Ontogenetic Changes in Root Nodule Subpopulations of Common Bean (Phaseolus vulgaris L.): II. PROTEIN CONTENT AND CARBOHYDRATE POOLS

Root nodules of common bean (Phaseolus vulgaris L. GN 1140)show different ontogenetic patterns of nitrogen fixation indifferent parts of the root system. The objective of the presentstudy was to search for nodule physiological factors which couldexplain these differences. Plants were grown to maturity undercontrolled environment conditions. Root nodules were regularlyharvested from two zones of the root system, and assayed forsoluble cytosolic content and carbohydrate status. Nodule concentrationsof soluble carbohydrates, glucose, fructose or sucrose, didnot change significantly during the pod-filling decline in nitrogenaseactivity. Nodule concentrations of sucrose appeared more relatedto nodule ontogeny than to the ontogenetic stage of the shoot.Total soluble protein and leghaemoglobin concentrations werehighly correlated to nitrogenase activity. In the top zone ofthe root system proteolytic capacity increased during pod-filling,indicating a role in nodule senescence. Proteolytic capacityalso increased in the mid zone, but to a lesser extent, andwithout correlation to nitrogenase activity. The two nodulesubpopulations showed different nitrogenase activity in relationto their leghaemoglobin and carbohydrate status. We suggestthat physiological differences within the root nodule subpopulations,possibly in combination with the distance from the shoot, makesthe top zone more susceptible to a systemic increase in proteolyticcapacity during rapid pod-filling. Key words: Nitrogen fixation, protein, carbohydrate, ontogeny, Phaseolus vulgaris     

Ontogenetic Changes in Root Nodule Subpopulations of Common Bean (Phaseolus vulgaris L.): I. NITROGENASE ACTIVITY AND RESPIRATION DURING POD-FILLING

Nitrogenase activity is commonly measured on a whole plant basis,or only on parts of the root system. In the present paper, activityin different root nodule subpopulations was followed throughoutreproductive growth, in order to characterize the pod-fillingdecline in nitrogenase activity. Inoculated common bean plantswere grown to maturity under controlled environment conditions.Nitrogenase activity (H₂ evolution in air) and nodule respiration(CO₂ evolution) were measured in three separate zones of theroot system with a non-destructive, open flow, gas-exchangesystem. Nitrogenase activity in the top zone drastically declinedat the initiation of pod-filling, whereas nitrogenase activityin the mid zone was stable during the same period. Hence, thepod-filling decline was limited to a certain nodule subpopulationand not of a systemic type. Nodule respiration showed a similar,but less pronounced pattern. The sharp decline in nitrogenaseactivity was not paralleled in nitrogenase specific activity.Nitrogenase activity is not likely to be limited by the availabilityof oxygen or carbohydrates at the onset of pod-filling becausespecific nodule respiration did not change significantly atthis time. In the top zone, nitrogenase specific activity declinedgradually throughout the measurements, whereas in the mid-partof the root system specific activity peaked and gradually declined2-4 weeks later. The dissimilarities between specific and totalnitrogenase activity were explained by differences in nodulegrowth rates. The data suggest that the oldest nodule populationloses activity at the onset of pod-filling. At the same time,nodules grow and nitrogenase activity increases in younger distalparts of the root system. Estimating total nitrogen fixationin this symbiosis by partial sampling of nodulated root systemsis likely to be very misleading. Key words: Nitrogen fixation, respiration, pod-filling decline, Phaseolus vulgaris, ontogeny     

Nitrogenase activity in root nodule homogenates ofAlnus incana

Summary Root nodule homogenates of actinorhizal plants may representFrankia in a symbiotic stage but released from environmental influence of the host plant. Anaerobic homogenization with a blender in buffer supplied with sucrose, polyvinylpyrrolidone and reducing substances gave three times higher yields of nitrogenase activity (C₂H₂-reduction) than crushing the nodules in liquid nitrogen. The activity in the homogenates was very reproducible and was, on average, nearly twice as high as the activity in excised nodules and c. 10% of the activity in intact plants. The difference in activity between excised nodules and intact plants was, roughly by halves, due to removal of the root system from the pot and to excision of the nodules. The nitrogenase activity in the homogenates was slightly higher when nodule excision was done in Ar or under water as well as after treatment of the homogenate with toluene or Triton X-100 or osmotic shock. These gains in activity were considered too small to outweigh the increased complications of preparing homogenates for routine use. Due to the reproducible recovery of nitrogenase in the homogenates the technique seems useful for physiological studies on nitrogen fixation inAlnus incana.     

