Isozymes of Glutamine Synthetase in Phaseolus vulgaris L. and Phaseolus lunatus L. Root Nodules

The glutamine synthetase (GS) isozymes in the plant fraction of nodule extracts from 62 cultivars of Phaseolus vulgaris L. and one cultivar of Phaseolus lunatus L. were analyzed by polyacrylamide gel electrophoresis. All P. vulgaris nodule extracts displayed two GS activity bands: a nodule-specific band (GS_n1) and a band (GS_n2) similar to the single band (GS_r) present in root extracts. In nodule extracts of P. lunatus, the GS_n1 band was detected, but the GS_n2 band was barely detectable. In contrast to P. vulgaris, the GS_n2 band and the GS_r band of P. lunatus appeared to be different. The electrophoretic mobility of the GS_n1 band in P. vulgaris was governed by both the plant cultivar and the development stage of the nodule. In nodule extracts of P. vulgaris and P. lunatus, the zone of GS_n1 activity coincided with six to nine distinct protein bands as revealed after treatment of gels, which had previously been stained for GS activity, with Coomassie blue. All these protein bands were shown to consist of polypeptides of identical molecular weight (approximately 47,000 daltons) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Our results indicate that P. vulgaris continuously generates isozymes of GS_n1 of increasing electrophoretic mobility during the course of nodule development.     

Mechanism of Nitrogenase Inhibition in Soybean Nodules : Pulse-Modulated Spectroscopy Indicates that Nitrogenase Activity Is Limited by O(2)

A novel, pulse-modulated spectroscopic system for measuring fractional leghemoglobin oxygenation and infected cell O₂ concentration (O_i) in intact attached nodules of soybean (Glycine max) is described. The system is noninvasive and uses a pulsed (1000 Hertz) light-emitting diode coupled to an optical fiber to illuminate the nodule with light at 660 nanometer. A second optical fiber receives a portion of the light reflected from the nodule and directs this to a photodiode. A lock-in amplifier measures only the signal from the photodiode which is in phase with the pulsed light from the light-emitting diode, and the voltage output from the amplifier, proportional to reflectance, is used to calculate fractional leghemoglobin oxygenation and the nanomolar concentration of free O₂ in the infected cells of the nodule (O_i). The system was used to show that inhibition of nitrogenase activity in soybean nodules by NO₃⁻ treatment, stem-girdling, continuous darkness, or nodule disturbance is caused by a reduction in O_i and limitation of respiration in support of nitrogenase activity. A plot of nitrogenase activity (measured as peak H₂ evolution in Ar:O₂) versus O_i for the various treatments was consistent with the concept that O_i limits in vivo nitrogenase activity in legume nodules under adverse conditions. The potential for using O_i to estimate nitrogenase activity in laboratory and field-grown legumes is discussed.     

Stress-Induced Legume Root Nodule Senescence. Physiological, Biochemical, and Structural Alterations

Nitrate-fed and dark-stressed bean (Phaseolus vulgaris) and pea (Pisum sativum) plants were used to study nodule senescence. In bean, 1 d of nitrate treatment caused a partially reversible decline in nitrogenase activity and an increase in O₂ diffusion resistance, but minimal changes in carbon metabolites, antioxidants, and other biochemical parameters, indicating that the initial decrease in nitrogenase activity was due to O₂ limitation. In pea, 1 d of dark treatment led to a 96% decline in nitrogenase activity and sucrose, indicating sugar deprivation as the primary cause of activity loss. In later stages of senescence (4 d of nitrate or 2–4 d of dark treatment), nodules showed accumulation of oxidized proteins and general ultrastructural deterioration. The major thiol tripeptides of untreated nodules were homoglutathione (72%) in bean and glutathione (89%) in pea. These predominant thiols declined by approximately 93% after 4 d of nitrate or dark treatment, but the loss of thiol content can be only ascribed in part to limited synthesis by γ-glutamylcysteinyl, homoglutathione, and glutathione synthetases. Ascorbate peroxidase was immunolocalized primarily in the infected and parenchyma (inner cortex) nodule cells, with large decreases in senescent tissue. Ferritin was almost undetectable in untreated bean nodules, but accumulated in the plastids and amyloplasts of uninfected interstitial and parenchyma cells following 2 or 4 d of nitrate treatment, probably as a response to oxidative stress.     

