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PubMed1984

Auxin Conjugation by Tobacco Mesophyll Protoplasts : Correlations between Auxin Cytotoxicity under Low Density Growth Conditions and Induction of Conjugation Processes at High Density.

Auxin induction of the proliferation of Nicotiana tabacum (cv Xanthi) mesophyll protoplasts and of protoplast-derived cells was studied. The growth-promoting properties and cytotoxicities at high concentrations of IAA and naphthaleneacetic acid were strongly affected by cell density. The induction of growth by 2,4-dichlorophenoxyacetic acid and picloram was not affected by cell density. The comparison of catabolism of these [(14)C]-labeled auxins by protoplasts showed that IAA and naphthalene-acetic acid were rapidly accumulated and conjugated unlike 2,4-dichlorophenoxyacetic acid and picloram. The major catabolite derived from naphthaleneacetic acid was identified as naphthaleneacetyl-l-aspartate. The biosynthesis of this conjugate in protoplasts was inducible by naphthaleneacetic acid concentrations found to be cytotoxic under low density growth conditions. However, although it was taken up by cells, the conjugate was not cytotoxic at concentrations as high as 0.2 mm under low density growth conditions. The relationship between conjugation processes and auxin cytotoxicity is discussed.

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PubMed1984

Determination of compartmented metabolite pools by a combination of rapid fractionation of oat mesophyll protoplasts and enzymic cycling.

In vivo pool sizes of a range of metabolites have been determined in subcellular fractions of darkened and illuminated mesophyll protoplasts of Avena sativa L. These estimations were made by combining a method of rapid protoplast fractionation with enzymic cycling techniques. Results are given for reduced and oxidized pyridine nucleotides, triose phosphates, 3-phosphoglycerate, inorganic phosphate, aspartate, malate, oxaloacetate, glutamate, 2-oxoglutarate, and citrate, from chloroplasts, mitochondria, and a fraction representing the remainder of the protoplast. The results indicate distinct differences of compartmented levels of certain metabolites between darkened and illuminated protoplasts.

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PubMed1984

Auxin-induced regulation of protein synthesis in tobacco mesophyll protoplasts cultivated in vitro: I. Characteristics of auxin-sensitive proteins.

The presence of auxin (2,4-D), in the culture medium of tobacco (Nicotiana tabacum var Maryland) mesophyll protoplasts is necessary both for cell wall regeneration and for passage of the cells from phase G(0) to phase G(1) of the cell cycle. Among about 250 proteins synthesized by protoplasts and characterized by their migration in a two-dimensional electrophoresis gel, 2,4-dichlorophenoxyacetic acid affects the synthesis of 11.Nine proteins are synthesized at a reduced level in the presence of the hormone, of which three are rapidly labeled and short-lived, while the others, which are long-lived, become detectable only after 2 hours of radioactive labeling, suggesting that they undergo slow posttranslational maturation. These nine proteins are proline-rich but the proline radicals are not strongly hydroxylated. The synthesis of these proteins is no longer inhibited by auxin if dichlorobenzonitril, a weed-killer which inhibits cell wall reformation of tobacco protoplasts, is added to the culture medium.Two proteins are only synthesized if protoplasts are cultivated in an auxin-containing medium. These polypeptides are rapidly labeled, and are long-lived. The inhibition of cell wall reformation by dichlorobenzonitril does not modify their synthesis.These results suggest that proteins whose synthesis is reduced by auxin are related to cell wall reformation and that they do not play a role in the induction of the cell cycle. In contrast, proteins whose synthesis is stimulated in the presence of auxin are good candidates for a role in the induction of the cell cycle.

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PubMed1984

Subcellular localization of spermidine synthase in the protoplasts of chinese cabbage leaves.

