Purification and Properties of Arginase from Soybean, Glycine max, Axes

Arginase (EC 3.5.3.1) was purified to homogeneity from cytosol of soybean, Glycine max, axes by chromatographic separations on Sephadex G-200, DEAE-sephacel, hydroxyapatite, and arginine-affinity columns. The molecular weight of the enzyme estimated by pore gradient gel electrophoresis was 240,000, while sodium dodecyl sulfate polyacrylamide gel electrophoresis gave a single band at the molecular weight of 60,000. The optimal pH for activity was 9.5 and the K_m value was 83 millimolar. The enzyme was stimulated by polyamines such as putrescine.     

Plant Regeneration from Protoplasts of Soybean (Glycine max L.)

Protoplasts were isolated enzymatically from immature cotyledons of soybean. The protoplasts divided to form calli in the K8P liquid medium. The calli further grew to 2–3 mm on the solid K8 medium and were transferred onto the MSB medium (MS minerals+B5 organic components+0.5–1.0 mg/l 2,4-D+0.2–0.5 mg/l BA) to obtain compact and nodular calli. Shoot formation was initiated on M1 medium (MSB medium with 0.15 mg/1 NAA, and BA, KT and ZT, 0.5 mg/l of each, 500 mg/1 CH). Differentiation frequency was 13.6–24.2%. Plants have been regenerated from protoplasts of immature cotyledons in 2 cultivars, and normal pods were obtained from them.     

Purification and Some Properties of the Nitrogenase From Soybean (Glycine max Merr.) Nodules

The nitrogenase system in cell-free extracts of soybean nodule bacteroids was fractionated into 2 components by use of protamine sulfate or polypropylene glycol precipitation followed by chromatography on DEAE-cellulose. Iron and molybdenum were concentrated in 1 fraction and iron in the other. Combination of fractions resulted in a striking stimulation in activity relative to the activity of individual fractions. The effect of different proportions of the 2 fractions on specific activities was studied. The ratios of the rates of reduction of acetylene and N₂ by extracts or fractions of different purities were relatively constant. Extracts or fractions retained most of their nitrogenase activities when stored in liquid N₂.     

Nitrate Reductases from Wild-Type and nr(1)-Mutant Soybean (Glycine max [L.] Merr.) Leaves : I. Purification, Kinetics, and Physical Properties

NADH:nitrate reductase (EC 1.6.6.1) and NAD(P)H:nitrate reductase (EC 1.6.6.2) were purified from wild-type soybean (Glycine max [L.] Merr., cv Williams) and nr₁-mutant soybean plants. Purification included Blue Sepharose- and hydroxylapatite-column chromatography using acetone powders from fully expanded unifoliolate leaves as the enzyme source.

Two forms of constitutive nitrate reductase were sequentially eluted with NADPH and NADH from Blue Sepharose loaded with extract from wild-type plants grown on urea as sole nitrogen source. The form eluted with NADPH was designated c₁NR, and the form eluted with NADH was designated c₂NR. Nitrate-grown nr₁ mutant soybean plants yielded a NADH:nitrate reductase (designated iNR) when Blue Sepharose columns were eluted with NADH; NADPH failed to elute any NR form from Blue Sepharose loaded with this extract. Both c₁NR and c₂NR had similar pH optima of 6.5, sedimentation behavior (s_20,w of 5.5-6.0), and electrophoretic mobility. However, c₁NR was more active with NADPH than with NADH, while c₂NR preferred NADH as electron donor. Apparent Michaelis constants for nitrate were 5 millimolar (c₁NR) and 0.19 millimolar (c₂NR). The iNR from the mutant had a pH optimum of 7.5, s_20,w of 7.6, and was less mobile on polyacrylamide gels than c₁NR and c₂NR. The iNR preferred NADH over NADPH and had an apparent Michaelis constant of 0.13 millimolar for nitrate.

Thus, wild-type soybean contains two forms of constitutive nitrate reductase, both differing in their physical properties from nitrate reductases common in higher plants. The inducible nitrate reductase form present in soybeans, however, appears to be similar to most substrateinduced nitrate reductases found in higher plants.

