Glycine Max Suspension Cultures Research Articles

Intracellular location of NADP+-linked malic enzyme in C3 plants

The aim of this work was to determine the maximum catalytic activity and intracellular location of NADP(+)-linked malic enzyme (EC 1.1.1.40) in C3 plants. Appreciable activities, ranging from 80 to 712 nmol · (gFW)(-1) · min(-1), were found in a wide range of tissues (roots and leaves of Pisum sativum L., cotyledons of Cucurbit a pepo Alef., developing seeds of Brassica napus L., mesocarp of Persea americana Gaertn., and suspension cultures of Glycine max L.). Overall, activity showed a rough positive correlation with biosynthesis. Differential and density-gradient fractionation of extracts of the cotyledons of germinating marrow (C. pepo) and lysates of protoplasts of suspension cultures of G. showed that the enzyme had the same distribution as the plastid marker enzymes. It is suggested that in C3 plants NADP(+)-linked malic enzyme is confined to the plastids and involved in biosynthesis.

Partial purification and immunological characterization of 1,3-?-glucan synthase from suspension cells of Glycine max

The plasma-membrane-localized 1,3-β-glucan synthase (EC 2.4.1.34) from suspension cultures of Glycine max (L.) Merr. was greatly enriched by a three-step purification procedure. Starting with a microsomal preparation, a six- to eightfold enrichment of the enzyme was achieved by isolating plasma-membrane vesicles in a polyethyleneglycol/dextran two-phase system. The enzyme was solubilized with the nonionic detergent digitonin and further purified 12-fold by successive centrifugations on two linear sucrose density gradients. The most purified enzyme preparation showed enrichment in a 31-kilodalton (kDa) polypeptide and was used to raise polyspecific antibodies which precipitated 1,3-β-glucan synthase activity. These antibodies were purified by affinity chromatography against immobilized membrane protein fractions of lower molecular weight which were devoid of 1,3-β-glucan synthase activity. The purified antibodies specifically labelled a single polypeptide of 31 kDa in the 1,3-β-glucan-synthase-containing heavy fractions of the first sucrose gradient indicating that this polypeptide represents part of the active enzyme complex.

Relationship between Pyrophosphate : Fructose-6-phosphate 1-phosphotransferase, Sucrose Breakdown, and Respiration

Summary The aim of this work was to discover the role of pyrophosphate : fructose-6-phosphate 1-phosphotransferase [PFK(PP;)]. Substrates and enzymes of sucrose breakdown were measured during starch accumulation by the endosperm of wild type and the sh 1 shrunken mutant of Zea mays L. The results suggested that the mutant depended primarily on alkaline invertase to break down sucrose, and showed that the activities of PFK(PPi) and UDPglucose pyrophosphorylase and contents of pyrophosphate and fructose2, 6-bisphosphate (Fru-2,6-P 2 ) were no lower in the mutant than in the wild type. Appreciable activities of PFK(PPi), which were not responsive to Fru-2,6-P 2 , were found in the following red algae, which lack sucrose: Audouninella purpurea, Polysiphonia sp., Mastocarpus stellatus, Rhodymenia pseudopalmata. No PFK(PP;) was found in the bacterium Paracoccus denitrificans. Addition of sucrose to starved suspension cultures of Glycine max L. led to increases in oxygen uptake and Fru-2,6-P2 content. Glucose produced the same effects. It is argued that, although one of the functions of PFK(PP;) is production of pyrophosphate for sucrose breakdown via sucrose synthase, the key role of the enzyme is the maintenance of the cytosolic concentration of pyrophosphate according to the supply and demand of the latter.

Purification and properties of S-adenosyl- l-methionine:Nicotinic acid- N-methyltransferase from cell suspension cultures of Glycine max L.

