Back to the Root The Role of Botany and Plant Physiology in Cannabis Testing, Part III: Genetic and Environmental Factors Associated with Terpene Synthesis in Plants

Terpenes are a class of compounds that have become of increasing interest to a broad swath
of the cannabis industry because of their desirable consumer appeal and undesirable potential
to act as interfering compounds in certain analytical assays. In Part III of "Back to the Root"
we explore the role that terpenes play in plant immunity, defense, and signaling.
A discussion of synthesis pathways will broaden our understanding of how and where these molecules are produced within plant tissues, as well as how they are expressed in plant defense
and immunity. Finally, this article investigates environmental and genetic factors that
influence terpene production in plants and the implications for cannabis.

As the body of research on cannabis continues to grow, terpenes have become of increasing interest to the cannabis industry as a whole from grower to scientist. Terpenes are a broad class of organic compounds classified by the number of 5-carbon isoprene units composing the molecule. Monoterpenes are composed of two isoprene units, sesquiterpenes have three, and diterpenes have four. Terpenes are secondary metabolites, which is just a way of saying that they are not involved in primary metabolic functions related to growth, development, or reproduction. This does not mean, however, that secondary metabolites are any less important to plants simply because their role is outside of primary metabolism. Terpenes in particular play an important role in plant immuno-defense systems, acting both directly as a primary defense and indirectly as communication molecules as we will see later.

Plant Defense Systems

To properly begin our discussion of terpenes, we need to consider their botanical importance, which means we need to understand a little bit about plant defense. In part II of "Back to the Root," we began to explore plant defense systems as they relate to the movement of systemic pesticides in plants (1). As a reminder, plant defenses can fall into two primary categories; induced defenses, which occur in response to stimuli that indicates a threat, and constitutive defenses, which are ever present regardless of attack (2). However, distinguishing between these categories can be a little murky; hypothetically plants that experience a consistent level of threat are thought to develop constitutive defenses while plants that experience varying levels of threat are more likely to rely on induced defenses (2). For example, plants in environments with a consistent level of herbivory, high or low, should develop constitutive defenses to defend them from herbivores. On the other hand, plants living in environments where levels of herbivory vary—say due to seasonal variations in the activity of herbivores—hypothetically will develop induced responses to conserve resources during times of lower threat. Keep in mind however, that these two strategies are not mutually exclusive and most plants tend to have both induced and constitutive defenses.

In cases of induced defenses, the plant needs to first perceive an imminent threat to trigger a defense response. Some examples of threatening stimuli include detection of insect footsteps or eggs, mechanical damage, introduction of hydrogen peroxide (H2O2) from insect feeding secretions, or fragments of cell walls from pathogens (2). We will begin by exploring the detection of pathogenic microorganisms. Plants detect pathogens at the cellular level; embedded in the plasma membrane of plant cells are receptors, proteins that recognize microbe-associated molecular patterns (MAMPs) (2). Some examples of MAMPs include oligosaccharides, peptides, and enzymes (2). These patterns tend to be highly conserved in the microbes’ genome and allow for the detection of entire groups of microbes that share a recognized molecular pattern (2). These patterns are highly conserved because they are usually critical to a microbe’s ability to survive, reproduce, or form colonies making them unlikely to be easily changed without serious consequences. Once a specific molecular pattern is recognized by its corresponding receptor, a signal pathway is triggered that induces a defense response.

As with all immuno-defense responses, this amounts to an arms race driven by selection and evolution. Over time, selection will favor those pathogens that possess a trait that improves their chances to avoid detection by the host, survive, and reproduce, resulting in the evolution of resistant microbe populations. We refer to these traits as effectors, some examples of which include changes to the identifying pattern that are just enough to make it unrecognizable while remaining functional or behaviors such as shedding an identifiable structure upon entering a host (2). In turn the hosts have evolved a way to still detect pathogens that have avoided detection by the more generalist receptors. In plants this amounts to a second, more specific level of defense involving specialized plant receptors called R proteins (R for resistance) that detect specific effectors and trigger a defense response (2). By combining broad detection of large groups of microbes with recognition of more specific effectors, plants have what amounts to a multilayered pathogen detection system. 

