Uncovering the mechanisms of phosphorus recycling and lipid class remodeling under P limitation by integration of transcriptomics and lipidomics data of Nannochloropsis oceanica.

In the present study the effect of phosphorus (P) limitation was investigated in the microalgae Nannochloropsis oceanica. Algal cultures were analysed for transcriptomic and lipidomic changes during P-limited growth, with sample time points: 24h, 48h, 53h, 58h and 70h. The transcriptome data identified unique P acquiring genes and the results were also supported by corresponding changes in the lipidome dataset, which showed a lipid class shift from phospholipids to phosphorus-free lipids under P-limitation. During the exponential growth phase a gradual 6-fold reduction of the cellular P level induced RNA transcripts encoding proteins acquiring P from inorganic and organic sources. For this process 6 novel purple acid phosphatases and 5 unique Pi transporters were identified. Additionally, increased gene expression of 2 triose-phosphate transporters (TPTs) indicated the importance of the carbon-to-phosphorus balance for the plastidial anabolic processes. Phospholipid degradation was shown to be an early response to P limitation, by overexpression of sets of 2 novel glycerophosphoryl diester phosphodiesterases (GDPDs), and 3 patatin-like phospholipases of type A. To compensate for phospholipid degradation, the transcriptome and lipidome level showed increased synthesis of sulfoquinovosyl diacylglycerol (SQDG), and diacylglyceroltrimethylhomoserine (DGTS). Phosphorus limitation triggered the lipid class shift in both the exponential growth phase and the triacylglycerol (TAG) accumulation period to release P and increase the organic C:P ratio. Axenic pre-cultures of Nannochloropsis oceanica CCMP1779 were grown in f/2 medium without silica in three 2 L Nalgene optically clear flasks (Thermo Fisher Nalgene®) under constant light (350 µmoL photons m-2 s-1), at 23°C. The Nalgene optically clear flasks were aerated with sterile-filtered air. The pre-cultures were grown to the exponential growth phase (OD750nm= 0.40, ~8*106 cells / mL), mixed, harvested, and washed with sterile seawater by centrifugation (4,700xg, 20 min). The experimental cultures were inoculated to an OD750nm of 0.06 (~2.5*106 cells / mL) in either f/2 medium without silica (36.38 μM PO43-, +P cells) or f/2 medium without silica with a reduced phosphate concentration (2.79 μM PO43-, -P cells). Three biological replicates of the algal cultures were established for each of the mentioned growth conditions. Samples of phosphate depleted cells were harvested at 24h, 48h, 54h, 58h and 72h, while samples from phosphate replete cultures were harvested after 24h.