The Influence of Abscisic Acid, Adenosine 3', 5' Cyclic Phosphate, and Gibberellic Acid on the Induction of Isocitrate Lyase Activity in the Endosperm of Germinating Castor Bean Seeds

Isocitrate lyase, one of the key enzymes of the glyoxylate cycle,provides a convenient marker for the development of metabolicactivity in castor bean endosperm. Gibberellin GA₇ (0.3 mM)was equally as effective as GA₃ in stimulating both the rateof appearance and total activity of the enzyme. Physiologicallyhigh levels of adenosine 3', 5' cyclic phosphate (1 mM and 5mM) had the same effect as gibberellins, but lower concentrationsdid not produce a response greater than the water controls.Application of 0.03 mM abscisic acid markedly inhibited germinationof the seeds and the induction of isocitrate lyase activitybut its action could be relieved by simultaneous addition ofequimolar concentrations of GA₃.     

Organelle Membranes from Germinating Castor Bean Endosperm: II. ENZYMES, CYTOCHROMES, AND PERMEABILITY OF THE GLYOXYSOME MEMBRANE

Glyoxysome ghosts were isolated from germinating castor bean endosperms using established methods. Electron microscopic examination showed that some matrix material was retained within the glyoxysomal membrane. Two cytochrome reductases and phosphorylcholine glyceride transferase co-sedimented with the alkaline lipase, a known component of the glyoxysome membrane, in sucrose gradient centrifugation of osmotically shocked glyoxysomes. The activities of these enzymes in the glyoxysome membranes were compared to those in the endoplasmic reticulum relative to phospholipid content. On this basis, the phosphorylcholine glyceride transferase was 10-fold more active in the endoplasmic reticulum, whereas the lipase was 50-fold more active in the glyoxysome membrane. The cytochrome reductases were only 2-fold more active in the endoplasmic reticulum, indicating that they are components of the two membranes. Difference spectroscopy of the glyoxysome membrane suspension revealed the presence of a b5-type cytochrome similar to that found in the endoplasmic reticulum. Since the glyoxysome membrane is apparently derived from the endoplasmic reticulum, components of the endoplasmic reticulum such as these are likely to be incorporated into the glyoxysome membrane during biogenesis.     

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

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.     

ATP Citrate Lyase from Germinating Castor Bean Endosperm: Localization and some Properties

ATP citrate lyase (EC 4.1.3.8) has been found in crude extracts from endosperm tissue of germinating castor bean and shows its maximum activity in 4- to 5-day-old seedlings. A strict requirement for coenzyme A and adenosine 5′-triphosphate was demonstrated. The pH optimum for the reaction is around 7.5. The unstable enzyme can be stabilized by freezing and addition of citrate and glycerol. (−)-Hydroxycitrate is a potent inhibitor. The molecular weight is about 400,000. The adenosine 5′-triphosphate citrate lyase is localized in the plastids, where it possibly plays a role in providing acetyl coenzyme A for lipid biosynthesis.     

Temperature-induced Phase Changes in the Membranes of Glyoxysomes, Mitochondria, and Proplastids from Germinating Castor Bean Endosperm

Sucrose-gradient purified mitochondria, glyoxysomes, and proplastids from germinating castor bean (Ricinus communis L.) endosperm were examined by electron-spin resonance spectroscopy. A temperature-induced phase change was demonstrated in all of these organelles, their derivative membranes, and in micelles formed from the membrane phospholipids. The apparent transition temperature of the membrane lipids varied slightly between the samples, but in all cases, fell within the temperature range around 10 C where physiological and biochemical changes in the response to temperature for most chilling-sensitive plants occur.     

Early Actions of Gibberellic Acid on the Embryo and on the Endosperm of Avena fatua Seeds

Gibberellic acid at 0.1 μm stimulates amylase synthesis in dormant Avena fatua seeds without inducing germination; at 0.5 mm it enhances biosynthesis of proteins and RNA in both the embryo and the endosperm and utilization of the endosperm sugars by the embryo. These events occur in early hours (0-14th hour) and prior to germination, which begins 24 hours after gibberellic acid application. These observations are in agreemeent with the concept that in cereal grains gibberellic acid has two morphological sites of actions: the embryo and the endosperm, and that germination (radicle protrusion) is not caused by gibberellic acid-induced amylase synthesis in the endosperm.     

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.     

