Soybean Protoplasts Research Articles

Monoclonal antibodies directed against protoplasts of soybean cells: analysis of the lateral mobility of plasma membrane-bound antibody MVS-1.

A monoclonal antibody (MVS-1) was used to monitor the lateral mobility of a defined component (Mr approximately 400,000) of the plasma membrane of soybean protoplasts prepared from suspension cultures of Glycine max (SB-1 cell line). The diffusion coefficient (D) of antibody MVS-1 bound to its target was determined (D = 3.2 X 10(-10) cm2/s) by fluorescence redistribution after photobleaching. Pretreatment of the protoplasts with soybean agglutinin (SBA) resulted in a 10-fold reduction of the lateral mobility of antibody MVS-1 (D = 4.1 X 10(-11) cm2/s). This lectin-induced modulation could be partially reversed by prior treatment of the protoplasts with either colchicine or cytochalasin B. When used together, these drugs completely reversed the modulation effect induced by SBA. These results have refined our previous analysis of the effect of SBA on receptor mobility to the level of a defined receptor and suggest that the binding of SBA to the plasma membrane results in alterations in the plasma membrane such that the lateral diffusion of other receptors is restricted. These effects are most likely mediated by the cytoskeletal components of the plant cell.

Cell-fractionation analysis of glucan synthase I and II distribution and polysaccharide secretion in soybean protoplasts

The organelles of soybean (Glycine max (L.) Merr.) protoplasts were separated using a recently developed procedure which allows rapid (3-h) recovery of a fraction enriched for coated vesicles (CVs). As determined by marker-enzyme enrichment and ultrastructural analysis of isolated membrane fractions, endoplasmic reticulum, Golgi membranes, glucan-synthase-II (EC 2.4.1.34)-containing membranes (putative plasma membrane), mitochondria, and CVs were enriched in separate fractions in a sucrose density gradient. Glucan synthase I (EC 2.4.1.12) had the highest specific activity in the Golgi-enriched and CV-enriched fractions and was found to comigrate with CVs upon rate-zonal centrifugation of a CV-enriched fraction. For further elucidation of the role of these latter organelles in cell-wall regeneration, freshly isolated protoplasts were pulsed with [(3)H]glucose for 20 min, and the disappearance of label from the organelles was followed for the ensuing 1 h. Although a CV-enriched fraction contained glucan synthase I, it contained very small amounts of labelled polysaccharide during the period of study. Pulse-chase experiments with [(3)H]glucose helped to confirm the role of the Golgi apparatus in secretion of matrix polysaccharides by protoplasts.

Phytoalexin Production by Isolated Soybean Protoplasts

Abstract Protoplasts isolated enzymatically from suspension-cultured cells of soybean (Glycine max L. Merr., cv. Harosoy 63) were used to study the production of the isoflavonoid-derived phytoale­xin, glyceollin. A large enhancement in the in vivo rates of synthesis and catalytic activities of two of the enzymes associated with glyceollin biosynthesis, phenylalanine ammonia-lyase and chalcone synthase, preceded phytoalexin accumulation during early stages of culture of isolated protoplasts while cell wall regeneration occurred. A glucan elicitor from cell walls of the fungus Phytophthora megasperm a f. sp. glycinea, an effective inducer of the phytoalexin response in cultured cells, was not capable of enhancing phytoalexin formation in protoplasts. Lack of respon­siveness of the protoplasts to the glucan elicitor could either be associated with their stressed metabolic state in which the response system is already saturated or with the removal from cultured cells of an essential factor of the glucan elicitor-mediated phytoalexin induction during protoplast isolation. At least two components of the protoplast isolation medium, the osmoticum and the fungal endopolygalacturonase, have the potential to initiate the observed phytoalexin synthesis during protoplast isolation. Our results indicate that under the methods employed isolated soybean protoplasts display a stress response which other types of soybean cells show following microbial attack or treatment with elicitor.

