## 3 Discussion  
With the deepening of research on the pharmacological effects of _S. milliorrhiza_[2; 38; 39; 40], its commercial value is being gradually recognized, and the market demand is constantly increasing. However, wild resources and existing artificial cultivation yield and quality are unable to meet the demand [41]. Expanding the planting area of _S. milliorrhiza_ and increasing the content of medicinal active ingredients are very necessary. Drought stress is a common type of stress faced by plants [42], and planting _S. milliorrhiza_ in arid areas is one of the important means to alleviate its resource shortage problem. As an important enzyme gene for carotenoid catalysis and ABA synthesis, research has shown that the CCO gene family plays an important role in regulating plant resistance to stress [43; 44]. Studying the molecular mechanism of drought resistance and the accumulation mechanism of secondary metabolites in _S. milliorrhiza_ is a prerequisite and a foundation for improving the yield and quality of _S. milliorrhiza_.  
_S. milliorrhiza_ is a model plant among medicinal plants [45]. Its growth is often affected by various types of environmental stress [46; 47; 48]. Therefore, in-depth research on the regulatory mechanism of stress resistance genes in _S. milliorrhiza_ is of great practical significance. The CCO gene family can regulate plant responses to various types of abiotic stress and has been extensively studied in various plants [49; 50; 51]; the identification and characterization of CCO based on the whole-genome sequence of _S. milliorrhiza_ were reported for the first time. In this study, 26 CCO members were characterized in _S. milliorrhiza_. Compared with _A. thaliana_[19], _Glycine max_[52], and _Malus domestica_[49], _S. milliorrhiza_ has a larger CCO family. CCO is a type of double-cleavage diooxygenase that contains the RPE65 domain and generates secondary metabolites during carotenoid metabolism [53]. The secondary metabolites are crucial for carotenoid metabolism and aroma volatiles. Fe\({}^{2+}\) can activate the catalytic activity of the enzyme [54; 55]. The four conserved histidine residues in CCO can regulate their binding to Fe\({}^{2+}\). In addition, several studies have confirmed that the conservation of CCO protein sequences is not high. Carotenoids are precursors of important plant hormones, such as ABA [56]. The latter plays an important role in many aspects of the plant life cycle, especially in the response of plants to various types of environmental stress [57].  
Figure 6: Detection of contents of abscisic acid (**A**) and MDA (**B**), H\({}_{2}\)O\({}_{2}\) accumulation (**C**), and CAT (**D**) and SOD activity (**E**) in WT and overexpression (OE) strains. All data represent means \(\pm\) SDs of three independent experiments. Statistical significance was assessed with Student’s \(t\)-test (**\(p<0.01\); * \(p<0.05\)).  
Most studies on the function of the _CCD4_ gene focus on plant coloration [58; 59; 60; 61]. However, there is almost no information on the regulation of medicinal active ingredients and drought resistance by _CCD4_ in medicinal plants. Therefore, this study first cloned the coding sequence of _SmCCD4_ in _S. milliorrhiza_ and found through RT-qPCR that _SmCCD4_ responds to ABA induction and PEG stress. Secondly, an overexpression vector for _SmCCD4_ was constructed, and a total of eight transgenic strains were obtained. By measuring the content of salvianolic acids and tanshinones in different strains, the _SmCCD4_ gene in _S. milliorrhiza_ can upregulate key genes in the biosynthesis pathways of these two secondary metabolites, thereby promoting the accumulation of the compounds. PEG was used to simulate drought stress conditions, and it was found that the ABA content in _SmCCD4_ OE strains was significantly increased, and compared with WT strains, the OE strains showed better drought resistance.