     

大豆遗传转化研究进展

综述了大豆遗传转化体系及其优缺点,转基因大豆的研究成果、生产状况和生物安全性评价,分析了大豆遗传转化中存在的一些问题及其解决办法,展望了未来大豆遗传转化的发展前景。     

Improving Soybean (Glycine max L.) N2 Fixation under Stress

Soybean is a major leguminous plant that has the ability to establish a symbiotic association with the N-fixing bacteria, Bradyrhizobium japonicum. Soils are usually subjected to stress including salinity, drought, acidity, and suboptimal root zone temperature, adversely affecting the symbiotic process between soybean and the bacteria. One of the important processes affecting the performance of soybean under stress is the inhibited exchange of symbiosis-related signaling molecules, specifically genistein, between the host legume and B. japonicum during the initiation of symbiosis. Interestingly, inoculation of B. japonicum with the signal molecule genistein can partially or completely alleviate the stress. Understanding the techniques and the precise molecular pathways, which may be influenced by the signaling molecules during the stress, can be useful to determine parameters that enhance the plant’s ability to cope with stress. For example, the use of proteomic techniques to identify proteins expressed under stress can help characterize those proteins and their involvement in stress. Biotechnological-genetic techniques, either breeding or transformation, are also among the effective methods of improving soybean’s ability to fix N₂ under stress. This can be achieved by identifying the genes, which may be expressed under stress in tolerant bacterial and plant species, and inserting them into the non-tolerant species. This article highlights some important advances in soybean N₂ fixation under different stress conditions, and reviews some of the techniques used to improve the ability of plants and bacteria to efficiently fix N₂ under stress.     

Molecular Genetic Identification and Certification of Soybean (Glycine max L.) Cultivars

An approach to certification of soybean genotypes has been developed. The procedure employs three methods of DNA analysis based on polymerase chain reaction (PCR): PCR with arbitrary primers (AP PCR), simple sequence repeat polymorphism (SSRP) analysis, and inter-simple sequence repeat (ISSR) analysis. The approach to certification proposed may be used in both genetic and breeding research and seed production. A certificate form that reflects the unique characteristics of each cultivar studied is proposed. The results of molecular genetic analysis of allele distribution in genotypes of soybean from different ecological geographic zones permit estimation of the adaptive significance of individual alleles.     

Dehydration Injury in Germinating Soybean (Glycine max L. Merr.) Seeds

The sensitivity of soybean (Glycine max L. Merr. cv Maple Arrow) seeds to dehydration changed during germination. Seeds were tolerant of dehydration to 10% moisture if dried at 6 hours of imbibition, but were susceptible to dehydration injury if dried at 36 hours of imbibition. Dehydration injury appeared as loss of germination, slower growth rates of isolated axes, hypocotyl and root curling, and altered membrane permeability. Increased electrolyte leakage due to dehydration treatment was observed only from isolated axes but not from cotyledons, suggesting that cotyledons are more tolerant of dehydration. The transition from a dehydration-tolerant to a dehydration-susceptible state coincided with radicle elongation. However, the prevention of cell elongation by osmotic treatment in polyethylene glycol (−6 bars) or imbibition in 20 micrograms per milliliter cycloheximide did not prevent the loss of dehydration tolerance suggesting that neither cell elongation nor cytoplasmic protein synthesis was responsible for the change in sensitivity of soybean seeds to dehydration. Furthermore, the rate of dehydration or rate of rehydration did not alter the response to the dehydration stress.     

Sulphate Transport into Plants and Excised Roots of Soybean (Glycine max L.)

The rates of sulphate transport into intact and excised rootsof soybean (Glycine max L.) were not significantly differentin the first hour and were maximal at pH 7. However, intactroots accumulated four times as much sulphate as excised rootsin 24 h, because of a marked reduction in the rate of transportby excised roots. The continued high rates of transport intointact roots were observed in plants kept in the light, andobserved in darkened plants growing in 1 per cent sucrose. Similarly,sulphate accumulation by excised roots was stimulated 2-foldby 1 per cent sucrose. The characteristics of sulphate accumulation by roots were notuseful in predicting sulphate translocation to the leaves. Transportto the leaves was maximal at pH 2–3, was almost totallylight-dependent and was not enhanced by growing plants in sucrose. Sulphate transport, Glycine max L., soybean, excised roots     

Aluminium Stress Affects Nitrogen Fixation and Assimilation in Soybean (Glycine max L.)

