Isoflavonoids in root and hypocotyl of soybean seedlings (Glycine max,Fabaceae)

Isoflavonoids, some of which are highly fluorescent, are produced by soybean [Glycine max (L.) Merr.] and serve as chemical signals for certain aspects of nitrogen fixation and microbial resistance. This study was conducted to determine whether soybean mutants with nonfluorescent roots contained abnormal concentrations of isoflavonoids. Isoflavonoids were extracted from the root and hypocotyl of 4-d-old wild-type soybean seedlings (cv. Hark) having fluorescent roots and from four nonallelic mutant, near isogenic lines of Hark having nonfluorescent roots. In addition, isoflavonoids were extracted from the root and hypocotyl of 4-d-old seedlings of near isogenic lines of Hark harboring two pairs of the mutant alleles for nonfluorescent roots. Malonyl daidzin, daidzin, malonyl genistin, and genistein were the most abundant isoflavonoids extracted from either the root or hypocotyl of seedlings with either fluorescent or nonfluorescent roots. Extracted malonyl daidzin, malonyl genistin, and malonyl glycitin decomposed readily, yielding daidzin, genistin, and glycitin, respectively. The concentrations of malonyl genistin and genistein, two highly fluorescent compounds, were similar in both fluorescent and nonfluorescent roots. Thus, root fluorescence was not correlated with abundance per se of fluorescent isoflavonoids in roots. In addition, the abundance of isoflavonoids extractable from the hypocotyl did not correlate with root fluorescence.     

Genistein accumulation in soybean (Glycine max [L.] Merr.) root systems under suboptimal root zone temperatures

Genistein, as a plant-to-bacteria signal, plays an importantrole in the establishment of the soybean (Glycine max [L.] Merr.)-Bradyrhizobiumjaponicum nitrogen-fixing symbiosis. It is essential to thedevelopment of effective root nodules and responsible for inducingthe nod genes of B. japonicum. Because sub-optimal root zonetemperature (RZT) delays infection and early nodule development,and decreases plant nodule number, and genistein addition overcomessome of this, it is reasonable to hypothesize that suboptimalRZT disrupts the inter-organismal signal exchange by inhibitinggenistein synthesis. Four experiments were conducted to testthese hypotheses. The results of these studies indicated that:(1) when soybean plants were germinated and maintained at RZTsranging from 13 to 17C, root genistein concentration and contentper plant were lower than those of plants with roots maintainedat RZTs above 17C; (2) when plants were germinated at an optimalRZT (25 C) then transferred to RZTs below 17C, and acclimatedfor a few days, root genistein concentration and content perplant were higher than those of plants with roots maintainedeither at optimal RZT, or transferred to RZT above 17 C, althoughby the end of the experiment, the genistein concentration ofroot systems at below 17C RZT appeared to be declining to valuesbelow those of plants with above 17 C RZT; (3) the root genisteinconcentration increased before the onset of nitrogen fixationand decreased thereafter; and (4) part of the effect of RZTson genistein content per plant root system was from reductionsin genistein concentration at lower RZT5, and part was due todecreased plant root growth. Key words: Genistein, Glycine max, suboptimal temperature     

Infection threads in the root hairs of soybean (Glycine max) plants inoculated withRhizobium japonicum

Summary The mode of infection leading to nodulation was studied in soybean (Glycine max) plants inoculated withRhizobium japonicum strains 61A76, 3I1b83, 3Ilb142, and 3Ilb143 and a commercial inoculum. Infection threads were noticed in the root hairs of plants grown in small field plots, Leonard bottle-jar assemblies and on agar slants. Two infection threads per root hair were commonly observed. Root hairs with infection threads were persistent on the nodules. The maximum number of infection threads per plant was observed in Leonard bottle-jar assemblies.Contribution No. 603 from Charles F. Kettering Research Laboratory.     

Effects of exogenous application and stem infusion of ascorbate on soybean (Glycine max) root nodules

Numerous biochemical and physiological studies have demonstrated the importance of ascorbate (ASC) as a reducing agent and antioxidant in higher plant metabolism. Of special note is the capacity of ASC to eliminate damaging activated oxygen species (AOS) including O₂^−· and H₂O₂. N₂-fixing legume nodules are especially vulnerable to oxidative damage because they contain large amounts of leghaemoglobin which produces AOS through spontaneous autoxidation; thus, ASC and other components of the ascorbate–reduced glutathione (ASC–GSH) pathway are critical antioxidants in nodules. In order to establish a meaningful correlation between concentrations of ASC and capacity for N₂ fixation in legume root nodules, soybean ( Glycine max ) plants were treated with excess ASC via exogenous irrigation or continuous intravascular infusion through needles inserted directly into plant stems. Treatment with ASC led to striking increases in nitrogenase activity (acetylene reduction), nodule leghaemoglobin content, and activity of ASC peroxidase, a key antioxidant enzyme. The concentration of lipid peroxides, which are indicators of oxidative damage and onset of senescence, was decreased in ASC-treated nodules. These results support the conclusion that ASC is critical for N₂ fixation and that elevated ASC allows nodules to maintain a greater capacity to fix N₂ over longer periods.     

