Subcellular distribution of gluconeogenetic enzymes in germinating castor bean endosperm

The intracellular distribution of enzymes capable of catalyzing the reactions from oxaloacetate to sucrose in germinating castor bean endosperm has been studied by sucrose density gradient centrifugation. One set of glycolytic enzyme activities was detected in the plastids and another in the cytosol. The percentages of their activities in the plastids were less than 10% of total activities except for aldolase and fructose diphosphatase. The activities of several of the enzymes present in the plastids seem to be too low to account for the in vivo rate of gluconeogenesis whereas those in the cytosol are quite adequate. Furthermore, phosphoenolypyruvate carboxykinase, sucrose phosphate synthetase, and sucrose synthetase, which catalyze the first and final steps in the conversion of oxaloacetate to sucrose, were found only in the cytosol. It is deduced that in germinating castor bean endosperm the complete conversion of oxaloacetate to sucrose and CO₂ occurs in the cytosol. The plastids contain some enzymes of the pentose phosphate pathway, pyruvate dehydrogenase and fatty acid synthetase in addition to the set of glycolytic enzymes. This suggests that the role of the plastid in the endosperm of germinating castor bean is the production of fatty acids from sugar phosphates, as it is known to be in the endosperm during seed development.     

Phycomyces: discovery of the aiming error in the avoidance response

Vacuoles were prepared from germinating castor bean endosperm (Ricinus communis var Hale) and purified by filtration through a cotton layer under physiological osmolarity. The purity of vacuoles prepared by this method was comparable with that prepared by a sucrose step gradient centrifugation reported in a previous paper (Nishimura, Beevers 1978 Plant Physiol 62: 44-48). It was shown by assays of marker enzymes that the final preparation contained trace contamination of other organelles (glyoxysomes, mitochondria, and endoplasmic reticulum) and the cytosol. The isolated vacuoles were stained with neutral red, indicating that the intravacuolar pH is acidic. Intravacuolar pH of isolated vacuoles was determined by measuring the distribution of [¹⁴C]methylamine in the vacuoles and by directly measuring the pH of vacuolar extracts. The pH of isolated vacuolar extracts was 5.7 to 5.9. Similar values were obtained by the methylamine method and it was shown that intravacuolar pH increased as the pH of the medium was increased.     

Heterogeneous distribution of glycosyltransferases in the endoplasmic reticulum of castor bean endosperm

Endoplasmic reticulum membranes stripped of attached ribosomes were isolated from homogenates of germinating castor bean (Ricinus communis L.) endosperm by sucrose density gradient centrifugation. The isolated endoplasmic reticulum fraction was further separated into two major membrane subfractions by centrifugation on a flotation gradient. Both subfractions appeared to be derived from the endoplasmic reticulum inasmuch as they share several enzymic markers including cholinephosphotransferase, NADH-cytochrome c reductase, and glycoprotein fucosyl-transferase and phase separation of membrane polypeptides using Triton X-114 revealed a striking similarity in both their hydrophilic and hydrophobic protein components. The endoplasmic reticulum membrane subfractions contain glycoproteins which were readily labeled by incubating intact endosperm tissue with radioactive sugars prior to fractionation.

Castor bean endosperm endoplasmic reticulum apparently exhibits a degree of enzymic heterogeneity, however, since the enzymes responsible for the synthesis of dolicholpyrophosphate N-acetylglucosamine and dolicholmonophosphate mannose together with their incorporation into the oligosaccharide-lipid precursor of protein N-glycosylation were largely recovered in a single endoplasmic reticulum subfraction.

