Unlocking the secrets of cannabis…

30 November 2019 - By: Caroline Wood

Unlocking the secrets of cannabis…

A maligned medicinal aid or a societal scourge? Cannabis sativa, also known as marijuana or hemp, is certainly a versatile plant, having been used over millennia as a source of oil and fibre source, besides psychoactive compounds. Traditionally, this plant has been highly restricted in Western economies, however new, non-psychoactive varieties and recent relaxes in legislation have opened up a thriving area of research. In particular, the purported medicinal benefits of certain cannabinoid compounds have garnered significant media attention and spawned a new economy in hemp-infused products. Nevertheless, further uses for cannabis are continually being revealed including being a model species for investigating questions in plant evolution and a potential source of more sustainable construction and packaging materials. With this current high interest, it will be an opportune time for the cannabis research community to come together during a dedicated session at SEB Prague 2020. Caroline Wood gives a preview of the discoveries that will be discussed during “Challenges and opportunities in cannabis research”.

A new plant for a new niche

“Cannabis is a really interesting platform for studying the interaction between human culture and plant evolution, since it was domesticated during at least two independent events for very different purposes” says Chris Grassa (Harvard University, USA).  Marijuana varieties were originally bred in Afghanistan and South Asia to develop plants that produce high quantities of intoxicating cannabinoids (particularly tetrahydrocannabinol, THC). Hemp cultivars, on the other hand, were bred in Europe and East Asia and as a food and fibre source, and produce a non-psychoactive cannabinoid called cannabidiol (CBD). This compound has attracted considerable medical interest, since CBD has shown effectiveness in treating epilepsy and can give a calming, pain-relieving effect without the hallucinatory side effects of THC. Unfortunately, hemp plants only produce CBD in low quantities, since cannabinoid content was not selected for during their domestication. In recent years, however, high-CBD, low-THC cannabis plants have emerged, opening up a booming market in ‘healthful CBD-infused products’ including everything from bath salts and skin creams to coffee and cookies. But where did these ‘miracle’ plants suddenly come from?

To investigate this, Chris sequenced the genomes of marijuana, hemp, and high-CBD plants using advanced long-read sequencing technologies. With the help of colleagues at the University of Wisconsin, he also accessed a marijuana x hemp genetic mapping population to identify quantitative trait loci (QTL) associated with cannabinoid production. “We found that the qualitative trait of whether THC or CBD is produced is determined by a single locus in the genome” Chris says. In the high-CBD plants, this gene was the same as hemp, indicating that the trait was developed by introducing a hemp gene into a marijuana background. “These cultivars are effectively mosaics, with 90% of the genome being marijuana-like and 10% being hemp-like”.

This fits the theory that a change in USA law opened up a ‘new ecological niche’ for these plants to be developed. In December 2018, the legal definition of cannabis changed from prohibiting all plants containing either CBD or THC to specifically outlawing cultivars containing more than 0.3% THC on a dry-weight basis. High-CBD plants, therefore, are legally classed as hemp, despite being more like marijuana plants in appearance. “It’s said that the first high-CBD cultivar, Charlotte’s Web, was developed to treat a young girl suffering with a severe form of epilepsy called Dravet syndrome” Chris says. “In plant breeding terms, this occurred incredibly quickly in only about 8 generations”.

Interestingly, although this locus is fixed for domesticated hemp and marijuana plants, according to Chris there does not appear to be a selective pressure in wild cannabis populations. “In feral plants, we find that this locus sorts to Hardy-Weinberg principles: the plants don’t seem to care which allele they carry”. These discoveries have opened up a wealth of new questions that Chris is eager to explore, using a collection of 2,000 cannabis samples from around the world that spans five centuries. “I’d like to understand how global trade affected the cannabis genome, especially when populations were exposed to new environments” he says. “Another interesting question is how law has acted as a barrier to gene flow between marijuana and hemp – did the war on drugs, for instance, limit the effective population sizes of marijuana?”

“It’s sometimes a challenge to have people take my work seriously. One strategy I have is to avoid naming my system at first and talk in terms of population genetics and diversification, before revealing that I work on cannabis” Chris concludes. Despite the taboo, his work undoubtedly shows the usefulness of Cannabis sativa to answer fundamental evolutionary questions.

