Protein Modification and Utilization of Starch in Soybean (Glycine max L. Merr.) Seed Maturation

Seeds of soybean (Glycine max L. Merr.) harvested at variousstages of development and allowed to dry in intact pods undergoa maturation process and are viable. Defatted powders of seedharvested 24–66 d after flowering were extracted to yieldbuffer-soluble and alkali-soluble proteins. Imposition of amaturation process increased the level of buffer-soluble proteinsbut had no effect on the disulflde content of these proteins.After undergoing maturation, seeds showed an accumulation ofbuffer-soluble polypeptides in the molecular weight range of43–94 kD. Maturation may be associated with the synthesisof specific polypeptides having a molecular weight of approximately85 kD. Alkali-soluble proteins, which represents the storageproteins, did not show any responses to maturation. Their quantityincreased substantially during seed development and the disulfidelevel was only half that of buffer-soluble proteins, attaininga maximum value of 10.9 mol S per 10⁵ g protein. Matured seedat all harvest dates had a final starch content close to thatof normal seed, 10–20 mg g^–1, and soluble sugarswere maintained at quite high levels, 51–83 mg g^–1.The metabolic program for synthesis and degradation of starchseems quite rigidly followed and is independent of harvest dateor of attachment to the parent plant. Soybean seeds retain considerablesoluble proteins and soluble sugars throughout maturation, andthese collectively may be important in maintaining a desiccationresistant structure.     

Nitrate Reduction by Roots of Soybean (Glycine max [L.] Merr.) Seedlings

Studies were conducted with 9 to 12 day-old soybean (Glycine max [L.] Merr. cv. Williams) seedlings to determine the contribution of roots to whole plant NO(3) (-) reduction. Using an in vivo -NO(3) (-) nitrate reductase (NR) assay (no exogenous NO(3) (-) added to incubation medium) developed for roots, the roots accounted for approximately 30% of whole plant nitrate reductase activity (NRA) of plants grown on 15 mm NO(3) (-).Nitrogen analyses of xylem exudate showed that 53 to 66% of the total-N was as reduced-N, depending on the time of day of exudate collection. These observations supported enzyme data that suggested roots were contributing significantly to whole plant NO(3) (-) reduction. In short-term feeding studies using (15)N-NO(3) (-) significant and increasing atom percent (15)N excess was found in the reduced-N fraction of xylem exudate at 1.5 and 3 hours after feeding, respectively, which verified that roots were capable of reducing NO(3) (-).Estimated reduced-N accumulation by plants based on in vivo -NO(3) (-) NR assays of all plant parts substantially over-estimated actual reduced-N accumulation by the plants. Thus, the in vivo NR assay cannot be used to accurately estimate reduced-N accumulation but still serves as a useful assay for relative differences in treatment conditions.     

De Novo Purine Synthesis in Nitrogen-Fixing Nodules of Cowpea (Vigna unguiculata [L.] Walp.) and Soybean (Glycine max [L.] Merr.)

Partially purified, cell-free extracts from nodules of cowpea (Vigna unguiculata L. Walp. cv. Caloona) and soybean (Glycine max L. Merr. cv. Bragg) showed high rates of de novo purine nucleotide and purine base synthesis. Activity increased with rates of nitrogen fixation and ureide export during development of cowpea plants; maximum rates (equivalent to 1.2 micromoles N₂ per hour per gram fresh nodule) being similar to those of maximum nitrogen fixation (1-2 micromoles N₂ per hour per gram fresh nodule). Extracts from actively fixing nodules of a symbiosis not producing ureides, Lupinus albus L. cv. Ultra, showed rates of de novo purine synthesis 0.1% to 0.5% those of cowpea and soybean. Most (70-90%) of the activity was associated with the particulate components of the nodule, but up to 50% was released from this fraction by osmotic shock. The accumulated end products with particulate fractions were inosine monophosphate and aminoimidazole carboxamide ribonucleotide. Further metabolism to purine bases and ureides was restricted to the soluble fraction of the nodule extract. High rates of inosine monophosphate synthesis were supported by glutamine as amide donor, lower rates (10-20%) by ammonia, and negligible rates with asparagine as substrate.     