Nitrogen fixation and assimilation in nodules and roots were studied in soybean (Glycine max L.) exposed to different levels of aluminium (Al) stress (0, 50, 200 and 500 μM). Al at 500 μM induced oxidative stress, which became evident from an increase in lipid peroxidation accompanied by a concomitant decline in antioxidant enzyme activities and leghaemoglobin breakdown. Consequently, there was also a reduction in nitrogenase activity. However, the leghaemoglobin levels and nitrogenase activity were unexpectedly found to be higher in nodules when the plants were treated with 200 μM Al. Of the enzymes involved in nitrogen assimilation, the activity of glutamate dehydrogenase-NADH was reduced in nodules under Al stress, but it was significantly higher in roots at 500 μM Al as compared to that in the control. In nodules, the glutamine synthetase/glutamate synthase-NADH pathway, assayed in terms of activity and expression of both the enzymes, was inhibited at >50 μM Al; but in roots this inhibitory effect was apparent only at 500 μM Al. No significant changes in ammonium and protein contents were recorded in the nodules or roots when the plants were treated with 50 μM Al. However, Al at ≥200 μM significantly increased the ammonium levels and decreased the protein content in the nodules. But these contrasting effects on ammonium and protein contents due to Al stress were observed in the roots only at 500 μM Al. The results suggest that the effect of Al stress on nitrogen assimilation is more conspicuous in nodules than that in the roots of soybean plants.     

Composition and Distribution of Adenylates in Soybean (Glycine max L.) Nodule Tissue

Adenylates (ATP, ADP, and AMP) may play a central role in the regulation of the O2-limited C and N metabolism of soybean nodules. To be able to interpret measurements of adenylate levels in whole nodules and to appreciate the significance of observed changes in adenylates associated with changes in O2-limited metabolism, methods were developed for measuring in vivo levels of adenylate pools in the cortex, plant central zone, and bacteroid fractions of soybean (Glycine max L. Merr cv Maple Arrow x Bradyrhizobium japonicum strain USDA 16) nodules. Intact nodulated roots were either frozen in situ by flushing with prechilled Freon-113(-156[deg]C) or by rapidly (<1 s) uprooting plants and plunging them into liquid N2. The adenylate energy charge (AEC = [ATP + 0.5 x ADP]/[ATP + ADP + AMP]) of whole-nodule tissue (0.65 [plus or minus] 0.01, n = 4) was low compared to that of subtending roots (0.80 [plus or minus] 0.03, n = 4), a finding indicative of hypoxic metabolism in nodules. The cortex and central zone tissues were dissected apart in lyophilized nodules, and AEC values were 0.84 [plus or minus] 0.04 and 0.61 [plus or minus] 0.03, respectively. Although the total adenylate pool in the lyophilized nodules was only 41% of that measured in hydrated tissues, the AEC values were similar, and the lyophilized nodules were assumed to provide useful material for assessing adenylate distribution. The nodule cortex contained 4.4% of whole-nodule adenylates, with 95.6% being located in the central zone. Aqueous fractionation of bacteroids from the plant fraction of whole nodules and the use of marker enzymes or compounds to correct for recovery of bacteroids and cross-contamination of the bacteroid and plant fractions resulted in estimates that 36.2% of the total adenylate pool was in bacteroids, and 59.4% was in the plant fraction of the central zone. These are the first quantitative assessments of adenylate distribution in the plant and bacteroid fractions of legume nodules. These estimates were combined with theoretical calculations of rates of ATP consumption in the cortex (9.5 nmol g-1 fresh weight of nodule s-1), plant central zone (38 nmol g-1 fresh weight of nodule s-1), and bacteroids (62 nmol g-1 fresh weight of nodule s-1) of soybean nodules to estimate the time constants for turnover of the total adenylate pool and the ATP pool within each nodule fraction. The low values for time constant (1.6-5.8 s for total adenylate, 0.9-2.5 s for ATP only) in each fraction reflect the high metabolic activity of soybean nodules and provide a background for further studies of the role of adenylates in O2-limited nodule metabolism.     

