## 3 Discussion
Consistently, studies of other cultivars in a similar flowering stage also found genes encoding enzymes in the MEP pathway to be more highly expressed than those in the MVA pathway [47; 48]. Genes in the latter pathway are instead more highly expressed during the early stage of flowering [48], in line with the larger sesquiterpene output reported during this time [49]. In addition, the exchange of isoprenoid precursors between two pathways is known to be possible [27], which further raises the possibility of the MEP pathway replenishing the MVA pathway for sesquiterpene biosynthesis, subject to the limited physical capacity of the chloroplast. Given the intense selection pressure for secondary metabolite production in the cultivars, such an adaptation would also benefit from the higher stoichiometric yield [50; 51] and lower oxygen requirement [26] of the MEP pathway. The coordination of the pathways has also been previously proposed in a study, which established a negative correlation between _HMGR_ expression in the MVA pathway and the MEP module comprising _CMK_, _MCS_, and _HDS_[52], which is consistent with the findings in this work. HMGR is further known to be a rate-limiting enzyme for the MVA pathway in many plants, whereas DXS and DXR are similarly so in the MEP pathway [53; 54]. Although the bottlenecked production of limiting enzymes may be overcome by their over-expression, as demonstrated in many plants, the relevant research has not yet been carried out for Cannabis, to the best of our knowledge. To illustrate the application of the current findings for crop selection to achieve high yields, a comparison of the expressions of pivotal enzyme genes, which were identified in commonly used cultivars, was pursued. There were large variations in their expressions and consequently in the implied production capacities of the cultivars. For example, there were 3.3-, 1.9-, 2.6-, and 1.8-fold differences in expression for _OAC_, _APT_, _THCAS_, and _CBDAS_ among five cultivars (Figure 4a), thus exemplifying the need to profile cultivars molecularly. Although the WS and TH cultivars were easily identified as high and low producers, respectively, based on their highly consistent expression profiles, it is less clear how the other cultivars ought to be classified--a situation further exacerbated by the lack of chemical profiles for evaluation. Thus, further studies are needed to determine the relative utilities of key identified enzymes in discerning cultivar outputs. Transcriptomics data are also highly useful for mining uncharacterized but highly expressed TPSs, which may contribute to the characteristic terpene repertoire of cultivars. Of the three genes picked out in this study, CsTPS5 TH was experimentally verified to be an (E)-nerolidol synthase. On the other hand, CsTPS3 GT and CsTPS4 WS were initially described as mono-TPS1 (MTS1) and TPS9 in the cs10 Cannabis reference genome. However, in this work, CsTPS3 GT and CsTPS4 WS were found to synthesize a sesquiterpene (Z)-\(\gamma\)-bisabolene and a monotperone (R)-linalool, respectively. Such misannotation of MTS1 underscores the challenge of using in silico assays for evaluating TPS function. Previously, an apple TPS was also mischaracterized as being most similar to mono-TPS due to the presence of the 'RR(X8)W' motif near the N-terminus [55]. However, it lacked the plastid transit peptide associated with mono-TPS, just like CsTPS3 GT. Similarly, CsTPS4 WS was initially considered to be capable of monoterpene cyclization because of the presence of the 'RR(X8)W' motif. However, it was found that it could only produce an acyclic monoterpene alcohol, (R)-linalool, using both in vitro and in vivo assays in this study. Given that the two natural enantiomers of linalool have distinct olfactory qualities, CsTPS4 WS may contribute to the lavelander-like smell of Cannabis with its production of (R)-linalool [56], rather than the floral and petitgrain-like scent of (S)-linalool [57].