Diethylene glycol disulfide from castor bean cell suspension cultures

Diethylene glycol disulfide was isolated from castor bean cell suspension cultures. Incubation of suspension cultures with Na₂³⁵SO₄ resulted in the incorporation of radioactivity into the isolated diethylene glycol disulfide. Diethylene glycol disulfide was detected in cells (430 nmol/g cells) and in cell-free growth medium (41.5 nmol/ml).     

Isolation of organelles from carrot cell suspension cultures

Summary Organelles isolated from carrot cell suspension cultures by density gradient centrifugation and identified by their specific marker enzymes were found at the following mean densities on the sucrose gradient: microbodies 1.25 g/cm³ (catalase), mitochondria 1.18 (fumarase), endoplasmic reticulum 1.09 g/cm³ (NADH-cytochrome c reductase). Further enzyme assays were done for characterization of microbodies from carrot cultures.This work was supported by the Deutsche Forschungsgemeinschaft.     

Characteristics of galacturonic Acid oligomers as elicitors of casbene synthetase activity in castor bean seedlings

The metabolism of [¹⁴CH₃]2-(2,4-dichlorophenoxy)isobutyric acid (DIB) was studied in plants and cell suspension cultures of Lycopersicon esculentum Mill. sp. `Lukullus'. Both plants and cells in suspension culture showed a rapid uptake of DIB from nutrient media. The metabolites, isolated by extraction with methanol and separated by chromatographic methods, were identified by enzymic, chemical, and spectrometric methods. Two conjugates of the carboxyl with 2 and 3 moles glucose per mole DIB and, to a smaller extent, its β-d-glucopyranosyl ester, were formed in both intact plants and cell suspension cultures, but there were quantitative differences.     

Isolation of intact plastids from protoplasts from castor bean endosperm

Protoplasts were prepared from castor bean (Ricinus communis) endosperm by treatment with a mixture of the commercial enzymes Macerozyme R-10 and Cellulose “Onozuka” R-10. The protoplasts were gently ruptured by forcing the suspension through a hypodermic needle and the homogenate centrifuged on a linear sucrose gradient. From such a homogenate the mitochondria are recovered at their typical isopycnic density of 1.18 g/ml, but the glyoxysomes are retained, with other membranes, at a density of 1.13. The plastids reach their typical density of 1.22 on the gradient and are thus clearly separated from other organelles. Moreover, since essentially all of the ribulose bisphosphate carboxylase activity on the gradient is present in this fraction it can be concluded that the plastids are intact and have been recovered in high yield.     

Elicitation of lignin biosynthesis and isoperoxidase activity by pectic fragments in suspension cultures of castor bean

Suspension cultures of castor bean (Ricinus communis L.) which have been treated with pectic fragment elicitor rapidly accumulate lignin as measured by derivatization with thioglycolic acid. The responsiveness of cultured cells to elicitor is dependent on the stage of culture growth. In 6-day (maximally responsive) cultures, increases in lignin are first evident 3 hours after addition of pectic fragment elicitor with maximal rates of lignin synthesis between 4 and 10 hours. The abundance of lignin in cultures after 12 hours of elicitor treatment is 10- to 20-fold higher than in untreated control cultures and can thereby account for as much as 2% of the dry cell weight. Only intermediate sizes of pectic oligomer are active as elicitors of lignin. Half-maximal accumulation of lignin occurs at 250 to 300 micrograms per milliliter of an optimal elicitor preparation with an average degree of polymerization of seven. We consider the synthesis of lignin in elicited cultures to be a mechanism of plant disease resistance which is induced by the elicitor. Plant peroxidases have been proposed to catalyze the last enzymatic steps in the biosynthesis of both lignin and hydrogen peroxide. Six extracellular isoenzymes of peroxidase (two anionic, designated A1 and A2, and four cationic, designated C2, C3, C4, and C7) are detectable in healthy suspension cultures of castor bean by native gel electrophoresis. Treatment of cultures with elicitor causes substantial changes in the activity of four of these species (A1, C2, C3, and C7). Elicitor treatment also results in the appearance of three new peroxidase isoenzymes that are not readily detectable in healthy cultures (C1, C5, and C6). Increases in the activities of these isoenzymes are concurrent with or slightly precede the accumulation of lignin in elicited 6-day cultures. By 12 hours after addition of elicitor, C1 becomes the most abundant extracellular isoperoxidase. The differential regulation of expression of peroxidase isoenzymes following elicitor treatment suggests that individual isoenzymes of peroxidase may have specific functional roles in the biosynthesis of disease-lignin.     

