Regulation of nitrogenase activity in Bradyrhizobium japonicum/soybean symbiosis by plant N status as determined by shoot C:N ratio

Regulation of nitrogenase is not sufficiently understood to engineer symbioses that achieve a high N₂ fixation rate under high levels of soil N. In the present hydroponic growth chamber study we evaluated the hypothesis that nitrogenase activity and the extent of its inhibition by NO₃⁻ may be related to both N and carbohydrate levels in plant tissues. A wide range of C:N ratios in various plant tissues (8.5 to 41.0, 1.9 to 3.7, and 0.8 to 1.8, respectively, in shoots, roots, and nodules) was generated through a combination of light and CO₂ levels, using two soybean genotypes differing in C and N acquisition rates. For both genotypes, N concentration in shoots was negatively correlated to nitrogenase activity and positively correlated to the extent of nitrogenase inhibition by NO₃⁻. Furthermore, nitrogenase activity was positively correlated to total nonstructural carbohydrates (TNC) and C:N ratio in shoot and nodules for both genotypes. Nitrogenase inhibition by NO₃⁻ was negatively correlated to TNC and C:N ratio in shoots, but not in nodules for both genotypes. At the onset of nitrogenase inhibition by NO₃⁻, C:N ratio declined in shoots but not in nodules. These results indicate that both C and N levels in plant tissues are involved in regulation of nitrogenase activity. We suggest that the level of nitrogenase activity may be determined by (1) N needs (as determined by shoot C:N) and (2) availability of carbohydrates in nodules. Modulation of the nitrogenase activity may occur through sensing changes in plant N, i.e. changes in shoot C:N ratio, possibly through some phloem translocatable compound(s).     

The feedback mechanism of nitrate inhibition of nitrogenase activity in soybean may involve asparagine and/or products of its metabolism

A feedback mechanism which involves sensing of change in phloem N concentration has been proposed to control nodulation and dinitrogen fixation in the presence of external combined N. Whether this control is in response to a change in total N or in some specific signal compound(s) is not known. In the present study we reevaluated the hypothesis that control of nodulation and N₂ fixation involves sensing of change in tissue N composition and attempted to identify potential signal molecule(s) involved. Two soybean (Glycine max [L.] Merr.) genotypes (Williams 82 and NOD1-3) differing in nodule number and tolerance to nitrate were germinated in sand trays. Seven-day-old seedlings were inoculated with a solution of Bradyrhizobium japonicum and grown for 28 days in growth chambers, using a hydroponic system with limited N supply to promote nodulation. Half of 28-day-old plants were treated with 15 mM NO₃^?, then control and treated plants were sampled at the onset of nitrogenase inhibition (24 h following NO₃^?, treatment) for evaluation of nitrogenase activity and tissue concentration of total N and of each individual free amino acid. Phenylisothiocyanate-(PITC) amino acid derivatives were separated and quantified using HPLC. The decline in nitrogenase activity following the short-term nitrate treatment was associated with a dramatic asparagine concentration increase in the shoot and an increase in nodule aspartate and glutamate in both genotypes. Asparagine concentration in the shoot increased 35 times from a barely detectable level of 95 to 3 327 nmol g^?1 fresh weight in Williams 82, and more than tripled from 509 to 1 753 nmol g^?1 fresh weight in NOD1-3. Increase in levels of free Asn and in total free amino acids in the shoot following the short-term nitrate treatment was more pronounced in Williams 82 than in its partially nitrate-tolerant mutant NOD1-3. These results indicate that the feedback control of nodule activity may involve sensing changes in shoot asparagine levels and/or products of its metabolism (aspartate and glutamate) in the nodule. These results also indicate that partial-nitrate tolerance of nodulation in the hypernodulated NOD1-3 mutant is associated with a lesser change in tissue N following nitrate treatment.     

Productivity of soybean-rhizobium symbiosis after modification of root nodule bacteria activity with exogenous proteins

Plant growth experiments were conducted to assess symbiotic efficiency, photosynthetic rates, and the development of soybean (Glycine max (L.) Merrill) seedlings after seed inoculation with active and inactive strains of root nodule bacteria Bradyrhizobium japonicum preincubated in the presence homologous and heterologous proteins. The properties of active and inactive symbiotic strains were differentially modulated by homologous soybean lectin, which had a marked influence on plant physiological condition. The incubation of active rhizobia with a homologous lectin, i.e., lectin of the respective plant, increased the nitrogen-fixing activity of nodules and, consequently, elevated photosynthetic rates and weight increments in soybean plants. At the same time, the homologous lectin suppressed the symbiotic properties of inactive strain of nodule bacteria. The preincubation of rhizobia with a heterologous pea lectin had virtually no effect on functioning of symbiotic apparatus and photosynthetic rate, whereas the preincubation of root nodule bacteria with human albumin exerted an effect similar to that induced by a homologous lectin on symbiotic productivity.     

