## 3 Discussion  
CYP716A is the major subfamily that exhibits catalytic activity in the conversion of \(\beta\)-amyrin to oleanonic acid [35]. Ever since CYP716A12 was first described as a triterpenoxidizing enzyme [17], the CYP716 family has been systematically identified and named in plants such as _Aralia elata_ and has been reported to play complex roles in different plants [36]. _P. grandiflora_ contains more than 70 saponin monomers, which mainly belong to oleanane-type saponins [29]. However, systematic identification and nomenclature for the CYP716 family has not been performed. A total of 22 _PgCYP716_ genes were identified and classified into seven subfamilies in this study. Multiple sequence alignment results indicated that all 22 genes contained the complete P450 helix K region sequence and heme binding motif (Figure 1A). Through the analysis of the evolutionary tree, the CYP716U, CYP716D, and CYP716 BV subfamilies were clustered with the CYP716A subfamily, which catalyzes the synthesis of oleanolic acid from \(\beta\)-amyrin (Figure 1B). It was hypothesized that these three gene families also have the same catalytic function. In addition, the _PgCYP716s_ subfamily was clustered with PgCYP716A53v2 from _P. ginseng_ (Figure 1B). The function of PgCYP716A53v2 is to catalyze the formation of protopanaxatriol from protopanaxadiol. Therefore, the PgCYP716S subfamily in _P. grandiflora_ may be involved in catalyzing the formation of protopanaxatriol from protopanaxadiol (Figure 1B) [28]. _P. ginseng_, _P. grandiflora_, and _P. quinatefolium_ are traditional Chinese medicinal plants, with saponins as their main active ingredients [37]. No collinearity was observed between the _CYP716_ genes in _P. grandiflora_ and those in the traditional Chinese medicines _P. pseudoginseng_ and _P. ginseng_; however, collinearity was observed in the model plant A. thaliana and a woody plant _A. eleta_ (Figure 3B), presumably due to the different types of saponin compounds. Various abiotic stresses such as drought, temperature fluctuations, UV radiation, and heavy metals are common challenges in plant growth environments. These environmental factors enhance plant resistance to external stresses by regulating the levels of phytohromones (e.g., methyl jasmonate (MeJA), abscisic acid (ABA), and salicylic acid (SA)) in plants. Phytohromones, as important signaling molecules, play a key role in regulating the synthesis of bioactive compounds in medicinal plants and are also one of the important mechanisms by which external biotic and abiotic stresses promote the accumulation of secondary metabolites in medicinal plants [38]. Analysis of the _cis_-acting elements of the promoter of the _PgCYP716_ genes revealed a large number of abiotic stress responses, including cold and drought stress, and hormone response elements, such as MeJA and SA (Figure 4). The analysis of the expression of _PgCYP716s_ under MeJA treatment indicated that MeJA could induce the expression of _PgCYP716s_ (Figure 5C; Figure 6C). In addition to abiotic stresses, transcription factors play an important role in regulating the biosynthesis of active ingredients in medicinal plants [39]. In _Artemisia annua_, the activation of _AaMYB15_, an R2R3-MYB transcription factor, was triggered by darkness and MeJA treatment. Moreover, _AaMYB15_ suppresses the biosynthesis of artemisinin by directly interacting with the promoter of _AaORA_[40]. The promoters of _PgCYP716s_ contained a large number of MYB and MYC transcription factor binding sites, suggesting that MYB and MYC transcription factors may affect the accumulation of saponins by regulating the transcription level of _PgCYP716s_ (Figure 4). Five _PgCYP716_ genes were identified and functionally verified in _P. grandiflora_; they catalyze the synthesis of oleanolic acid from \(\beta\)-amyrin (Figure 1B). For more comprehensive understanding of the functions of the remaining genes in this family, we cloned the remaining 17 genes of this family, successfully isolating 11 genes.