Light Regulation of beta-Tubulin Gene Expression during Internode Development in Soybean (Glycine max [L.] Merr.)

The relationship between tubulin gene expression and cell elongation was explored in developing internodes of Glycine max (L.) Merr., using light as a variable to alter the rate of elongation. First internodes of etiolated seedlings elongated two to three times more rapidly than did those of seedlings growing under a 12 hour diurnal light/dark cycle. Furthermore, light slowed or completely halted internode elongation in the etiolated seedlings, depending upon the age of the seedlings at the time of the light treatment. Steady state levels of β-tubulin mRNA were determined in Northern blots and by solution hybridization of poly(A)⁺RNA with a probe derived from the coding region of a previously characterized soybean β-tubulin gene. (MJ Guiltinan, DP Ma, RF Barker, MM Bustos, RJ Cyr, R Yadegari, DE Fosket [1987] Plant Mol Biol 10: 171-184). Internodes of light-grown seedlings exhibited levels of β-tubulin mRNA that differed by a factor of three, and varied concomitantly with the elongation rate. Illumination of 10-day-old etiolated seedlings not only stopped first internode elongation, but also brought about a 80% decrease in the steady state level of β-tubulin mRNA over the course of the subsequent 12 hours. This strong down regulation of β-tubulin mRNA occurred without significant changes in the size of the soluble tubulin pool and it was accompanied by a marked increase in chlorophyll a/b binding protein mRNA.     

Changes in Soybean (Glycine max [L.] Merr.) Glycerolipids in Response to Water Stress

Soybean (Glycine max [L.] Merr.) plants with the first trifoliate leaf fully expanded were exposed to 4 and 8 days of water stress. Leaf water potentials dropped from −0.6 megapascal to −1.7 megapascals after 4 days of stress; then to −3.1 megapascals after 8 days without water. All of the plants recovered when rewatered. The effects of short-term drought stress on triacylglycerol, diacylglycerol, phospholipid, and galactolipid metabolism in the first trifoliate leaves was determined. Leaf triacylglycerol and diacylglycerol content increased 2-fold during the first 4 days of stress and returned to control levels 3 days after rewatering. The polar lipid fraction, which contained phospholipids and galactolipids, changed little during this time. The linolenic acid (18:3) content of the triacylglycerol and diacylglycerol increased 25% during stress and the polar lipid 18:3 content decreased 15%. The pattern of glycerolipid labeling, after applying [2-¹⁴C]acetate to intact leaves was altered by water stress. After 4 days of water stress the radioactivity of phosphatidic acid + phosphatidylinositol, phosphatidylcholine, triacylglycerol, and diacylglycerol increased between 4 and 9% (compared to control plans) while radioactivity of phosphatidylethanolamine, monogalactosyldiglyceride, and digalactosyldiglyceride decreased 2 to 11%. These data indicated that increased levels of triacylglycerol and diacylglycerol observed during water stress were attributed to de novo synthesis rather than breakdown or reutilization of existing glycerolipids and fatty acids.     

低磷对大豆主根伸长生长的影响

文章采用卷纸培养和分层琼脂培养的方法,研究磷对大豆主根伸长影响的结果表明:低磷[0.2 μmol(KH2PO4)·L-1]显著促进大豆主根伸长,特别是延长大豆主根根尖至最新侧根间的距离;组织切片表明,低磷对主根伸长的促进主要是通过延迟主根伸长区的分化实现的,并且低磷对主根的促进作用不受亚磷酸盐的影响.琼脂分层培养的结果表明,在磷分布不均匀的条件下,低磷影响主根的仲长生长,上层或下层不施磷的大豆主根伸长均有增加.     