Nitrogen fixation and respiration by root nodules ofAlnus rubra Bong.: Effects of temperature and oxygen concentration

Summary Using a root nodule cuvette and a continuous flow gas exchange system, we simultaneously measured the rates of carbon dioxide evolution, oxygen uptake and acetylene reduction by nodules ofAlnus rubra. This system allowed us to measure the respiration rates of single nodules and to determine the effects of oxygen concentration and temperature on the energy cost of nitrogen fixation. Energy cost was virtually unchanged (2.8–3.5 moles of carbon dioxide or oxygen per mole of ethylene) from 16 to 26°C (pO₂=20 kPa) while respiration and nitrogenase activity were highly temperature dependent. At temperatures below 16°C, nitrogenase activity decreased more than did respiration and as a result, energy cost rose sharply. Acetylene reduction ceased below 8°C. Inhibition of nitrogenase activity at low temperatures was rapidly reversed upon return to higher temperatures. At high temperatures (above 30°C) nitrogenase activity declined irreversibly, while respiration and energy cost increased.Energy cost was nearly unchanged at oxygen partial pressures of 5 to 20 kPa (temperature of 20°C). Respiration and nitrogenase activity were strongly correlated with oxygen tension. Below 5 kPa, acetylene reduction and oxygen uptake decreased sharply while production of carbon dioxide increased, indicating fermentation. Fermentation alone was unable to support nitrogenase activity. Acetylene reduction was independent of oxygen concentration from 15 to 30 kPa. Nitrogenase activity decreased and energy cost rose above 30 kPa until nearly complete inactivation of nitrogenase at 70–80 kPa. Activity declined gradually, such that acetylene reduction at a constant oxygen concentration was stable, but showed further inactivation when oxygen concentration was once again increased. Alder nodules appear to consist of a large number of compartments that differ in the degree to which nitrogenase is protected from excess oxygen.Supported by United States Department of Agriculture Grant 78-59-2252-0-1-005-1     

木麻黄和桤木离体根瘤和立地土壤的固氮酶与N2O还原酶活性的研究

为了解非豆科固氮树种的固氮酶和N_2O还原酶(Nos)活性,采用乙炔还原法和乙炔抑制技术对细枝木麻黄(Casuarina cunninghamiana)和江南桤木(Alnus trabeculosa)离体根瘤及立地土壤的两种酶活性进行了研究。结果表明,离体根瘤只在厌氧条件下有固氮酶活性,在好氧条件下有Nos活性。根瘤区根际土和非根瘤区根际土的固氮酶活性在好氧条件大于厌氧条件,Nos活性只表现在厌氧条件下。在好氧条件下,根瘤区根际土和非根瘤区根际土的固氮酶活性无显著差异;根瘤区根际土的Nos活性显著大于非根瘤区根际土。除离体根瘤在好氧条件下不表现固氮酶活性外,细枝木麻黄和桤木的离体根瘤、根瘤区根际土和非根瘤区根际土的固氮酶活性均都大于Nos活性。好氧条件下根瘤区根际土的固氮酶活性与非根瘤区根际土的呈极显著正相关,而厌氧条件下根瘤的固氮酶活性与好氧条件下根瘤区根际土和非根瘤区根际土固氮酶活性、好氧条件下根瘤的Nos活性与厌氧条件下根瘤区根际土和非根瘤区根际土Nos活性均呈极显著负相关。这为研究弗兰克氏菌结瘤植物共生固氮体系对N2O汇强度的影响和调控奠定基础。     

