## 3 Discussion
### Roles of Terpenoid Biosynthesis Genes in Resin Yield  
Resin is composed of terpenoids, and terpenoid biosynthesis plays a central role in the resin yield. The resin biosynthesis of conifer derives from the two isoprenoid molecules isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) which originate from the MEP and MVA pathways. Two or more of these five-carbon (C5) metabolites are conjugated by isoprenyl diphosphate synthases (IDS) to produce the longer-chain (C10, C15, C20) substrates for TPS. MEP and MVA pathway genes and their functions are extensively conserved [46], and many genes of these pathways have been described in conifers. In this study, a total of 25 genes, that covered the entire MEP and MVA pathways were identified (Figure 8A). The majority of genes involved in MEP and MVA pathways exhibited significant changes at the transcriptional and proteomic levels. In the MEP pathway, the majority of genes were significantly up-regulated at the proteomic level (including _DXR_, _CMK_, _MDS_, _HDR1_, and _GGPPS1_), and two _GPPS_s (including _GPPS1_ and _GPPS2_) and _FPPS_ were greatly up-regulated at the transcriptional level, indicating that increased expression levels of genes involved in MEP pathway contributed to the increase of resin yield. However, most members in the MVA pathway exhibited different changes from the MEP pathway. The majority of genes related to the MVA pathway were dramatically down-regulated at the transcriptional and proteomic levels (including _HMGS_, _HMGR4_, _MK_, _PMK_, and _MVD_). The above results indicated that the MEP pathway played a more important role in the increase of resin yield than the MVA pathway. Remarkably, _PMK_ and _MVD_ showed consistent expression changes both at the mRNA and protein levels, indicating that these two genes were a negative correlation with resin yield. _IDSs_ (including _GPPS_, _FPPS_, and _GGPPS_), which produce GPP, FPP, and GGPP as precursors of monoterpenoids, sesquiterpenoids, and diterpenoids, respectively, are key genes for terpenoid biosynthesis. These genes were greatly up-regulated in high resin yield, which was consistent with previous reports [36; 37], indicating their involvement in resin yield. The TPS generates much of the huge chemical diversity found in conifer resin [5; 47; 48]. Conifer TPS genes include _MonoTPS_, _SesquiTPS_, and _DiTPS_, which use DMAPP, GPP, FPP, and GGPP, respectively, as substrates for resin. In the present study, we found five _MonoTPSs_, three _SesquiTPSs_, and one _DiTPS_ in the needles, and _MonoTPSs_ and _SesquiTPSs_ exhibited greater differences than _DiTPS_ between high and common resin yield, indicating that there were significant differences in the monoterpene and sesquiterpene biosynthesis in needles from high and common resin yield. Cytochrome P450 enzymes of the CYP720B subfamily play a central role in the DRAs biosynthesis, which has been identified in different spruce and pine genomes and transcriptomes. The known CYP720B sequences are classified into four distinct clades I-IV. Of these known spruce and pine CYP720B8, four have been proven to be involved in the biosynthesis of DRAs, two in clade I (CYP720B2, CYP720B12) and two in clade III (CYP720B1, CYP720B4) [49; 50; 51]. In a recent study, the _PmCYP720B11v2_ gene was also proven to be involved in the metabolic processes of diterpene biosynthesis in the stem as a positive part of the defense against pinewood nematode (PWN) attack in masson pine [52]. In the present study, three genes in clade I (_CYP720B2_, _CYP720B10_, _CYP720B11_), one gene in clade III (_CYP720B1_), and one gene in clade IV (_CYP720B8_) were identified in needles. These _CYP720B_ genes did not exhibit significant differences at the transcript level, only _CYP720B8_, _CYP720B10_, and _CYP720B11_ differed at the protein level, indicating that there were no significant differences in DRAs biosynthesis in needles from high and common resin yield.