Analysis of polyamine metabolism in soybean seedlings using 15N-labelled putrescine

The translocation and metabolism of polyamines during soybean germination were studied using 15N-labelled putrescine as a precursor. Both 15N-labelled and unlabelled polyamines were simultaneously detected using a novel application of ionspray ionization-mass spectrometry. 15N-putrescine was rapidly transported to the shoots and roots, where it was converted to spermidine and spermine. The main 15N-polyamine that accumulated in the root was 15N-spermine. It was found that there were differences in the way endogenous putrescine and exogenous 15N-putrescine were metabolized in soybean seedlings.     

Lectin in five soybean cultivars previously considered to be lectin-negative

Hemagglutinating proteins were isolated by affinity chromatography from seeds of each of five cultivars of soybeans (Clycine max (L.) Merr.) previously reported to lack detectable lectin (S.P. Pull et al., 1978; Science 200, 1277). Quantities were between 1,000 and 10,000 times less than that found in the seeds of the reference cultivar, Chippewa. The sensitivity of the hemagglutinating assay was 0.05 g ml^-1. Hemagglutinating activity was demonstrated in affinity-purified fractions from bulk seeds and seeds from individual plants in two cultivars, 30–70% ammonium-sulfate-precipitable fractions of seeds from individual plants of all five cultivars, and in whole crude extracts of individual seeds from each cultivar. In all instances, hemagglutinating activity was inhibited by galactose, anti-soybean agglutinin (SBA), and lectin-binding polysaccharide produced by Rhizobium japonicum. Affinity-purified lectin from seeds of a single Columbia plant was labeled with fluorescein isothiocyanate (FITC) and observed by fluorescence microscopy to bind to R. japonicum cells with specificity, intensity and localization indistinguishable from FITC-SBA. Lectins from distinguishable from FITC-SBA. Lectins from three cultivars in sufficiently high concentration for study had molecular properties very similar to Chippewa SBA.Abbreviations FITC fluorescein isothiocyanate - IgG immunoglobulin G - SBA soybean agglutinin     

Molecular and structural analysis of electrophoretic variants of soybean seed storage proteins

Soybean (Glycine max L.) storage proteins are composed mainly of two major components, beta-conglycinin and glycinin. Electrophoretic variants of the beta subunit of beta-conglycinin and the A3 polypeptide of glycinin were detected on SDS-PAGE, and designated them as beta* and A3*, respectively. beta* and A3* exhibited higher and lower mobilities, respectively, than the common beta subunit and A3 polypeptide. The N-terminal nine and 10 amino acid sequences of beta* and A3* were completely identical to the previously reported sequences of the beta subunit and the A3 polypeptide, respectively. Analysis using concanavalin A-horseradish peroxidase and treatment with N-glycosidase indicated that glycans were not responsible for the difference in electrophoretic mobility of beta* or A3*. Furthermore, five clones of beta* or beta and three clones of A3*, respectively, were sequenced but we could not detect deletions and insertions except for a single or a few amino acid substitutions as compared with the common beta subunit and A3 polypeptide. These results indicate that a single or a few amino acid substitution affects the electrophoretic mobilities of beta* and A3*.     

Comparison of protein solubilization methods suitable for proteomic analysis of soybean seed proteins

Extraction of soybean seed proteins for two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry analysis is challenging and inconsistent. In this study, we compared four different protein extraction/solubilization methods-urea, thiourea/urea, phenol, and a modified trichloroacetic acid (TCA)/acetone-to determine their efficacy in separating soybean seed proteins by 2D-PAGE. In all four methods, seed storage proteins were well separated by 2D-PAGE with minor variations in the intensity of the spots. The thiourea/urea and TCA methods showed higher protein resolution and spot intensity of all proteins compared with the other two methods. In addition, several less abundant and high molecular weight proteins were clearly resolved and strongly detected using the thiourea/urea and TCA methods. Protein spots obtained from the TCA method were subjected to mass spectrometry analysis to test their quality and compatibility. Fifteen protein spots were selected, digested with trypsin, and analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and liquid chromatography mass spectrometry (LC-MS). The proteins identified were beta-conglycinin, glycinin, Kunitz trypsin inhibitor, alcohol dehydrogenase, Gly m Bd 28K allergen, and sucrose binding proteins. These results suggest that the thiourea/urea and TCA methods are efficient and reliable methods for 2D separation of soybean seed proteins and subsequent identification by mass spectrometry.     