Carbon cost of nitrogenase activity in Frankia–Alnus incana root nodules

The carbon cost of nitrogenase activity was investigated to determine symbiotic efficiency of the actinorhizal root nodule symbiosis between the woody perennial Alnus incana and the soil bacterium Frankia. Respiration (CO₂ production) and nitrogenase activity (H₂ production) by intact nodulated root systems were continuously recorded in short-term assays in an open-flow gas exchange system. The assays were conducted in N₂:O₂, thus under N₂-fixing conditions, in all experiments except for one. This avoided the declines in nitrogenase activity and respiration due to N₂ deprivation that occur in acetylene reduction assays and during extended Ar:O₂ exposures in H₂ assays. Two approaches were used: (i) direct estimation of root and nodule respiration by removing nodules, and (ii) decreasing the partial pressure of O₂ from 21 to 15% to use the strong relationship between respiration and nitrogenase activity to calculate CO₂/H₂. The electron allocation of nitrogenase was determined to be 0.6 and used to convert the results into moles of CO₂ produced per 2e^– transferred by nitrogenase to reduction of N₂. The results ranged from 2.6 to 3.4mol CO₂ produced per 2e^–. Carbon cost expressed as gC produced per gN reduced ranged from 4.5 to 5.8. The result for this actinorhizal tree symbiosis is in the low range of estimates for N₂-fixing actinorhizal symbioses and crop legumes. Methodology and comparisons of root nodule physiology among actinorhizal and legume plants are discussed.     

Nitrogenase activity of two genotypes of Acacia mangium as affected by phosphorus nutrition

The specific nodulation, nitrogenase activity (acetylene reduction) and budgets of carbon allocation to respiration by nodulated roots were examined in two provenances of Acacia mangium Willd. grown in a glasshouse for 17 weeks to investigate the effects of soil phosphorus and genotypes of the host plant on symbiotic nitrogen fixation. Application of phosphorus (0–80 mg P kg^-1 soil) increased specific nodulation (g nodule dry weight g^-1 plant dry weight) of provenance Ma11 by two-fold and the percentage of nodulated root respiration allocated to nitrogenase by 50%, but had no effect on specific activity of nitrogenase or specific respiration coupled with nitrogenase activity. Improved phosphorus nutrition increased the specific nitrogenase activity of provenance Ma9 by 2-fold, the percentage of nodulated root respiration allocated to nitrogenase, and specific nitrogenase-linked respiration by 50%, respectively, but had no effect on the specific nodulation. The percentage of respiration coupled with nitrogenase activity in nodulated root respiration by provenance Ma9 was 60–70% higher than that in provenance Ma11, regardless of phosphorus levels applied. At the optimal level of phosphorus addition (10 mg P kg^-1 soil), provenance Ma9 had a lower dry mass than provenance Ma11. This was accompanied by a lower nodulated root respiration and a higher percentage of nodulated root respiration allocated to nitrogenase activity in provenance Ma9.     

Nodule structure and nitrogenase activity ofCoriaria arborea in response to varying pO2

When excised root nodules ofCoriaria arborea are assayed for nitrogenase activity at various pO₂ they show a broad optimum between 20 and 40 kPa O₂, with some evidence for adaptation. Continuous flow assays of nodulated root systems of intact plants indicate that Coriaria shows an acetylene induced decline in nitrogenase activity. When root systems were subject to step changes in pO₂ nitrogenase activity responded with a steep decline followed by a slower rise in activity both at lower and higher than ambient pO₂. Thus Coriaria nodules are able to adapt rapidly to oxygen levels well above and well below ambient. Measurement of nodule diffusion resistance showed that the adaptation is accompanied by rapid increase in resistance at above ambient pO₂ and decrease in resistance at below ambient pO₂. Plants grown with root systems at pO₂ from 5–40 kPa O₂ did not differ in growth or nodulation. The anatomy of Coriaria nodules shows they have a dense periderm which encircles the nodule and also closely invests the infected zone. The periderm is both thicker and more heavily suberised in nodules grown at high pO₂ than at low pO₂. Vacuum infiltration of India ink indicates that oxygen diffusion is entirely through the lenticel and via a small gap adjacent to the stele.     

