Characterising NADPH oxidase in marine diatoms

Abstract

NADPH oxidase (NOX) is a widespread enzyme that catalyses the transmembrane reduction of oxygen, generating extracellular reactive oxygen species (ROS). NOX-derived production of ROS is best characterised as a defence mechanism, but a number of functions have been ascribed across eukaryotes, including intercellular signalling, facilitating cell-wall development and nutrient acquisition. Though extracellular ROS (eROS) production in unicellular algae is increasingly researched, characterisation of an enzyme source is relatively limited. This thesis addressed this by examining NOX distribution and function in marine diatoms, reporting several key findings. Firstly, a detailed screen of diatoms was carried out to identify NOX protein distribution. Compared to established NOX proteins in plants and animals, diatom NOX proteins are shown to be unusually diverse, with three structurally and phylogenetically distinct Classes of NOX protein and an atypical NOX-like Class. Secondly, the dynamics of eROS production and transmembrane electron transport were examined in three ecologically distinct marine diatoms. Baseline production rates differed significantly between species and changes to light intensity generated species-specific effects. NOX activity was inferred to be responsible for ROS production in two of the species tested. Finally, the function for NOX in the model diatom Phaeodactylum tricornutum was investigated. Following chemical inhibition of NOX, significant reductions to growth and photophysiology were observed, alongside an increase in cytosolic H2O2 (detected using a genetically encoded biosensor roGFP2-Orp1). This thesis proposes that NOX acts as a photoprotective mechanism by dissipating excess electrons and preventing over-reduction of chloroplast photosystems. Together, this study greatly improves understanding of NOX proteins in diatoms. By focusing on an understudied group, compared to animals or plants, knowledge of NOX distribution is expanded, with implications for NOX evolution. Furthermore, active eROS production can be beneficial to marine algae, and an electron dissipation function may explain the widespread use of eROS production by phytoplankton. While enzymatic sources of eROS are surprisingly diverse in diatoms, NOX likely represents the most common source. Thus, greater understanding of NOX function and distribution in diatoms may provide insights into understanding unique diatom photoprotection mechanisms, helping explain how diatoms can respond to changing environmental conditions.

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ORCID for Christopher Moore: orcid.org/0000-0002-9541-6046

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