## 4 Discussion  
Linalool is an important secondary metabolite in linalool-type _Cinnamomum camphora_[5]. In our study, the linalool content was significantly increased during leaf development stages (Supplement Figure S2). This result suggested that the linalool is continuously synthesized and accumulated during the development of the leaf, which is consistent with the previous study [28]. The content of linalool in 65-day-old leaves was significantly higher than that in 15-day-old leaves. To elucidate the molecular mechanisms in proteomics underlying the regulation of isoprenoid biosynthesis at different development stages in _C. camphora_, we performed proteomic analysis of 15 d and 65 d leaves and identified 11,503 proteins, of which 11,076 proteins were annotated. Among these proteins, most proteins functioned in metabolism (Supplement Figure S3). Further analysis of differentially expressed proteins revealed that the expressions of 1245 proteins were induced; meanwhile, 1941 proteins were inhibited in 65d (Figure 2a). Previous research has verified that translation, RNA processing and DNA organization, energy generation, transport, and metabolic processes play important roles in regulating the leaf growth of _Arabidopsis thaliana_[29]. In our study, significant changes were also found in the processes of photosynthesis and metabolism at different development stages (Figure 2b,c). We detected some essential enzymes related to the biosynthesis of isoprenoids in _C. camphora_. Based on KEGG pathway enrichment analysis, in the category of biosynthesis of secondary metabolites, 21 proteins participated in the biosynthesis of isoprenoids, as catalytic enzymes were expressed differentially at different stages (Figure 3, Table S3). Notably, the expression of MCT, MDS, and AACT was significantly induced in the 65 d. Consistent with our result, a previous study has also verified that the overexpression of MDS promoted the accumulation of linalool content in _Osmanthus fragrans_[30]. By combining the metabolic analysis with the proteomic approach, we hypothesized that the expression changes of isoprenoid-related synthases at the protein level might be a major cause for the significant accumulation of linalool that we detected in the 65 d.  
Figure 4: Analysis of protein-protein interaction network. (**a**) Protein-protein co-expression network of differentially expressed candidate synthases related to isoprenoid synthesis. (**b**) Protein-protein interaction network of differentially expressed transcription factors. Circles represent differentially expressed transcription factors, and triangles represent other differentially expressed proteins. The protein-protein interaction network was then constructed to illustrate the internal connection among the isoprenoid-related synthases. We found that the DXR, IPPI, FPPS, VTE4, AACT, GGPPS, VTE1, crtlSO, PDS, NCED, ABA2, ZEP, MCT, HDR, and HDS were involved in the co-expression network, suggesting that these synthases constitute a complicated network and play crucial regulatory roles in the biosynthesis of isoprenoid. (Figure 4a). Moreover, in the proteome-wide protein-protein interaction analysis, we noticed a network that consisted of several transcription factors and other DEPs. Transcription factors, including BES1, bHLH, C3H, FAR1, MYB, NF-YC, Nin-like, WRKY, Whirly, and ZF-HD occupied the core position of the network and formed a sophisticated network by linking with many other DEPs (Figure 4b). This finding suggested that these transcription factors may act as candidate regulators in the leaf development of _C. camphora_. Consistent with this result, many studies have confirmed the important roles that BES1 [31], C3H [32], MYB [33], NF-YC [34], Nin-like, WRKY [35], ZF-HD [36], and bHLH [37; 38] families play in plant growth and development. In addition, the FAR1 [39], WRKY [40; 41], Whirly [42], and ZF-HD [43] families have been described as regulators of leaf senescence in previous research.