phosphate and oxaloacetate (OAA). Thus, it is intimately involved in C4-dicarboxylic acid metabolism in these organisms. It also plays an anaplerotic role in most non-photosynthetic organs of plants, C3 leaves and microalgae (including diatoms), providing OAA and/or malate to replenish citric acid cycle intermediates consumed in other primary metabolic pathways, notably ammonia assimilation . PEPC is considered to be a key enzyme for carbon fixation in diatoms [18,24]. In fact, the moderate up-regulation of the two PEPCs here was basically in accord with the other reports that P. tricornutum PEPC transcripts showed no significant increase under low CO2 conditions [7,25]. Although the carbon fixation pathway in P. tricornutum has not been fully elucidated, the deduced pathway in some diatoms [18,25-27] and increases in transcript levels of both PEPCs observed here suggest that it can take up inorganic carbon and possesses a CO2 concentration mechanism (CCM) (Additional file 4: Figure S2).
Additional file 4: Figure S2. Expression alteration in some metabolic pathways. A) nitrogen fixation pathway; B) carbon fixation pathway; C) TCA cycle. Genes were mapped to KEGG pathways through annotation records in KEGG as well as homology map. Numbers in the boxes represent the EC number of the gene. Red indicates increase in expression level and blue indicates decrease. The intensity of the color bar is linearly correlated to the expression level change (log2 fold).
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In addition, transcript levels of a predicted malic enzyme (NADP+-dependent ME; EC220.127.116.11, Phatrdraft_51970), a component of the putative carbon fixation pathway, with a predicted chloroplast localization (chlo: 8.0, cyto: 6.0, by WoLF PSORT), dramatically increased, over 7-fold (Additional file 4: Figure S2; Additional file 3: Table S1d). ME catalyzes a rate-limiting step for fatty acid biosynthesis; the irreversible decarboxylation of malate to pyruvate with the formation of NADPH from NADP+, hence providing essential 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