Effect of the Nitrogen Supply on the Activities of Isoenzymes of NADH-dependent Glutamate Synthase and Glutamine Synthetase in Root Nodules of Phaseolus vulgaris L.

This paper has investigated the regulation of the activitiesof glutamine synthetase (GS) and NADH-dependent glutamate synthase(NADH-GOGAT) of Phaseolus vulgaris in relation to the nitrogensupply. The activity of NADH-GOGAT II, which is the most abundantisoenzyme of glutamate synthase in root nodules of P. vulgaris,was either absent or barely detectable in other organs of thisspecies. Moreover, its activity in roots could not be inducedby ammonium. In nodules NADH-GOGAT II activity was detectedin nodules grown under an atmosphere of 80% argon: 20% oxygenand in nodules formed with a Fix- Rhizobium mutant. However,in these non-fixing nodules the activity of this isoenzyme attainedless than 15% of the activity in fixing nodules and switchingargon/oxygen grown nodules to nitrogen/oxygen led to an increasein this isoenzyme within 24 h. This effect could not be mimickedby the addition of exogenous ammonium. Ammonium addition, however,promoted nodule senescence and also led to a decrease in theactivities of nitrogenase and plant GS. In particular, the nodule-enhancedGS isoenzyme but not the GSß isoenzyme was affectedby these changes and in a manner similar to the changes in NADH-GOGATII. The activity of the NADH-GOGAT I isoenzyme was detectablein other organs of P. vulgaris and in nodules its activity alsoshowed some changes in response to the rate of dinitrogen fixation. Key words: Glutamate synthase, glutamine synthetase, nitrogen fixation, nodule metabolism, Phaseolus vulgaris     

Expression of Two Different Glutamine Synthetase Polypeptides during Root Development in Phaseolus vulgaris L

Immunoprecipitation and two-dimensional gel electrophoresis analysis of the glutamine synthetase (GS) polypeptides (α and β) during Phaseolus vulgaris root development shows that the α polypeptide is the main component of the enzyme in the embryo and in up to 5 day old roots. From 5 days on, the β polypeptide becomes the root predominant GS monomer. The α/β ratio of the in vitro translated GS polypeptides from the total polysomal RNA isolated at different root ages correlates with the α/β ratio observed in the root extracts. These results suggest that the two root GS polypeptides are encoded by different mRNA species in Phaseolus vulgaris.     

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.     

Common Bean (Phaseolus vulgaris L.) Mutants Defective in Root Nodule Formation: II GENETIC ANALYSIS

Genetic analysis of crosses between two induced, ineffectivelynodulating mutants of common bean, NOD238 and NOD109, revealedthat their mutated nodulation phenotype is under the controlof the same locus in both mutants. The two mutants also resultedallelic for poor pod fertility, the other trait common to themutants. F₁ plants from crosses with their wild types nodulatedeffectively and had wild type pod fertility. Ineffective nodulationand poor pod fertility traits co-segregated in the F₂generationin which plants with the mutant nodulation and pod fertilityphenotypes represented 12.5% of the total population. Analysisin F₃confirmed that these plants were homozygous for both mutatedcharacters. The results indicated both mutant traits studiedare determined by a single recessive allele, named sym-2, whoseinheritance is negatively affected by its pleiotropic effecton pod fertility determining a deficit of ineffectively nodulatingcombinations. In an allelism test with the non-nodulating mutantof common bean NOD125 it was found that ineffective nodulationis controlled at a different locus and that the two loci arenot linked. Key words: Phaseolus valgaris, nitrogen fixation, nodulation mutants, genetics     

Isoenzymes of Superoxide Dismutase in Nodules of Phaseolus vulgaris L., Pisum sativum L., and Vigna unguiculata (L.) Walp

The activity and isozymic composition of superoxide dismutase (SOD; EC 1.15.1.1) were determined in nodules of Phaseolus vulgaris L., Pisum sativum L., and Vigna unguiculata (L.) Walp. formed by Rhizobium phaseoll 3622, R. Ieguminosarum 3855, and Bradyrhizobium sp. BR7301, respectively. A Mn-SOD was present in Rhizobium and two in Bradyrhizobium and bacteroids. Nodule mitochondria from all three legume species had a single Mn-SOD with similar relative mobility, whereas the cytosol contained several CuZn-SODs: two in Phaseolus and Pisum, and four in Vigna. In the cytoplasm of V. unguiculata nodules, a Fe-containing SOD was also present, with an electrophoretic mobility between those of CuZn- and Mn-SODs, and an estimated molecular weight of 57,000. Total SOD activity of the soluble fraction of host cells, expressed on a nodule fresh weight basis, exceeded markedly that of bacteroids. Likewise, specific SOD activities of free-living bacteria were superior or equal to those of their symbiotic forms. Soluble extracts of bacteria and bacteroids did not show peroxidase activity (EC 1.11.1.7), but the nodule cell cytoplasm contained diverse peroxidase isozymes which were readily distinguishable from leghemoglobin components by electrophoresis. Data indicated that peroxidases and leghemoglobins did not significantly interfere with SOD localization on gels. Treatment with chloroform-ethanol scarcely affected the isozymic pattern of SODs and peroxidases, and had limited success in the removal of leghemoglobin.     

