Dynamic Regulation of Extracellular Superoxide Production by the Coccolithophore Emiliania huxleyi (CCMP 374)

Abstract

In marine waters, ubiquitous reactive oxygen species (ROS) drive biogeochemical cycling of metals and carbon. Marine phytoplankton produce the ROS superoxide (O 2 − ) extracellularly and can be a dominant source of O 2 − in natural aquatic systems. However, the cellular regulation, biological functioning, and broader ecological impacts of extracellular O 2 − production by marine phytoplankton remain mysterious. Here, we explored the regulation and potential roles of extracellular O 2 − production by a noncalcifying strain of the cosmopolitan coccolithophorid Emiliania huxleyi , a key species of marine phytoplankton that has not been examined for extracellular O 2 − production previously. Cell-normalized extracellular O 2 − production was the highest under presumably low-stress conditions during active proliferation and inversely related to cell density during exponential growth phase. Removal of extracellular O 2 − through addition of the O 2 − scavenger superoxide dismutase (SOD), however, increased growth rates, growth yields, cell biovolume, and photosynthetic efficiency ( F v /F m ) indicating an overall physiological improvement. Thus, the presence of extracellular O 2 − does not directly stimulate E. huxleyi proliferation, as previously suggested for other phytoplankton, bacteria, fungi, and protists. Extracellular O 2 − production decreased in the dark, suggesting a connection with photosynthetic processes. Taken together, the tight regulation of this stress independent production of extracellular O 2 − by E. huxleyi suggests that it could be involved in fundamental photophysiological processes.

Spatial Heterogeneity in Particle‐Associated, Light‐Independent Superoxide Production Within Productive Coastal Waters

“…These correlations implicate eukaryotic phytoplankton as important contributors to particle‐associated ROS. This is consistent with previous studies that have demonstrated that eukaryotic phytoplankton produce extracellular superoxide at the highest rates of any widespread marine microbe and are known to produce extracellular superoxide in the dark (Diaz et al, 2019; Plummer et al, 2019; Rose et al, 2008b; Sutherland et al, 2019). Interestingly, Synechococcus abundance across all stations does not correlate to superoxide production rate (Figure 5), suggesting that eukaryotic phototrophs play a much more dominant role in ROS production in this coastal setting.…”

“…Microbes produce extracellular superoxide via membrane bound and transmembrane oxidoreductase enzymes. This avenue of extracellular ROS production has been documented across the domains of Bacteria and Eukarya, including phototrophs and heterotrophs (Diaz et al, 2013Hansel et al, 2016;Plummer et al, 2019;Rose et al, 2008b;Sutherland et al, 2019). Within eukaryotic phytoplankton, the transmembrane NOX family NADPH-oxidases have been implicated in extracellular ROS production (Anderson et al, 2016;Kustka et al, 2005;Saragosti et al, 2010).…”

“…Phytoplankton and heterotrophs both contribute to marine ROS production (Diaz et al, 2013; Sutherland et al, 2019), with phytoplankton in the surface ocean accounting for a significant proportion of marine superoxide production (Diaz & Plummer, 2018; Godrant et al, 2009; Rose et al, 2008b; Sutherland et al, 2020). Although the production of light‐independent, extracellular superoxide is ubiquitous among photosynthetic microorganisms, extracellular superoxide production by these organisms is enhanced by photosynthetically active radiation (PAR, Diaz et al, 2019; Plummer et al, 2019; Schneider et al, 2016; Yuasa et al, 2020). Multiple studies suggest that light‐mediated extracellular superoxide production in phototrophs is directly related to the accumulation of NADPH, implicating extracellular superoxide in maintaining redox homeostasis during photosynthesis (Diaz et al, 2019; Yuasa et al, 2020).…”

Dark biological superoxide production as a significant flux and sink of marine dissolved oxygen

“…The dark superoxide production rates compiled in this study, with the exception of coccolithophores, were measured from cells grown to midexponential phase under ideal growth conditions using a flow-injection chemiluminescent approach (9). The study investigating the cell-specific superoxide production rate of coccolithophores measured their production rate throughout their growth curve, which we converted to a time-weighted average using trapezoidal integration of the cell superoxide production rate as a function of time (18). Cell-normalized superoxide production rates presented in the scientific literature are either presented as net or gross superoxide production rates, the latter requiring an exogenous spike of superoxide to determine the proportion of extracellular ROS that is enzymatically degraded by the organisms.…”

“…Yet, dark (lightindependent) particle-associated superoxide production accounts for a significant fraction of superoxide measured in natural waters within both the photic and aphotic zone (9,10). Dark, extracellular superoxide production is in fact prolific among marine heterotrophic bacteria, cyanobacteria, and eukaryotes (9,(11)(12)(13)(14)(15)(16)(17)(18). Production of extracellular superoxide proceeds via a one-electron transfer initiated by transmembrane, outer membrane-bound, or soluble extracellular enzymes thought to belong generally to NAD(P)H oxidoreductases (19), and more recently to heme peroxidases and glutathione reductases (9,20,21).…”