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Diatoms are important components of marine ecosystems and contribute greatly to the world's primary production. Despite their important roles in ecosystems, the molecular basis of how diatoms cope with oxidative stress caused by nutrient fluctuations remains largely unknown. Here, an isobaric tags for relative and absolute quantitation (iTRAQ) proteomic method was coupled with a series of physiological and biochemical techniques to explore oxidative stress- and cell fate decision-related cellular and metabolic responses of the diatom Thalassiosira pseudonana to nitrate (N) and inorganic phosphate (P) stresses. A total of 1151 proteins were detected; 122 and 56 were significantly differentially expressed from control under N- and P-limited conditions, respectively. In N-limited cells, responsive proteins were related to reactive oxygen species (ROS) accumulation, oxidative stress responses and cell death, corresponding to a significant decrease in photosynthetic efficiency, marked intracellular ROS accumulation, and caspase-mediated programmed cell death activation. None of these responses were identified in P-limited cells; however, a significant up-regulation of alkaline phosphatase proteins was observed, which could be the major contributor for P-limited cells to cope with ambient P deficiency. These findings demonstrate that fundamentally different metabolic responses and cellular regulations are employed by the diatom in response to different nutrient stresses and to keep the cells viable.

Investigation of how diatoms cope with the rapid fluctuations in iron bioavailability in marine environments may facilitate a better understanding of the mechanisms underlying their ecological success, in particular their ability to proliferate rapidly during favorable conditions. In this study, using in vivo biochemical markers and whole-cell iTRAQ-based proteomics analysis, we explored the cellular responses associated with reactive oxygen species (ROS) production and cell fate decision during the early response to Fe limitation in the centric diatom Thalassiosira pseudonana. Fe limitation caused a significant decrease in Photosystem (PS) II photosynthetic efficiency, damage to the photosynthetic electron transport chain in PS I, and blockage of the respiratory chain in complexes III and IV, which could all result in excess ROS accumulation. The increase in ROS likely triggered programmed cell death (PCD) in some of the Fe-limited cells through synthesis of a series of proteins involved in the delicate balance between pro-survival and pro-PCD factors. The results provide molecular-level insights into the major strategies that may be employed by T. pseudonana in response to Fe-limitation: the reduction of cell population density through PCD to reduce competition for available Fe, the reallocation of intracellular nitrogen and Fe to ensure survival, and an increase in expression of antioxidant and anti-PCD proteins to cope with stress.

Silicon is a critical element for diatom growth; however our understanding of the molecular mechanisms involved in intracellular silicon responses are limited. In this study, an iTRAQ-LC-MS/MS quantitative proteomic approach was coupled with an established synchrony technique to reveal the global metabolic silicon-response in the model diatom Thalassiosira pseudonana subject to silicon starvation and readdition. Four samples, which corresponded to the time of silicon starvation, girdle band synthesis, valve formation, and right after daughter cell separation (0, 1, 5, 7 h), were collected for the proteomic analysis. The results indicated that a total of 1,831 proteins, representing 16% of the predicted proteins encoded by the T. pseudonana genome, could be identified. Of the identified proteins, 165 were defined as being differentially expressed proteins, and these proteins could be linked to multiple biochemical pathways. In particular, a number of proteins related to silicon transport, cell wall synthesis, and cell-cycle progress could be identified. In addition, other proteins that are potentially involved in amino acid synthesis, protein metabolism, and energy generation may have roles in the cellular response to silicon. Our findings provide a range of valuable information that will be of use for further studies of this important physiological response that is unique to diatoms.