 under N deprivation. These findings suggest that TCA cycle genes were markedly up-regulated under N deprivation, thus increases in fluxes through the TCA cycle may compensate for the loss of assimilatory power for carbon fixation due to the down-regulation of photosynthesis.
The transcript abundance of isocitrate lyase (Phatrdraft_14401, EC22.214.171.124) and malate synthase (Phatrdraft_54478), two glyoxylate cycle enzymes identified in the P. tricornutum genome, decreased 5- and 2.5-fold, respectively. This implies that the metabolic flux through the glyoxylate cycle could be reduced under N deprivation, thereby further inhibiting overproduction of oxaloacetate and gluconeogenesis. Similarly, transcripts encoding phosphoenolpyruvate carboxykinase (Phatrdraft_55018), which catalyzes the committed step of gluconeogenesis, decreased by 20%.
Multiple sources for TAG accumulation and reductions in lipid catabolism
Transcript levels of genes associated with TAG biosynthesis were up-regulated by N-deprivation (Additional file 3: Table S1f). Notably, mRNA levels of Phatrdraft_9794, encoding diacylglycerol acyltransferase (DGAT, EC126.96.36.199), increased by 30%, according to both the RNA-Seq and qPCR analyses (Table 2). DGAT catalyzes the final committed step of TAG biosynthesis, thus the increase in its mRNA abundance under N deprivation may have increased TAG levels somewhat, but not as much as the observed increase in neutral lipid contents. Similarly, in C. reinhardtii there are two DGAT homologs, and transcripts encoding (DGTT2) are reportedly present at consistently low levels under all tested conditions, including N deprivation, while mRNA of the other (DGTT3) is present at low levels and only increases slightly under N deprivation . The ability of increases in DGAT expression to raise TAG levels has been demonstrated by heterologous expression of P. tricornutum DGAT (PtDGAT1, Phatrdraft_9794) in a Saccharomyces cerevisiae neutral lipid-deficient quadruple mutant strain, which restored TAG and lipid body formation, 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 (