est the differential expression. The RNA-Seq measurements are highly consistent with the quantitative PCR results, confirming their robustness. For example, Phatrdraft_27877, encoding an ammonium transporter protein, was up-regulated 5.46 (log2)-fold and 116-fold under N-deprivation, according to the RNA-Seq and qPCR analyses, respectively (Table 2).
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Additional file 2: Figure S1. Distribution of the number of reads across different mRNA sequence size. Each box plot depicts the numbers of reads for each gene (log base 10) in an mRNA sequence size bin (0–5,000 bp, bin size of 500 bp). The line shows the range from minimum to maximum. The box and bar shows the quantiles. All mRNAs of 5,000 or more nucleotides are classified as 5,000 and above. A) nitrogen deprived condition. B) normal condition.
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Table 2. qPCR of differentially expressed genes following N deprivation
General transcriptional changes under N deprivation
To elucidate the molecular basis of the observed accumulation of fatty acids under N deprivation, genes encoding all known enzymes in the P. tricornutum genome were mapped in KEGG pathways, together with log2-fold differences in RNA expression between the control and N-deprived cultures. Global changes in major categories of genes involved in various pathways, reflecting general transcriptional responses to N deprivation, are depicted in Figure 4 and listed in Additional file 3: Table S1a. Genes encoding photosynthesis, gluconeogenesis, glyoxylate cycle, chrysolaminarin synthesis and sucrose metabolism enzymes were, on average, significantly down-regulated under N deprivation, while genes involved in nitrogen fixation, carbon fixation, glycolysis and the TCA cycle were generally up-regulated.
Figure 4. Proposed general transcriptional changes under N deprivation. Schematic diagram showing the putative localization of central metabolic pathways of P. tricornutum. Changes in the transcript abundance of associated genes are indicated with the following symbols representing fold changes: ++++ > 5; 5 > +++ > 3; 3 > ++ > 1; 1 > + > 0; 0 < − < −1; -1 < −−− < −3; -3 < −−− < −5; −−− < −5. The symbol denotes the overall changes of predicted isoenzymes. Some co-substrates or co-products of some reactions are omitted for clarity. The one “+” in the fatty acid synthesis in plastids denotes the overall transcriptional changes of the enzymes involved. Detailed fold changes are listed in Additional file 3 and Additional file 1. Enzymes were based on the genome annotation and prediction. Locus tags of some ambiguous isoenzymes are indicated. The localization of most of the enzymes are based on prediction and have not been determined experimentally.
The primary effect of N deprivation is the reduced availability of nitrogen, an essential nutrient for de novo synthesis of amino acids, nucleic acids and various other cellular constituents. Unsurprisingly, therefore, it strongly affected transcript levels of genes involved in nitrogen assimilation and metabolism (Additional file 3: Table S1b). Notably, marked increases were observed in transcripts encoding five of at least six ammonium transporters present in the diatom, which transport ammonium ions across the cell membrane and are known to be activated by N deprivation . The rise in the level of one of them was also confirmed in our qPCR test (Phatrdraft_27877, 45-fold increase). In addition, there was a 7-fold increase in transcripts of glutamine synthase (GS, Phatrdraft_22357, EC 22.214.171.124), a key enzyme in ammonia assimilation in both plants and Gram-negative microorganisms. GS plays an important role in the efficient use of nitrogen sources and nitrogen metabolism. Other critical nitrogen metabolism enzymes, such as nitrate reductase (Phatrdraft_54983, EC126.96.36.199), a molybdoenzyme that reduces nitrate to nitrite, and ferredoxin-nitrite reductase (Phatrdraft_12902, EC188.8.131.52), which catalyzes conversion of nitrite to ammonia, also showed significant increases (4-fold and 33-fold, respe