The Glycine-Glomus-Bradyhizobium symbiosis. VIII. Phosphorususe efficiency of CO2 and N2 fixation in mycorrhizal soybean

Soybean [ Glycine max (L.) Merr. cv. Hobbit] plants nodulated by Bradyhizobium japonicum strain USDA 110 were grown in pot cultures in severely P- and N-deficient soil and either colonized by the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus mosseae (Nicol. & Gerd.) Gerd. and Trappe or fertilized with a high (HP) or low (LP) level of KH₂PO₄ (0.6 or 0.3 m M , respectively), After 7 weeks of growth, nodule and chloroplast activities (C₂H₂ reduction and CO₂ exchange rate) were determined. Photosynthetic P-use efficiency of CO₂ fixation was significantly higher in VAM than in HP plants, while that of nitrogenase activity was lower. The LP plants were intermediate in both respects. The ratio of nodule to chloroplast activity [mol C₂H₂ reduced (mol CO₂ fixed)⁻¹] was highest in HP and lowest in VAM plants. Root colonization by the VAM fungus significantly increased nodule number and dry weight and reduced nodule specific mass and activity in comparison to HP plants. In spite of lower nodule activity, VAM plants were significantly larger and had higher N concentrations than the HP plants. The results suggest nonnutritional. VAM-elicited and host-mediated effects on the symbiotic functions of the legume association.     

Oxygen limitation of N2 fixation in stem-girdled and nitrate-treated soybean

The effects of increasing rhizosphere pO₂on nitrogenase activity and nodule resistance to O₂diffusion were investigated in soybean plants [Glycine max (L.) Merr. cv. Harosoy 63] in which nitrogenase (EC 1.7.99.2) activities were inhibited by (a) removal of the phloem tissue at the base of the stem (stem girdling), (b) exposure of roots to 10 mM NO₃over 5 days (NO₃-treated), or (c) partial inactivation of nitrogenase activity by an exposure of nodulated roots to 100 kPa O₂(O₂-inhibitcd). In control plants and in plants which had been treated with 100 kPa O₂, increasing rhizosphere O₂concentrations in 10 kPa increments from 20 to 70 kPa did not alter the steady-state nitrogenase activity. In contrast, in plants in which nitrogenase activities were depressed by stem girdling or by exposure to NO₃, increasing rhizosphere pO₂resulted in a recovery of 57 or 67%, respectively, of the initial, depressed rates of nitrogenase activity. This suggests that the nitrogenase activity of stem-girdled and NO₃-treated soybeans was O₂-limited. For each treatment, theoretical resistance values for O₂diffusion into nodules were estimated from measured rates of CO₂exchange, assuming a respiratory quotient of 1.1 and 0 kPa of O₂in the infected cells. At an external partial pressure of 20 kPa O₂, the stem-girdled and NO₃^--treated plants displayed resistance values which were 4 to 8.6 times higher than those in the nodules of the control plants. In control and O₂-inhibited plants, increases in pO₂from 20 to 70 kPa in 10 kPa increments resulted in a 2.5- to 3.9-fold increase in diffusion resistance to O₂, and had little effect on either respiration or nitrogenase activity. In contrast, in stem-girdled and NO₃^--treated plants, increases in external pO₂had little effect on diffusion resistance to O₂, but resulted in a 2.3- to 3.2-fold increase in nodule respiration and nitrogenase activity. These results are consistent with stem-girdling and NO₃^--inhibition treatments limiting phloem supply to nodules causing an increase in diffusion resistance to O₂at 20 kPa and an apparent insensitivity of diffusion resistance to increases in external pO₂.     

