More than 2000 years ago, Aristotle wondered about “a cold fire,” illuminating from the woods. He referred it as “glowing, rotting tree bark” although the light he saw actually came from fungi.
The glowing fungi are an enchanting and bizarre phenomenon, truly occurring in nature. They commonly reside in forests, scattered from the temperate regions to the tropical regions.
In many cultures around the world, the eerie glow from these fungi in the dark have caused fear—viewing these fungi as “ghost mushrooms”.
For Pokémon fans, these fungi remind us of Morelull, a mushroom-like Pokémon creature from the “Detective Pikachu” movie released in 2019. Similar to Morelull, the glowing fungi can make their mushroom cap glow.
So let’s uncover what's behind this strange phenomenon, shall we?
What are glowing fungi?
Glowing fungi are fungi emitting green light with maximum intensity in the range of 520-530 nm (Chew et al., 2015). All glowing fungi form mushrooms and produce white spores. The glowing parts of the fungi can come from the mycelia, the mushrooms, or both parts.
Bioluminescent Fungi. Fungi produce an eerie green light on the mushroom caps.
So far, scientists have discovered at least 81 out of 100,000 fungi worldwide with this remarkable ability. Therefore, only a small portion of known fungi produces light.
The glowing fungi use a lot of energy to produce constant light, but scientists are still unsure about the benefit of the light synthesis for the fungi.
Why do fungi glow?
Scientists have come up with two possible theories as to why fungi produce light. The first theory: the fungi use the light to lure insects. Fungi are unable to move, disperse the spores, and colonize new areas in the forest. Therefore, they must rely on the wind or animals, such as insects, to carry their spores elsewhere.
The second theory is the light could be an accidental by-product of metabolism. In this case, the light production has no benefits at all for the fungi.
Scientists investigated the first theory by using plastic mushrooms with green LED-emitting light, similar to the fungal bioluminescence in the Brazilian Coconut Forest (Oliviera et al., 2015). They observed the light by the plastic mushrooms attracted a variety of insects, which could potentially disseminate spores.
However, in another study, a group of researchers in Australia reported the light from the ghost fungus did not attract any potential spore dispersing insects (Weinstein et al., 2016).
Hence, the glow may attract insects for some fungi, but it may also be a useless by-product of metabolism for the others.
How do glowing fungi produce light?
The glowing fungi make light by using a chemical reaction, which involves luciferin, a luciferase enzyme, and molecular oxygen. This chemical reaction, called bioluminescence, is almost similar to how fireflies produce light. The difference is fungi use a unique metabolic pathway with some extra enzymes in addition to luciferase.
In a recent study, scientists identified the genes associated with the fungal bioluminescent pathway in the glowing fungus Neonothopanus nambi (Kotlobay et al. 2018).
They also identified three important steps in the fungal bioluminescence pathway:
- The synthesis of hispidin from the substrate, caffeic acid. This process uses the first enzyme, hispidin synthase or HispS.
- The synthesis of the fungal luciferin from hispidin by using the second enzyme, H3H.
- The catalysis of the luciferin by fungal luciferase or Luz to produce light.
In the study, the researchers engineered yeasts expressing those key enzymes in the fungal bioluminescent pathway and added an additional gene, npgA. The npgA gene is important for many biological processes.
Interestingly, when the researchers grew the yeasts in a medium containing caffeic acid, they observed green light glowing in the medium. Caffeic acid is a compound found naturally in many vascular plants, which produces lignin and other metabolites. To perform a similar type of assay, try GoldBio’s caffeic acid.
Discovering about the fungal bioluminescence pathway is exciting because it leads us to many promising applications by transferring this pathway to a new system and different species.
Using the fungi bioluminescent pathway to create glow-in-the-dark plants
Producing bioluminescence in plants was nearly impossible due to various hurdles: the difficult strategy to create uniform glow in plant tissues, the expensive cost to maintain the glow, the inconvenient method to produce continuous light, and the inefficient way to deliver the substrate (Reuter et al., 2020).
To overcome these hurdles, two groups of researchers successfully incorporated the fungal bioluminescence pathway to engineer glowing plants (Mitiouchkina et al., 2020; Khakhar et al., 2020). In both studies, the substrate, caffeic acid, came directly from the plants.
To transform Agrobacterium competent cells, those researchers used DNA cassettes with codon-optimized versions of fungal genes: nnluz (luciferase), nnhisps (hispidin synthase), and nnh3h (hispidin-3-hydroxylase).
In addition to the three genes encoding the key enzymes in the pathway, they also included another gene, nncph (caffeoyl pyruvate hydrolase), to recycle the final metabolite on the pathway back into caffeic acid. Therefore, it resulted in a long-term luminescence.
In the first study, researchers transformed and integrated Agrobacterium containing the fungal genes at a random site of tobacco plants (Mitiouchkina et al., 2020). They reported that the tobacco plants were able to express the pathway and glow.
Watch a time-lapse video of glowing tobacco plants below:
Another group of researchers used the same approach, improved the system, and extended the plant list (Khakhar et al., 2020).
To improve the uneven levels of caffeic acid between plant tissues, they included a T-DNA in the Agrobacterium cells to express some bacterial enzymes, including tyrosine ammonia lyase. These enzymes catalyze the synthesis of caffeic acid from tyrosine, which is widely available in the plant tissues. Upon the infiltration of this system into tobacco leaves, they detected a significant increase in bioluminescence on different sections of the same leaf.
They then tested the system in tomato and some flowering plants, such as roses and periwinkle. From the petals of roses and periwinkle, they detected luminescence signal, although it diminished within a day after detaching the flowers from the plants.
Glowing periwinkle. Periwinkle expressed enzymes from the fungal bioluminescent pathway and produced green light (Photo used with permission |image credit: James Chamness/University of Minnesota, Dept. of Genetics, Cell Biology & Development).
They also built a system by using the promoter of the ODORANT1 gene from petunia together with the fungal luciferase gene. This promoter drives the gene expression to be diurnal, peaking in the evening. As a result, they observed the luminescence increased at the transition between day and night.
Overall, uncovering the mystery of the glowing fungi turns out to bring an exciting innovation in plant research. This discovery also opens a new door to many potential applications. As an example, this system can become a powerful reporter technology to study gene expression on a whole plant. Otherwise, it can also be a valuable tool to study the signaling events of plant hormones.
Perhaps, we can enjoy glowing flowers at night in our gardens in the future, but for now, we can only dream about it.
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