     

A Vacuolar Processing Enzyme in Maturing and Germinating Seeds: Its Distribution and Associated Changes during Development

Proprotein precursors of vacuolar components are transportedfrom endoplasmic reticulum to the dense vesicles, and then targetedto the vacuoles, where they are processed proteolytically totheir mature forms by a vacuolar processing enzyme. Immunoelectronmicroscopy of the maturing endosperm of castor bean (Ricinnscommunis) revealed that the vacuolar processing enzyme is selectivelylocalized in the dense vesicles as well as in the vacuolar matrix.This indicates that the vacuolar processing enzyme is transportedto vacuoles via dense vesicles as does IIS globulin, a majorseed protein. During seed maturation of castor bean, an increasein the activity of the vacuolar processing enzyme in the endospermpreceded increases in amounts of total protein. The enzymaticactivity reached a maximum at the late stage of seed maturationand then decreased during seed germination concomitantly withthe degradation of seed storage proteins. We examined the distributionof the enzyme in different tissues of various plants. The processingenzyme was found in cotyledons of castor bean, pumpkin and soybean,as well as in endosperm, and low-level processing activity wasalso detected in roots, hypocotyls and leaves of castor bean,pumpkin, soybean, mung bean and spinach. These results suggestthat the proprotein-processing machinery is widely distributedin vacuoles of various plant tissues. (Received July 11, 1993; Accepted August 17, 1993)     

Developmental changes in endosperm of germinating castor bean independent of embryonic axis

Dry castor bean (Ricinus communis) seeds were cut transversely into halves and the half without the embryonic axis was placed in moist vermiculite at 30 C for 5 days. The development of the endosperm in the half-bean was found to be qualitatively similar to that in the whole seedling in the appearance of various enzymes of gluconeogenesis, the accumulation of glucose and sucrose as the end products of fat utilization, and the development of subcellular structure. It is concluded that during germination of castor bean, the embryonic axis does not directly control the developmental changes in the endosperm.     

Biosynthesis of the Macrocyclic Diterpene Casbene in Castor Bean (Ricinus communis L.) Seedlings : Changes in Enzyme Levels Induced by Fungal Infection and Intracellular Localization of the Pathway

Casbene is a macrocyclic diterpene hydrocarbon that is produced in young castor bean (Ricinus communis L.) seedlings after they are exposed to Rhizopus stolonifer or other fungi. The activities of enzymes that participate in casbene biosynthesis were measured in cell-free extracts of 67-hour castor bean seedlings (a) that had been exposed to R. stolonifer spores 18 hours prior to the preparation of extracts, and (b) that were maintained under aseptic conditions throughout. Activity for the conversion of mevalonate to isopentenyl pyrophosphate does not change significantly after infection. On the other hand, the activities of farnesyl pyrophosphate synthetase (geranyl transferase), geranylgeranyl pyrophosphate synthetase (farnesyl transferase), and casbene synthetase are all substantially greater in infected tissues in comparison with control seedlings maintained under sterile conditions. The subcellular localization of these enzymes of casbene biosynthesis was investigated in preparations of microsomes, mitochondria, glyoxysomes, and proplastids that were resolved by centrifugation in linear and step sucrose density gradients of homogenates of castor bean endosperm tissue from both infected and sterile castor bean seedlings. Isopentenyl pyrophosphate isomerase and geranyl transferase activities are associated with proplastids from both infected and sterile seedlings. Significant levels of farnesyl transferase and casbene synthetase are found only in association with the proplastids of infected tissues and not in the proplastids of sterile tissues. From these results, it appears that at least the last two steps of casbene biosynthesis, geranylgeranyl pyrophosphate synthetase and casbene synthetase, are induced during the process of infection, and that the enzymes responsible for the conversion of isopentenyl pyrophosphate to casbene are localized in proplastids.     

Studies on the Development and Localization of Catalase and H(2)O(2)-generating Oxidases in the Endosperm of Germinating Castor Beans

During germination of castor bean seeds (Ricinus communis var. Hale), the changes of activity of catalase, uricase, and α-hydroxyacid oxidase of the endosperm follow a rise and fall pattern with a peak between day 4 and 5 similar to that observed for the glyoxylate cycle enzymes. After 3 days of germination, most of the activities of these enzymes are recovered from the glyoxysomal fraction separated by isopycnic sucrose density gradient centrifugation.     