Regeneration from long-term cell suspension cultures of tepary bean (Phaseolus acutifolius)

Plant regeneration has been achieved from long-term cell suspension cultures established from leaf derived callus of tepary bean (Phaseolus acutifolius). The proportion of densely cytoplasmic cells in suspension culture increased when cultured in the L-6 medium with 54 M NAA and 2 M KN. Filtration of the cells at each of five consecutive subcultures resulted in the isolation of a plant regenerating cell line (TB 686), which is being maintained in L-6 medium with 4.5 M 2,4-D and 2.3 M zeatin. Differentiated green cell aggregates were obtained when cells from maintenance medium were transferred to the same medium with 10 M BA. Embryo-like structures developed from these aggregates on L-6 medium with 2.3 M zeatin, 0.69 M GA₃ and 1.5 M NAA. Plantlets regenerated from these structures when they were cultured on L-6 medium with 7.0 M NAA and 1.0 M KN. Plant regeneration from the cell line remained relatively constant for 270 days. Regenerated plants were grown to maturity in the greenhouse.Abbreviations BA Benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ Gibberellic acid - IPA Isopentenyladenine - KN Kinetin - NAA Naphthaleneacetic acid - AA Amino acid medium (Toriyama and Hinita, 1985) The research was sponsored by United States Agency for International Development, Washington D.C., Cooperative Agreement DAN-4137-A-00-4053-00     

Enucleation of protoplasts derived from suspension cultures of winged bean and from a crown gall cell line ofParthenocissus tricuspidata

Protoplasts derived from suspension cultures of the winged bean and a crown gall lineof Parthenocissus tricuspidata were enucleated by centrifugation on iso-osmotic discontinuous Percoli gradients and on discontinuous sucrose-mannitol density gradients. Enucleation was achieved according to the buoyant density of the protoplasts and preparations of both cytoplasts and miniprotoplasts were recovered. The Percoll gradients gave more satisfactory results than the sucrose-mannitol gradients. Enucleation was increased by raising the centrifugal force from 20 kg to 60 kg. The presence of nuclei was determined by the DNA specific probe 4′6-diamidino-2-phenylindole (DAPI). Membrane integrity, as measured by staining with fluorescein diacetate, showed that some 85 % of the cytoplasts were bounded by an intact plasma membrane.     

Solubilization, partial purification, and immunodetection of squalene synthetase from tobacco cell suspension cultures

Squalene synthetase, an integral membrane protein and the first committed enzyme for sterol biosynthesis, was solubilized and partially purified from tobacco (Nicotiana tabacum) cell suspension cultures. Tobacco microsomes were prepared and the enzyme was solubilized from the lipid bilayer using a two-step procedure. Microsomes were initially treated with concentrations of octyl-β-d-thioglucopyranoside and glycodeoxycholate below their critical micelle concentration, 4.5 and 1.1 millimolar, respectively, to remove loosely associated proteins. Complete solubilization of the squalene synthetase enzyme activity was achieved after a second treatment at detergent concentrations above or at their critical micelle concentration, 18 and 2.2 millimolar, respectively. The detergent-solubilized enzyme was further purified by a combination of ultrafiltration, gel permeation, and Fast Protein Liquid Chromatography anion exchange. A 60-fold purification and 20% recovery of the enzyme activity was achieved. The partially purified squalene synthetase protein was used to generate polyclonal antibodies from mice that efficiently inhibited synthetase activity in an in vitro assay. The apparent molecular mass of the squalene synthetase protein as determined by immunoblot analysis of the partially purified squalene synthetase protein separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 47 kilodaltons. The partially purified squalene synthetase activity was optimal at pH 6.0, exhibited a K_m for farnesyl diphosphate of 9.5 micromolar, and preferred NADPH as a reductant rather than NADH.     

Fatty Acid synthesis in endosperm of young castor bean seedlings

Enzyme assays on organelles isolated from the endosperm of germinating castor bean (Ricinus communis) by sucrose density gradient centrifugation showed that fatty acid synthesis from [¹⁴C]malonyl-CoA was localized exclusively in the plastids. The optimum pH was 7.7 and the products was mainly free palmitic and oleic acids. Both NADH and NADPH were required as reductants for maximum activity. Acetyl-CoA, and acyl-carrier protein from Escherichia coli increased the rate of fatty acid synthesis, while low O₂ levels suppressed synthesis. In the absence of NADPH or at low O₂ concentration, stearic acid became a major product at the expense of oleic acid. Fatty acid synthesis activity was highest during the first 3 days of germination, preceding the maximum development of mitochondria and glyoxysomes. It is proposed that the plastids are the source of fatty acids incorporated into the membranes of developing organelles.     