Unravelling the mystery of the sexes…

Outside the world of cannabinoids, cannabis plants are proving a useful tool to explore another evolutionary mystery: the evolution of plant sex chromosomes. “There have been several independent transitions to dioecy, where plants have separate male and female sexes. But the genetic transition is not well understood” says Djivan Prentout (Laboratory of Biometry and Evolutionary Biology, Lyon, France). Dioecy is much less common in plants than animals, being found in only 5-6% of flowering species. Plant sex chromosomes also evolved much more recently than those of animals: mammalian sex chromosomes, for instance, originated 150-180 million years ago, whilst those of plants studied so far are less than 10-15 million years old. This has made it challenging to decipher how the sex chromosomes evolve after the initial transition to dioecy. Preliminary work, however, suggested that cannabis plants have considerably older sex chromosomes than all previously studied plant species, which attracted Djivan’s original interest in the plant.

One of his main research aims was to understand why and how the Y chromosome often degenerates over time, for instance due to the accumulation of deleterious mutations. It is generally thought that the transition to dioecy begins with mutations on a pair of chromosomes that form sex determining genes: possibly, these may inhibit the formation of the male/female reproductive organs. The emergence of these genes suppresses meiotic recombination, where chromosome pairs exchange DNA sequences and repair damaged sequences. As the Y chromosome is now effectively isolated from its former twin, it cannot repair itself and degenerates over time as genes become non-functional.

“Our work found that cannabis has the oldest sex chromosomes ever identified in plants: over 27 million years old” says Djivan. Using RNA-sequencing to measure the quantity of genes that are directly expressed, he found that up to 70% of the X chromosome genes are not expressed by the Y counterpart. “What is interesting is that despite this difference in gene expression, the X and Y chromosomes in cannabis are still the same size” Djivan says. “This makes cannabis a very curious model because a large non-recombining region is normally associated with heteromorphic sex chromosomes. Further analysis on the genomic content of the Y chromosome would help to understand this paradox”.

Djivan now hopes his results could be practically applied to develop genetic markers for cannabis sex chromosomes. “Only the female plants produce cannabinoids such as THC and CBD” he says. “For industrial cannabis production, it is a considerable waste of energy and water if you have to wait for several months before you can detect the male plants”. At the SEB Prague session, he hopes to find collaborators to develop genetic sex markers and to identify sex-determining genes in cannabis and potentially other crops. But why only female cannabis plants produce cannabinoids is itself a mystery.

In the meantime, Djivan’s next research interest could benefit an entirely different industry. “Hop is a sister species of cannabis, and we believe that the speciation event occurred after the evolution of the sex chromosomes” he says. “Since only female hop plants produce the molecules that give beer a bitter quality, our genetic markers could also benefit beer producers”.

Hemp-infused honey?

Even as the market for CBD-containing products grows exponentially, regulation over the levels of cannabinoid compounds in food and cosmetic products has not matched this pace. “The legal management of CBD and CBD-containing products in Europe is still confused and there is a need for proper legislation” says medicinal chemist Federica Pellati (University of Modena and Reggio Emilia, Italy). She argues that products containing low levels of CBD should be regulated as a food supplement or over-the-counter pharmaceutical product. This would depend, however, on a cheap, fast and reliable method to accurately quantify cannabinoids in a range of different products. For the past twenty years, Federica has applied her extensive experience in natural products chemistry and analysis to test a range of methods which could address this challenge. “We decided to focus our attention on cannabis, since the remarkable diffusion of this plant and its derived products presented the issue of possible dietary consumption of bioactive compounds from this plant” says Federica, who will be presenting her results at the SEB Prague meeting.

One of her projects specifically investigated whether cannabinoids could accidentally be incorporated into honey. Since the greatest proportion of non-psychoactive cannabinoids occurs in the inflorescences, there is a real chance they could be transferred to bees foraging on the pollen of hemp flowers. “Bee products are ubiquitously diffused and consumed and since they are produced by bees that could use a variety of floral sources, including hemp, it was reasonable to check the presence of cannabinoids” Federica says. For the study, her group tested six different commercially available honey brands, including a ‘hemp honey’ produced by bees whose main floral sources were hemp and alfalfa. Inflorescences and pollen samples from the hemp plants themselves were also tested.