Previous studies on the presence of spermidine synthase (EC 2.5.1.16) in the protoplasts of Chinese cabbage (Brassica pekinensis var Pak Choy) leaves had detected a small but significant fraction of the enzyme in a crude chloroplast fraction (Cohen, Balint, Sindhu 1981 Plant Physiol 68: 1150-1155). To establish whether this enzyme is truly a chloroplast component, we have isolated purified intact chloroplasts from protoplasts by density gradient centrifugation in silica sols (Ludox AM). Such chloroplasts contained all of the diaminopimelate decarboxylase (EC 4.1.1.20) of the protoplasts, but were essentially devoid of spermidine synthase. Control experiments showed that the latter had not been inactivated under conditions of isolation, purification, and assay of the intact chloroplasts. Isolation and assay of protoplast vacuoles in a further examination of the supernatant fluid containing the enzyme revealed a significant fraction of the enzyme in the vacuole fraction. However this fraction was found to contain similar proportions of a soluble enzyme, glucose 6-phosphate dehydrogenase. It has been concluded that vacuolar fractions are difficultly separable from soluble cytoplasmic material, which is probably the only compartment containing spermidine synthase.

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PubMed1985

Dicyclohexylamine-induced shift of biosynthesis from spermidine to spermine in plant protoplasts.

An improved analytical method, based on high pressure liquid chromatography, has been developed for the simultaneous determination of the polyamines and S-adenosyl-containing compounds in extracts of plant protoplasts. The method involves simple procedures for sample preparation and permits quantification of 1 picomole or less for all the compounds. This method has been used to study the effects of dicyclohexylamine, an inhibitor of plant spermidine synthase (Sindhu, R. K., S. S. Cohen 1984 Plant Physiol 74: 645-649), on biosynthesis of polyamines and 1-aminocyclopropane-1-carboxylate in protoplasts derived from Chinese cabbage leaves. Dicyclohexylamine effectively inhibits spermidine synthase in vivo. Inhibition of the synthesis of spermidine by dicyclohexylamine resulted in a stimulation of spermine synthesis, without significant effect on the synthesis of 1-aminocyclopropane-1-carboxylate. Decarboxylated S-adenosylmethionine is present in control Chinese cabbage protoplasts at approximately 10(-18) moles per cell, and dicyclohexylamine caused an increase of this metabolite of up to 10-fold in a 4-hour period. The increase in decarboxylated S-adenosylmethionine permitted an increased synthesis of spermine. These findings suggest that the availability of decarboxylated S-adenosylmethionine may be rate-limiting for the synthesis of spermine in plant protoplasts.

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PubMed1986

Assay of photosynthetic oxygen evolution from single protoplasts.

A semiquantitative assay for light-dependent O(2) evolution by a single mesophyll protoplast is described. The assay indicator is the density of aerotactic bacteria (Pseudomonas aeruginosa, ATCC 10145; ;Engelmann experiment') attracted to the protoplast. Quantification is by dark field microphotometry. The sensitivity is about 50 femtomoles O(2) per protoplast per minute. The results demonstrate the biphasic nature of O(2) evolution of a single protoplast during photosynthetic induction. Computerized data acquisition yields traces which, until a steady state of photosynthetic O(2) evolution is reached, are identical to ordinary O(2) electrode traces.

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PubMed1986

Potassium transport in suspension culture cells and protoplasts of carrot.

The properties of potassium transport in carrot (Daucus carota L.) suspension culture cells and their isolated protoplasts were examined. Cells cultured in Murashige and Skoog (MS) medium (Plant Physiol 15: 473-497) were potassium saturated and, consequently, they exhibited little net potassium accumulation. Cells that transport and accumulate potassium were derived from the MS-grown cells by culturing them in a potassium-free modified medium. The transport properties of the modified medium cells included: (a) smooth nonsaturating kinetics with 80% of the maximum rates occurring at 0.1 millimolar KCl, (b) linear transport for at least 75 min, (c) alkaline pH optimum, (d) little accompanying anion uptake with increased malate concentrations balancing net increases in positive charge, and (3) little effect on transport by plasmolysis. Potassium transport activity appeared to be 50% lower in protoplasts isolated from the modified medium cells. Nevertheless, the protoplasts exhibited essentially the same kinetics, time course, pH response, and malate adjustment as the intact cells. We concluded from these results that the low potassium cells and their isolated protoplasts are ideally suited to investigating potassium transport at the cell level without the complications associated with multilayered and highly differentiated tissues.

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PubMed1987

Replication of cauliflower mosaic virus DNA in leaves and suspension culture protoplasts of cotton.