Nodule Activity and Allocation of Photosynthate of Soybean during Recovery from Water Stress

Nodulated soybean plants (Glycine max [L.] Merr. cv Ransom) in a growth-chamber study were subjected to a leaf water potential (Ψ_w) of −2.0 megapascal during vegetative growth. Changes in nonstructural carbohydrate contents of leaves, stems, roots, and nodules, allocation of dry matter among plant parts, in situ specific nodule activity, and in situ canopy apparent photosynthetic rate were measured in stressed and nonstressed plants during a 7-day period following rewatering. Leaf and nodule Ψ_w also were determined. At the time of maximum stress, concentration of nonstructural carbohydrates had declined in leaves of stressed, relative to nonstressed, plants, and the concentration of nonstructural carbohydrates had increased in stems, roots, and nodules. Sucrose concentrations in roots and nodules of stressed plants were 1.5 and 3 times greater, respectively, than those of nonstressed plants. Within 12 hours after rewatering, leaf and nodule Ψ_w of stressed plants had returned to values of nonstressed plants. Canopy apparent photosynthesis and specific nodule activity of stressed plants recovered to levels for nonstressed plants within 2 days after rewatering. The elevated sucrose concentrations in roots and nodules of stressed plants also declined rapidly upon rehydration. The increase in sucrose concentration in nodules, as well as the increase of carbohydrates in roots and stems, during water stress and the rapid disappearance upon rewatering indicates that inhibition of carbohydrate utilization within the nodule may be associated with loss of nodule activity. Availability of carbohydrates within the nodules and from photosynthetic activity following rehydration of nodules may mediate the rate of recovery of N₂-fixation activity.     

Interactions between Nitrogen Fixation, Mycorrhizal Colonization, and Host-Plant Growth in the Phaseolus-Rhizobium-Glomus Symbiosis

Bean (Phaseolus vulgaris L. cv. Dwarf) roots were inoculated with Rhizobium phaseoli and colonized by the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus fasciculatum Gerd. and Trappe or left uncolonized as controls. The symbiotic associations were grown in an inert substrate using 0, 25, 50, 100, or 200 milligrams hydroxyapatite (HAP) (Ca₁₀[PO₄]₆[OH]₂) per pot as a P amendment. Plant and nodule dry weights and nodule activity increased for both VAM and control plants with increasing P availability, but values for VAM plants were significantly lower in all parameters than for controls. Inhibition of growth and of N₂ fixation in VAM plants was greatest at the lowest and highest P regimes. It was smallest at 50 milligrams HAP, where available P at harvest (7 weeks after planting) was 5 micrograms P per gram substrate. At this level of P availability, the association apparently benefited from increased P uptake by the fungal endophyte. Percent P values for shoots, roots, and nodules did not differ significantly (p > 0.05) between VAM and control plants. The extent of colonization, fungal biomass, and the fungus/association dry weight ratio increased several fold as HAP was increased from 0 to 200 milligrams. It is concluded that intersymbiont competition for P and photosynthate was the primary cause for the inhibition of growth, nodulation, and nodule activity in VAM plants. Impaired N₂ fixation resulted in N stress which contributed to inhibition of host plant growth at all levels of P availability.     

Carbon Dioxide Fixation by Lupin Root Nodules: I. Characterization, Association with Phosphoenolpyruvate Carboxylase, and Correlation with Nitrogen Fixation during Nodule Development

In vivo CO₂ fixation and in vitro phosphoenolpyruvate (PEP) carboxylase levels have been measured in lupin (Lupinus angustifolius L.) root nodules of various ages. Both activities were greater in nodule tissue than in either primary or secondary root tissue, and increased about 3-fold with the onset of N₂ fixation. PEP carboxylase activity was predominantly located in the bacteroid-containing zone of mature nodules, but purified bacteroids contained no activity. Partially purified PEP carboxylases from nodules, roots, and leaves were identical in a number of kinetic parameters. Both in vivo CO₂ fixation activity and in vitro PEP carboxylase activity were significantly correlated with nodule acetylene reduction activity during nodule development. The maximum rate of in vivo CO₂ fixation in mature nodules was 7.9 nmol hour⁻¹ mg fresh weight⁻¹, similar to rates of N₂ fixation and reported values for amino acid translocation.     