Purification and Characterization of Arginine Decarboxylase from Soybean (Glycine max) Hypocotyls

Arginine decarboxylase (EC 4.1.1.19 [EC] ) was purified from soybean,Glycine max, hypocotyls by a procedure which includes ammoniumsulfate fractionation, acetone precipitation, gel filtrationchromatography, and affinity chromatography. Using this procedure,ADC was purified to one band in non-denaturing PAGE. The purifiedADC has an M_r of 240 kDa based on gel filtration chromatographyand is a trimer of identical subunits which has an estimatedM_r of 74 kDa based on SDS-PAGE. ADC is active between 30 and50°C and has a K_m value of 46.1 µM. ADC is very sensitiveto agmatine or putrescine but not to spermidine or spermine.In the presence of 0.5 mM agmatine (or putrescine), the enzymeactivity was inhibited by 70%. However, at the same concentrationof spermidine (or spermine), the enzyme activity was inhibitedby only 10–20%. (Received April 2, 1997; Accepted August 18, 1997)     

Purification and Characterization of Membrane-Bound Inositol Phospholipid-Specific Phospholipase C from Suspension-Cultured Rice (Oryza sativa L.) Cells (Identification of a Regulatory Factor)

A membrane-bound inositol phospholipid-specific phospholipase C was solubilized from rice (Oryza sativa L.) microsomal membranes and purified to apparent homogeneity using a series of chromatographic separations. The apparent molecular mass of the enzyme was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 42,000 D, and the isoelectric point was 5.1. The optimum pH for the enzyme activity was approximately 6.5, and the enzyme was activated by both Ca2+ and Sr2+. The chemical and catalytic properties of the purified membrane-bound phospholipase C differed from those of the soluble enzyme reported previously (K. Yotsushima, K. Nakamura, T. Mitsui, I. Igaue [1992] Biosci Biotech Biochem 56: 1247-1251). In addition, we found a regulatory factor for the phosphatidylinositol-4,5-bisphosphate (PIP2) hydrolyzing activity of phospholipase C from rice cells. The regulatory factor was dissociated from the catalytic subunit of phospholipase C during the purification. The regulatory factor was necessary to induce PIP2-hydrolyzing activity of both membrane-bound and -soluble phospholipase C; these purified enzymes had no activity alone. Because the plasma membranes isolated from rice cells could also act as a regulatory factor, the regulatory factor seems to be localized in the plasma membranes. Regulation of inositol phospholipid turnover in rice cells is discussed.     

大豆中L34基因的克隆与表达分析

目的:采用分子手段试图分离与大豆种子低温吸胀相关的基因.方法:利用cDNA-AFLP(cDNA-amplified fragment length polymorphism)和RACE技术从大豆种子胚轴中克隆到一个编码132 kD的全长核糖体蛋白基因,命名为SOL34.结果:序列分析表明,SOL34和苜蓿(gi | 113205273 |)、茄科植物(gi | 48057670 |)及拟南芥(gi | 2500376 |)中L34蛋白基因的同源性分别为95%、95%和90%.半定量RT-PCR结果表明:大豆种子在4℃下吸胀24h内,胚轴中的SOL34被诱导表达,其中当种子低温吸胀6h,SOL34表达量升高非常明显,18h的表达量最大,24h表达量和18h相似;SOL34在大豆不同部位的表达不同,4叶期的大豆幼苗经过低温处理后,根尖中SOL34的表达比非处理材料增强约5倍,同时比胚轴中高约2倍;但是在叶片中,SOL34表达量并没有受到温度的影响,表达量也很弱,说明SOL34在叶片中是组成型表达.结论:结果分析表明,SOL34可能和大豆根的代谢有关.     

l-Arginine and l-Canavanine Metabolism in Jack Bean, Canavalia ensiformis (L.) DC. and Soybean, Glycine max (L.) Merr

Studies have been conducted with the arginase (l-arginine amidinohydrolase, EC 3.5.3.1) of two legumes: jack bean, Canavalia ensiformis (L.) DC., a l-canavanine-containing plant and soybean, Glycine max, a canavanine-free species. Analyses of the arginase obtained from gradient-purified mitochondria of these legumes revealed that the arginine-dependent (ADA) and canavanine-dependent activities (CDA) were localized within this organelle.     

