NADPH-dependent extracellular superoxide production is vital to photophysiology in the marine diatom Thalassiosira oceanica

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.

Keywords: biogeochemistry; oxidative stress; photosynthesis; reactive oxygen species.

Fig. 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.

Fig. 2.

Extracellular superoxide production by T. oceanica cells in the presence of inhibitors and stimulants. (A) Rates of net extracellular superoxide production, average ± SD of 4 (control, NADPH) or 3 biological replicates (all other treatments). Treatments that are significantly different from the “no addition” control are indicated by an asterisk (Dunnett test, P < 0.05). (B) Time course of extracellular superoxide concentrations reflecting the effect of GSSG in a typical FeLume run. Shaded regions correspond to (i) baseline signal of MCLA reagent + carrier solution with clean syringe filter inline, (ii) pump stopped while cells are loaded onto the filter, (iii) stabilization of cell-derived superoxide signal, (iv) superoxide levels with the addition of GSSG, and (v) SOD. Arrows indicate the point when inhibitors were added.

Fig. 3.

Light dependence of extracellular superoxide production by T. oceanica cells and impacts on photosynthetic health. (A) Net rates of extracellular superoxide production by cells as a function of irradiance in 3 biological replicates. Data were fit using a double exponential model modified from Platt et al. (38). Model results are presented in SI Appendix, Table S9. (B) Net rates of extracellular superoxide production in A were binned and averaged according to irradiance (I) levels above or below the minimum saturation (E_k) of photosynthesis, 400 μmol photons m⁻² s⁻¹ (39). Error bars represent the SE of 12 observations. Results were compared using a 2-sample t test. (C) The average efficiency of PSII in T. oceanica cells exposed to chemical inhibitors of extracellular superoxide production under a range of light levels (μmol photons m⁻² s⁻¹): dark (0), ambient light (∼10), and high light (2,250). The efficiency of PSII is a ratio that reflects the amount of light energy used in photosynthesis relative to the total amount of light absorbed. The negative value for the DPI treatment under high light reflects a lack of chlorophyll fluorescence due to damage of the photosynthetic apparatus (SI Appendix, Supplemental Methods). Error bars represent the SD of 3 biological replicates. Treatments not connected by the same letter are significantly different (Tukey HSD, P < 0.05).

Fig. 4.

Distribution of putative homologs of T. oceanica GR in representative phytoplankton genomes and the global ocean. (A) Genomes of the marine eukaryotic phytoplankton species Thalassiosira pseudonana, Phaeodactylum tricornutum, Fragilariopsis clyindrus, Ostreococcus tauri, Micromonas pusilla, Emiliania huxleyi, and Symbiodinium microadriaticum were searched for putative homologs to ToGR1, canonical NADPH oxidase from human (HsNox3), and respiratory burst oxidases from C. reinhardtii (CrRbo2) and Arabidopsis thaliana (AtRboB). The overall score for the top hit in each search is shown. Full results are presented in SI Appendix, Table S10. Relative abundance (normalized to total sequences) of putative ToGR1 homologs among eukaryotic phytoplankton sequences from (B) the surface ocean and (C) deep chlorophyll maximum. Stations are shown with an X. Size and color of circles indicate the relative abundance of phytoplankton hits with high sequence similarity to ToGR1.