The regulation of nodulation,nitrogen fixation and ammonium assimilation under a carbohydrate shortage stress in the Discaria trinervis-Frankia symbiosis

N₂-fixation is sensitive to limitation in the availability of newly synthesised carbohydrates for the nodules. We decided to explore the response of the D. trinervis - Frankia symbiosis to a transient decrease in carbohydrate supply to nodules. Feedback inhibition of nodulation as well as nodule growth was not released by a 6-day dark stress in D. trinervis nodulated plants. However, nitrogen fixation and assimilation were affected by the imposed stress. Nitrogenase activity was totally inhibited after 4 days of darkness although high levels of nitrogenase components were still detected at this time. Degradation of FeMo and Fe nitrogenase subunits – both at similar rates – was observed after 6 days of dark stress, revealing the need for inactivation to precede enhancement of protein turnover. Glutamine synthetase (GS), malate dehydrogenase (MDH) and asparagine synthetase (AS) polypeptides were also degraded during the dark stress, although at a lower rate than nitrogenase. ARA and nitrogenase were totally recovered 8 days after resuming normal illumination. It seems that current nitrogenase activity and ammonium assimilation are not, or are only weakly linked with the feedback control of nodulation in D. trinervis. These observations give support to the persistence of an autoregulatory signal in mature nodules that is not sensitive to transient shortages of carbon supply and sustains the inhibition of nodulation in the transient absence of N₂ fixation.     

Ammonium assimilation in root nodules of actinorhizal Discaria trinervis. Regulation of enzyme activities and protein levels by the availability of macronutrients (N,P and C)

Asparagine was found to be the main N compound exported from Discaria trinervis nodules. Aspartate (Asp), glutamate (Glu), alanine (Ala) and serine (Ser) were also detected in root xylem sap, but at lower concentrations. A comparable picture is found in nodulated alfalfa. We hypothesized that a similar set of enzymes for Asn synthesis was present in D. trinervis nodules. We demonstrate the expression of most of the enzymes involved in the synthesis of Asn from NH⁺ ₄ and oxoacids, in nodules – but not in roots – of fully symbiotic D. trinervis. By complementation of enzyme assays (^A) and immunodetection (^I) we detected glutamane-synthetase (GS^{A, I}), Asp-aminotransferase (AAT^A), malate-dehydrogenase (MDH^{A, I}, at least two isoforms), Glu-dehydrogenase (GDH^A), Glu-synthase (GOGAT^I) and Asn-synthetase (AS^I). PEP-carboxylase (PEPC) activity was not detected. We previously shown that N acts as a negative regulator of nodulation and nodule growth, while P is a strong stimulator for nodule growth. We present data on the regulation of nodule N metabolism by altering, during 4 weeks, the availability of N, P and light in symbiotic D. trinervis. NH₄NO₃ (2 mM) induced inactivation and degradation of nodule GS, MDH and AS, but activation of GDH and AAT; the amount of nitrogenase components was not affected. A 10-fold increase in P supply did not greatly affect activity and amount of enzymes, suggesting that N metabolism is not P-limited in nodules. On the other hand, suppression of P supply induced an important reduction of nodule GS, GOGAT, MDH and AS protein levels, although nitrogenase was not affected. GDH was the only measured activity that was stimulated by limiting P supply. Shading plants did result in complete degradation of nitrogenase and partial degradation of GS, AS and nodule-specific MDH isoform, but GDH and AAT were activated. These results are discussed in connection with the regulation of nodulation and nodule growth in D. trinervis.     

