## 2 Results
### PgCYP716s Overexpression Elevated the Saponin Content  
Yeast heterologous expression analysis revealed that all four enzymes catalyzed the conversion of \(\beta\)-amyrin into oleanolic acid. To further confirm the function of these enzymes in _P. grandiflora_ ontogeny, their functions were validated using the hairy root transformation established in our laboratory [34]. The transgenic hairy roots of four _PgCYP716_ genes were generated via _Agrobacterium_-mediated transformation (Figure 8A; Supplementary Figure S1). The metabolite content of the transgenic hairy roots was evaluated. The four genes could increase the content of \(\beta\)-amyrin, total saponin, platycoside E, platycodin D3, and platycodin  
Figure 7: In vivo enzymatic activity assay in yeast. (**A**) Detection of the yeast products using the vanillin-perchloric acid method indicated that the four enzymes could catalyze the production of oleanolic acid from \(\beta\)-amyrin. The top figure shows yeast-induced products, and the bottom figure shows oleanolic acid detected using the vanillin-perchloric acid method. (**B**) Schematic representation of the construction of PgbAS1 and the yeast heterologous expression vectors with four PgCYP716s. (**C**) HPLC analysis of yeast cultures indicated that PgCYP716A264, PgCYP716A391, PgCYP716A291, and PgCYP716 BWv3 could produce oleanolic acid. (**D**) A summary of the biochemical reactions catalyzed by four PgCYP716 enzymes.  
D (Figure 8B-E). Oleanolic acid content in the transgenic hairy roots with overexpressed _PgCYP716A264_, _PgCYP716A391_, and _PgCYP716A291_ exhibited an increase, whereas that in the transgenic hairy roots with overexpressed _PgCYP716 BWv3_ exhibited a decreasing trend (Figure 8E). The macromolecules, namely, soluble sugars and starch, were the key upstream substrates for the synthesis of saponins. In the four transgenic hairy roots, the content of soluble sugars and starch exhibited a decreasing trend compared with the control group. This indicated that in the process of producing downstream specialized metabolites, the upstream macromolecules such as soluble sugars and starch underwent degradation and transformation, leading to reduced content (Figure 8B-E). qRT-PCR was performed to detect the expression levels of functional genes in the saponin synthesis pathway (_PgSS_, _PgGPP52_, _PgGMS_, _PgHMGS_, and _PgSE_). The results showed that the overexpression of _PgCYP716A264_, _PgCYP716A391_, and _PgCYP716A291_ and _PgCYP716 BWv3_ genes could promote the expression of saponin synthesis pathway genes (Figure 8F-I). These data revealed that four PgCYP716 enzymes participated in catalyzing the production of oleanolic acid from \(\beta\)-amyrin, ultimately contributing to the accumulation of saponin content.  
Figure 8: _PgCYP716A264_, _PgCYP716A391_, _PgCYP716A291_, and _PgCYP716 BWv3_ genes were functionally validated in the hairy roots of _P. grandiflora_. (**A**) GFP fluorescence was detected in the hairy roots overexpressing _PgCYP716A264_, _PgCYP716A391_, _PgCYP716A291_, and _PgCYP716 BWv3_ genes. (**B–E**) Content of platycoside E, platycodin D3, \(\beta\)-amyrin, oleanolic acid, platycodin D, total saponin, soluble sugar, and starch in the WT and transgenic hairy roots. (**F–I**) qRT-PCR analysis of the expression of saponin synthesis pathway genes in overexpressed hairy roots. Asterisks indicated significant differences (\(p<0.05\)).