Synthesis and deposition of zein in protein bodies of maize endosperm

The origin of protein bodies in maize (Zea mays L.) endosperm was investigated to determine whether they are formed as highly differentiated organelles or as protein deposits within the rough endoplasmic reticulum. Electron microscopy of developing maize endosperm cells showed that membranes surrounding protein bodies were continuous with rough endoplasmic reticulum membranes. Membranes of protein bodies and rough endoplasmic reticulum both contained cytochrome c reductase activity indicating a similarity between these membranes. Furthermore, the proportion of alcohol-soluble protein synthesized by polyribosomes isolated from protein body or rough endoplasmic reticulum membranes was similar, and the alcohol-soluble or -insoluble proteins showed identical [¹⁴C]leucine labeling. These results demonstrated that protein bodies form simply as deposits within the rough endoplasmic reticulum.

Messenger RNA that directed synthesis of only the smaller molecular weight zein subunit was separated from mRNA that synthesized both subunits by sucrose gradient centrifugation. This result demonstrated that separate but similar sized mRNAs synthesize the major zein components. In vitro translation products of purified mRNAs or polyribosomes were approximately 2,000 daltons larger than native zein proteins, suggesting that the proteins are synthesized as zein precursors. When intact rough endoplasmic reticulum was placed in the in vitro protein synthesis system, proteins corresponding in molecular weight to the native zein proteins were obtained.

     

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.     

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.     

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.     

Membrane lipid metabolism in germinating castor bean endosperm

Castor bean (Ricinus communis L. var. Hale) endosperms, excised after 2 days germination at 30 C, were incubated 5 min to 8 hr with ¹⁴C-acetate and ³H-glycerol. Homogenates were fractionated by sucrose gradient centrifugation. Organelles found to be active in lipid synthesis were the lipid bodies and the endoplasmic reticulum. The products of incorporation in the lipid bodies were ³H-diglycerides containing ¹⁴C-fatty acids of more than 20 carbons. In contrast, the endoplasmic reticulum produced ³H-phospholipids as well as ³H-diglycerides rich in ¹⁴C-linoleate. The phospholipids synthesized and their acyl contents were of the types known to be the major components of organelle membranes in this tissue. Phospholipids and diglycerides containing ¹⁴C and ³H were found in the glyoxysomes and mitochondria subsequent to their appearance in the endoplasmic reticulum. The results show that germinating castor bean endosperm synthesizes membrane lipids de novo from acetate rather than reutilizing stored lipid components directly. It is also apparent that the endoplasmic reticulum is responsible for several steps in membrane lipid production.     

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.     

Serological and developmental relationships between endoplasmic reticulum and glyoxysomal proteins of castor bean endosperm

Glyoxysomes isolated from the endosperm of castor bean (Ricinus communis L.) by sucrose density gradient centrifugation were fractionated into their matrix protein and membrane components. Antisera were raised in rabbits against both the matrix proteins and sodium dodecyl sulphate (SDS)-solubilized membrane proteins. SDS-polyacrylamide gel electrophoresis (PAGE) analysis established that such antisera precipitate all major polypeptide components present in their respective glyoxysomal mixedantigen preparations. Furthermore, when soluble constituents recovered from the microsomal vesicles or solubilized microsomal membranes were challenged with the appropriate glyoxysomal antiserum, serological determinants were again found to be present. Intact endosperm tissue was incubated with [³⁵S]methionine and the kinetics of ³⁵S-incorporation into protein recovered in immunoprecipitates when the glyoxysomal matrix fraction or the soluble fraction released from the microsomes were incubated with anti-glyoxysomal matrix serum were followed. [³⁵S]antigens rapidly appeared in the microsomal fraction whereas a lag period preceded their appearance in glyoxysomes. Interupting such kinetic experiments by the addition of an excess of unlabelled methionine resulted in a rapid decrease in the microsomal content of [³⁵S]antigens and a concomitant increase in glyoxysomal content.Abbreviations SDS sodium dodecyl sulphate - PAGE polyacrylamide gel electrophoresis - ER endoplasmic reticulum     

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.     

