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

## TL;DR

This study explores how the microalga Nannochloropsis oceanica adapts to nitrogen stress by regulating protein phosphorylation, revealing two distinct phases in its response and the role of the TOR signaling pathway.

## Contribution

The study provides a comprehensive phosphoproteomic analysis of Nannochloropsis oceanica under nitrogen stress, revealing new insights into the regulatory mechanisms of lipid accumulation.

## Key findings

- Two distinct regulatory phases were identified in response to nitrogen stress: nitrogen conservation followed by lipid accumulation.
- Phosphorylation events in the TOR signaling pathway suggest it acts as a nitrogen sensor in Nannochloropsis oceanica.
- Integration of phosphoproteomic data with transcriptomic and metabolomic datasets reveals a systems-level view of nitrogen stress adaptation.

## Abstract

Microalgae called Nannochloropsis oceanica turn sunlight and carbon dioxide into valuable oil, which has huge potential for bio-energy application, but only when exposed to nitrogen-limiting conditions. How they sense the nitrogen shortage and trigger oil accumulation remains unclear. For this, the cellular protein network was examined with a focus on protein phosphorylation, since it is well known that decoration of certain sites in proteins with phosphate affects their individual function and potentially the whole network. In total, 1371 phosphorylation sites on more than 800 proteins were identified. Two clear waves appeared in the network: first the cells saved nitrogen, then they boosted the oil-producing machinery. Moreover, phosphorylation of the Target of Rapamycin (TOR) signaling pathway, which is a master growth control pathway, was regulated, suggesting that this pathway was the nitrogen sensor. These results support future design of strains to become better oil producers to supply more and cheaper renewable fuel towards a greener economy.

Microalgae of the genus Nannochloropsis 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 Nannochloropsis oceanica 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 N. oceanica 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.