forms (THC and CBD) upon heating or storage (Sirikantaramas et al. 2004 ). There
also exists many lower abundant and lesser-known metabolic intermediates that are
piquing interest as potential pharmaceutical drugs (Hill et al. 2012 ; see also
Sect.16.4.3.3). Key enzymes that form the major cannabinoids have been identified
(Fig.16.1). However, there is still much to learn about the genetics and bio-
chemistry of cannabinoid biosynthesis (van Bakel et al. 2011 ).
Gene transfer technologies are assisting with the characterization of steps
involved in THCA biosynthesis by facilitating the identification of key enzymes,
their reaction mechanisms and their cellular and subcellular locations. As a result,
an increased understanding of the cannabinoid pathway enables the exploration of
metabolic engineering as a means to producing THCA and other cannabinoids. As
stable transgenic Cannabis plants cannot yet be recovered in an efficient manner,
nor has there been demonstration of transient gene expression inC. sativa, the study
and manipulation of the cannabinoid biosynthetic pathway is being performed in
other organisms.
16.4.1 Localization of Pathway Intermediates
The primary site of cannabinoid biosynthesis is glandular trichomes that form on
femaleflowers (Happyana et al. 2013 ). Glandular trichomes are epidermal hairs that
contain specialized cells for the synthesis, accumulation and secretion of products
such as secondary metabolites, nectar and mucilage (Lange and Turner 2013 ).
Cannabis possesses three types of glandular trichomes, but the capitate-stalked
glandular hairs are the main sites of cannabinoid and essential oil production
(Happyana et al. 2013 ). Capitate-stalked trichomes are mushroom-shaped hairs
consisting of two parts, the gland and the stem (Mahlberg and Kim 2004 ). The
gland is composed of disc cells whose outer walls split to form a storage cavity at
the top of the gland. Disc cells release secretory vesicles into the storage cavity
which enlarges as secretions accumulate.
The cellular and subcellular locations of cannabinoid pathway enzymes have
been revealed with the assistance of gene transfer technologies. Using gene cloning
methods, genes encoding fluorescent labels such as greenfluorescent protein
(GFP) are fused to genes encoding selected Cannabis biosynthetic enzymes and then
transferred to plant cells using methods such asAgrobacterium-mediated transfor-
mation or agroinfiltration (Day and Davidson 2009 ; Miyawaki 2011 ). Fluorescence
or confocal microscopy are used to detect thefluorescently labeled proteins.
16.4.1.1 Tetrahydrocannabinolic Acid Synthase is Secreted
into the Storage Cavity of Glandular Trichomes
In Cannabis tissues, thefinal biosynthetic step in the production of THC is the
conversion of cannabigerolic acid (CBGA) to tetrahydrocannabinolic acid (THCA)
350 M. Feeney and Z.K. Punja