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     

Common Bean (Phaseolus vulgaris L.) Mutants Defective in Root Nodule Formation: I. PHYSIOLOGICAL CHARACTERIZATION

Two ineffectively nodulating, allelic mutants of the commonbean (Phaseolus vulgaris L.) lines RIZ30 and RIZ36 were studied.In both mutants the nodulation phenotype was characterized bythe formation of tumour-like swellings when inoculated withbean Rhizobium strains. Late formation of pink nodules was observedin mutant plants grown in pots with soil or in hydroponic conditionswith perlite. In the absence of mineral nitrogen, mutant growthand nodulation were poor; a low peak of acetylene reductionactivity being detected 45 d after inoculation. Mutant growthin the presence of mineral nitrogen was similar to that of thewild type. The mutants were also characterized by poor pod fertility(seed per pod). The nodulation and pod fertility phenotypesare specifically controlled at the root and shoot levels, respectively,as assessed with grafting experiments. Mutant shoots graftedon wild type, nitrogen-fixing roots produced a number of seedssimilar to the wild type despite the expression of the poorpod fertility phenotype. Nitrogen and seed yields of wild typeshoots, grafted on mutant, ineffectively nodulating roots wereseverely reduced when compared with control wild type plantsalthough nitrogen accumulation during vegetative phase was similar. Key words: Phaseolus valgaris, nitrogen fixation, nodulation mutants, grafting, nitrogen assimilation     

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     

Subunit Composition of Glutamine Synthetase Isozymes from Root Nodules of Bean (Phaseolus vulgaris L.)

Glutamine synthetase from bean nodules can be separated into two isoforms, GS_n1 and GS_n2. A purification protocol has been developed. It included protamine sulfate precipitation, ammonium sulfate fractionation, anthranilate-affinity chromatography, Dye-Matrex (Orange A) chromatography, and diethylaminoethyl-cellulose ion-exchange chromatography. GS_n1 and GS_n2 have been purified to homogeneity. Subunit structure analysis using two-dimensional polyacrylamide gel electrophoresis revealed that GS_n1 was composed of two different types of subunit polypeptides. They differed in isoelectric points (6.0 and 6.3) but had the same molecular weights (46,000 Daltons). GS_n2 was composed of only one type of subunit polypeptide. It had an isoelectric point of 6.0 and a molecular weight of 46,000 Daltons. It was apparently identical to one of the polypeptides found in GS_n1. Glutamine synthetase holoenzyme consisted of eight subunits. In the nodule there are two different types of glutamine synthetase subunit polypeptides. Random combinations of the polypeptides should generate nine different isozymes. Our electrophoretic analysis revealed that GS_n2 was but one of the isozymes, and GS_n1 was a composite of the other eight. Hence, nodule glutamine synthetase isozymes were homo-octameric as well as hetero-octameric.     

Carbon Partitioning among Leaves, Fruits, and Seeds during Development of Phaseolus vulgaris L

Development of vegetative and floral buds was found to be a key factor in establishing the way carbon is distributed among growing leaves and fruits in Phaseolus vulgaris L. plants. Leaves emerged principally during a period 14 to 32 days after planting while flowers were produced during a 10- to 12-day period near the end of leaf emergence. Timing of anthesis established the sigmoidal time course for dry weight accumulated by the composite of all fruits on the plant. During the first 12 days following anthesis, fruit growth mainly consisted of elongation and dry weight accumulation by the pod wall. Thereafter, seed dry weight increased for about 1 week, decreased markedly for several days, and then increased again over the next 2 weeks. Accumulation of imported carbon in individual seeds, measured by steady-state labeling, confirmed the time course for dry weight accumulation observed during seed development. Seed respiration rate initially increased rapidly along with dry weight and then remained nearly steady until seed maturation. A number of developmental events described in the literature coincided with the different phases of diauxic growth. The results demonstrated the feasibility of relating current rates of carbon import in individual seeds measured with tracer ¹⁴C to the rates of conversion of imported sucrose and use of the products for specific developmental processes. The resulting data are useful for evaluating the roles of conversion and utilization of imported sucrose in regulating import by developing seeds.     

