Release of Calcium from Suspension-Cultured Glycine max Cells by Chitosan, Other Polycations, and Polyamines in Relation to Effects on Membrane Permeability

Treatment with chitosan of suspension-cultured Glycine max cells labeled with ⁴⁵Ca²⁺ caused a rapid release of calcium, which was complete much earlier than the chitosan-induced leakage of intracellular electrolytes and probably reflects calcium loss primarily from the cell wall and/or plasma membrane. A linear correlation was found between calcium release from chitosan-treated whole cells or isolated cell walls and the amount of chitosan bound. Other polycations (poly-l-lysine, histone, DEAE-dextran, and protamine sulfate), low molecular weight polyamines (spermine, spermidine, and putrescine) and polyanions (polygalacturonate and poly-l-aspartate, which act as chelating agents) also released calcium from whole cells and isolated cell walls; however, only the polycations increased membrane permeability. Poly-l-lysines of differing molecular weight showed a similar ability to release calcium, but their effect on membrane permeability increased with increasing molecular weight. The results suggest that the effect of polycations on permeability is not the direct result of calcium displacement from the cell surface but is probably due to cross-linking of surface components. The order of effectiveness of inorganic cations in displacing calcium from whole cells and isolated cell walls was Ca²⁺, Ba²⁺, Sr²⁺ > Mg²⁺ > K⁺, Na⁺.     

Subculture-induced protein synthesis in tissue cultures of Glycine max and Phaseolus vulgaris

The effects of subculture of tissue cultures on the levels of certain mRNAs have been investigated, and the action of cytokinins on the disposition of certain mRNAs between possible non-translating and translating pools has been determined. mRNA preparations were assayed by cell free translation with message-dependent reticulocyte lysate and the in vitro products resolved by polyacrylamide gel electrophoresis. Subculture of the cells caused a rapid stimulation of polysome formation. It also increased the translatable levels of a small group of mRNAs, one of which was present in both bean and soybean cultures. Cytokinins caused a slight increase in polysome levels after subculture, but had no effect on the levels of particular mRNAs, nor on the distribution of mRNAs between a non-translating and translating pool, nor on polysome levels in the absence of subculture.Abbreviations EDTA ethylenediaminetetra acetic acid - EGTA 1,2-di-(2-aminoethoxy)ethane-NNNN-tetra acetic acid - IEF isoelectric focusing - SDS sodium dodecyl sulphate     

Synergetic Cultures of Glycine max Root Cells and Rhizobia Separated by Membrane Filters

When suspension cultures of actively growing soybean (Glycine max L.) root cells were separated by two or three membrane filters from suspension cultures of the bacteria, a synergetic (cooperative) activation of nitrogenase was observed in the Rhizobium japonicum used in the bacterial side. Either plant cells or plant cell-conditioned medium was needed for this activation to take place. Both acetylene reduction and hydrogen evolution by the activated R. japonicum persisted for several days after removal from the apparatus when (a) a suitable carbon source was provided, (b) oxygen supply was limited, and (c) growth of bacteria was suppressed by lowering of ammonia and nitrate concentrations. Activation could also take place when the bacteria were placed in media to which plant cell-conditioned medium was added. The advantages of this method for studies on symbiosis are discussed.     

Phospholipase D from Soybean (Glycine max L.) Suspension-Cultured Cells: Purification, Structural and Enzymatic Properties