Pre-incubation of Bradyrhizobium japonicum with jasmonates accelerates nodulation and nitrogen fixation in soybean (Glycine max) at optimal and suboptimal root zone temperatures

Jasmonic acid (JA) and methyl jasmonate, collectively known as jasmonates, are naturally occurring in plants; they are important signal molecules involved in induced disease resistance and mediate many physiological activities in plants. We studied the effect of JA and its methyl ester, methyl jasmonate (MeJA), on the induction of nod genes in Bradyrhizobium japonicum GG4 (USDA3) carrying a plasmid with a translational fusion between B. japonicum nodY and lacZ of Escherichia coli, and the expression activity was measured by β-galactosidase activity. Both JA and MeJA strongly induced the expression of nod genes. They have little or no deleterious effects on the growth of B. japonicum cells, while genistein (Gen) showed inhibitory effects. We further studied the effect of JA- and MeJA-induced B. japonicum on soybean nodulation and nitrogen fixation under optimal (25°C) and suboptimal (17°C) root zone temperature (RZT) conditions. B. japonicum cells were grown in liquid yeast extract mannitol media and induced with a range of Gen, JA, and MeJA concentrations, including a treatment control with no inducer added. Soybean seedlings were grown at 25 or 17°C RZT with a constant air temperature (25°C) and inoculated, at the vegetative cotyledonary stage, with various B. japonicum induction treatments. Addition of Gen or jasmonates to B. japonicum, prior to inoculation, enhanced nodulation, nitrogen fixation, and plant growth at suboptimal RZT conditions. A higher concentration of Gen was inhibitory at 25°C, while this same concentration was stimulatory at 17°C. Interestingly, pre-incubation of B. japonicum with JA and MeJA enhanced soybean nodulation and nitrogen fixation under both optimal and suboptimal RZTs. We show that jasmonates are thus a new class of signaling molecules in the B. japonicum-soybean symbiosis and that pre-induction of B. japonicum with jasmonates can be used to enhance soybean nodulation, nitrogen fixation, and early plant growth.     

Occurrence of ferredoxin-dependent glutamate synthase in plant cell fraction of soybean root nodules (Glycine max)

Ferredoxin-dependent glutamate synthase (EC 1.4.7.1) and NADH-dependent glutamate synthase (EC 1.4.1.14) have been identified in the plant cells of soybean nodules. Ferredoxin-dependent glutamate synthase is 2-fold more active than NADH-dependent enzyme in vitro. Ferredoxin-dependent glutamate synthase cross-reacts with IgG against ferredoxin-dependent glutamate synthase of rice green leaves, whereas NADH-dependent glutamate synthase does not recognize the IgG, indicating that there are two distinct enzyme proteins. Ferredoxin-dependent glutamate synthase is composed of polypeptide chain(s) of 165 kDa and has a high affinity to spinach leaf ferredoxin as an electron carrier.     

大豆PEBP基因家族的分析

磷脂酰乙醇胺结合蛋白(PEBP,phosphatidyl ethanolamine-binding protein)基因家族在动物、植物和微生物中广泛存在,在控制植物开花和种子休眠中起重要作用。本研究对大豆PEBP基因家族进行了分析,发现了27个大豆PEBP基因的候选序列,其中16个具有完整PEBP结构域的全长序列被认为是大豆Gm PEBP家族基因。Gm PEBP基因分布在9条染色体上,基因结构高度保守。通过系统发生分析,可将大豆Gm PEBP基因家族成员分为FT-like、TFL1-like和MFT-like 3个亚族,并且发现Gm PEBP家族成员数目按照大豆物种特异性的方式进行了扩张。对重复基因的Ks分析表明,绝大多数重复基因主要由5900万年前和1300万年前的大豆基因组复制所致。     

Studies of histidine residues in soybean (Glycine max) urease

Soybean urease has been investigated extensively to reveal the presence of histidine residue (s) in the active site and their potential role in the catalysis. The spectrophotometric studies using diethylpyrocarbonate (DEP) showed the modification of 11.76 ± 0.1 histidine residues per mole of native urease. Therefore, the results are indicative of the presence of twelve histidine residues per urease molecule. It is presumed that the soybean urease, being a hexameric protein possess two histidine residues per subunit. Correlation plot showed that the complete inactivation of soybean urease corresponds to the modification of 1.97 histidine residues per subunit. Further, double logarithmic plot of kapp versus DEP concentration has resulted in a linear correlation and thereby demonstrating that only one of the two histidine residues per subunit is catalytically essential. Significant protection has been observed against inactivation when urea or acetohydroxamate (AHA) is incubated with DEP treated urease. The studies have demonstrated the presence of one histidine residue at the active site of soybean urease and its significance in catalysis.     