Proteomic analysis of soybean nodule cytosol

An isolation procedure for soybean (Glycine max L. cv Williams 82) nodule cytosol proteins was developed which greatly improved protein resolution by two-dimensional polyacrylamide gel electrophoresis. The most abundant proteins were selected and analyzed by mass spectrometry. The identified proteins were categorized by function (% of total proteins analyzed): carbon metabolism (28%), nitrogen metabolism (12%), reactive oxygen metabolism (12%) and vesicular trafficking (11%). The first three categories were expected based on the known physiological functions of the symbiotic nitrogen fixation process. The number of proteins involved in vesicular trafficking suggests a very active exchange of macromolecules and membrane components. Among the 69 identified proteins were the enzymes of the three carbon portion of glycolysis, which were further characterized to support their roles in the sucrose synthase pathway to provide malate for the bacteroids. Proteomic analysis provides a functional tool by which to understand and further investigate nodule function.     

Potential defense-related prenylated isoflavones in lactofen-induced soybean

An integrated LC-MS and NMR metabolomic study was conducted to investigate metabolites whose formation was induced by lactofen (1), a soybean (Glycine max L.) disease resistance-inducing herbicide. First, LC-MS analyses of control and lactofen (1)-induced soybean extracts were performed. The LC-MS raw data were then processed by a custom designed bioinformatics program to detect the induced metabolites so formed. Finally, structures of unknown induced metabolites were determined on the basis of their 1D and 2D NMR spectroscopic data. Structure of two previously unreported compounds, 7,8-dihydroxy-4′-methoxy-3′-prenylisoflavone (2) and 7-hydroxy-4′,8-dimethoxy-3′-prenylisoflavone (3) were elucidated together with four known prenylated compounds, 3′-prenyldaidzein (4), 8-prenyldaidzein (5), 3′-prenylgenistein (6), and 4-prenylcoumestrol (7). Compounds (2-6) are reported for the first time in soybean, as are the ¹³C chemical shift assignments for compound (7). Formation of these six prenylated compounds was also induced by the primary defense glucan elicitor from the cell wall of the pathogen Phytophthora sojae (Kauf. and Gerde.), further suggesting a potential role in soybean defense. These results highlight the metabolic flexibility within soybean secondary product pathways and suggest that prenylation may be associated with defense responses. Moreover, this study demonstrates a promising future approach using metabolomics on elicitor-induced plants for discovery of unknown compounds even in relatively well studied plants.     

Effect of chaotropic agents on reversible unfolding of a soybean (Glycine max) seed acid phosphatase

In this work we examined the effect of urea and guanidinium chloride on the structural stability of a single isoform of soybean seed acid phosphatase, based on the intensity of tryptophan fluorescence as a function of denaturant concentration. The free energy of unfolding, DeltaGu, was calculated at 25 degrees C as a function of the concentrations of both chaotropic agents; the conformational stability, DeltaG (H2O), was determined to be 2.48 kcal mol(-1). Center of mass, determined from analysis of fluorescence data, was used as a parameter to assess conformational changes. Our results indicate that complete enzyme inactivation occurred before full enzyme unfolding in both cases, and suggest that there are differences between the conformational flexibility of the active-site and that of the macromolecule as a whole.     

大豆异黄酮浸种对盐胁迫大豆幼苗的生理效应

大豆异黄酮( Soybean isoflavones)是在大豆生长过程中形成并在成熟种子和叶片中积累较多的一类具有生物活性的次生代谢物,通常可作为人们日常生活中的一类营养保健品.研究了外源大豆苷或染料木苷溶液(0.01 mg/L)浸种处理对盐胁迫栽培大豆(N23674品种)和滩涂野大豆(BB52种群)及其经逐代耐盐性筛选的杂交后代(4076株系,F5)幼苗叶片伤害率、光合作用、Na+含量和Na+/K+值、活性氧清除酶活性及内源大豆异黄酮含量等生理指标的影响.结果表明:盐胁迫下,两种外源大豆异黄酮浸种处理均可显著抑制叶片相对电解质渗透率和硫代巴比妥酸反应物(TBARS)含量的上升及净光合速率(Pn)的下降,降低Na+含量和Na+/K+值,增强超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)活性,提高内源大豆异黄酮含量,从而表现对盐害的缓解效应,其中对耐盐性较弱的栽培大豆N23674品种效应更明显.这为大豆异黄酮在大豆耐盐育种、化学调控和盐碱地种植利用等提供了理论依据.     