Effect of spring flooding on endophyte differentiation,nitrogenase activity,root growth and shoot growth inMyrica gale

Summary Spring flooding was investigated as a possible limiting factor in the development of nitrogenase activity, root growth, and shoot growth inMyrica gale. Dormant, one year oldMyrica gale plants were placed in a greenhouse in early April and given three treatments: control (not flooded), flooded-water (flooded with water to 2.5 cm above the soil level) and flooded-peat (flooded with water-saturated peat to 4.0 cm above the soil level). Nitrogenase activity was absent at budbreak but appeared concurrently with the differentiation of vesicles by theFrankia sp. endophyte. Flooding delayed the onset of nitrogenase activity, substantially reduced the specific nitrogenase activity of the nodules, and also severely limited the production of the new nodule biomass. Consequently by 67 days past budbreak nitrogenase activity was much greater in the control plants (5.55±0.42 mol C₂H₄/plant.h; ± SE; N=9) than in the flooded-water (1.18±0.29) and flooded-peat (0.15±0.05) plants. Production of new secondary roots was substantially reduced in the flooded plants but adventitious roots were rapidly produced along the flooded portion of the stem in the better aerated zone near the surface. New nodules formed on several adventitious roots by 67 days indicating that the plants are able to replace their largely nonfunctional deeply flooded nodules with new nodules in the aerobic zone. Initially shoot growth was unaffected by flooding but by 67 days the flooded plants had substantially less leaf biomass, lower leaf and stem nitrogen concentrations, and less total shoot nitrogen content than the control plants.     

Carbohydrate supply and n(2) fixation in soybean : the effect of varied daylength and stem girdling

When arrival of shoot supplied carbohydrate to the nodulated root system of soybean was interrupted by stem girdling, stem chilling, or leaf removal, nodule carbohydrate pools were utilized, and a marked decline in the rates of CO₂ and H₂ evolution was observed within approximately 30 minutes of treatment. Nodule excision studies demonstrated that the decline in nodulated root respiration was associated with nodule rather than root metabolism, since within 3.5 hours of treatment, nodules respired at less than 10% of the initial rates. Apparently, a continuous supply of carbohydrate from the shoot is required to support nodule, but not root, function. Depletion of nodular carbohydrate pools was sufficient to account for the (diminishing) nodule respiration of girdled plants. Of starch and soluble sugar pools within the whole plant, only leaf starch exhibited a diurnal variation which was sufficient to account for the respiratory carbon loss of nodules over an 8 hour night. Under 16 hour nights, or in continuous dark, first the leaf starch pools were depleted, and then nodule starch reserves declined concomitant with a decrease in the rates of CO₂ and H₂ evolution from the nodules. Nodule soluble sugar levels were maintained in dark treated plants but declined in girdled plants. The depletion of starch in root nodules is an indicator of carbohydrate limitation of nodule function.     

Nonphotosynthetic CO(2) Fixation by Alfalfa (Medicago sativa L.) Roots and Nodules

The dependence of alfalfa (Medicago sativa L.) root and nodule nonphotosynthetic CO₂ fixation on the supply of currently produced photosynthate and nodule nitrogenase activity was examined at various times after phloem-girdling and exposure of nodules to Ar:O₂. Phloemgirdling was effected 20 hours and exposure to Ar:O₂ was effected 2 to 3 hours before initiation of experiments. Nodule and root CO₂ fixation rates of phloem-girdled plants were reduced to 38 and 50%, respectively, of those of control plants. Exposure to Ar:O₂ decreased nodule CO₂ fixation rates to 45%, respiration rates to 55%, and nitrogenase activities to 51% of those of the controls. The products of nodule CO₂ fixation were exported through the xylem to the shoot mainly as amino acids within 30 to 60 minutes after exposure to ¹⁴CO₂. In contrast to nodules, roots exported very little radioactivity, and most of the ¹⁴C was exported as organic acids. The nonphotosynthetic CO₂ fixation rate of roots and nodules averaged 26% of the gross respiration rate, i.e. the sum of net respiration and nonphotosynthetic CO₂ assimilation. Nodules fixed CO₂ at a rate 5.6 times that of roots, but since nodules comprised a small portion of root system mass, roots accounted for 76% of the nodulated root system CO₂ fixation. The results of this study showed that exposure of nodules to Ar:O₂ reduced nodule-specific respiration and nitrogenase activity by similar amounts, and that phloem-girdling significantly reduced nodule CO₂ fixation, nitrogenase activity, nodule-specific respiration, and transport of ¹⁴C photoassimilate to nodules. These results indicate that nodule CO₂ fixation in alfalfa is associated with N assimilation.     

