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E M Herman

Publications and source records attributed to E M Herman.

32 records · Page 2Linked to original sources
PubMed1989

An abundant, highly conserved tonoplast protein in seeds.

We have isolated the membranes of the protein storage vacuoles (protein bodies) from Phaseolus vulgaris cotyledons and purified an integral membrane protein with M(r) 25,000 (TP 25). Antiserum to TP 25 recognizes an abundant polypeptide in the total cell extracts of many different seeds (monocots, dicots, and a gymnosperm), and specifically labels the vacuolar membranes of thin-sectioned soybean embryonic axes and cotyledons. TP 25 was not found in the starchy endosperm of barley and wheat or the seed coats of bean but was present in all seed parts examined that consist of living cells at seed maturity. The abundance of TP 25 was not correlated with the amount of storage protein in seed tissue, and the protein was not found in leaves that accumulate leaf storage protein. On the basis of its abundance, evolutionary conservation, and distribution in the plant, we propose that TP 25 may play a role in maintaining the integrity of the tonoplast during the dehydration/rehydration sequence of seeds.

Journal Article↗
PubMed1989

In vitro mutated phytohemagglutinin genes expressed in tobacco seeds: role of glycans in protein targeting and stability.

Phytohemagglutinin is a glycoprotein that accumulates in the protein storage vacuoles of bean seeds. The mature glycoprotein has a high-mannose and a complex glycan. We describe here the use of site-directed mutagenesis and expression of the mutated genes in transgenic tobacco to study the role of glycans in intracellular targeting. The reading frame for phytohemagglutinin-L was mutated so that either one or both of the glycosylation signals were disrupted to specifically prevent the attachment of asparagine-linked glycans. Expression of these genes with the beta-phaseolin promoter in the seeds of transgenic tobacco plants showed that phytohemagglutinin-L with only one glycan or without glycans was correctly targeted to the protein storage vacuoles of the seeds. Furthermore, the absence of either the complex glycan or the high-mannose glycan did not alter the processing of the other glycan. On the basis of these results, we propose that the targeting signal of this vacuolar protein is contained in its polypeptide domain and not in its glycans.

Amino Acid Sequence↗
PubMed1988

Bark and Leaf Lectins of Sophora japonica Are Sequestered in Protein-Storage Vacuoles.

The leguminous tree Sophora japonica contains a family of closely related, but distinct, lectins. Different members of this family are independently expressed in seeds, leaves, and bark (CN Hankins, J Kindinger, LM Shannon 1987 Plant Physiol 83: 825-829; 1988 Plant Physiol 86: 67-10). The inter-, and intracellular distribution of the bark and leaf lectins was studied by indirect postembedding immunogold electron microscopy. Aldehyde fixed bark and leaves postifixed with OsO(4) and embedded in LR White resin permitted sensitive and specific immunogold labeling while maintaining cellular ultrastructure. The leaf and bark tissue cells contain protein-filled storage vacuoles which occupy most the cell's interior volume. The leaf and bark vacuoles closely resemble the protein bodies, or protein storage vacuoles, of seed cotyledons. The leaf and bark lectins were found to be exclusively sequestered in the protein-storage vacuoles of these tissues.

Journal Article↗
PubMed1987

Synthesis and protein body deposition of maize 15-kd zein in transgenic tobacco seeds.

The maize 15-Kd zein structural gene was placed under the regulation of French bean beta-phaseolin gene flanking regions. Agrobacterium tumefaciens-mediated transformation was used to insert the chimeric phaseolin-zein gene into the tobacco genome. Transgenic plants synthesized zein in a tissue-specific manner during the latter half of seed development. Transcription of the chimeric gene was initiated in phaseolin-derived sequences, and was terminated within the phaseolin gene 3' flanking region. Both zein- and phaseolin-derived polyadenylation signals were used in the processing of zein RNA in transgenic plant seeds. Zein accumulation, though subject to an 80-fold variation among 19 plants tested, could reach as much as 1.6% of the total seed protein in several plants. In developing tobacco seeds, zein was correctly processed by the removal of a 20-amino-acid signal peptide. Electron microscope immunogold localization of the zein expressed in embryo and endosperm tissue indicates that the monocot protein accumulates in the crystalloid component of vacuolar protein bodies. The density of gold label over the protein bodies is several fold greater in the embryo than the endosperm. Zein is found in roots, hypocotyls and cotyledons of germinating transgenic tobacco seeds.

