Diatoms are ubiquitous marine photosynthetic eukaryotes responsible for approximately 20% of global photosynthesis. Little is known about the redox-based mechanisms that mediate diatom sensing and acclimation to environmental stress. Here we used a quantitative mass spectrometry-based approach to elucidate the redox-sensitive signaling network (redoxome) mediating the response of diatoms to oxidative stress. We quantified the degree of oxidation of 3,845 cysteines in the Phaeodactylum tricornutum proteome and identified approximately 300 redox-sensitive proteins. Intriguingly, we found redox-sensitive thiols in numerous enzymes composing the nitrogen assimilation pathway and the recently discovered diatom urea cycle. In agreement with this finding, the flux from nitrate into glutamine and glutamate, measured by the incorporation of 15 N, was strongly inhibited under oxidative stress conditions. Furthermore, by targeting the redox-sensitive GFP sensor to various subcellular localizations, we mapped organellespecific oxidation patterns in response to variations in nitrogen quota and quality. We propose that redox regulation of nitrogen metabolism allows rapid metabolic plasticity to ensure cellular homeostasis, and thus is essential for the ecological success of diatoms in the marine ecosystem.phytoplankton | redox proteomics | roGFP | marine diatoms A erobic organisms produce reactive oxygen species (ROS) as a byproduct of oxygen-based metabolic pathways, such as photosynthesis, photorespiration, and oxidative phosphorylation (1). Perturbations in oxygenic metabolism under various stress conditions can induce oxidative stress from overproduction of ROS (2, 3). Because ROS are highly reactive forms of oxygenic metabolites, critical mechanisms for ROS detoxification have evolved consisting of ROS-scavenging enzymes and small molecules, including glutathione (GSH) (4). As the most abundant low molecular weight thiol antioxidant, GSH has critical roles in maintaining a proper cellular thiol-disulfide balance and in detoxifying H 2 O 2 via the ascorbate-GSH cycle (5).Although classically ROS were considered toxic metabolic byproducts that ultimately lead to cell death, it is now recognized that ROS act as central secondary messengers involved in compartmentalized signaling networks (1, 6-8). Modulation of various cell processes by ROS signaling is mediated largely by posttranslational thiol oxidation, whereby their physical structure and biochemical activity are modified upon oxidation (9). Thus, the redox states of these proteins possess crucial information needed for cell acclimation to stress conditions (10, 11). The emergence of advanced redox proteomic approaches, such as the OxICAT method (12), has created new opportunities to identify redox-sensitive proteins (e.g., redoxome) on the system level and to quantify their precise level of oxidation on exposure to environmental stress conditions. Marine photosynthetic microorganisms (phytoplankton) are the basis of marine food webs. Despite the fact that their biomas...

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Diatoms are ubiquitous marine photosynthetic eukaryotes that are responsible for about 20% of global photosynthesis. Nevertheless, little is known about the redox-based mechanisms that mediate diatom sensing and acclimation to environmental stress. Here we used a redox-sensitive green fluorescent protein sensor targeted to various subcellular organelles in the marine diatom Phaeodactylum tricornutum, to map the spatial and temporal oxidation patterns in response to environmental stresses. Specific organelle oxidation patterns were found in response to various stress conditions such as oxidative stress, nutrient limitation and exposure to diatom-derived infochemicals. We found a strong correlation between the mitochondrial glutathione (GSH) redox potential (EGSH) and subsequent induction of cell death in response to the diatom-derived unsaturated aldehyde 2E,4E/Z-decadienal (DD), and a volatile halocarbon (BrCN) that mediate trophic-level interactions in marine diatoms. Induction of cell death in response to DD was mediated by oxidation of mitochondrial EGSH and was reversible by application of GSH only within a narrow time frame. We found that cell fate can be accurately predicted by a distinct life-death threshold of mitochondrial EGSH (−335 mV). We propose that compartmentalized redox-based signaling can integrate the input of diverse environmental cues and will determine cell fate decisions as part of algal acclimation to stress conditions.

Diatoms are photosynthetic microorganisms of great ecological and biogeochemical importance, forming vast blooms in aquatic ecosystems. However, we are still lacking fundamental understanding of how individual cells sense and respond to diverse stress conditions, and what acclimation strategies are employed during bloom dynamics. We investigated cellular responses to environmental stress at the single-cell level using the redox sensor roGFP targeted to various organelles in the diatom Phaeodactylum tricornutum. We detected cell-to-cell variability using flow cytometry cell sorting and a microfluidics system for live imaging of oxidation dynamics. Chloroplast-targeted roGFP exhibited a light-dependent, bi-stable oxidation pattern in response to H2O2 and high light, revealing distinct subpopulations of sensitive oxidized cells and resilient reduced cells. Early oxidation in the chloroplast preceded commitment to cell death, and can be used for sensing stress cues and regulating cell fate. We propose that light-dependent metabolic heterogeneity regulates diatoms’ sensitivity to environmental stressors in the ocean.

Marine bacteria contribute substantially to nutrient cycling in the oceans and can engage in close interactions with microalgae. Many microalgae harbor characteristic satellite bacteria, many of which participate in N-acyl homoserine lactone (AHL) mediated quorum sensing. In the diffusion-controlled phycosphere, AHLs can reach high local concentrations, with some of them transforming into tetramic acids, compounds with a broad bioactivity. We tested a representative AHL, N-(3-oxododecanoyl) homoserine lactone, and its tetramic acid rearrangement product on the diatom Phaeodactylum tricornutum. While cell growth and photosynthetic efficiency of photosystem II were barely affected by the AHL, exposure to its tetramic acid rearrangement product had a negative effect on photosynthetic efficiency and led to growth inhibition and cell death in the long term, with a minimum inhibitory concentration between 20 and 50 μΜ. These results strengthen the view that AHLs may play an important role in shaping the outcome of microalgae–bacteria interactions.