Pools of non-structural carbohydrates in soybean root nodules during water stress

The aim of this study was to examine how the pools of non-structural carbohydrates in soybean nodules are affected under water stress conditions depending on the nature of the symbiont strains with particular emphasis on the plant-borne carbohydrates sucrose and pinitol, and on trehalose, a compatible solute synthesized by the bacteroids. Soybean ( Glycine max [L.] Merr. cv. Maple Arrow) plants were inoculated with the nitrogen-fixing strains Bradyrhizobium japonicum 61-A-101 or USDA 110 spc4 and cultivated axenically under conditions in which nodules formed in an upper soil compartment while roots for water supply grew into a compartment filled with nutrient solution. When the nodules were well established (1 month post inoculation), 10% (w/v) PEG 6000 was added to the nutrient solution. This led to a slowly progressing, moderate water stress, as determined by measuring the decrease of transpiration, and to a decrease in nitrogen fixation. The pool sizes of the major non-structural nodule carbohydrates changed during progression of water stress. Sucrose, the major soluble carbohydrate in nodules of unstressed plants (2 and 4%, respectively of nodule dry weight depending on symbiont strain), strongly increased in nodules of stressed plants, reaching nearly 10% of dry weight. The activities of two major sucrose-consuming enzymes, sucrose synthase and alkaline invertase, decreased markedly in nodules of stressed plants. Starch decreased only transiently upon water stress. Pinitol, a cyclitol serving as compatible solute in many plants, increased more than 4 times, reaching about 1% of nodule dry weight during the stress. Trehalose, the major soluble carbohydrate synthesized by the bacteroids, increased in nodules colonized by USDA 110 spc4 from about 0.2 to 0.8% of nodule dry weight, while in nodules colonized by 61-A-101 it amounted to more than 1.5% of dry weight both with and without stress.     

The Glycine-Glomus-Bradyrhizobiun symbiosis. XI. Nodule gas exchange and efficiency as a function of soil and root water status in mycorrhizal soybean

Soybean [ Glycine max (L.) Merr. cv. Hobbit] plants were inoculated with a HUP− strain of Bradyrhizobium japonicum (Nitragin 61A118) and either colonized by the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus mosseae (Nicol & Gerd.) Gerd. and Trappe or fertilized with KH₂PO₄ (nonVAM). They were grown for 50 days in a growth chamber and harvested over a 4-day drought period during which available soil water decreased to 0. Nodule P concentrations and P-use efficiency declined linearly with soil and root water content during the harvest period in both VAM and nonVAM plants. Nitrogenase activity, estimated from H₂ evolution and C₂H₂ reduction data, was also a linear function of declining nodule P concentrations and CO₂-exchange rates and showed simular patterns in both treatments. Hydrogen evolution and the relative efficiency of N₂ fixation, on the other hand, reacted differently to increasing drought in VAM and nonVAM plants. Differences in the responses of nodule activity in VAM and nonVAM plants to drought are interpreted in terms of demand for nodule P and carbohydrates and of the effects of dehydration on O₂ diffusion through nodule tissue.     

Nitrogen fixation and growth of soybean as influenced by varying the methods of inoculation with Bradyrhizobium japonicum

The effects of inoculating soil with a water suspension of Bradyrhizobium japonicum (i) at seeding, (ii) 7, or (iii) 14 days after planting (DAP), (iv) seed slurry inoculation and (v) seed slurry supplemented with postemergence inoculation of a water suspension of Bradyrhizobium at 7 or (vi) 14 DAP, on nodulation, N₂ fixation and yield of soybean (Glycine max. [L.] Merrill) were compared in the greenhouse. The ¹⁵N isotope dilution technique was used to quantify N₂ fixed at flowering, early pod filling and physiological maturity stages (36, 52 and 70 DAP, respectively). On average, the water suspension inoculation formed the greatest number of nodules, and seed plus postemergence inoculation formed slightly more nodules than the seed-only inoculated plants (27, 19 and 12 nodules/plant respectively at physiological maturity). Seed slurry inoculation followed by postemergence inoculation at 14 DAP gave the highest nodule weight, with the plants fixing significantly more (P<0.05) N₂ (125 mg N plant⁻¹ or 56% N) than any other treatment (mean, 75 mg plant⁻¹ or 35% N). However, the higher N₂ fixation was not translated into higher N or dry matter yields. Estimates of N₂ fixed by the ostemergence Bradyrhizobium inoculations as well as plant yield were not significantly different from those of the seed slurry inoculation. Thus, delaying inoculation (e.g., by two weeks as in this study) did not reduce the symbiotic ability of soybean plants.     