Characterization of a Membrane Fraction Containing a b-type Cytochrome

The various components obtained from etiolated corn (Zea mays L.) coleoptiles were fractionated by differential or sucrose gradient centrifugation. The endoplasmic reticulum, proplastids, Golgi, and mitochondria were localized by enzymic or other markers in the various fractions. A fifth fraction was also characterized. It contains glucan synthetase II activity, binding sites for N-naphthylphthalamic acid, NADH dehydrogenase activity which is both antimycin A- and cyanide-insensitive, and a b-type cytochrome. It is possible that this fraction is plasma membrane and that it may contain the blue-ultraviolet photoreceptor for phototropism in corn.     

Electron transport in glyoxysomal membranes

Glyoxysomes were isolated from germinating castor bean endosperm by equilibrium density gradient centrifugation in a vertical rotor. To recover the membranes, glyoxysome ghosts were prepared by osmotic shock and then subjected to differential centrifugation. The glyoxysomal membranes and the endoplasmic reticulum (ER), isolated by the same methods, were assayed for electron transport components. Both organelles contained NADH ferricyanide reductase, NADH cytochrome c reductase, and cytochromes b₅ and P-420. The ER also contained cytochrome P-450. Pyridine hemochrome derivatives of the organelle membranes and hemin produced coincident difference spectra, indicating that only b-type cytochromes are present in glyoxysomal and ER membranes. The maximal activities of ferricyanide reductase and cytochrome c reductase in glyoxysomes, 2.19 and 0.33 μmol min^?1 mg membrane protein^?1, respectively, represent 30 and 18% of the activities in the ER. The cytochrome b₅ content of the glyoxysomal membrane is 0.108 nmol mg^?1, 31% of the level found in ER. The reductases from both organelles were resistant to solubilization by salt (0.2 m KCl) and were easily solubilized by detergent (1% Triton X-100). Flavin analysis of the organelles from germinating castor beam endosperm confirmed spectral evidence that the flavin content of glyoxysomes is quite high, 100 pmol mg protein^?1, more than twice that of mitochondria. Three-quarters of the glyoxysomal flavin was solubilized by KCl, but even after salt treatment the glyoxysomal membrane flavin content, 98 pmol mg membrane protein^?1, is three times greater than that of the ER.     

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.     

Incorporation of D-[14C]galactose into organelle glycoprotein in castor bean endosperm

Excised casto bean (Ricinus communis L.) endosperm tissue supplied with [¹⁴C]galactose incorporates radioactivity into particulate cell components. Fractionation of homogenates established that ¹⁴C-labeled trichloroacetic acid-insoluble material was located primarily in the microsomal and glyoxysomal fractions. The capacity of the tissue to incorporate [¹⁴C]galactose into organelle glycoprotein varied during seedling development, increasing during the first 3 days of germination and subsequently declining. The kinetics of incorporation into the major organelle fractions of 2-day old endosperm tissue showed that the endoplasmic reticulum was immediately labeled whereas a lag period preceded the labeling of glyoxysomes. Sub-fractionation of the isolated organelles established that the greatest proportion of the [¹⁴C]-galactose labeled glycoprotein was located in the membrane, although a significant incorporation into the matrix protein was also observed.The results indicate that the addition of the carbohydrate moiety to the polypeptide cores occurs in the endoplasmic reticulum during or immediately after their synthesis on membrane-bound ribosomes.Abbreviations ER endoplasmic reticulum - SDS sodium dodecyl sulphate - TCA trichloroacetic acid     

Auxin-binding Sites of Maize Coleoptiles Are Localized on Membranes of the Endoplasmic Reticulum

Sites in maize (Zea mays L.) coleoptile homogenates that reversibly bind naphthalene-1-acetic acid with high affinity and may represent receptor sites for auxins are located primarily on cellular membranes that show the enzymic and buoyant density characteristics of membranes of the rough endoplasmic reticulum. The sites remain attached to the endoplasmic reticulum (ER) membranes after the ribosomes have been stripped off them. Binding sites for naphthylphthalamic acid, an inhibitor of auxin transport, are located on membranes different from those that carry the naphthalene-1-acetic-acid (NAA)-binding sites, and which are probably plasma membrane. The two kinds of binding sites can be largely separated by appropriate density gradient centrifugation. The results raise the possibility that primary auxin action occurs at ER membranes and could represent facilitation of the transfer of hydrogen ions and nascent secretory protein into the ER lumen followed by secretory transport of these products to the cell exterior via the Golgi system.     

