Nodule Enzyme Research Articles

Presence and characteristics of nadp-malic enzyme in soybean nodule bacteroids

The localization and characteristics of NADP-malic enzyme [EC 1.1.1.40], a key enzyme for succinate oxidation in nodule bacteroids, were surveyed in soybean nodules and bacteroids. Protoplasts prepared from nodules were separated into cortex cell, infected cell, and uninfected cell groups following the determination of NADP-malic enzyme activity in those cells. The enzyme activity was localized in the cortex cells and the infected cells. In the infected cells the malic enzyme activity was not localized in the cytosol but in the bacteroids. The uninfected cells showed no detectable enzyme activity. The NADP-malic enzyme activity in the bacteroids prepared by Percoll density gradient centrifugation or by differential centrifugation was almost identical with that of the infected cells (mg protein basis). These data indicate that NADP-malic enzyme in soybean nodules was localized both in the cortex cells and in the bacteroids. Staining for the detection of the NADP-malic enzyme activity after polyacrylamide gel electrophoresis and the enzyme activity assay after isoelectrofocusing revealed that the malic enzyme in the bacteroids exhibited a unique Rf value from that in the whole nodules, indicating that the NADP-malic enzyme in Bradyrhizobium japonicum J501 bacteroids was from that in the cortex cells. The NADP-malic enzyme in the bacteroids was partially purified, and the characteristics were surveyed. At least two NADP-malic enzymes were present in the nodule bacteroids, and a major malic enzyme activity in the bacteroids was purified 127 fold. The enzyme showed an optimum pH at 7.5, a K m value at 109µM for malic acid and absolute requirement of NADP for the enzyme reaction. Strong inhibition of the enzyme activity by citrate, malonate, and glyoxylate was observed, suggesting a possible regulatory role of the malic enzyme in the organic acid metabolism in bacteroids.

Dinitrogen Fixation Technologies for Alfalfa Improvement

The energy crisis of the 1970s stimulated renewed research on the use of forage legumes as inexpensive sources of N in croping systems. New research programs were begun to improve the N assimilation of forage legumes, and to convert scientific knowledge about legume N2 fixation into technological choices that would support a profitable and sustainable agriculture. We assess the status of selected technologies that have resulted from this research. Four technologies, viz. alfalfa germplasm with enhanced activities of nodule enzymes, alfalfa germplasm unable to fix N2, alfalfa germplasm with improved N accumulation in herbage or roots, and improved rhizobial strains for inoculum have reached the developmental stage of field testing. Selection for activity of nodule enzymes resulted in experimental populations with enhanced or with reduced enzyme activity, but without differences in N2 fixation in the field. Several alfalfa germplasms incapable of N2 fixation have been released for use as non-N2 fixing controls in field experiments and in breeding programs. Nondormant alfalfa has been developed for enhanced N accumulation and storage, and one special purpose cultivar has been released. Molecular biology methods applied to inoculum improvement have yielded at least one rhizobial strain that enhanced yield in the greenhouse. These technologies can provide new cultivars for the forage legume seed industry, new controls for use in forage legume cultivar evaluation studies, and germplasm for use in testing models of soil nutrient transformations and soil management. These technologies will influence future crop management systems and soil conservation practices.

Variation for Activity of Nodule Nitrogen and Carbon Assimilating Enzymes in Alfalfa1

