TAG is the major storage lipid in oleaginous microalgae and in this study nitrogen limitations induced a five-fold increase in its intracellular con­tent. Several genes involved in TAG biosynthesis displayed changes in their expression in response to nitrogen limitation. Biosynthesis of TAG in the chloroplast begins with two consecutive acyl transfers from acyl-CoA to positions 1 and 2 of glycerol-3-phosphate to form phosphatidic acid (PA), which is subsequently dephosphorylated to form 1,2-diacylglycerol (DAG) (Figure 5B). These reactions are catalyzed by enzymes glycer­ol-3-phosphate acyltransferase (GPAT), acyl-glycerol-3-phosphate acyl — transferase (AGPAT), and phosphatidate phosphatase (PP), respectively. The last step in the pathway, catalyzed by diacylglycerol acyltransferase (DGAT), involves the transfer of third acyl group to the DAG 3 position. This final reaction is the only dedicated step in TAG synthesis since the preceding intermediates (i. e. PA and DAG), are also substrates for the syn­thesis of membrane lipids. Our results indicated that the expression of genes encoding GPAT and AGPAT was up-regulated in response to nitro­gen starvation. However, the expression of gene encoding PP and DGAT remained relatively unchanged.

Though TAG biosynthesis in microalgae is believed to occur mainly through the glycerol pathway as described above, an alternative route known as the acyl CoA-independent mechanism has also been reported to take place in some plants and yeast [29]. In this mechanism, phospholipid is utilized as the acyl donor in the last step of TAG formation and the reac­tion is catalyzed by phospholipid:diacylglycerol acyltransferase (PDAT). We have recently found homologues of gene encoding for PDAT in the D. tertiolecta transcriptome, suggesting that the PDAT route could also play a role in microalgae TAG biosynthesis [14]. We did not however identify such homologues in the transcriptome of N. oleoabundans, making it un­clear if PDAT contributes to TAG biosynthesis in this organism.