6.3. Model plants and candidate genes: Arabidopsis, soybean and the hunt for flowering time genes in Cannabis

While important efforts to determine the environmental stimuli impacting floral induction in Cannabis have been undertaken, the genetic pathways and loci underlying the environmental responsiveness still require elucidation (Salentijn et al., 2019). Huge diversity exists for flowering time in Cannabis with phenotypes generally categorised as early-, mid- or late-flowering (Salentijn et al., 2019). Furthermore, photoperiod-insensitive (also known as day-neutral or auto-flowering) cultivars exist (Small, 2018). A recent study suggests that female floral initiation occurs independently of the photoperiod in some Cannabis cultivars, while in others shorter photoperiods were required for flower maturation and development (Spitzer-Rimon et al., 2019). Further research is required to substantiate the molecular basis of those observations, and research on model plants may serve as an important primer to understand the gene regulatory network controlling flowering time in Cannabis.
Among the model plant species for which comprehensive analyses of flowering time have been conducted are the long-day plantArabidopsis thaliana and the short-day plant Oryza sativa(rice). In Arabidopsis, the complex flowering time network is well-characterised with several pathways described including the vernalisation, autonomous, photoperiod, circadian clock, age, ambient temperature and gibberellin pathways (Blümel et al., 2015). One of the key integrators of floral inductive signals in Arabidopsis is FLOWERING LOCUS T (FT ), the protein product of which is known as florigen (Turck et al., 2008).
As mentioned previously, Cannabis is particularly sensitive to alterations in the photoperiod and as such the photoperiodic pathway of Arabidopsis warrants some more detailed discussion. The photoperiodic flowering pathway depends on cross-talk between light perception and the circadian clock, which coordinate to control the expression of the main integrator FT(Cao et al., 2017). The first step in the photoperiodic pathway is the perception of light by the photoreceptors (phytochromes and cryptochromes). Phytochromes exist in inactive (Pr) and active (Pfr) forms. Pr is synthesised in the dark, and upon red-light perception is activated to Pfr which translocates to the nucleus. Pfr can interact with transcription factors and induce large-scale transcriptional alterations in response to light (Legris et al., 2019). Pfr then reverts to Pr by far-red light absorption or by light-independent thermal reversion (Klose et al., 2020). Phytochromes have a myriad of roles in regulating plant development and several phytochromes exist in angiosperms. The Brassicaceae possess five phytochromes: phyA to phyE. In Arabidopsis phyA and phyB are functionally the most important (Legris et al., 2019).
The photoreceptors subsequently transmit signals to the central node of the photoperiodic pathway: the GIGANTEA-CONSTANS-FT(GI-CO-FT ) signalling cascade. Briefly, the action of the GI-CO-FT module in Arabidopsis is as follows: the active Pfr form of phyA promotes the stability of the nuclear transcription factor CONSTANS (CO) which activates transcription of FT(Putterill et al., 1995; Samach et al., 2000). From the FT locus, florigen is produced, a small mobile protein which travels via the phloem from the leaves to the shoot apical meristem to induce the transition from vegetative to reproductive growth (Corbesier et al., 2007). The circadian clock gene GIGANTEA (GI ) allows the degradation of transcriptional repressors that repress the expression ofCO thus indirectly promoting FT(Sawa et al., 2007). The MADS-box transcription factor gene SOC1 is indirectly upregulated by CO via florigen. SOC1, in turn, activates the floral meristem identity gene LEAFY, thus promoting flowering (Lee et al., 2008; Yoo et al., 2016).
Importantly, SOC1 is a major floral integrator of different flowering pathways in Arabidopsis. For example, another MADS-box gene, FLOWERING LOCUS C (FLC ), which is involved in the vernalization pathway directly binds to the SOC1 promoter, and blocks SOC1 transcriptional activation by CO (Hepworth et al., 2002). FLC also represses FT transcription in the leaves and blocks florigen transport thus inhibiting flowering (Searle et al., 2006).
GI, CO and FT seem to be conserved in flowering pathways in many crops, such as wheat, barley, grapevine, pea, tomato, onion and cucurbits (Watanabe et al., 2011 and references therein). Thus, these genes are promising candidates for flowering time control in Cannabis. However, the gene functions and mechanisms controlling the flowering pathways may differ between species, and thus must be elucidated in Cannabis.
Several of these key regulators of flowering time have been demonstrated to have pleiotropic effects on agronomically valuable characteristics, further emphasising the importance of elucidating the role of these regulators in crop species (Blümel et al., 2015).
Given that Cannabis is a eudicot, short-day plant, the commonly used models- the long-day Arabidopsis or the monocot rice - may not be the most applicable for comparative analysis. Glycine max(soybean) is a short-day crop that belongs to the Fabales and is, therefore, more closely related to Cannabis (Rosales) than rice (Poales) or Arabidopsis (Brassicales) (Figure 3). Flowering time control in soybean is well studied and may provide important clues about how flowering is regulated in Cannabis.
In soybean, the E genes and the JUVENILE (J ) gene are involved in flowering time control (Figure 8) (Copley et al., 2018 and references therein). J is also named GmELF3, it is orthologous to Arabidopsis EARLY FLOWERING3 (ELF3 ), which is an important part of the circadian clock (Lu et al., 2017). Individuals that carry loss-of-function alleles for E1 toE4 exhibit photoperiod insensitive flowering as higher transcript levels of the FT genes are present (Figure 8) (Xu et al., 2013).E1 is a legume specific transcription factor and the remaining genes are orthologous to those involved in flowering time control in Arabidopsis: E2 (also named GmGIa ) is an ortholog of GI, and E3 (GmPhyA3 ) and E4(GmPhyA2 ) are orthologous to PhyA.