ATP Sulfurylase Activity in the Soybean [Glycine max (L.) Merr.

ATP sulfurylase activity was assayed in soybean leaf extracts. A simple, rapid assay system using molybdate as an analogue of sulfate was developed. The assay was coupled to inorganic pyrophosphatase. The high pyrophosphatase level in soybean leaf extracts obviated the necessity of adding this enzyme to the assay system. ATP sulfurylase has a pH maximum above 7.5, uses molybdate and ATP as substrates, and requires magnesium ions for activity.     

Factors Affecting Shedding of Flowers in Soybean (Glycine max (L.) Merrill)

Flower shedding in soybean, Glycine max (L.) Merrill, was studiedusing cultivar ‘Clark’, isoline E₁t, which has relativelylong racemes for convenient identification and observation ofindividual flowers. On each raceme studied, pod set was greatestat the proximal (basal) positions, whereas shedding was greatestat the most distal positions. When proximal flowers were removedas they reached anthesis, pod set increased at the more distalpositions. Pod set was increased in some instances by applicationof water directly to the ovaries as a drop in the calyx cup.Peroxidase activity changed in parallel with ovary development,increasing rapidly in growing pods but not in shedding flowers.Increases in flower peroxidase was mainly in ovary walls. Flowerstaken at or near anthesis from positions with high percent podset could be grown in vitro with especially good ovary enlargement,whereas ovaries in flowers taken from positions of low pod setdid not enlarge in culture. Unidentified substances were extracted from young pods which,when incorporated into lanolin and tested in an in situ bioassay,could mimic the effect of proximal flowers in inducing sheddingof distal flowers. Indole-3-acetic acid resembled the extractedmaterials in inducing shedding, but differed by eliciting side-effectsthat extracts did not. The growth substances abscisic acid,gibberellic acid, and benzyladenine did not promote sheddingin the in situ test. The evidence was taken to indicate that soybean flower sheddingis induced in distal flowers by substances from the more proximal,fertilized ovaries, and that this is possibly due to interferencewith some of the intense metabolic changes that follow pollinationand fertilization.     

Soybean (Glycine max. L.) and bacteroid glyoxylate cycle activities during nodular senescence

Soybean (Glycine max. L.) nodular senescence results in the dismantling of the peribacteroid membrane (PBM) and in an increase of soybean isocitrate lyase (ICL; EC 4.1.3.1) and malate synthase (MS; EC 4.1.3.2) mRNA and protein levels. This suggests that in senescing soybean nodular cells, the specific glyoxylate cycle enzyme activities might be induced to reallocate carbon obtained from the PBM degradation. In order to evaluate as well the carbon metabolism of the nitrogen-fixing Bradyrhizobium japonicum endosymbiotic bacteroids during nodular senescence, their glyoxylate cycle activities were also investigated. To this end, partial DNA sequences were isolated from their icl and ms genes, but the corresponding mRNAs were not detected in the microorganisms. It was also observed that the bacteroid ICL and MS activities were negligible during nodular senescence. This suggests that glyoxylate cycle activities are not reinitiated in the bacteroids under these physiological conditions. In case the microorganisms nevertheless feed on the PBM degradation products, this might occur via the citric acid cycle exclusively.     

大豆GmCOL8基因的克隆及表达分析

从大豆品种‘垦农18’中克隆了一个CONSTANS—like基因,命名为GmCOL8。进化树分析表明它属于CONSTANS—like亚家族1的成员。mRNA表达分析显示,GmCOL8在短日下具有明显的生物钟节律性表达特性,其表达高峰出现在凌晨。GmCOL8主要在复叶中表达,表达高峰出现在开花时期。