Cassius Stevani has foraged for glow-in-the-dark fungi in remote rain forests for 17 years. Looking down at these bioluminescent fungi in a dark forest can often feel like gazing out over the lights of a big city from an airplane window. It’s a strange and magical experience that has captured people’s imaginations for literally thousands of years, dating back to Aristotle’s description of foxfire.

In this tradition of scientific curiosity, Stevani and his colleagues have spent years investigating the chemical building blocks of these strange and magical mushrooms. Scientists often suspected that the 80 known species of bioluminescent fungi shared similar chemical properties with bioluminescent insects, bacteria, and some underwater animals, and now they have a much better idea of what’s really going on inside the cells of these glowing fungi. In addition to gaining a greater understanding of how the natural world functions, with this knowledge, scientists could use the chemicals in bioluminescent mushrooms to develop new imaging technologies for biology and chemistry research.

In a study published Wednesday in the journal Science Advances, Stevani and his colleagues describe the chemical mechanism that makes bioluminescent mushrooms glow. The researchers involved in this study already know the chemicals that seem to be involved in this process: luciferin and its associated enzyme, luciferase. But with this latest study, they show how the mushrooms use tiny amounts of these chemicals to achieve stunning results. Simply put, bioluminescent fungi recycle their active chemicals.

bioluminescent mushroom
Glow, baby, glow! ('Neonothopanus gardneri' glowing in the dark)

“Our results indicate there’s no need for the fungus to produce and store larger amounts of either hispidin (luciferin precursor) or luciferin, as luciferin is recycled over and over,” Stevani tells Inverse. To figure this out, the researchers examined the bioluminescent process of cultures grown from Neonothopanus gardneri (native to Brazil) and Neonothopanus nambi (native to Vietnam) in a liquid chromatograph.

In the mushrooms, luciferase catalyzes a reaction between atmospheric oxygen and luciferin. This produces oxyluciferin, a highly excited state of luciferin. In order to return to its ground state, the oxyluciferin releases the oxygen. This energy release is what produces the glowing effect we observe. When the luciferin returns to its ground state, it isn’t lost or destroyed, so the process can continue as long as there is oxygen present.

luciferin pathway
This shows how luciferase catalyzes luciferin to react with oxygen. This produces excited oxyluciferin, which produces the mushroom's characteristic glow when the chemical returns to its ground state. 

While the hypothesis for how bioluminescent mushrooms produce their light was proposed in the 1960s, research into this area took off in 2009, when Stevani’s lab established the role of enzymes in the process. In 2012, he and his colleagues showed that all fungi demonstrate the same mechanism of illumination. Then in 2015, Ilia Yampolsky, a fellow scientist who focuses on bioluminescent fungi, and his colleagues discovered fungal luciferin.

four colored mushrooms
Scientists can alter luciferase to make mushrooms glow different colors than its true green.

This new study builds on a body of knowledge dating back decades, but a long-overdue collaboration between Stevani’s and Yampolsky’s labs has accelerated our understanding of this highly specific research area.

“Personally, this work was very important to me as I began to study this system in 2000,” Stevani says. “I wandered in the Brazilian Rain Forest in the night to get bioluminescent fungi to initiate cultures. We have been working on this system for more than one decade. Hence, I’m very proud of this work. I wish I could have started the collaboration with Yampolsky earlier. We work very well and efficiently together.”

Stevani says this research could help scientists perform imaging research by using luciferase as a reporter gene, a gene that attaches to an area of a genetic sequence that researchers are interested in. He also notes that scientists can produce synthetic luciferins with interesting properties.

“Synthetic luciferins can be synthesized, adjusting color and the intensity of emission,” he says. This could help scientists use fungal luciferins to create a range of color emissions for imaging cells and genes. Stevani and his colleagues hope to explore this further in the near future, and their newfound collaboration with the Yampolsky lab should yield results quickly.

Abstract

Bioluminescent fungi are spread throughout the globe, but details on their mechanism of light emission are still scarce. Usually, the process involves three key components: an oxidizable luciferin substrate, a luciferase enzyme, and a light emitter, typically oxidized luciferin, and called oxyluciferin. We report the structure of fungal oxyluciferin, investigate the mechanism of fungal bioluminescence, and describe the use of simple synthetic a-pyrones as luciferins to produce multicolor enzymatic chemiluminescence. A high-energy endoperoxide is proposed as an intermediate of the oxida- tion of the native luciferin to the oxyluciferin, which is a pyruvic acid adduct of caffeic acid. Luciferase promiscuity allows the use of simple a-pyrones as chemiluminescent substrates.

Photos via Cassius V. Stevani/IQ-USP, Brazi, Kaskova et al., Sci. Adv. 2017;3:e1602847  , Cassius V. Stevani/IQ-USP, Brazil