Journal Article↗
PubMed1985

Accumulation and Subcellular Localization of alpha-Galactosidase-Hemagglutinin in Developing Soybean Cotyledons.

We have investigated the accumulation and intracellular localization of soybean (Glycine max [L.] Merr. cv Forrest) alpha-galactosidase-hemagglutinin during seed development. Cotyledon tissue was embedded in Lowicryl K4M and immunocytochemical localization was accomplished through treating thin sections with alpha-galactosidase antisera followed by indirect labeling with protein A coupled to colloidal gold. Gold particles were localized on the Golgi apparatus and protein bodies. We interpret this to indicate that alpha-galactosidase-hemagglutinin is transferred to and transported through the Golgi apparatus and finally deposited within the protein body by a Golgi apparatus-mediated process.

Journal Article↗
PubMed1981

Uptake and apparent digestion of cytoplasmic organelles by protein bodies (protein storage vacuoles) in mung bean cotyledons.

The large protein bodies of the storage parenchyma cells of mung bean (Vigna radiata) cotyledons contain vesicles measuring 0.2 to 2.0 mum in diameter. The vesicles contain ribosomes, ribosomes, membranous elements which may be derived from the endoplasmic reticulum and occasionally Golgi bodies and mitochondria. The vesicles can be seen by transmission electron microscopy in thin sections of plastic embedded specimens and in replicas of freeze-fractured preparations. Serial sections show that the vesicles are completely separated from the protein body membrane and are not invaginations of that membrane. Vesicles with cytoplasmic structures are seen most frequently in 2 to 4 day old seedlings. The vesicles may be formed when undulations of the protein body membrane are so deep as to permit the pinching-off of a portion of the cytoplasm, resulting in its subsequent isolation from the cytoplasm within the protein body. The digestion of the storage protein in the protein body is accompanied by the disappearance of the ribosomes and the membranous elements in the vesicles. We interpret this disappearance of the cytoplasmic structures in the vesicles as being due to their digestion by the protein body hydrolases (ribonuclease, proteinase and lipolytic enzymes). The uptake of cytoplasmic structures by the protein bodies continues after the reverse proteins have been digested. Cytochemical staining shows that the protein bodies and especially the vesicles are rich in acid phosphatase, a known marker of lytic activity in cells. The evidence presented here indicates that the protein bodies are the intracellular sites at which the digestion of cytoplasmic structure occurs. Protein bodies should therefore be considered not only as compartments for the hydrolysis of the stored protein, but also as autophagic organelles involved in the degradation of cytoplasmic macromolecules. The term protein bodies is well established, but the term protein storage vacuoles may describe these organelles more precisely.

Cytoplasm↗
PubMed1980

Protein bodies of mung bean cotyledons as autophagic organelles.

We present evidence that protein bodies constitute the principal lytic compartment in storage parenchyma cells of mung bean cotyledons and propose that they play a role in cellular autophagy. We developed a method to isolate protein bodies by incubating tissue slices with cell wall-degrading enzymes and fractionating the cellular organelles on a Ficoll gradient. About 75-80% of the protein bodies present in the protoplasts were recovered intact in a band at the 5/25% Ficoll interface. This band contained a similar proportion of the cellular alpha-mannosidase, N-acetyl-beta-glucosaminidase, ribonuclease, acid phosphatase, phosphodiesterase, and phospholipase D. beta-Amylase was present in the cells but not in the protein bodies. Ultrastructural observations showed that on the 3rd day of seedling growth protein bodies contain small vesicles (0.3-1.0 mum) with a cytoplasmic content (ribosomes, membrane vesicles, mitochondria). Later in seedling growth these vesicles appeared empty. We believe that these are autophagic vesicles resulting from invaginations of the protein body membrane and that their cytoplasmic contents are digested by the acid hydrolases present in the protein bodies.