Subcellular Localization of Oxygen Defense Enzymes in Soybean (Glycine max [L.] Merr.) Root Nodules

Soybean (Glycine max [L.] Merr.) root nodules contain the enzymes of the ascorbate-glutathione pathway to minimize oxidative damage. In the present study, fractionation and immunocytochemistry were used to determine the subcellular location of the enzymes of this pathway. All four enzymes (ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, and glutathione reductase) were present in the soluble fraction from nodule plant cells and in isolated mitochondria. No activity was detected in peroxisomes. Bacteroids contained glutathione reductase but not the other enzymes of this pathway. Immunogold localization indicated that ascorbate peroxidase was present in the cytosol of infected and uninfected cells but not in the peribacteroid space. Results of immunogold and immunofluorescence studies indicated that monodehydroascorbate reductase was located primarily in the cell wall, suggesting that ascorbate regeneration in the cytoplasm may proceed primarily through the action of dehydroascorbate reductase. The possible roles of monodehydroascorbate reductase in cell wall metabolism are discussed.     

A Pod Leakage Technique for Phloem Translocation Studies in Soybean (Glycine max [L.] Merr.)

Radioactive photosynthetic assimilates, translocated to a soybean (Glycine max [L.] Merr. `Fiskeby V') pod can be measured directly by excising the stylar tip of the pod under 20 mm ethylenediaminetetraacetate solution (pH 7.0) and allowing the material to leak into the solution. Pods at the source node received approximately 50% of the ¹⁴C exported from the source leaf to the pod and leaked approximately 1 to 3% of this into the solution. More than 90% of the ¹⁴C that leaked from the pods was found in the neutral fraction and, of this, about 93% was in sucrose. Fifteen amino acids were identified in the leakage including: alanine, arginine, asparagine, γ-aminobutyric acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, phenylalanine, serine, threonine, tyrosine, and valine. The majority of the ¹⁴C in the basic fraction was found in serine (30%) and asparagine (23%). The inorganic ions K, Ca, P, Mg, Zn, and Fe were found in the leakage component. Nitrate was not detectable in the collected leakage solution. The absence of NO₃⁻ and the large proportion of the label in sucrose suggest a possible phloem origin for most of the material. The technique provides an uncomplicated, reproducible means of analyzing the material translocated into and through the soybean pod, as well as following the time course of label arrival at the pod.     

Sequence Level Analysis of Recently Duplicated Regions in Soybean [Glycine max (L.) Merr.] Genome

A single recessive gene, rxp, on linkage group (LG) D2 controlsbacterial leaf-pustule resistance in soybean. We identifiedtwo homoeologous contigs (GmA and GmA') composed of five bacterialartificial chromosomes (BACs) during the selection of BAC clonesaround Rxp region. With the recombinant inbred line populationfrom the cross of Pureunkong and Jinpumkong 2, single-nucleotidepolymorphism and simple sequence repeat marker genotyping wereable to locate GmA' on LG A1. On the basis of information inthe Soybean Breeders Toolbox and our results, parts of LG A1and LG D2 share duplicated regions. Alignment and annotationrevealed that many homoeologous regions contained kinases andproteins related to signal transduction pathway. Interestingly,inserted sequences from GmA and GmA' had homology with transposaseand integrase. Estimation of evolutionary events revealed thatspeciation of soybean from Medicago and the recent divergenceof two soybean homoeologous regions occurred at 60 and 12 millionyears ago, respectively. Distribution of synonymous substitutionpatterns, K_s, yielded a first secondary peak (mode K_s = 0.10–0.15)followed by two smaller bulges were displayed between soybeanhomologous regions. Thus, diploidized paleopolyploidy of soybeangenome was again supported by our study.     