The mechanism of herbivore detection is thought to be similar to that of pathogen detection; plants possess receptor proteins able to recognize herbivore-associated molecular patterns (HAMPs) (2). Some molecular patterns identified by research include chemicals released when an insect walks across a plant surface, as well as insect secretions from both feeding and egg-laying (2). While many of the receptors involved in microbial detection are well understood, less is known about the receptor proteins that detect herbivore-associated molecular patterns. Molecular and chemical cues are not the only way that plants detect insect threats. Plants have been shown to be highly sensitive when it comes to detecting the pitter patter of insect feet; very slight disruptions of the epidermis by the claws of caterpillar feet are enough to trigger a defense response in plants (2). This shows that in the case of footsteps at least, plants are sometimes responding to mechanical damage, rather than chemical cues released by the insects as they walk.

In the case of feeding insects, detection cues that trigger a defense response can be either chemical, through molecular recognition by a receptor, or mechanical and seem to vary amongst plants. Some plants respond similarly to wounding by feeding insects as they do to simulations such as leaf clipping, suggesting that mechanical cues are triggering a defense response (2). Other plants treat these as two different threats that elicit different responses, indicating that they are using chemical cues such as feeding secretions to distinguish between threats (2). To the best of my knowledge at this time, little has been done regarding insect wounding studies on Cannabis subspecies (ssp.) (see footnote below) though there has been some indication that insect wounding increases resin production (3). This is an important knowledge gap because as any grower knows, pests are a major source of crop damage and loss for both indoor and outdoor cannabis grow facilities. A better understanding of how cannabis plants detect insect damage might allow growers to better tap into native plant defense systems.

Broadening our understanding of the cues that trigger different defense responses, the receptors that detect those cues, and how cannabis responds to threats would enhance our knowledge of the chemical ecology of cannabis. Discovering both microbe-associated and herbivore-associated molecular patterns that cannabis plants use to detect threats might allow for a sort of immunization of plants, called systemic acquired resistance (SAR). Additionally, plants can inherit immunity and resistance to pathogens and pests, making selective breeding another highly viable option for improving crop health as genetic research on cannabis advances. As I have advocated previously, until we have a better understanding of the relationships of the varied chemotypes in the genus Cannabis (high-cannabidiol [CBD], high-tetrahydrocannabinol [THC], fiber producing, and so on) each should be investigated individually since so much variation among plant responses can and does exist.

The Role of Terpenes in Plant Defense

Now that we have an idea of how plants identify threats through molecular and physical cues, we’re ready to move on to a discussion of the role that terpenes play in plant defense. So far we have predominantly talked about the detection of cues that result in induced defenses, wherein the plant is responding to a threat in response to some cue. Another way to think about plant defense is to consider whether the defense is direct, attacking the threat organism outright, or indirect. Terpenes can act in both indirect and direct defense. As direct defense, many terpenes have antifungal or antibacterial action while others are repellent or outright toxic to insects (2,4). Terpenes can also act as a direct defense by deterring insect egg-laying (2,5). If the eggs are not laid, the caterpillars won’t hatch and consume the plant; really a rather proactive approach.

Indirect defenses are perhaps the more interesting, from a behavioral standpoint, because they require communication: intraplant (from one part of the plant to another), plant to plant, or plant to insect. Keep in mind that while it’s common to think of communication generally as sound or movement, plant communication is done through cell-signaling and the emission of volatile organic compounds (VOCs). When a plant is attacked, communicating the need to ramp up defenses to all tissues at risk is crucial. Plants generally communicate this need using salicylic acid in the case of microbial attack and jasmonic acid in the case of insect attack. These acids are transported through the plant via the phloem. Translocation of these compounds is relatively slow and is further compounded by the mosaic of connectivity of plant vascular systems (2). This means that the leaves immediately next to a leaf under attack might not have a direct vascular connection, but would surely benefit from a ramped up defense response.