The nodule enzymes glutamate synthase (GOGAT) and phosphoenolpyruvate carboxylase (PEPC) are important in the assimilation of symbiotically fixed N in alfalfa (Medicago sativa L.). The objectives of this study were: to evaluate several alfalfa germplasm sources for variability in nodule GOGAT and PEPC activities, and to determine the relationships between N2 fixation and plant yield traits with nodule GOGAT and PEPC enzyme activities. Approximately 160 plants from each of six alfalfa germplasms were grown in sand culture in the glasshouse and assayed for nodule GOGAT and PEPC enzyme activities. Twenty‐four genotypes (four from each germplasm) were selected to represent a range in GOGAT and PEPC activities. These genotypes were evaluated in glasshouse and field clonal studies for GOGAT and PEPC activities per gram nodule fresh weight, acetylene reduction activity (ARA), nodule mass, nodule color, nodule soluble protein concentration, and plant yield. Variability for nodule GOGAT and PEPC activities within each of the six germplasms was large and normally distributed. The 12 high GOGAT genotypes (2 from each germplasm) were 44% greater GOGAT activity than the 12 low genotypes. The 12 high PEPC genotypes were 27% greater in PEPC activity than the 12 low genotypes. Both groups of high enzyme activity genotypes were significantly greater than the low genotypes in ARA, nodule soluble protein concentration, and field shoot yield. Both GOGAT and PEPC activities were positively correlated (r=0.61**, significant at the 0.01 probability level). Also, GOGAT and PEPC were each correlated with ARA (r=0.43*, significant at the 0.05 probability level, and 0.45*, respectively), nodule color score (r=0.46** and 0.73**) soluble protein concentration (r= 0.77** and 0.90**). Plant yield and N2 fixation measured in the field were not correlated with nodule GOGAT and PEPC activities measured in the glasshouse. The relationships between GOGAT and PEPC with other plant traits indicated that these enzymes are associated with the host‐Rhizobium meliloti Dang. symbiosis and should be explored as possible selection criteria to include in an alfalfa breeding program.

Purification, Properties, and Distribution of Ascorbate Peroxidase in Legume Root Nodules

All aerobic biological systems, including N(2)-fixing root nodules, are subject to O(2) toxicity that results from the formation of reactive intermediates such as H(2)O(2) and free radicals of O(2). H(2)O(2) may be removed from root nodules in a series of enzymic reactions involving ascorbate peroxidase, dehydroascorbate reductase, and glutathione reductase. We confirm here the presence of these enzymes in root nodules from nine species of legumes and from Alnus rubra. Ascorbate peroxidase from soybean nodules was purified to near homogeneity. This enzyme was found to be a hemeprotein with a molecular weight of 30,000 as determined by sodium dodecyl sulfate gel electrophoresis. KCN, NaN(3), CO, and C(2)H(2) were potent inhibitors of activity. Nonphysiological reductants such as guaiacol, o-dianisidine, and pyrogallol functioned as substrates for the enzyme. No activity was detected with NAD(P)H, reduced glutathione, or urate. Ascorbate peroxidation did not follow Michaelis-Menten kinetics. The substrate concentration which resulted in a reaction rate of (1/2) V(max) was 70 micromolar for ascorbate and 3 micromolar for H(2)O(2). The high affinity of ascorbate peroxidase for H(2)O(2) indicates that this enzyme, rather than catalase, is responsible for most H(2)O(2) removal outside of peroxisomes in root nodules.

Symbiotic properties of Lotus pedunculitis root nodules induced by Rhizobium loti and Bradyrhizobium sp. (Lotus)

Vance, C. P., Reibach, P. H. and Pankhurst, C. E. 1987. Symbiotic properties of Lotus pedunculatus root nodules induced by Rhizobium loti and Bradyrhizobium sp. (Lotus).Symbiotic properties of root nodules were evaluated in glasshouse‐grown Lotus pedunculatus Cav. cv. Maku inoculated with either a fast‐growing Rhizobium loti strain NZP2037 or a slow‐growing Bradyrhizobium sp. (Lotus) strain CC814s. Although the nodule mass of plants inoculated with NZP2037 was twice that of plants inoculated with CC814s, the yield of NZP2037 shoots and roots was 50% that of CC814s shoots and roots. Nodules induced by Bradyrhizobium fixed substantially more N than nodules induced by R. loti. Glucose requirements [mol glucose (mol N2 fixed)‐1] of nodules induced by CC814s and NZP2037 were 7.1 and 16.6, respectively. Nodule enzymes of carbon and nitrogen assimilation reflected the disparity of the two sym‐bioses. Xylem sap of the symbiosis with the higher yield contained a higher concentration of asparagine [9.86 μmol (ml xylem sap)‘] than did the lower yielding symbiosis [5.80 umol (ml xylem sap)“’]. Nodule CO2 fixation was directly linked to nodule N assimilation in both symbioses. The results indicate that the difference between the two symbioses extend to nodule N and C assimilation and whole plant N transport. The data support a role for host plant modulation of bacterial efficiency and assimilation of fixed N.

