Soybean Cell Suspension Cultures Research Articles

Rapid induction of phenylalanine ammonia-lyase and chalcone synthase mRNAs during fungus infection of soybean (Glycine max L.) roots or elicitor treatment of soybean cell cultures at the onset of phytoalexin synthesis

The differential regulation of the activities and amounts of mRNAs for two enzymes involved in isoflavonoid phytoalexin biosynthesis in soybean was studied during the early stages after inoculation of primary roots with zoospores from either race 1 (incompatible, host resistant) or race 3 (compatible, host susceptible) of Phytophthora megasperma f.sp. glycinea, the causal fungus of root rot disease. In the incompatible interaction, cloned cDNAs were used to demonstrate that the amounts of phenylalanine ammonia-lyase and chalcone synthase mRNAs increased rapidly at the time of penetration of fungal germ tubes into epidermal cell layers (1-2 h after inoculation) concomitant with the onset of phytoalxxin accumulation; highest levels were reached after about 7 h. In the compatible interaction, only a slight early enhancement of mRNA levels was found and no further increase occurred until about 9 h after inoculation. The time course for changes in the activity of chalcone synthase mRNA also showed major differences between the incompatible and compatible interaction. The observed kinetics for the stimulation of mRNA expression related to phytoalexin synthesis in soybean roots lends further support to the hypothesis that phytoalexin production is an early defense response in the incompatible plant-fungus interaction. The kinetics for the enhancement of mRNA expression after treatment of soybean cell suspension cultures with a glucan elicitor derived from P. megasperma cell walls was similar to that measured during the early stages of the resistant response of soybean roots.

Use of Soybean (Glycine max) and Velvetleaf (Abutilon theophrasti) Suspension-Cultured Cells to Study Bentazon Metabolism

Metabolism and phytotoxicity of bentazon by suspension-cultured cells of soybean and velvetleaf were compared. Growth of suspension cells of both species was reduced when the cells were exposed to increasing concentrations of bentazon. However, soybean plants were tolerant and velvetleaf plants were susceptible to postemergence applications of bentazon. After incubation with 1 μM14C-bentazon for 6 h, soybean and velvetleaf cells in the log phase of the culture growth cycle contained similar levels of14C (6 nmol/g fresh weight). Of the total14C in the soybean cells, 57 to 92% was present as the glucosyl conjugates of 6-OH- and 8-OH-bentazon with the remainder present as bentazon; the percentage depended on the phase of the culture growth cycle. Bentazon metabolism was greatest in the stationary phase of growth. Thin, transverse sections of soybean hypocotyl metabolized bentazon to the same two metabolites as soybean suspension cells did. The ratio of 6-O-glucosyl-bentazon to 8-O-glucosyl-bentazon was always greater than 1:1 for both the hypocotyl sections and the suspension cells. Bentazon metabolites were not detected in the velvetleaf cells, the velvetleaf hypocotyl sections, or the media of either species. Soybean suspension-cultured cells appear to be a valid and advantageous system for studying the hydroxylation and glucosylation of bentazon, the primary reactions believed to be responsible for detoxication of the herbicide in tolerant plants.

Growth and nicotinate biotransformation in batch cultured and airlift fermenter grown soybean cell suspension cultures

Fermentation of a heterotrophic cell suspension culture of soybean (Glycine max) in a 15 l airlift fermenter for 10 to 12 days yielded appr. 4.67 kg fr. wt. (233.5 g dr. wt.) of biomass starting with an inoculum of 300 g fr. wt. Growth limiting factors were the conditions of preculturing the cells and the available amounts of phosphate and oxygen in the fermenter culture medium. Comparison of the metabolic activity of cells grown in 200 ml Erlenmeyer flasks and in the fermenter was performed by measuring the enzyme activities of nicotinamide-amidohydrolase and SAM: nicotinic acid-N-methyltransferase both in the presence and the absence of exogenous nicotinic acid during the biotransformation of nicotinate to trigonelline. The applied nicotinic acid was quantitatively taken up by the cells and an enhanced production of trigonelline could be observed. The activities of the aforementioned soybean enzymes were the same both in the batch cultured and in the airlift fermenter grown cells indicating good physiological conditions of the cells from the fermenter process.

