G galactosyltransferases, occurred in the N-deprived cells.
To further assess the contribution of membrane remodeling to lipid accumulation, total lipid extracts were separated into various lipid classes (Additional file 5: Figure S3) and analyzed by ESI/MS. The results showed that there were nine main classes of lipids in the P. tricornutum cells, and peak area measurements indicated that the proportions of MGDG and DGDG in the cells markedly decreased 50% under N deprivation, while the proportion of TAG (the major component of neutral lipids, mixed with photosynthetic pigments) appeared doubled.
Additional file 5: Figure S3. Separation and identification of lipid classes. Chromatograms showing (A) Total lipids of control cells, (B) Total lipids of N-deprived cells. Peak identifications are described in the text.
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We also analyzed the acquired transcription dataset to identify down-regulated genes that were annotated to the chloroplast. Out of 50 genes annotated to thylakoids, 45 were down-regulated, and only five were up-regulated. Our results demonstrate that lipid remodeling provides potential pathways for converting membrane lipids to substrates for TAG accumulation in P. tricornutum’s responses to N deprivation.
Moreover, electron-dense osmiophilic globuli previously reported as plastoglobuli  were detected in the stroma of both N-deprived and control cells, but there were fewer in the former (Figure 5). The functions of plastoglobuli and triggers for their formation are not well understood. However, protein profiles of plastoglobuli indicate that metabolic enzymes accumulate in them and they have distinct lipoprotein structures . In addition, overexpressing the plastoglobule structural protein fibrillin in plants results in increased frequencies of plastoglobuli , suggesting that fibrillin is involved in their formation. Transcripts encoding fibrillins in P. tricornutum (Phatrdraft_48066 and Phatrdraft_55153, encoding PAP-fibrillin-1 and PAP-fibrillin-2) decreased 4- and 5-fold, respectively. This could at least partially explain the decrease of plastoglobuli in N-deprived cells.
The overall goal of this study was to use genomic information and various analytical techniques to obtain an overview of the molecular mechanisms responsible for neutral lipid accumulation in the model diatom P. tricornutum induced by N deprivation. N-deprived cultures of the diatom showed up to 2.4-fold increases in neutral lipid contents per cell, slightly higher cell density than controls and exhibited ultrastructural changes. In addition, the lipid composition of the N-deprived cells was more suitable for biodiesel production as they had higher proportions of saturated fatty acids.
Although the level of transcription is not directly proportional to the enzyme and the catalytic activity, recent study on another diatom T. pseudonana has shown a good correlation of proteomic and transcriptomic data . In combination with biochemical investigations, our results could provide a global view of biosynthetic metabolic fluxes and unraveled significantly altered key metabolic pathways in the diatom following N deprivation. As summarized in Figure 6, it induced genes involved in carbon fixation, the TCA cycle and glycerolipid metabolism, but inhibited genes involved in light harvesting and photosynthesis. Energy resources necessary for carbon fixation and energy demand associated with the neutral lipid synthesis could be replenished from other sources such as elevated TCA cycle. A possible catabolism of carbohydrate reserves including chrysolaminarin may largely explain the neo-synthesis of TAG, in accordance with the up-regulation of glycolysis, coupled with the suppression of critical enzymes, mostly involved in lipid catabolism. The gene expression analyses, in combination with the ultrastructural and biochemical investigations, indicate that additional fatty acids could be produced by membrane remodeling. Thus, the findings provide indications of se