 fact, Fourier transform infrared spectrometer (FTIR) assay conducted according to Jiang et al.  showed a slight increase of carbohydrate under N deprivation (data not shown), which is basically in line with the previous findings in P. tricornutum and T. pseudonana. Furthermore, the 2-fold increase of C:N ratio during N depletion according to Valenzuela et al.  together with the slight increase of carbohydrate implicate that the carbon is challenged into the production of storage lipid during -N.
P. tricornutum appears to possess at least five isoforms of fructose-1,6-bisphosphatase (FBP, EC22.214.171.124), a key regulatory enzyme of carbon metabolism (particularly the Calvin cycle and gluconeogenesis), which catalyzes the conversion of fructose-1,6-biphosphate to fructose-6-phosphate (Additional file 3: Table S1d). Transcript levels of genes encoding four of these isoforms, Phatrdraft_2793, Phatrdraft_9359, Phatrdraft_31994 and Phatrdraft_8744, declined under N deprivation, 45-, 17-, 3- and 3.5-fold, respectively, while those of the other (Phatrdraft_23247) slightly increased, by 50%. Therefore, gluconeogenesis was markedly inhibited and carbon flux was re-directed towards TAG accumulation under N deprivation. In contrast, two genes encoding phosphofructokinase (EC126.96.36.199), which catalyzes the committed step in the glycolytic pathway (conversion of fructose-6-phosphate to fructose-1,6-bisphosphate) were up-regulated: Phatrdraft_55126 and Phatrdraft_16844, by 36% and 50%, respectively. The up-regulation of glycolysis should theoretically direct carbon flux to the formation of pyruvate. Moreover, levels of transcripts encoding a precursor of the pyruvate dehydrogenase E1 component beta subunit (Phatrdraft_20183, EC188.8.131.52) and the alpha subunit (Phatrdraft_55035) increased 3-fold and slightly (by 15%), respectively. These findings propose that conversion of pyruvate to acetyl-CoA may be activated under N deprivation, thereby providing substrate for the citric acid (TCA) cycle following the conversion of 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