Dinoflagellate Bioluminescence

Introduction

Dinoflagellates are a group of unicellular eukaryotes who mostly live in marine environments. They are typically classified as algae, but only half of the group is photosynthetic, with many being heterotrophic or mixotrophic. Two flagella extend from their membrane to provide locomotion via a tumbling motion through the water. Due to the relative difficulty to grow them in captivity, they are not very useful for biotechnological purposes, so have not been studied as much as some other groups; however, dinoflagellates can cause harm to human health via their synthesis of cyanotoxins. When their numbers increase in large, seasonal blooms, these toxins can have a major effect on the ecosystem and can be toxic to humans. With climate change affecting weather patterns, recent studies suggest that these blooms could become year round, and their toxic effects which are normally mitigated by periods without blooms, could become much more severe. Dinoflagellate blooms are frequently called the red tide, because their reddish color can tint the water red. Even more famous however, are the blue tides some species create at night through their bioluminescence.

Figure 1. An image of the blue wave created by dinoflagellate luminescence. Taken in 2023 off the Scripps pier at UC San Diego by Erik Jepsen. [2].

Introduction to Bioluminescence in Dinoflagellates

Dinoflagellate species, especially belonging to the Gonyaulacales, Noctilucales, and Peridiniales orders, exhibit bioluminescence when physically agitated by anything from predators to the surf, which can turn the ocean a glowing blue during large blooms. There are two main theories as to why bioluminescence in dinoflagellates evolved. The first is that the flashes of light act to confuse and startle predators. Studies have shown a correlation where an increase in the intensity of bioluminescence leads to a decrease in the total number of cells being consumed by a predator. The second main theory, also supported by experimental data, is that the flashes of light act as a metaphorical burglar alarm, where they attract the attention of a predator of the organism praying on the dinoflagellates. Both of these mechanism would have evolved via kin selection, where individual cells may sacrifice themselves by flashing, but are more likely to save nearby cells. This theory is especially appealing this case because dinoflagellates reproduce asexually, so neighboring cells are likely to be near clones. It is probably a combination of these two theories, and possibly other factors, that led to the evolution of bioluminescence in dinoflagellates. The mechanism of bioluminescence in this phylum is different from most other organisms, making it very interesting to study.

Genetics

The dinoflagellate genome is very unique among eukaryotes for several reasons. First, their genomes can be more than 80 times larger than the human genome, being mostly composed of repeated copies of many genes. The reason for this is unknown, but because of it genome sequencing is very difficult and not very useful simply because of the shear size of DNA sequences. A more useful method of study has been transcriptomic sequencing, where the sequences of RNA transcripts are analyzed instead of the original DNA, in order to parse out the repeated genes. Another unique facet of the dinoflagellate genome is their lack of true histone proteins. Instead, they wrap DNA around histone-like proteins and dinoflagellate viral nucleoproteins.

The evolution of dinoflagellates is not well understood, partially due to the complexity of their genomes. The story is much further complicated by the addition of several genes, including some histone-like proteins, via horizontal gene transfer from bacteria. Dinoflagellates also cannot be cleanly split between heterotrophs and autotrophs, because recent studies have found that photosynthesis has been lost at least 21 times in different species, so this cannot be used as a synapomorphy.

Section 1 Luciferin and Luciferase

Luciferase

The evolution of bioluminescence in dinoflagellates is largely due to the appearance of the dinoflagellate luciferase gene (lcf), which encodes the enzyme luciferase. Luciferase catalyzes the reaction that produces light. All bioluminescent dinoflagellates have the lcf gene, but there is a lot of variation between genera in the exact nucleotide sequence. At least in the species Gonyaulax polyedra, the gene is a sequence of tandem repeats that seem to have promoters in between.

Figure 2. One of the domains of L. polyedra luciferase. The alpha helices are shown in red and the beta barrel is shown in blue. (b) The four histidines which are thought to play a role in pH regulation. Created in 2020 at Uppsala University in Uppsala, Sweden by Natasha Kamerlin, Mickaël G. Delcey, and Roland Lindh [3].

The mechanism for transcription of the luciferase gene is unknown. Dinoflagellate chromosomes are unique because they do not have histones and are all always condensed, so the overall mechanism of transcription is likely different from that of other eukaryotes. Regulation of the gene coding for luciferase is particularly interesting because its expression is regulated by a circadian rhythm, with increased expression at night when bioluminescence can actually be seen. Despite this interest, the mechanism is still unknown, but some circadian regulation is thought to be done post-transcriptionally.

Structurally, dinoflagellate luciferase differs between species in some ways, but appears to function similarly amongst all species. There is variety in the number of active domains in luciferase, with some species having three while others only having one. In the species Lingulodinium Polyedra, the enzyme has three domains which are all homologous and independently enzymatically active. Each domain has two main components: the β-barrel and alpha-helix bundle. The beta barrel is the catalytic part of the enzyme, while the alpha helices play a role in pH regulation (Figure 1).

Luciferin

Luciferin is the generic name for the molecule that is oxidized by luciferase in order to produce bioluminescence. Less is known about dinoflagellate luciferin than luciferase, with the actual source of the compound still being unclear. Luciferin does have a very similar structure to chlorophyll, so one theory is that this is its source. Many dinoflagellates are autotrophic, so they could convert chlorophyll for photosynthesis and luciferin for bioluminescence back and forth. Another source of chlorophyll for non photosynthetic species could be photosynthetic prey they consume. This theory is not fully supported however, because one study has shown that heterotrophic dinoflagellates did not lose their bioluminescent ability when cultured on a medium of only rice flour. This result suggests they can synthesize their own luciferin, though the mechanism is unknown

Section 2 Mechanism of Bioluminescence

Bioluminescence in dinoflagellates is is a pH regulated process and occurs in specialized organelles unique to the taxa called scintillons. It is triggered by physical agitation, which causes an influx of protons into the scintillons. Scintillons will protrude into very acidic vacuoles, so when stress is applied a mechanotransduction pathway, where mechanical stimuli are converted to biochemical signals, is activated, causing an action potential along the vacuole and scintillon membranes. This action potential triggers the opening of voltage gated proton channels, allowing protons to flow from the acidic vacuole into the scintillons, lowering their pH dramatically.

Dinoflagellates only produce light when a luciferase enzyme catalyzes the oxidation of a luciferin. In high pH environments the alpha-helices of luciferase block access of the luciferin substrate to the catalytic beta-barrel where the oxidation occurs (Figure 2). However at a low pH, such as that caused by the influx of protons into the scintillons, the alpha-helices move out of the way, allowing the reaction to proceed. The exact mechanism of this is unproven, but the leading hypothesis suggests that it is due to the protonation of several histidines in the polypeptide, as well as a lysine residue. These protonations cause electrostatic repulsion between the histidine-cations, leading to the conformational change uncovering the active site in the beta-barrel. Once the active site is uncovered, luciferin is oxidized by luciferase. This reaction causes the release of photons with a wavelength of about 475 nm, which is perceived by humans as a flash of blue light. How the actual production of light works, and why light is released instead of other types of energy, is not well understood.

A second form of regulation, possibly relating to the circadian rhythm which prevents luminescence during the day but encourages it during the night, is the luciferin binding protein (LBP). In some species, this protein binds to luciferin and only unbinds at low pH, creating a similar regulatory effect as described above.

References

Edited by Dylan Ryznar, student of Joan Slonczewski for BIOL 116, 2024, Kenyon College.