QTL mapping of domestication-related traits in soybean (Glycine max)

BACKGROUND AND AIMS: Understanding the genetic basis underlying domestication-related traits (DRTs) is important in order to use wild germplasm efficiently for improving yield, stress tolerance and quality of crops. This study was conducted to characterize the genetic basis of DRTs in soybean (Glycine max) using quantitative trait locus (QTL) mapping. METHODS: A population of 96 recombinant inbred lines derived from a cultivated (ssp. max) x wild (ssp. soja) cross was used for mapping and QTL analysis. Nine DRTs were examined in 2004 and 2005. A linkage map was constructed with 282 markers by the Kosambi function, and the QTL was detected by composite interval mapping. KEY RESULTS: The early flowering and determinate habit derived from the max parent were each controlled by one major QTL, corresponding to the major genes for maturity (e1) and determinate habit (dt1), respectively. There were only one or two significant QTLs for twinning habit, pod dehiscence, seed weight and hard seededness, which each accounted for approx. 20-50 % of the total variance. A comparison with the QTLs detected previously indicated that in pod dehiscence and hard seededness, at least one major QTL was common across different crosses, whereas no such consistent QTL existed for seed weight. CONCLUSIONS: Most of the DRTs in soybeans were conditioned by one or two major QTLs and a number of genotype-dependent minor QTLs. The common major QTLs identified in pod dehiscence and hard seededness may have been key loci in the domestication of soybean. The evolutionary changes toward larger seed may have occurred through the accumulation of minor changes at many QTLs. Since the major QTLs for DRTs were scattered across only six of the 20 linkage groups, and since the QTLs were not clustered, introgression of useful genes from wild to cultivated soybeans can be carried out without large obstacles.     

Identification and characterization of SSRs from soybean (Glycine max) ESTs

Microsatellites, or simple sequence repeats (SSRs), are very useful molecular markers for a number of plant species. We used a new publicly available module (TROLL) to extract microsatellites from the public database of soybean expressed sequence tag (EST) sequences. A total of 12,833 sequences containing di- to penta-type SSRs were identified from 200,516 non-redundant soybean ESTs. On average, one SSR was found per 7.25?kb of EST sequences, with the tri-nucleotide motifs being the most abundant. Primer sequences flanking the SSR motifs were successfully designed for 9,638 soybean ESTs using the software primer3.0 and only 59 pairs of them were found in earlier studies. We synthesized 124 pairs of the primers to determine the polymorphism and heterozygosity among eight genotypes of soybean cultivars, which represented a wide range of the cultivated soybean cultivars. PCR amplification products with anticipated SSRs were obtained with 81 pairs of primers; 36 PCR products appeared to be homozygous and the remaining 45 PCR products appeared to be heterozygous and displayed polymorphism among the eight cultivars. We further analysed the EST sequences containing 45 polymorphic EST-SSR markers using the programs BLASTN and BLASTX. Sequence alignment showed that 29 ESTs have homologous sequences and 15 ESTs could be classified into a Uni-gene cluster with comparatively convincing protein products. Among these 15 ESTs belonging to a Uni-gene cluster, 9 SSRs were located in 3'-UTR, 4 SSRs were located in the intron region and 2 SSRs were located in the CDS region. None of these SSRs was located in the 5'-UTR. These novel SSRs identified in the ESTs of soybean provide useful information for gene mapping and cloning in future studies.     