Genetic Mapping of Soybean [Glycine max (L.) Merr.] Using Random Amplified Polymorphic DNA (RAPD)

Random amplified polymorphic DNA (RAPD) is based on DNA amplification by polymerase chain reaction (PCR) of random DNA segments using single arbitrary nucleotide sequences. We have adapted the assay to soybeans by using Stoffel Fragment DNA polymerase and by optimizing the reaction conditions. To increase the percentage of RAPD polymorphisms, the DNA template was digested with restriction enzymes before amplification. The combination of twenty-four primers and five DNA template treatments (Undigested, DraI, EcoRI, HindIII, and TaqI digested) revealed 94 polymorphic DNA fragments differing between soybean lines PI437654 and BSR101. Many polymorphic DNA bands were found unreliable or non-scoreable after re-screening of primers and verification of marker-allele segregation with 20 recombinant inbred lines (RILs). However, 28 RAPD markers were consistently polymorphic between the parental lines and followed Mendelian expectations. The use of DNA templates digested with DraI, EcoRI, HindIII or TaqI increased three times the number of RAPD markers compared to undigested DNA template alone. The 28 RAPD markers obtained were further screened with 72 RILs and placed on an existing RFLP map.     

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.     

Plant Growth-promoting Rhizobacteria and Soybean [ Glycine max (L.) Merr.] Growth and Physiology at Suboptimal Root Zone Temperatures

Application of plant growth-promoting rhizobacteria (PGPR) hasbeen shown to increase legume growth and development under optimaltemperature conditions, and specifically to increase nodulationand nitrogen fixation of soybean [Glycine max (L.) Merr.] overa range of root zone temperatures (RZTs). Nine rhizobacteriaapplied into soybean rooting media were tested for their abilityto reduce the negative effects of low RZT on soybean growthand development by improving the physiological status of theplant. Three RZTs were tested: 25, 17.5, and 15 °C. At eachtemperature some PGPR strains increased plant growth and development,but the stimulatory strains varied with temperature. The strainsthat were most stimulatory at each temperatures were as follows:15 °C—Serratia proteamaculans 1–102; 17.5 °C—Aeromonashydrophila P73, and 25 °C—Serratia liquefaciens 2–68.Because enhancement of plant physiological activities were detectedbefore the onset of nitrogen fixation, these stimulatory effectscan be attributed to direct stimulation of the plant by thePGPR rather than stimulation of plant growth via improvementof the nitrogen fixation symbiosis. Legume; nitrogen fixation; nodulation; root zone temperature; PGPR     

RFLP mapping using near-isogenic lines in the soybean [Glycine max (L.) Merr.]

Summary A molecular marker analysis of a near-isogenic line (NIL), its donor parent (DP), and its recurrent parent (RP) can provide information about linkages between molecular markers and a conventional marker introgressed into the NIL. If the DP and RP possess different alleles for a given molecular marker, and if the NIL possesses the same allele as the DP, then it is reasonable to presume a linkage between that molecular marker and the introgressed marker. In this study, we examined the utility of RFLPs as molecular markers for the NIL genemapping approach. The allelic status of fifteen RFLP loci was determined in 116 soybean RP/NIL/DP line sets; 66 of the Clark RP type and 50 of the Harosoy RP type. Of the 1740 possible allelic comparisons (116 NILs x 15 RFLP loci), 1638 were tested and 462 (33.9%) of those were informative (i.e., the RP and DP had different RFLP alleles). In 15 (3.2%) of these 462 cases the NIL possessed the DP-derived RFLP allele, leading to a presumption of linkage between the RFLP locus and the introgressed conventional marker locus. Two presumptive linkages, pK-3 — and pK-472 — Lf _i, were subsequently confirmed by cosegregation linkage analysis. Although not yet confirmed, two other associations, pk-7 ab and pK-229 — y ₉ seemed to be plausible linkages, primarily because the pk-7 — ab association was detected in two independently derived NILs and both markers of the pK-229 — y ₉ association were known to be linked to Pb. The data obtained in this investigation indicated that RFLP loci were useful molecular markers for the NIL gene-mapping technique.Published as Paper no. 9101, Journal Series, Nebraska Agric. Res. Div. Project no. 12-091. Research partially funded by a grant from the Nebraska Soybean Development, Utilization, and Marketing Board     

Cryopreservation of the SB-M photosynthetic soybean [Glycine max (L.) Merr.] suspension culture