Enzymes of glutathione metabolism as biochemical markers during hepatocarcinogenesis.

Enzymes of glutathione metabolism, particularly gamma-glutamyltransferase (GGT) and glutathione S-transferase (GST), play a role in multistage hepatocarcinogenesis. The enhanced expression of these enzymes in preneoplastic altered hepatic foci, nodules, and hepatocellular carcinomas has been demonstrated after treatment with a variety of initiating and promoting agents. Glutathione is necessary for the detoxification of xenobiotics and carcinogens and for cell replication. Induction of GGT in altered hepatocytes may permit these cells to utilize extracellular glutathione to preserve their internal glutathione levels. GST induction allows glutathione utilization for the protection of the altered hepatocyte in an environment of exposure to xenobiotics, such as promoting agents. Thus, the combined effects of GGT and GST, in a toxic environment, may provide for the enhanced proliferation observed in preneoplastic hepatocytes. New clinical and research opportunities may involve the use of GGT and the placental isozyme of GST (PGST) as markers of preneoplasia and neoplasia in humans. Many factors, such as hormones, diet, and exposure to initiating and promoting agents, influence GGT and GST expression. The recent cloning of cDNAs to GGT and PGST offers opportunities for the study of factors involved in the genetic expression of these two enzymes. Coupled with the use of hepatocyte culture and transplantation, the factors involved at the molecular level in the creation of hepatocellular neoplasia may be discovered.

Soil fertility effects on growth, nitrogen fixation, nodule enzyme activity and xylem exudates of lablab purpureus (L.) sweet, grown on a typic eutrustox

Abstract The Lablab or Hyacinth bean, Lablab purpureus (L.) Sweet, is a drought tolerant grain‐forage legume widely grown within a wide range of neotropical regions of the world. It has been an important cultivated food grain crop for many centuries within extensive warm humid‐subhumid climatic areas and grown on widely different Ultisol and Oxisol soil types. The objective of these studies was to determine soil fertility effects of Ca, P, and K factorial combinations applied to a Typic Eutrustox on growth, nodulation, and nitrogen fixation of Lablab bean. Stem and nodule xylem components were determined along with soil pH and nitrification within the nodule rhizosphere. Highly significant increases resulted with P fertilization for stem, leaf, seed, nodulation, and nitrogenase activity. With the exception of nitrogenase, all were also increased with K additions. Both stem and seed were significantly increased with Ca treatments. Highly significant increases for total plant N content resulted with P, K, C...

Effect of applied nitrate on enzymes of ammonia assimilation in nodules ofCicer arietinum L.

The relationship between N2-fixation, nitrate reductase and various enzymes of ammonia assimilation was studied in the nodules and leaves ofC. arietinum. In the nodules of the plants growing on atmospheric nitrogen, maximum activities of glutamine synthetase (GS), glutamate synthase (GOGAT), glutamate dehydrogenase (GDH), asparagine synthetase (AS) and aspartate aminotransferase (AAT) were recorded just prior to maximum activity of nitrogenase. In nitrate fed plants, the first major peak of GDH and AS coincided with that of nitrate reductase in the nodules. With the exception of AS, application of nitrate decreased the activities of all these enzymes in nodules but not in leaves. Activities of GS, GOGAT and AAT were affected to much greater extent than that of GDH. On comparing the plants grown without nitrate and those with nitrate, the ratios of the activities of GDH/GS and GDH/GOGAT in nitrate given plants, increased by 4 and 12 fold, respectively. The results presented in this paper suggest that in nodules of nitrate fed plants, assimilation of ammonia via GDH assumes much greater importance.

