catalysed by the enzyme tetrahydrocannabinolic acid synthase (THCAS)
(Fig.16.1). THCA was shown to accumulate in the secretory cavity of Cannabis
glandular trichomes (Mahlberg and Kim 2004 ). Adding to this knowledge,
Sirikantaramas et al. ( 2005 ) used gene transfer technologies to establish that the
biosynthetic enzyme, THCAS, is exclusively expressed in secretory cells and is
secreted into the storage cavity where it catalyses the synthesis of THCA. As
Cannabis is not easily transformed, tobacco, which also bears glandular trichomes
(Cui et al. 2011 ; Lange and Turner 2013 ), was used as an alternate host in the
following experiments. To localize the enzyme, the CannabisTHCASgene was
cloned and transformed into tobacco BY-2 cells using A. tumefaciens
(Sirikantaramas et al. 2005 ). Enzyme assays demonstrated that most of the activity
was present in the BY-2 culture medium, suggesting that THCAS is trafficked from
the endoplasmic reticulum to the outside of the cell. Next,CsTHCASwas fused to
GFPand theTHCAS-GFP fusion was stably expressed in tobacco plants by
Agrobacterium-mediated transformation. Using fluorescence microscopy, the
THCAS-GFP fusion was observed to accumulate in the storage cavity of tobacco
glandular trichomes. Taken together, these experiments, and others, revealed that
Cannabis glandular trichomes secrete THCA and its biosynthetic enzyme. A series
of experiments were then carried out to understand the rationale for THCAS
secretion. Cannabis glandular trichomes are known to accumulate THCA as a
defense mechanism against insect predators (Taura et al. 2007 ). However
Sirikantaramas et al. ( 2005 ) demonstrated that THCA is toxic to plant cells as well.
Indeed, incubation of THCA with BY-2 and Cannabis suspension cultures induced
cell death. Thus, trafficking THCAS to the storage cavity is thought to compart-
mentalize the biosynthesis of THCA, avoiding cellular damage.
16.4.1.2 Hexanoyl-CoA Synthetase (CsAAE1)
is Localized to the Cell Cytoplasm
Hexanoyl-CoA is a metabolic intermediate that feeds into the early steps of the
cannabinoid pathway (Fig.16.1). Stout et al. ( 2012 ) identified anacyl-activating
enzyme (CsAAE1) as the enzyme responsible for catalysing the synthesis of
hexanoyl-CoA. Analysis of the Cannabis trichome cell transcriptome revealed two
candidate genes encoding acyl-activating enzymes,CsAAE1andCsAAE3, that were
possibly involved in synthesizing hexanoyl-CoA. To localize the enzymes, their
genes were fused to theyellowfluorescentprotein(YFP) gene and the fusions were
transiently expressed in N. benthamiana by agroinfiltration. Using confocal
microscopy, YFP-AAE1 and YFP-AAE3 were localized to different subcellular
compartments, the cytosol and the peroxisome, respectively. Their subcellular
location was then compared to the enzyme thought to catalyse the next step in the
cannabinoid pathway, olivetol synthase (OLS; Taura et al. 2009 ; Fig.16.1). The
OLSgene was fused to thecyanfluorescentprotein(CFP) gene and co-infiltrated
with theAAE-YFPgene candidates inN. benthamianaleaves. Fluorescent signals
for both YFP-AAE1 and OLS-CFP co-localized to the same compartment, the
16 The Role ofAgrobacterium-Mediated and Other Gene-Transfer... 351