Associative effect ofAzospirilium brasilense withRhizobium japonicum on nodulation and yield of soybean (Glycine max)

Summary Azospirillum was associated with nodules of soybean. In general, seed inoculation with a broth culture ofAzospirillum brasilense alone significantly increased nodulation and grain yield of soybean grown in pots in unsterilized soil with different levels of urea ranging from 0 to 80 kg N/ha. This trend was significantly reproducible in a second experiment when a carrier based inoculant of the bacterium was used for seed inoculation.Inoculation withRhizobium japonicum andA. brasilense in combination generally increased grain yield in both the experiments, although the data were not significant.     

Perception of Bradyrhizobium japonicum Nod factor by soybean [Glycine max (L.) Merr.] root hairs under abiotic stress conditions

Suboptimal growth conditions, such as low rhizosphere temperature, high salinity, and low pH can negatively affect the rhizobia-legume symbioses, resulting in poor nodulation and lower amounts of nitrogen fixed. Early stages of the Bradyrhizobium japonicum-soybean [Glycine max (L.) Merr.] symbiosis, such as excretion of genistein (the plant-to-bacteria signal) and infection initiation can be inhibited by abiotic stresses; however, the effect on early events modulated by Nod factors (bacteria-to-plant signalling), particularly root hair deformations is unknown. Thus, the objective of this study was to evaluate the perception of Nod factor by soybean root hairs under three stress conditions: low temperature, low pH, and high salinity. Three experiments were conducted using a 1:1 ratio of Nod Bj-V (C(18:1), MeFuc) and Nod Bj-V (Ac, C(16:0), MeFuc). Nod factor induced four types of root hair deformation (HAD), wiggling, bulging, curling, and branching. Under optimal experimental conditions root hair response to the three levels of Nod factor tested (10(-6), 10(-8), and 10(-10) M) was dose-dependent. The highest frequency of root hair deformations was elicited by the 10(-6) M level. Root hair deformation decreased with temperature (25, 17, and 15 degrees C), low pH, and high salinity. Nod factor concentration did not interact with either low temperature or pH. However, salinity strongly inhibited HAD responses to increases in Nod factor concentration. Thus, the addition of higher levels of Nod factor is able to overcome the effects of low pH and temperature stress, but not salinity.     

Characterization of aluminium-induced citrate secretion in aluminium-tolerant soybean (Glycine max) plants

Recently, we showed that secretion of citrate in an aluminium (Al) tolerant cultivar soybean (Glycine max) (cv. Suzunari) is a specific response to Al stress [Yang et al. (2000) Physiol Plant 110: 72–77]. Here we investigated the intrinsic mechanisms behind the secretion of citrate induced by Al. The amount of citrate secreted during the 24‐h Al treatment period increased with increasing concentration of Al (0–70 μM). We analysed citrate secretion basically under 3 conditions: (1) by varying light‐exposure, (2) with intact or excised shoots and (3) by using a divided chamber technique. Further, the content of organic acids in the tissue and the activity of enzymes involved in organic acid metabolism were analysed and evaluated. The results indicate that high rate of citrate secretion in soybean requires a 4‐h induction period. Al had a continuous effect on the citrate secretion when Al was removed from the treatment solution. Citrate secretion increased steadily under exposure to continuous light. However, when the shoots were excised the citrate secretion rate dropped to 3–6 times that of their control counterparts. Results of root manipulation experiments revealed that citrate secretion required the direct contact of Al. In other words, only the Al‐treated root portions secreted citrate. All these observations suggest that the shoots play a role in Al‐induced citrate secretion. Although shoots may not supply citrate for the secretion upon Al treatment, it seems that they may provide the carbon source and/or energy for citrate synthesis in the root. On the other hand, the root organic acid content (1‐cm apex) indicated that malate might contribute to citrate secretion by keeping the balance between citrate synthesis and release in the root apices. Quantification of enzymes involved in organic acid metabolism showed only a 16% increase in citrate synthase activity upon Al treatments (6 h) with no differences in other enzymes. Hence, we could not rule out completely the potential contribution of citrate from shoots and the results are discussed in the light of shoots contributing either energy or citrate itself for enhanced citrate secretion in the Al‐tolerant plant roots.     

Comparative proteomic profiles of the soybean (Glycine max) root apex and differentiated root zone

The root apical meristem (RAM) is responsible for the growth of the plant root system. Because of the importance of root architecture in the performance of crop plants, we established a proteome reference map of the soybean root apex and compared this with the proteome of the differentiated root zone. The root apex samples contained the apical 1?mm of the root, comprising the RAM, quiescent center and root cap. We identified 342 protein spots from 550 excised proteins (～62%) of root apex samples by MALDI-TOF MS/MS analysis. All these proteins were also present in the differentiated root, but differed in abundance. Functional classification showed that the most numerous protein categories represented in the root were those of stress response, glycolysis, redox homeostasis and protein processing. Using DIGE, we identified 73 differentially accumulated proteins between root apex and differentiated root. Proteins overrepresented in the root apex belonged primarily to the pathways for protein synthesis and processing, cell redox homeostasis and flavonoid biosynthesis. Proteins underrepresented in the root apex were those of glycolysis, tricarboxylic acid metabolism and stress response. Our results highlight the importance of stress and defense response, redox control and flavonoid metabolism in the root apex.     

