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Greenwood et al. 10.1073/pnas.0409429102. |
Fig. 8. Ricinosomes in the senescing nucellar cells at 10 DAP are larger and structurally compound compared with ricinosomes in senescing nucellar cells at 20 DAP (this paper) and ricinosomes in endosperm cells of germinating seeds (1, 2). Ricinosomes were isolated from senescing nucellar cells (10 DAP) on a 44–65% sucrose gradient (2). Twenty-four 1.25-ml fractions were harvested (fraction 1, lower part, and fraction 24, upper part of the gradient) and analyzed by SDS/PAGE followed by Coomassie blue staining (A), silver staining (C), or Western blotting using the primary antibodies anti-CysEP (D), anti-glyoxysomal malate dehydrogenase (E), or anti-mitochondrial malate dehydrogenase (not shown). MM, molecular mass markers. Primary antibodies were decorated with alkaline phosphatase-coupled goat anti-rabbit IgG and developed as described (3). Western blotting revealed that the lowermost fractions (fractions 1-9) contained the 45-kDa proCysEP indicative for ricinosomes (D), which are well separated from other organelles such as glyoxysomes (E) or mitochondria (not shown) (fractions 15-18). Coomassie blue staining of the acrylamide gel (A) revealed that the protein composition of ricinosomes from maturing seeds (10 DAP) is different from ricinosomes of germinating seeds (see B for comparison), where the ricinosomes in the three lowermost fractions of the sucrose gradient contained the 45-kDa proCysEP as a major matrix enzyme (2). Silver staining of the acrylamide gel (C) revealed a complex protein composition indicating many additional matrix enzymes besides the proCysEP.
1. Schmid, M., Simpson, D., Kalousek, F. & Gietl, C. (1998) Planta 206, 466-475.
2. Schmid, M., Simpson, D. J., Sarioglu, H., Lottspeich, F. & Gietl, C. (2001) Proc. Natl. Acad. Sci. USA 98, 5353-5358.
3. Schmid, M., Simpson, D. & Gietl, C. (1999) Proc. Natl. Acad. Sci. USA 96, 14159-14164.
Fig. 9. Electron micrograph of an endosperm cell adjacent to the crushed walls of nucellar cells. Enlargements of the regions B (Upper) and C (Lower) as depicted in Fig. 7 are shown. (Upper) Cell wall portions with plasmodesmata (arrows). (Lower) Cell wall portion with wall ingrowths.