Knockout of an <i>SPX</i>-related gene for polyphosphate synthetase accelerates phosphate starvation responses in the oleaginous microalga <i>Nannochloropsis oceanica</i>

SPX domain-containing proteins are important for phosphate (Pi) signaling and homeostasis in various eukaryotes. Genomic and transcriptomic analyses of the oleaginous microalga Nannochloropsis oceanica identified four genes encoding SPX family members with distinct domain architectures: SPX (NoSPX1), SPX-VTC (NoSPX2), SPX-EXS (NoSPX3), and SPX-MFS (NoSPX4). These NoSPX genes responded differentially to Pi deprivation, with NoSPX1 and NoSPX2 expression being significantly up-regulated. NoSPX1 encodes an essential nuclear protein of unknown function and NoSPX2 encodes a vacuolar or acidocalcisomal protein homologous to the vacuolar transporter chaperone 4 that catalyzes polyphosphate production for vacuolar phosphorus storage. Disruption of NoSPX2 diminished polyphosphate levels and slowed the net uptake of external Pi, confirming its role in polyphosphate biosynthesis and Pi homeostasis. Furthermore, Nospx2 mutant cells accumulated significantly more triacylglycerol and greater biomass, and had enlarged lipid droplets under Pi deficiency compared with control cells. Together with these augmented Pi starvation responses, we observed a marked up-regulation of genes involved in autophagy and lipid transfer across organellar membranes and the down-regulation of photosynthesis-related genes, probably reallocating resources toward lipid biosynthesis. These results suggest that inadequate polyphosphate storage accelerates Pi starvation responses in microalgae, providing potential strategies for enhancing microalgal triacylglycerol accumulation and lipid productivity.