What is a plant to do? Both salicylic and jasmonic acid can be converted to volatile methyl esters, which when released can communicate a threat to other parts of the plant that have limited vascular connectivity (2). Usually release is precipitated by wounding in the case of jasmonates, the group of compounds that include jasmonic acid and methyl jasmonate (2). Methyl jasmonate has even been shown to induce the synthesis of defense compounds in neighboring plants in leaf wounding studies wherein wounded plants are grown in the vicinity of nonwounded plants and both are measured for defense responses (2). Less is known about the ability of methyl salicylate to confer systemic microbial resistance to neighboring plants (2). Jasmonates can also regulate the emission of other stress induced volatile organic compounds including terpenes (4). It is yet unknown how exactly plants perceive these volatile signaling molecules. Interestingly, terpenes themselves can stimulate the biosynthesis of jasmonic acid indicating that there is a very close molecular interplay between these compounds (2). It makes sense for defense signaling molecules to have a reciprocal response to each other, since that amounts to what is effectively a two-way communication channel.

Once released, terpenes can induce the expression of a multitude of herbivore- and pathogen-defense genes (2,4). Riedlmeier and colleagues showed in the model organism Arabidopsis thaliana that a- and b-pinene induced defense responses, as well as stimulating the expression of several genes related to salicylic acid production and SAR (6). Further, both pinenes and camphene elicited a defense response in neighboring plants, showing that these terpenes can act as communication molecules between plants (6). Evidently, monoterpenes in particular play an important role in
intra- and interplant communication.

Another interesting way that terpenes and jasmonates can function in indirect defense is by attracting herbivore predators and parasites (2,4). When a plant has been wounded, the released terpenes and other volatiles act as a distress signal that has wasps and other insect predators buzzing in. The mixture of volatiles emitted by a plant in response to herbivory can be specific to the plant and the attacking herbivore, meaning that insects, allies, and threats alike can tell who is being attacked and by whom (2). Increasingly, some cannabis growers have opted to use biological controls such as beneficial insects over traditional chemical pesticides. Understanding more about the specific volatile signature of cannabis plants under various insect stressors could prove useful by enhancing the effectiveness of biological controls.

It is not well understood how exactly insects, both herbivores and predators of herbivores alike, are distinguishing between these volatile molecular cocktails, which can sometimes contain hundreds of compounds. Much debate surrounds the topic, though it seems most likely that insects are relying on different ratios of compounds for identification rather than identifying species-specific volatiles (2). It is worth mentioning briefly that terpenes can also play a role in attracting pollinators, though this doesn’t apply in the case of cannabis which is a wind pollinated species.

Terpenes and Abiotic Stress

Cannabis ssp. can produce an extensive assortment of terpenes, with more than 150 unique terpenes identified in the resin of various cannabis chemotypes (7). We now have a good idea of the role terpenes play in plant defense, so let’s talk about abiotic stress. Abiotic stress is any nonliving factor that negatively impacts a plant’s ability to survive, grow, or reproduce. Some examples of abiotic stress include nutrient deficiencies, drought, salinity, temperature, and damage from reactive oxygen species. Because plants are sessile (they can’t run away), being able to adapt to abiotic stress is crucial to their survival. It has been demonstrated that plants emit terpenes at higher levels when they experience heat stress (8). Terpene emissions should be expected to increase as temperature increases because heat makes them more volatile, resulting in a higher vapor pressure. However, more terpenes are emitted under temperature stress than can be accounted for by vapor pressure changes alone, making the case that biosynthesis has increased as well. Further, research has shown that plants fumigated with certain monoterpenes recover more rapidly from high temperature exposure, as measured by photosynthesis rates, than untreated controls (8). This indicates that terpene exposure beneficially impacts recovery time for plants exposed to heat stress.

Another abiotic stress that terpenes can alleviate is oxidative stress. Oxidative stress can result in damage to cells and biomolecules such as DNA, making the control of reactive oxygen species critical. Further, oxidative stress in plants is an additional consequence of many other biotic and abiotic stresses including herbivory, temperature, and light stress. Monoterpenes and sesquiterpenes both have been shown to decrease reactive oxygen species in fumigation and genetic modification studies (8). It has also been shown that plants produce high levels of sesquiterpenes when exposed to ozone (O3), a common reactive oxygen species (4). Alternatively, when monoterpene biosynthesis was inhibited, plants showed higher levels of oxidative damage and reduced photosynthesis (8). The evidence that terpene presence improves recovery from oxidative stress while terpene suppression results in greater damage suggests that terpenes play an important role in oxidative stress responses.