Aryl hydroxylation of isopropyl-3-chlorocarbanilate by soybean plants

Isopropyl-3-chlorocarbanilate-phenyl UL-¹⁴C (CIPC-¹⁴C) is absorbed, translocated and metabolized by soybean plants. Water-soluble metabolites in root and shoot were purified and the root major metabolite characterized. The acetylated aglucones from the β-glucosidase hydrolysis and the esters from the direct acetylation of CIPC-¹⁴C polar metabolites were purified by GLC and analysed by mass spectrometry. The data showed that the phenyl riong of CIPC-¹⁴C was hydroxylated by both root and shoot tissues. Isopropyl-5-chloro-2-hydroxycarbanilate (hydroxy-CIPC) was the predominant aglucone liberated by β-glucosidase from polar metabolites in root and shoot. The o-glucoside of hydroxy-CIPC was shown to be present, by direct acetylation and characterization. In shoot tissue the major metabolites were dechlorinated hydroxy-CIPC and were not hydrolysed by β-glucosidase. These data show that soybean root or shoot tissues hydroxylate the phenyl ring of CIPC-¹⁴C but do not alter either the isopropyl alcohol moiety or the earbamate bond.     

The genetic base of Brazilian soybean cultivars: evolution over time and breeding implications

Genetic diversity is essential for crop breeding and one way to estimate it is through the concept of genetic base, which can be defined as the number of ancestors and their relative genetic contributions (RGC) to each cultivar. The RGC can be estimated through the coefficient of parentage between the ancestors and cultivars. Previous studies determined that the genetic base of Brazilian soybean was very narrow. The objective of this work was to evaluate the pedigree of 444 Brazilian soybean cultivars to estimate their genetic base. The cultivars were divided according to their release dates and according to their origin (public or private), and the genetic base for each group was also estimated. We found 60 ancestors, of which the top four (CNS, S-100, Roanoke and Tokyo, respectively) contribute 55.3% of the genetic base. Only 14 ancestors have an RGC over 1.0%, and they represent 92.4% of the genetic base. Analysis of the release dates indicated that there has been an increase in the number of ancestors over time, but the four main ancestors were the same over all periods, and their cumulative RGC increased from 46.6% to 57.6%, indicating a narrowing of the genetic base.     

An efficient extraction method to enhance analysis of low abundant proteins from soybean seed

Large amounts of the major storage proteins, β-conglycinin and glycinin, in soybean (Glycine max) seeds hinder the isolation and characterization of less abundant seed proteins. We investigated whether isopropanol extraction could facilitate resolution of the low abundant proteins, different from the main storage protein fractions, in one-dimensional polyacrylamide gel electrophoresis (1D-PAGE) and two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). 1D-PAGE of proteins extracted by different concentrations (10%, 20%, 30%, 40%, 50%, 60%, 70% and 80%) of isopropanol showed that greater than 30% isopropanol was suitable for preferential enrichment of low abundant proteins. Analysis of 2D-PAGE showed that proteins which were less abundant or absent by the conventional extraction procedure were clearly seen in the 40% isopropanol extracts. Increasing isopropanol concentration above 40% resulted in a decrease in the number of less abundant protein spots. We have identified a total of 107 protein spots using matrix-assisted laser desorption/ionization time of flight mass spectrophotometry (MALDI-TOF-MS) and liquid chromatography-mass spectrometry (LC-MS/MS). Our results suggest that extraction of soybean seed powder with 40% isopropanol enriches lower abundance proteins and is a suitable method for 2D-PAGE separation and identification. This methodology could potentially allow the extraction and characterization of low abundant proteins of other legume seeds containing highly abundant storage proteins.     

Molecular markers associated with seed weight in two soybean populations

Seed weight (SW) is a component of soybean, Glycine max (L.) Merr., seed yield, as well as an important trait for food-type soybeans. Two soybean populations, 120 F₄-derived lines of YoungxPI416937 (Pop1) and 111 F₂-derived lines of PI97100xCoker 237 (Pop2), were mapped with RFLP makers to identify quantitative trait loci (QTLs) conditioning SW across environments and populations. The genetic map of Pop1 consisted of 155 loci covering 973 cM, whereas Pop2 involved 153 loci and covered 1600 cM of map distance. For Pop1, the phenotypic data were collected from Plains, GA., Windblow, N.C., and Plymouth, N.C., in 1994. For Pop2, data were collected from Athens, GA., in 1994 and 1995, and Blackville, S.C., in 1995. Based on single-factor analysis of variance (ANOVA), seven and nine independent loci were associated with SW in Pop1 and Pop2, respectively. Together the loci explained 73% of the variability in SW in Pop1 and 74% in Pop2. Transgressive segregation occurred among the progeny in both populations. The marker loci associated with SW were highly consistent across environments and years. Two QTLs on linkage group (LG) F and K were located at similar genomic regions in both populations. The high consistency of QTLs across environments indicates that effective marker-assisted selection is feasible for soybean SW.     