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.

Induction of phytoalexin synthesis in soybean: Enzymatic cyclization of prenylated pterocarpans to glyceollin isomers

A microsome preparation from elicitor-challenged soybean cell suspension cultures catalyzed an NADPH-dependent and oxygen-dependent cyclization of a mixture of 2- and 4-dimethylallylglycinols to the glyceollin isomers I–III. This is the last committed step in glyceollin biosynthesis. The cyclase was inhibited in a light-reversible manner by carbon monoxide in the presence of oxygen. Cyclase was also inhibited by cytochrome c, NADP +, and a number of inhibitors of cytochrome P-450 enzymes. NADH in the presence of low concentrations of NADPH had a synergistic effect. On a Percoll gradient, the position of cyclase coincided with marker enzymes for the endoplasmic reticulum. These properties identify the cyclase as a cytochrome P-450-dependent monooxygenase. Unstimulated soybean cell culture did not contain detectable cyclase activity. Challenge with either a glucan elicitor from Phytophthora megasperma f.sp. glycinea or with yeast extract caused strong stimulation of cyclase activity with a maximum at about 24 h after elicitor addition.

Cell expansion and single-cell separation induced by colchicine in suspension-cultured soybean cells

Single plant cells have been obtained without the preparation of protoplasts by culturing pieces of soybean callus tissue with colchicine. Cell expansion and separation were evoked by colchicine (1 mM) within a week of culture. Microscopic observation showed that cells took on a spherical shape in the presence of colchicine and then separated into single cells. Addition of colchicine to the culture medium did not affect the composition of cell wall polysaccharides, but a uronic acid-rich extracellular polysaccharide appeared during cell expansion and separation. Addition of microtubule stabilizers, glycerol (300 mM) or dimethyl sulfoxide [3% (vol/vol)], inhibited the secretion of the polysaccharide as well as cell expansion and separation. The extracellular polysaccharide elicited by colchicine was isolated by ion-exchange chromatography on DEAE-Sepharose and gel filtration on Sepharose CL-6B from the conditioned medium of colchicine-treated soybean cells. The purified 18-kDa polysaccharide immediately enhanced cell expansion and separation when added to soybean callus tissue cultured in medium containing colchicine, even at low concentrations (0.1 mM). The polysaccharide was composed of galacturonic acid and, after digestion with a pectinase preparation, had no effect on the cells. Methylation analysis suggests that the polysaccharide consists of approximately 100 sequential alpha-1,4-galacturonic acids. The galacturonan increased the viability of separated cells cultured in medium containing colchicine, and the single cells obtained did not produce a wound-response callose. (Aminoethoxyvinyl)glycine, a specific inhibitor of ethylene production, extensively decreased the cell expansion and separation but did not inhibit the formation of the extracellular polysaccharide, suggesting that the polysaccharide may exert its effect by stimulating ethylene production.

Comparison of photosynthetic characteristics of two photoautotrophic cell suspension cultures of soybean