Nitrogen Fixation by Subterranean Clover at Varying Stages of Nodule Dehydration: I. CARBOHYDRATE STATUS AND SHORT-TERM RECOVERY OF NODULATED ROOT RESPIRATION

The nodule water potential (_nod) of subterranean clover (Trifoliumsubterraneum L.) cv. Seaton Park incubated in a flow-throughgas-exchange system was induced to decline independently ofleaf water potential (₁) by passing a continuous dry airstreamover the nodulated roots of intact well-watered plants. Reducedtranspiration by plants whose nodules had become dehydratedwas hypothesized to be related to the decline in nitrogen fixation.Whole-plant and nodule soluble carbohydrates increased as _noddeclined. Throughout an 8 d period of continual nodule dehydration,the gaseous diffusion resistance of nodules increased and theoptimum pO₂ for nitrogenase activity declined from 52 to 28kPa. Following rehydration of the nodulated roots between days4 and 5 and between days 7 and 8, nodulated root respirationincreased to or above pre-stress levels whereas nitrogenaseactivity did not recover. Re-establishment of initial ratesof nodulated root respiration was due to the stimulation ofgrowth and maintenance respiration, not to the respiration coupledto nitrogenase activity. Although no recovery of nitrogenaseactivity occurred, the elapsed time from the introduction ofacetylene into the gas stream flowing past the nodules untilmeasurement of the acetylene-induced decline in nitrogenaseactivity, decreased substantially. This was characteristic ofan increase in the permeability of the nodules to gaseous diffusionupon rehydration. However, calculated values of nodule diffusionresistance after the 24 h periods of rehydration did not indicateany recovery of gaseous diffusion resistance based on measurementsof the respiration coupled to nitrogenase activity. Hence, useof a diffusion analogue (i.e. Fick's Law) in conjunction withnodule respiratory CO₂ efflux was unable to predict changesin permeability of the variable barrier of legume nodules followingnodule dehydration and recovery. Key words: Subterranean clover, gaseous diffusion, respiration, carbohydrates, drought     

Nitrogenase activity of pea bacteroids as affected by carbohydrates and ammonium chloride

Summary Regulation and efficiency of the nitrogen-fixing system of the rhizobium-pea symbiosis were investigated. Acetylene reduction of detached root nodules was measured with various substrates added. Succinate, fumarate and malate were most effective in stimulating nitrogenase activity; glucose, pyruvate and citrate were also active. Acetylene reducing activity of detached nodules was inhibited by the addition of NH₄Cl, irrespective of the substrate present. Nitrogenase activity of isolated bacteroids was not influenced by NH₄Cl.Respiration of detached nodules was not significantly stimulated by the addition of substrates. Ammonium chloride did not influence respiration. With detached nodules and isolated bacteroids a consumption of about 16 g of carbohydrate per g of nitrogen fixed could be calculated. Detached nodules produced more hydrogen relative to the acetylene reduced than did isolated bacteroids and intact plants.Results obtained indicate that the regulation of nitrogenase activity and the efficiency of substrate consumption depend on environmental conditions.     

