In VivoP NMR Spectroscopic Studies of Soybean Bradyrhizobium Symbiosis: I. Optimization of Parameters.

(31)P NMR spectroscopy was used to study in vivo the symbiotic state established between soybean (Glycine max [L.] Merr. cv Williams) and Bradyrhizobium japonicum (USDA 110 and 138). Different experimental conditions were used to maintain perfused, respiring detached or attached nodules in an NMR magnet. The pH of the perfusion medium affected the cytoplasmic pH and the resolution of the spectra. The internal Pi content and distribution were assessed as a function of nodule age and green-house growth conditions and the rate of glucose and 2-deoxyglucose uptake into nodules in split and intact states. The major metabolites (glucose-6-P, fructose-1,6-diP, P-choline, Pi, NTP, UDP-glc, and NAD) were readily identified from (31)P NMR spectra of perchloric acid extracts of nodules with the exception of one unknown phosphorus metabolite. Nodules stressed by glucose deprivation demonstrated movement of Pi between the vacuole and cytoplasmic compartments not previously observed in (31)P NMR studies.

Endogenous ethylene production is a potential problem in the measurement of nitrogenase activity associated with excised corn and sorghum roots.

Endogenous ethylene production was evaluated as a source of ethylene during acetylene reduction assays with freshly collected roots of field-grown corn, Zea mays L. cv Funks G-4646, and sorghum, Sorghum bicolor (L.) Moench. cv CK-60A. Ethylene production was not detected when roots were incubated in air without acetylene. The presence of endogenous ethylene production was confirmed when roots were incubated anaerobically and in the presence of 40 millimolar sodium hydrosulfite. Ethylene oxidase activity was also associated with excised roots. The rate of ethylene oxidation was higher than the rates of ethylene accumulation during either acetylene reduction assays or anaerobic incubations. These results indicate that the procedure of incubating roots of grasses in air to monitor endogenous ethylene production is not a valid control in acetylene reduction studies with grasses. The presence of endogenous ethylene production during acetylene reduction assays was demonstrated by using either CO to inhibit nitrogenase activity or chloramphenicol to inhibit nitrogenase synthesis in freshly excised roots.

Relationship between Ureide N and N(2) Fixation, Aboveground N Accumulation, Acetylene Reduction, and Nodule Mass in Greenhouse and Field Studies with Glycine max L. (Merr).

The relationship between ureide N and N(2) fixation was evaluated in greenhouse-grown soybean (Glycine max L. Merr.) and lima bean (Phaseolus lunatus L.) and in field studies with soybean. In the greenhouse, plant N accumulation from N(2) fixation in soybean and lima bean correlated with ureide N. In soybean, N(2) fixation, ureide N, acetylene reduction, and nodule mass were correlated when N(2) fixation was inhibited by applying KNO(3) solutions to the plants. The ureide-N concentrations of different plant tissues and of total plant ureide N varied according to the effectiveness of the strain of Bradyrhizobium japonicum used to inoculate plants. The ureide-N concentrations in the different plant tissues correlated with N(2) fixation. Ureide N determinations in field studies with soybean correlated with N(2) fixation, aboveground N accumulation, nodule weight, and acetylene reduction. N(2) fixation was estimated by (15)N isotope dilution with nine and ten soybean genotypes in 1979 and 1980, respectively, at the V9, R2, and R5 growth stages. In 1981, we investigated the relationship between ureide N, aboveground N accumulation, acetylene reduction, and nodule mass using four soybean genotypes harvested at the V4, V6, R2, R4, R5, and R6 growth stages. Ureide N concentrations of young stem tissues or plants or aboveground ureide N content of the four soybean genotypes varied throughout growth correlating with acetylene reduction, nodule mass, and aboveground N accumulation. The ureide-N concentrations of young stem tissues or plants or aboveground ureide-N content in three soybean genotypes varied across inoculation treatments of 14 and 13 strains of Bradyrhizobium japonicum in 1981 and 1982, respectively, and correlated with nodule mass and acetylene reduction. In the greenhouse, results correlating nodule mass with N(2) fixation and ureide N across strains were variable. Acetylene reduction in soybean across host-strain combinations did not correlate with N(2) fixation and ureide N. N(2) fixation, ureide N, acetylene reduction, and nodule mass correlated across inoculation treatments with strains of Bradyrhizobium spp. varying in effectiveness on lima beans. Our data indicate that ureide-N determinations may be used as an additional method to acetylene reduction in studies of the physiology of N(2) fixation in soybean. Ureide-N measurements also may be useful to rank strains of B. japonicum for effectiveness of N(2) fixation.