Cauliflower mosaic virus (CaMV) replicated in protoplasts and in inoculated leaves of the non-host, cotton (Gossypium hirsutum, L.). Protoplasts prepared from suspension-cultured cotton cells were infected by incubation with liposome-encapsulated CaMV virions. During a 1-week culture period the amount of CaMV nucleic acid as detected by nucleic acid hybridization in the protoplasts increased significantly regardless of whether or not the protoplasts contained vacuoles. In leaves inoculated with CaMV virions or CaMV DNA, viral DNA sequences were found by leaf skeleton hybridization to be located in small circular areas. DNA extracted from ultracentrifugal pellets of homogenates of inoculated leaves contained circular, gapped CaMV DNA only when inocula contained CaMV virions, CaMV DNA, or partial nested dimer CaMV plasmid DNA. When plants had been heavily watered, the CaMV DNA recovered contained degraded CaMV DNA. The results suggest that the host range limitation for CaMV is not due to an inability to replicate or spread locally in inoculated leaves.

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PubMed1987

Chilling sensitivity of cucumber cotyledon protoplasts and seedlings.

Cucumber (Cucumis sativus L.) seedlings are more sensitive to chilling stress when transferred to low temperature from the night cycle than from the day cycle. However, greater damage occurs when chilling is carried out in light than in dark. Freshly isolated protoplasts are extremely sensitive to damage when chilled at 4 degrees C in light, but suffer significantly less injury when chilled in dark. If freshly isolated protoplasts are pre-chill conditioned at 27 degrees C in either light or dark for a few hours prior to exposure to various chilling stresses, subsequent chilling damage is markedly reduced. Damage to chilled protoplasts also is reduced if cultures are placed in dark instead of light immediately following removal from low temperature. Experiments utilizing the cell wall synthesis inhibitor, dichlorobenzonitrile, showed that cell wall regeneration during the pre-chill conditioning period at 27 degrees C does not appear to be associated with the enhanced chilling tolerance observed in these cultures. The results obtained in this investigation suggest that the physiological properties of cucumber cotyledon protoplasts accurately reflect those of intact seedlings, and hence provide a good system for studies into the mechanism of chilling damage in plants.

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PubMed1987

Factors affecting the activation state and the level of total activity of ribulose bisphosphate carboxylase in tobacco protoplasts.

The relationship between the activation state and the level of total activatable activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) was examined in tobacco protoplasts. When darkened protoplasts were illuminated, both activation and total activity increased, but at different rates; the t((1/2)) were 2.3 and 6.7 minutes, respectively. The light response of rubisco activation state and total activity, measured after 15 minutes of illumination, were similar but their responses to light transitions and photosynthetic inhibitors were different. When irradiance was reduced from saturating to subsaturating, deactivation of rubisco in protoplasts was immediate, whereas there was little change in total activity during the first 20 minutes following the transition. The light-induced increases in activation state and total activity were inhibited by nigericin, but activation was more sensitive exhibiting a response similar to that of photosynthesis. Treatment of tobacco protoplasts and leaves with methyl viologen at limiting irradiance increased rubisco activation, but inhibited the light-induced increase in total activity. These results indicate that light activation of rubisco is mechanistically distinct from the light-dependent changes in total activity in tobacco, a species containing carboxyarabinitol 1-phosphate, an endogenous inhibitor of total rubisco activity.

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PubMed1988

Plant Plasma Membrane Proteins : II. Biotinylation of Daucus Carota Protoplasts and Detection of Plasma Membrane Polypeptides after Sds-Page.

The ability of two biotinylating reagents, sulfosuccinimidobiotin and sulfosuccinimidyl 2-(biotinamido)ethyl-1,3'-dithiopropionate, to label plasma membrane proteins was examined. These compounds form covalent bonds with the free amino groups of proteins and label the proteins with biotin. Biotinylated proteins can be detected with avidin-peroxidase staining. Protoplasts isolated from embryogenic Daucus carota suspension cells were labeled with biotin and the membranes were separated on linear sucrose gradients. The conditions used for labeling the protoplasts did not cause protoplast rupture or loss of viability. The distribution of the biotin label in these linear sucrose gradients was analyzed and compared to the distribution of vanadate-sensitive ATPase activity, a marker for the plasma membrane. Both the biotin label and the vanadate-sensitive ATPase activity were strongly localized in the gradient at peak density of 1.16 gram per cubic centimeter. When the protoplast surface was labeled, biotinylated polypeptides were detected after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and polypeptides of 153, 94, 51, 30, 20, 17, and 14 kilodaltons were shown to be plasma membrane in origin. When a crude membrane pellet was labeled, numerous biotinylated polypeptides were distributed throughout the gradient. Because the position of the biotin label in the gradient is strongly correlated with the distribution of vanadate-sensitive ATPase, it is concluded that these biotinylating reagents are effective and reliable labels for proteins of the plant plasma membrane. Furthermore, these labels permit the positive identification of plasma membrane proteins after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and can serve as convenient markers for solubilization and purification of these proteins.

