Figure 1.
Effect of nitrogen-deprivation on the cell proliferation and lipid accumulation in Symbiodinium.
(A) Growth of Symbiodinium cells cultivated in control versus nitrogen-deprivation media. The data represents mean ± SD (n = 3). (B) The visualization of neutral lipid accumulation using BODIPY 493/503 in control vs. nitrogen-deprived cultures. Scale bar, 10 µm.
Figure 2.
The TLC analysis of lipids extracted from Symbiodinium spp. cells.
The comparison between control and nitrogen-deprivation treated cells (A). The lipid content of the purified LDs from Symbiodinium after five days of nitrogen deprivation is shown in (B).
Table 1.
TAGs and CEs accumulation in Symbiodinium spp.
Figure 3.
The change of fatty acid compositions in TAGs of Symbiodinium after five days of nitrogen deprivation.
Relative amounts (%) of fatty acid compositions in purified TAGs from total Symbiodinium were determined (see the “Materials and methods” section). The data represents mean ± SD (n = 3).
Figure 4.
The ultrastructural examination of morphological changes and LD formation in Symbiodinium after nitrogen deprivation.
Transmission electron micrographs of Symbiodinium in control (A, B) and nitrogen-deprivation media (five days: C–D; seven days: E–F). Insets in A, C and D were magnified as B, D and F, respectively. Arrows in A, C, and E indicated cell walls, while arrowheads in F indicated the OsO4-negative “inclusion bodies”. Abbreviations: LD, lipid droplet; Ch, chloroplast; S, starch granule; P, pyrenoids; N, nucleolus.
Table 2.
Influence of duration after nitrogen depletion on Symbiodinium cells.
Figure 5.
Light microscopy of the LDs purified from Symbiodinium cells after different treatments.
The LDs were suspended in the (A) pH 7.5 grinding buffer, (B) pH 6.5 grinding buffer or (C) treated by the trypsin digestion. (D) Phospholipid analyses by TLC showing the presence of phospholipids (PLs) in purified LDs (the top layer during the centrifugation) but not lower layer fractions after detergent (0.1% Triton X-100) washing.
Figure 6.
LDs purification and protein analyses.
(A) SDS-PAGE analyses of isolated LDs fraction and the LD purity assessment by RuBisCO western blotting. Symbiodinium spp. cells harvested after five days of nitrogen deprivation were homogenized and fractionated to purify LDs as shown in the “Materials and methods” section. The purity of LDs was examined based on the absence of RuBisCO contamination by western blotting. Proteins bands 1 to 5 were excised for mass spectrometric analysis. (B) BODIPY 493/503 staining of isolated LDs displayed the abundance of neutral lipid.
Table 3.
Identification of lipid droplet proteins in Symbiodinium spp.