The complexity of phytocannabinoid synthases does not end there, though.
Copy number variation of CBDA and THCA synthase genes might be involved
in phytocannabinoid level and composition
(Vergara et al.,
2019) and most likely, the number of synthase (pseudo)genes might be
different for each cultivar sequenced
(Grassa et al., 2018;
Laverty et al., 2019;
McKernan et al.,
2020).
High throughput assays for BT and BDmarkers have been developed and show that many plants actually contain
both loci (Cascini et
al., 2019; McKernan et al., 2020; Toth et al., 2020). Moreover, many
BD/BD plants, especially those with
higher CBDA levels, have THCA levels of above 0.3 % of dry flower mass,
despite the absence of a functional BT allele
(Toth et al., 2020).
This residual THCA is probably at least to some extent a by-product of
the CBDA synthase itself. The THCA and CBDA synthase have a relatively
high sequence similarity (83.85 %, Figure 5) and process the same
precursor molecule, CBGA (Figure 4). In vitro studies have shown
that the CBDA synthase produced CBDA and THCA at roughly a ratio of 20:1
(Zirpel et al.,
2018). This is similar to ratios observed in planta in high-CBD
hemp varieties as well
(Toth et al., 2020;
Weiblen et al.,
2015). This potentially results in the problem that, if CBDA production
is increased, THCA also increases as a by-product, even if plants do not
express a functional THCA synthase. Cannabis varieties with very
high CBD levels may thus be at risk of exceeding legal THC thresholds.
Understanding the exact genetics underlying the different chemotypes
will be important for future targeted breeding approaches. Tight
restrictions across the world make it difficult for farmers to grow
chemotype III, IV and V varieties, because the presence of residual THC
creates regulatory problems and uncertainties. Especially type III
plants often have THCA/THC levels slightly above the legal THC limit
(Aizpurua-Olaizola et
al., 2016; Toth et al., 2020). Hence, one important breeding goal is
going to be the generation of zero-THC lines which still produce high
levels of CBD in the range of 15 to 20 % of dry flower mass. Whether
this is possible to achieve is difficult to say, since even in the
absence of a THCA synthase, CBDA synthases produce THCA as a by-product
(Toth et al., 2020;
Zirpel et al., 2018). This will, therefore, require identification of a
CBDA synthase that does produce only very low or no amounts of THCA.In vitro experiments show that point mutations can alter the
amount of by-products
(Zirpel et al.,
2018). Natural variation in synthase genes exists and have been linked
to altered phytocannabinoid compositions
(Onofri et al.,
2015). Hence, naturally occurring or artificially generated CBDA
synthase varieties could be used for targeted breeding in this
direction.
In addition, Cannabis varieties used for fibre or seed production
could be selectively bred and genotyped to have 0 % overall
phytocannabinoids (chemotype V), as currently even the farming of these
kinds of varieties is heavily restricted in many countries.
Other phytocannabinoids like CBG(A) and CBC(A) as well as the manifold
variants of terpenes produced in Cannabis flowers are
increasingly coming into focus in the medical research fields (reviewed
in Booth and Bohlmann,
2019; Deiana, 2017; Pollastro et al., 2018), hence generating lines
with specific phytocannabinoid profiles might be of interest in further
research.
4. A hairy topic: Flower development and morphology in Cannabis
The flower is the reproductive structure of flowering plants
(angiosperms), which represent one of the most successful and diverse
groups of organisms on this planet
(Krizek and Fletcher,
2005). While the characteristic shape of the Cannabis leaf is
often used as a symbol for the whole plant, Cannabis female
flowers are of particular interest because they are the main site of
production of pharmacologically active compounds (phytocannabinoids)
(Spitzer-Rimon et al.,
2019). Understanding the morphology of Cannabis flowers and
their developmental genetics is therefore especially important.
The typical angiosperm flower consists of four different organ types,
which are organized in concentric whorls: sepals, petals, stamens and
carpels (Endress,
1992; Krizek and Fletcher, 2005). Sepals are in the outermost whorl and
usually green and leaflike in appearance. Petals are in the second whorl
and often coloured to attract pollinators. Petals together with sepals
are termed the perianth and constitute the non-reproductive part of a
flower. Stamens are typically located in the third floral whorl. They
are the male reproductive organs and are composed of an anther and a
filament. The anthers grow on top of the stalk-like filaments and are
the site of pollen production. Finally, carpels develop in the fourth
and central whorl of a typical flower. Carpels are the reproductive
organs that contain an ovary inside which ovules develop. The tip of the
carpel, the stigma, receives the pollen. The style connects the stigma
to the ovary (Becker,
2020; Endress, 1992; Krizek and Fletcher, 2005).
Notably, the number, arrangement, and morphology of the floral organs
varies substantially between different species of flowering plants
(Endress, 2011;
Theissen and Melzer, 2007). Most flowers contain, as described above,
both carpels and stamens, and are therefore termed bisexual flowers
(Renner, 2014).
However some 15 % of flowering plant species are monoecious or
dioecious and have unisexual flowers that develop only stamens or
carpels (Renner,
2014). In dioecious plants, female and male flowers develop on separate
individuals. In contrast, in monoecious plants male and female flowers
develop on the same individual
(Renner, 2014).