Heterogeneity of Glutamine Synthetase Polypeptides in Phaseolus vulgaris L.

Glutamine synthetases from roots, nodules, and leaves of Phaseolus vulgaris L. have been purified to homogeneity and their polypeptide composition determined.The leaf enzyme is composed of six polypeptides. The cytosolic fraction contains two 43,000 dalton polypeptides and the chloroplastic enzyme is formed by four 45,000 dalton polypeptides. Root glutamine synthetase consists only of the same two polypeptides of 43,000 dalton that are present in the leaf enzyme. The nodule enzyme is formed by two polypeptides of 43,000 dalton, one is common to the leaf and root enzyme but the other is specific for N(2)-fixing nodule tissue. The two glutamine synthetase forms of the nodule contain a different proportion of the 43,000 dalton polypeptides.

A multiple-trait breeding program for improving the symbiosis for N2 fixation betweenMedicago sativa L. andRhizobium meliloti

The goal of breeding alfalfa for increased N2 fixation potential is addressed. A chronological progression of breeding, physiological, microbiological, and plant pathological research is described. Studies describing the interrelationships among plant morphological, plant physiological, andRhizobium effectiveness traits are summarized. It was concluded that N2 fixation in alfalfa is affected by coordinated responses among many physiological and biochemical traits. The simultaneous improvement of many factors in the symbiosis requires a comprehensive multiple-step breeding program. The current program includes selection in the glasshouse for seedling vigor,Rhizobium preference, shoot growth, nodule mass, root growth, nitrogenase (as measured by acetylene reduction), and nodule enzyme activity. The inclusion of additional selection traits is anticipated. Field evaluations of N2 fixation potential of alfalfa populations are made with15N isotope dilution techniques. Plant germplasm sources used in the breeding program include several heterogeneous populations which have good combining ability and pest resistance when they are intercrossed. Significant progress has been made in achieving the goal of breeding alfalfa for improved N2 fixation.

Host Plant Nodule Enzymes Associated with Selection for Increased N2 Fixation in Alfalfa1

In legumes symbiotically fixed N2 is assimilated into C skeletons to form amino acids as a prerequisite for enhancing plant growth. No attempts have been made to determine which nodule enzymes of ammonia and C assimilation are related to N2 fixation in alfalfa (Medicago sativa L.). The objective of this study was to assess if nodule enzymes of ammonia and C assimilation were associated with acetylene reduction acitivity in alfalfa genotypes previously selected for various traits associated with N2 fixation. Nodules from eight subpopulations derived by bi‐directional selection within each of two alfalfa germplasm sources, an intercross between the two germplasm sources and three alfalfa cultivars were used to measure nodule soluble protein, and in vitro specific activities of the plant enzymes glutamine synthetase (GS), glutamate synthase (GOGAT) glutamate dehydrogenase (GDH), and phosphoenolpyruvate carboxylase (PEPC). Acetylene reduction rate was measured as an indicator of nitrogenase activity. Significant genotypic differences were observed among subpopulations for all variables tested. The GOGAT and PEPC activities were correlated with acetylene reduction activity in one germplasm source but not the other. Phenotypic correlation coefficients among variables showed that GOGAT and PEPC activities were frequently coupled. Germplasm differences in nodule enzyme activity suggest that nodule carbon and N assimilation may be more closely associated with N2 fixation potential in one germplasm than the other. The GOGAT and PEPC activity may be useful traits to include in breeding programs to enhance N2 fixation in alfalfa.