Enzymes of phospholipid metabolism in the endoplasmic reticulum of castor bean endosperm

The intracellular location of several enzymes concerned with phospholipid metabolism was investigated by examining their distribution in organelles separated on sucrose gradients from total homogenates of castor bean (Ricinus communis var. Hale) endosperm. The enzymes phosphatidic acid phosphatase, CDP-diglyceride-inositol transferase, and phosphatidyletha-nolamine-l-serine phosphatidyl transferase were all primarily or exclusively confined to membranes of the endoplasmic reticulum. These results and those reported previously on lecithin synthesis establish a major role of the endoplasmic reticulum in phospholipid and membrane synthesis in plant tissues.     

Protein Bodies from the Endosperm of Castor Bean: Subfractionation, Protein Components, Lectins, and Changes during Germination

Protein bodies from the storage endosperm of dry castor bean (Ricinus communis L.) were isolated by successive nonaqueous linear density gradient centrifugation. The isolated protein bodies were lysed by the addition of water, and the various structural components of the organelles were separated by sucrose gradient centrifugation. The matrix protein remained at the top of the gradient while the membrane, the crystalloids, and the globoids migrated to densities 1.15 g/cm³, 1.30 g/cm³, and > 1.46 g/cm³, respectively. The protein of the protein bodies was distributed evenly between the crystalloids and the matrix, and little protein was present in the globoids or the membrane.     

Mixed function oxidases from germinating castor bean endosperm

Homogenates from germinating castor bean endosperm were fractionated by sucrose density gradient centrifugation and examined for mixed function oxidase activity. Activity of cinnamic acid 4-hydroxylase and p-chloro-N-methylaniline N-demethylase was highest in the endoplasmic reticulum fraction. Activity of both enzymes is dependent on NADPH and on molecular oxygen; both activities are inhibited by carbon monoxide. When challenged with a number of potential inhibitors the enzymes responded in ways fairly typical of mixed function oxidases from other plants and animals. The N-demethylase appears to be specific for N-methylarylamines. In the absence of NADPH, cumene hydroperoxide is able to support N-demethylation. The mechanistic significance of this activity is discussed.     

Enzymes of the Glycolytic and Pentose Phosphate Pathways in Proplastids from the Developing Endosperm of Ricinus communis L

The metabolism of sucrose to long chain fatty acids in the endosperm of developing castor bean (Ricinus communis L.) seeds requires a combination of cytosolic and proplastid enzymes. The total activity and the subcellular distribution of the intermediate enzymic steps responsible for the conversion of sucrose to pyruvate have been determined. Hexose phosphate synthesis from sucrose occurs in the cytosol along with the first oxidative step in the pentose phosphate pathway, glucose-6-phosphate dehydrogenase. The proplastids contain the necessary complement of glycolytic enzymes to account for the in vivo rates of acetate synthesis from glucose 6-phosphate. These organelles also contain the majority of the cellular 6-phosphogluconate dehydrogenase, transketolase, and transaldolase activities.     

Enzymes of glycerol metabolism in the storage tissues of Fatty seedlings

Various enzymes of glycerol metabolism in the extracts of 5-day-old eastor bean (Ricinus communis L. var. Hale) endosperm and 4-day-old peanut (Archis hypogaea L.) cotyledon were studied. NAD-glycerol dehydrogenase and NAD-α-glycerolphosphate dehydrogenase were not detected. Glycerol kinase was detected in the soluble fractions and an α-glycerolphosphate oxidoreductase was found in the particulate fractions. The particulate fractions were separated into various organelle fractions by sucrose gradient centrifugation and the α-glycerolphosphate oxidoreductase was shown to be present in the mitochondria. The properties of the castor bean mitochondrial α-glycerolphosphate oxidoreductase resembled those of a similar enzyme present in the mitochondria of many animal tissues. A survey showed that the α-glycerolphosphate oxidoreductase was present in great amount only in the storage tissues of fatty seedlings but not in other nonfatty plant tissues. It is concluded that in the storage tissues of fatty seedlings, the soluble glycerol kinase and the mitochondrial cytochrome-linked α-glycerolphosphate oxidoreductase are the two enzymes responsible for the initial conversion of glycerol to hexose.     