Phospholipase D (phosphatidylcholine phosphatidohydrolase EC3.1.4.4 [EC] ) from soybean (Glycine max L.) suspension-cultured cellwas purified around 1,200-fold to homogeneity by acetone precipitation,Macro-Prep High Q anion exchange, and octyl-Sepharose CL-4Baffinity chromatography. The purified enzyme released 1,600µmol of choline per min per mg of protein. The enzymeis monomeric with a molecular mass of 92 kDa, as estimated bySDS-PAGE. One of the most interesting characteristics of thepurified soybean phospholipase D was the dependence of the pHoptimum on the Ca²⁺ ion concentration in the assay. With 10mM, 20 mM and 40 mM Ca²⁺ ions, the optima were at pH 7.5, 6and 5.5, respectively. The specific adsorption of phospholipaseD onto octyl-Sepharose gel suggests that the molecule becomesmore hydrophobic in the presence of Ca²⁺ ions. The amino acidsequence of the first 18 N-terminal residues of soybean phospholipaseD revealed a high degree of homology with those previously publishedfor cabbage leaf and castor bean endosperm enzymes. Westernblots of the soybean phospholipase D showed an immunoreactivitywith antibodies raised against a synthetic peptide correspondingto the 15 N-terminal aminoacid residues of phospholipase D fromcabbage leaves. (Received March 13, 1995; Accepted May 29, 1995)     

The glycosylated seed storage proteins of Glycine max and Phaseolus vulgaris. Structural homologies of genes and proteins

Considerable information is now available concerning the 7 S seed storage proteins of legumes and the genes that encode them. Our study compares the gene encoding a beta-type subunit of phaseolin (Pvu beta), the 7 S protein of common bean (Phaseolus vulgaris), with the gene encoding an alpha'-subunit of beta-conglycinin (Gma alpha'), the 7 S protein of soybean (Glycine max). The comparison involves 2880 base pairs of Pvu beta and 3636 base pairs of Gma alpha' and includes approximately 1 kilobase pair of 5'-flanking sequences, and 5' and 3' untranslated sequences, as well as the six exons and five introns that are found to occur in similar positions in both genes. Conserved sequences in the 5'-flanking regions of these genes are discussed in light of their potential regulatory role. Published sequences for 7 S genes of pea (Pisum sativum) permit the inference of the nature and direction of evolutionary change and, in particular, show that the major size difference between the large Gma alpha' polypeptide and the smaller polypeptides of pea and common bean is due to a large insertion in the first exon of Gma alpha'. Comparisons of protein primary structure, potential glycosylation sites, and predicted protein hydropathy show that strongly conserved features of 7 S proteins cut across exon boundaries and that nonconserved regions exist that may have potential for protein modification.     

Induction of Glutamine Synthetase Activity in Nonnodulated Roots of Glycine max, Phaseolus vulgaris, and Pisum sativum

Nitrate or ammonium fertilization significantly increased glutamine synthetase (GS) activity in nonnodulated roots of French bean (Phaseolus vulgaris), soybean (Glycine max), and pea (Pisum sativum). Western analysis revealed substantial GS antibody-positive protein in root extracts that had minimal GS activity, indicating that an inactive form of GS may be present in nonfertilized plants.     

A Relationship between Membrane Permeability and Ethylene Production at High Temperature in Leaf Tissue of Phaseolus vulgaris L.

Leaf discs cut from primary leaves of Phaseolus vulgaris L cvMasterpiece were incubated at temperatures higher than the growthtemperature of 25 °C Both basal and wound ethylene productionincreased up to temperatures of 35–37 5 °C, thereafterdeclining rapidly There was no detectable ethylene productionat temperatures above 42 5 °C Exposure of leaf discs tohigh temperature for 60 mm resulted in a large production ofwound ethylene when they were returned to 25 °C The magnitudeof ethylene production was related to the initial incubationtemperature as was the length of the lag period before maximumproduction was achieved The results are discussed in relationto the requirement for continued membrane integrity for ethyleneproduction ethylene, temperature, membrane permeability, Phaseolus vulgaris L, dwarf bean     

Effect of Glyphosate on Intact Bean Plants (Phaseolus vulgaris L.) and Isolated Cells

Whole bean (var. “Eastern Butterwax”) plants and isolated cells were used to investigate possible mechanisms of action of glyphosate [N-(phosphonomethyl)glycine]. Results showed that glyphosate was quickly absorbed by the whole plant but not by individual cells and that it caused a rapid reduction in leaf dry matter accumulation, leaf expansion, leaf angle, and stomatal aperture without affecting the water status of the plant. Glyphosate also caused a rapid reduction in cellular uptake of ⁸⁶Rb and ³²P which preceded its detrimental effects on photosynthesis, RNA and protein synthesis, and respiration of isolated cells. This reduction in ion absorption was not due to a loss of membrane integrity, decrease in energy supply or chelation of ions. It was concluded that glyphosate was directly inhibiting the ion absorption process of bean leaf cells.     