Carbon Metabolism in Alfalfa Root Nodules: Developmental Patterns of Host Plant Enzymes Before and After Shoot Removal1

Dinitrogen fixation in the nodules of perennial forage legumes usually decreases rapidly after the harvest of shoots and then increases as new shoots are produced. In this study the activities of enzymes of carbon metabolism which may indirectly support dinitrogen fixation and subsequent assimilation were measured in the plant fractions of alfalfa (Medicago sativa L.) nodules before and after shoot removal. Nodule enzymes of sucrose and glucose metabolism (invertase, glucose‐6‐phosphate dehydrogenase) and organic acid metabolism (malate dehydrogenase and NADP‐isocitrate dehydrogenase) decreased rapidly in activity (gram fresh weight basis) after shoot removal. Except for NADP‐isocitrate dehydrogenase, the decreases in specific activity of these enzymes after shoot removal were similar to that for soluble protein suggesting general proteolytic degradation. Amylase activity showed no clear response to shoot removal. These data suggest that when alfalfa shoots are removed, root nodule capacity to metabolize some hexoses and organic acids is reduced while the capacity to degrade nodule starch is not greatly influenced.

Root Nodule Enzymes of Ammonia Metabolism from Medicago sativa L. as Influenced by Nitrate Levels

Plants of alfalfa (Medicago sativa L.) were subjected to six levels of nitrate ranging from 0 to 200 mM. Then, nodule content of soluble protein and activities of the nodule enzymes, nitrogenase (C(2)H(2) reduction activity), glutamine synthetase (EC 6.3.1.2), glutamate synthase (EC 1.4.1.14), and NADH/NAD(+)-glutamate dehydrogenases (EC 1.4.1.2) were determined. Decline of glutamate synthase activity closely paralleled that of nitrogenase. In contrast, glutamine synthetase activity reached a minimum at 20mM nitrate but began to recover at higher nitrate concentrations. Patterns of NADH-(aminating) and NAD(+)-(deaminating) glutamate dehydrogenase activities were opposed with a great increase (2- to 5-fold) in the aminating direction at highest levels of nitrate. Results indicate a main role of glutamine synthetase/glutamate synthase pathway in the assimilation of fixed N. In addition, increase of the activities of glutamine synthetase and NADH-glutamate dehydrogenase concomitant with an almost fully suppressed nitrogenase and decreased NAD(+)-glutamate dehydrogenase activities at above 20mM nitrate reflect: (a) rapid induced senescence of nodules and (b) the production of ammonia by alternative means as nitrate reduction or oxidative deamination of amino acids.

Metabolism of 14C-Labeled Photosynthate and Distribution of Enzymes of Glucose Metabolism in Soybean Nodules

The metabolism of translocated photosynthate by soybean (Glycine max L. Merr.) nodules was investigated by (14)CO(2)-labeling studies and analysis of nodule enzymes. Plants were exposed to (14)CO(2) for 30 minutes, followed by (12)CO(2) for up to 5 hours. The largest amount of radioactivity in nodules was recovered in neutral sugars at all sampling times. The organic acid fraction of the cytosol was labeled rapidly. Although cyclitols and malonate were found in high concentrations in the nodules, they accumulated less than 10% of the radioactivity in the neutral and acidic fractions, respectively. Phosphate esters were found to contain very low levels of total label, which prohibited analysis of the radioactivity in individual compounds. The whole nodule-labeling patterns suggested the utilization of photosynthate for the generation of organic acids (principally malate) and amino acids (principally glutamate).The radioactivity in bacteroids as a percentage of total nodule label increased slightly with time, while the percentage in the cytosol fraction declined. The labeling patterns for the cytosol were essentially the same as whole nodule-labeling patterns, and they suggest a degradation of carbohydrates for the production of organic acids and amino acids. When it was found that most of the radioactivity in bacteroids was in sugars, the enzymes of glucose metabolism were surveyed. Bacteroids from nodules formed by Rhizobium japonicum strain 110 or strain 138 lacked activity for phosphofructokinase and NADP-dependent 6-phosphogluconate dehydrogenase, key enzymes of glycolysis and the oxidative pentose-phosphate pathways. Enzymes of the glycolytic and pentose phosphate pathways were found in the cytosol fraction.In three experiments, bacteroids contained about 10 to 30% of the total radioactivity in nodules 2 to 5 hours after pulse-labeling of plants, and 60 to 65% of the radioactivity in bacteroids was in the neutral sugar fraction at all sampling times. This strongly suggests some absorption and metabolism of sugars by bacteroids in spite of the lack of key enzymes. Bacteroids did possess enzymes for the formation of hexose phosphates from glucose or fructose. Radioactivity in alpha,alpha-trehalose in bacteroids increased until, after 5 hours, trehalose was a major labeled compound in bacteroids. Thus, trehalose synthesis may be a major fate of sugars entering bacteroids.

