Effect of Chitosan on Membrane Permeability of Suspension-Cultured Glycine max and Phaseolus vulgaris Cells

Treatment of suspension-cultured Glycine max cv Harosoy 63 cells with soluble chitosan (20-500 micrograms per milliliter) increased membrane permeability as shown by leakage of electrolytes, protein, and UV absorbing material. Severe damage to the cell membrane by chitosan (100 and 500 μg/ml) was also indicated by reduced staining with fluorescein diacetate and the leakage of fluorescein from preloaded cells. Other basic polymers (poly-l-lysine, histone, DEAE-dextran, protamine sulfate, and glycol chitosan) also increased permeability, whereas the basic monomers l-lysine and d-glucosamine, and acidic or neutral polymers were not active. Chitosan-induced leakage was inhibited by divalent cations, the order of effectiveness being Ba²⁺ > Ca²⁺ > Sr²⁺ > Mg²⁺. Na polygalacturonate and Na poly-l-aspartate also reduced polycation-induced leakage, probably by formation of polycation-polyanion complexes. A chitosan-polygalacturonate complex precipitated on mixing solutions of the two polymers containing approximately equal numbers of galacturonate and glucosamine residues, but not with either polymer in excess. A similar concentration-dependent precipitation of chitosan by Na poly-l-aspartate was found. Leakage from Phaseolus vulgaris cv Grandessa cells was also induced by chitosan, and was inhibited by Ca²⁺ and Na polygalacturonate.     

Electrogenic ATPase Activity on the Peribacteroid Membrane of Soybean (Glycine max L.) Root Nodules

Electrogenic ATPase activity on the peribacteroid membrane from soybean (Glycine max L. cv Bragg) root nodules is demonstrated. Membrane energization was monitored using suspensions of intact peribacteroid membrane-enclosed bacteroids (peribacteroid units; PBUs) and the fluorescent probe for membrane potential (ΔΨ), bis-(3-phenyl-5-oxoisoxazol-4yl) pentamethine oxonol. Generation of a positive ΔΨ across the peribacteroid membrane was dependent upon ATP, inhibited by N,N′-dicyclohexyl-carbodiimide and vanadate, but insensitive to N-ethylmaleimide, azide, cyanide, oligomycin, and ouabain. The results suggest the presence of a single, plasma membrane-like, electrogenic ATPase on the peribacteroid membrane. The protonophore, carbonyl-cyanide m-chlorophenyl hydrazone, completely dissipated the established membrane potential. The extent of reduction in the steady state membrane potential upon addition of ions was used to estimate the relative permeability of the peribacteroid membrane to anions. By this criterion, the relative rates of anion transport across the peribacteroid membrane were: NO₃⁻ > NO₂⁻ > Cl⁻ > acetate⁻ > malate⁻. The observation that 10 millimolar NO₃⁻ completely dissipated the membrane potential was of particular interest in view of the fact that NO₃⁻ inhibits symbiotic nitrogen fixation. The possible function of the ATPase in symbiotic nitrogen fixation is discussed.     

Dynamics of Galactolipids and Plastids in Nonphotosynthetic Cells of Glycine max Suspension Cultures : A Morphological and Biochemical Study

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. [³H]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.     

Ethylene Production by Root Nodules and Effect of Ethylene on Nodulation in Glycine max

Nodulated soybean roots produced more ethylene and contained more 1-aminocyclopropane-1-carboxylic acid than uninoculated roots. Nodules produced more ethylene and contained more 1-aminocyclopropane-1-carboxylic acid per gram of material than roots. Almost all of the ethylene produced by the nodules was produced by the plant fractions of the nodules. Ethylene, at physiological concentrations, did not inhibit nodulation in soybeans.     

