Sanchez, A. O.; Antolin, I.; Do Nascimento, M.; Sanchez Rizza, L.; Mechaly, A. S.; Lopez-Garcia, A.; Gonzalez-Bodi, S.; Huerta Cepas, J.; Flores, E.; Rubio, L. M.; Curatti, L.

Earths nitrogen cycle is central to sustaining ecosystem productivity and global biogeochemical balance. Although biological N2 fixation is well characterized in prokaryotes and plant symbioses, in other eukaryotic lineages it remains poorly understood. Diatoms of the family Rhopalodiacea harbor diazoplasts, endosymbiotic spheroid bodies specialized for N2 fixation. This makes these diatoms genuine N2 fixing eukaryotes that represent a unique model for organelle evolution, parallel but distinct from haptophyte nitroplasts. Here, we report the isolation and stable cultivation of an Epithemia adnata strain, the sequencing of its diazoplast genome and its proteomic profile when growing diazotrophically in the light or darkness, or upon exposure to ammonium. Our analyses reveal that ammonium induced broad down regulation of diazoplast proteins, particularly those linked to N2 fixation, ATP synthesis, and central carbon metabolism underscoring a general regulatory commitment toward diazotrophic metabolism tightly coupled to host carbon and nitrogen status. The pentose phosphate pathway and ferredoxin NADP+; oxidoreductase appear as likely source of reductant to nitrogenase. A striking enrichment of chaperones, peroxiredoxins, bacterioferritin-like proteins, and DpsA might stabilize nitrogenase and buffer against oxidative stress during light-driven diazotrophy. Importantly, we identified a plasmid-encoded GlpF as a putative glycerol transporter, pointing to glycerol-mediated host symbiont metabolic integration in the extant symbiosis and possibly a crucial innovation during the early evolutionary stages of its establishment. Thus, diazoplast activity is not autonomous but requires integration with host carbon and nitrogen status, establishing glycerol transport, reductant supply, stress mitigation, and nutrient-responsive regulation as pivotal mechanisms of nitrogenase activity and host integration. These findings have broad implications for biogeochemical cycling, organellogenesis, and synthetic biology strategies aimed at engineering N2 fixation in crop plants.