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.     

Cadmium-induced senescence in nodules of soybean (Glycine max L.) plants

The relationship between cadmium-induced oxidative stress and nodule senescence in soybean was investigated at two different concentrations of cadmium ions (50 and 200 μM), in solution culture. High cadmium concentration (200 μM) resulted in oxidative stress, which was indicated by an increase in thiobarbituric acid reactive substances content and a decrease in leghemoglobin levels. Consequently, nitrogenase activity was decreased, and increases in iron and ferritin levels were obtained. Senescent parameters such as ethylene production, increased levels of ammonium and an increase in protease activity were simultaneously observed. Glutamate dehydrogenase activity was also increased. Peroxidase activity decreased at the higher cadmium concentration while the lower cadmium treatment produced changes in peroxidase isoforms, compared to control nodules. Ultrastructural investigation of the nodules showed alterations with a reduction of both bacteroids number per symbiosome and the effective area for N₂-fixation. These results strongly suggest that, at least at the higher concentration, cadmium induces nodule senescence in soybean plants.     

O-antigen from Bradyrhizobium japonicum lipopolysaccharide inhibits intercellular (symplast) communication between soybean (Glycine max) cells

The technique of fluorescence redistribution after photobleaching was utilized to measure intercellular movement of low molecular weight fluorescent hydrophilic substances across the cell wall/membrane interface between contiguous soybean (Glycine max (L.) Merr. cv. Mandarin) root cells (SB-1 cell line) in tissue culture. Lipopolysaccharide (LPS) purified from Bradyrhizobium japonicum R110d, a Gram-negative bacterium that normally infects and induces nodulation in soybean roots in vivo, inhibits intercellular communication between the soybean cells in a dose-dependent manner. In contrast, LPS from noninfecting strains failed to yield the same effect. The inhibitory activity of the LPS was localized to the O-antigen region of the LPS.     

Proteomic analysis of soybean root hairs after infection by Bradyrhizobium japonicum

Infection of soybean root hairs by Bradyrhizobium japonicum is the first of several complex events leading to nodulation. In the current proteomic study, soybean root hairs after inoculation with B. japonicum were separated from roots. Total proteins were analyzed by two-dimensional (2-D) polyacrylamide gel electrophoresis. In one experiment, 96 protein spots were analyzed by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) to compare protein profiles between uninoculated roots and root hairs. Another 37 spots, derived from inoculated root hairs over different timepoints, were also analyzed by tandem MS (MS/MS). As expected, some proteins were differentially expressed in root hairs compared with roots (e.g., a chitinase and phosphoenolpyruvate carboxylase). Out of 37 spots analyzed by MS/MS, 27 candidate proteins were identified by database comparisons. These included several proteins known to respond to rhizobial inoculation (e.g., peroxidase and phenylalanine-ammonia lyase). However, novel proteins were also identified (e.g., phospholipase D and phosphoglucomutase). This research establishes an excellent system for the study of root-hair infection by rhizobia and, in a more general sense, the functional genomics of a single, plant cell type. The results obtained also indicate that proteomic studies with soybean, lacking a complete genome sequence, are practical.     

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.     

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.     

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.     

Polyamine synthesis in plants. Purification and properties of amidinotransferase from soybean (Glycine max) axes

Three-day-old soybean (Glycine max) seedlings were exposed to 0.4 M sorbitol solution for 4 h to induce amidinotransferase activity, with the corresponding enzyme being purified to homogeneity by chromatographic separation on DEAE-Sephacel, Sephacryl S-300 and L-arginine Sepharose 4B. The purified enzyme used L-arginine and L-glycine as the major donor/acceptor of the amidino group, respectively, with formation of guanidinoacetic acid and ornithine products being confirmed by ESI-MS. The enzyme is a tetrameric protein having a molecular mass of 240,000 Da, whose thiol group is needed for enzymatic activity. The K(M)s for arginine and glycine were 3.8 and 0.89 mM, respectively, with optimal temperature and pH being 37 degrees C and 9.5, respectively. The soybean amidinotransferase could be indirectly involved in nitrogen metabolism, as suggested by the observation that arginine:glycine amidinotransferase in soybean axes is indirectly involved in putrescine biosynthesis and displays feedback control at high levels of an endogenous regulator, putrescine.     

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.     

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.     

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.     

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.