Lateral diffusion of phospholipids in the plasma membrane of soybean protoplasts: Evidence for membrane lipid domains

Fluorescent lipid and phospholipid probes were incorporated at 4 degrees C into soybean protoplasts prepared from cultured soybean (SB-1) cells. Fluorescence microscopy showed that the plasma membrane as well as the nucleus were labeled. Fluorescence redistribution after photobleaching (FRAP) analysis was performed on these cells at 18 degrees C to monitor the lateral mobility of the incorporated probes. After labeling at low concentrations (40 mug/ml) of phosphatidyl-N-(4-nitrobenzo-2-oxa-1,3-diazolyl)ethanolamine (NBD-PtdEtn), a single mobile component was observed with a diffusion coefficient (D) of approximately 3 x 10(-9) cm(2)/sec. After labeling at higher probe concentrations (>/=100 mug/ml), two diffusing species were observed, with diffusion coefficients of approximately 3 x 10(-9) cm(2)/sec ("fast") and approximately 5 x 10(-10) cm(2)/sec ("slow"). Similar results were observed with fluorescent derivatives of phosphatidylcholine and fatty acids. In contrast to these results, parallel analysis of 3T3 fibroblasts, using the same probes and conditions, yielded only a single diffusion component. These results suggest that the soybean plasma membrane may contain two distinct lipid domains in terms of lipid mobility. Consistent with this idea, experiments with soybean protoplasts yielded a single diffusion component under the following conditions: (i) labeling with NBD-PtdEtn (100 mug/ml), FRAP analysis at 37 degrees C (D = 1.1 x 10(-8) cm(2)/sec); (ii) labeling with NBD-PtdEtn (100 mug/ml), FRAP analysis at 18 degrees C in the presence of 2 mM EGTA (D = 4.2 x 10(-9) cm(2)/sec); (iii) labeling with 5-(N-dodecanoyl)aminofluorescein (a short-chain lipid probe), FRAP analysis at 18 degrees C or 37 degrees C (D = 2.5 x 10(-8) cm(2)/sec). These results suggest that the plasma membrane of soybean cells may contain stable immiscible domains of fluid and gel-like lipids.

The isolation of coated vesicles from protoplasts of soybean

Fractions enriched in coated vesicles were obtained from protoplasts derived from suspension cultured Glycine max (L.) Merr. cells. Initial enrichment was achieved by isopycnic centrifugation of a protoplast homogenate through a linear sucrose gradient in a vertical rotor. The coated-vesicle fractions from this gradient were pooled and centrifuged through a second linear sucrose gradient in a rate zonal fashion to remove the larger contaminating membrane vesicles. The most prominent polypeptide in the coated-vesicle fractions, plant "clathrin", had a relative molecular mass of approx. 190 kdalton as determined by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Other enriched polypeptides included bands at 105, 100, 96, 64, 50, 38 and 32 kdalton. This method was compared with a procedure utilizing sucrose step gradients for preparing coated vesicles from soybean protoplasts. The effectiveness of the isopycnic-rate zonal centrifugation procedure was also tested for the preparation of bovine-brain coated vesicles.

Endocytosis of cationized ferritin by coated vesicles of soybean protoplasts

Soybean (Glycine max (L.) Merr.) protoplasts have been surface-labelled with cationized ferritin, and the fate of the label has been followed ultrastructurally. Endocytosis of the label occurs via the coated-membrane system. The pathway followed by the label, once it has been taken into the interior of the protoplast, appears to be similar to that found during receptor-mediated endocytosis in animal cells. Cationized ferritin is first seen in coated vesicles but rapidly appears in smooth vesicles. Labelled, partially coated vesicles are occasionally observed, indicating that the smooth vesicles may have arisen by the uncoating of coated vesicles. Structures which eventually become labelled with cationized ferritin include multivesicular bodies, dictyosomes, large smooth vesicles, and a system of partially coated reticula.