Drought stress and salt stress are also abiotic stressors that have been shown to increase terpene emission by plants, but inconsistently (8). This suggests that a terpene response to these stressors may be species specific, indicating that they might induce terpene release in Cannabis ssp. or they might not. More research is needed to determine what effects drought and salt stress might have on terpene emissions in cannabis, if any. Low nitrogen availability appears to have an impact on isoprene emissions in plants in general, though whether or not it impacts terpene emission is not well understood. Because isoprene is the building block of terpenes this relationship bears further investigation. In hemp, there has been some evidence that nitrogen metabolism-related genes and genes involved in secondary metabolism are coregulated (9). However, more research is needed to determine if terpene genes specifically are coregulated with genes related to nitrogen-metabolism.

Terpenes in Cannabis

In Cannabis ssp. terpenes are both produced and stored in glandular trichomes found on all aerial parts of the plant, but female flowers possess the greatest quantity of trichomes. Cannabis flowers have three morphologically different types of trichomes: bulbous, which are the smallest and produce few secondary metabolites; sessile, which sit on a short stalk, topped with a round disk of secretory cells and have a storage compartment that extends below the surface; and stalked, which are structurally similar to sessile trichomes but with a larger head and a longer stalk (10). Vegetative leaves and anthers do not possess stalked trichomes, but do have sessile trichomes (10). Trichome morphology alone cannot tell us about any chemical differences or the developmental relationship between these different trichome types.

Livingston and colleagues sought to greatly broaden our understanding of cannabis trichome structure and chemical composition, finding evidence that on female flowers, stalked glandular trichomes develop from immature, “sessile-like” trichomes (10). These immature trichomes differ from “true” sessile trichomes found on leaves and anthers in several key ways. True sessile trichomes were shown to have a greater ratio of sesquiterpenes to monoterpenes and to sit directly on the surface of the epidermis (10). In contrast, stalked glandular trichomes and their immature, sessile-like precursors have a greater ratio of monoterpenes to sesquiterpenes and sit on a stalk that lengthens as the trichome develops (10). The two can also be distinguished by the number of cells they have and their fluorescence (10). Which cannabinoids the trichomes produced was not found to vary across trichome types but the quantity of cannabinoids did, with stalked trichomes having the highest cannabinoid content (10). This study is especially noteworthy because Livingston and colleagues tested both the model hemp variety “Finola” and two “marijuana-type” (high THC) varieties: “Purple Kush” and “Hindu Kush.” Because they were so thorough they were able to show that their findings on trichome structure were consistent across these chemotypes.

It is well known that terpenes and cannabinoids alike are produced and stored in various glandular trichomes; more recently researchers have begun to examine terpene synthesis pathways and their related genetic components in Cannabis ssp. As mentioned earlier, terpenes are composed of isoprene units, the number of which determine the type of terpene (mono, sesqui, di, and so on). Terpenes are synthesized via two primary metabolic pathways: via the mevalonic pathway from acetyl-CoA or via the methylerythritol phosphate pathway from pyruvate (2). Special enzymes called terpene synthasescatalyze the chemical reactions that convert precursors into terpenes; the diversity of terpenes found in cannabis is reflective of the diversity of genes related to terpene synthase enzymes in the cannabis genome (7). Many of these terpene synthases can produce more than one type of terpene, possibly explaining why certain terpenes in cannabis tend to co-occur (7). Monoterpenes and cannabinoids share a precursor molecule, 10-carbon geranyl diphosphate (GPP C10), though sesquiterpenes have a different 15-carbon precursor (7). The fact that monoterpenes and cannabinoids share a precursor is logical because, as we have learned, cannabinoids are produced in greater quantities within stalked trichomes that have a greater proportion of monoterpenes.

