## 2 Results
### An Uncovered Pathway for Meeting NAD(P)H Demand in Trichomes  
Besides GAPN, the current analysis further implicated the transcriptional amplification of four consecutive reactions catalyzed by malate synthase (MS), malic enzyme [NADP+ dependent] (ME [NADP+]), pyruvate decarboxylase 1 (PDC1), and aldehyde dehydrogenases (ALDH) to further meet the demand of NAD(P)H cofactors for secondary metabolite production in the trichomes (Figure 2c). The pathway is as follows: the MS enzyme first transfers the acetyl moiety of AcCoA to glyoxylate for the formation of malate. In turn, malate, which is also well replenished by the TCA cycle due to enhanced _CS_ expression (Figure S3), is oxidized by ME (NADP+) in the second reaction to form pyruvate, as well as the first NADPH molecule produced by the pathway. In the third reaction, pyruvate, which is also well provided by glycolysis, is then oxidized by PDC1 to form acetaldehyde. The resulting aldehyde molecule can then be oxidized by ALDH (ALDH2C4 (ALDH family 2 member C4) and ALDH3F1 (ALDH family 3 member F1)) to generate the second NADH molecule as well as acetate (Figure 2c). Besides being major cellular sources of reducing equivalents through NAD(P)H production [25], ALDHs are also required for generating hexanoic acid, a key precursor for cannabinoid production [4]. Importantly, the pathway prioritized NADPH production over NADH, which was similarly observed with the relative regulation of GAPN, GAPDH, and PGK, as described previously. For example, while the fold-change value of the pathway enzymes was generally large (log\({}_{2}\)FC = 3.8 to 8.3) compared to stem tissue, the ME (NADP+) enzyme stood out in having an increased expression (D) that was at least 50 times those of the three individual pathway enzymes (1.2 \(\times\) 10\({}^{6}\) versus 1.7 \(\times\) 10\({}^{4}\)-2.3 \(\times\) 10\({}^{4}\)) and close to a hundred times that of GAPN (1.23 \(\times\) 10\({}^{4}\)) (Figure S3). This observation thus implicates ME (NADP+) as another major contributor to NADPH production in trichome cells, besides GAPN. Figure 2: Transcriptional regulation of central metabolism in the trichomes of Cannabis cultivars with respect to stem tissue. (**a**) M-D plot of mean expression values (TMM) of genes encoding enzymes involved in the central metabolism (the glycolytic pathway, pyruvate metabolism, the pentose phosphate pathway, and the tricarboxylic acid cycle). Filled orange circles denote differentially upregulated genes of interest, while filled blue circles represent all other genes in the central metabolism. It should be noted that _PEPC_ is significantly down-regulated. (**b**) Pathway diagram of the central metabolism with corresponding enzyme names marked in red/orange. Note that ICL had very low expression. (**c**) Similar diagram for NAD(P)H replenishment pathway. Refer to Table S8 for corresponding gene IDs of enzymes. **Enzyme abbreviations in numeric and alphabetical order**: ACAT1 (acetyl-CoA acetyltransferase cytosolic 1); ALDH2C4 (aldehyde dehydrogenase family 2 member C4, NADH producing [-like]); ALDH3F1 (aldehyde dehydrogenase family 3 member F1, NADH producing); ALDO6 (fructose-bisphosphate aldolase 6, cytosolic); ALDO5 (fructose-bisphosphate aldolase 5, cytosolic); CS (ATP-citrate synthase); GAPDH (glyceroldehyde-3-phosphate dehydrogenase); GAPN (NADP-dependent glyceraldehyde-3-phosphate dehydrogenase); GPI-like (glucose-6-phosphate isomerase, chloroplastic-like); ICL (isocitrate lyase); ME (NADP+) (NADP-dependent malic enzyme); MS (malate synthase, glycosylomal); PDC1-like (pyruvate decarboxylase 1-like); PDH (pyruvate dehydrogenase, chloroplastic); PEP (phosphoenolpyruvate carboxylase, housekeeping isozyme); PGK (phosphoglycerate kinase); PK (pyruvate kinase, cytosolic); PK2 (plastitial pyruvate kinase 2).