## 3 Discussion  
In this study we identified _CsMIXTA_, the first characterized cannabis TF that could potentially influence cannabis CSG development. _CsMIXTA_ is highly expressed in the female cannabis flowers, with expression patterns that are coordinated with cannabinoid biosynthesis and the development of infloresences. Phylogenetic analysis indicated that _CsMIXTA_ is closely related to _Arabidopsis thaliana_ AtMYB16 and _Artemisia annua AaMIXTA_ (Figure S1). MIXTA-like proteins have been shown to coordinate cuticle deposition in tomato and _Arabidopsis thaliana_[20; 21]. Modification of _AaMIXTA_ expression altered the biosynthesis of wax and cutin monomers, components of the cuticle [12], and MIXTA-like proteins identified in liverworts have implicated MIXTA in the early evolution of cuticle formation and therefore the colonization of land by plants [22]. Because cutin is an important structural component of the trichome, _CsMIXTA_ may coordinate the biosynthesis of this compound in the trichome. Our previous study demonstrated that expression of cannabinoid biosynthetic genes peaked in weeks 4 or 5 [15]; _CsMIXTA_ expression showed a similar pattern. It has been shown that trichomes produce most cannabinoids during the latter capitate-stalked glandular phase. Therefore, it is reasonable to propose that the development of the stalk cells also occurs around week 4 of floral development, in correlation with the expression of _CsMIXTA_. There is a strong positive correlation between _CsMIXTA_ expression and the expression of both _GPPS_ and _OAC_, enzymes which synthesize the necessary precursors (GPP and olivetolic acid, respectively) [15] for both cannabinoids and, in the case of _GPPS_, monoterpenses. Sessile (non-stalked) glandular trichomes produce minimal monoterpenes or cannabinoids before maturation, possessing a sesquiterpene-dominant chemotype. However, after the sessile trichomes develop into CSGs, cannabinoid and monoterpene biosynthesis takes place. Previous studies have shown that _GPPS_ was more highly expressed in stalked trichomes, but _OAC_ expression was similar among different glandular trichome types [10]. The heterologous overexpression of _CsMIXTA_ in tobacco has shown a significant impact on trichome development with increased density, enlarged size and more branching (Figure 4). The overexpression of _CsMIXTA_ in tobacco showed more distinct trichome phenotypes than that of _AmMIXTA_ overexpression [14; 23]. The overexpression of known MIXTA or MIXTA-like genes did not show a similar glandular trichome phenotype caused by overexpression of _CsMIXTA_, indicating that _CsMIXTA_ may have unique functions in glandular trichome morphogenesis in cannabis. Figure 5 quantifies a modest (twofold) increase in trichome glands which can be seen in the Fluorescence Microscopy images  
Figure 5: Tobacco transgenic lines overexpressing _CsMIXTA_ generated more trichomes than wt. (**A**) Average number of glands on the glandular trichomes per mm\({}^{2}\), observed by intrinsic fluorescence as shown in Figure 4G–I. (**B**) Average trichome number per mm\({}^{2}\), obtained from SEM images in Figure 4A–C. Averages taken from 3 biological reps (\(n=3\)) for each transgenic line and wt. * indicates \(p<0.05\), which was determined using Student t-test by comparing the two transgenic lines to wt. (Figure 4G-I). What cannot be seen in the fluorescence images, however, is the forest of trichomes and trichome branches, which becomes evident in the SEM photos (Figure 4A-C). The increase in trichome density (threefold) as well as trichome branching indicates that _CsMIXTA_ may also function to regulate the initiation and development of trichome types other than glandular stalked trichomes. According to these findings, genetic engineering of _CsMIXTA_ may have the potential to promote cannabinoid production by increasing glandular trichome formation. Our data provide evidence associating _CsMIXTA_ with trichome development in cannabis.