Alpha-Mannosidase II Isoenzyme in the Peribacteroid Space of Glycine max Root Nodules

Kinnback, A., Mellor, R. B. and Werner, D. 1987. Alpha-mannosidaseII isoenzyme in the peribacteroid space of Glycine max rootnodules.—J. exp. Bot. 38: 1373–1377. Three isoenzymes of alpha-mannosidase from soybean tissue wereseparated by isoelectric focussing. The vacuomal isoenzymesI and II accounted typically for over 90% of the activity inextracts from roots whether or not they were infected with Bradyrhizobiumjaponicum, tissue culture and effective nodules. Ineffectivenodules, on the other hand, contained up to 62% of the typicallyextracellular located isoenzyme III. The compartment betweenthe symbiotic partners (the peribacteroid space) contained onlyisoenzyme II. The bearing of isoenzyme distribution upon modelsof peribacteroid membrane biogenesis is discussed. Key words: Aipha-mannosidase, Bradyrhizobium japonicum, Glycine max, membrane flow/recycling, peribacteroid space, symbiosis     

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⁺.     

费氏中华根瘤菌HN01DL在大豆根圈能定殖动态与结瘤研究

以发光酶基因luxAB为标记,在根盒缩影条件下研究了费氏中华根瘤菌HN01DL在大豆根圈的定殖动态、分布范围及其结瘤情况．结果表明,HN01DL在根盒．灭菌土壤和非灭菌土壤缩影中的大豆全根系定殖动态与水平明显不同,前者在第12d时达到最高定殖密度(8．65log cfu·g^-1),而后者的早期定殖数量下降较快,且于第15d时达到最高定殖密度(6．88log cfu·g^-1)．HN01DL在大豆播种5d后在大豆根部的A(0～4cm)区根段上达到最高定殖密度(7．05log cfu·g^-1),然后开始缓慢下降,至第19d时仍维持相对稳定,在第33d时又开始回升．至播种后第46d时HN01DL可散布至种子下方16cm处的根段部位．HN01DL在A区根段的定殖密度持续较高,所形成的发光根瘤总数(16．3个)及发光比例(68．8％)最高,且发光根瘤主要集中于该区段主根上．发光根瘤比例沿A-E区段逐渐下降,在E区段未检测到发光根瘤．     

Plant regeneration by organogenesis in Glycine max

A procedure for the regeneration of fertile plants by organogenesis from tissue cultures of soybeans, Glycine max is described. Seeds were germinated on reduced inorganic salt MS medium containing 5M BA. Cotyledonary nodes were excised and cultured on the same medium. Presence of BA in the medium during seed germination and culture of nodal explants was required for multiple shoot and shoot-bud formation. Histological analyses established the de novo nature of shoot regeneration. Separate reduction of the concentration of inorganic salts or substitution of sucrose for fructose during culture had minimal effects on the regeneration response. Conversely, if the BA was reduced, the inhibition response could not be overcome by increased salt concentration or altered carbon source.Abbreviations BA benzyladenine - IAA indoleacetic acid - SAS secondary axillary shoots - MS Murashige and Skoog (1962) medium     

Hydrogen Sulfide Promotes Root Organogenesis in Ipomoea batatas, Salix matsudana and Glycine max

In this report, we demonstrate that sodium hydrosulfide (NaHS), a hydrogen sulfide (H2S) donor, promoted adventitious root formation mediated by auxin and nitric oxide (NO). Application of the H2S donor to seedling cuttings of sweet potato (Ipomoea batatas L.) promoted the number and length of adventltious roots in a dose-dependent manner. It was also verified that H2S or HS- rather than other sulfur-containing components derived from NariS could be attributed to the stimulation of adventitious root formation. A rapid Increase In endogenous H2S, indole acetic acid (IAA) and NO were sequentially observed in shoot tips of sweet potato seedlings treated with HallS. Further investigation showed that HzS-mediated root formation was alleviated by N-l-naphthylphthalamic acid (NPA), an IAA transport inhibitor, and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), an NO scavenger. Similar phenomena in H2S donor-dependent root organogenesis were observed in both excised willow (Sallx matsudana var. tortuosa Vilm) shoots and soybean (Glycine max L.) seedlings. These results indicated that the process of H2S-induced adventitious root formation was likely mediated by IAA and NO, and that H2S acts upstream of IAA and NO signal transduction pathways.     

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     

Scanning Electron Microscopy of Microbial Cells on Membrane Filters

Scanning electron micrographs of a Pseudomonas species, Staphylococcus aureus, and Bacillus subtilis on two membrane filtration systems are compared.     

