Development of Microbodies in Sunflower Cotyledons and Castor Bean Endosperm during Germination

In cotyledons of sunflower seedlings glyoxysomal and peroxisomal enzymes exhibit different rates of development during germination. The total activity of isocitrate lyase, a glyoxysomal marker enzyme, rapidly increased during the first 3 days, and then decreased 89% by day 9. Exposure to light accelerated this decrease only slightly. The specific activity of glyoxysomal enzymes (malate synthetase, isocitrate lyase, citrate synthetase, and aconitase) in the microbody fraction from sucrose density gradients increased between days 2 and 4 about 2- to 3-fold, and thereafter it remained about constant in light or darkness.     

Proteases and Peptidases of Castor Bean Endosperm: Enzyme Characterization and Changes during Germination

The endosperm of castor bean seeds (Ricinus communis L.) contains two —SH-dependent aminopeptidases, one hydrolyzing l-leucine-β-naphthylamide optimally at pH 7.0, and the other hydrolyzing l-proline-β-naphthylamide optimally at pH 7.5. After germination the endosperm contains in addition an —SH-dependent hemoglobin protease, a serine-dependent carboxypeptidase, and at least two —SH-dependent enzymes hydrolyzing the model substrate α-N-benzoyl-dl-arginine-β-naphthylamide (BANA). The carboxypeptidase is active on a variety of N-carbobenzoxy dipeptides, especially N-carbobenzoxy-L-phenylalanine-l-alanine and N-carbobenzoxy-l-tyrosine-l-leucine. The pH optima for the protease, carboxypeptidase, and BANAase acivities are 3.5 to 4.0, 5.0 to 5.5, and 6 to 8, respectively.     

Development of Endoplasmic Reticulum and Glyoxysomal Membrane Redox Activities during Castor Bean Germination

Redox activities, NADH:ferricyanide reductase, NAD(P)H:cytochrome reductases, and NADH:ascorbate free-radical reductase, are present in endoplasmic reticulum (ER) and glyoxysomal membranes from the endosperm of germinating castor bean (Ricinus comminus L. var Hale). The development of these functions was followed in glyoxysomes and ER isolated on sucrose gradients from castor bean endosperm daily from 0 through 6 days of germination. On a per seed basis, glyoxysomal and ER protein, glyoxysomal and ER membrane redox enzyme activities, and glyoxylate cycle activities peaked at day 4 as did the ER membrane content of cytochrome P-450. NADH:ferricyanide reductase was present in glyoxysomes and ER isolated from dry seed. This activity increased only about twofold in glyoxysomes and threefold in ER during germination relative to the amount of protein in the respective fractions. The other reductases, NADH:cytochrome reductase and NADH:ascorbate free-radical reductase, increased about 10-fold in the ER relative to protein up to 4 to 5 days, then declined. NADPH:cytochrome reductase reached maximum activity relative to protein at day 2 in both organelles. The increases in redox activities during germination indicate that the membranes of the ER and glyoxysome are being enriched with redox proteins during their development. The development of redox functions in glyoxysomes was found to be coordinated with development of the glyoxylate cycle.     

Hydrolysis of Crystalloid Storage Protein in Castor Bean Seed Endosperm During and Following Germination

Hydrolysis of the insoluble crystalloid storage proteins ofcastor bean endosperm during germination released buffer-solublepolypeptides with molecular weights in the presence of sodiumdodecyl sulphate of 30000–40000. These polypeptides appearto be dimers since the addition of 2-mercaptoethanol decreasestheir molecular weights to 15000–22000. Hydrolysis ofthe crystalloid proteins was detected 12–18 h after seedimbibition (HAI), which is before the completion of germination;maximum rates were attained at 30 HAI. During this period, parallelincreases in free amino acids were observed. Hydrolysis of thecrystalloid proteins during early germination was insensitiveto cycloheximide treatment and therefore did not require newlysynthesized proteases. Hydrolysis was effected by proteaseswhich were made in an inactive form during seed developmentand activated upon seed imbibition. Key words: Castor bean, crystalloid storage protein hydrolysis, seed germination, endosperm     

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.     

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.     

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     

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.     

Evidence for No Proteolytic Processing during Transport of Isocitrate Lyase into Glyoxysomes in Castor Bean Endosperm

The amino-terminal sequence of isocitrate lyase purified fromcastor bean endosperm glyoxysomes was compared with that deducedfrom the nucleotide sequence of cDNA for the enzyme [Plant Mol.Biol. (1987) 8: 471]. The isolated active enzyme lacked sixamino acid residues in the amino terminus, although the enzymeimmunoselected from a tissue homogenate with trichloroaceticacid had the amino-terminal part. Thus the six amino acid residuesseem to be eliminated during enzyme purification and the enzymeis transported into glyoxysomes without proteolytic processing. (Received August 31, 1987; Accepted November 30, 1987)     

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.     

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     

Multiple Forms of Phospholipase D following Germination and during Leaf Development of Castor Bean