“The method we ultimately developed allows the analysis of the main bioactive cannabinoids in less than twenty minutes” says Federica. This coupled high performance liquid chromatography under reversed-phase conditions to tandem-mass spectrometry. “Importantly, since honey is a complex matrix, we also developed an effective sample preparation procedure based on the usage of QuEChERS” says Federica. This method – standing for ‘quick, easy, cheap, effective, rugged and safe’- is a solid phase extraction technique used to detect pesticide residues in food.

For most of the samples, no cannabinoids were detected at all with the exception being the hemp-honey where CBD was detected at 4.2 ng/g. Crucially, psychoactive THC was not present, which was to be expected since the hemp plants were certified as having less than 0.2% THC content. “The cannabinoid profile of this sample matched that of the inflorescences, with the compound cannabidiolic acid (CBDA) being most abundant” says Federica. “This shows that the bees do not chemically alter the cannabinoids while making honey”. Cannabinoids were also detected in the hemp pollen, raising the possibility that these could be transferred to bees and incorporated into apiary products, such as bee pollen powders.

“Considering the trace amount of non-psychoactive cannabinoids in hemp honey, this product can be considered as safe to consume” says Federica. “Nevertheless, it should be taken into account if consumed by children or on a daily basis over a long period of time”. In the meantime, she hopes this work could be extended towards other compounds. “With suitable changes to the analytical parameters and the sample preparation procedure, this method could be applied to detect many plant cannabinoids within different samples” Federica concludes.

Fantastic fibres

Hemp has many uses, however, beyond being a source of CBD since the stems provide economically valuable fibres. “As a cell wall biologist, I find hemp a particularly interesting crop” says Luisa Trindade (Wageningen University & Research, The Netherlands). Since hemp plants are more efficient in the use of water than cotton, they offer a more sustainable source of fibres to satisfy the growing human demand. Besides traditional uses in textiles and furniture, hemp fibres are increasingly being applied in the construction industry, including to strengthen cement and to give concrete enhanced acoustic properties. There has also been much interest in using them to develop novel bioplastic materials.

Despite this versatility, there is room for improvement in the hemp fibre industry. “The biggest bottleneck is the extraction of the fibres” says Luisa. “This is done in a process called retting, which separates the high-quality ‘bast’ fibres from the rest of the layers in the stem, such as the cortex and epidermis”. Bast fibres support the conductive cells of the phloem and are made of overlapping cellulose fibres held together with pectin gum. In traditional ‘field retting’, the cut hemp stalks are left in the field where they are colonised by fungi and yeast that produce polysaccharide-degrading enzymes. More modern techniques rely on chemical or mechanical methods. However, different hemp cultivars show immense variation in the ease at which the bast fibres detach. Luisa’s recent work has focused on identifying genetic markers for this, besides other fibre traits such as fineness.

“At the conference, I will be presenting the genetic diversity explicit in hemp for fibre quality and how this is correlated with other agronomic and morphological traits” Luisa says. This should provide a valuable tool for the hemp fibre industry, as it will help breeders to understand how selecting for certain traits may affect other aspects of the fibres. To maximise fibre production, for instance, hemp plants are typically bred to be tall and relatively unbranched, and to have a low seed output. But this could affect other aspects of fibre quality if they show close genetic linkage. “We found that to a large extent fibre quality is affected by flowering time” says Luisa. “Certain varieties flower very early, so they stop growing and produce a higher proportion of secondary fibres, which are of a lower quality”.

Currently, Luisa is working to fine-tune the genetic markers, so that the candidate genes responsible for the traits can be identified. But for future work, she has her eye on an even bigger challenge: using genetics for sex determination. “Hemp plants are naturally dioecious but the male and female plants have very different morphologies, which makes it difficult to get a homogenous crop” Luisa explains. “Although some monoecious varieties have been developed that combine the male and female flowers, often male plants appear again in the progeny. Currently, we do not understand how this happens”. Luisa’s group are hoping to use molecular techniques to uncover this process.

“I hope at the conference we can synergise the research from the hemp industry with the medicinal side of cannabis, so that we can learn from each other about the different aspects of this fascinating crop” she concludes.

Whether you are now considering cannabis for your next research model, or simply curious to learn more about this fascinating plant, why not join us in Prague to delve further into the secrets of sativa?