A soluble enzyme which catalyzes the transfer of the methyl group from S-adenosyl- l-methionine to the nitrogen atom of pyridine-3-car☐ylic acid (nicotinic acid) could be detected in protein preparations from heterotrophic cell suspension cultures of soybean ( Glycine max L.). Enzyme activity was enriched nearly 100-fold by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography to study kinetic properties. S-Adenosyl- l-methionine:nicotinic acid- N-methyltransferase (EC 2.1.1.7) showed a pH optimum at pH 8.0 and a temperature optimum between 35 and 40 °C. The apparent K M values were determined to be 78 μ m for nicotinic acid and 55 μ m for the cosubstrate. S-Adenosyl- l-homocysteine was a competitive inhibitor of the methyl-transferase with a K I value of 95 μ m. The native enzyme had a molecular mass of about 90 kDa. The catalytic activity was inhibited by reagents blocking SH groups, whereas other divalent cations did not significantly influence the enzyme reaction. The purified methyltransferase revealed a remarkable specificity for nicotinic acid. No other pyridine derivative was a suitable methyl group acceptor. To study a potential methyltransferase activity with nicotinamide as substrate, an additional purification step was necessary to remove nicotinamide amidohydrolase activity from the enzyme preparation. This was achieved by affinity chromatography on S-adenosyl- l-homocysteine-Sepharose thus leading to a 580-fold purified enzyme which showed no methyltransferase activity toward nicotinamide as substrate.

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.

Alkaline inorganic pyrophosphatase and starch synthesis in amyloplasts

The aim of this work was to see if amyloplasts contained inorganic pyrophosphatase. Alkaline pyrophosphatase activity, largely dependant upon MgCl2 but not affected by 100 μM ammonium molybdate or 60-100 mM KCl, was demonstrated in exracts of developing and mature clubs of the spadix of Arum maculatum L. and of suspension cultures of Glycine max L., but not in extracts of the developing bulb of Allium cepa L. The maximum catalytic activity of alkaline pyrophosphatase in the above tissues showed a positive correlation with starch synthesis, and in the first two tissues was shown to exceed the activity of ADPglucose pyrophosphorylase. Of the alkaline pyrophosphatase activity in lysates of protoplasts of suspension cultures of Glycine max, 57% was latent. Density-gradient centrifugation of these lysates showed a close correlation between the distribution of alkaline pyrophosphatase and the plastid marker, nitrite reductase. It is suggested that much, if not all, of the alkaline pyrophosphatase in suspension cultures of Glycine max is located in the plastids.

Nucleotide sugars and starch synthesis in spadix of Arum maculatum and suspension cultures of Glycine max

The aim of this work was to determine the relative contributions of ADPglucose and UDPglucose to starch synthesis in two non-photosynthetic tissues, the developing club of the spadix of Arum maculatum and suspension cultures of Glycine max. Rates of starch accumulation during growth are compared with estimates of the maximum catalytic activities in vitro of ADPglucose starch synthase, ADPglucose pyrophosphorylase, UDPglucose pyrophosphorylase and UDPglucose starch synthase. The latter could only be measured at high concentrations (10–30 mM) of UDPglucose. Clubs of Arum and cells of Glycine contained 292 and 6.8 nmol UDPglucose per gram fresh weight, respectively. The corresponding figures for ADPglucose were 29 and 0.4. From the above data it is argued that in both Arum club and Glycine cells the activity of UDPglucose starch synthase is too low to make any quantitatively significant contribution to starch synthesis. The activities of ADPglucose starch synthase and pyrophosphorylase were high enough to mediate the observed rates of starch accumulation. It is suggested that starch synthesis in these tissues is via ADPglucose.

Labelling of carbohydrate by [ 14C]glycerol supplied to suspension cultures of soybean

Suspension cultures of Glycine max were incubated for 4, 12 and 24 hr in [U- 14C]glycerol in 0.2 M potassium dihydrogen phosphate, in [U- 14C

Plant cell suspension cultures as model systems for investigating growth regulating compounds

Several plant growth regulators were investigated for their activity in cell suspension cultures of Glycine max, Gossypium hirsutum and Zea mays. The effect on the growth of the cell cultures was traced by means of cell counting and determining packed cell volume and turbidity of the suspensions. The growth retardant 5-(4-chlorophenyl)-3,4,5,9,10-pentaaza-tetracyclo-5,4,10(2,6) ,0(8,11)-dodeca-3,9-diene (NDA) and, to a slightly lesser extent, ancymidol proved to be the compounds with the greatest inhibitory action on cell division growth of all three cell cultures. In the case of cotton this effect was accompanied by increased synthesis and secretion of cell-wall material. Staining methods showed that, especially in the case of NDA, a high percentage of cells could be considered as viable, and showed thus that NDA inhibits the cell division process while the cells remain metabolically active. The effects of 1,1-Dimethyl-piperidiniumchloride (DPC), a genuine growth retardant of cell propagation, and, with less efficiency, N-trimethyl-(β-chloroethyl)-ammoniumchloride (CCC) in cotton, the triazole LAB 117 682 in soybean and maize, and, to a lesser extent, (2-isopropyl-5-methyl-4-trimethyl-ammoniumchloride)-phenyl-l-piperidiniumcarboxylate (AM0-1618) in soybean can be regarded as species-specific. Otherwise, CCC and particularly daminozide exhibited no action at the concentrations used. A comparison of the data from hydroculture studies with soybean and maize seedlings showed considerable agreement with the effectiveness of the substances in the corresponding cell cultures. Thus, cell cultures can be used to identify and screen substances with growth-influencing activity, and may also offer new ways to elucidate the mode of action of plant growth regulators.

