5 and C22:6 were 43% and 40% lower, respectively. The changes in fatty acid composition were largely due to dramatic increases in palmitic and palmitoleic acids, with accompanying losses of eicosapentaenoic acid, which is generally one of the most abundant fatty acids in P. tricornutum.
Observation of oil bodies by confocal microscopy
The size and number of oil bodies in sampled cells were observed every day during the culture cycle using a laser scanning confocal microscope. Substantially more oil bodies were observed in the N-deprived cells than in controls, but their sizes were similar (Figure 3), with diameters ranging from 0.2 to 2.0 μm. Thus, the total volume of oil bodies was clearly higher in the N-deprived cultures than in the controls, in accordance with the neutral lipid increase, as shown in Figure 2.
Figure 3. Representative confocal microscope images of the diatoms, showing oil bodies. Cells sampled during a 7-day culture cycle were stained with Nile Red and photographed under the confocal microscope daily. –N, cells subjected to the N deprivation treatment five days after subculture; Control, cells grown in f/2-Si medium and switched to fresh medium five days after subculture. Bar = 2 μm.
High-throughput analysis of differential gene expression
To characterize the effects of N deprivation further, we examined differences in gene expression before and after N-deprivation, using RNA-Seq. The complete dataset is listed in Additional file 1, and also available at http://guanlab.ccmb.med.umich.edu/data/Yang_N_deprivation/ webcite, but briefly 1213 genes were found to be up-regulated, with an FDR cutoff of 0.1%, and 4527 genes were down-regulated, indicating that the deprivation induced wide re-programming of regulation. Distribution of the number of reads across different mRNA sequence size indicates a significant correlation (Additional file 2: Figure S1). To validate the findings we applied quantitative RT-PCR (qPCR) to a randomly selected set of genes, including β-actin as a housekeeping marker, to test 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).
Format: XLSX Size: 1.5MB Download file
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.
Format: TIFF Size: 451KB Download file
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 indic