Using Firefly Genes to Understand the Biology of Cannabis

Light it up: Using Firefly Genes to Understand the Biology of Cannabis

Yi Ma with cannabis plants in the CAHNR Greenhouse. Credit: Jason Sheldon/UConn Photo

Cannabis, a plant that is gaining more attention for its extensive medicinal properties, contains dozens of compounds known as cannabinoids.

One of the best-known cannabinoids is cannabidiolic acid (CBD), which is used to treat pain, inflammation, nausea and more.

Cannabinoids are produced by trichomes, small pointed projections on the surface of cannabis flowers. Aside from this fact, scientists know very little about how cannabinoid biosynthesis is controlled.

Yi Ma, research assistant professor, and Gerry Berkowitz, professor in the College of Agriculture, Health and Natural Resources, explored the underlying molecular mechanisms behind trichrome development and cannabinoid synthesis.

Berkowitz and Ma, along with former graduate students Samuel Haiden and Peter Apicella, discovered transcription factors responsible for trichome initiation and cannabinoid biosynthesis. Transcription factors are molecules that determine whether a piece of an organism’s DNA will be transcribed into RNA and thus expressed.

In this case, the transcription factors cause epidermal cells on the flowers to turn into trichomes. The team’s discovery was recently published as an editorial in Plants† Related trichome research has also been published in: Plant immediately† Because of the gene’s potential economic impact, UConn has filed a provisional patent application on the technology.

Building on their results, the researchers will continue to investigate how these transcription factors play a role in trichome development during flower maturation.

Berkowitz and Ma will clone the promoters (the part of the DNA to which transcription factors bind) of interest. They will then place the promoters in the cells of a model plant, along with a copy of the gene that makes fireflies light up, known as firefly luciferase; the luciferase is fused to the cannabis promoter, so when the promoter is activated by a signal, the luciferase reporter will generate light. “It’s a useful way to evaluate signals that orchestrate cannabinoid synthesis and trichome development,” Berkowitz says.

The researchers will load the cloned promoters and luciferase into a plasmid. Plasmids are circular DNA molecules that can replicate independently of the chromosomes. This allows the scientists to express the genes of interest even though they are not part of the plant’s genomic DNA. They will deliver these plasmids into the plant leaves or protoplasts, plant cells without a cell wall.

When the promoter controlling luciferase expression comes into contact with the transcription factors responsible for trichome development (or triggered by other signals such as plant hormones), the luciferase “reporter” will produce light. Ma and Berkowitz will use an instrument called a luminometer, which measures how much light is coming out of the sample. This will tell the researchers whether the promoter regions they’re looking at are controlled by: transcription factors responsible for increasing trichome development or modulating genes encoding biosynthetic cannabinoid enzymes. They can also learn whether the promoters respond to hormonal signals.

In previous work that underpinned the rationale for this experimental approach, Ma and Berkowitz along with graduate student Peter Apicella found that the enzyme that makes THC in cannabis trichomes may not be the critical limiting step that regulates THC production, but rather the generation of the precursor for THC (and CBD) production and the transporter-facilitated commute from the precursor to the extracellular sphere may be important determinants in developing high THC or CBD cannabis strains.

Most cannabis farmers grow hemp, a variety of cannabis with naturally lower THC levels than marijuana. Currently, most high-CBD hemp strains also contain unacceptably high levels of THC. This is probably because the hemp plants are still making the enzyme that produces THC. If the plant contains more than 0.3% THC, it is considered federally illegal and must be destroyed in many cases. A better understanding of how the plant produces THC means scientists can selectively turn off the enzyme that synthesizes THC using genome editing techniques such as CRISPR. This would produce plants with lower or no THC content.

“We envision that the fundamental knowledge gained can be translated into new genetic tools and strategies to improve the cannabinoid profile, help hemp farmers with the common problem of THC overproduction, and benefit human health‘, say the researchers.

On the other hand, this knowledge could lead to the production of cannabis plants that produce more of a desired product cannabinoidmaking it more valuable and profitable.

The frostier the flower, the more potent the cannabis

More information:
Samuel R. Haiden et al, Overexpression of CsMIXTA, a transcription factor of Cannabis sativa, increases glandular trichome density in tobacco leaves, Plants (2022). DOI: 10.3390/plants11111519

Peter V. Apicella et al, Delineation of genetic regulation of cannabinoid biosynthesis during female flower development in Cannabis sativa, Plant immediately (2022). DOI: 10.1002/pld3.412

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