A new photoautotrophic soybean suspension culture (SB1-P) was initiated within a 14-month period, and was characterized for the purpose of comparing the physiology with that of a established soybean photoautotrophic strain, SB-P. SB1-P was found to have comparable Chl levels (1500–1700 μg g −1 fresh weight), cell size (near 30μm in diameter) cell aggregate size (150–190 μm diameter), chromosomes number ( 2n = 40), rates of growth (cell division time 4.5–5.0 days), net photosynthetic CO 2 fixation (19 and 36 μmol mg −1 Chl h −1 at atmospheric levels of CO 2 and 21 and 2% O 2, respectively) and oxygen evolution (74–76 μmol mg −1 Chl h −1), light-dependent and dark 14CO 2 fixation (49–54 and 3.5–3.7 μmol mg −1 Chl h −, respectively) and oxygen inhibition of photosynthesis (47% inhibition at 21% O 2 compared to photosynthesis at 2% O 2 with atmospheric levels of CO 2). SB1-P was similar to SB-P with regard to the activation level of RuBPcase (40% activated in cells grown at 5% CO 2), low in vitro RuBPcase activity (25–30 μmol CO 2 fixed mg −1 Chl h −1), and a low ratio of RuBPcase(initial)/PEP case (0.28–0.85). Traits shared by SB1-P and SB-P are a low photosynthetic capacity, an inability to grow at air levels of CO 2, high dark respiration, an elevated CO 2 compensation concentration in culture, and RuBPcase deactivation at air CO 2 levels. From these observations it was concluded that many of the characteristics of photoautotrophic soybean cultures are caused by the conditions employed in culture, and are not due to the specific genotype of soybean cultured or to chance occurence during initiation. The cultures which are actively growing exhibit several traits similar to those of developing leaf cells (active cell division, low photosynthetic capacity, elevated respiration, a low ratio of RuBPcase(initial)/PEPcase. This indicates the photoautotrophic cell physiology is more comparable to that of developing leaf cells, rather than to that of mature leaf cells.

Rapid elicitor-induced chemiluminescence in soybean cell suspension cultures

Between 20 and 30 min after exposure to an elicitor from Phytophthora megasperma f.sp. glycinea, soybean cell suspensions exhibit enhanced chemiluminescence in the presence of luminol. The enhanced chemiluminescence is inhibited by added catalase (EC 1.11.1.6) and superoxide dismutase (EC 1.15.1.1) as well as by the peroxidase inhibitors salicylhydroxamic acid and cyanide. It is apparent that cells produce hydrogen peroxide and O 2 − as an early reponse to elicitor, and likely that peroxidase (EC 1.11.1.7) is involved in the light generating reactions.

Uptake and metabolism of triclopyr by soybean cell suspension cultures: a comparison with (2,4-D) 2,4-dichlorophenoxyacetic acid

The mode of action of triclopyr (3,5,6-trichloro-2-pyridinyloxyacetic acid), a broadleaf herbicide, is unknown. No data concerning the fate of triclopyr in plants have yet been published. This communication summarizes a comparison of the uptake and metabolism of triclopyr and 2,4-D (2,4dichlorophenoxyacetic acid) using cell suspension cultures of soybean, Glycine max, var. Harcor. Soybean cells were maintained in aseptic shake culture, on the following media: Gamborg’s B5 (Gamborg, 1975) supplemented with 2,4-D (2.3 PM), indole-3-acetic acid ( 2 . 9 p ~ ) , kinetin ( 4 . 6 ~ ~ ) and sucrose (2% w/v) at pH 6.5, or Miller’s medium (Miller, 1963) supplemented with naphthylI-acetic acid (1 1 PM), kinetin ( 2 . 6 ~ ~ ) and sucrose (2% w/v) at pH 5.8. Cells were subcultured (10ml into 40ml) at 14-16 day intervals. Seven days after subculturing, [2,6-’4CZ]triclopyr (0.82 pCi, 15.6 mCi/mmol) or 2,4-dichlorophenoxy[2-’4C]acetic acid (0.85 pCi, 13.4mCi/mmol, diluted from 55mCi/mmol, Amersham) was added in 50% aq. ethanol. After 6 h, 3 days, 7 days and 21 days, cultures were filtered under vacuum through nylon mesh. Cells were ground (liquid N,) and extracted (80% methanol, 3 x 15 ml), cell debris was sedimented by centrifugation and the supernatant solutions were taken. The I4C recovered from each culture (cells and medium) was quantified by liquid scintillation counting. Cell extracts and media were examined by t.1.c. Radioactive zones were located by radiography, cut from t.1.c. plates, suspended in scintillant and quantified by liquid scintillation counting. All incubations were performed in duplicate. In Miller’s medium: (i) 2,4-D was taken up slightly faster than triclopyr. (ii) Metabolites of both herbicides were largely retained in the cells, except the 2,4-D metabolites nos. 1 1 and 12 (see Fig. l), up to 30% of which appeared in the medium after 21 days. (iii) Metabolism of both herbicides continued until 21 days when no 2,4-D, and ca. 10% triclopyr, remained. (iv) Triclopyr was metabolized to two major compounds (Fig. l), having the following properties: (a) ion-exchange chromatographic behaviour and derivatization by diazomethane showed these metabolites to be acids; (b) acid hydrolysis (2 M-HCl, 80°C, 6 h) yielded triclopyr as sole product; (c) /-glucosidase treatment (citrate phosphate buffer, pH 4.5, 37”C, 18 h) left both metabolites unchanged; ( d ) the metabolites co-eluted on both silica gel t.1.c. and C,,-reverse phase h.p.1.c. with authentic triclopyraspartate (major) and triclopyr-glutamate (minor). Under the same conditions as above, 2,4-D was metabolized to several compounds (Fig. 1). By their chromatographic properties, the products of acid and P-glucosidase treatments, comparison with the triclopyr metabolites and standards described above, and precedents in the literature (Mumma & Hamilton, 1978), these metabolites were probably 2,4-D-glutamate (no. 8), 2,4-D-aspartate (no. 9), 4OH-2,5-D-O-glucoside (no. 10) and 4-OH-2,3-D-0glucoside (no. 11). The behaviour of cells grown on B5 differed significantly from that of cells grown on Miller’s medium in the following respects: (i) B5 cells rapidly released unchanged 2,4-D, but not triclopyr, after initial herbicide uptake. At 3 days and

