## 3 Discussion
In _Citrus unshiu_, the expression of four clones, CitMTSE1, CitMTS3, CitMTS61, and CitMTS62 coding for D-limonene synthase, \(\gamma\)-terpinene synthase, and \(\beta\)-pinene synthase, respectively, appeared mainly in the peel at an early stage of fruit development, whereas they decreased or disappeared in later stages of development [32]. However, the expression level of other genes were consistent with the content. The expression of several compounds, including camphene, carophyllene, \(\alpha\)-pinene, \(\beta\)-pinene, \(\beta\)-myrcene, D-limonene, geraniol (the content were above 1%), were decreased from 60 DAF to 135 DAF (Table S3), which was accompanied by a reduction in RPKM of the related genes during the fruit development (Table S4). The phenomenon was also found in other species. In _Humulus lupulus_ L, the expression of _HISTS1_ encoding for the sesquiterpene synthases which catalyse the formation of carophyllene and humulenewas abundant in those tissues with high contents of humulene and caryophyllene [33]. The higher amounts of artemisinin, which is produced by _Artemisia annua_, was mainly a result of higher expression of amorpha-4,11-diene synthase (_ADS_) and artemisinin aldehyde, was a result of higher expression of the amorpha-4,11-diene synthase (ADS) and artemisinin aldehyde \(\Delta\)11-13 reductase (_DBR2_) genes [34]. In the second phase of terpenoid biosynthesis, IPP and DMAPP are used to produce the immediate precursors of monoterpenes, sesquiterpenenes and diterpenes, which are namely geranyl diphosphate (GPP), farnesyl diphosphate (FPP), geranyl geranyl diphosphate (GGPP) by geranyl diphosphate synthase (GPPS), farnesyl diphosphate synthase (FPPS), and geranyl geranyl diphosphate synthase (GGPPS), and the immediate precursors of monoterpenes, sesquiterpenes and diterpenes, respectively. In our research, these three enzymes were found to be differentially expressed during _L. cubebe_ fruit development, and most of them were down-regulated. For example, g 5372 (for GPPS) was up-regulated compared to 60 and 75 DAF, but then down-regulated compared to 60 vs. 90 DAF, 60 vs. 105 DAF, and 60 vs. 135 DAF contrast groups. Additionally, g 4493 and g 21893 (for FPPS) and g 50359 and g 57414 (for GGPPS) were down-regulated in four respective groups. These results suggested that the terpenoid biosynthesis might ocurr during the young stage of fruit development and the products stored during maturation. However, the changes in there DEGs were not consistent during the fruit development, which suggests that terpenoid biosynthesis is a complex pathway. For example, GPP exists in both the homodimeric and heterodimeric forms in both angiosperms and gymnosperms [35; 36], and two types of subunits constitute heterodimeric GPPS: a large subunit (LSU) and a small subunit (SSU) [37]. These two subunits have different expression parttens for the expression of _GPPS_. In _Salvia militorrhiza_, _GPPS.LSU_ is less tissue specific compared with that of _GPPS.SSU_ and the two types have different gene organization [21]. Fourteen enzymes are involved in the first phase of terpenoid biosynthesis, namely AACT HMGS, HMGR, mevalonate kinase (MVK), phosphomevalonate kinase (PMK), and mevalonate diphosphate decarboxylase (MVD), isopentenyl diphosphate isomerase (IDI), 1-deoxy-D-xylulose 5-phosphate synthase (DXS), 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), 2-C-methyl-D-erythritol 4-phosphate cytidyltransferase (MCT), 4-(cytidine 50-diphospho)-2-C-methyl-D-erythritol kinase (CMK), 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MDS), (_E_)-4-hydroxy-3-methyl-but-2-enyldiphosphate synthase (HDS), (_E_)-4-hydroxy-3-methyl-but-2-enyl diphosphateoreductase (HDR). Studies have suggested that these enzymes could affect the yield of essential oil [38; 39]. Therefore, we should focus on expression change of these enzymes. In this research, most genes for these enzymes were detected, and the change trends of those were complicated during the development stage of fruit.