Multiple molecular forms of phospholipase D (PLD; EC 3.1.4.4) were identified and partially characterized in endosperm of germinated seeds and leaves of castor bean (Ricinus communis L. var Hale). The different PLD forms were resolved by nondenaturing polyacrylamide gel electrophoresis, isoelectric focusing, and size-exclusion chromatography. PLD was detected with both a PLD activity assay and immunoblots with PLD-specific antibodies. There were three major forms of PLD, designated types 1, 2, and 3, based on their mobility during nondenaturing polyacrylamide gel electrophoresis. Molecular masses of the PLD variants were estimated at 330, 230, and 270 kD for the types 1, 2, and 3, respectively. Isoelectric points of the native type 1, 2, and 3 PLDs were approximately 6.2, 4.9, and 4.8. Under the in vitro assay conditions used, the three forms of PLD exhibited the same substrate specificity, hydrolyzing phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylglycerol (PG) but not phosphatidylserine (PS) and phosphatidylinositol (PI). The three forms of PLD differed in their substrate preferences, and the order of activities was: PLD 1, PE > PG = PC; PLD 2, PE > PG > PC; PLD 3, PE = PG = PC. The Km values of PLDs 1, 2, and 3 for PC were 1.92, 2.62, and 5.18 mM, respectively. These PLDs were expressed differentially following seed germination and during leaf development. Type 1 was found in the early stages of seedling growth and in young leaves, type 2 was present in all the tissues and growth stages examined, and type 3 was expressed in senescent tissues. The PLDs shifted from largely cytosolic to predominantly membrane-associated forms during leaf development. The present studies demonstrate the structural heterogeneity of plant PLD and growth stage-specific expression of different molecular forms. The possible role for the occurrence of multiple molecular forms of PLD in cellular metabolism is discussed.     

Acid Lipase of Castor Bean Lipid Bodies : Isolation and Characterisation

The acid lipase of castor endosperm lipid bodies has been studied using colorimetric assay based on the measure of the hydrolytic activity of p-nitrophenyl ester of palmitate and other acyl derivatives. These substrates are compatible with the natural triacylglycerols for the measure of lipolytic activities. The subcellularly-surveyed acid lipolytic activity in the germinated castor bean endospermal tissue was found to be enhanced in the lipid bodies. The lipase, which is partially latent and tightly associated with lipid bodies, is an exceptionally stable enzyme with an optimum activity at pH 4.5 and displays an inverse relationship between its activity and the acyl chain length of its substrate. To facilitate isolation of the acid lipase, a procedure has been developed to solubilise the membrane-bound enzyme in an active form. The detergent-solubilised acid lipase after two chromatographic steps yielded an eight-fold active preparation which after gel permeation resolved as heterogeneous aggregate in excess of 500 kD. Lipase-enriched preparations showed consistent presence of 14 and 60 kD proteins which constituted the most abundant species of the lipid bodies. Although it has not been possible to obtain an active lipase preparation in a state free of either the 14 or 60 kD protein, the lipase activity in the detergent extracts of lipid bodies was immunoprecipitable with antibodies raised against the 60 kD component.     

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.     

Phosphatidylcholine Synthesis in Castor Bean Endosperm : Occurrence of an S-Adenosyl-l-Methionine:Ethanolamine N-Methyltransferase

The methylation steps in the biosynthesis of phosphatidylcholine by castor bean (Ricinus communis L.) endosperm have been studied by pulse-chase labeling. Endosperm halves were incubated with [methyl-¹⁴C]S-adenosyl-l-methionine, [2-¹⁴C]ethanolamine, [¹⁴C]ethanolamine phosphate, or [¹⁴C]serine phosphate. The kinetics of appearance were followed in the free, phospho-, and phosphatidyl-bases. The initial methylation utilized ethanolamine as a substrate to form methylethanolamine, which was then converted to dimethylethanolamine, choline, and phosphomethylethanolamine. Subsequent methylations occurred at the phospho-base and, to a lesser extent, the phosphatidyl-base levels, after which the radioactivity either remained constant or decreased in these compounds and accumulated in phosphatidylcholine. Although the precursors tested did support the synthesis of choline, the kinetics of the labeling make them unlikely to be the major sources of free choline to be utilized for the nucleotide pathway. A model with two pools of choline is proposed, and the implications of these results for the pathways leading to phosphatidylcholine biosynthesis are discussed.     

Biosynthesis of Cytidine 5'-Diphosphate-diacylglycerol in Endoplasmic Reticulum and Mitochondria of Castor Bean Endosperm

Cytidine 5′-triphosphate (CTP):phosphatidate cytidyltransferase from the endoplasmic reticulum and mitochondria of Ricinus communis L. var Hale was characterized. The endoplasmic reticulum enzyme has a pH optimum of 6.5 and a divalent cation is required, Mn²⁺ being preferred and giving maximum activity at 2.5 millimolar. The estimated K_m for CTP is 16.7 micromolar, but that for phosphatidate could not be determined accurately. The activity was inhibited by both deoxycholate and Triton X-100 at concentrations as low as 0.01% (w/w).

The mitochondrial enzyme has a pH optimum of 6.0 and a divalent cation requirement similar to that of the endoplasmic reticulum. Maximum stimulation of the reaction by substrates occurred with 1.5 millimolar phosphatidate (from egg phosphatidylcholine) and about 400 micromolar CTP. The apparent K_m for phosphatidate could not be estimated accurately since activity was obtained in the absence of added lipid, apparently utilizing endogenous substrate. The K_m estimated for CTP was altered by the presence of the detergent Triton X-100; in its absence the value was 33.3 micromolar, but in its presence the value was 66.7 micromolar. Inclusion of 0.6% (w/w) Triton X-100 in the assay mixture stimulated the activity about 2.5-fold.

     

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.     

Spherosomes of Castor Bean Endosperm: MEMBRANE COMPONENTS, FORMATION, AND DEGRADATION

The membrane components of the castor bean spherosomes were characterized. The storage triacylglycerols of isolated spherosomes were extracted with diethyl ether, and the membrane was isolated by sucrose gradient centrifugation. It had an apparent equilibrium density of 1.12 grams per cubic centimeter, and possessed an antimycin A-insensitive NADH cytochrome c reductase and an acid lipase. Phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol in roughly equal amounts were the major phospholipids. The membrane proteins were resolved into several major and minor protein bands of molecular weights ranging from 10,000 to 70,000 by acrylamide gel electrophoresis, and the protein pattern in the gel was different from those of the endoplasmic reticulum, mitochondrial, and glyoxysomal membranes.