Illuminating luciferin bioluminescence in dinoflagellates

Jan Janouskovec, University of Southampton; Matthew Terry, UoS; Kenneth Mertens, IFREMER; Glen Wheeler, MBA
Rationale: 

Noctiluca scintillans (the sea sparkle) and other dinoflagellates are responsible for most bioluminescence in European marine environments and familiar blue-glow blooms. The ecological role of dinoflagellate bioluminescence is currently not clear and its apparent importance in marine food webs [2,3] highlight the need to understand its environmental regulation and molecular underpinning. Fundamental gaps in knowledge include how bioluminescence varies within natural populations [3] and how dinoflagellates produce luciferin [1]. We hypothesize that (1) Bioluminescence intensity and gene expression vary among individual cells and are circadian-regulated; (2) Luciferin is generated by an oxygenase enzyme from a chlorophyll-related molecule. The investigation of these issues will help illuminate the environmental role of bioluminescence and accelerate the industrial use of dinoflagellate luciferin. Three innovative approaches will be used:

 

  • Studying non-photosynthetic Noctiluca which, unlike other bioluminescent dinoflagellates, lacks endogenous photosynthetic chlorophylls and has non-bioluminescent strains, both advantages when analyzing luciferin tetrapyrroles [1,2].

 

  • Single-cell sequencing to profile expression and circadian regulation of bioluminescence genes in natural populations on a cell-by-cell basis.

 

  • Identifying new bioluminescence-associated genes by their unique day/night expression profile [2], specifically oxygenases that could synthesize dinoflagellate luciferin in a single catalytic step, followed by their functional assessment after heterologous expression.

 

Methodology: 

The project offers an exciting mix of dry lab, wet lab, and computational approaches. Sampling under Nagoya Protocol declarations will occur from April to October in the English Channel and Bay of Biscay where Noctiluca is ubiquitous and its blooms are increasingly common and easily identifiable [3]. Cell isolation and culturing will follow published protocols [2].

 

Sequencing know-how, single-cell platforms (SmartSeq3, DropSeq) and funding are available in the supervisor’s laboratory. Cellular luminescence will be measured by fluorescent microscopy. Expression of genes known to participate in bioluminescence or tetrapyrrole biosynthesis and controls will be profiled by transcriptomics in cells and culture/bloom samples over the day/night cycle. A specific ALA synthase inhibitor will be used to test whether the luciferin is derived from tetrapyrrole biosynthesis. Findings will encompass luminescence variation among cells, diversity of luciferases and luciferin-binding proteins and candidate bioluminescence genes (identified by their circadian expression).

 

Oxygenases with expression patterns similar to luciferase will be cloned into vectors for heterologous expression in Synechocystis PCC6803, Phaeodactylum tricornutum and Escherichia coli. Transformants will be tested for luminescence (FluoStar OPTIMA plate reader), luciferin and modified tetrapyrroles (HPLC). Purified enzymes or cell extracts will be assayed in vitro with the luciferin precursor pyropheophorbide a.

 

Location: 
University of Southampton
Training: 

The INSPIRE DTP programme provides comprehensive personal and professional development training alongside extensive opportunities for students to expand their multi-disciplinary outlook through interactions with a wide network of academic, research and industrial/policy partners. The student will be registered at the University of Southampton and hosted by the School of Biological Sciences. Specific training will include:

  • In-person training to recognize plankton species (KM).
  • Organizing sample collection in and outside the UK (all collaborators & their networks).
  • Cell culturing and live cell imaging techniques (JG, JJ, GW).
  • Training in high-throughput sequencing of culture/bloom samples and individual cells (JJ).
  • Creating bioinformatic pipelines for transcriptomic and statistical analysis (JJ).
  • Experimental design for and training in gene cloning and transformation of model species (GW, JJ, JG).
  • Opportunities to network and proactively develop the project in new directions.
  • Opportunities to communicate findings in the form of presentations, research articles, and public outreach.

 

Eligibility & Funding Details: 

Please see https://inspire-dtp.ac.uk/how-apply for details.

 

Background Reading: 

[1] Janouškovec J, Gavelis GS, Burki F, Dinh D, Bachvaroff TR, Gornik SG, Bright KJ, Imanian B, Strom SL, Delwiche CF, Waller RF, Fensome RA, Leander BS, Rohwer FL, Saldarriaga JF. 2017. Major transitions in dinoflagellate evolution unveiled by phylotranscriptomics. Proceedings of the National Academy of Sciences 114:E171–E180. doi:10.1073/pnas.1614842114

[2] Valiadi M, de Rond T, Amorim A, Gittins JR, Gubili C, Moore BS, Iglesias‐Rodriguez MD, Latz MI. 2019. Molecular and biochemical basis for the loss of bioluminescence in the dinoflagellate Noctiluca scintillans along the west coast of the U.S.A. Limnol Oceanogr 64:2709–2724. doi:10.1002/lno.11309

[3] Ollevier A, Mortelmans J, Aubert A, Deneudt K, Vandegehuchte MB. 2021. Noctiluca scintillans: Dynamics, Size Measurements and Relationships With Small Soft-Bodied Plankton in the Belgian Part of the North Sea. Frontiers in Marine Science 8. doi:10.3389/fmars.2021.777999