Purification and Properties of Chalcone Synthase from Cell Suspension Cultures of Soybean

Incubation of soybean cell suspension cultures in B-5 medium containing 0.4 ᴍ sucrose caused induction of chalcone synthase (CHS) up to a specific activity of 8 µkat/kg. CHS from these cultures was purified to apparent homogeneity by a 5-step procedure. Isoelectric focussing of pure CHS gave a major band at pH 5.45 and two weaker bands at pH 5.35 and 5.5. At least the bands at pH 5.35 and 5.45 had CHS activity. Analysis of pure CHS by two-dimensional electrophoresis gave a set ot six proteins with a M, of 40 kDa and pIs between 6.0 and 6.6. One-dimensional PAGE of CHS under non-denaturing conditions gave three closely spaced protein bands. A specific antibody was raised against soybean CHS which cross-reacted with parsley CHS. Attempts to find synthesis of deoxychalconc or of the corresponding 7.4′-dihydroxyflavanonc with CHS of different purification stages and with various cofactors failed.

Partial purification and characterization of beta-mannosyltransferase from suspension-cultured soybean cells.

The beta-mannosyltransferase that catalyzes the synthesis of Man-beta-GlcNAc-GlcNAc-PP-dolichol from GDP-mannose and dolichyl-PP-GlcNAc-GlcNAc was solubilized from microsomes of suspension-cultured soybean cells by treatment with 1.5% Triton X-100 and was purified about 700-fold by chromatography on DEAE-cellulose, hydroxylapatite, and a GDP affinity column. The purified enzyme was reasonably stable in the presence of 20% glycerol and 0.5 mM dithiothreitol. The enzyme required either detergent (Triton X-100 or NP-40) or phospholipid for maximum activity, but the effects of these two were not additive. Thus, either phosphatidylcholine or Triton X-100 could give maximum stimulation. In terms of phospholipid stimulation, both the head group and the acyl chain appeared to be important since phosphatidylcholines with 18-carbon unsaturated fatty acids were most effective. The purified enzyme had a sharp pH optimum of 6.9-7.0 and required a divalent cation. Mg2+ was the best metal ion with optimum activity occurring at 6 mM, but Mn2+ was reasonably effective while Ca2+ was slightly stimulatory. The Km for GDP-mannose was calculated to be 1.7 X 10(-6) M and that for dolichyl-PP-GlcNAc-GlcNAc about 9 X 10(-6) M. The enzyme was inhibited by a number of guanosine nucleotides such as GDP-glucose, GDP, GMP, and GTP, but various uridine and adenosine nucleotides were without effect. The purified enzyme was apparently free of alpha-1,3-mannosyltransferase (and perhaps other mannosyltransferases) and dolichyl-P-mannose synthase since the only product seen from dolichyl-PP-GlcNAc-GlcNAc and GDP-mannose was Man-beta-GlcNAc-GlcNAc-PP-dolichol.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of Ca 2+ on phytoalexin induction by fungal elicitor in soybean cells