Subcellular localization of mannosyl transferase and glycoprotein biosynthesis in castor bean endosperm

Differential and sucrose density gradient centrifugation have shown that the mannosyl transferase present in germinating castor bean endosperm cells which catalyses the synthesis of mannosyl-phosphoryl-polyisoprenol is exclusively located in the endoplasmic reticulum membrane. This intracellular location was confirmed using both ribosome-denuded microsomes isolated in the presence of EDTA and rough-surfaced microsomes isolated in the presence of excess Mg²⁺ added to maintain ribosome-membrane attachment. Separation of organelles following the incubation of crude particulate fractions with GDP[¹⁴C]mannose demonstrated that most of the mannolipid thus formed remained associated with the microsomal fraction. When organelles were isolated from intact tissue which had previously been incubated with GDP[¹⁴C]mannose, [¹⁴C]glycoprotein was found to be associated with other cellular fractions in addition to the microsomes, in particular the glyoxysomes. The kinetics of radioactive labelling of these organelles suggest that [¹⁴C]glycoprotein appears initially in the microsomal fraction and subsequently accumulates in the glyoxysomes. Subfractionation of isolated, [¹⁴C]glycoprotein-labelled glyoxysomes established that over 80% of the total radioactivity was present in the membrane, while sodium dodecyl sulphate-polyacrylamide gel electrophoresis of solubilized glyoxysomal membranes showed that the [¹⁴C]sugar moiety was associated with several, but not all, constituent polypeptides.Abbreviations ER endoplasmic reticulum - TCA trichloroacetic acid - SDS sodium dodecylsulphate - GDP guanosine diphosphate     

Isolation of Smooth Endoplasmic Reticulum and Tonoplast from Mung Bean Hypocotyls (Vigna radiata [L.] Wilczek) Using a Ficoli Gradient and Two-Polymer Phase Partition

A new method for the isolation of smooth endoplasmic reticulumand tonoplast from etiolated mung bean hypocotyls (Vigna radiata[L.] Wilczek) has been developed. After centrifugation in aFicoli density gradient [5.5% (w/w) in 15% (w/w) sucrose] ofa crude microsomal membrane fraction (10,000–156,000?gpellet) which had been prepared and resuspended in buffer systemsthat contained 0.25 M sorbitol, more than 80% of the total amountsof smooth endoplasmic reticulum and tonoplast were co-bandedat the interface between the sample load and the Ficoll layer,while most of the other cellular membranes, including plasmamembrane, Golgi membranes and yellow-colored membrane materials,which were presumably the etioplast envelopes, were sedimentedthrough the Ficoli layer. Smooth endoplasmic reticulum and tonoplastwere separated from each other to a high degree of enrichmentby a subsequent two-polymer phase partitioning. The separationis based on the principle that mung bean tonoplast has a highpartition coefficient for the polyethylene glycol-enriched upperphase and the smooth endoplasmic reticulum has a high partitioncoefficient for the Dextran-enriched lower phase. Assessed interms of degree of contamination by activities of membrane markerenzymes, the isolated smooth endoplasmic reticulum and tonoplastfractions were found to be highly purified. An ATPase sensitiveto neutral detergent and vanadate was found to be specificallyassociated with the smooth endoplasmic reticulum. ¹Contribution No. 3172 from the Institute of Low TemperatureScience (Received April 22, 1988; Accepted September 28, 1988)     

Sterol biosynthetic capability of purified membrane fractions from maize coleoptiles

