## 3 Discussion  
This study provides clear evidence at the transcriptome level that illuminates the holistic and concerted adaptations of metabolism in Cannabis trichomes to facilitate cannabinoid and terpene production. For example, our findings on the importance of enhanced and coordinated AcCoA production in Cannabis trichomes is supported by studies in various organisms; an increase in AcCoA supply via overexpression of _PDH_ and _AcCoA synthetase_ (_ACS_) improved IPP precursor biosynthesis through the MVA pathway [24], while the enhancement of pyruvate level by up-regulating pyruvate kinase expression (e.g., _PK_ and _PK_2) led to improved AcCoA availability, promoting terpene production [36]. The excessive demand of NADPH for secondary metabolite production was also supported by the increase in expression (D-value) of the gene encoding the ME (NADP+) enzyme (Figure 2a), which was the largest among the primary pathways. Consequently, the MDH reactions that compete for the same malate substrate to generate NADH instead were likely reduced. The phenomena of swapping NADH for NADPH production in Cannabis trichomes was repeated with the four-fold increase in the expression of the gene for the  
Figure 7: Functional characterization of CsTPSs. In vitro (**a**–**d**) and in vivo (**e**–**g**) characterization of CsTPS3 GT, CsTPS4 WS, and CsTPSSTH. The products were confirmed by comparison of retention times and mass spectra with those of standards (Figure S7). TIC: total ion chromatogram; EIC: extracted ion chromatogram; RT: retention time. GAPN enzyme, which re-routed G3P away from the GAPDH and PGK reactions that produce NADH and ATP as an alternative. The findings further indicate that, if inappropriately dealt with, NADPH deficiency can be a major production bottleneck in the Cannabis plant. In this regard, the provision of NADPH and the other uncovered carbon substrates (G3P, pyruvate, and AcCoA) has been repeatedly demonstrated to result in high terpenoid productivity by engineered _Escherichia coli_, with a mass yield of up to 45% [36; 37; 38; 39]. Thus, the current analysis newly demonstrates similar convergence in the adaptations of the Cannabis plant to enhance secondary metabolite yields. In addition, it was also found that the NAD(P)H-producing pathway, with enhanced _ME (NADP+)_ expression (Figure 2c), possibly constitutes a modified acetate fermentation pathway. Firstly, PDH, PDC, and ALDH have functional roles in the pathway [40], and PDC is known to be induced in an anoxic environment [41]. When the fate of acetate (as the end metabolite of the pathway) was investigated, there was no indication of its being recycled to regenerate AcCoA [42] due to low _ACS_ expression. Thus, the metabolite could be secreted in a fermentative manner. Furthermore, the glandular trichomes of the peppermint plant were also recently shown to exhibit fermentative metabolism [43], thus suggesting a similar feature in the Cannabis plant, for which the glandular trichomes of female flowers are the major sites of cannabinoid and terpene production [44]. This suggests that an anaerobic environment may not be required for the fermentation, as the trichome cells could just be prioritizing usage of their enzymatic capacity [45; 46] for secondary metabolite production, over acetate recycling. Furthermore, the recycling is unnecessary, as carbon supplies are expected to be plentiful, given the productive role of the trichomes. Furthermore, the current analysis highlights the heightened capacity of the MEP pathway compared to the MVA pathway in the trichomes, as suggested by the up-regulation of more genes encoding biosynthetic enzymes catalyzing rate-limiting reactions, and further characterized by larger fold changes and increased expressions (Figure 3a). This could be explained by the increased requirement for GPP as a precursor for monoterpene as well as cannabinoid biosynthesis in the fully matured flowers.