Journal Article↗
PubMed1980

Characteristics and subcellular localization of phospholipase d and phosphatidic Acid phosphatase in mung bean cotyledons.

Mixed micelles of (32)P-labeled phosphatidylcholine or phosphatidic acid (PA) and the nonionic detergent octylphenol polyethylene oxide (NP-40 Nonidet) were used to assay the activities of phospholipase D and PA phosphatase in crude extracts of mung bean (Vigna radiata) cotyledons. Together these enzymes degrade phosphatidylcholine to free choline, inorganic phosphate, and sn-1,2-diacylglycerol. Both enzymes have pH optima around 5.0. The enzymes are present in fully imbibed cotyledons and increase in activity during seedling growth. Fractionation of cotyledon extracts on sucrose gradients showed that the cells contain two PA phosphatases. One enzyme with a pH optimum of 7.5 has the same distribution on sucrose gradient as the endoplasmic reticulum marker enzyme NADH-cytochrome c reductase. The other, PA phosphatase, with a pH optimum of 5.0, was present in a protein body-rich fraction and in the load portion of the gradient. Fractionation of broken protoplasts on Ficoll gradients (a method which allows for the isolation of a high proportion of intact protein bodies) indicates that most of the cellular phospholipase D and PA phosphatase (pH 5.0) are associated with the protein bodies. Using column chromatography (DEAE-cellulose and Sephadex G-200), PA phosphatase (pH 5.0) was found to be a different enzyme from the major acid phosphatase in the cotyledons. Apparent molecular weights of phospholipase D and PA phosphatase were 150,000 and 37,000, respectively. The activity of phospholipase D was not affected by free choline, but was markedly inhibited by the choline analog and plant growth retardant isopropyl 4'-(trimethylammonium chloride-5'-methylphenyl piperidine-1-carboxylate (AMO 1618). The finding that these acid hydrolases are located in the protein bodies supports the conclusion that protein bodies form the general lytic compartment in the storage parenchyma cells.

Journal Article↗
PubMed1979

Rapid degradation and limited synthesis of phospholipids in the cotyledons of mung bean seedlings.

Seedling growth of mung bean is accompanied by the rapid catabolism of the three major phospholipids in the cotyledons (phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol). The decline starts 24 hours after the beginning of imbibition and by the 4th day of growth more than 50% of the phospholipids have been catabolized. Extracts of cotyledons of 24-hour-imbibed beans contain enzymes capable of degrading membrane-associated phospholipids in vitro. This degradation involves phospholipase D and phosphatase activity.Studies with radioactive acetate, glycerol, and orthophosphate indicate that the three major phospholipids are also synthesized in the cotyledons. Incorporation of glycerol and acetate into phospholipids of cotyledons is relatively constant throughout seedling growth, while the incorporation of [(32)P]orthophosphate steadily declines from a high value 24 hours after the start of imbibition. The newly synthesized phospholipids become associated with membranous organelles, especially the endoplasmic reticulum, and have an in situ half-life of 2 to 2.5 days.Determination of the activities of two enzymes involved in phospholipid biosynthesis (phosphorylcholine-glyceride transferase and CDP-diglyceride-inositol transferase) shows that the enzymes have their highest activities 12 hours after the start of imbibition. High activities for both enzymes were found in cotyledons of beans incubated at 1 C, indicating that the enzymes may preexist in the dry seeds.The experiments demonstrate that cotyledons start synthesizing new phospholipids immediately after imbibition, but that the rate of phospholipid catabolism far exceeds the rate of synthesis long before the cotyledons start to senesce.

Journal Article↗