Molecular and whole-plant responses to selection for enzyme activity in alfalfa root nodules: evidence for molecular compensation of aspartate aminotransferase expression

Summary The enzyme aspartate aminotransferase (AAT) plays a key role in the assimilation of fixed-N in alfalfa (Medicago sativa L.) root nodules. AAT activity in alfalfa nodules is due to the activity of two dimeric isozymes, AAT-1 and AAT-2, that are products of two distinct genes. Three forms of AAT-2 (AAT-2a, -2b, and-2c) have been identified. It was hypothesized that two alleles occur at the AAT-2 locus, giving rise to the three AAT-2 enzymes. In a prior study bidirectional selection for root nodule AAT and asparagine synthetase (AS) activities on a nodule fresh weight basis in two diverse alfalfa germ plasms resulted in high nodule enzyme activity subpopulations with about 20% more nodule AAT activity than low enzyme activity subpopulations. The objectives of the study presented here were to determine the inheritance of nodule AAT-2 production and to evaluate the effect of bidirectional selection for AAT and AS on AAT-2 allelic frequencies, the relative contributions of AAT-1 and AAT-2 to total nodule activity, nodule enzyme concentration, and correlated traits. Two alleles at the AAT-2 locus were verified by evaluating segregation of isozyme phenotypes among F₁ and S₁ progeny of crosses or selfs. Characterization of subpopulations for responses associated with selection was conducted using immunoprecipitation of in vitro nodule AAT activity, quantification of AAT enzyme protein by ELISA, and AAT activity staining of native isozymes on PAGE. Results indicate that selection for total AAT activity specifically altered the expression of the nodule AAT-2 isozyme. AAT-2 activity was significantly greater in high compared to low activity subpopulations, and high AAT subpopulations from both germ plasms had about 18% more AAT-2 enzyme (on a nodule fresh weight basis). No significant or consistent changes in AAT-2 genotypic frequencies in subpopulations were caused by selection for AAT activity. Since changes in AAT activity were not associated with changes in AAT-2 genotype, selection must have affected a change(s) at another locus (or loci), which indirectly effects the expression of nodule AAT.Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture, and does not imply its approval to the exclusion of other products or vendors that might also be suitable     

Appearance of a novel form of plant glutamine synthetase during nodule development in Phaseolus vulgaris L.

The activities of glutamine synthetase (GS), nitrogenase and leghaemoglobin were measured during nodule development in Phaseolus vulgaris infected with wild-type or two non-fixing (Fix^-) mutants of Rhizobium phaseoli. The large increase in GS activity which was observed during nodulation with the wild-type rhizobial strain occurred concomitantly with the detection and increase in activity of nitrogenase and the amount of leghaemoglobin. Moreover, this increase in GS was found to be due entirely to the appearance of a novel form of the enzyme (GS_n1) in the nodule. The activity of the form (GS_n2) similar to the root enzyme (GS_r) remained constant throughout the experiment. In nodules produced by infection with the two mutant strains of Rhizobium phaseoli (JL15 and JL19) only trace amounts of GS_n1 and leghaemoglobin were detected.Abbreviations DEAE-Sephacel diethylaminoethyl-Sephacel - GS glutamine synthetase     

Development of nodules of Glycine max infected with an ineffective strain of Rhizobium japonicum

Bacteroids in ineffective (nitrogenase negative) nodules of Glycine max, infected with Rhizobium japonicum 61-A-24, as compared to those in effective nodules are characterized by reduced specific activities of alanine dehydrogenase to 15%, of 3-hydroxybutyrate dehydrogenase to 50%, and an increase of glutamine synthetase to 400%. In the plant cytoplasm of ineffective nodules, glutamine synthetase activity is reduced to 10–30%, glutamate dehydrogenase to 50–70%, and the aspartate aminotransferase and alanine aminotransferase are enhanced to 120–200%, depending on the age of the nodules. The total pool of soluble amino acids is reduced to 52 mol per g nodule fresh weight, as compared to 186 mol in effective nodules, with a replacement of asparagine (42 mol% of the amino acids) by an unknown amino compound. This compound is absent in nitrogenase, repressed and derepressed, free-living Rhizobium japonicum cells and in the uninfected root tissue. In nitrogenase derepressed, as compared to the repressed free-living cells of Rhizobium japonicum 61-A-101, arginine shows the most obvious change with a reduction to less than one tenth. The ultrastructure of the ineffective nodule is different from the effective organ even in the early stages. The membrane envelopes of the infection vacuoles are decomposing in heavily infected cells within 18 to 20 d after infection. In lightly infected cells very large vacuoles develop with only a few bacteroids inside. No close associations of cristae-rich mitochondria with amyloplasts are observed as in effective nodules. The uninfected cells keep their large starch granules even 40 d after infection. Some poly--hydroxybutyrate accumulation in the bacteroids is observed but only in the early stages, and it is almost absent in old nodules (40 d). At this age the infected cells are obviously compressed by uninfected cells, whereas in effective nodules with nitrogenase activity and leghaemoglobin formation, the infected cells have a much higher osmotic pressure than the neighbouring uninfected cells.Abbreviations PHBA poly--hydroxybutyric acid Prof. Dr. A. Pirson on the occasion of his 70th birthday     

