## 3 Discussion  
### In Vitro GT Assay Proof of Concept  
_In vitro_ experiments have previously been carried out on isolated peppermint GTs to study how carbon flux in these cells affect terpenoid biosynthesis; these experiments used small amounts of trichome heads which were incubated in a liquid culture for only 1 h [44, 45]. In this study, we wanted to understand phytohormonal control of cannabis GTs in _in vitro_ isolated conditions to remove the potential effects of other signalling pathways that would be active in _in planta_ conditions. Unlike [PERSON] et al. [45], solid plate culturing was chosen since initial attempts of culturing in liquid media proved unsuccessful. Cannabis GT cells hyperaccumulated polyphenic pigments when in liquid, indicating a high level of cell stress. We surmised that this was due to the relatively anoxic environment of the liquid media compared to the solid plate culture. The GT disc cells were incubated for 24 h to give enough time for turnover of the proteome and changes in CBGA content in response to treatments. As an intermediate cannabinoid, CBGA experiences high flux, and abundance can change in as little as 6 h. Changes in abundance can be used as a broad gauge for cannabinoid pathway activity [33]. The cells were incubated in constant light to mimic daytime conditions, which is when cannabis GTs are most metabolically active for cannabinoid biosynthesis [33]. We initially expected that GT disc cells would experience some level of stress in the _in vitro_ assay, but proteomic data suggested otherwise. Separated from their stalks that are the efficient conduits supplying photoassimilates [10], the GTs were in an artificial environment where nutrient availability varies from what is supplied when attached on the plant. Some increased DAPs (Supplementary Figure S3) such as a sucrose synthase (SUS3) and several aminotransferases and synthetases (ALAAT2, ATCS-C, and ASN1) may indicate to sugar starvation [48]. Yet, changes in other key regulators indicate that the cells were not metabolically stressed. The structural subunit d of mitochondrial ATP synthase (ATPd) increased in abundance (Figure 1D), indicative of greater ATP generation [49, 50]. While not enzymatic, ATPd is an essential subunit for the ATP synthase complex to be in functional conformation [51]. The subunit has a regulatory role in ATP generation [49] as higher expression directly results in a higher ATP/ADP ratio in cotton ovule fibre cells (a type of trichome) [50]. Importantly, ACLA-1 also increased in abundance (Figure 1D). By being a non-redundant subunit of ATP citrate lyase (ACL), ACLA-1 acts as a regulator of ACL activity and controls the available pool of cytosolic acetyl-CoA in the cells and how resources are allocated to different metabolic pathways [52, 53, 54]. A decrease in ACLA-1 in arabidopsis induces metabolic stress and upregulation of stress response genes, causing the plant to reallocate the limited pool of acetyl-CoA to specific metabolic pathways, such as anthocyanin biosynthesis [53]. Overexpression of ACLA-1 in arabidopsis leads to elevated ACL activity, producing a greater pool of acetyl-CoA and a different shift in resource allocation whereby cutin and cuticular wax biosynthesis is upregulated rather than other lipid metabolites like glycerolipids and sterols [52]. Thus, an increase in ACLA-1 would indicate that GT disc cells were not stressed and have carbon resources available. In addition, we observed that the cells responded accordingly to lower levels of high light stress and osmotic stress. DEG7 (Figure 1D), a plastidial DegP protease that responds to high light conditions and damage by photoinhibition [55], and RD22 (Figure 1D), a key positive regulator of drought stress response [56], both decreased in abundance. Lastly, microbial contamination was likely not a stressor for GT disc cells since none of the DAPs were enriched for biotic stress response at 24 h (Figure 1B) and microbial contamination was not apparent until 48 h in initial time course trials (Supplementary Figure S4).