J. Biochem, 1997, Vol. 121, No. 4 762-768
© 1997 Japanese Biochemical Society
research-article |
A New Method for Seed Oil Body Purification and Examination of Oil Body Integrity Following Germination1
Graduate Institute of Agricultural Biotechnology, National Chung-Hsing University Taichung, Taiwan 40227
2To whom correspondence should be addressed: Phone: +886-4-2840328, Fax: +886-4-2861905 E-mail: TCTZEN{at}dragon.nchu.edu.tw
Plant seeds store triacylglycerols as energy sources for germination and postgerminative growth of seedlings. The triacylglycerols are preserved in small, discrete, intracellular organelles called oil bodies. A new method was developed to purify seed oil bodies. The method included extraction, flotation by centrifugation, detergent washing, ionic elution, treatment with a chaotropic agent, and integrity testing by use of hexane. These processes subsequently removed non-specifically associated or trapped proteins within the oil bodies. Oil bodies purified by this method maintained their integrity and displayed electrostatic repulsion and steric hindrance on their surface. Compared with the previous procedure, this method allowed higher purification of oil bodies, as demonstrated by SDS-PAGE using five species of oilseeds. Oil bodies purified from sesame were further analyzed by two-dimensional gel electrophoresis and revealed two potential oleosin isoforms. The integrity of oil bodies in germinating sesame seedlings was examined by hexane extraction. Our results indicated that consumption of triacylglycerols reduced gradually the total amount of oil bodies in seedlings, whereas no alteration was observed in the integrity of remaining oil bodies. This observation implies that oil bodies in germinating seeds are not degraded simultaneously. It is suggested that glyoxisomes, with the assistance of mitochondria, fuse and digest oil bodies one at a time, while the remaining oil bodies are preserved intact during the whole period of germination.
1The work was supported by grants from the National Science Council, Taiwan, ROC (NSC 85-2321-B-005-042 and NSC 86-2313-B-005-048 to JTC Tzen).
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