Nitrate Reductase Activity in Soybeans (Glycine max [L.] Merr.): I. Effects of Light and Temperature

The optimum in vivo nitrate reductase (NR) assay medium for soybean (Glycine max [L.] Merr.) leaves was 50 mm KNO(3), 1% (v/v) 1- propanol, and 100 mm potassium phosphate buffer (pH 7.5).Loss of in vivo NR activity from leaves of soybeans exposed to dark was fastest at 40 C and slowest at 20 C. However, by the end of a 16-hr dark period, even those plants exposed to the lowest (20 C) temperature had lost 95% of the initial activity. Upon re-exposure to light, following a 16 hr-30 C dark period, in vivo NR activity increased rapidly to maximum levels after 4 hr light. The rate of increase was proportional to light intensity (6, 16, and 45 klux) and independent of temperature (20, 30, and 40 C).Studies with field-grown soybeans indicated that mighttime temperature (16-27 C) had no effect on the subsequent in vivo NR activity in sunlight at ambient temperature. There was a marked decrease in in vivo NR activity in late afternoon with the field-grown plants. This decrease continued throughout the night with elevated temperature (27 C) while NR activity increased when a cooler (16 C) night temperature was imposed.The changes in in vivo NR activity in response to light and dark treatments were quite rapid and thought to be related to energy limitations as well as enzyme level.     

Canopy and Seasonal Profiles of Nitrate Reductase in Soybeans (Glycine max L. Merr.)

Nitrate reductase activity of soybeans (Glycine max L. Merr.) was evaluated in soil plots and outdoor hydroponic gravel culture systems throughout the growing season. Nitrate reductase profiles within the plant canopy were also established. Mean activity per gram fresh weight per hour of the entire plant canopy was highest in the seedling stage while total activity (activity per gram fresh weight per hour times the total leaf weight) reached a maximum when plants were in the full bloom to midpod fill stage. Nitrate reductase activity per gram fresh weight per hour was highest in the uppermost leaf just prior to full expansion and declined with leaf position lower in the canopy. Total nitrate reductase activity per leaf was also highest in the upper-most fully expanded leaf during early growth stages. Maximum total activity shifted to leaf positions lower in the plant canopy with later growth stages.     

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.     

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.     

Flower and Pod Development in Water-Deficient Soybeans (Glycine max L. Merr.)

Water deficits during flowering decrease the number of seed-bearingpods in soybean (Glycine max L. Merr.). Failure to set podsmay indicate an inherent sensitivity to low tissue water potential(     

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     

Nitrate Reductases from Wild-Type and nr(1)-Mutant Soybean (Glycine max [L.] Merr.) Leaves : II. Partial Activity, Inhibitor, and Complementation Analyses

Soybean (Glycine max [L.] Merr.) leaves have been shown to contain three forms of nitrate reductase (NR). Two of the forms, which are present in leaves of wild-type (cv. Williams) plants grown in the absence of NO₃⁻, are termed constitutive and designated c₁NR and c₂NR. The third form, which is present in NO₃⁻-grown mutant (nr₁) plants lacking the constitutive forms, is termed inducible and designated iNR. Samples of c₁NR, c₂NR, and iNR obtained from appropriately treated plants were analyzed for the presence of partial activities, response to inhibitors, and ability to complement a barley NR which lacks the molybdenum cofactor (MoCo) but is otherwise active.

The three forms were similar to most assimilatory NR enzymes in that they (a) exhibited NADH-cytochrome c reductase, reduced flavin mononucleotide-NR, and reduced methyl viologen-NR partial activities; (b) were inhibited by p-hydroxymercuribenzoate at the site of initial electron transport through each enzyme; (c) were more inhibited by CN⁻ in their reduced enzyme state as compared with their oxidized state; and (d) complemented a MoCo-defective NR (e.g. contained cofactors with characteristics similar to the MoCo found in barley NR and commercial xanthine oxidase). However, among themselves, they showed dissimilarities in their response to treatment with HCO₃⁻ and CN⁻, and in their absolute ability to complement the barley NR. The site of effect for these treatments was the terminal cofactor-containing portion of each enzyme. This indicated that, although a terminal cofactor (presumably a MoCo) was present in each form, structural or conformational differences existed in the terminal cofactor-protein complex of each form.