Autoanalytical procedure for the determination of allantoin and allantoic Acid in soybean tissue.

Ureide analyses of soybean (Glycine max L.) tissues were accomplished with a modified and simplified automated analysis used to determine allantoin concentration in rat urine. The length of the circuit and flow rates of the solutions were reduced, and NaOH was used for color development at room temperature. Keto-acids did not significantly interfere with the determinations of ureides except for glyoxylic acid in extracts of fresh soybean tissue. The interference caused by glyoxylic acid was avoided by adding phenylhydrazine HCl to the solution of NaOH used for alkaline hydrolysis of allantoin.

Interaction of Combined Nitrogen with the Expression of Root-Associated Nitrogenase Activity in Grasses and with the Development of N(2) Fixation in Soybean (Glycine max L. Merr.).

Soluble root N concentrations of corn, sorghum, pearl millet, rice, wild rice, and soybeans were determined and related to measurements of nitrogenase activity and changes in availability of combined N to plants. In corn, sorghum, and pearl millet, applications of fertilizer N increased soluble root N concentrations, but root-associated nitrogenase activity was negligible in control and treated plants. Applications of NH(4)NO(3) to rice increased the water soluble root N concentrations and inhibited root-associated nitrogenase activity. In wild rice, root-associated nitrogenase activity was absent during vegetative growth and developed at the reproductive growth stage. The soluble root N concentration decreased progressively as wild rice grew indicating that the availability of combined N in the root environment declined. Therefore, development of nitrogenase activity in wild rice is associated with the change in availability of combined N in the root environment. The development of nitrogenase activity in wild rice was probably not due to colonization of roots by N(2)-fixing bacteria because most probable numbers of recovery did not significantly vary throughout the plants' growth cycle. In field-grown soybeans with or without fertilizer N application, we also observed a relationship between a decrease in soluble root N concentration and the development of nitrogenase activity.

Enumeration and Localization of N(2)-Fixing Bacteria Associated with Roots of Spartina alterniflora Loisel.

Numbers and possible locations of N(2)-fixing bacteria were investigated in roots of Spartina alterniflora Loisel, which support nitrogenase activity in the undisturbed native habitat. N(2)-fixing bacteria were recovered in cultures both from S. alterniflora roots and from the surrounding sediment, and they formed a greater proportion of the bacteria recovered from root homogenates than from salt-marsh sediment. N(2)-fixing bacteria were recovered in high numbers from the rhizoplane of S. alterniflora after roots were treated with 1 or 5% chloramine-T for 1 h or with 1% NaOCl for 1 or 2 h. Immersing S. alterniflora roots in 5% NaOCl for 1 h was more effective in distinguishing bacteria inside the roots since this treatment nearly eliminated N(2)-fixing bacteria recoverable from the rhizoplane, although high numbers of N(2)-fixing bacteria were recovered from homogenates of roots treated with 5% NaOCl for 1 h. However, this treatment was less effective with roots of Zea mays L. (Funks G4646) and Sorghum bicolor (L.) Moench (CK-60 A), indicating that techniques to surface sterilize roots should be evaluated for different plants. Bacteria were observed by light and electron microscopy inter- and intracellularly in the cortex and in the aerenchyma of S. alterniflora roots. This study clearly shows that bacteria, including N(2) fixers, colonize the interior of roots of S. alterniflora growing in a Chesapeake Bay, Maryland, salt marsh.

Physiology of Root-Associated Nitrogenase Activity in Oryza sativa.

An intact method for measuring immediately linear rates of acetylene reduction was used to investigate the relationship between temperature, pH, O(2) concentration, and light intensity with the rate of root-associated nitrogenase activity in rice (Oryza sativa L.). Nitrogenase activity varied over a temperature range of 10 to 50 degrees C and optimal rates of acetylene reduction were recorded at 35 degrees C. Nitrogenase activity was also influenced by the pH of the liquid surrounding the roots prior to assay. Maximal rates of acetylene reduction were recorded over a pH range from 5.8 to 7.5. Nitrogenase activity was significantly reduced by concentrations of O(2) 0.5% (v/v) or more when the intact plant assay method was used, and no optimum was detected. However, when the plant tops were removed and the cut ends sealed from the atmosphere for 4 hours, acetylene reduction rates were maximal at 0.25% O(2) (v/v). When plants were moved from sunlight (1,400 microeinsteins per square meter per second) to shade (9.6) root-associated nitrogenase activity at 35 degrees C significantly decreased 15 min later to one-fourth the rate and recovered upon return to sunlight. When the light intensity reaching the leaf canopy was progressively reduced from 1,050 to 54 microeinsteins per square meter per second the rate of root-associated nitrogenase activity decreased from 550 +/- 135 to 192 +/- 55 nanomoles ethylene per gram dry root per hour. The study suggests that the rate of root-associated nitrogenase activity in rice at constant temperature may well be mediated by variations in the concentration of O(2) resulting from changes in the rate of photosynthesis as well as variations in the rate of transport of photosynthate.