An increasingly popular way to clarify the relationships surrounding terpene synthesis, terpene synthases, and various terpene synthesis-related genes is through transcriptomic analysis. Transcriptomic analysis looks at the transcriptome, or total set of RNA transcripts produced by the genome in a specific cell or under specific conditions. It has become increasingly popular with researchers of secondary metabolites because it reflects the genes that are actively being expressed at any given time, allowing for the identification of comprehensive sets of genes involved in the synthesis of compounds of interest. Several research groups have recently applied this methodology to illuminating terpene synthesis in cannabis. Booth and colleagues used transcriptomic analysis to identify nine major cannabis terpene synthases in the “Finola” hemp variety (11). The products of these synthases are responsible for most of the terpenes present in “Finola” resin including β-myrcene, (E)-β-ocimene, (-)-limonene, (+)-α-pinene, β-caryophyllene, and α-humulene (11). This was a significant contribution to understanding terpene synthesis pathways in cannabis because these terpenes are frequently detected at significant levels in cannabis and hemp, in my experience.

The researchers were also able to compare their discovered genes with the recently sequenced genome of “Purple Kush,” a “marijuana-type” (high-THC chemotype) cannabis strain. Some terpene synthase-related genetic overlap between the varieties was observed, but not all of the genes identified were present in both chemotypes. The researchers speculated that some genes might have evolved to have different functions over time in different cannabis varieties (11). For example, they found a gene involved in α-pinene synthesis in “Finola” but that gene was not present in the “Purple Kush” genome, a strain that tends to have high levels of α-pinene (11). This implies that some other gene, or combination of genes, might be responsible for α-pinene synthesis in “Purple Kush.” This highlights the need to research as many chemotypes as is practical because genes can evolve over time, resulting in multiple synthesis pathways to the same product.

Building off the work of Booth and colleagues, Zager and colleagues did a transcriptomic analysis of nine recreational cannabis strains, including the high-CBD recreational strain “Canna-tsu.” They found that the genes identified by Booth and colleagues for β-myrcene, (2)-limonene, α -pinene, β-caryophyllene, and α-humulene, were expressed at high levels across all strains (12). They also identified a gene that codes for a terpene synthase responsible for producing linalool and nerolidol in these strains (12). This makes a strong argument for the close relationship of recreational cannabis chemotypes and also identifies another terpene (linalool) commonly found at significant levels in cannabis. Nerolidol on the other hand, is relatively rare in my experience, making the fact that it shares a synthase with linalool interesting.

In addition to characterizing chemical differences in different trichome types across different cannabis varieties, Livingston and colleagues also did a transcriptomic analysis of different flower trichome types from the model hemp variety “Finola.” They found that there was no significant difference in gene expression between stalked trichomes and their immature prestalk precursors (10). Livingston and colleagues also identified two previously uncharacterized terpene synthases in “Finola,” which produced terpinolene and β-ocimene in recombinant studies (10). Prior to this study, no terpene synthase producing terpinolene had been identified.

Conclusion

It’s exciting to see so much novel, molecular research exploring terpene synthesis pathways in both hemp and recreational cannabis chemotypes. As the body of molecular and genetic knowledge accumulates, we will be better equipped to understand the ecological role of terpenes in cannabis; the study of which will provide benefits beyond knowledge alone. We will also be able to move closer to clarifying the relationship between Cannabis ssp. and the genetic factors that characterize strains of recreational cannabis. In the next and final article of this series we will explore cannabinoids, specifically what we know about them botanically and what we don’t. 

References

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About the Author

Gwen Bode, B.S., is an aspiring doctoral candidate and botanist with a strong chemistry background. As an undergraduate at Eastern Washington University she investigated the vitamin content of a wild edible plant via HPLC. She has since worked at the front line of the cannabis testing industry, integrating her botanical knowledge with the practical aspects of analytical testing. Direct correspondence to: gwenbode@gmail.com​

Footnote: In trying to find a concise but accurate way of expressing the uncertainty surrounding the speciation of cannabis, I have chosen to refer to the genus (Cannabis) followed with the abbreviation for subspecies, because this best reflects the formal taxonomy of cannabis, which recognizes only one species (Cannabis sativa L.), while encompassing all varieties and or chemotypes.

How to Cite this Article

G. Bode, Cannabis Science and Technology 3(7), 26-31 (2020).