Location of two isoenzymes of NADH-dependent glutamate synthase in root nodules of Phaseolus vulgaris L.

The two isoenzymes of NADH-dependent glutamate synthase (NADH-GOGAT; EC 1.4.1.14), previously identified in root nodules of Phaseolus vulgaris L., have both been shown to be located in root-nodule plastids. The nodule specific NADH-GOGAT II accounts for the majority of the activity in root nodules, and is present almost exclusively in the central tissue of the nodule. However about 20% of NADH-GOGAT I activity is present in the nodule cortex, at about the same specific activity as this isoenzyme is found in the central tissue. Glutamine synthetase (GS; EC 6.3.1.2) occurs predominantly as the polypeptide in the central tissue, whereas in the cortex, the enzyme is represented mainly by the polypeptide. Over 90% of both GS and NADH-GOGAT activities are located in the central tissue of the nodule and GS activity exceeds NADH-GOGAT activity by about twofold in this region. Using the above information, a model for the subcellular location and stoichiometry of nitrogen metabolism in the central tissue of P. vulgaris root nodules is presented.Abbreviations Fd-GOGAT ferredoxin-dependent glutamate synthase - GOGAT glutamate synthase - GS glutamine synthetase - NADH-GOGAT NADH-dependent glutamate synthase - IEX-HPLC ion-exchange high-performance liquid chromatography     

Purification, Characterization, and Immunological Properties of NADH-Dependent Glutamate Synthase from Rice Cell Cultures

To obtain a monospecific antibody against NADH-dependent glutamate synthase (NADH-GOGAT; EC 1.4.1.14), the enzyme was purified to homogeneity from cultured rice cells (Oryza sativa) with column chromatography using Butyl Toyopearl 650M, Sephacryl S-300, Blue Sepharose CL-6B, and Butyl Toyopearl 650S. The specific activity at the final stage of the purification was 9.8 micromoles of glutamate formed per minute per milligram of protein. The yield was 6.1% and purification was 815-fold. Analysis by denaturing gel electrophoresis revealed a single polypeptide with an apparent molecular weight of 196,000, similar to the value of 194,000 estimated for the native protein. Apparent K_m values for l-glutamine, 2-oxoglutarate, and NADH were 811, 76, and 3.0 micromolar, respectively. Neither NADPH nor l-asparagine substituted for NADH and l-glutamine, respectively. The enzyme had its absorption maxima at 273, 373, and 440 nanometers with a shoulder at 475 nanometers, suggesting that the rice NADH-GOGAT is a flavoprotein. Monospecific antibody raised against NADH-GOGAT purified from the rice cells was obtained as the first instance for the enzyme in higher plants. Immunological analyses showed that the antibody for rice cell NADH-GOGAT reacted with only the enzyme in extracts from the cells. The anti-NADH-GOGAT antibody did not recognize the ferredoxin-GOGAT purified from rice leaves, and likewise the anti-rice leaf ferredoxin-GOGAT antibody did not react with the NADH-GOGAT purified from the cultured rice cells.     

Effects of Root Pruning on Translocation of Photosynthates in Phaseolus vulgaris L

Root pruning increased the level of ethanol soluble sugars inred kidney bean plants (Phaseolus vulgaris L. ) grown in aeratednutrient solution. However, the concentration gradient of thesesugars down the stem and its translocation velocity remainedunchanged. Removal of 50% of the roots had no effect on thetotal photosynthates exported from source leaves but the finaldistribution pattern of photosynthates was altered; less movingtoward the upper plant parts, and accumulation occurring inthe lower stems. Translocation velocity of photosynthates towardthe upper plant parts was drastically reduced by root pruning. Key words: Phaseolus vulgaris, Photosynthate translocation, Root pruning     

Changes in the Number, Viability, and Amino-acid-incorporating Activity of Rhizobium Bacteroids during Lupin Nodule Development

Between days 10 and 21 after inoculation of Lupinus angustifolius seedlings with Rhizobium NZP 2257, the average nodule fresh weight increased 3-fold and the number of bacteroids per nodule increased more than 10-fold.     

