 acetyl-CoA to citrate as well as building up precursors to ACCase for fatty acid biosynthesis. And we found that the predicted cytosolic isocitrate dehydrogenase (Phatrdraft_14762) increased 16-fold, which is involved in the conversion of citrate to α-ketoglutarate. During this process, NADP+ is reduced to NADPH which is a critical cofactor for many enzymatic reactions in lipid biosynthesis .
Diatoms do not possess starch-forming enzymes, but store fixed carbon as a complex, soluble carbohydrate called chrysolaminarin (β-1, 3-glucan) in vacuoles. Transcript levels of phosphoglucomutase (Phatrdraft_50445), which catalyzes the rate-limiting step of carbohydrate synthesis, decreased 3.7-fold under N deprivation. This is consistent with both the observations of neutral lipid accumulation and recent reports that N-deprived C. reinhardtii mutants with phosphoglucomutase deficiencies store neutral lipids in lipid bodies [33,34]. Furthermore, since chrysolaminarin and TAG syntheses share common 3-carbon photosynthate precursors, mutations or reductions in the expression of phosphoglucomutase could affect chrysolaminarin biosynthesis and redirect carbon fluxes to TAG accumulation. The decrease in carbohydrate contents under N deprivation is consistent with previous findings that high lipid contents in P. tricornutum are accompanied by low total carbohydrate contents .
Almost all of the genes involved in the TCA cycle—which converts 2-oxoglutarate, coenzyme A and NAD(+) to succinyl-CoA, NADH and carbon dioxide—were activated under N deprivation (Additional file 4: Figure S2; Additional file 3: Table S1e). Levels of transcripts encoding two potentially rate-limiting enzymes of the TCA cycle—citrate synthase (Phatrdraft_30145, EC184.108.40.206) and isocitrate dehydrogenase (EC220.127.116.11, Phatrdraft_30578) increased 3- and 22-fold, respectively. In addition, those of a third TCA cycle enzyme, oxoglutarate dehydrogenase (Phatrdraft_29016, EC18.104.22.168), which converts 2-oxoglutarate to succinyl-CoA , slightly increased 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 format