Julia M. Diaz, Sydney Plummer, Colleen M. Hansel, Peter F. Andeer, Mak A. Saito, and Matthew R. McIlvin

PNAS August 13, 2019 116 (33) 16448-16453

Environmental Sciences

Significance

Superoxide and other reactive oxygen species (ROS) are commonly regarded as harmful progenitors of biological stress and death, but this view has been changing. Indeed, many phytoplankton actively generate extracellular superoxide under ideal growth conditions for reasons that are mysterious. Results from this study suggest that extracellular superoxide production by the marine diatom Thalassiosira oceanica may promote photosynthetic health by modulating the oxidation state of the cellular NADP⁺/NADPH pool. The key enzyme implicated in this process is present in other representative marine phytoplankton and global ocean metagenomes. Overall, these findings transform the perceived role of superoxide in the health and functioning of phytoplankton and present implications for redox balance, biogeochemistry, and ecology in the future ocean.

Abstract

Reactive oxygen species (ROS) like superoxide drive rapid transformations of carbon and metals in aquatic systems and play dynamic roles in biological health, signaling, and defense across a diversity of cell types. In phytoplankton, however, the ecophysiological role(s) of extracellular superoxide production has remained elusive. Here, the mechanism and function of extracellular superoxide production by the marine diatom Thalassiosira oceanica are described. Extracellular superoxide production in T. oceanica exudates was coupled to the oxidation of NADPH. A putative NADPH-oxidizing flavoenzyme with predicted transmembrane domains and high sequence similarity to glutathione reductase (GR) was implicated in this process. GR was also linked to extracellular superoxide production by whole cells via quenching by the flavoenzyme inhibitor diphenylene iodonium (DPI) and oxidized glutathione, the preferred electron acceptor of GR. Extracellular superoxide production followed a typical photosynthesis-irradiance curve and increased by 30% above the saturation irradiance of photosynthesis, while DPI significantly impaired the efficiency of photosystem II under a wide range of light levels. Together, these results suggest that extracellular superoxide production is a byproduct of a transplasma membrane electron transport system that serves to balance the cellular redox state through the recycling of photosynthetic NADPH. This photoprotective function may be widespread, consistent with the presence of putative homologs to T. oceanica GR in other representative marine phytoplankton and ocean metagenomes. Given predicted climate-driven shifts in global surface ocean light regimes and phytoplankton community-level photoacclimation, these results provide implications for future ocean redox balance, ecological functioning, and coupled biogeochemical transformations of carbon and metals.

See https://www.pnas.org/content/116/33/16448

Figure 1: Superoxide production by T. oceanica concentrated extracellular proteins in the presence of inhibitors and stimulants. (A) NBT reduction to monoformazan (MF) is driven by superoxide production by bulk concentrated extracellular proteins (average ± SD of 3 biological replicates). SOD treatments show some superoxide-independent NBT reduction, which is eliminated by boiling or incubation with DPI. Superoxide production is therefore represented after accounting for SOD controls and applying the MF:superoxide reaction stoichiometry (1:2) (28). Treatments not connected by the same letter are significantly different (Tukey HSD, P < 0.01). (B–D) Activity gels reflecting superoxide production by concentrated extracellular proteins separated by native PAGE and incubated in 5 mM Tris (pH = 8) with (B) no amendment (C) NADPH, or (D) NADPH and SOD. Lane i is a prestained protein ladder, and each subsequent lane (ii–iv) represents a biological replicate.