Association of the glyoxylate cycle enzymes in a novel subcellular particle from castor bean endosperm

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 ...

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“…3. Ricinosomes are clearly visible in the endosperm cytoplasm after the seed has been germinating 4 to 6 days (see arrow).…”

Section: Discussionmentioning

confidence: 99%

“…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.…”

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confidence: 99%

Studies on Seeds

Mollenhauer

Totten

1970

Plant Physiol.

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“…The tissue (20 g of peanut cotyledons or 12 g of maize scutella) was minced with a razor blade and gently homogenized with a mortar and pestle with 3 volumes of the grinding medium described by Breidenbach and Beevers (2). Hepes (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) was used, however, instead of tris as a buffering agent.…”

Section: Methodsmentioning

confidence: 99%

“…They have been isolated by means of density gradient centrifugation from castor bean, watermelon, and peanut seeds (2,4). The two key enzymes of the glyoxylate cycle, isocitratase and malate synthetase, which are localized chiefly in the glyoxysomes, undergo striking changes in activity during germination (5,13).…”

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confidence: 99%

The Development of Glyoxysomes in Peanut Cotyledons and Maize Scutella

Longo¹,

Longo²

1970

Plant Physiol.

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The development of glyoxysomes during germination has been studied in isolated peanut (Arachis hypogaea L.) cotyledons and in maize (Zea mays L.) scutella. In peanut cotyledons isocitratase, malate synthetase, and protein associated with the glyoxysomal fraction increase simultaneously from the 3rd to the 8th day of incubation. In scutella of germinating maize seeds the specific activities of isocitratase, malate synthetase, and catalase associated with the glyoxysomes rise until the 4th day of germination and then decline while the total amount of protein present in the fraction stays constant during the first 5 days. If the peanut cotyledons are cultured in 2% glucose, the development of isocitratase and malate synthetase is severely inhibited, but the level of the glyoxysomal protein is not measurably affected.Glyoxysomes are subcellular organelles, present in fat-storing tissues of higher plants, which contain the enzymes necessary for the operation of the glyoxylate cycle. They have been isolated by means of density gradient centrifugation from castor bean, watermelon, and peanut seeds (2, 4). The two key enzymes of the glyoxylate cycle, isocitratase and malate synthetase, which are localized chiefly in the glyoxysomes, undergo striking changes in activity during germination (5, 13). They are present at very low levels in the dry seeds, increase greatly during the first 4 to 5 days of germination, and then decline again. It has been shown that the increase of isocitratase and malate synthetase activity in peanut cotyledons is due to synthesis de novo of the two enzymes (8, 11), but comparatively little is known about the development of the particles themselves (7). It may be that the glyoxysomal particles are assembled at the same time that the glyoxylate cycle enzymes are synthesized. Alternatively, one could thing that newly synthesized enzymes are introduced into preexisting particles. This paper provides some information about the development of glyoxysomes in peanut cotyledons and maize scutella. MATERIALS AND METHODSPeanut seeds (Arachis hypogaea L.) were of the strain Virginia R-56, 1966 crop, maize seeds (Zea mays L.) of the strain

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“…Isocitrate lyase and malate synthetase, key enzymes of the glyoxylate pathway, are present in such fatty seedlings when lipid stores are being metabolized in the endosperm (3,30,45) or the cotyledons (8,43,57) and in fact appear to be restricted in higher plants to this one stage of growth (3). Using castor bean endosperm, Beevers and co-workers (6,7) localized these enzymes in a subcellular particle, the glyoxysome, which is now also known to be the site of fatty acid oxidation (12,26). Since their discovery in endosperm, it has been reported that glyoxysomes are the site of the glyoxylate pathway in a number of fatty cotyledons, including those of watermelon (11,28,39), peanut (11,35), cucumber (53), and sunflower (22).…”

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confidence: 99%