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Lobosphaera incisa is a freshwater oleaginous microalga comprises high proportions of omega-6 LC-PUFA arachidonic acid (ARA) in membrane lipids and triacylglycerols (TAG). In this work, we set to provide novel insights into the role of ARA in L. incisa, using two novel mutants that were isolated during transformant screening and compared to WT and the better studied Δ5 desaturase mutant. The strain M2-35 with a downsized ARA content appeared to be most vulnerable to nitrogen starvation and high-light, showed impaired growth and compromised restoration of photosynthetic apparatus from N-deprivation. GLA cannot reconstitute the function of LC-PUFA in cellular lipids. The presence of LC-PUFA in chloroplast membrane lipids of L. incisa is important for photosynthetic performance and development of NPQ. Taken together, our findings indicate that LC-PUFA influence a variety of physiological responses in L. incisa and impact the efficiency of light utilization for growth and carbon allocation for storage products.