Aggregated Forms of Malate and Citrate Synthase are Localized in Endoplasmic Reticulum of Endosperm of Germinating Castor Bean

The endosperm of 3-day germinated seedlings of Ricinus communis was homogenized in the presence or absence of Mg²⁺. When the Mg²⁺ -containing homogenate was fractionated on linear, 20 to 40% sucrose gradients, the endoplasmic reticulum (ER) reached equilibrium at a density of 1.146 grams per cubic centimeter. Absence of Mg²⁺ in the grinding medium resulted in displacement of the ER in the gradient from a density of 1.146 to 1.138 grams per cubic centimeter. At either density, the activities of both malate and citrate synthase were found to overlap the activity of NADH-cytochrome c reductase (an ER marker) in the gradient. Furthermore, this overlap of activities was observed whether the gradients were centrifuged for 3 or 19 hours. An analysis of sedimentation characteristics of the solubilized enzymes revealed that they exist, predominantly, as a 5.2S (s_20,w × 10⁻¹³) form (malate synthase) and a 6.8S form (citrate synthase) in the glyoxysomes and cytosol. When the two enzymes were released from the ER, they appeared as aggregate forms of 70S and 55S, respectively. These results support the conclusion that the synthases are associated with the ER.     

Evidence that a Proliferation of the Endoplasmic Reticulum Precedes the Formation of Glyoxysomes and Mitochondria in Germinating Castor Bean Endosperm

Excised castor bean endosperm halves incubated with CDP-[Me-¹⁴C]cholineactively incorporated this compound into membrane phosphatidylcholine.The capacity of the tissue to synthesize phosphatidyl-[¹⁴C]cholineincreased during the first 3 d of germination and subsequentlydeclined. At the onset of germination phosphatidyl-[^l4C]cholinewas exclusively recovered in the ER membrane fraction. The rateof incorporation into the ER membranes increased strikinglyduring the first 24 h of germination while that into mitochondriaand glyoxysomes remained low. At later developmental stagesan increasing proportion of the newly synthesized phosphatidyl-[¹⁴C]cholinewas present in mitochondria and glyoxysomes; the rate of incorporationinto the membranes of these organelles increased while thatinto the ER membrane began to level off. The kinetics of CDP-[¹⁴C]cholineincorporation into membrane phosphatidylcholine of the majororganelle fractions of 3-d-old endosperm tissue showed thatthe ER was immediately labelled, whereas a lag period precededthe labelling of mitochondria and glyoxysomes. Assuming that the incorporation of CDP-[¹⁴C]choline into phosphatidylcholineserves as a reliable indicator of membrane synthesis, the resultsobtained suggest that a proliferation of ER membranes precedesthe formation of glyoxysomes and mitochondria in germinatingcastor bean endosperm. A comparison of developmental changesin (a) total ER and glyoxysomal phospholipid content and (b)ER and mitochondrial NADH cytochrome c reductase activity providedadditional evidence supporting this conclusion.     

Physiological Effects of Gibberellic Acid. IX. Recovery of Gibberellic Acid Following Incubation with Endosperm

To determine the fate of gibberellic acid (GA₃), solutions were incubated for 24 hours with or without barley endosperm and were subsequently applied to dwarf maize seedlings. Hormone activity appeared to increase as a result of incubation with endosperm. This apparent increase in GA₃ concentration was probably due to a synergistic interaction between GA₃ and endosperm constituents, particularly carbohydrate, released during the incubation period. It is concluded that relatively little hormone is inactivated during the initiation of endosperm mobilization.     

N-Acetylglucosamine Transfer Reactions and Glycoprotein Biosynthesis in Castor Bean Endosperm

N-Acetyl-[³H]glucosamine supplied to intact 3 d old castor beanendosperm tissue was incorporated into TCA-insoluble productpresumed to be glycoprotein. After an incubation time of 2 hthe major paniculate location of this product within the cellwas the endoplasmic reticulum. Cell-free preparations containingparticulate enzymes transferred N-acetyl-[¹⁴C]glucosamine fromUDP-N-acetyl-[¹⁴C]glucosamine into a fraction soluble in chloroform/methanol(2: 1, by vol), a fraction soluble in chloroform/methanol/water(10: 10: 3, by vol.), and an insoluble residue. Mild acid hydrolysisreleased the saccharide moieties from the lipids. Paper chromatographicanalysis of the released saccharides established that the C/M-solubleproducts contained both N-acetyl-[¹⁴C]glucosamine and N, N'-diacetyl-[¹⁴C]chitobiose.In contrast, N-acetyl-[¹⁴C]glucosamine released from the C/M/W-solubleproduct was contained in an oligosaccharide, probably in associationwith unlabelled mannose residues. The stimulatory effect ofdolichol monophosphate and the inhibitory effect of tunicamycinon saccharide-lipid synthesis indicated that N-acetyl-glucosamineis transferred to a glycopolymer by the established reactionsof the dolichol monophosphate pathway. The enzymes catalysingthe constituent reactions of this pathway were exclusively locatedin the ER.     