消除共生质粒的费氏中华根瘤菌HND29SR在大豆根圈的定殖动态

比较研究了费氏中华根瘤菌（Sinorhizobium fredii)HN01(出发菌）、发光酶基因标记菌HNO1L（参照菌）、消除HN01共生质粒的菌株HND29SR在无菌砂培条件下的大豆根圈定殖动态。供试菌单独接种时：HN01、HN01L和HND29SR的定殖动态基本一致,其早期定殖密度下降较快,播种后第16天时HN01和HN01L分别达到较高的定殖水平6.49logcfu/g鲜根和6.78logcfu/g鲜根,然后维持相对稳定的定殖水平。但HND29SR的定殖密度持续下降到播种后第16天时才开始上升,至第35天时仍维持相对稳定的定殖密度6.94logcfu/g鲜根。等量混合接种时供试菌在根圈定殖群体中各自定殖密度在测定过程中基本相等。结果表明消除HN01的共生质粒对其在大豆根圈中定殖能力无显著影响。     

Chromosomal features revealed by comparison of genetic maps of Glycine max and Glycine soja

Recombination is a crucial component of evolution and breeding. New combinations of variation on chromosomes are shaped by recombination. Recombination is also involved in chromosomal rearrangements. However, recombination rates vary tremendously among chromosome segments. Genome-wide genetic maps are one of the best tools to study variation of recombination. Here, we describe high density genetic maps of Glycine max and Glycine soja constructed from four segregating populations. The maps were used to identify chromosomal rearrangements and find the highly predictable pattern of cross-overs on the broad scale in soybean. Markers on these genetic maps were used to evaluate assembly quality of the current soybean reference genome sequence. We find a strong inversion candidate larger than 3 Mb based on patterns of cross-overs. We also identify quantitative trait loci (QTL) that control number of cross-overs. This study provides fundamental insights relevant to practical strategy for breeding programs and for pan-genome researches.     

Characteristics of Modified Leghemoglobins Isolated from Soybean (Glycine max Merr.) Root Nodules

Hemoprotein derivatives of an abundant soybean (Glycine max Merr.) root nodule leghemoglobin, Lba, were studied for their modified spectral characteristics and physical properties. Three modified hemoprotein derivatives of Lba (Lbam1, Lbam2, and Lbam3) were purified by preparative isoelectric focusing. The ferric forms of these pigments were green and exhibited anomalous spectra in the visible region as compared to the Lba3+ forms. These modified pigments showed a hypochromic shift of 10 nm for the charge transfer absorption maximum; however, differences were not apparent in the Soret region. Upon binding with nicotinate, the [alpha] and [beta] bands were shifted significantly into the red region as compared to the Lba3+ nicotinate complex. The three Lbam fractions were reduced by dithionite or by NADH in the presence of riboflavin. Lbam2+ also bound nicotinate and displayed absorption spectra indistinguishable from those of Lba2+ nicotinate. In contrast to Lba2+, Lbam2+ displayed aberrant spectra when bound with either O2 or CO. These complexes exhibited a prominent charge transfer band at approximately 620 nm and failed to exhibit spectra characteristic of Lba2+O2 and Lba2+CO. The protein moiety of these modified pigments was intact because their tyrosine/tryptophan ratios and their amino acid compositions were identical with those of Lba, nor were differences observed in the peptide profiles resulting from trypsin digests of purified Lba and Lbams. Automated Edman degradation of selected peaks further confirmed the intactness of the protein backbone including the absence of deamination. Pyridine hemochromogen for heme from Lbams could be formed, and the spectra displayed distinct differences compared to those of Lba. A new peak at 580 nm and a loss of a peak at 480 nm were observed for all three Lbams.     