Membrane Depolarization Induced by N-Acetylchitooligosaccharide Elicitor in Suspension-Cultured Rice Cells

N-acetylchitooIigosaccharides (fragments of chitin) elicit defenseresponses, including phytoalexin production, in suspension-culturedrice cells. They induced rapid and transient membrane depolarizationaccompanied by a transient increase in net CP^–-efflux.The membrane depolarization was not affected by anaerobiosisor azide, suggesting that the major part of the depolarizationwas mediated by ion channels, not by energy-dependent ion pumps.Depolarization was partly inhibited in the presence of Ca²⁺-or Cl^–-channel blockers and highly inhibited by depletionof Ca²⁺ in the extracellular medium. A calcium ionophore, A23187 [GenBank] ,caused a transient depolarization but not an increase in Cl^–efflux, while it did not inhibit the elic-itor-induced transientdepolarization and Cl^– efflux. These suggest that theinflux of Ca²⁺ from the extracellular space to the cytoplasmis necessary as an initial trigger but not sufficient for membranedepolarization, Cl^– efflux, and the following signalingprocesses. (Received November 2, 1996; Accepted May 12, 1997)     

Effects of Temperature on Development of Heterodera glycines on Glycine max and Phaseolus vulgaris

Soybean cyst nematode resistant ''Fayette'' and susceptible ''Williams 79'' soybeans (Glycine max) and resistant ''WIS (RRR) 36'' and susceptible ''Eagle'' snap beans (Phaseolus vulgaris) were used in determining the effects of host and temperature on the development, female production, sex ratios, and host response to Heterodera glycines. Temperatures were maintained constant at 16, 20, 24, 28, and 32 C using water-filled tanks. The most rapid development and greatest female production occurred between 20 and 28 C. The equation DS = 5(10⁻⁶)x²y² - 3(10⁻⁴)x²y - 2.8(10⁻³)x² - 1.94(10⁻²)y² + 0.4288x + 1.0220y - 12.7185, where DS = developmental stage, X = time, and Y = temperature, predicted the developmental stage of the nematode and accounted for 84% of the variation. Male : female ratios did not differ within this range and were generally less than one. At all temperatures the resistant soybean produced the greatest number of necrotic responses to H. glycines infection, followed by the resistant snap bean. The susceptible soybean and snap bean produced the fewest necrotic responses.     

Effect of Potassium on Photosynthate Unloading from Seed Coats of Phaseolus vulgaris L. Specificity and Membrane Location

Rates of ¹⁴C-photosynthate unloading from excised seed-coathalves of Phaseolus vulgaris L. plants were stimulated by externalKCI concentrations in excess of 10 mM with an optimal responseat 100–150 mM KCI. The cellular pattern of ¹⁴C-photosynthatemetabolism was not altered by KCI but the treatment preferentiallystimulated the release of sucrose from the seed-coats. Photosynthateunloading was insensitive to Cl^– and was stimulated bya range of membrane-permeable cations (Na⁺, Mg²⁺ and tetraphenylphosphoniumion) in addition to K⁺. The K⁺ ionophore, valinomycin, abolishedthe K⁺ stimulation of ¹⁴C-photosynthate unloading. A switchto a wash solution containing K⁺ elicited a rapid burst of ¹⁴C-photosynthateunloading; the rate constant for the final phase of ¹⁴C-efflux(probably across the tonoplast) was unaffected by K⁺. The KCItreatment did not change the passive permeability of eitherthe plasmalemma or tonoplast. While sucrose influx across theplasmalemma was insensitive to K⁺, sucrose transfer to the vacuolewas slowed. The results obtained support the postulate thatK⁺ (and other membrane permeable cations) preferentially stimulatesucrose efflux across the plasmalemma of the unloading cellsby serving to carry positive charge in the opposite direction. Phaseolus vulgaris, bean, photosynthate unloading, potassium stimulation, seed-coat     