Summary The photosynthetic cell suspension culture of soybean [Glycine max (L.) Merr. cv. Corsoy] (SB-M) was successfully cryopreserved in liquid nitrogen using a preculture and controlled freezing to −40° C (two-step) freezing method. The effective method included a preculture treatment with gradually increasing levels of sorbitol added to the 3% sucrose already present in the medium. The cells were then placed in a cryoprotectant solution [10% DMSO (dimethylsulfoxide) and 9.1% sorbitol, or 10% DMSO and 8% sucrose], incubated for 30 min at 0° C, cooled at a rate of 1° C/min to −40° C, held at −40° C for 1 h, and then immersed directly into liquid nitrogen. The cells were thawed at 40° C and then immediately placed in liquid culture medium. The cell viabilities immediately after thawing were 75% or higher in all cases where cell growth resumed. The original growth rate and chlorophyll level of the cells was recovered within 40 to 47 d. If the sorbitol level was not high enough or the preculture period too short, growing cultures could not be recovered. Likewise, survival was not attained with cryoprotectant mixtures consisting of 15% DMSO, 15% glycerol, and 9.1% sucrose or 15% glycerol and 8% sucrose. The successful method was reproducible, thus allowing long-term storage of this and certain other unique photosynthetic suspension cultures in liquid nitrogen.     

Efficiency of Nitrogen Assimilation by N(2)-Fixing and Nitrate-Grown Soybean Plants (Glycine max [L.] Merr.)

Nodulated and non-nodulated (not inoculated) soybeans (Glycine max [L.] Merr. cv Wells) were grown in controlled environments with N₂ or nonlimiting levels of NO₃⁻, respectively, serving as sole source of nitrogen. The efficiency of the N₂-fixing plants was compared with that of the nitrate-supplied plants on the basis of both plant age and plant size. Efficiency evaluations of the plants were expressed as the ratio of moles of carbon respired by the whole plant to the moles of nitrogen incorporated into plant material.

Continuous 24-hour CO₂ exchange measurements on shoot and root systems made at the beginning of flowering (28 days after planting) indicated that N₂-fixing plants respired 8.28 moles of carbon per mole of N, fixed from dinitrogen, while nitrate-supplied plants respired only 4.99 moles of carbon per mole of nitrate reduced. Twenty-one-day-old nitrate-supplied plants were even more efficient, respiring only 3.18 moles of carbon per mole of nitrate reduced. The decreased efficiency of the N₂-fixing plants was not due to plant size since, on a dry weight basis, the 28-day-old N₂-fixing plants were intermediate between the 28- and 21-day-old nitrate-supplied plants.

The calculated efficiencies were predominantly a reflection of root-system respiration. N₂-fixing plants lost 25% of their daily net photosynthetic input of carbon through root-system respiration, compared with 16% for 28-day-old nitrate-supplied plants and 12% for 21-day-old nitrate-supplied plants. Shoot dark respiration was similar for all three plant groups, varying between 7.9% and 9.0% of the apparent photosynthate.

The increased respiratory loss by the roots of the N₂-fixing plants was not compensated for by increased net photosynthetic effectiveness. Canopy photosynthesis expressed on a leaf area basis was similar for 28-day-old N₂-fixing plants (15.5 milligrams CO₂ square decimeter per hour) and 21-day-old nitrate-supplied plants (14.5 milligrams CO₂ square decimeter per hour). Both were similar in total canopy leaf area. The larger nitrate-supplied plants (28-day-old) had lower photosynthetic rates (12.5 milligrams CO₂ square decimeter per hour), presumably due to self-shading of the leaves.

These data indicate that, during the early stages of plant development, dependence solely on N₂-fixation is an expensive process compared to nitrate reduction in nitrate-supplied plants, since the N₂-fixing plants retained 8% to 12% less of their photosynthate as dry matter.

     

Genetics and cytology of a new genic male-sterile soybean [Glycine max (L.) Merr.]