Osmotic stress inhibits thymidine incorporation into soybean protoplast DNA

DNA synthesis in protoplasts isolated from soybean cell suspension cultures has been investigated by [(3)H] thymidine uptake and incorporation kinetics. Initial rates of incorporation in exponential and 5-fluorodeoxyuridine synchronized protoplasts are inhibited by increased osmolarities of the medium. The inhibition was not readily reversible during 3 h culture in low osmotic medium. Velocity sedimentation analyses of replicating DNA from such protoplasts shows a complex pattern of inhibition. The inhibition probably effects replicon initiation as well as strand elongation and ligation of replication intermediates.

Infection of Soybean Protoplasts from Cell Suspension Culture with Bean Pod Mottle Virus

SUMMARY Protoplasts were isolated enzymically from soybean (Glycine max L. ‘Harosoy 63’) callus continuously maintained as suspension cultures. The protoplasts were inoculated with bean pod mottle virus (BPMV) using a medium consisting of 0.4 m-sorbitol, virus, poly-l-ornithine (PLO), buffer and inorganic salts. A concentration of 0.5 × 105 to 1.0 × 105 protoplasts per ml was optimum for efficient virus infection. Although PLO was not essential for infection, it was stimulatory. A combination of PLO and CaCl2 had what appeared to be a synergistic effect in enhancing infection, especially at low virus concentrations. When virus was preincubated for 15 min with CaCl2 or MgCl2 prior to inoculation, both at 0.5 mm or with potassium phosphate buffer at 10 mm pH 5.6, infection of protoplasts was significantly increased over virus which had not been preincubated. The infection process was independent of temperature under the conditions tested. BPMV and the previously introduced cowpea mosaic virus, both comoviruses, showed some contrasting differences in requirements for PLO, pH optimum and temperature.

Cellulose and 1,3-glucan synthesis during the early stages of wall regeneration in soybean protoplasts

Protoplasts isolated from cultured soybean cells (Glycine max (L.) Merr., cv. Mandarin) were used to study polysaccharide biosynthesis during the initial stages of cell wall-regeneration. Within minutes after the protoplasts were transferred to a wall-regeneration medium containing [(14)C]glucose, radioactivity was detected in a product which was chemically characterized as cellulose. The onset and accumulation of radioactivity into cellulose coincided with the appearance fibrils on the surface of protoplasts, as seen under the electron microscope. At these early stages, a variety of polysaccharide-containing polymers other than cellulose were also synthesized. Under conditions where the protoplasts were competent to synthesize cellulose from glucose, uridine diphosphate-[(14)C]glucose and guanosine diphosphate-[(14)C]glucose did not serve as effective substrates for cellulose synthesis. However, substantial amounts of label from uridine diphosphate glucose were incorporated into 1,3-glucan.

イネ・ダイズのプロトプラストの培養および融合に関する研究(年次講演会要旨)

イネ・ダイズのプロトプラストの培養および融合に関する研究(年次講演会要旨)

Infection of Protoplasts from Soybean Cell Culture with Southern Bean Mosaic and Cowpea Mosaic Viruses

SUMMARY Soybean protoplasts, isolated from liquid suspension culture, were successfully infected with cowpea mosaic virus (CPMV) and with southern bean mosaic virus (SBMV). Poly-l-ornithine (PLO) was required for infection with either virus. As detected by fluorescent antibody, 70 to 90% of the protoplasts were infected by CPMV when the inoculation medium contained 0.4 m-sorbitol, 0.5 µg virus/ml, 1.5 µg PLO/ml, 10 mm-potassium phosphate buffer, pH 6.3, and 0.5 mm-CaCl2, and inoculation was performed at 23 °C. This maximum level of infection was decreased sixfold when CaCl2 was omitted. Inoculation at temperatures below 10 °C also decreased infection substantially. With SBMV a maximum of 30 to 35% of the protoplasts were infected when 0.4 m-sorbitol, 2 to 2.5 µg virus/ml, 2 µg PLO/ml, 10 mm-tris-HCl buffer, pH 8.0, 1 mm-MgSO4 and 0.5 mm-CaCl2 were present in the inoculum. Less than 5% of the protoplasts were infected when magnesium and calcium salts were omitted. The major advantage of the use of soybean cell suspension culture for plant virus studies is that it can provide a reliable and continuous source of cells for protoplast isolation. These soybean protoplasts allow synchronous infection and replication under sterile conditions without antibiotics and also a high degree of reproducibility.