A glucan elicitor from the cell walls of the fungus Phytophthora megasperma f.sp. glycinea caused increases in the activities of the phytoalexin biosynthetic enzymes, phenylalanine ammonia-lyase and chalcone synthase, and induced the production of the phytoalexin, glyceollin, in soybean ( Glycine max) cell suspension cultures when tested in culture medium containing 1.2 mmol/liter Ca 2+. Removal of extracellular Ca 2+ by treatment with ethylene glycol bis(β-aminoethyl ether)- N,N′-tetraacetic acid followed by washing the cells with Ca 2+-free culture medium abolished the elicitor-mediated phytoalexin response. This suppression was largely reversed on readdition of Ca 2+. Elicitor-mediated enhancement of biosynthetic enzyme activities and accumulation of glyceollin was strongly inhibited by La 3+; effective concentrations for 50% inhibition were (μmol/liter) 40 for phenylalanine ammonia-lyase, 100 for chalcone synthase, and 30 for glyceollin. Verapamil caused similar effects only at concentrations higher than 0.1 mmol/ liter, whereas trifluoperazine and 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate did not affect enzyme induction by the elicitor in the concentration range tested. Uptake of α-amino isobutyric acid into soybean cells, which was rapidly inhibited in the presence of the glucan elicitor, was not affected by La 3+ nor was uptake inhibition by the elicitor relieved by La 3+. The Ca 2+ ionophore, A23187, enhanced phytoalexin biosynthetic enzyme activities and glyceollin accumulation in a dose-dependent manner, with 50% stimulation (relative to the elicitor) occurring at about 5 μmol/liter. The results suggest that the glucan elicitor causes changes in metabolite fluxes across the plasma membrane of soybean cells, among which changes in Ca 2+ fluxes appear to be important for the stimulation of the phytoalexin response.

Photosynthetic Characteristics of a Photoautotrophic Cell Suspension Culture of Soybean

A soybean suspension culture (SB-P) which can grow photoautotrophically in 5% CO(2) will not grow in ambient CO(2) levels. This elevated CO(2) requirement seems to be due to the additive effects of a number of factors. The in vivo activity of ribulose-1,5-bisphosphate carboxylase (RuBPcase) is much lower in the SB-P cells, compared to soybean plants. This may be due to the low light intensity used to culture the cells, which has been shown to decrease both the amount and activity in whole plants, resulting in a low rate of net photosynthesis. The RuBPcase activation level is also lowered in air CO(2) levels. The presence of the liquid medium raises the cells CO(2) compensation concentration (the CO(2) concentration reached when the rates of CO(2) fixed by photosynthesis and the CO(2) respired by the cells are equal). These factors, coupled with the high respiratory loss of CO(2) all contribute to reduced net photosynthesis in air, resulting in a photosynthetic capacity that is inadequate for cell survival. Active cell division, low photosynthetic capacity, elevated respiration, and a low ratio of RuBPcase(initial)/phosphoenolpyruvate carboxylase are traits that SB-P cells share with young leaf cells, indicating SB-P cell physiology may be comparable to that of young expanding leaves rather than to that of mature leaves.

