Fertilization of Cannabis decreases phytocannabinoids accumulation and alters the accumulation of terpenoids from distinct families.

Cannabis sativa L. (Cannabis) has been known as a medicinal plant since ancient times (Bonini et al., 2018). During the last two decades, many studies added to the growing evidence for its therapeutic effects in a wide range of conditions such as neurodegenerative disorders (Fernández-Ruiz, 2019; Cassano et al., 2020), pain (Starowicz and Finn, 2017), epilepsy (Franco et al., 2021), multiple sclerosis (Rice and Cameron, 2017), and others (for review, see Gonçalves et al., 2019). These therapeutic abilities have been attributed to the secondary metabolites biosynthesized in Cannabis (Andre et al., 2016). Around 500 different secondary metabolites have been identified (ElSohly and Slade, 2005; Flores-Sanchez and Verpoorte, 2008). These belong to several groups of compounds including phytocannabinoids, terpenoids and flavonoids. The most characterized to date are the phytocannabinoids, lipophilic compounds made of isoprene units (five-carbon building blocks) (Hanuš et al., 2016), which are almost exclusive to Cannabis (Gülck and Møller, 2020). More than 140 different phytocannabinoids are accumulated to various extents in glandular trichomes that are located in the aerial parts of the plant and mostly on the female flowers, which are arranged in a cluster on the stem of the inflorescence (Hanuš et al., 2016). The phytocannabinoids can be classified into several subclasses according to their chemical structures, including the (-)-Δ⁹-trans-tetrahydrocannabinol (THC) and cannabidiol (CBD) families as well as cannabinol (CBN), cannabigerol (CBG), cannabichromene (CBC), and others (Flores-Sanchez and Verpoorte, 2008; Berman et al., 2018). A second large group of metabolites is the terpenoids, which are also found in many other plants. These metabolites are closely related to phytocannabinoids, sharing the same isoprenoid precursor and built up by branched isoprene units (Booth et al., 2020). Terpenoids are responsible for the fragrance and taste of the plant and are suggested to also have defensive roles. They also contribute to the therapeutic effects attributed to Cannabis (Russo and Marcu, 2017). Another group of metabolites worth mentioning is flavonoids. Among this group, which is widespread in the plant kingdom, there are three specific prenylated flavonoids, termed Cannflavins A–C, which are unique to Cannabis and show potent anti-inflammatory abilities (Radwan et al., 2008; Rea et al., 2019; Erridge et al., 2020).

Ongoing research is focused on matching specific metabolites found in the plant and their therapeutic capabilities. To this end, specialized analytical methods have been developed in order to obtain precise knowledge on all the components of the plant and the effects they are responsible for. Currently, more than 90 phytocannabinoids and 100 terpenoids are routinely identified and quantified to obtain an overall chemical profile of each chemovar used for a medicinal purpose (Berman et al., 2018; Shapira et al., 2019). In parallel to the search for specific biological activities of the secondary metabolites, broad ongoing research is focused upon the elucidation of in planta metabolites’ biosynthesis, transport and accumulation pathways. Genome, transcriptome and proteome data have been published since 2011 (van Bakel et al., 2011; Laverty et al., 2019; Vincent et al., 2019; Livingston et al., 2020; McGarvey et al., 2020), and have been integrated into a genomic database for Cannabis (CannabisGDB) (Cai et al., 2021). Biosynthetic pathways are being unraveled, and recently more than 30 Cannabis specific terpenoid synthases have been characterized (Booth et al., 2017, 2020; Allen et al., 2019; Zager et al., 2019; Livingston et al., 2020). In addition, the environmental and developmental factors that affect metabolite accumulation are also studied, such as light (Hawley et al., 2018; Magagnini et al., 2018; Namdar et al., 2019), soil and harvest time (Meier and Mediavilla, 1998; Bernstein et al., 2019; Chandra et al., 2020). The increasing information on the impact of these different factors on metabolite accumulation has the prospect of developing specific chemovars harboring a pre-planned group of metabolites (Romero et al., 2020).