Deposition of Matrix and Crystalloid Storage Proteins during Protein Body Development in the Endosperm of Ricinus communis L. cv. Hale Seeds

Protein bodies within the endosperm of castor bean (Ricinus communis L. cv. Hale) seeds arise from numerous small vacuoles which progressively become filled with storage protein, of which the crystalloid proteins make up approximately 70%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) shows that the crystalloids are a family of at least four proteins which reduce to two complementary groups after 2-mercaptoethanol treatment. The matrix, which comprises the remainder, has two major components, the soluble albumins and the lectins. The lectins are the only glycoproteins within the mature protein body. Both cytochemical staining and SDS-PAGE indicate that the synthesis of the crystalloid and the majority of matrix proteins begins some 20 days after pollination. Additionally, the crystalloid proteins are synthesized concurrently, whereas there is temporal variation in the synthesis of matrix proteins.     

Effects of leupeptin on proteinase and germination of castor beans

Leupeptin, a tripeptide inhibitor of some proteinases, was shown previously to maintain the stability of several enzymes (isocitrate lyase, fumarase, and catalase) in crude extracts of castor bean endosperm. This reagent is now shown to inhibit the breakdown of water-soluble and crystalloidstorage proteins of the protein bodies isolated from castor beans by the SH-proteinase and it also inhibits the endopeptidase from mung beans. When suitably introduced into the endosperm of dry castor beans it strongly inhibits germination and seedling development. Application of leupeptin to endosperm halves removed from the seed prevents the normal development of enzymes concerned with gluconeogenesis from fat and drastically curtails sugar production. The results suggest that the SH-proteinase is intimately involved in the mobilization of storage proteins.     

Glutamate dehydrogenase in developing endosperm, chloroplasts, and roots of castor bean

All the glutamate dehydrogenase activity in developing castor bean endosperm is shown to be located in the mitochondria. The enzyme can not be detected in the plastids, and this is probably not due to the inactivation of an unstable enzyme, since a stable enzyme can be isolated from castor bean leaf chloroplasts. The endosperm mitochondrial glutamate dehydrogenase consists of a series of differently charged forms which stain on polyacrylamide gel electrophoresis with both NAD⁺ and NADP⁺. The chloroplast and root enzymes differ from the endosperm enzyme on polyacrylamide gel electrophoresis. The amination reaction of all the enzymes is affected by high salt concentrations. For the endosperm enzyme, the ratio of activity with NADH to that with NADPH is 6.3 at 250 millimolar NH₄Cl and 1.5 at 12.5 millimolar NH₄Cl. K_m values for NH₄⁺ and NAD(P)H are reduced at low salt concentrations. The low K_m values for the nucleotides may favor a role for glutamate dehydrogenase in ammonia assimilation in some situations.     

Effetto di Inibitori Della Sintesi Proteica Sull'aumento di Attività Enzimatiche e sul Risveglio del Metabolismo Nell'Endosperma di Semi di Ricino in Germinazione

Abstract

Effects of inhibitors of protein synthesis on the development of metabolic activity in the endosperm during the germination of castor bean seeds. — The effect of chloramphenicol, streptomycin and actinomycin-C on the increase of the activities of glyceroaldehyde-phosphate dehydrogenase, aldolase, glucose-6-phosphate dehydrogenase, fructose 1–6 diphosphate-1-phosphatase, phosphomonoesterase, in the endosperm of germinating castor bean seeds was investigated.