Inhibition of Elicitor-Induced Phytoalexin Formation in Cotton and Soybean Cells by Citrate

Addition of an elicitor preparation from Verticillium dahliae to soybean or cotton cell suspension cultures induces the formation of the phytoalexins, glycelollin or sesquiterpene aldehydes, respectively. Recent work (PS Low, PF Heinstein 1986 Arch Biochem Biophys 249: 472-479) has shown that the induction of phytoalexin biosynthesis in these cells is preceded by rapid changes in the plant cell membrane which can be conveniently monitored by membrane associated fluorescent probes. Using this elicitation assay, we have found that citrate, a common metabolite of higher plants, acts as a potent inhibitor of elicitation when added prior to treatment with elicitor. The citrate concentration required to obtain a 50% inhibition of the elicitor-induced fluorescence transition in cultured cotton cells was found to be about 2 millimolar, while the concentration of citrate observed to inhibit elicitor-induced sesquiterpene aldehyde formation in the same cell suspensions was also 2 millimolar. Curiously, in the presence of elicitor, citrate at less than ID(50) concentrations increased cell mass accumulation significantly above control incubations without elicitor. A similar inhibition of glyceollin formation with an increase in cell mass accumulation was also observed upon addition of 1 to 5 millimolar citrate to soybean cell suspension cultures. The physiological significance of the inhibition by citrate of phytoalexin formation in plant cell suspensions was supported by the observation that a similar inhibition of sesquiterpene aldehyde formation occurs in cotton plantlets elicited by cold shock or V. dahliae stress. The specificity of citrate as an inhibitor of phytoalexin formation was demonstrated by data showing that other di- and tricarboxylic-hydroxy acids did not inhibit, with the exception of malate which inhibited phytoalexin formation in soybean cells with roughly half the potency of citrate. These experiments not only demonstrate that citrate can act as a specific inhibitor of elicitation, but they further confirm the validity of monitoring elicitation and its modulation with fluorescent probes.

Calcium ions and polyamines activate the plasma membrane-located 1,3-?-glucan synthase

Sucrose-density-gradient centrifugation and partitioning in a polyethylene glycol/dextran two-phase system were used to isolate plasmamembrane vesicles from microsomal preparations of soybean cell suspension cultures. Both methods resulted in the enrichment of the activity of a 1,3-β-glucan synthase which forms a polymer consisting of more than 99% of 1,3-linked glucose (callose). Digitonin increases the 1,3-β-glucan synthase activity in the various membrane fractions to a different degree, supporting the suggestion that this enzyme is vectorially arranged in the plasma membrane. The enzyme is greatly activated either by poly-L-ornithine or synergistically by Ca(2+) and spermine, indicating that the same enzyme is affected and exhibits the regulatory properties necessary for callose synthesis.

A Calcium-Dependent but Calmodulin-Independent Protein Kinase from Soybean

A calcium-dependent protein kinase activity from suspension-cultured soybean cells (Glycine max L. Wayne) was shown to be dependent on calcium but not calmodulin. The concentrations of free calcium required for half-maximal histone H1 phosphorylation and autophosphorylation were similar ( approximately 2 micromolar). The protein kinase activity was stimulated 100-fold by >/=10 micromolar-free calcium. When exogenous soybean or bovine brain calmodulin was added in high concentration (1 micromolar) to the purified kinase, calcium-dependent and -independent activities were weakly stimulated (</=2-fold). Bovine serum albumin had a similar effect on both activities. The kinase was separated from a small amount of contaminating calmodulin by sodium dodecyl sulfate polyacrylamide gel electrophoresis. After renaturation the protein kinase autophosphorylated and phosphorylated histone H1 in a calcium-dependent manner. Following electroblotting onto nitrocellulose, the kinase bound (45)Ca(2+) in the presence of KCl and MgCl(2), which indicates that the kinase itself is a high-affinity calcium-binding protein. Also, the mobility of one of two kinase bands in SDS gels was dependent on the presence of calcium. Autophosphorylation of the calmodulin-free kinase was inhibited by the calmodulin-binding compound N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7), showing that the inhibition of activity by W-7 is independent of calmodulin. These results show that soybean calcium-dependent protein kinase represents a new class of protein kinase which requires calcium but not calmodulin for activity.