Lactofen induces isoflavone accumulation and glyceollin elicitation competency in soybean

Lactofen, the active ingredient of the soybean disease resistance-inducing herbicide, Cobra, induces large accumulations of isoflavone conjugates and aglycones in soybean tissues. The predominant isoflavones induced in cotyledon tissues are daidzein (and its conjugates) and formononetin and glycitein aglycones. The latter two isoflavones are usually present only at very low levels in soybean seedling tissues. In leaves, the predominant lactofen-induced isoflavones are daidzein and formononetin aglycones and the malonyl-glucosyl conjugate of genistein. Isoflavone induction also occurs in cells distal to the point of treatment, but is only weakly systemic. Lactofen also induces elicitation competency, the capacity of soybean cells to accumulate the pterocarpan phytoalexin glyceollin in response to glucan elicitors from the cell wall of the pathogen Phytophthora sojae. Comparison of the activity of a series of diphenyl ether herbicides demonstrated that while all diphenyl ethers tested induced some degree of elicitation competency, only certain ones induced isoflavone accumulation in the absence of glucan elicitor. As a group the diphenyl ethers are thought to inhibit protoporhyrinogen oxidase, eventually leading to singlet oxygen generation. Another singlet oxygen generator, rose bengal, also induced elicitation competency, but little isoflavone accumulation. It is hypothesized that diphenyl ether-induced activated oxygen species mimic some aspects of hypersensitive cell death, which leads to elicitation competency in infected tissues.     

Separation and identification of soybean leaf proteins by two-dimensional gel electrophoresis and mass spectrometry

To establish a proteomic reference map for soybean leaves, we separated and identified leaf proteins using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry (MS). Tryptic digests of 260 spots were subjected to peptide mass fingerprinting (PMF) by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS. Fifty-three of these protein spots were identified by searching NCBInr and SwissProt databases using the Mascot search engine. Sixty-seven spots that were not identified by MALDI-TOF-MS analysis were analyzed with liquid chromatography tandem mass spectrometry (LC-MS/MS), and 66 of these spots were identified by searching against the NCBInr, SwissProt and expressed sequence tag (EST) databases. We have identified a total of 71 unique proteins. The majority of the identified leaf proteins are involved in energy metabolism. The results indicate that 2D-PAGE, combined with MALDI-TOF-MS and LC-MS/MS, is a sensitive and powerful technique for separation and identification of soybean leaf proteins. A summary of the identified proteins and their putative functions is discussed.     

Comparison of somatic embryogenesis and embryo conversion in commercial soybean cultivars

Six commercially important soybean cultivars and one control cultivar were compared for differences in induction-efficiency of somatic embryogenesis, primary embryo yield, and embryo conversion. Cotyledons from immature seeds of similar developmental stage for all soybean cultivars were used for embryo induction. The experiments utilized a Latin square design to exclude the effect of differential lighting and position due to plate location in the growth chamber on the embryogenesis process. Results indicated that the efficiency of embryo induction and yield of primary somatic embryos were genotype-dependent. In contrast, no dependence on genotype was observed for the conversion of embryos to form roots and shoots. The percentage of cotyledons that gave a positive embryogenic response ranged from 26 to 89% for the soybean cultivars tested. The average number of primary globular-stage embryos per responding cotyledon after one month on induction medium ranged from 6 to 13 among the seven cultivars. Conversion frequencies for all genotypes ranged from 27 to 45%.     

