l reducing power for fatty acid biosynthesis via fatty acid synthase. Another previously reported putative decarboxylating enzyme of the malic enzyme family encoded by Phatrdraft_56501 , was predicted to be mainly chloroplast location (chlo: 11.0, mito: 2.0, by WoLF PSORT). Its presence is also consistent with reports of a single-cell C4 pathway in marine diatoms, including P. tricornutum, involving OAA entering the chloroplast . Wynn et al. have investigated the role of ME in filamentous fungi  and found that its overexpression increases NADPH production, thereby providing reducing power and cofactors for reactions catalyzed by enzymes involved in TAG synthesis such as ACCase, fatty acid synthase (FAS), ultimately leading to increases in TAG accumulation. Similarly, overexpression of two ME genes from Mucor circinelloides and Mortierella alpine in Mucor circinelloides reportedly increased ME activity 2- to 3-fold, and increased lipid contents from 12% to 30% of total biomass . Therefore, the dramatic increase in ME transcription we observed under N deprivation could contribute substantially to the accumulation of neutral lipids in P. tricornutum. In contrast, levels of pyruvate-phosphate dikinase (EC18.104.22.168, Phatrdraft_21988) transcripts decreased 7.5-fold. Pyruvate-phosphate dikinase is a key enzyme in the C4 photosynthetic pathway , thus reduction in its expression could reduce photosynthesis rates. Under such circumstances, increases in PEPC expression or activities in P. tricornutum could optimize use of the available phosphoenolpyruvate for carbon fixation, while saving most pyruvate for acetyl-CoA formation. Further, decreases in pyruvate-phosphate dikinase activity may prevent possible activation of alternative gluconeogenic pathways, such as that reportedly observed in Rhizobium (Sinorhizobium) meliloti. Taken together, the transcriptional changes in carbon assimilation under N deprivation may substantially increase carbon influxes, providing a rich source of substrate for fatty acid production. In 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