## 3 Discussion
Terpenoids, including monoterpenes (C10), sesquiterpenes (C15), and polyterpenes, are the most diverse natural products in nature and are also the most intensively studied compounds around the world. Plants first synthesize the terpenoid precursors dimethylallyl diphosphate (FPP) and isoprene pyrophosphate (GPP) through two pathways: mexulonic acid (MVA) and methylerythritol (MEP) [11; 12]. FPP is further used to synthesize sesquiterpenoid and triterpenoids, while GPP further serves to synthesize monomers and diterpenoids. The differential metabolites in the IFS and FFS in this study were mainly sesquiterpenoids and monoterpends, and all terpenoids were up-regulated except for (E, E)-Farnesol. HMGCS, HMGR, MK, and PPMD are the key enzymes of the MVA pathway for the synthesis of terpenoid skeletons [11], while DXS, DXR, MCT, CMK, MDS, HDS, and HDR are the key enzymes for the synthesis of terpenoid skeletons in the MEP pathway [11]. The expression levels of these key enzyme genes gradually increase during flowering and decrease after full flowering [35; 36; 37]. In this study, the expression levels of differentially expressed genes in the terpenoid skeleton synthesis pathway were up-regulated in the FFS except for DXS. These results indicate that the synthesis of terpenoids gradually increased during the flowering process of chestnut, further confirming the conclusion that terpenoids are the main components of chestnut volatiles. _DXS_ is the first transketolase in the MEP pathway and also the highest rate-limiting enzyme [38]. The overexpression or inhibition of _DXS_ can cause changes in the contents of downstream metabolites. Silencing the _SIDXS2_ gene in tomato caused a decrease in the \(\beta\)-phellandrene content [39]. However, the activity of _DXS_ is also subject to feedback regulation by 3-isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). IPP and DMAPP will competitively bind to the active center of the DXS enzyme and disengage TPP, thereby inhibiting the activity of the DXS enzyme [40]. In this study, there was no significant difference in each product of the terpenoid backbone synthesis pathway from the IFS to FFS; however, the contents of sesquiterpenoids, monomers, and synthetic genes were up-regulated in the FFS (Figure 6). This suggests that substances in the DMEP pathway do not decrease the activity of the DXS enzyme during chestnut blooming, which may be the reason for the decreased expression of _DXS_ in the FFS. Figure 6: Schematic diagram of terpenoid synthesis pathway from the first flowering stage to the full flowering stage of chestnut. Red indicates the up-regulated genes or VOCs. Blue indicates the down-regulated genes or VOCs. Acetophenone has a bactericidal function and has been applied in the disease control of plant cultivation and harvested agricultural products. After fumigation with 100 \(\upmu\)L/L acetophenone for 12 h, it showed good antifungal activity against the anthracose pathogen in postharvest chili fruit [41]. Acetophenone showed antifungal activity to _Penicillium italicum_[42]. The currently used acetophenone mainly comes from artificial synthesis. In fact, acetophenone could be biosynthesized through styrene metabolic pathway in bacteria such as the _Azaroxus_-like strain EbN1 and _Streptomyces globisporus_ JK-1 [13; 42]. However, the endogenous biosynthesis pathway of acetophenone in plants is still unclear. Acetophenone was not detected in the IFS period of chestnut, and the styrene content was less than 0.01%. However, during the FSS period, the styrene and acetophenone contents increased sharply, to more than 10% and 5%, respectively. This result indicates that acetophenone can also be synthesized in plants. During the FFS stage, acetophenone is the most abundant VOCs, suggesting that acetophenone may be one of the main active ingredients in the sterilization and disinfection function of chestnut infloresances. Ethylbenzene is catalyzed by _nahAa/b/c/d_ to form styrene or 1-Phenylethanol.