     

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.     

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.     

Cytokinin Regulation of Flower and Pod Set in Soybeans (Glycine max(L.) Merr.)

Exogenous application of cytokinin to raceme tissues of soybean(Glycine max(L.) Merr.) has been shown to stimulate flower productionand to prevent flower abortion. The effects of these hormoneapplications have been ascertained for treated tissues, butthe effects of cytokinins on total seed yields in treated plantshave not been evaluated. Our objectives were to examine theeffects of systemic cytokinin applications on soybean yieldsusing an experimental line of soybeans, SD-87001, that has beenshown to be highly sensitive to exogenous cytokinin application.Soybeans were grown hydroponically or in pots in the greenhouse,and 6-benzylaminopurine (BA) was introduced into the xylem streamthrough a cotton wick for 2 weeks during anthesis. After theplants had matured, the number of pods, seeds per pod, and thetotal seed weight per plant were measured. In the greenhouse,application of 3.4 x 10^-7 moles of BA resulted in a 79% increasein seed yield compared with controls. Results of field trialsshowed much greater variability within treatments, with consistent,but non-significant increases in seed number and total yieldsof about 3%. Data suggest that cytokinin levels play a significantrole in determining total yield in soybeans, and that increasingcytokinin concentrations in certain environments may resultin increased total seed production. Copyright 2001 Annals ofBotany Company Glycine max, soybean, flower abortion, cytokinin, 6-benzylaminopurine, hydroponic, seed yield, wicking     

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     

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.     

Protein Synthesis during Natural and Precocious Soybean Seed (Glycine max [L.] Merr.) Maturation

Protein synthesis was studied during precocious and natural soybean seed (Glycine max [L.] Merr.) maturation. Developing seeds harvested 35 days after flowering were precociously matured through controlled dehydration. Total soluble proteins and proteins labeled with [³⁵S]methionine were extracted from control, developing seeds and from precociously and naturally matured seeds and were analyzed by one-dimensional PAGE and fluorography. The results demonstrated that several polypeptides which were designated “mature polypeptides,” were synthesized de novo during precocious and natural seed maturation. Two of these polypeptides, 31 and 128 kilodalton in mass, also stained intensely with Coomassie blue, suggesting their abundant accumulation during seed maturation. Results from in vitro translation experiments showed that the mRNAs corresponding to these “maturation polypeptides” accumulated during precocious maturation and in naturally matured seeds, but not in seeds freshly harvested 35 days after flowering (control). The role of the “maturation polypeptides” is currently unknown; however, their presence and that of their corresponding mRNAs was coincident with the ability of matured seeds to establish seedling growth. This study has demonstrated that precocious seed maturation treatments may be extremely useful for investigations of metabolic events and molecular control mechanisms affecting soybean seed maturation.     

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.     

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.     

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.     

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.     

Factors underlying genotypic differences in the induction of photosynthesis in soybean [Glycine max (L.) Merr.]

Crop leaves are subject to continually changing light levels in the field. Photosynthetic efficiency of a crop canopy and productivity will depend significantly on how quickly a leaf can acclimate to a change. One measure of speed of response is the rate of photosynthesis increase toward its steady state on transition from low to high light. This rate was measured for seven genotypes of soybean [Glycine max (L.) Merr.]. After 10 min of illumination, cultivar ‘UA4805’ (UA) had achieved a leaf photosynthetic rate (P_n) of 23.2 μmol · m^?2 · s^?1, close to its steady‐state rate, while the slowest cultivar ‘Tachinagaha’ (Tc) had only reached 13.0 μmol · m^?2 · s^?1 and was still many minutes from obtaining steady state. This difference was further investigated by examining induction at a range of carbon dioxide concentrations. Applying a biochemical model of limitations to photosynthesis to the responses of P_n to intercellular CO₂ concentration (C_i), it was found that the speed of apparent in vivo activation of ribulose‐1:5‐bisphosphate carboxylase/oxygenase (Rubisco) was responsible for this difference. Sequence analysis of the Rubisco activase gene revealed single nucleotide polymorphisms that could relate to this difference. The results show a potential route for selection of cultivars with increased photosynthetic efficiency in fluctuating light.