Figure 1.
A. Fluorescence microscopy images, showing the 1- to 32-cell stages, and the flagellated stage. DIC: differential interference contrast image; SYBR: SYBR Green I-stained cells (green); CHL: chlorophyll autofluorescence (red); and Overlay: overlaid images of SYBR and CHL. B. Illustration of life cycle of H. pluvialis. Refresh: when old cultures are transplanted into fresh medium, coccoid cells undergo cell division to form flagellated cells within the mother cell wall. Germination: Flagellated cells settle and become coccoid cells. Continuous and/or strong light accelerate the accumulation of astaxanthin during encystment (red arrows).
Figure 2.
Transmission electron micrographs of green coccoid cells in H. pluvialis.
A. General ultrastructure. The cell wall is surrounded by extracellular matrix (arrowheads). Arrows indicate astaxanthin granules. B. Chloroplast and pyrenoid. C. High-magnification view of astaxanthin granules (arrows). D, E. One-layer thylakoids with a regular arrangement. C, chloroplast; CW, cell wall; N, nucleus; P, pyrenoid. Scale bars in A and B–E: 5 µm and 1 µm, respectively.
Figure 3.
Transmission electron micrographs of intermediate H. pluvialis cells.
A. General ultrastructure. B. High-magnification view of astaxanthin oil droplets. C. Partial degradation of thylakoids (arrow). D. High-magnification view of thylakoid degradation (arrows). C, chloroplast; CW, cell wall; N, nucleus; OD; oil droplet; P, pyrenoid; SC, starch capsule; SG, starch grain. Scale bars in A and B–D: 5 µm and 1 µm, respectively.
Figure 4.
Transmission electron micrographs of H. pluvialis cyst cells.
A. General ultrastructure of cyst cells, showing small granules that contain astaxanthin. B. General ultrastructure of a cyst cell, showing astaxanthin accumulation in oil droplets. C. General ultrastructure of a cyst cell, showing large oil droplets. Chloroplasts localize in the interspace between oil droplets (arrows). D. Some oil droplets are fused. E. High-magnification view of chloroplasts. C, chloroplast; N, nucleus; OD, oil droplet. Scale bars in A–D and E: 5 µm and 0.5 µm, respectively.
Figure 5.
3D TEM images of subcellular components.
A, C, E, G, I, and K represent a green coccoid cell; B, D, F, H, J, and L represent a cyst cell. A and B. 3D reconstruction of chloroplasts with pyrenoids, mitochondria, and/or starch grains. C and D. 3D reconstruction of astaxanthin distribution. E and F. 3D reconstruction of starch grains with the nucleus. G and H. 3D reconstruction of Golgi bodies with the nucleus. I and J. 3D reconstruction of mitochondria (with the nucleus in J). K and L. 3D reconstruction of pyrenoids and starch capsules. All subcellular components are denoted by different colors as indicated in the color legends. Scale bar in all images: 5 µm.
Figure 6.
A. Cut-away image of a green coccoid cell. B. Cut-away image of a cyst cell. All subcellular components are denoted by different colors as indicated in the color legends. Scale bar: 5 µm. (See also Movies S1 and S2 for supporting information.)
Figure 7.
interference contrast (DIC) image and subcellular localization of lipids, astaxanthin, and chlorophyll in an astaxanthin-rich H. pluvialis cell.
Nile Red (NR) signal, astaxanthin (AXT), and chlorophyll (CHL) autofluorescence are yellow, red, and green, respectively. Two overlaid images (NR+AXT and NR+AXT+CHL) are shown. Note that the Nile Red signals are colocalized with astaxanthin, shown in orange. Scale bar: 10 µm.
Figure 8.
Relative volumes of H. pluvialis subcellular components.
Subcellular components are indicated by colors in pie charts. A. Relative volumes of subcellular components in a green coccoid cell. B. Relative volumes of subcellular components in a cyst cell.