32-fold). These results are in line with the higher MUFA and lower PUFA proportions of fatty acids under N deprivation (Table 1).
In accordance with the general increases in transcript levels of genes involved in TAG synthesis, levels of those involved in lipid catabolism generally decreased under N deprivation, those of acyl-CoA oxidase (Phatrdraft_19979) and 3-oxoacyl-CoA thiolase (ATO1) most dramatically; 11-fold and more than 3-fold, respectively.
Lipases are enzymes that de-esterify carboxyl esters, such as TAGs and phospholipids. More than 28 genes encoding putative lipases are present in the P. tricornutum genome (Additional file 3: Table S1g) and their responses to N deprivation varied markedly. Since TAG accumulated markedly under N deprivation, TAG lipases would be expected to be down-regulated. Among the putative lipase genes, eight (28%) showed increased transcript abundance but mainly only slightly, and transcript levels of most of the others decreased more than 2-fold. For example: Phatrdraft_44231, which encodes a putative triacylglycerol lipase, decreased 5-fold; Phatrdraft_50397 decreased 4-fold; while 43593 increased nearly 4-fold. These lipase genes may play important roles in membrane turnover, and their overall reduction in transcription may protect the TAG from degradation, thereby promoting remodeling and TAG accumulation under N deprivation.
Effects of N deprivation on the subcellular ultrastructure
Sections of cells were observed by transmission electron microscopy to assess the impact of N deprivation on their ultrastructure. Numerous oil bodies, of various sizes, were present in both the N-deprived and control cells. However, their total volumes were substantially higher in the former (Figure 5). The oil bodies in the N-deprived cells were globular, compact, and mostly 0.1 to 0.3 μm in diameter, but a few (2 to 5 per section) were much larger, up to 2.0 μm. In addition to causing significant changes in oil body contents, N deprivation strongly affected chloroplast ultrastructure. Diatoms 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 DGD