Dynamics of Galactolipids and Plastids in Nonphotosynthetic Cells of Glycine max Suspension Cultures

The age-dependent interrelationship of galactolipids and plastids in heterotrophic cell suspension cultures of Glycine max (soybean) was studied with regard to aging of nonphotosynthetic cells. Cells were propagated in the dark and under illumination with white light, and were harvested at days 7 (end of logarithmic phase), 14, and 21 (extended stationary phase). Electron microscopy revealed in dark-grown cells a proliferating decay of the amyloplast-type plastids, which could be correlated to a decrease of galactolipids. This trend was dramatically reversed in irradiated cultures, where the plastids of day 21 cells appeared rejuvenated. A concomitant increase of galactolipid content in the cells was observed, yet chlorophyll synthesis and photosynthetic activity were not induced. The dynamics of galactolipid contents did not correlate with total lipid contents in dark-grown as well as in irradiated cultures. [(3)H]Galactose served as a radioactive probe for the subcellular localization of galactolipids by electron microscopic autoradiography. Apart from plastids, galactolipids may also be constituents of the plasma membrane. The results render the heterotrophic cell suspension culture a suitable model to study the impact of senescence on plastids of nonphotosynthetic cells.

Biotransformation of 3-Methyl-2-buten-1-ol and 3-Methyl-3-buten-1-ol by the Suspension Cultures of Glycine max

The biotransformation of 3-methyl-2-buten-1-ol and 3-methyl-3-buten-1-ol by the suspension cultures of Glycinc max (soy bean) under various conditions has been investigated. 3-Methyl-2-buten-1-ol was biotransformed into 3-methyl-2-butenal by the cultures at a high conversion ratio, but not into 3-methyl-3-buten-1-ol, while 3-methyl-3-buten-1-ol was biotransformed, although at a low conversion ratio, into 3-methyl-2-buten-1-ol and 3-methyl-2-butenal. More than 99% of the substances detected by GLC were found in the medium, and only a trace amount was found in the callus. The effects of auxins, light, shaking, and the amount of callus inoculum have been investigated. Throughout these experiments, no monoterpene formation was observed.

Stimulation of fatty acid biosynthesis in glycine max suspension cultures by acyl carrier protein

Fatty acid synthesis in disrupted proplatids of Glycine max (soybean) cell suspension cultures is stimulated by E. coli acyl carrier protein (ACP). From a cell free homogenate of G. max cultures the fatty acid synthetase was precipitated by ammonium sulfate fractionation between 30–70% saturation. In this fraction, fatty acid synthesis, as determined by incorporation of malonylCoA into fatty acids, was enhanced by the addition of E. coli ACP and, to a lesser degree, by an acid precipitate obtained from the supernatant of the G. max cell free homogenate saturated with 70% ammonium sulfate.

The Occurrence of Phenylalanine Ammonia-Lyase and Cinnamic Acid p-Hydroxylase on the Endoplasmic Reticulum of Cell Suspension Cultures of Glycine max

Abstract 1. The time course of activity of soluble and microsomal phenylalanine ammonia-lyase (PAL) was studied in dark grown cell cultures of soybean (Glycine max). A distinct activity increase of PAL in the soluble and microsomal fraction occurred prior to the stationary phase of the cell culture. Cinnamic acid p-hydroxylase and NADH : cytochrome c reductase, too, exhibited maximal activity in the log phase, 5 days after the transfer of soybean cells to fresh culture medium. 2. Upon subfractionation of the once washed microsomal fraction by sedimentation velocity centrifugation on a sucrose gradient, membranes of the endoplasmic reticulum could be separated from fractions containing mainly membranes from the Golgi apparatus or plasma membranes, respectively. PAL and cinnamic acid p-hydroxylase were found in fractions of endoplasmic reticulum whereas no activity of either enzymes could be detected in fractions containing Golgi apparatus or plasma membranes. 3. Repeated washing of microsomal fractions led to a residual membrane-bound PAL representing about 1% of the total PAL activity of the cells. This residual membrane-bound activity could be solubilized almost completely by Triton X-100 or digitonin at concentrations of 0.5 - 5%.