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PubMed1990

A polyethylene glycol-mediated protoplast transformation system for production of fertile transgenic rice plants.

We have established an efficient procedure for protoplast transformation and regeneration of fertile transgenic plants of rice (Oryza sativa L.) cultivars Nipponbare and Taipei 309. Protoplasts were mixed with a plant-expressible hygromycin resistance gene and treated with 25% (w/v) polyethylene glycol. Stringent selection of transformed colonies was applied to 14-day-old regenerated protoplasts in the presence of 95 micromolar of hygromycin B for 12 days. After selection, 450 and 200 resistant colonies were recovered per million treated Taipei 309 and Nipponbare protoplasts, respectively. Southern hybridization analysis of hygromycin-resistant cell lines and regenerated plants indicated that 1 to 10 copies of transferred DNA were integrated at 1 to 4 loci of the rice genome. Southern DNA analysis suggests that the introduced plasmid DNA may form concatemers by intermolecular recombination prior to integration. Four Taipei 309 and 39 Nipponbare transgenic rice plants were regenerated and grown to maturity in the greenhouse. Two Taipei 309 and 35 Nipponbare plants set viable seeds. Agronomic traits of Taipei 309 transgenic plants and inheritance of the hygromycin resistance trait by progeny of the selfed transgenic plants were analyzed.

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PubMed1991

On the Role of Mitochondrial Oxidative Phosphorylation in Photosynthesis Metabolism as Studied by the Effect of Oligomycin on Photosynthesis in Protoplasts and Leaves of Barley (Hordeum vulgare).

Low concentrations of oligomycin, which strongly inhibit mitochondrial oxidative phosphorylation but do not affect chloroplast photophosphorylation, caused an inhibition of photosynthesis by 30 to 40% in barley (Hordeum vulgare L.) leaf protoplasts. This inhibition is reversed and the full rate of photosynthesis is regained when the protoplasts are ruptured so as to leave the chloroplasts intact. Oligomycin fed into barley leaves by the transpiration stream inhibited photosynthesis in these leaves by up to 60%. The measurement of metabolites in protoplast and leaf extracts showed that oligomycin caused a decrease in the ATP/ADP ratio and an increase in the content of glucose- and fructose 6-phosphate. Subcellular analysis of protoplasts revealed that the decrease in ATP/ADP ratio in the cytosol was larger than in the stroma and that the increase in hexose monophosphates was restricted to the cytosol, whereas the stromal hexosemonophosphates decreased upon the addition of oligomycin. Moreover, oligomycin caused an increase in the triosephosphate-3-phosphoglycerate ratio. It is concluded from these results that during photosynthesis of a plant leaf cell mitochondrial oxidative phosphorylation contributes to the ATP supply of the cell and prevents overreduction of the chloroplast redox carriers by oxidizing reductive equivalents generated by photosynthetic electron transport.

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PubMed1983

Formation, Fusion, and Regeneration of Protoplasts from Wild-Type and Auxotrophic Strains of the White Rot Basidiomycete Phanerochaete chrysosporium.

A preparation of two commercial enzymes was used to liberate protoplasts from 16-h-old mycelium of Phanerochaete chrysosporium. Regeneration frequencies of up to 5% were attained when the protoplasts were plated in a medium containing 10% sorbose and 3% agar. Fusion of protoplasts from different auxotrophic strains in polyethylene glycol-Ca produced heterokaryons. Separation of the heterokaryons into their constituent homokaryotic strains could be effected through protoplast release and formation of colonies on regeneration agar.

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PubMed1984

Protoplast Fusion of Trichoderma reesei, Using Immature Conidia.

