cv. Zifengyu without 2-PE, as well as from _P. lactisiflora_ cv. Liantai with a high amount of 2-PE. As expected, _PIAADCI_ from _P. lactisiflora_ cv. Zifengyu encoded an AADC protein with a C-terminal truncation compared to that from _P. lactisiflora_ cv. Liantai (Figure 6E), possibly resulting from the alternative splicing of the same gene (Figure S3). Despite the lack of robust evidence, we tentatively inferred that the truncated form of AADC might block PALd formation. Interestingly, the cloned _PIAADCI_ from _P. lactisiflora_ cv. Liantai shared the same sequence with isoform_341577 from _P. lactisiflora_ cv. Zifengyu, suggesting that different splicing transcripts co-existed and the ratio between the full-length and truncation transcripts might determine the final PALd content. As expected, the highly expressed _AADC_ form in these two representative cultivars differed as depicted in Figure 6E using reverse transcription quantitative PCR (RT-qPCR). Consequently, the selective splicing of _AADC_ and the relative abundance of differential transcripts might explain the difference in 2-PE content among peny cultivars, at least between _P. lactisiflora_ cv. Zifengyu and _P. lactisiflora_ cv. Liantai. However, further studies on the precise functions of these genes and their interactions within the biosynthetic pathway is crucial for deciphering the genetic mechanisms underlying 2-PE absence in specific peny cultivars.