DNA replication in soybean protoplasts and suspension-cultured cells: Comparison of exponential and fluorodeoxyuridine synchronized cultures

Cell-suspension cultures of soybean (Glycine max (L.) Merr., line SB-1) have been used to study DNA replication. Cells or protoplasts incorporate either radioactive thymidine or 5-bromodeoxyuridine (BUdR) into DNA. The DNA has been extracted as large molecules which can be visualized by autoradiography. Nuclei were isolated and lysed on slides thus avoiding degradation of DNA by a cytoplasmic endonuclease. The autoradiograms demonstrated that DNA synthesis occurs at several sites tandemly arranged on single DNA molecules separated by center to center distances ranging from 10 to 30 μm. Velocity sedimentations through alkaline gradients confirm the lengths of the replicated regions seen in autoradiograms. By using velocity sedimentation it also has been possible to demonstrate that replication proceeds by the synthesis of very small (4-6S) DNA intermediates which join to form the larger, replicon-size pieces seen in autoradiograms. Both small (4-6S) and large (20-30S) intermediates are observed in synchronized and exponential cultures. However, after synchronization with fluorodeoxyuridine (FUdR) the rate of DNA synthesis is reduced. Since the size of intermediates is not reduced by FUdR treatment, it is concluded that the slower rate of replication results from a reduction in the number of tandem replication units but not in the rate at which they are elongated. After FUdR treatment, the density analogue of thymidine, BUdR, can be substituted for almost all of the thymidine residue in DNA, resulting in a buoyant density increase (in CsCl) from 1.694 to 1.747 g/cm(3). Using this density analogue it is possible to estimate the amount of template DNA attached to new replication sites. When this is done, it can be shown that synchronized cells initiate replication at about 5,000 different sites at the beginning of S. (Each such site will replicate to an average length of 20 μm.) Use of BUdR also substantiates that at early stages of replication, very small replicated regions (<8S) exist which are separated by unreplicated segments of DNA which replicate at a later time. Most of these conclusions agree with the pattern of DNA replication established for animal cells. However, a major difference appears to be that after prolonged inhibition of soybean cell replication with FUdR, very small, as well as replicon-size intermediates accumulate when replication is restored. This indicates that regulation of replication in these cells may be different from animal cells.

The isolation of plasma membrane from protoplasts of soybean suspension cultures.

A procedure for the isolation of plasma membranes from protoplasts of suspension-cultured soybean is described. Protoplasts were prepared by enzymic digestion of the cell wall and the plasma membrane was labelled with radioactive diazotized sulphanilic acid. The membrane systems from broken protoplasts were separated by continuous isopycnic sucrose gradient centrifugation. Radioactivity was localized in a band possessing a buoyant density of 1-14 g ml-1. The activities of NADPH- and NADH-cytochrome c reductase, fumarase, Mg2+-ATPase, IDPase and acid phosphodiesterase in the various regions of the density gradient were determined. A plasma membrane fraction was selected which was relatively uncontaminated with membranes derived from endoplasmic reticulum, tonoplasts and mitochondria. The results indicated that Mg2+-ATPase and possibly acid phosphodiesterase were associated with the plasma membrane.

Chromosomal behaviour in somatic hybrids of soybean-Nicotiana glauca

Protoplasts of soybean and N. glauca were induced to fuse with polyethylene glycol (PEG 1540). Up to 39% of the protoplasts in the treated population were heterokaryocytes. When the heterokaryocytes were isolated and individually cultivated they divided indefinitely and each produced many millions of cells within 2–3 months.