Transient Energy Coupling Between Rhizobia and Legume Cells Mediated by the Peribacteroid Membrane ATPase Proton Pump

We present a model for the metabolic coupling between rhizobia and plant cell in the nitrogen-fixing legume root nodules. The symbiosome, an organelle-like structure formed by the modified rhizobia (the bacteroids) enclosed by a plant cell derived peribacteroid membrane, is an unique structure in which two energized membranes are closely packed: the inner bacteroid membrane and the peribacteroid membrane that possesses an ATPase proton pump. The model is based on the following points: (i) The permeability for hydrogen ions of the outer membrane of the rhizobia. (ii) The reversibility of the ATPase proton pump of the peribacteroid membrane [Szafran, M. M. and Haaker, H. (1995) Plant Physiol. 108, 1227–1232]. (iii) The relative affinites for oxygen of the bacteroid and plant mitochondria terminal oxidases, and the prevailing oxygen concentration inside the nodule, which results in aerobic metabolism for the bacteroid, but in quite fermentative catabolism for the host plant cell. We propose that the bacteroid can transiently supply free energy to the plant cell in the form of protonmotive force by the movement of hydrogen ions from the bacteroid periplasmic space to the plant cytoplasm through the peribacteroid membrane ATPase. The proposed hydrogen ion flux could be dependent on the phosphorylation potential in both the plant cell cytoplasm and the bacteroid, and the simultaneous ion movements to avoid the development of opposite . It could be important in situations of transient ATP depletion inside plant cell, which involves the block of ammonia assimilation and, subsequently, the inhibition of bacteroid nitrogenase.     

Detoxification of Formaldehyde by the Spider Plant (Chlorophytum comosum L.) and by Soybean (Glycine max L.) Cell-Suspension Cultures

The phytotoxicity of formaldehyde for spider plants (Chlorophytum comosum L.), tobacco plants (Nicotiana tabacum L. cv Bel B and Bel W3), and soybean (Glycine max L.) cell-suspension cultures was found to be low enough to allow metabolic studies. Spider plant shoots were exposed to 7.1 [mu]L L-1 (8.5 mg m-3) gaseous [14C]-formaldehyde over 24 h. Approximately 88% of the recovered radioactivity was plant associated and was found to be incorporated into organic acids, amino acids, free sugars, and lipids as well as cell-wall components. Similar results were obtained upon feeding [14C]formaldehyde from aqueous solution to aseptic soybean cell-suspension cultures. Serine and phosphatidylcholine were identified as major metabolic products. Spider plant enzyme extracts contained two NAS+-dependent formaldehyde dehydrogenase activities with molecular mass values of about 129 and 79 kD. Only the latter enzyme activity required glutathione as an obligatory second cofactor. It had an apparent Km value of 30 [mu]M for formaldehyde and an isoelectric point at pH 5.4. Total cell-free dehydrogenase activity corresponded to 13 [mu]g formaldehyde oxidized h-1 g-1 leaf fresh weight. Glutathione-dependent formaldehyde dehydrogenases were also isolated from shoots and leaves of Equisetum telmateia and from cell-suspension cultures of wheat (Triticum aestivum L.) and maize (Zea mays L.). The results obtained are consistent with the concept of indoor air decontamination with common room plants such as the spider plant. Formaldehyde appears to be efficiently detoxified by oxidation and subsequent C1 metabolism.     

费氏中华根瘤菌HN01DL在大豆根圈的定殖动态与结瘤研究

以发光酶基因luxAB为标记,在根盒缩影条件下研究了费氏中华根瘤菌HN01DL在大豆根圈的定殖动态、分布范围及其结瘤情况.结果表明,HN01DL在根盒灭菌土壤和非灭菌土壤缩影中的大豆全根系定殖动态与水平明显不同,前者在第12d时达到最高定殖密度(8.65logcfu·g^-1),而后者的早期定殖数量下降较快,且于第15d时达到最高定殖密度(6.88logcfu·g^-1).HN01DL在大豆播种5d后在大豆根部的A(0～4cm)区根段上达到最高定殖密度(7.05logcfu·g^-1),然后开始缓慢下降,至第19d时仍维持相对稳定,在第33d时又开始回升.至播种后第46d时HN01DL可散布至种子下方16cm处的根段部位.HN01DL在A区根段的定殖密度持续较高,所形成的发光根瘤总数(16.3个)及发光比例(68.8%)最高,且发光根瘤主要集中于该区段主根上.发光根瘤比例沿A-E区段逐渐下降,在E区段未检测到发光根瘤.