The Antiranspirant Effect of Acetylsalcylic Acid on Phaseolus vulgaris

A series of experiments were carried out in order to elucidate whether acetylsalicylic acid (ASA) fed via petioles to beans could affect transpiration rate. In comparison with the water control it was found that a 10^?3M ASA reduces transpiration as much as 43%, a reduction equivalent to 5 × 10^?5M abscisic acid (ABA). Preliminary kinetics on the ABA, ASA and the water control on transpiration rate are presented and the results discussed.     

铜、砷及其复合污染对黄豆（Glycine max）影响的初步研究

通过水培实验研究了Cu、As单一及复合污染对黄豆种子萌发、幼苗生长及部分酶活性的影响,结果表明,Cu、As污染明显抑制黄豆种子萌发、幼苗生长,对黄豆种子萌发时的呼吸强度、蛋白酶活性有显著的抑制作用,且随着Cu、As浓度的增加,抑制作用增强,呈负相关;而OD活性则随污染物浓度的增加而增加,呈正相关,Cu、As污染共同存在时,随二者投加比例不同出现不同程度的拮抗效应,可以降低和缓解单一污染物的毒害作用。     

Studies on Fertilization in Glycine max

This work studies the whole process of fertilization in Glycine max. The results are summarized as follows: 1. The type of ripened pollen grain of soybean was two-cell type. The generative cell was divided mitotically into two spermatids within the pollen tube. 2. In the 6th hour after self-pollination, the pollen tube entered into the embryo sac and released two sperms. Before the fusion of the male and female nuclei, the cytoplasmic sheath of the spermatids falled off. The cytoplasm of the male gamete did not fuse with that of the egg cell. 3. In the 6th hour after self-pollination, one spermatid nucleus come in contact with the egg nucleus and the other with the secondary nucleus, The contact of the sperimatid nucleus with the egg nucleus was a little earlier than that with the secondary nucleus. 4. The spermatid nucleus entered into the egg nucleus; the chromatic granules of the spermatid despiralized. After the complete fusion of the spermatid nucleus with the egg nucleus, the egg nucleus was darkly stained and the chromonemata increased, afterward the male nucleus appeared. 5. In the 28th hour after self-pollination, the zygote begun the first mitotic division. It took about 20 hours to fuse the male and female gametes, and to form the zygote untile before first mitotic division. It means that the zygote dormancy stage of soybean was about twenty hours. 6. In the 7th hour after self-pollination, the spermatid nucleus fused with the secondary nucleus. The process was similar to that of the spermatid nucleus with the egg nucleus. The chromatic granules gradually despiralized within the secondary nucleus, and the male nucleoli appeared. The velocity of the fusion of the spermatid nucleus with the secondary nucleus was faster than that of the other spermatid nucleus with the egg nucleus. 7. In the 10th hour after self-pollination, the secondary nucleus begun the first mitotic division. 8. The fertilization of soybean was the premitotic type.     

Cytokinin-Mediated Translational Control of Protein Synthesis in Cultured Cells of Glycine max

Polyribosome formation was stimulated by cytokinin treatmentof cultured cells of Glycine max cv. Funk Delicious. When suspensioncultures were given 0·5 µM zeatin after 24 h inculture in medium lacking a cytokinin, a nearly 2-fold increasein the polyribosome/monoribosome ratio occurred over the subsequent3 h. The effect of actinomycin D and of 5-fluorouridine on RNAsynthesis and on the polyribosome/monoribosome ratios of thesecells was examined. Actinomycin D at 5 and 20 µg/ml^–1inhibitedtotal RNA synthesis by 39 and 60%, respectively, as measuredby [³H]uridine incorporation into acid-precipitable material.The degree of inhibition of precursor incorporation into polyribosomalRNA was similar. At 0·1 mM, 5-fluorouridine inhibited[³H]uridine incorporation by 76%, and [³H]guanosine incorporationby 66% into polyribosomal RNA after 3 h of treatment. Fractionationof the polyribosomal RNA by oligo(dT)-cellulose chromatographydemonstrated that low concentrations of both actinomycin D (5µg ml^–1) and 5-fluorouridine (0·1 mM) inhibitedthe synthesis of ribosomal RNA to a greater extent than thepoly(A)-containing fraction of the messenger RNA. Synthesisof the poly(A)-containing RNA was inhibited by 24% with 5µgml^–1 actinomycin D and by 30% with 0·1 mM 5-fluorouridine.At the above concentrations, these two inhibitors reduced thepolyribosome/monoribosome ratio of the cytokinin-deprived cellsover a 3 h period, but they did not prevent cytokinin-inducedpolyribosome formation. These results provide further evidencethat cytokinin regulates polyribosome levels through an effecton protein synthesis at the translational level     