This study examined the effect of an additional factor, the fertilization of Cannabis flowers following pollination of the pistil. Fertilization of flowers is a key step in the plant life cycle. Successful pollination activates a series of events followed by fertilization and embryogenesis. This includes the development of an ovary on one hand, together with senescence and abscission of floral organs, degradation of macromolecules, and recycling of different nutrients on the other hand (O’Neill, 1997; Tripathi and Tuteja, 2007; Borghi and Fernie, 2020). Cannabis is a dioecious plant, harboring either female or male reproductive organs. It is also a wind-pollinated plant, in which the pollination of flowers is not dependent on specific animal pollinators. Phytocannabinoids are most abundant in the female flower inflorescences (Flores-Sanchez and Verpoorte, 2008). Fertilized flowers, harboring seeds, are considerably less potent. Hence the term “sinsemilla,” Spanish for “without seed,” that defines plants associated with high psychoactive effects (Potter et al., 2018). In addition, it is a common work practice by Cannabis growers to eliminate male plants growing in a field to maintain the unfertilized inflorescences and maximize the phytocannabinoid concentrations. Therefore, it is likely that the content of secondary metabolites such as phytocannabinoids and terpenoids changes following the pollination and fertilization of Cannabis inflorescences. However, although mentioned in a few studies (Meier and Mediavilla, 1998; Potter, 2009; Russo and Marcu, 2017), this phenomenon was not studied in depth. In the last few years, an increasing number of Cannabis growers are moving from using cuttings from female “mother plants” to seeds. Even though the seeds are usually feminized, around 5–10% will be males, and thus the question about the effect of pollination on the phytocannabinoids and terpenoids expression becomes critical.

In order to gain more insight into the Cannabis metabolite regulation pathway, this work studied the effect of flower fertilization on the plant’s secondary metabolite accumulation. We used indoor growing methods together with analytical procedures in order to investigate the effect of fertilization on metabolite composition and concentration in Cannabis inflorescences, and specify which metabolites are affected and to what extent.

The present study was designed to examine the influence of flower fertilization on the accumulation of Cannabis secondary metabolites. The primary outcome is the significant overall decrease in phytocannabinoid metabolites upon fertilization. This decrease was evident in almost all phytocannabinoids measured, regardless if those were the abundant ones or the relatively low accumulating components (Figure 2). Though the altogether amount of phytocannabinoids is drastically reduced, the ratio between the different compounds is kept and their profile in the plant remains principally unchanged.

Terpenoid concentrations mostly decreased but varied. While monoterpenoids had a similar decrease as portrayed by the phytocannabinoids, sesquiterpenoids exhibited a more diverse pattern, some of which increased and some decreased upon fertilization (Figure 3). However, examining specific metabolites can point to several phytocannabinoids or terpenoids that have an individual trend, suggesting a more complex regulatory network (Figures 4, 5).

First, these results confirm that when the objective is to maintain high levels of phytocannabinoids, fertilization must be avoided. Apart from a physical separation between female and male flowers or vegetative reproduction, this goal could be achieved using advanced genetic manipulations that target female fertilization pathways (Huang et al., 2016; Jung et al., 2020; Zhu et al., 2020).

Second, this study revealed the resemblance between monoterpenoids and phytocannabinoids accumulation patterns. Both secondary metabolite species are decreased upon fertilization, while sesquiterpenoids are differently influenced. Possible explanations for this similarity are common intracellular regulation pathways or shared morphological structures. From a cellular perspective, monoterpenoids and phytocannabinoids share the common biosynthetic precursor Geranyl diphosphate (GPP) and are both biosynthesized in the plastid (Booth et al., 2020; Romero et al., 2020). In contrast, sesquiterpenoids are synthesized in the cytosol from a different precursor (Farnesyl pyrophosphate—FPP). This suggests that phytocannabinoids and monoterpenoids may share a common regulation mechanism, involving an enzymatic step upstream to GPP, such as GPP synthase (illustrated in Figure 7).

