Integrative Multi-Omics Analyses Reveal the Global Regulation Network of the Microalga Nannochloropsis oceanica Under Nitrogen Stress Adaptation

Microalgae of the genus <i>Nannochloropsis</i> are known for their ability to accumulate large amounts of lipids, particularly triacylglycerides (TAGs), when exposed to nitrogen-limiting conditions. This trait makes them promising candidates for biofuel production. While previous studies have used transcriptomics and metabolomics to explore how these organisms respond to nutrient stress, the role of post-translational modifications-especially protein phosphorylation-remains poorly understood. To address this gap, we conducted a comprehensive analysis of protein phosphorylation events in <i>Nannochloropsis oceanica</i> under both nitrogen-replete and nitrogen-depleted conditions over a time-course experiment. Using mass spectrometry-based phosphoproteomics, we identified 1371 phosphorylation sites across 884 proteins. Temporal clustering of these phosphorylation events revealed two distinct regulatory phases: an early response aimed at conserving nitrogen resources, and a later phase that promotes lipid accumulation. Notably, we identified 11 phosphorylated proteins associated with the Target of Rapamycin (TOR) signaling pathway, suggesting that this conserved regulatory network plays a key role in coordinating the cellular response to nitrogen deficiency. By integrating our phosphoproteomic result with previously published transcriptomic and metabolomic datasets, we provide a more complete view of how <i>N. oceanica</i> adapts to nitrogen stress at the molecular level. This systems-level approach highlights the importance of protein phosphorylation in regulating metabolic shifts and offers new insights into engineering strategies for enhancing lipid production in microalgae.