s are protists that contain typical secondary plastids surrounded by four membranes. Thus, they differ strongly from those of the algae sensu stricto (in particular, green algae such as Chlamydomonas) and vascular plants such as Arabidopsis, which contain simple plastids bounded by two membranes). In the control cells, although the cultures were in stationary phase, the chloroplast and thylakoid membranes were still highly organized. In contrast, the four enclosing membranes and thylakoid membrane system tended to be dispersed and poorly organized under N deprivation.
Figure 5. Effects of N deprivation on P. tricornutum ultrastructure. Transmission electron micrographs showing subcellular structures of N-deprived (−N) and control P. tricornutum cells. OB, oil body; Chl, chloroplast; Pg, plastoglobule; boxed areas are magnified and showed to the right, demonstrating the dispersed thylakoid membranes under -N. Bar = 200 nm.
The main membrane lipids in plant chloroplasts are monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG). In Arabidopsis, these galactolipids are critical for the biogenesis of photosynthetic membranes, and provide sources of PUFA for the whole cell and phospholipid surrogates under phosphorus-limited conditions. Further, dramatic accumulations of DGDG accompanied by major reductions in membrane phospholipids have been observed in phosphorus-limited Arabidopsis, suggesting that the plant can substitute DGDG for phospholipids during phosphate starvation . These galactolipids are synthesized by MGD and DGD galactosyl-transferases/synthases, which also mediate membrane lipid remodeling . Three genes encoding putative monogalactosyldiacylglycerol synthases (EC 220.127.116.11) were detected in the P. tricornutum genome: Phatrdraft_9619, Phatrdraft_54168 and Phatrdraft_14125, which showed 6-, 6- and 9-fold decreases in transcript levels, respectively, under N deprivation. These results imply that membrane lipid remodeling, mediated by reductions in the expression of MGDG and/or DGDG 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.
Format: PPT Size: 181KB Download file
This file can be viewed with: Microsoft PowerPoint Viewer
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, enc