dicating that the stress impaired PSII reaction centers, reduced the chlorophyll fluorescence yield, and decreased both the primary light energy conversion efficiency and potential photochemical activity of the PSII reaction centers . The inhibition of genes associated with photosynthesis by N deprivation observed in this study is consistent with reported responses of P. tricornutum and green alga Chlamydomonas[12,22], and can be considered a generic response to nutrient deficiencies. A practical implication of the findings for industrial biodiesel production is that light may not be required for neutral lipid accumulation under N deprivation. However, a previous report indicates that polysaccharides are produced at high rates during light periods and consumed in the dark, while the total lipid contribution to dry weight is positively associated with light intensity in N-deficient P. tricornutum. Thus, the effects of light on lipid accumulation in diatoms, and its interactive effects with other environmental factors, clearly require further characterization.
Table 3. Measurement of photosynthetic activity
Increased carbon assimilation and carbon fluxes towards TAG accumulation
Two genes encoding putative isoforms of phosphoenolpyruvate carboxylase (PEPC, EC184.108.40.206), Phatrdraft_27976 and Phatrdraft_51136, are present in the P. tricornutum draft genome and both were up-regulated moderately under N deprivation, 1.5-fold and 2-fold, respectively (Additional file 3: Table S1d). The two PEPCs were predicted to be localized in the chloroplast (Phatrdraft_27976; mito: 7.0, chlo: 7.0; Phatrdraft_51136, chlo: 9.0, cyto: 5.0, by WoLF PSORT) implicating the presence of C4 photosynthetic pathway. Two green microalgae, C. reinhardtii and Selenastrum minutum, also reportedly possess at least two distinct PEPC isoforms, which differ significantly from their plant and prokaryotic counterparts . PEPC catalyzes the irreversible β-carboxylation of phosphoenolpyruvate (PEP) in the presence of HCO3- and Me2+ to yield inorganic 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 essentia