Membrane fractions were isolated from etiolated maize coleoptiles by differential and sucrose density gradient centrifugation. Specific membrane components were identified by using marker enzyme activities. Fractions were also tested for α-naphthylacetic acid (NAA).and N-naphthylphthalamic acid (NPA) binding activities. Evidence is presented for the isolation of a plasma-membrane fraction containing specific binding sites for NPA, a high concentration of sterols, and most of the total UDP-glucose sterol glucosyltransferase activity. A fraction rich in endoplasmic reticulum is shown to contain most of the binding sites for NAA and all of the activity of both S-adeno-Syl-L-methionine-Δ24 cycloartenol methyltransferase and cycloeucalenol-obtusifoliol isomerase.     

Isolation and characterization of oat aleurone and starchy endosperm protein bodies

To compare oat (Avena sativa L. cv Froker) aleurone protein bodies with those of the starchy endosperm, methods were developed to isolate these tissues from mature seeds. Aleurone protoplasts were prepared by enzymic digestion and filtration of groat (caryopsis) slices, and starchy endosperm tissue was separated from the aleurone layer by squeezing slices of imbibed groats followed by filtration. Protein bodies were isolated from each tissue by sucrose density gradient centrifugation. Ultrastructure of the isolated protein bodies was not identical to that of the intact organelles, suggesting modification during isolation or fixation. Both aleurone and starchy endosperm protein bodies contained globulin and prolamin storage protein, but minor differences in the protein-banding pattern by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were evident. The amino acid compositions of the protein body fractions were similar and resembled that of oat globulin. The aleurone protein bodies contained phytic acid and protease activity, which were absent in starchy endosperm protein bodies.     

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.     

ENDOPLASMIC RETICULUM AS THE SITE OF LECITHIN FORMATION IN CASTOR BEAN ENDOSPERM

The properties of a discrete membranous fraction isolated on sucrose gradients from castor bean endosperm have been examined. This fraction was previously shown to be the exclusive site of phosphorylcholine-glyceride transferase. The distribution of NADPH-cytochrome c reductase and antimycin insensitive NADH-cytochrome c reductase across the gradient followed closely that of the phosphorylcholine-glyceride transferase. This fraction also had NADH diaphorase activity and contained cytochromes b₅ and P 450. On sucrose gradients containing 1 mM EDTA this fraction had a mean isopycnic density of 1.12 g/cm³ and sedimented separately from the ribosomes; electron micrographs showed that it was comprised of smooth membranes. When magnesium was included in the gradients to prevent the dissociation of membrane-bound ribosomes, the isopycnic density of the membrane fraction with its associated enzymes was increased to 1.16 g/cm³ and under these conditions the electron micrographs showed that the membranes had the typical appearance of rough endoplasmic reticulum. Together these data show that the endoplasmic reticulum is the exclusive site of lecithin formation in the castor bean endosperm and establish a central role for this cytoplasmic component in the biogenesis of cell membranes.     

Organelle membranes from germinating castor bean endosperm

Summary Endoplasmic reticulum, mitochondria, and glyoxysomes were obtained from germinating castor bean endosperm,Ricinus communis, by sucrose gradient centrifugation. When each of the three organelle preparations was diluted in 150 mM KCl and centrifuged, all of the component membrane material, measured as phospholipid, was sedimented. Also, the respective membrane enzymes, phosphorylcholine-glyceride transferase, cytochrome c oxidase and alkaline lipase were recovered. The endoplasmic reticulum retained most (60%) of its protein. The mitochondria lost almost no protein while the glyoxysomes lost much of their soluble contents.The isolated endoplasmic reticulum was in the form of vesicles, 0.02 to 1 m, lacking bound ribosomes. The size, 0.5 to 0.8 m, and the structure of the mitochondria were unchanged by the purification procedure. The mitochondria were contracted, whereas the glyoxysomes were distended. The diameter of the glyoxysomes remained 0.4 to 1.5 m, but they lost much of their internal matrix. The small amount of matrix that survived was not especially associated with the membrane. The glyoxysome membrane was about the same thickness as that of the endoplasmic reticulum, 70 Å.