Intercellular location of glycoprotein in soybean nodules: effect of altered rhizosphere oxygen concentration

Abstract. A glycoprotein which occludes intercellular spaces in the inner cortex of legume nodules may be involved in controlling oxygen diffusion into rhizobial-infected cells. Here we investigated this possibility by localizing the glycoprotein using monoclonal antibodies and immunogold labelling in nodulated roots of soybean cv. Clarke inoculated with Bradyrhizobium japonicum strain RCR3442 exposed to atmospheres with either 10, 21 (control) or 40% oxygen for 28d. Infected cells showed evidence of premature senescence when grown in above or below ambient pO₂ particularly at 10% oxygen, although cortical cells appeared to be little altered by oxygen treatment. In the inner cortical cells, more glycoprotein was seen to be occluding intercellular spaces of those nodules subjected to 40% oxygen and less in those nodules exposed to 10% oxygen, when compared to controls. This observation, made at the light microscope level (using silver enhancement) was confirmed under the TEM using immunogold labelling. Therefore, it is suggested that intercellular space glycoprotein is one of the structural components of the diffusion resistance in the cortex of legume nodules.     

缺硼对大豆根瘤结构和功能的影响

在营养液培养条件下以普通结结瘤大豆Ｂｒａｇｇｃｖ．「Ｇｌｙｃｉｎｅｍａｘ（Ｌ．）Ｍｅｒｒ」及其超结瘤突变体ｎｔｓ３８２为实验材料,运用光学显微方法研究了硼对大豆根瘤结构的影响,并测定了根瘤固氮酶活性结果表明,缺硼使根瘤结构受到严重破坏,并使固氮酶活性显著下降,缺硼使根瘤结构受到破坏是导致固氮酶活性下降的可能原因。     

Mineral and lipid composition of Glycine - Glomus - Bradyrhizobium symbioses

Soybean [Glyeine max (L.) Merr. cv. Amsoy 71] plants were inoculated with either the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus fasciculatum. with a strain of Bradyrizobobium Japonicum. or with both endophytes in combintion. Noninoculated controlplantes were fertilized with levels of N and P previously found to compensate for nutrient input following infection by Bradyhizobium or Glomus Temporal differences in N and P assimilation in nodulated or mycorrhizal plants indicated that Glomus was most effective during early vegetative growth and Bradyrhizobion was active until the mid-pod-fill stage in soybean. In general. soybeans colonized by Glomus contained more Cu but less Mn and P than corresponding P-fertilized plants. Soyubean roots infected with G. fasciculaum contained five unusual fatty acids: [16: 1 (11c): 8:3 (6c, 9c, 12c): 20:3 (8c, 11c, 14c): 20:4 (5c, 8c, 11c, 14c): 20:5 (5c, 8c, 11c, 14c, 17c)] that were absent in non-infected roots. Fatty acid 16:1 (11c) comprised 43% of total fatty acids in Glomus-infected roots at week 9 and 29% of total root fatty acids at week 15. This isomer of hexadecenoic acid was positively correlated with vesicle number (r = 0.92**). and 16:1 (11c) was probably the principalstorage fatty acid in fungal vesicles. These five unusual fatty acids were not found in the leaves. pods or seeds of either VAM or non-VAM plants. Specific leaf area increased with time in nodulated soybeans. but these plants contained lessCu than corresponding N-fertilized plants. Soybeans nodulated with Bradyrthizobium contained more total lipid and proportionately more fatty acid 16:0 than N-fertilized plants. Infection by Glomus or Bradyrhizobium also altered the fatty acid composition of above-ground plant parts, although these changes were subtle compared to the markedly different fatty acid found in Glomus-infected roots. These findings suggest that seed quality may be altered due to the physiological changes resulting from infection by N²-fixing bacteria and/or endomycorrhizal fungi. Observed differences in the plant nutrition of inoculated soybeans could not be replicated by fertilizer addition alone.     

The Glycine-Glomus-Bradyrhizobium symbiosis. IX. Nutritional, morphological and physiological responses of nodulated soybean to geographic isolates of the mycorrhizal fungus Glomus mosseae.