Dolichylphosphate-dependent Glycosyl Transfer Reactions in the Endoplasmic Reticulum of Castor Bean Endosperm

In the endosperm of Ricinus communis (castor bean) a number of glycosyl transferases were found to be present during germination. They catalyze the incorporation of mannose from guanosine diphosphate mannose and of N-acetylglucosamine from uridine diphosphate N-acetylglucosamine into a glycolipid fraction, which had all of the properties of dolichylphosphate and pyrophosphate sugars, respectively. The sugar moiety of dolichylphosphate mannose is transferred to a lipid-oligosaccharide, containing more than 6 hexose units. When the membranes are preincubated with nonradioactive guanosine diphosphate mannose and uridine diphosphate N-acetylglucosamine, radioactivity from dolichylphosphate [¹⁴C]mannose is also transferred to a glycopolymer. In addition, the formation of radioactive glycoproteins from guanosine diphosphate [¹⁴C]mannose has been demonstrated using a combination of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autofluorography.     

Fat Metabolism in Higher Plants. XXXVII. Characterization of the beta-Oxidation Systems From Maturing and Germinating Castor Bean Seeds

In the maturing castor bean seed (Ricinus communis), maximum β-oxidation appears at 28 days after flowering and in the germinating seed, 4 days after germination. Highest specific activities for both β-oxidation systems and their component enzymes are associated with cytosomal particles banding at a density of 1.25 g/ml in a sucrose gradient. Substrate specificity studies indicate that of several fatty acids, ricinoleate is oxidized most rapidly by the preparation from the maturing seed (28 days after flowering) while palmitate and linoleate are oxidized most rapidly by extracts obtained from tissue germinated for 4 days. The β-oxidation activities observed in both systems reflect the expression of activity of at least 3 of the component enzymes, crotonase, β-hydroxyacyl dehydrogenase and β-keto-thiolase, which rise and fall co-ordinately. Acyl thiokinase does not appear to play a limiting role in regulating β-oxidation per se under the conditions employed here.     

Vacuolation and Storage Protein Breakdown in the Castor Bean Endosperm following Imbibition

Cytochemical staining of sections prepared for light microscopy,electron microscope sections, and sodium dodecyl sulphate-polyacrylamidegel electrophoresis reveal that, following imbibition, storageproteins are mobilized from the protein bodies of the endospermof castor bean (Ricinus communis L. cv. Hale). This is accompaniedby fusion of protein bodies to form a central vacuole, beforeall the protein is hydrolyzed. Mobilization of the US crystalloidprotein complex and of the 2S albumin fraction commences 2 dafter imbibition and is completed within 2 d. This loss of proteinis accompanied by an increase in activity of three proteolyticenzymes, one carboxypeptidase and two -SH-dependent aminopeptidases.In contrast to the 11S and 2S protein fractions the lectins,located within the protein body, are mobilized only slowly andare present after the other proteins have been completely brokendown. Hence lectins may have a role other than as storage proteins. Key words: Castor bean, Protein breakdown, Storage protein, Lectin, Vacuolation, Seed germination     

Deterioration of Mitochondrial Membranes from Castor Bean Endosperm by Hydrogen Peroxide

Loss of membrane integrity by hydrogen peroxide (H₂O₂) was studiedin isolated mitochondria of castor bean. Incubation of mitochondriawith H₂O₂ resulted in the release of fumarase with a concomitantloss of phospho-lipids. Degradation of membrane was found tooccur independent of lipid peroxidation. H₂O₂-treated mitochondriawere able to degrade exogenous radiolabelled phospho-lipids. ³Corresponding author; fax 82-42-821-2391     

Phosphatidylcholine Synthesis in Castor Bean Endosperm : I. Metabolism of l-Serine

Three levels of free amines and the activities of their biosynthetic enzymes were measured in subcellular fractions of two cell lines of Nicotiana tabacum L. cv Xanthi. The TX4 cell line, a p-fluorophenylalanine resistant culture which accumulates high levels of cinnamoylamides, was compared to the wild-type culture TX1. In cells harvested on day 6 of the growth cycle, nearly all free putrescine, spermidine, and tyramine was found in the supernatant fraction of both cell lines. Although a consistent portion of ornithine decarboxylase activity was detected in the nuclear-enriched fractions of TX1 and TX4, the largest levels of activity were in the supernatants of both lines. In TX1, arginine decarboxylase activity was low relative to that of ornithine decarboxylase, but, in the TX4 line arginine decarboxylase levels in the cytosol were substantially elevated. Tyrosine decarboxylase was not detected in 6-day-old TX1 cells, but significant amounts of activity were measured in the 1000g and supernatant fractions of TX4. S-Adenosylmethionine decarboxylase activity was low in both cell lines and was located predominantly in the supernatant.