Nuclear-cytoplasmic interaction in chlorophyll-deficient soybean,Glycine max (Fabaceae)

In higher plants, plastids and mitochondria are the predominant carriers of extrachromosomal genetic information. There is interplay between the plastids, the mitochondria, and the nuclear genome. In soybean, Glycine max (L.) Merr., both nuclearly and maternally inherited chlorophyll-deficient mutants have been described. Conditional lethality previously was reported in soybean when maternally inherited chlorophyll-deficient mutant (Genetic Type T275) was crossed with nuclearly inherited yellow foliar malate dehydrogenase null mutants (Genetic Types T253 and T323). Our objective was to test for conditional lethality when maternally inherited yellow foliar mutants T278, T314, T315, T316, T319, and T320 were female parents and nuclearly inherited yellow foliar malate dehydrogenase null mutants T253 and T323 were male parents. Our results indicated conditional lethality in the F₂ generation when any of the six cytoplasmically inherited yellow foliar mutants were female parents and either T253 or T323 were male parents. The physiological nature of conditional lethality is not known. Data indicate a common basis in soybean for conditional lethality among the cytoplasmically inherited yellow foliar mutants when crossed with the nuclearly inherited yellow foliar malate dehydrogenase null mutants. No interactions were observed between cytoplasmically inherited or nuclearly inherited green seed embryo mutants as female parents and either T253 or T323 as male parents.     

Shoot-applied polyamines suppress nodule formation in soybean (Glycine max)

In legumes, the number of root nodules is controlled by a mechanism called autoregulation. Recently, we found that the foliar brassinosteroid (BR), a plant growth-regulating hormone, systemically regulates the nodule number in soybean plants. In the present study we report that such down-regulation of root nodule formation by a BR may occur through a change of the polyamine contents, with the experimental evidence as follows. The foliar contents of both spermidine (Spd) and spermine (Spm) in the super-nodulating soybean mutant, En6500, were always lower than those in its parent line, Enrei. This lower Spd and Spm content accompanied a striking accumulation of putrescine (Put) in the former plant. This finding indicates that Spd and Spm biosynthesis from their precursor Put is repressed in En6500. The foliar treatments with Spd or Spm of En6500 led to a reduction of both nodule number and root growth. On the other hand, foliar treatment with MDL74038, a specific inhibitor of Spd biosynthesis, apparently increased the root nodule number in Enrei. Foliar application of brassinolide (BL) of En6500 increased the leaf Spd level and reduced the nodule number. These results suggested that BL-induced Spd synthesis in shoots might suppress the root nodule formation.     

Nodulin gene expression during soybean (Glycine max) nodule development

In vitro translation products of total RNA isolated from soybean nodules at successive stages of nodule development were analyzed by two-dimensional gel electrophoresis. In that way the occurrence of over 20 mRNAs specifically transcribed from nodulin genes was detected. The nodulin genes could be divided into two classes according to the time of expression during nodule development. Class A comprises at least 4 nodulin mRNAs which are found when a globular meristem is present in the root cortex. These class A nodulin genes have a transient expression. Class B nodulin genes are expressed when the formation of a nodule structure has been completed. Bradyrhizobium japonicum nod ⁺ fix^-mutants, with large deletions spanning the nif H,DK region, still induced nodules showing normal expression of all nodulin genes, indicating that the nif H,DK region is not involved in the induction of nodulin genes. In nodules induced by Bradyrhizobium japonicum nod ⁺ fix^-mutant HS124 the bacteria are rarely released from the infection thread and the few infected cells appear to be collapsed. All class A and class B nodulin genes are expressed in HS124 nodules with the exception of 5 class B genes.     

Mechanical impedance increases aluminium tolerance of soybean (Glycine max) roots

The effect of aluminium (Al) on root elongation was studied in solution culture and sand culture. Compared to solution culture, in sand culture a ten times higher Al supply was necessary to inhibit root elongation to a comparable degree. This was due to a much lower Al uptake into the 5 mm root tips in sand culture. Fe concentrations in root tips were also lower in sand culture. Ca concentrations were higher and less depressed by Al, whereas Mg and K concentrations were not affected by the culture substrate. Regressions of Al concentrations in root tips versus inhibition of root elongation by Al revealed root damage at lower Al concentrations in sand culture. The effect of culture substrate on Al tolerance was independent of N source and could also be shown in flowing solution culture with and without sand. The results indicate that mechanical impedance in sand culture decreased Al uptake. This may be due to enhanced exudation of organic complexors thus reducing activites of monomeric Al species.     