Nitrogenase Activity in Protoplasts Isolated from Root Nodules of Glycine max

Root nodule protoplasts, isolated from soybeans under anaerobic conditions, showed nitrogenase activity. Alternatively, rhizobia present in the root nodules ( Rhizobium japonicum ATCC No 10324), kept under the same conditions in pure culture showed no nitrogenase activity. Attempts to culture agglutinated and fusion products of root nodule and normal soybean protoplasts failed due to contaminations of rhizobia set free from burst protoplasts.

Labelling of concanavalin A sites on the plasma membrane of soybean protoplasts

The plasma membranes of protoplasts from cultured soybean cells bind haemocyanin after treatment with concanavalin A. On unfixed protoplasts the binding sites were unevenly distributed. In thin section and in Pt/Pd replicas, the haemocyanin molecules were seen in clusters. After 16 hours incubation, the haemocyanin appeared in tighter clusters and newly synthesized cellulose microfibrils were present. In contrast, the protoplasts prefixed before concanavalin A treatment exhibited a very even distribution of haemocyanin. These observations are discussed in relation to membrane structure and the fluid mosaic model based on analogous studies of animal cells.

Cell Division of Intergeneric Protoplast Fusion Products

Protoplasts from soybean cell suspension cultures were fused with those isolated from leaves of two Pisum sativum varieties, four Nicotiana species and Colchicum autumnale. Fusion products of all combinations underwent cell division. The first division of fusion products occurred on day 2 or 3 of the culture period. Parental soybean protoplasts started to divide on day 1, leaf protoplasts on day 5 or later. The capacity to divide proved the protoplast combinations tested to be compatible when fused.

Electron-microscope observations of mitosis and cytokinesis in multinucleate protoplasts of soybean.

Multinucleate soybean protoplasts produced by spontaneous fusion during enzyme digestion of the cell wall initiated cell division after approximately 40 h in culture. The structure of these protoplasts during mitosis and cytokinesis was studied with both light and electron microscopes. Most nuclei did not fuse but divided synchronously. Interphase nuclei was commonly connected by short narrow nuclear bridges. At prophase and metaphase the nuclei appeared typical of those in most higher plants; technical difficulties prevented an adequate examination of protoplasts at anaphase. Telophase was characterized by cytokinesis involving phragmoplast and cell plate formation; however, complete partitioning of the cytoplasm by cell plants was not observed. Numerous coated vesicles were present near to or continuous with the cell plate and plasmalemma. The presence of a few dividing protoplasts with at least double the normal chromosome number suggests that some nuclear fusion occurred prior to mitosis. Very little cell wall material was detected at the margin of the dividing protoplasts.

The Effect of Canavanine on the Growth of Cells from Suspension Cultures and on Intergeneric Heterokaryocytes of Canavanine Sensitive and Tolerant Plants )

Through application of the polyethylene glycol method, protoplasts of canavanine sensitive soybean cell cultures were fused with canavanine tolerant protoplasts from alfalfa, Caragana, and sweet clover. Within several days of culture the fusion products, heterokaryocytes, divided and developed into small cell clusters. The heterokaryocytes exhibited a sensitivity to canavanine as strong as soybean protoplasts. The results indicate that protoplasts from cultured soybean cells and from mature leaves of alfalfa, Caragana,and sweet clover when fused do not generate an incompatibility which would prevent cell division. The reaction pattern of the heterokaryocytes appeared to be determined by the soybean partner.

The effects of ultra-violet irradiation on survival and on nucleic acids and protein synthesis in plant protoplasts

Protoplasts were subjected to increasing doses of ultra-violet irradiation to determine the effect on nucleic acid and protein synthesis, and also on survival and cell colony forming ability. The ability of soy bean protoplasts to form cell colonies was destroyed by exposure to u.v.-irradiation exceeding 192 ergs/mm 2 . However, as measured by vital staining, over 90 per cent of the protoplasts remained viable for at least 24 hr after exposure to u.v.-irradiation up to 1920 ergs/mm 2 . While DNA and RNA synthesis were sharply reduced by increasing u.v.-dose, protein synthesis was inhibited to a lesser extent.