Glyoxysomes, a form of microbody, are present in castor bean endosperm during the first 8 days of seed germination.They have a "typical" microbody form and are shown histochemically to contain catalase. The catalase label is distributed throughout the microbody and is not an exclusive feature of the crystalline or amorphous core.Castor bean endosperm contain a second cytosome, only slightly larger than the glyoxysomes, which is bound by a rough-surfaced membrane and which does not label for catalase. We have not observed these cytosomes in other tissues, suggesting that they may have a specific cellular function characteristic of castor bean endosperm.Glyoxysomes are a morphologically and biochemically unique component of many cells (3,4,12,13). They are a form of microbody containing enzymes of the glyoxylate cycle. Like peroxisomes and other types of microbodies, they also contain catalase and glycolic acid oxidase (4).Microbodies are present in castor bean endosperm throughout seed development, dormancy, and germination, but enzymes of the glyoxylate cycle do not reach maximal activity until the 4th or 5th day of seed germination (5, 12). There is no indication from ultrastructural studies that microbodies disappear, reform, or change significantly in number, in relation to the appearance of enzymes of the glyoxylate cycle. It is assumed, therefore, that glyoxysomes are somehow evolved from microbodies by enzyme activation or by gradual replacement of existing microbodies with those of the glyoxysome type.We have isolated microbodies, as well as glyoxysomes, from a number of seeds and have found that the glyoxysome fractions from castor bean endosperm sometimes contain a contaminating particle that is similar in size to glyoxysomes. Studies in vivo of germinating castor bean confirm that a microbody-like particle does exist in the endosperm and is most easily recognized when enzymes of the glyoxylate cycle are active. We thought initially that these new particles might be related, or equivalent, to glyoxysomes and, therefore, initiated these studies. This report describes these new particles and compares them structurally and histochemically with microbodies. 2"Studies on Seeds, I through IV" will appear in the Journal of Cell Biology. MATERIALS AND METHODSCastor bean (Hale variety and Baker variety 296 obtained from The Baker Castor Oil Co., La Mesa, Calif.) seeds were scarified and the seeds were soaked in water for 2 to 24 hr. The seeds were germinated for 3 to 8 days in the dark at 28 C in vermiculite moistened with distilled water.Slices of endosperm were prefixed in a mixture of 2% glutaraldehyde-2 % paraformaldehyde in 0.05 M collidine buffer containing 0.06 M sucrose, pH 7.3 to 7.4 for 2 hr, cold. After prefixation, the tissues were rinsed in six 5-to 10-min changes of buffer. Unless otherwise indicated, the buffer used was 0.05 M collidine with 0.06 M sucrose, pH 7.3 to 7.4. The tissues were either postfixed (see below) or allowed to warm to room temperature in the last buffer rinse and then ...
Several fixation procedures were studied to determine those most suitable for preservation of seeds during late stages of development and early stages of germination . These are the periods when the tissues are partially dehydrated and are most difficult to fix for electron microscopy . It was found that a prefixation with a mixture of glutaraldehyde, reconstituted formaldehyde (i .e. paraformaldehyde), and acrolein, followed by a postfixation in Os04 or KMnO 4 , gives very acceptable images . The results also indicate that glutaraldehyde is necessary for preservation of cell shape, paraformaldehyde for stabilization of reserve proteins, and acrolein for rapid penetration of tissues . Phosphate, cacodylate, and collidine are all acceptable buffers, although collidine gives the most consistent results .
Lipid content has been determined for two types of lipid-rich vesicles isolated from bush bean cotyledon at 24 hr of germination . The larger, nonassociating vesicles are four to six times richer in triglyceride than the smaller vesicles which associate strongly among themselves, as well as with smooth membranes in the cell . The larger vesicles contain about 640 µmoles of phospholipid per gram of protein, while the smaller vesicles have only onehalf to two-thirds as much phospholipid per gram of protein . The ratio of individual phospholipids in both kinds of vesicles is close to 20% phosphatidylethanolamine, 60% phosphatidylcholine, and 20% phosphatidylinositol . The fatty acid composition of all phospholipids is similar, and quite different from that of triglyceride, which contains twice as much linolenic acid and less than one-fourth as much palmitic acid . Pea cotyledon has quantitatively the same lipid content as bean cotyledon .
At least two kinds of lipid vesicles are present in pea and bean cotyledons which can be recognized at seed maturity on the basis of whether they do or do not interassociate into lipid vesicle sheets . Those that do interassociate into sheets are also characterized by (a) their association with plastids or plasma membranes during dormancy, and (b) the unique transformation into flattened saccules that they undergo during the first few days of seed germination . These interassociated (or composite) lipid vesicles have been found in only a few seeds and may be restricted to certain classes of plants and/or certain states of cellular development .Lipid vesicle-to-saccule transformation is predominantly confined to the germinating seed . However, some lipid vesicle-derived saccules are already present in some cells even before the seed reaches maturity . These partially transformed vesicles and saccules remain unchanged over dormancy, and then resume their transformation when the seed is germinated . This suggests that some stages of seed germination are already underway before the seed reaches maturity and are only resumed at seed germination .The lipid vesicles that do not interassociate into sheets (i.e ., the simple lipid vesicles) are present in all tissues at all states of cellular development . These vesicles do not undergo any conspicuous structural changes during development .
Two structurally distinct lipid vesicles are present in pea and bean cotyledons during the first few days of germination . Both were isolated by sucrose density gradient centrifugation without significant morphological changes .Lipid vesicles of one type were elongated into a sausage-like or flattened-saccular shape, and were interassociated into sheets which were usually one vesicle thick . These sheets remained intact during homogenization and centrifugation, because some of the lipid vesicles in the sheet were interconnected through their bounding membranes, and because there seemed to be a bonding substance between adjacent vesicles . These vesicles were called "composite" lipid vesicles to distinguish them from the more usual, or "simple," lipid vesicles of other plant and animal tissues .Lipid vesicles of the other type were usually larger than the composite lipid vesicles and were always spherical in form . These vesicles remained single and did not interassociate into sheets . They were probably equivalent to the simple lipid vesicles of other tissues.
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