The procyanidins of Douglas fir seedlings,callus and cell suspension cultures derived from cotyledons

While cotyledons of Douglas fir seedlings contain only 2–3% of their dry weight as procyanidins (mainly in an insoluble form), callus cultures and cell suspension cultures derived from them contain up to 40%. About 70–85% of the procyanidins isolated from these cell suspension cultures are soluble in 70% methanol, but insoluble in ethyl acetate. They can be separated into a minimum of 4 fractions, all of which have apparent molecular weights greater than that of a tetramer. Dimers, trimers or tetramers are absent or present in only trace amounts, but large amounts of catechin, and lesser amounts of epicatechin, are found in the ethyl acetate-soluble fraction.     

Isolation and characterization of organelles from soybean suspension cultures

Whole homogenates from cells of Glycine max grown in suspension culture were centrifuged on linear sucrose gradients. Assays for marker enzymes showed that distinct peaks enriched in particular organelles were separated as follows: endoplasmic reticulum (density 1.10 g/cm³, NADH-cytochrome-c reductase), Golgi membranes (density 1.12 g/cm³, inosine diphosphatase), mitochondria (density 1.18—1.19 g/cm³, fumarase, cytochrome oxidase) and microbodies (density 1.21—1.23 g/cm³, catalase). In cells which had ceased to grow (stationary phase) only a single symmetrical catalase peak at density 1.23 g/cm³ was observed on the sucrose gradient. During the phase of cell division and expansion a minor particulate catalase component of lighter density was present; its possible significance is discussed.     

Isolation and characterization of a naturally occurring inhibitor from mung bean (Vigna radiata) seedlings for serine hydroxymethyltransferase.

A naturally occurring inhibitor of serine hydroxymethyltransferase (EC 2.1.2.1) in mung bean seedlings extracts was purified by ammonium sulphate precipitation, phenyl-Sepharose chromatography followed by heating to release the inhibitor bound to the protein. The inhibitor had an absorption maximum at 200 nm, was not precipitated by trichloroacetic acid, was dialysable and resistant to inactivation by heating at 98 degrees C for 4 hr, protease and ribonuclease digestion; but was acid labile. The chromatographically pure preparation inhibited both mung bean and sheep liver SHMT. Qualitative and quantitative analyses indicated that it contained a carbohydrate moiety, an O-amino and vicinal diol groups. Paper electrophoresis at pH 4.3 suggested that the inhibitor was positively charged.     

Isolation of protoplasts and vacuoles from cell suspension cultures of Coleus blumei Benth.

In order to study the accumulation and transport of rosmarinic acid in suspension cells of Coleus blumei we established an efficient method to isolate protoplasts and vacuoles. Protoplasts were disrupted by an osmotic shock in a medium with basic pH containing ethylenediamine tetraacetic acid. The resulting vacuoles were purified on a two-step Ficoll gradient. The comparison of the rosmarinic acid contents of cells, protoplasts and vacuoles showed that the depside is localized in the vacuole. Data concerning the yield and purity of the vacuoles are presented. In addition we show that at the physiological pH of the cytoplasm rosmarinic acid is present almost exclusively as an anion and cannot pass a membrane by simple diffusion. We therefore propose a carrier system for the transport of rosmarinic acid into the vacuole.Abbreviations EDTA ethylenediamine tetraacetic acid - HEPES 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethane sulfonic acid - HPLC high performance liquid chromatography - MES morpholinoethane sulfonic acid - NADP⁺ ß-nicotinamide adenine dinucleotide phosphate - PEG polyethylene glycol - RA rosmarinic acid - Tris Tris(hydroxymethyl)aminomethane     

Isolation and identification of a growth inhibitor from blue light-illuminated cress seedlings

A plant growth inhibitor was isolated from blue light-illuminated cressseedlings and identified as 4-hydroxy-2,3-dimethyl-2-nonen-4-olide from¹H and ¹³C NMR and ESI-MS spectra. It inhibited thehypocotyl growth of cress seedlings at concentrations higher than 100M.     