Non-target impacts of soybean rust fungicides on the fungal entomopathogens of soybean aphid

Soybean aphid, Aphis glycines, has caused serious economic damage to soybean across the North Central US since its introduction to North America in 2000. The management of another invasive soybean pest, Asian soybean rust, Phakopsora pachyrhizi, using foliar fungicide applications has the potential to impact soybean aphid populations by suppressing beneficial fungal entomopathogens. In 2005 and 2006, we applied recommended soybean rust fungicide treatments, consisting of strobilurin and triazole fungicides, to small soybean plots in two locations to assess if such applications might suppress aphid fungal epizootics. In Lamberton, MN, in 2005, during the epizootic, fungicide-treated plots averaged 2.0 ± 0.7% (mean ± SE) disease prevalence while untreated plots averaged 14.2 ± 5.6%. In 2007, we applied strobilurin and strobilurin-triazole mix fungicides to single-plant microplots either before or after release of Pandora neoaphidis, the most commonly observed aphid pathogen in 2005 and 2006. Treatments that contained a mixture of two active ingredients significantly lowered peak and cumulative aphid disease prevalence in both early and late reproductive stage soybeans indicating that fungicide mixtures used to manage soybean rust can negatively impact an aphid-specific fungal pathogen. However, no consistent soybean aphid population response was observed in these studies of low levels of aphid fungal infection.     

Genetic structure and a selected core set of Brazilian soybean cultivars

Soybean is one of the most valuable and profitable oil crop species and a thorough knowledge of the genetic structure of this crop is necessary for developing the best breeding strategies. In this study, a representative collection of soybean cultivars recommended for farming in all Brazilian regions was genotyped using 27 simple sequence repeat (SSR) loci. A total of 130 alleles were detected, with an average allelic number of 4.81 per locus. These alleles determined the core set that best represented this soybean germplasm. The Bayesian analysis revealed the presence of two clusters or subgroups within the whole collection (435 soybean cultivars) and the core set (31 entries). Cultivars of similar origin (ancestral) were clustered into the same groups in both analyses. The genetic diversity parameters, based on the SSR loci, revealed high similarity between the whole collection and core set. Differences between the two clusters detected in the core set were attributed more to the frequency of their ancestors than to their genetic base. In terms of ancestry, divergent groups were presented and a panel is shown which may foster efficient breeding programs and aid soybean breeders in planning reliable crossings in the development of new varieties.     

Insights into the role and structure of plant ureases

The broad distribution of ureases in leguminous seeds, as well as the accumulation pattern of the protein during seed maturation, are suggestive of an important physiological role for this enzyme. Since the isolation and characterization of jack bean urease by Sumner in 1926, many investigations have been dedicated to the structural and biological features of this enzyme; nevertheless, many questions still remain. It has been reported that ureases from plants (jack bean and soybean seeds) display biological properties unrelated to their ureolytic activity, notably a high insecticidal activity against Coleoptera (beetles) and Hemiptera (bugs), suggesting that ureases might be involved in plant defense. Besides the insecticidal activity, canatoxin, a jack bean urease isoform, causes convulsions and death in mice and rats, induces indirect hemagglutination (hemilectin activity) and promotes exocytosis in several cell types. Not only plant ureases but also some microbial ureases (found in Bacillus pasteurii and Helicobacter pylori) are able to induce activation of platelets in a process mediated by lipoxygenase-derived metabolites. This review summarizes the biological and structural properties of plant ureases, compares them with those displayed by bacterial ureases, and discusses the significance of these findings.     

Pseudomonas asturiensis sp. nov., isolated from soybean and weeds

Five strains of gram negative bacteria, isolated from soybean (LPPA 221^T, 222 and 223) and weeds (LPPA 816 and 1442), were analyzed by a polyphasic approach. The isolates showed variation in their phenotypic traits and were placed in the Pseudomonas fluorescens lineage, based on 16S rRNA gene sequence phylogeny, as a single but well separated cluster. MLSA analysis based on gyrB and rpoD sequences clustered the strains in a single branch in the Pseudomonas syringae group, and revealed P. viridiflava as closest relative. DNA–DNA hybridizations showed medium levels of DNA–DNA relatedness with the type strain of P. viridiflava (50%) and lower levels (<32%) with other type strains of the P. syringae group, supporting classification within a novel species of the genus Pseudomonas. The strains can be distinguished from species of the P. syringae group by the fatty acid C_17:0 cyclo that is present in a low amount (2.5%) and from P. viridiflava by their inability to assimilate d-tartrate and d-sorbitol, and by the formation of red colonies on TTC medium. For this new species, the name Pseudomonas asturiensis sp. nov. is proposed. The type strain is LPPA 221^T (=LMG 26898^T = CECT 8095^T).