Localisation and characterization of the fatty acid synthesizing system in cells of Glycine max (soybean) suspension cultures

In course of a study of fatty acid synthetase in higher plants, non-green cell suspension cultures of Glycine max (soybean) served as model tissues. For the first time, a fatty acid synthesizing system was characterized in cell cultures of higher plants and was found to be solely located in proplastids of the cells. Optimum activity of the fatty acid synthesizing system in proplastids was observed between pH 8.0 and 8.2; with [1- 14C]acetate as substrate, cofactors required were CoA, ATP, Mn 2+, Mg 2+, HCO 3 −, NADH and NADPH. The system was more sensitive towards NADH than NADHP. [1- 14C]Acetate, [2- 14C]-malonate and [3- 14C]pyruvate served as precursors for fatty acids, indicating the presence of pyruvate dehydrogenase activity in proplastids. In disrupted proplastids, [2- 14C]malonylCoA was a better precursor than [1- 14C]acetylCoA. After incubation of proplastids with [2- 14C]malonate, a small shift, from palmitic acid to higher homologs, of label incorporated was observed, as compared to incorporation of label from [1- 14C]acetate and [3- 14C]pyruvate. Under the conditions of the experiment, only small amounts of polyunsaturated fatty acids, the main fatty acid components of this organelle, were synthesized. In respect to fatty acid synthesis, the non-green cell suspension culture resembles photosynthetic leaf tissue.

Cell-cycle parameters of soybean (Glycine max L.) cells growing in suspension culture: Suitability of the system for genetic studies

Suspension cultures of Glycine max (L.) Merr. were grown at 22 and 33°. The doubling times of dividing cells were 35 and 25 h, respectively. G2 was 6.2 and 6.7 h, and S was 13.8 and 6.5 h. G1 was calculated as 13 and 10 h, respectively. These values were determined by labeling cells with (3)H thymidine and measuring the appearance of radioactive mitotic figures. Treatment with 5-fluorodeoxyuridine (FudR) inhibited DNA synthesis and, as a result, cells accumulated in S. Such cells were viable and, upon removal of the FudR, proceeded synchronously into mitosis. Treatment with 5-bromodeoxyuridine, following FudR synchronization, sensitized the cells to white light. Thus cells capable of synthesizing DNA could be killed. 20-30% of the cells in suspension cultures growing at 22 or 33° were not able to synthesize DNA. Nevertheless, these non-dividing (Q) cells were able to synthesize RNA and protein at a reduced rate. The proteins synthesized appear to be a particular subset of the proteins made by normal cells. The results are analyzed in relation to the use of this suspension cell culture system for isolating conditional lethal mutants of plant cells.

Katabolismus von 4′,6-dihydroxyauron in pflanzlichen zellsuspensionskulturen

Cell suspension cultures of Glycine max, Phaseolus aureus, Cicer arietinum and Petroselinum hortense were shown to catabolize (α - 14C) - 4′,6-dihydroxyaurone as measured by 14CO 2 production and isolation of ( 14C)- p-hydroxybenzoic acid. Aurone catabolism in plants is thus comparable with the degradation of chalcones flavanones and flavonols because in all cases the B-ring is liberated as a substituted benzoic acid.

An Extracellular Macromolecular Complex from the Surface of Soybean Suspension Cultures

A cell-free, insoluble cell wall fraction is described which floats on the surface of suspension cultures of Glycine max L. Merrill var. Acme cells. Its accumulation is governed by both the shaking speed and the medium volume, a shaking speed of 110 to 120 revolutions per minute with a medium volume of about 100 to 120 milliliters in a 250-milliliter flask being optimal. Various factors which could control the accumulation of the complex were tested and are discussed, and scanning electron micrographs of the complex being released from the cell surface are presented.The composition of the complex by weight is 46% galacturonic acid, 36% protein, 11% lignin (apparent), 4.4% arabinose, 2% ash, and 0.5% methyl ester. Evidence for an intimate relationship between the uronic acid and protein fractions is presented. The protein contains hydroxyproline, and the bulk of it is tightly bound to the complex, although a portion can be removed with high salt treatments.