Tissue-specific expression of the alternative oxidase in soybean and siratro

Alternative oxidase activity (cyanide-insensitive respiration) was measured in mitochondria from the shoots, roots, and nodules of soybean (Glycine max L.) and siratro (Macroptilium atropurpureum) plants. Activity was highest in the shoots and lowest in the nodules. Alternative oxidase activity was associated with one (roots) or two (shoots) proteins between 30 and 35 kilodaltons that were detected by western blotting with a monoclonal antibody against Sauromatum guttatum alternative oxidase. No such protein was detected in nodule mitochondria. Measurements of oxygen uptake by isolated soybean root and nodule cells in the presence of cyanide and salicylhydroxamic acid indicated that alternative oxidase activity was confined to the uninfected cortex cells of the nodule. Immunoprecipitation of translation products of mRNA isolated from soybean shoots revealed a major band at 43 kilodaltons that is assumed to be the precursor of an alternative oxidase protein. This band was not seen when mRNA from nodules was treated in the same fashion. The results indicate that tissue-specific expression of the alternative oxidase occurs in soybean and siratro.     

Reversible o(2) inhibition of nitrogenase activity in attached soybean nodules

Various forms of stress result in decreased O₂ permeability or decreased capacity to consume O₂ in legume root nodules. These changes alter the nodule interior O₂ concentration (O_i). To determine the relationship between O_i and nitrogenase activity in attached soybean (Glycine max) nodules, we controlled O_i by varying external pO₂ while monitoring internal H₂ concentration (H_i) with microelectrodes. O_i was monitored by noninvasive leghemoglobin spectrophotometry (nodule oximetry). After each step-change in O_i, H_i approached a new steady state, with a time constant averaging 23 s. The rate of H₂ production by nitrogenase was calculated as the product of H_i, nodule surface area, and nodule H₂ permeability. H₂ permeability was estimated from O₂ permeability (measured by nodule oximetry) by assuming diffusion through air-filled pores; support for this assumption is presented. O_i was nearly optimal for nitrogenase activity (H₂ production) between 15 and 150 nm. A 1- to 2-min exposure to elevated external pO₂ (40-100 kPa) reduced H_i to zero, but nitrogenase activity recovered quickly under air, often in <20 min. This rapid recovery contrasts with previous reports of much slower recovery with longer exposures to elevated pO₂. The mechanism of nitrogenase inhibition may differ between brief and prolonged O₂ exposures.     

Effects of carbohydrate on the internal oxygen concentration, oxygen uptake, and nitrogenase activity in detached pea nodules

The interaction between carbon substrates and O₂ and their effects on nitrogenase activity (C₂H₂) were examined in detached nodules of pea (Pisum sativum L. cv “Sparkle”). The internal O₂ concentration was estimated from the fractional oxygenation of leghemoglobin measured by reflectance spectroscopy. Lowering the endogenous carbohydrate content of nodules by excising the shoots 16 hours before nodule harvest or by incubating detached nodules at 100 kPa O₂ for 2 hours resulted in a 2- to 10-fold increase in internal O₂, and a decline in nitrogenase activity. Conversely, when detached nodules were supplied with 100 millimolar succinate, the internal O₂ was lowered. Nitrogenase activity was stimulated by succinate but only at high external O₂. Oxygen uptake increased linearly with external O₂ but was affected only slightly by the carbon treatments. The apparent diffusion resistance in the nodule cortex was similar in all of the treatments. Carbon substrates can thus affect nitrogenase activity indirectly by affecting the O₂ concentration within detached nodules.     

Regulation of Nodule Glutamine Synthetase by CO(2) Levels in Bean (Phaseolus vulgaris L.)

Nodulated bean (Phaseolus vulgaris) plants were grown for 17 days after infection in normal (0.02%) CO₂ and from day 8 to 17 in high (0.1%) CO₂ in order to increase nitrogen fixation and define how nodule glutamine synthetase (GS) isoforms are regulated by the ammonia derived from the bacteroid. Nitrogenase activity was detected by day 10, and by day 17 activity was over twofold higher in 0.1% of CO₂ compared with plants grown in 0.02% CO₂ and inoculated with Rhizobium wild-type strain CE3. Likewise, plant fresh weight increased in response to increased CO₂, particularly in plants inoculated with the Rhizobium phaseoli mutant strain CFN037. Glutamine synthetase specific activity increased 2.5- to 6.5-fold from day 11 to 17. However, increased CO₂ did not appear to have an effect on GS specific activity. Analysis of the nodule GS polypeptide composition revealed that the γ polypeptide was significantly reduced in response to high CO₂, whereas the β polypeptide was not affected. The significance of this result in relation to the regulation of GS isoforms and their role in the assimilation of ammonia in the nodule is discussed in this paper.     

