Molecular Regulation of Glandular Trichome Initiation and Morphogenesis in Cannabis sativa L.

<p dir="ltr">A thesis submitted in total fulfilment of the requirement for the degree of Doctor of Philosophy to the School of Agriculture, Biomedicine and Environment, La Trobe University, Victoria, Australia.</p><p dir="ltr">Abstract:</p><p dir="ltr"><i>Cannabis sativa </i>L. (cannabis), a dioecious angiosperm in the Order Rosales, is a rich source of bioactive specialised metabolites and prolific producer of a class of pharmaceutically valuable compounds referred to as phytocannabinoids (PCs). PCs are predominantly synthesised in stalked glandular trichomes (SGTs) that are concentrated on female flowers and may function as a chemical and physical defence against (a)biotic stress. Recent legislative shifts favouring the medicinal utilisation of PCs have increased licit demand for high PC-yielding cannabis plants. Despite the importance of SGT in PC production, the specific transcription factors (TFs) regulating cannabis SGT ontogenesis remain undefined. Myeloblastosis (MYB) TFs, like GLABRA1 (GL1) and MIXTA, play a pivotal role in governing epidermal cell fate, division, and differentiation to control trichome morphology and spatiotemporal distribution in model angiosperm taxa. This thesis aims to characterise cannabis MYBs and the transcriptional networks underpinning cannabis SGT ontogenesis. A transcriptomics approach evaluated <i>CsMYB </i>functions in trichome development and identified 16 <i>CsMYB </i>candidates with predicted functions in trichome cell fate and morphogenesis. Phylogenetic analysis identified expansion of the subgroup 9 MIXTA clade and an absence of GL1-like TFs within the R2R3-CsMYB subfamily. Overexpression experiments in transgenic <i>Arabidopsis </i>yielded a trichome-less phenotype by an R3-CsMYB, and increased trichome branching by an R2R3-CsMYB potentially involved in stress-responsive cuticular wax biosynthesis. Notably, <i>CsMIXTA </i>did not affect trichome development, suggesting that trichome regulatory pathways in cannabis and <i>Arabidopsis </i>are non-homologous. A novel time-course analysis using SGT-dense reduced cannabis leaves as a model organ showed that clusters of co-expressed genes correlating with SGT density were linked to floral organogenesis, phytohormone signalling, and cell wall organisation. A MIXTA-like R2R3-CsMYB and a HD-ZIP IV TF were identified as central components of gene regulatory networks that associated with SGT density.</p>