Alternatively, from a morphological perspective, previous studies have shown that although phytocannabinoids, monoterpenoids, and sesquiterpenoids are all biosynthesized and accumulated in the glandular trichomes, their distribution differentiates during trichome development and between trichome types. A recent study by Booth et al. (2020) showed an increase in the ratio of monoterpenoids relative to sesquiterpenoids when flowers are maturing. Another study (Livingston et al., 2020) showed that monoterpenoids are accumulated in both pre-stalked and stalked trichomes, while sesquiterpenoids are abundant in sessile trichomes. Phytocannabinoids are accumulated in both types of trichomes, but the stalked type composed 80–90% of the total trichomes in the mature flower. A common accumulation pattern of monoterpenoids and phytocannabinoids during flower development was also previously demonstrated (Aizpurua-Olaizola et al., 2016). Parallel accumulation and decrease of phytocannabinoids and monoterpenoids in contrast to sesquiterpenoids may suggest that trichome types are differently affected by fertilization, and hence the diversity in metabolite accumulation.

An additional major finding depicted in this study is the somewhat dependent outcome of the fertilization process on the pollen donor plant. Both THC- or CBD-rich male plants, whether naturally occurring or female-induced, had a different impact on the metabolite concentration in the female after fertilization. For instance, fertilization by the induced-male led to an increase of specific phytocannabinoids (Figure 2): THC and THCA in the CBD female and CBC, CBCA, CBCVA, and 373-15C in the THC female. The exact mechanism by which these phytocannabinoids are increased is not yet clear. It may be the result of altered regulation of synthesis enzymes, for example the upregulation of THCA synthase or CBCA synthase (Laverty et al., 2019). A previous study found over 10,000 genes are differentially expressed upon masculinization of female plants (Adal et al., 2021), but it is not clear how these genes are related to phytocannabinoid expression in the fertilized female plant. A donor-dependent effect was also detected in the specific increase in the Eudesmol family (α-, β-, and γ-Eudesmol) components, which were highly increased in the THC-rich female upon fertilization by the THC-rich male plant (Figure 6A) and a parallel specific increase in Linalool in the CBD-rich female fertilized by the induced male (Figure 6B). However, regardless of the type of male plant used for fertilization, the overall profile of the phytocannabinoids in the fertilized female plant remained unaltered, i.e., no new phytocannabinoids that were not expressed in the unfertilized plant were discovered and the relative ratio between the different phytocannabinoids was mostly kept. Interestingly, though the density of phytocannabinoids and terpenoids in males is minor (data not shown) compared to the female flowers, with high potency female plants showing 10–20-times more THC than corresponding males (Clarke and Merlin, 2016), male plants also possess a distinct profile of these compounds.

Here, we used highly advanced analytical methods to thoroughly assess the composition of 95 phytocannabinoids and 113 terpenoids in the inflorescences of female plants fertilized by different males, including the female plant itself induced to develop male pollen sacs. We found that fertilization significantly decreased phytocannabinoids content, while terpenoids were differentially affected. To further elucidate the effect of fertilization on the secondary metabolite accumulation, future studies that follow the gene expression of enzymes upstream to GPP after fertilization may allow exposing master regulators of the biochemical pathways. In addition, better characterization of the morphological changes following fertilization may shed light on how different trichome types are affected by fertilization. Finally, the variance in metabolites observed by fertilization with different male plants may suggest that the pollen itself or the developing embryo influence the female sporophyte.

Altogether, one must remember that these specialized secondary metabolites have an important role in planta, increasing the plant fitness to the environment (Huchelmann et al., 2017). The substantial decrease in phytocannabinoids and terpenoids after fertilization may point to their functional roles in the plant. The actual functions of phytocannabinoids and terpenoids in Cannabis were only sparsely studied, mainly suggesting roles in defense against biotic or abiotic factors (Potter, 2009), protection from UV radiation (Eichhorn Bilodeau et al., 2019), prevention of desiccation (Gülck and Møller, 2020), or induction of cell death in leaves (Morimoto et al., 2007). The observed dynamics of the accumulation of these metabolites during flower development and fertilization may point to their different roles along the plant’s life cycle.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

PB, MF, and DM: conception and design. OC, IK, and RP: plant maintenance, manipulations, fertilization, and harvest. CL: acquisition and ESI-LC/MS analysis and interpretation of data. AS: acquisition of GC data and development of semi-quantitative GC methodology. OG: extraction and sample preparation of Cannabis samples and UHPLC/UV analysis. CL, AS, and SP: figure preparation. CL, SP, PB, MF, and DM: writing, review, and revision of the manuscript. MF and DM: study supervision. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.