NODULE ROOT and COCHLEATA Maintain Nodule Development and Are Legume Orthologs of Arabidopsis BLADE-ON-PETIOLE Genes

During their symbiotic interaction with rhizobia, legume plants develop symbiosis-specific organs on their roots, called nodules, that house nitrogen-fixing bacteria. The molecular mechanisms governing the identity and maintenance of these organs are unknown. Using Medicago truncatula nodule root (noot) mutants and pea (Pisum sativum) cochleata (coch) mutants, which are characterized by the abnormal development of roots from the nodule, we identified the NOOT and COCH genes as being necessary for the robust maintenance of nodule identity throughout the nodule developmental program. NOOT and COCH are Arabidopsis thaliana BLADE-ON-PETIOLE orthologs, and we have shown that their functions in leaf and flower development are conserved in M. truncatula and pea. The identification of these two genes defines a clade in the BTB/POZ-ankyrin domain proteins that shares conserved functions in eudicot organ development and suggests that NOOT and COCH were recruited to repress root identity in the legume symbiotic organ.     

Effect of Atmospheric CO(2) Enrichment on Growth, Nonstructural Carbohydrate Content, and Root Nodule Activity in Soybean

The objective of this study was to determine whether the supply of current photosynthate was limiting root nodule activity. Both short-term (36 hours) and long-term (16 days) periods of CO₂ enrichment were imposed on vegetative, growth chamber-grown soybean plants (Glycine max. [L.] Merr. cv. `Clay') to increase the supply of current photosynthate and to observe the effects on photosynthate partitioning in the plants, plant growth, and root nodule activity.     

Regulatory Patterns of a Large Family of Defensin-Like Genes Expressed in Nodules of Medicago truncatula

Root nodules are the symbiotic organ of legumes that house nitrogen-fixing bacteria. Many genes are specifically induced in nodules during the interactions between the host plant and symbiotic rhizobia. Information regarding the regulation of expression for most of these genes is lacking. One of the largest gene families expressed in the nodules of the model legume Medicago truncatula is the nodule cysteine-rich (NCR) group of defensin-like (DEFL) genes. We used a custom Affymetrix microarray to catalog the expression changes of 566 NCRs at different stages of nodule development. Additionally, bacterial mutants were used to understand the importance of the rhizobial partners in induction of NCRs. Expression of early NCRs was detected during the initial infection of rhizobia in nodules and expression continued as nodules became mature. Late NCRs were induced concomitantly with bacteroid development in the nodules. The induction of early and late NCRs was correlated with the number and morphology of rhizobia in the nodule. Conserved 41 to 50 bp motifs identified in the upstream 1,000 bp promoter regions of NCRs were required for promoter activity. These cis-element motifs were found to be unique to the NCR family among all annotated genes in the M. truncatula genome, although they contain sub-regions with clear similarity to known regulatory motifs involved in nodule-specific expression and temporal gene regulation.     

Bradyrhizobia from Wild Phaseolus, Desmodium, and Macroptilium Species in Northern Mexico

rRNA genetic markers were analyzed in 97 isolates of nodule bacteria from six legume species in Chihuahua, Mexico. The most common genotypes were widely shared across host species and had 16S rRNA sequences identical to those of strains from an eastern North American legume (Amphicarpaea) that are closely related to Bradyrhizobium elkanii.     

Effect of form and level of applied nitrogen on nitrogenase and nitrate reductase activities in faba beans

The effects of nitrogen applied at increasing levels of 0, 4, 8, 16 and 32 mM N (KNO₃ or NH₄Cl) were studied in faba bean (Vicia faba) nodulated byRhizobium leguminosarum bv.viceae RCR lool. Nitrogenase activity was higher at 4 and 8 mM N than the zero N treatment (control), but 16 and 32 mM N significantly reduced the efficiency of nodule functions. Nitrate reductase activities (NRA) of leaves, stems, roots, nodules and nodule fractions (bacteroid and cytosol) were increased with rising the NO₃ ^? or NH₄ ⁺ levels. NRA decreased in the order of nodules>leaves>stems>roots. Cytosolic NR was markedly higher than that recorded in the bacteroid fractions. Nitrate levels were linearly correlated to NRA of nodules. Accumulation of NO₂ ^? within nodules suggests that NO₂ ^? inhibits nodule’s activity after feeding plants with NO₃ ^? or NH₄ ⁺.     