Uptake of Abscisic Acid by Suspension-Cultured Phaseolus coccineus L. Cells: Evidence for Carrier Participation

The uptake of abscisic acid (ABA) by suspension-cultured Phaseoluscoccineus L. cv. ‘Prizewinner’ (runner bean) cellshas been studied. In addition to non-mediated diffusive uptakeof ABAH, a saturable component of uptake occurs which is demonstrableas inhibition of uptake of submicromolar concentrations of [2-¹⁴C]ABAby increasing concentrations of nonradioactive ABA to a constantplateau level. Saturation is not due to competition for extracellularbinding sites or cytoplasmic acidification and can only be partiallyaccounted for by saturation of metabolic enzymes. It is consideredlargely to represent carrier-mediated ABA uptake. The maximumvelocity of the carrier is greater at pH 4.0 than at pH 5.0,although the apparent Michaelis constants are similar (about2.0 mmol m^–3). No carrier activity was detectable abovepH 6.0. The carrier is unaffected by indol-3-yl acetic acid(IAA), gibberellin A₃, benzyladenine or 2, 3, 5-triiodobenzoicacid (TIBA). Use of protein-modifying reagents suggested a roleof histidine residues in carrier activity. Reagents expectedto modify the transmembrane pH ( pH) and electrical ( E) gradientswere used to study the driving forces for ABA uptake. Therewas no apparent effect of E, indirectly monitored by effectson an electrogenic TIBA-sensitive component of IAA transport,on ABA uptake. Both diffusive and saturable components weremodified in proportion by changes in pH, suggesting a commonsensitivity to this driving force. The carrier is suggestedto act as an ABA^–/H⁺ symport. Key words: Abscisic acid, Phaseolus coccineus L., Transport, Suspension culture     

Soybean (Glycine max) Nodule Physical Traits Associated with Permeability Responses to Oxygen

Nodule permeability (P) controls the amount of O2 entering the nodule and is an important determinant of N2 fixation. Modulation of water volume in the intercellular spaces of the nodule cortex was hypothesized to change the effective thickness of a diffusion barrier and account for changes in P. This hypothesis was examined by evaluating physical traits of nodules that may affect P. The first test of the hypothesis was to determine whether alterations in P may result in changing both the density and the air space content of nodules as the water content of intercellular spaces was varied. Density of nodules exposed to 21 kPa O2 increased as the time following detachment from the plant increased from 5 to 60 min. Nodules from soybean (Glycine max [L.] Merr.) plants shaded for 48 h had a lower fractional air space content than nodules from control plants. Nodule detachment and prolonged shading decreased P, and the increase in density and decrease in fractional air space content associated with decreased P in these treatments supports the proposed hypothesis. The second test of the hypothesis was to determine whether nodules released water easily in response to water potential gradients. The intrinsic capacitance of nodules determined by pressure-volume analysis was 0.29 MPa-1 and indicated that the tissue can release relatively large amounts of water from the symplast with only small changes in total nodule water potential. Estimates of the bulk modulus of elasticity ranged from 0.91 to 2.60 MPa and indicated a high degree of elasticity. It was concluded that the physical properties of nodules were consistent with P modulation by the release and uptake of intercellular water in the nodule cortex.