## 2 Results
### CsMIXTA Promotes Standard Trichome Formation in Tobacco Leaves  
Our work monitoring _CsMIXTA_ expression in cannabis shows evidence of an associative relationship between _CsMIXTA_ expression during the female flower development and cannabinoid synthesis, presumably mediated by _CsMIXTA_-mediated control of trichome development. We undertook further work to examine if there is a causal relationship between _CsMIXTA_ expression and trichome development in a heterologous system. Cannabis is recalcitrant to genetic transformation; a few reports have been published recently [16; 17; 18], but their transformation efficiencies are not robust. In lieu of a reliable cannabis transformation protocol, we used a heterologous system to determine if there is a causal effect of _CsMIXTA_ expression on trichome morphogenesis. Tobacco (_Nicotiana tabacum_), like cannabis, has glandular stalked trichomes but in the case of tobacco, GSTs are abundant on the leaves [19]. We ectopically expressed _CsMIXTA_ (controlled by a constitutive (35S CaMV) promoter) in tobacco plants to evaluate if this cannabis TF could regulate glandular trichome initiation/development on tobacco leaves. Figure 3 displays a side view of leaves from wt and a transgenic tobacco line, L1. An increase in trichome size and branching is clearly observed on the transgenic tobacco leaf. In addition, the upper leaf surface of the transgenic tobacco L1 is noticeably \"hairier\" than that of the wt, indicating a greater density of leaf trichomes produced in the _CsMIXTA_ overexpressing tobacco line (Figure S3). Figure 1: Expression analysis of _CsMIXTA_. (**A**) Relative expression of _CsMIXTA_ in 5 different tissue types harvested from Cherry Wine. (**B**) Relative Expression of _CsMIXTA_ over 7 weeks of flowering. Results ((**A,B**)) are shown as means \(\pm\) SE (\(n\) = 4). Means separation between expression in various tissues compared to the level in roots (**A**) and at various times during flower development as compared to expression at week one (**B**) was evaluated using Student’s \(t\)-test; * indicates \(p\) < 0.05, and ** indicates \(p\) < 0.01. (**C**) Integrated expression analyses of _CsMIXTA_ and genes encoding cannabinoid biosynthetic enzymes along with cannabinoid levels monitored over the 7-week flowering period were evaluated using [PERSON]’s correlation coefficient analyses. Green boxes highlight _CsMIXTA_. Blue indicates a positive correlation, while red indicates a negative correlation. Size and shade of circles represent strength of correlation. Figures were generated using the R corrplot package ([[https://github.com/taiyun/corplot](https://github.com/taiyun/corplot)]([https://github.com/taiyun/corplot](https://github.com/taiyun/corplot))). (*, \(p\) < 0.05; **, \(p\) < 0.01; ***, \(p\) < 0.001.) Percentage data was arcsine transformed prior to statistical analysis. We further examined trichome morphology on tobacco leaves using SEM. Two transgenic lines (L1 and L3) were evaluated in this experiment (Figure 4). Both lines (Figure 4B,C) showed substantially higher trichome density compared to wt (Figure 4A). Typical wt trichomes possess only one gland and rarely branch [19]. However, we observed highly branched glandular stalked trichomes in both transgenic lines. In addition, trichomes in L1 showed higher density, size, and branching than L3, which could be due to the increased increased number of trichomes in the transgenic line. Figure 3: Trichomes on wt (**A**) and transgenic (**B**) tobacco leaf edges visualized using a dissection microscope. Samples were viewed at 38\(\times\) magnification. It is noticeable that the base of some trichomes has become enlarged and trichomes are exhibiting novel branching in the transgenic line (**B**). Scale bars: 200 \(\upmu\)m.  
Figure 2: Yeast transactivation assay and subcellular localization of CsMIXTA. **(A)** Top: yeast cells grown on SD medium lacking W. All cells grew normally, including cells transformed with the empty vector (EV).