Purification and Characterization of a Polygalacturonase-Inhibiting Protein from Phaseolus vulgaris L

Homogeneous endo-polygalacturonase (PG) was covalently bound to cyanogen-bromide-activated Sepharose, and the resulting PG-Sepharose conjugate was utilized to purify, by affinity chromatography, a protein from Phaseolus vulgaris hypocotyls that binds to and inhibits PG. Isoelectric focusing of the purified PG-inhibiting protein (PGIP) showed a major protein band that coincided with PG-inhibiting activity. PGIP formed a complex with PG at pH 5.0 and at low salt concentrations. The complex dissociated in 0.5 m Na-acetate and pH values lower than 4.5 or higher than 6.0. Formation of the PG-PGIP complex resulted in complete inhibition of PG activity. PG activity was restored upon dissociation of the complex. The protein exhibited inhibitory activity toward PGs from Colletotrichum lindemuthianum, Fusarium moniliforme and Aspergillus niger. The possible role of PGIP in regulating the activity of fungal PG's and their ability to elicit plant defense reactions are discussed.     

Changes in Cell Fine Structure in the Cotyledons of Phaseolus vulgaris L. during Germination

When germination begins, the storage cells of Phaseolus vulgariscotyledons are packed with starch grains and protein bodies.Digestion of these reserves starts in cells furthest away fromthe vascular bundles and is practically completed in eight daysat 25° C. After the reserves are hydrolysed, the storagecells die. The changes in fine structure during the processof digestion and protoplasmic breakdown are described. Vascularbundle and epidermal cells survive till the cotyledons absciss,but in these tissues also profound changes occur in cellularorganization. The observations on fine structure are discussedwith reference to the metabolic activities of the cotyledons.     

Consequences of Sporangial Development for Nodule Function in Root Nodules of Comptonia peregrina and Myrica gale

Frankia sp., the actinomycetous endophyte in nitrogen-fixing actinorhizal nodules, may differentiate two forms from its hyphae: vesicles and sporangia. In root nodules of Comptonia peregrina (L.) Coult. and Myrica gale L., sporangia may be either absent or present. Nitrogenase activity and symbiotic efficiency were contrasted in spore(+) and spore(−) nodules of these two host genera. Seedlings of C. peregrina nodulated with the spore(+) inoculum showed only 60% of the nitrogenase activity and 50% of the net size of their spore(−) counterparts after 12 weeks of culture. Measurements of acetylene reduction (i.e., nitrogenase activity) were coordinated with samplings of nodules for structural studies. Significant differences in acetylene reduction rates were discernible between spore(+) and spore(−) nodules commencing 4 weeks after nodulation, concomitant with the maturation of sporangia in the nodule. Spore(+) nodules ultimately reached less than half of the rate of nitrogenase activity of spore(−) nodules. Both types of nodules evolved only small amounts of molecular hydrogen, suggesting that both were equally efficient in recycling electrons lost to the reduction of hydrogen ions by nitrogenase. Respiratory cost of nitrogen fixation, expressed as the quotient of micromole CO₂ to micromole ethylene evolved by excised nodules, was significantly greater in spore(+) than in spore(−) nodules. M. gale spore(−) nodules showed variable effectivity, though all had low CO₂ to ethylene evolution ratios. M. gale spore(+) nodules resembled C. peregrina spore(+), with low effectivity and high respiratory cost for nitrogen fixation.     

Alfalfa Root Nodule Carbon Dioxide Fixation : II. Partial Purification and Characterization of Root Nodule Phosphoenolpyruvate Carboxylase

A nonphotosynthetic phosphoenolpyruvate carboxylase (EC 4.1.1.31) was partially purified from the cytosol of root nodules of alfalfa. The enzyme was purified 86-fold by ammonium sulfate fractionation, DEAE-cellulose, hydroxylapatite chromatography, and reactive agarose with a final yield of 32%. The enzyme exhibited a pH optimum of 7.5 with apparent K_m values for phosphoenolpyruvate and magnesium of 210 and 100 micromolar, respectively. Two isozymes were resolved by nondenaturing polyacrylamide disc gel electrophoresis. Subsequent electrophoresis of these isozymes in a second dimension by sodium dodecyl sulfate slab gel electrophoresis yielded identical protein patterns for the isozymes with one major protein band at molecular weight 97,000. Malate and AMP were slightly inhibitory (about 20%) to the partially purified enzyme. Phosphoenolpyruvate carboxylase comprised approximately 1 to 2% of the total soluble protein in actively N₂-fixing alfalfa nodules.