The objective of the work was to determine differences in plant response to geographic isolates of a vesicular-arbuscular mycorrhizal (VAM) fungus, and to demonstrate the need for such determinations in the selection of desirable host-endophyte combinations for practical applications. Soybean ( Glycine max (L.) Merr.) plants were inoculated with Bradyrhizobium japonicum and isolates of the VAM-fungal morphospecies Glomus mosseae (Nicol. & Gerd.) Gerd. and Trappe, collected from an arid (AR), semiarid (SA) or mesic (ME) area. Inoculum potentials of the VAM-fungal isolates were determined and the inocula equalized, achieving the same level of root colonization (41%, P >0.05) at harvest (50 days). Plants of the three VAM treatments (AR, SA and ME) were evaluated against von VAM controls. Significant differences in plant response to colonization were found in dry mass, leaf K, N and P concentrations, and in root/shoot, nodule/root, root length/leaf area and root length/root mass ratios. The differences were most pronounced and consistent between the AR and all other treatments. Photosynthesis and nodule activity were higher ( P <0.05) in all VAM treatments, but only the AR plants had higher ( P <0.05) photosynthetic water-use efficiency than the controls. Nodule activity, evaluated by H₂ evolution and C₂H₂ reduction, differed significantly between treatments. The results are discussed in terms of nutritional and non-nutritional effects of VAM colonization on the development and physiology of the tripartite soybean association in the light of intraspecific variability within the fungal endophyte.     

A metabolic connection between nitrogenase activity and the synthesis of ureides in nodulated soybean

Application of allopurinol (AP; 1H-pyrazolo-[3,5- d ]pyrimidine-4-o1) to intact nodulated roots of ureide-forming legumes causes rapid inhibition of NAD:xanthine dehydrogenase (XDH: EC 1.2.1.37), cessation of ureide synthesis and, subsequently, severe nitrogen deficiency (Atkins et al. 1988. Plant Physiology 88: 1229–1234). Nitrogen deficiency is a result of inhibited nitrogenase (EC 1.7.99.2) activity. Using an open gas exchange system to measure H₂ and CO₂ evolution, short term effects of AP application were examined in a Hup ⁻ soybean symbiosis [ Glycine max (L.) Merr. cv. Harosoy: USDA 16]. The onset of inhibition of nitrogenase was detected after ca 2 h exposure of the roots to AP. At the same time xanthine began to accumulate and ureide levels declined in nodules as a result of inhibition of XDH. The decline in H₂ evolution following AP application was not due to altered electron allocation between N₂ and H⁺ by nitrogenease but was coincident with increased gaseous diffusive resistance of nodules and a decline in intracellular oxygen concentration. A possible scheme for the intermediary metabolism of soybean nodules which might account for a direct connection between nitrogenase activity and ureide synthesis is proposed. The suggested mechanism envisages coupling production of reducing power by cytosolic enzymes of purine oxidation to synthesis of dicarboxylic acid substrates (malate and succinate) required for bacteroid respiration.     

Regulation of soybean nodule phosphoenolpyruvate carboxylase in vivo

The sensitivity of soybean ( Glycine max L. Merr, cv. PS47) nodule phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) to inhibition by L-malate in vitro increased when well-nodulated plants were subjected to decapitation (shoot removal). There was no effect of decapitation on the apparent K_m of the enzyme for its substrate PEP but the I₅₀ (L-malate) decreased from 4.2 to 1.7 m M. The total amount of PEP doubled and that of malate decreased by half in the nodules of decapitated plants relative to the control plants. This observation was consistent with a decrease in the activity of PEPC in vivo as a result of the increased malate sensitivity of the enzyme observed in vitro. Sucrose levels in the nodules declined in response to decapitation but there were no effects on the levels of glucose, fructose, pyruvate, 2-oxoglutarate, glutamine or glutamate. The results are discussed in terms of the role of protein phosphorylation in the regulation of PEPC activity in legume nodules.     

Regulation of nitrogenase activity by light in the azolla-anabaena symbiosis

Nitrogenase activity and the rate of photosynthesis were measured simultaneously in Azolla by a continuous gas flow system. The mode of interaction between light, photosynthesis and nitrogenase activity was analysed.Nitrogenase activity dropped off when either Azolla plants or the cyanobiont Anabaena were transferred from light to dark. This decline was immediate and was independent of length or intensity of the prior light phase. Reillumination restored nitrogenase activity.Nitrogenase activity did not depend on the rate of photosynthesis at light intensities below 10 μE m⁻² s⁻¹. Its activity was saturated at 200 μE m⁻² s⁻¹ while CO₂ fixation was saturated at a light intensity of 850 μE m⁻² s⁻¹. Azolla photosynthetic activity followed the absorption spectrum of chlorophyll a, while nitrogenase activity markedly increased between 690 and 710 nm. Inhibition of photosynthesis by DCMU was accompanied by an increase in nitrogenase activity. These results suggest direct light regulation of nitrogenase activity in Azolla independent of CO₂ fixation, and a possible inhibition of nitrogenase activity by the oxygen produced in photosynthesis.     

