ctively) in transcript abundance. These observations indicate that N deprivation induces homeostatic responses, including activation of the glutamine synthesis pathway and increases in the cells’ capacity to utilize trace amounts of nitrogen resources, and also the possible redistribution of intracellular nitrogen such as increased amino acid catabolism in accordance with the study in T. pseudonana, ultimately yielding acetyl-CoA or succinyl-CoA as the TCA cycle intermediates.
Transcripts of photosynthesis-related genes decreased
As shown in Additional file 3: Table S1c, levels of most transcripts encoding proteins associated with photosynthesis decreased under N deprivation, implicating that photosynthesis could be inhibited or at a standstill. Notably, only one encoding a light harvesting complex (LHC) component was up-regulated (Phatrdraft_16481, fucoxanthin chlorophyll a/c protein; up-regulated 10-fold), while most of the others were down-regulated more than 10-fold. As previously demonstrated in C. reinhardtii, reductions in the abundance of light-harvesting complexes may reduce photosynthetic rates , in accordance with theoretical expectations, unless reductions in transcript levels are balanced by changes at other levels. Transcript levels of ferredoxin-NADP+ reductase (Phatrdraft_43018), which catalyzes the last electron transfer, from photosystem I to NADPH during photosynthesis , also decreased (4.6-fold).
The reductions in mRNA levels of genes encoding photosynthetic proteins under N deprivation were consistent with expectations since various stresses can inhibit photosynthesis, and upregulated light harvesting can result in excess production of highly toxic reactive oxygen species (ROS) . To examine whether the reduced expression of photosynthetic genes was reflected in physiological changes, we also measured and calculated chlorophyll fluorescence parameters of the cultures, including Fo, Fm, Fv/Fm and Fv/Fo. As shown in Table 3, all of these parameters significantly decreased under N deprivation, indicating 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 