RNA sequencing analysis of salt tolerance in soybean (Glycine max)

Salt stress causes foliar chlorosis and scorch, plant stunting, and eventually yield reduction in soybean. There are differential responses, namely tolerance (excluder) and intolerance (includer), among soybean germplasm. However, the genetic and physiological mechanisms for salt tolerance is complex and not clear yet. Based on the results from the screening of the RA-452 x Osage mapping population, two F_4:6 lines with extreme responses, most tolerant and most sensitive, were selected for a time-course gene expression study in which the 250 mM NaCl treatment was initially imposed at the V1 stage and continued for 24 h (hrs). Total RNA was isolated from the leaves harvested at 0, 6, 12, 24 h after the initiation of salt treatment, respectively. The RNA-Seq analysis was conducted to compare the salt tolerant genotype with salt sensitive genotype at each time point using RNA-Seq pipeline method. A total of 2374, 998, 1746, and 630 differentially expressed genes (DEGs) between salt-tolerant line and salt-sensitive line, were found at 0, 6, 12, and 24 h, respectively. The expression patterns of 154 common DEGs among all the time points were investigated, of which, six common DEGs were upregulated and seven common DEGs were downregulated in salt-tolerant line. Moreover, 13 common DEGs were dramatically expressed at all the time points. Based on Log2 (fold change) of expression level of salt-tolerant line to salt-sensitive line and gene annotation, Glyma.02G228100, Glyma.03G226000, Glyma.03G031000, Glyma.03G031400, Glyma.04G180300, Glyma.04G180400, Glyma.05 g204600, Glyma.08G189600, Glyma.13G042200, and Glyma.17G173200, were considered to be the key potential genes involving in the salt-tolerance mechanism in the soybean salt-tolerant line.     

Distribution and remobilization of symbiotically fixed nitrogen in soybean (Glycine max)

Partitioning of nitrogen by soybeans ( Glycine max L. Merr. cv. Hodgson) grown in natural conditions was studied by successive exposures of root systems to ¹⁵N₂ and periodical measurements of ¹⁵N distribution. Nitrogen derived from the atmosphere was mainly found in the aerial parts of the plants, and the stage of development exerted a strong influence on the initial ¹⁵N distribution (measured one week after incorporation). Until day 69 after sowing, leaf blades contained 47 to 57% of the fixed N. After that, reproductive structures attracted increasing proportions, 10 to 60% between days 69 and 92. Around day 82, stems and petioles stored up to 30% of the newly fixed N. During pod development and pod filling and until maturity, fixed N was remobilized from vegetative tissues and pod walls to seeds. These transfers first concerned the newly incorporated N, but at maturity 80 to 90% of the total was recovered in the seeds. The high mobility of N originating from the atmosphere as compared to that coming from the soil (vegetative tissues exported only 50% of their total N) seems to indicate that fixed N was at least partially integrated in a special pool. This was certainly the case at the later stage of N₂ fixation, when a large portion of fixed N accumulated in the stems and petioles, probably in the form of storage compounds such as ureides for later transfer to the developing seeds. Further research is needed in order to investigate the nature and role of this pool in the nitrogen nutrition of soybeans.     

Temperature influences the expression of resistance of soybean (Glycine max) to the soybean aphid (Aphis glycines)

Resistance of plants to arthropods may be lost at low or high temperatures. I tested whether the relative resistance of five genotypes of soybean, Glycine max (L.) Merr., to three isolates of the soybean aphid, Aphis glycines Matsumura, was influenced by three temperatures, 14, 21 and 28°C, in no‐choice tests in the laboratory. The interaction between temperature and the genotype of soybean influenced the population sizes of two isolates of A. glycines. Two genotypes of soybean, LD05‐16611 and PI 567597C, which usually are resistant to isolate 1 and 3, became susceptible: LD05‐16611 at the low temperature and PI 567597C at the high temperature. The genotypes PI 200538 and PI 567541B usually are susceptible to isolate 3 but were resistant at 21 and 28°C. I can only speculate as to the reason why temperature influences resistance of some genotypes of soybean to A. glycines: A. glycines may be directly influenced by temperature or indirectly influenced by changes in the host plant. Nevertheless, my results suggest that temperature may be one factor that influences the expression of resistance of soybean to A. glycines, so genotypes of soybean should be screened for resistance to the aphid at multiple temperatures.     

Sugar uptake in soybean (Glycine max) cells in suspension culture

In chlorophylkras soybean ( Glycine max L.) cell suspensioo cultures glucose uptake has been studied using the analogue 3-O-methyIglucose. Uptake could be distinguished into: a) a high affinity phase with K_m= 0.06 m M and b) a low affinity phase with K_m 2.0 m M . The uptake of glucose was accompanied by H⁺-cotransport with a stoichiometry of 0.3 H⁺ per molecule 3-O-methylglucose. Experiments in which sugar uptake was measured in the presence of various inhibitors of respiration and photosynthesis demonstrated that the glucose uptake system was dependent on energy metabolism and the ATP-content of the cells. Efflux experiments in the presence of the uncoupler dinitrophenol confirmed this energy dependency. Glucose uptake did not decrease before the ATP-content of the cells had decreased considerably.