Short communication: Screening of native and foreign Bradyrhizobium japonicum strains for high N2 fixation in soybean

Four local rhizobia isolates selected after two screening experiments and five USDA Bradyrhizobium japonicum strains were estimated for N₂ fixation in soybean using the ¹⁵N isotope dilution technique. Strain USDA 110 was superior to the local isolates in nodulation and N₂ fixation when inoculated onto soybean cv TGX 1497-ID in a Nigerian soil and could therefore be used as an inoculant for enhanced N₂ fixation in soybean in Nigeria.     

Nutrient transfer between the root zones of soybean and maize plants connected by a common mycorrhizal mycelium

The objective of the study was to determine whether nutrient fluxes mediated by hyphae of vesicular-arbuscular mycorrhizal (VAM) fungi between the root zones of grass and legume plants differ with the legume's mode of N nutrition. The plants, nodulating or nonnodulating isolines of soybean [ Glycine max (L.) Merr.], were grown in association with a dwarf maize ( Zea mays L.) cultivar in containers which interposed a 6-cm-wide root-free soil bridge between legume and grass container compartments. The bridge was delimited by screens (44 μm) which permitted the passage of hyphae, but not of roots and minimized non VAM interactions between the plants. All plants were colonized by the VAM fungus Glomus mosseae (Nicol. & Gerd.) Gerd. and Trappe. The effects of N input to N-sufficient soybean plants through N₂-fixation or N-fertilization on associated maize-plant growth and nutrition were compared to those of an N-deficient (nonnodulating, unfertilized) soybean control. Maize, when associated with the N-fertilized soybean, increased 19% in biomass, 67% in N content and 77% in leaf N concentration relative to the maize plants of the N-deficient association. When maize was grown with nodulated soybean, maize N content increased by 22%, biomass did not change, but P content declined by 16%. Spore production by the VAM fungus was greatest in the soils of both plants of the N-fertilized treatment. The patterns of N and P distribution, as well as those of the other essential elements, indicated that association with the N-fertilized soybean plants was more advantageous to maize than was association with the N₂-fixing ones.     

Nitrogenase activity and ureide levels in a supernodulating soybean mutant: Effects of inoculum dose and nitrate treatment

The effects of nitrate on nitrogenase (EC 1.18.2.1) activity of soybean ( Glycine max [L.] Merr) cv. Bragg and its supernodulating mutant derivative, nts382, were compared. A short-term nitrate treatment was used to allow effects on nitrogenase activity to be studied in the absence of effects on nodule growth and a low inoculum dose, which prevented supernodulation of nts382, was employed to test for any interaction between supernodulation and the magnitude of the effect of nitrate on nitrogenase activity. At the usual inoculum dose, nitrogenase activity, per g nocule, of nts382 was lower than that of Bragg and was proportionally less affected by nitrate. Decreasing the inoculum dose increased nitrogenase activity of nts382 and also the proportional decline in response to nitrate. The decline in the ureide conentration in xylem exudate in response to nitrate was proportionally similar to the decline in nitrogenase activity per plant. However, although nitrogenase activity per plant of nts382 was several-fold less than that of Bragg, the ureide flux rate (ureide concentration x xylem sap exudation rate), was not different. At the usual inodulum dose, the ureide content of the nocules, stems plus petioles and leaves of nts382 was greater than that of Bragg. Decreasing the inoculum dose reduced the ureide content of the nodules of nts382 but not of Bragg. Ureide degradative capacity of the leaves was the same for Bragg and nts382. Low activities of 5-phosphoribosyl pyrophosphate amidotransferase (EC 2.4.2.14) and glutamine synthetase (EC 6.3.1.2) in the nodules reflected the low nitrogenase activity of nts382.     