Characterization of (1-4)-.ALPHA.-D-glucan synthesized by granulebound starch synthase from suspension cultured soybean cells.

Characterization of (1-4)-.ALPHA.-D-glucan synthesized by granulebound starch synthase from suspension cultured soybean cells.

Solubilization, affinity chromatography and Ca 2+/polyamine activation of the plasma membrane-located 1,3-β-D-glucan synthase

Plasma membranes from suspension-cultured soybean cells have been enriched with a dextran-polyethylene glycol two-phase system and were used to solubilize the 1,3-β-D-glucan synthase by digitonin. The soluble enzyme bound to spermine which was immobilized on Tresyl-activated Sepharose 4B and could be eluted by about 1 mM spermine ·4 HCl or 40 mM NaCl, indicating a preferential binding to the polyamine. Enzyme activity could be recovered from the column only in the presence of some digitonin. The chromatographed enzyme is still synergistically activated by Ca 2+ and polyamines or poly-L-ornithine, indicating a direct and specific interaction with these two types of effectors.

Effects of Haloxyfop on Corn (Zea mays) and Soybean (Glycine max) Cell Suspension Cultures

To investigate the mode of action of a grass-specific herbicide, haloxyfop {2-[4-[[3-chloro-5-(trifluoromethyl)-2-pyridinyl] oxy] phenoxy] propanoic acid} was added to corn and soybean cell suspension cultures containing metabolic intermediates such as sucrose [14C-U], sodium acetate [14C-1,2], or L-leucine [14C-U]. Death of the corn suspension cells occurred within 3 days after haloxyfop was added at a concentration of 1.0μM to supplemented Murashige and Skoog's medium. At 0.1μM haloxyfop,14C-sucrose uptake and14C loss due to respiration of the corn cells declined without a significant reduction in cell fresh weight, cell viability, cellular ATP level, free sugar content, cell wall materials, or level of glycolytic intermediates. The proportions of sucrose, glucose, and fructose in the free sugar fraction were not affected. However, the14C-labeled free amino acid level was increased. During a 24-h incubation period, incorporation of leucine [14C] as a precursor revealed that haloxyfop did not inhibit protein synthesis. Incorporation of acetate [14C-1,2] showed that total lipid synthesis was inhibited by 42% at 0.1μM haloxyfop (sublethal). Similar responses were observed in the soybean suspension cells; however, the calculated 50% lethal concentration (LC50) was 47 times higher than for corn cells. The results of this study suggest that the site of action of haloxyfop is located where metabolic intermediates enter the Kreb's cycle and lipid synthesis. Glycolysis is not inhibited.

Characteristics of the oleoyl- and linoleoyl-CoA desaturase and hydroxylase systems in cell fractions from soybean cell suspension cultures

Proplastid and microsome fractions of soybean cell cultures showed both Δ 12-desaturase and 3-hydroxylase activities. High thiolase activity for either 18:1-CoA or 18:2-CoA was present in both fractions, but this could be overcome by the addition of ATP, Mg 2+ and CoA. Requirement for NADPH or NADH could not be established conclusively for either enzyme activity; in fact NADH was found to be inhibitory for both enzyme systems. The desaturase and hydroxylase systems were similar to those of other plants with respect to an absolute requirement for oxygen, but differed with respect to their sensitivity to cyanide, which was inhibitory only at very high concentrations. Herbicides such as San 6706 and Destun were strongly inhibitory to both enzyme systems. All metabolic products formed from 18:1-CoA and 18:2-CoA were recovered almost entirely (about 90%) as acyl-CoAs, indicating that the enzyme systems used acyl-CoA as substrate and that acyltransferase activity was low.