Ontogenetic Variation of Nitrogenase, Nitrate Reductase, and Glutamine Synthetase Activities in Oryza sativa.

The relationship between the rates of nitrogenase, nitrate reductase, and glutamine synthetase activities, and plant ontogeny in rice (Oryza sativa L.), cultivar ;M9', grown in salt marsh sediment with and without nitrate treatment was studied. In both treatments, nitrogenase activity measured as the immediate linear rate of acetylene reduction by bacteria associated with the roots varied with plant age. In control plants, the nitrogenase activity developed during the vegetative stage, peaked during early reproductive growth and then declined. The application of 10 kilograms N per hectare as KNO(3) once every 2 weeks delayed the development of and decreased the nitrogenase activity. The nitrogenase activity in both treatments developed as leaf nitrate reductase activity declined. The per cent nitrogen of roots was negatively correlated with the rates of acetylene reduction during the life cycles of control and nitrate-treated plants. This suggests that the concentration of combined nitrogen in the plants controlled the development and rate of root-associated nitrogenase activity. During reproductive growth, no nitrate reductase activity was detected in the roots from either treatment. In control plants, the patterns of nitrogenase activity and glutamine synthetase activity in the roots were similar. Thus, rice roots have the potential to assimilate ammonia while fixing N(2). During the vegetative and early reproductive stages of growth, the development of maximal rates of nitrogenase activity coincided with an increase of total nitrogen of the plants in both treatments.

Comparing time course profiles of immediate acetylene reduction by grasses and legumes.

The time course profiles of C(2)H(2) reduction by intact Scirpus olneyi (bulrush), Oryza sativa (rice) and Spartina alterniflora (cordgrass) with roots in atmospheres of N(2) and 30-day-old Glycine max (soybean) in air were all immediately linear. This is the first report of immediately linear rates of C(2)H(2) reduction by grass roots removed from soil. The immediately linear profile of C(2)H(2) reduction by soil-free grass roots was achieved by preventing contact between the roots and air. Roots of soybeans and S. olneyi receiving pretreatments of O(2) above normal environmental levels for 15 min before assay exhibited a short delay in C(2)H(2) reduction. These initially nonlinear rates of C(2)H(2) reduction are attributable to transient O(2) inhibition of nitrogenase. Initial nonlinear rates of C(2)H(2) reduction were also observed with immature soybean plants and with intact plant assays of O. sativa and S. olneyi in which C(2)H(2) was injected into cylinders surrounding the plant tops. These results indicate that, apart from O(2) inhibition of nitrogenase, the diffusion of C(2)H(2) and C(2)H(4) between the nitrogen-fixing sites and the sampling ports may cause initial nonlinear rates of C(2)H(2) reduction. We conclude that in situ plant-associated nitrogenase activity should result in immediate reduction of C(2)H(2) and that linear rates are observed when the proper assay conditions are used. Our data suggest that nitrogen fixation is closely associated with the roots of S. olneyi, O. sativa, and S. alterniflora growing in salt marsh sediment.

Immediate acetylene reduction by excised grass roots not previously preincubated at low oxygen tensions.

Excised roots of Spartina alterniflora Loisel. and corn reduced acetylene in air without the previously reported period of zero activity lasting 8 to 18 hours. The profiles of acetylene-dependent ethylene accumulation by excised roots and intact plants of S. alterniflora were similar. No significant change in the number of bacteria associated with the roots was detectable during the assay. Most of the nitrogenase activity was detected in the roots and rhizomes of the plants. The salt marsh sediment also was capable of reducing acetylene. Additional damage to roots by washing and cutting increased the rate of acetylene reduction with samples incubated in air. Low concentrations of nitrate significantly inhibited the nitrogenase activity associated with the sediment and excised roots, but not with intact plants. Rates of acetylene reduction by excised corn roots were low. Oxidation and endogenous production of ethylene in the absence of acetylene were negligible. Measurements made with excised grass roots as described probably reflect the occurrence and magnitude of nitrogenase activity associated with the plants in the field.