 Genetic and cytological studies were conducted with a new male-sterile, female-fertile soybean [Glycine max (L.) Merr.] mutant. This mutant was completely male sterile and was inherited as a single-recessive gene. No differences in female or male gamete transmission of the recessive allele were observed between reciprocal cross-pollinations in the F₁ or F₂ generations. This mutant was not allelic to any previously identified soybean genic male-sterile mutants: ms1, ms2, ms3, ms4, ms5, or ms6. No linkage was detected between sterility and flower color (W1 locus), or between sterility and pubescence color (T1 locus). Light microscopic and cytological observations of microsporogenesis in fertile and sterile anthers were conducted. The structure of microspore mother cells (MMC) in male-sterile plants was identical to the MMCs in male-fertile plants. Enzyme extraction analyses showed that there was no callase activity in male-sterile anthers, and this suggests that sterility was caused by retention of the callose walls, which normally are degraded around tetrads at the late tetrad stage. The tapetum from male-sterile anthers also showed abnormalities at the tetrad stage and later stages, which were expressed by an unusual formation of vacuoles, and by accumulation of densely staining material. At maturity, anthers from sterile plants were devoid of pollen grains. Received: 13 May 1996 / Revision accepted: 19 August 1996     

Growth and Development of Soybean (Glycine max [L.] Merr.) Pods: CO(2) Exchange and Enzyme Studies

The rates of CO₂ exchange and ¹⁴CO₂ incorporation in the light and dark and the activities of several photosynthetic, photorespiratory, and respiratory enzymes of soybean (Glycine max [L.] Merr. cv. Wye) reproductive structures were determined at weekly intervals from anthesis to pod maturity. At all stages of pod development soybean reproductive structures were found to be incapable of net photosynthesis under the experimental conditions employed, but capable of gross photosynthesis and light-induced ¹⁴CO₂ uptake. Consistent with the lack of net photosynthesis throughout the development of the reproductive structure, the maximum in vitro activity of ribulose 1,5-bisphosphate carboxylase (EC 4.1.1.39) in pod tissue was only 3% of that in leaf extracts when expressed on a fresh weight basis. We concluded that the major role of the reproductive structure of the soybean with respect to photosynthetic carbon metabolism is the reassimilation of its respiratory CO₂.     

Haloxyfop Inhibition of the Pyruvate and the alpha-Ketoglutarate Dehydrogenase Complexes of Corn (Zea mays L.) and Soybean (Glycine max [L.] Merr.)

The grass-specific herbicide haloxyfop, ((±)-2-[4-((3-chloro-5-(trifluoromethyl)-2-pyridinyl)oxy)-phenoxy] propionic acid) has been shown to inhibit lipid synthesis and respiration, to cause the accumulation of amino acids, and not to affect cellular sugar or ATP levels. Thus studies were carried out with enzyme activities from corn (Zea mays L.) (haloxyfop sensitive) and soybean (Glycine max [L.] Merr.) (haloxyfop tolerant) to locate the possible inhibition sites among the glycolytic and tricarboxylic acid (TCA) cycle enzymes. Following along the oxidative metabolism pathway of sugars, the pyruvate dehydrogenase complex (PDC) was the first enzyme among the glycolytic enzymes that demonstrated noticeable inhibition by 1 millimolar haloxyfop. Kinetic studies with corn and soybean PDC from both purified etioplasts and mitochondria gave K_i values of from 1 to 10 millimolar. Haloxyfop also inhibited the activity of the TCA cycle enzyme, the α-ketoglutarate dehydrogenase complex (α-KGDC) which carries out the same reaction as PDC except for the substitution of α-ketoglutarate for pyruvate as one of the substrates. The K_i values were somewhat lower in this case (near 1 millimolar). The relatively high K_i values for both enzyme complexes would indicate that these may not be the herbicidal sites of inhibition, but it is possible that the herbicide could be concentrated in compartments and/or the substrate concentrations may be well below optimal. Likewise little difference was seen in the haloxyfop inhibition of the enzyme activities from the sensitive species, corn, and from the tolerant species, soybean, so the selectivity of the herbicide is not evident from these results. The inhibition of the PDC and α-KGDC as the mode of action of haloxyfop is, however, consistent with the observed physiological effects of the herbicide, and these are the only enzymic activities so far found to be sensitive to haloxyfop.