Nitrogen metabolism of soybeans: I. Effect of tungstate on nitrate utilization, nodulation, and growth

The effects of N source (6 mm nitrogen as NO₃⁻ or urea) and tungstate (0, 100, 200, 300, and 400 μm Na₂ WO₄) on nitrate metabolism, nodulation, and growth of soybean (Glycine max [L.] Merr.) plants were evaluated. Nitrate reductase activity and, to a lesser extent, NO₃⁻ content of leaf tissue decreased with the addition of tungstate to the nutrient growth medium. Concomitantly, nodule mass and acetylene reduction activity of NO₃⁻-grown plants increased with addition of tungstate to the nutrient solution. In contrast, nodule mass and acetylene reduction activity of urea-grown plants decreased with increased nutrient tungstate levels. The acetylene reduction activity of nodulated roots of NO₃⁻-grown plants was less than 10% of the activity of nodulated roots of urea-grown plants when no tungstate was added. At 300 and 400 μm tungstate levels, acetylene reduction activity of nodulated roots of NO₃⁻-grown plants exceeded the activity of comparable urea-grown plants.     

Nitrogenase activity, nodule respiration, and o(2) permeability following detopping of alfalfa and birdsfoot trefoil

Gas exchange measurements and noninvasive leghemoglobin (Lb) spectrophotometry (nodule oximetry) were used to monitor nodule responses to shoot removal in alfalfa (Medicago sativa L. cv Weevlchek) and birdsfoot trefoil (Lotus corniculatus L. cv Fergus). In each species, total nitrogenase activity, measured as H₂ evolution in Ar:O₂ (80:20), decreased to <50% of the initial rate within 1 hour after detopping, and net CO₂ production decreased to about 65% of the initial value. In a separate experiment in which nodule oximetry was used, nodule O₂ permeability decreased 50% within 5 hours in each species. A similar decrease in the O₂-saturated respiration rate (V_max) for the nodule central zone occurred within 5 hours in birdsfoot trefoil, but only after 24 hours in alfalfa. Lb concentration, also measured by oximetry, decreased after 48 to 72 hours. The decrease in permeability preceded the decrease in V_max in each species. V_max may depend mainly on carbohydrate availability in the nodule. If so, then the decrease in permeability could not have been triggered by decreasing carbohydrate availability. Both oximetry and gas exchange data were consistent with the hypothesis that, for the cultivars tested, carbohydrate availability decreased more rapidly in birdsfoot trefoil than in alfalfa nodules. Fractional Lb oxygenation (initially about 0.15) decreased during the first 24 hours after detopping but subsequently increased to >0.65 for a majority of nodules of each species. This increase could lead to O₂ inactivation of nitrogenase.     

Interactive effects of elevated CO2, phosphorus deficiency, and soil drought on nodulation and nitrogenase activity in Alnus hirsuta and Alnus maximowiczii

We examined the interactive effects of elevated CO₂, soil phosphorus (P) availability, and soil drought on nodulation, nitrogenase activity, and biomass allocation in Alnus hirsuta and Alnus maximowiczii. Potted seedlings were grown in either ambient or elevated CO₂ (36 Pa and 72 Pa CO₂), with different levels of P (7.7 and 0.77 mgP pot^?1 week^?1 for high-P and low-P, respectively) and water supply in a natural daylight phytotron. Measurements of nitrogenase activity by an acetylene reduction assay failed to reveal significant effects of the treatments in any species. In high-P, nodule biomass increased under elevated CO₂ and decreased under drought. In low-P, nodule biomass decreased substantially compared to high-P, but the effect of elevated CO₂ on nodule biomass was unclear. Soil drought increased the partitioning of biomass into nodules, especially in A. hirsuta. These results suggest that with high P availability, elevated CO₂ could promote N₂ fixation by increasing nodule biomass even under drought. On the other hand, if soil P is limiting, elevated CO₂ may not enhance N₂ fixation because of the suppression of growth.