Regulation of nodule formation in soybean-Bradyrhizobium symbiosis is controlled by shoot or/and root signals

Two strains of Bradyrhizobium japonicum wereevaluated with five commercial cultivars of soybean(Clark, Crauford, Davis, Centaur, and Nessen) and onehypernodulating mutant NOD1-3. The hypernodulatingNOD1-3 produced 30–50 times more nodules thancommercial cultivars either inoculated with B.japonicum strain USDA 123 or RCR 3409. The currentexperiments were extended to determine if therestricted nodulation of commercial cultivars could be overcome by grafting them to a hypernodulated shoot (NOD1-3). Grafting of NOD1-3 shoots to Clark and Davis roots induced hypernodulation on roots of Clark and Davis but did not enhance nodulation when grafted onto the roots of Crauford, Centaur, and Nessen. The shoots of Clark, Davis, Centaur and Nessen significantlyinhibited nodule formation on the root of NOD1-3,while Crauford shoots did not alter nodule formationon the roots of NOD1-3 as compared with self-grafts ofNOD1-3. It appears that the shoot of NOD1-3 has theability to alter autoregulatory control of nodulationof Clark and Davis cultivars, but did not withCrauford, Centaur and Nessen. The results suggestedthat the regulation of nodulation in soybean cultivarsClark and Davis is controlled by the shoot factors,while the Crauford was root controlled.Reciprocal-grafts between NOD1-3 and Centaur or Nessenindicate that both shoot and root factors involved inregulation of nodulation and the regulation ofnodulation did not depend on bradyrhizobial strains. Isoflavonoid analyses from extracts of grafted plantsshowed that NOD1-3 shoots had markedly higher rootisoflavonoid concentrations in roots of both Clark andNOD1-3. The shoot control of hypernodulation may becausally related to differential root isoflavonoidlevels, which are also controlled by the shoot. Thecurrent work was extended to investigate the effect ofapplication of an isoflavonoid (daidzein) on nodulationand nitrogen fixation of soybean cultivars Clark andCentaur as well as in vitro growth of Bradyrhizobium japonicum. Application of theisoflavonoid (daidzein) significantly enhanced thenodulation and nitrogenase activity of Clark but notof Centaur indicating that this character is notrelated to isoflavonoids. Therefore, autoregulationin Clark and Centaur plants may be separate events inlegume-rhizobia symbiosis and regulated by differentkinds of signals. Addition of daidzein to yeastmannitol broth medium promoted the growth of B.japonicum strain USDA 123 and RCR 3409. It seemsthat this compound is able to help the nodulation ofsoybean cv Clark by a Bradyrhizobium strain. Understanding the signaling pathways between rhizobiaand their host plants may allow modifications of thisinteraction to improve symbiotic performance.     

Laser‐ablation electrospray ionization mass spectrometry with ion mobility separation reveals metabolites in the symbiotic interactions of soybean roots and rhizobia

Technologies enabling in situ metabolic profiling of living plant systems are invaluable for understanding physiological processes and could be used for rapid phenotypic screening (e.g., to produce plants with superior biological nitrogen‐fixing ability). The symbiotic interaction between legumes and nitrogen‐fixing soil bacteria results in a specialized plant organ (i.e., root nodule) where the exchange of nutrients between host and endosymbiont occurs. Laser‐ablation electrospray ionization mass spectrometry (LAESI‐MS) is a method that can be performed under ambient conditions requiring minimal sample preparation. Here, we employed LAESI‐MS to explore the well characterized symbiosis between soybean (Glycine max L. Merr.) and its compatible symbiont, Bradyrhizobium japonicum. The utilization of ion mobility separation (IMS) improved the molecular coverage, selectivity, and identification of the detected biomolecules. Specifically, incorporation of IMS resulted in an increase of 153 differentially abundant spectral features in the nodule samples. The data presented demonstrate the advantages of using LAESI–IMS–MS for the rapid analysis of intact root nodules, uninfected root segments, and free‐living rhizobia. Untargeted pathway analysis revealed several metabolic processes within the nodule (e.g., zeatin, riboflavin, and purine synthesis). Compounds specific to the uninfected root and bacteria were also detected. Lastly, we performed depth profiling of intact nodules to reveal the location of metabolites to the cortex and inside the infected region, and lateral profiling of sectioned nodules confirmed these molecular distributions. Our results established the feasibility of LAESI–IMS–MS for the analysis and spatial mapping of plant tissues, with its specific demonstration to improve our understanding of the soybean‐rhizobial symbiosis.