ion, and promoted incorporation of saturated fatty acids into TAGs . Similarly, overexpression of Tropaeolum majus DGAT1 in Brassica napus and Arabidopsis thaliana resulted in 11–30% net increases in seed oil content .
Comparing with the much more increase of neutral lipid levels in P. tricornutum under N deprivation (2.4-fold), the slight increase of DGAT transcripts could be less effective. We examined further potential sources of substrates for TAG accumulation, and found that mRNA levels of another enzyme, responsible for the last step of TAG biosynthesis, phospholipid:diacylglycerol acyltransferase (PDAT, Phatrdraft_8860, EC188.8.131.52), increased 2-fold. Interestingly, in addition to the DGAT-catalyzed pathway, another acyl-CoA independent pathway catalyzed by PDAT for TAG synthesis has been discovered in Arabidopsis, in which acyl is directly transferred from phosphatidylcholine (PC) to DAG and thus TAG is synthesized without use of CoA as an intermediate . Phosphatidate phosphatase (PAP, EC184.108.40.206, Phatrdraft_40261) mRNA levels increased by 25%, which were responsible for the intermediate DAG for PDAT-catalyzed TAG accumulation. These findings suggest that the PDAT-mediated pathway or intracellular membrane remodeling may have contributed to the observed TAG accumulation.
The transcript abundance of genes involved in fatty acid elongation decreased under N deprivation. Delta 6 elongase (Phatrdraft_20508) and long chain acyl-CoA elongase (Phatrdraft_34485) levels decreased by 16% and 32%, respectively, which is consistent with observed reductions in C20:5 and C22:6 fatty acids, as shown in Table 1. Responses of genes encoding fatty acid desaturases varied. Transcript abundance of two desaturases catalyzing PUFA formation (delta 12 fatty acid desaturase, Phatrdraft_25769, and delta 9 fatty acid desaturase, Phatrdraft_28797) decreased, by 2.6- and 4-fold, respectively. In contrast, the expression of delta 5 fatty acid desaturase (Phatrdraft_46830), involved in MUFA synthesis, increased dramatically (32-fold). These results are in line with the higher MUFA and lower PUFA proportions of fatty acids under N deprivation (Table 1).
In accordance with the general increases in transcript levels of genes involved in TAG synthesis, levels of those involved in lipid catabolism generally decreased under N deprivation, those of acyl-CoA oxidase (Phatrdraft_19979) and 3-oxoacyl-CoA thiolase (ATO1) most dramatically; 11-fold and more than 3-fold, respectively.
Lipases are enzymes that de-esterify carboxyl esters, such as TAGs and phospholipids. More than 28 genes encoding putative lipases are present in the P. tricornutum genome (Additional file 3: Table S1g) and their responses to N deprivation varied markedly. Since TAG accumulated markedly under N deprivation, TAG lipases would be expected to be down-regulated. Among the putative lipase genes, eight (28%) showed increased transcript abundance but mainly only slightly, and transcript levels of most of the others decreased more than 2-fold. For example: Phatrdraft_44231, which encodes a putative triacylglycerol lipase, decreased 5-fold; Phatrdraft_50397 decreased 4-fold; while 43593 increased nearly 4-fold. These lipase genes may play important roles in membrane turnover, and their overall reduction in transcription may protect the TAG from degradation, thereby promoting remodeling and TAG accumulation under N deprivation.
Effects of N deprivation on the subcellular ultrastructure
Sections of cells were observed by transmission electron microscopy to assess the impact of N deprivation on their ultrastructure. Numerous oil bodies, of various sizes, were present in both the N-deprived and control cells. However, their total volumes were substantially higher in the former (Figure 5). The oil bodies in the N-deprived cells were globular, compact, and mostly 0.1 to 0.3 μm in diameter, but a few (2 to 5 per section) were much larger, up to 2.0 μm. In addition to causing significant changes in oil body contents, N deprivation strongly affected chloroplast ultrastructure. Diatom