Glyoxysomes of Castor Bean Endosperm and Their Relation to Gluconeogenesis*

Beevers, Harry

doi:10.1111/j.1749-6632.1969.tb43118.x

Cited by 194 publications

(99 citation statements)

References 17 publications

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“…The amount of RuDP carboxylase activity in castor bean endosperms is 25 % of soybean leaves and 50% of maize leaves. The developmental curve for RuDP carboxylase is similar to the developmental curves for malate synthetase and isocitric lyase in endosperms of germinating castor beans (3). At three different times during the development of RuDP carboxylase activity in the endosperms, the Vm.…”

Section: Methodsmentioning

confidence: 61%

Development of Ribulose 1,5-Diphosphate Carboxylase in Nonphotosynthetic Endosperms of Germinating Castor Beans

1976

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Previous reports have described the presence and properties of RuDP2 carboxylase in castor bean endosperms (4, 15). These are the only reports of the occurrence of this enzyme in nonphotosynthetic higher plant tissue. RuDP carboxylase has been localized in the proplastids of germinating castor beans (15) and the enzyme reaches a peak activity in the dark in 4 days (4). Dark-grown etiolated leaves contain low amounts of RuDP carboxylase activity which increases substantially only after exposure of the leaves to light (6, 10, 13). The purpose of this paper was to study the unique development of RuDP carboxylase and proplastids in the endosperms of germinating castor beans. were dusted with Arasan. The seeds were imbibed in H20 for 24 hr and germinated in moist vermiculite at 35 C in the dark. For developmental studies, the seedlings were harvested from 0 to 8 days. For antibiotic studies, the seedcoats were removed, and the seeds were imbibed in H20, 2 Ag/ml CHI, or 50 Atg/ml CAP. MATERIALS AND METHODSThe imbibed seeds were germinated at 35 C in the dark in Petri dishes in H20 or antibiotic solution. To study the effect of light on the development of RuDP carboxylase, the imbibed seeds, without seedcoats, were germinated in the light or dark at 35 C. The light source was General Electric cool-white 20-w fluorescent bulbs. The light intensity at the seed surface was 30 ,ueinsteins m-2 sec-1.Previously (4), it has been shown that RuDP carboxylase activity peaks in 4 day old germinating castor beans. We found that both the rate of growth of the castor bean seedlings and the rate of development of RuDP carboxylase in the seedlings are influenced by (a) removal of the hard seedcoat prior to germination and (b) the watering regime during the growth of the seedlings. Removing the seedcoats, prior to imbibing and germinating the seeds, increases the rate of seedling growth. In these seedlings growing in Petri dishes, RuDP carboxylase activity peaks 48 hr after germination. Depending on the watering regime, the peak carboxylase activity can be extended 1 to 2 days in seeds germinating in vermiculite. Because of these variables, in most experiments we have recorded the chronological age of the seedlings and the fresh weight of the endosperms. In most experiments, maximal RuDP carboxylase activity developed in seedlings with an endosperm weight between 0.60 and 0.84 g fresh weight/seed. Enzyme Isolation and Purification. RuDP carboxylase was isolated and partially purified from endosperms of 5-day-old seedlings. (0.75 g endosperm/seed). All steps were carried out at 4 C. The endosperms were separated from the testa, hypocotyl, epicotyl, and cotyledons and rinsed with deionized H20. The endosperms were homogenized for 60 sec in a prechilled Waring Blendor in 0.1 M tris buffer, pH 7.5, containing 0.1 mm GSH. The homogenizing medium contained 1 ml buffer/g fresh weight of endosperm. The homogenate was filtered through two layers of cheesecloth and centrifuged 15 min at 1OOOg in a RC-5 Sorvall refrigerated centrifuge. Th...

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Section: Methodsmentioning

confidence: 61%

Development of Ribulose 1,5-Diphosphate Carboxylase in Nonphotosynthetic Endosperms of Germinating Castor Beans

1976

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“…The mRNA had been isolated from castor bean endosperm tisue excised at a developmental stage when rapid synthesis of glyoxysomes was occurring (2). Accordingly, glyoxysomal proteins should account for a reasonable proportion of the total translation products.…”

Section: Resultsmentioning

confidence: 99%

“…The rapid synthesis of enzymes and organelles which occurs in the endosperm tissue during the first few days of growth of castor bean seedlings (2) indicates that this tissue is a potentially rich source of mRNA for organellar proteins. The endosperm tissue however has a relatively short active life during seedling development and rapidly senesces as fat utilization is replaced by photosynthetic carbon fixation as the mode of seedling nutrition.…”

Section: Discussionmentioning

confidence: 99%

Isolation and Cell-free Translation of Total Messenger RNA from Germinating Castor Bean Endosperm

Bowden-Bonnett

Lord

1979

Plant Physiol.

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Polyadenylated RNA was isolated from the total RNA fraction extracted from the endosperm tissue of 3-day-old castor bean seedlings by affinity chromatography on oligo(dT)-cellulose. This polyadenylated RNA was efficiently translated into protein when added to a messenger RNA-dependent cell-free system derived from rabbit reticulocytes. Characterization of the translational products by electrophoresis followed by autoradiography established that numerous discrete polypeptides were formed with molecular weights ranging from 10,000 to over 100,000. Immunoprecipitation in the presence of antiserum raised in rabbits against the total glyoxysomal matrix proteins showed that these proteins accounted for 15 to 20% of the total translational products.Attempts to reconstitute rough endoplasmic reticulum by the addition of washed castor bean microsomal membranes to the translational system were unsuccessful, these membranes severely inhibiting protein synthesis. Canine pancreatic microsomes could be added to endosperm messenger RNA-dependent reticulocyte lysates at relatively high concentrations while still allowing significant protein synthesis.The uptake of water by dry seeds initiates a rapid transcription of RNA and ensuing protein synthesis which permits metabolic activity during germination (11,12). In the endosperm of germinating castor bean seedlings, this metabolic activity is predominantly associated with the conversion of stored triglycerides into carbohydrate (1). We have used this tissue to examine events surrounding the synthesis of gluconeogenic enzymes and the organelles (in particular, the glyoxysomes) which house them (5-7). This tissue offers a particularly attractive system for the study of organelle biogenesis, not only because of the rapid and massive formation of the organelles, but also because these syntheses occur in the absence of cell division or net protein accumulation.In order to reach a better understanding of these cellular processes, the isolation of mRNAs and their subsequent translation in a cell-free protein-synthesizing system are potentially important. The control of translation of specific mRNAs and mechanisms of post translational modification can be studied and the relevance of such events in terms of organelle biogenesis and protein segregation can be assessed.In Proteinase K was added (200 jig/ml final concentration) and the mixture was stirred for 10 min at room temperature. An equal volume of phenol-chloroform (1:1) was added and after a further 10-min stirring the phases were separated by centrifugation and the aqueous phase was removed. The organic phase was extracted with a half-volume of 20 mm Tris-HCl (pH 9.0), 2 mM EDTA, and the combined aqueous phases were reextracted twice with equal volumes of phenol-chloroform. The final aqueous phase was adjusted to 0.3 M NaCl and the nucleic acids were precipitated with 2.5 volumes of ethanol at -20 C overnight. The nucleic acid pellet, collected by centrifugation at 10,000g and 2 C for 10 min, was visibly contaminated with ...

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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).…”

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confidence: 99%