Protoplast fusion of strains derived from Trichoderma reesei QM9414 and QM9136 and the segregation of the resulting fusants were studied. Combinations of protoplasts prepared from young conidia with double amino acid requirements, one of which was a common requirement and the other uncommon, were fused in the presence of polyethylene glycol 6000. Fusants were selected as regenerant colonies requiring only the commonly deficient amino acid. The frequency of fusion was 0.9 x 10 to 4.0 x 10 for the starting conidia and 3.0 x 10 to 4.9 x 10 for the regenerated protoplasts, which was significantly higher than the expected reversion frequencies by mutation. Conidia generated on the fusant colonies showed diverse phenotypes, i.e., parental types (40 to 80%) and nonparental types (20 to 60%). Colonies developed from single conidia of the nonparental phenotype contained special spots called "knobs" that have a higher density of mycelia. The phenotype of the knobs was again varied among prototrophs, parental types, and recombinant types; and their traits were inherited stably. The phenotype of the mycelia in the nonknob part was essentially the same as that of the original conidia and again formed knobs in colonies upon transfer of a piece of mycelia to a fresh medium. The conidial DNA content of the knob clone was almost the same as that of the parents, but that of the fusants was 1.2 to 2.0 times higher than that of the parents. From these results, we conclude that knobs are the segregants from the fusants. One knob clone showed twice the carboxymethyl cellulose hydrolyzing activity of the parents, suggesting the possibility of breeding T. reesei cells by the protoplast fusion technique.

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PubMed1986

Mutagenesis of Protoplasts and Regeneration of Mycelium in the Mushroom Volvariella volvacea.

Regenerating protoplasts were obtained from mycelial culture of the mushroom Volvariella volvacea by the action of the lytic enzyme Novozym 234 in the presence of 0.01 M phosphate buffer (pH 6.0) containing 0.6 M NaCl. Regeneration was found to be poor in liquid medium, but more than 50% regeneration was achieved on solid 2% agar medium overlaid with 0.5% agar. Protoplasts of V. volvacea were found to be highly sensitive to the killing action of both UV irradiation and N-methyl-N'-nitro-N-nitrosoguanidine. However, no morphological or auxotrophic mutants could be obtained from protoplasts by chemical mutagenesis. Four types of morphological mutants and one auxotrophic (adenine-negative) mutant were obtained from UV-irradiated protoplasts. The adenine-negative mutant of V. volvacea was found to be stable, not losing auxotrophy on repeated subculture.

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PubMed1987

Formation and regeneration of protoplasts of the actinorhizal nitrogen-fixing actinomycete frankia.

Procedures for forming and regenerating protoplasts of four Frankia strains are described. Cells obtained from growth medium containing 0.1% glycine were digested with lysozyme (250 mug/ml) in a medium containing 0.5 M sucrose, 5.0 mM CaCl(2), and 5.0 mM MgCl(2). Protoplasts were formed during 15 to 120 min of digestion at 25 degrees C. Optimum conditions for protoplast regeneration involved placing protoplasts on a layer of complex growth medium containing 0.3 M sucrose, 5.0 mM CaCl(2), and 5.0 mM MgCl(2) which was overlaid with a layer of 0.8% low-melting-point agarose containing 0.5 M sucrose, 5.0 mM MgCl(2), and 5.0 mM CaCl(2). The maximum regeneration efficiency was 36.9% for strain CpI1, 1.3% for strain ACN1, 27% for strain EAN1pec, and 20% for strain EuI1c.

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PubMed1981

Transformation of Bacillus thuringiensis protoplasts by plasmid deoxyribonucleic acid.

A method has been developed to transform plasmid deoxyribonucleic acid into protoplasts of the insect pathogen Bacillus thuringiensis. Protoplasts were formed by treatment of cells with lysozyme. The efficiency of formation of protoplasts was affected by the strain, the media, and the cell density. Deoxyribonucleic acid uptake was induced by polyethylene glycol. Deoxyribonucleic acid from the Staphylococcus aureus plasmid pC194 was used for transformation. Although this plasmid could not be isolated as a stable extrachromosomal element, its chloramphenicol resistance was transferred to the recipient protoplasts. This was confirmed by assay for the enzyme chloramphenicol acetyltransferase, which confers resistance to chloramphenicol. This suggested that pC194 acts as an insertion element in B. thuringiensis.

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