Effect of chilling temperatures on the activities of glyoxysomal and mitochondrial enzymes from castor bean seedlings

Castor bean (Ricinus communis L.) is included among the group of plants sensitive to chilling temperatures. Seedlings of this species were shown to exhibit visible symptoms of this injury, as well as impaired radicle growth and storage product mobilization. Respiration of intact seedlings and oxidation of succinate by isolated mitochondria displayed discontinuities in Arrhenius plots of their reaction velocities, characteristic of chilling species. However, gluconeogenic glyoxysomal enzymes do not display such discontinuities, indicating that there is probably no functional relationship between these enzymes and the glyoxysomal membrane.     

Glycoproteins in the matrix of glyoxysomes in endosperm of castor bean seedlings

The matrix of glyoxysomes from endosperm of castor bean (Ricinus communis cv Hale) seedlings has been analyzed for the presence of glycosylated proteins. Glyoxysome preparations were monitored for organelle homogeneity by electron microscopy and enzyme marker activities. Glyoxysomes were essentially free of endoplasmic reticulum, mitochondria, and protein bodies. At least eight glyoxysomal matrix glycopeptides ranging in size from 39 to 160 kilodaltons were identified by their affinity for concanavalin A. The glyoxysomal glycoproteins were shown to be radioactively labeled when endosperm was allowed to incorporate glucosamine. Incorporation of glucosamine was inhibited by tunicamycin under conditions which did not inhibit protein synthesis. Hydrolysis of glyoxysomal extracts and subsequent analysis by paper chromatography showed that the labeled precursor was incorporated into the glycoprotein without prior dispersion of the label into amino acids. The present data demonstrate the occurrence of N-linked, high mannose oligosaccharides on polypeptides of the glyoxysomal matrix. This finding is discussed in relation to pathways of protein maturation and transport during glyoxysomal biogenesis.     

Reduced protease activity in transformed rice cell suspension cultures expressing a proteinase inhibitor

In this study, we synthesized a synthetic serine proteinase inhibitor II gene (sPI-II) that harbored the chymotrypsin and trypsin inhibitor domains of the PI-II gene from Nicotiana alata. In an effort to reduce protease activity in a rice cell suspension culture, we first synthesized sPI-II using overlap PCR and then introduced the gene into a rice calli (Oryza sativa L. cv. Dongin) by particle bombardment-mediated transformation. The sPI-II gene was under the control of a rice alpha-amylase 3D promoter induced by sugar starvation. To verify the integration and expression of the sPI-II gene in the transformed rice cells, we employed genomic DNA PCR amplification and Northern blot analysis, respectively. The relative protease activity of the transformed cell suspension culture was reduced to approximately 23% when compared to the non-transformed culture. This indicates that a transformed suspension culture system expressing a proteinase inhibitor, may be a useful tool to protect against recombinant protein losses resulting from extracellular proteases.     

Synthesis and degradation of fructose 2,6-bisphosphate in endosperm of castor bean seedlings

The aim of this work was to examine the possibility that fructose 2,6-bisphosphate (Fru-2,6-P₂) plays a role in the regulation of gluconeogenesis from fat. Fru-2,6-P₂ is known to inhibit cytoplasmic fructose 1,6-bisphosphatase and stimulate pyrophosphate:fructose 6-phosphate phosphotransferase from the endosperm of seedlings of castor bean (Ricinus communis). Fru-2,6-P₂ was present throughout the seven-day period in amounts from 30 to 200 picomoles per endosperm. Inhibition of gluconeogenesis by anoxia or treatment with 3-mercaptopicolinic acid doubled the amount of Fru-2,6-P₂ in detached endosperm. The maximum activities of fructose 6-phosphate,2-kinase and fructose 2,6-bisphosphatase (enzymes that synthesize and degrade Fru-2,6-P₂, respectively) were sufficient to account for the highest observed rates of Fru-2,6-P₂ metabolism. Fructose 6-phosphate,2-kinase exhibited sigmoid kinetics with respect to fructose 6-phosphate. These kinetics became hyperbolic in the presence of inorganic phosphate, which also relieved a strong inhibition of the enzyme by 3-phosphoglycerate. Fructose 2,6-bisphosphatase was inhibited by both phosphate and fructose 6-phosphate, the products of the reaction. The properties of the two enzymes suggest that in vivo the amounts of fructose-6-phosphate, 3-phosphoglycerate, and phosphate could each contribute to the control of Fru-2,6-P₂ level. Variation in the level of Fru-2,6-P₂ in response to changes in the levels of these metabolites is considered to be important in regulating flux between fructose 1,6-bisphosphate and fructose 6-phosphate during germination.