Expression of nodule-specific glutamine synthetase genes during nodule development in soybeans

Summary A cDNA clone (pcPvNGS-01) to glutamine synthetase (GS) mRNA from root nodules of Phaseolus vulgaris showed cross-hybridization to GS and mRNA from soybean root nodules, thus allowing its use as a probe to study the expression of GS genes during root nodule development in soybeans. Hybrid-select translation of root and nodule RNA of soybean with DNA from pcPvNGS-01, followed by 2D gel electrophoresis, showed six peptides in the root and an additional four peptides in the nodule which represent nodule-specific glutamine synthetase (GS_n) gene products. The GS_n gene products appeared for the first time between day 11 and 12 after infection, either concomitant with the onset of nitrogenase activity or immediately following it. The levels of expression of the GS_n and leghemoglobin genes were not affected in young Fix^- nodules formed by Bradyrhizobium japonicum strains that are defective in nitrogenase activity, suggesting that the induction of these two sets of host genes take place independent of nitrogenase activity. However, in Fix^- nodules that are incapable of maintaining the peribacteroid membrane, GS_n gene products were not detected while 1ba, 1bc₂ and 1bc₃ appeared. In both the timing of appearance during root nodule development and the effect of different bacterial mutations on the expression, GS_n genes differ from most other nodulin genes examined (30), suggesting different regulatory mechanisms.     

Can a limitation in pholem supply to nodules account for the inhibitory effect of nitrate on nitrogenase activity in soybean?

Within 48 h of exposure of nodulated soybean [Glycine max (L.) Merr. cv. Harosoy 63 x Bradyrhizobium japonicum USDA 16] to 10 mM NO₃, significant decreases were observed in nodule-specific nitrogenase (EC 1.7.99.2) activity and CO₂ evolution and in the proportion of [¹⁴C]-labeled photosynthate partitioned to nodule biomass and respiration. These trends continued over the subsequent 3 days of the study period. Concomitant with these events was an 137% increase in the relative growth rate of the whole plant and a cessation in nodule growth. Although the concentration of total soluble sugar in nodules was not affected by NO₃ treatment, the concentration of starch declined to 13% of the control level after 2 days exposure to NO₃^?. In contrast to the effects of NO₃^?, nodules in which nitrogenase activity was partially inhibited by a 30 min exposure to 100% O₂, showed a 52% increase over control in the starch pool over a 72 h period. The results were compared with recent studies of NO₃^? inhibition of nitrogenase activity in legumes, and in contrast to these studies it was concluded that the inhibitory effects of NO₃^? could be accounted for by alterations in photosynthate partitioning to nodules. A hypothesis is proposed which attempts to account for the recent observation (J. K. Vessey, K. B. Walsh, and D. B. Layzell 1988. Physiol. Plant. 73: 113–121) that nitrogenase activity in phloem-limited and nitrate-inhibited nodules is limited by O₂ diffusion. This hypothesis separates the concepts of photosynthate partitioning and phloem supply from that of carbohydrate deprivation and related effects on the size of the carbohydrate pools in nodules.