In all cases, the protein synthesis inhibitors depressed the activation of the enzymes tested: in particular, actinomycin (50 μg/ml) completely suppressed the increase of the activities.

The development of the rate of oxygen uptake and the conversion of fats to sugars was strongly affected by the inhibitors.

These data suggest that the increase of the activities of several enzymes in the germinating endosperm is dependent on enzyme synthesis rather than on the conversion from the inactive to the active form of the enzymes.     

Lipase Activities in Castor Bean Endosperm during Germination

Two lipases were found in extracts from castor bean (Ricinus communis L.) endosperm. One, with optimal activity at pH 5.0 (acid lipase), was present in dry seeds and displayed high activity during the first 2 days of germination. The second, with an alkaline pH optimum (alkaline lipase), was particularly active during days 3 to 5. When total homogenates of endosperm were fractionated into fat layer, supernatant, and particulate fractions, the acid lipase was recovered in the fat layer, and the alkaline lipase was located primarily in the particulate fraction. Sucrose density gradient centrifugation showed that the alkaline lipase was located mainly in glyoxysomes, with some 30% of the activity in the endoplasmic reticulum. When glyoxysomes were broken by osmotic shock and exposed to KCl, which solubilizes most of the enzymes, the alkaline lipase remained particulate and was recovered with the glyoxysomal “ghosts” at equilibrium density 1.21 g/cm³ on the sucrose gradient. Association of the lipase with the gly-oxysomal membrane was supported by the responses to detergents and to butanol. The alkaline lipase hydrolyzed only monosubstituted glycerols. The roles of the two lipases in lipid utilization during germination of castor bean are discussed.     

Pyruvate dehydrogenase complex from germinating castor bean endosperm

Subcellular organelles from castor bean (Ricinus communis) endosperm were isolated on discontinuous sucrose gradients from germinating seeds which were 1 to 7 days postimbibition. Marker enzyme activities of the organelles were measured (fumarase, catalase, and triose phosphate isomerase) and the homogeneity of the organelle fractions was examined by electron microscopy. Pyruvate dehydrogenase complex activity was measured only in the mitochondrial fraction and attempts to activate or release the enzyme from the proplastid were not successful. A pathway is proposed for the most efficient use of endosperm carbon for de novo fatty acid biosynthesis that does not require the presence of the pyruvate dehydrogenase complex in the proplastid to provide acetyl-coenzymeA.     

Phospholipid Metabolism in Plant Mitochondria: II. SUBMITOCHONDRIAL SITES OF SYNTHESIS OF PHOSPHATIDYLCHOLINE AND PHOSPHATIDYLETHANOLAMINE

CDPcholine:1,2-diacylglycerol cholinephosphotransferase (EC 2.7.8.2) and CDPethanolamine:1,2-diacylglycerol ethanolaminephosphotransferase (EC 2.7.8.1) were detected in mitochondrial fractions from castor bean (Ricinus communis) endosperm. These activities were not due to contamination of the fractions with endoplasmic reticulum. The enzymes were localized on both the inner and outer mitochondrial membranes.     

Characterization of deoxyribonucleic Acid species from castor bean endosperm: inability to detect a unique deoxyribonucleic Acid species associated with glyoxysomes

Three DNA buoyant density species (nuclear, 1.692 g cm⁻³; mitochondria 1.705 g cm⁻³; and proplastid, 1.713 g cm⁻³) can be detected in extracts from castor bean endosperm. No other buoyant density species can be identified. DNA extracts from sucrose density gradient purified glyoxysomes exhibit varying amounts of each of the three identified DNAs but no other distinguishable DNA species. RNA synthesized in vitro by Escherichia coli RNA polymerase using purified castor bean nuclear DNA as a template, hybridizes equally well with its template and with the 1.692 g cm⁻³ species from glyoxysome fractions. These results are discussed in terms of their relevance to microbody biogenesis.