Effects of the Administration of Anticoagulants on the Activity of the Enzyme-Reduced NAD(P)H Dehydrogenase in Rat Livers, Hepatomas and Precarcinomatous Rat Liver Lesions

The anticoagulants dicumarol, warfarin and diphenadione, are in vitro inhibitors of the enzyme reduced NAD(P)H dehydrogenase of rat liver. These chemicals were administered by intragastric gavage to determine whether the same inhibitory effects could be observed in Sprague-Dawley male rats. Doses of 2 and 10 mg/100 g body weight were used. Our results indicate that only dicumarol inhibited the enzyme, whereas warfarin did not produce a significant effect, and diphenadione at large doses produced an increase in the activity of the enzyme. Dicumarol was further tested to see if it would alter the activity of the enzyme in hyperplastic nodules and liver hepatomas. A similar inhibitory effect was found. The three strains of rats tested in this work have different levels of reduced NAD(P)H dehydrogenase activity. Thus, our results indicate that dicumarol is the only anticoagulant that inhibits in vivo the reduced NAD(P)H dehydrogenase of rat liver and liver neoplasms.

Plant determined ineffective nodules in alfalfa (Medicago sativa): structural and biochemical comparisons

Nodule structure, nodule enzymes of ammonia assimilation, nodule phosphoenolpyruvate carboxylase, nitrogenase-dependent acetylene reduction, and soluble protein were studied in four plant-determined ineffective genotypes and in one effective genotype of alfalfa (Medicago sativa L.) grown in a glasshouse. Ineffective nodules that formed on genotypes MnAg(In) and MnSa(In) were similar to effective nodules in early development. However, these nodules had smaller bacteroids and senesced much more rapidly than effective nodules. In contrast, nodules that formed on ineffective genotypes MnNC-3226(In) and MnNC-3811(In) were tumorlike and had few infection threads and bacteroids, and host cells were filled with starch. Nodules from plant-determined ineffective genotypes had reduced glutamine synthetase, glutamate synthase, phosphoenolpyruvate carboxylase, and nodule soluble protein when compared with effective nodules. There were differences between genotypes for all biochemical parameters assayed.

Cluster Bean—A Ureide- or Amide-Producing Legume?

Xylem sap of cluster bean (Cyamopsis tetragonoloba L. cv FS-277) and pigeonpea (Cajanus cajan cv UPAS-120) were analyzed for total nitrogen, amide nitrogen, and ureide nitrogen at flowering stage. Nitrogenase, uricase, and allantoinase were compared in nodules of cluster bean and pigeonpea. Xylem sap of cluster bean exhibited higher amounts of amides as compared to ureides, and the activities of uricase and allantoinase (ureide-producing enzymes) in nodules were also low, whereas the reverse was the case for pigeonpea. Based on these investigations, it has been concluded that cluster bean is an amide-producing legume rather than ureide-producing as had been reported earlier.

MULTIPLE FORMS OF GLUTAMINE SYNTHETASE IN THE PLANT FRACTION OF PHASEOLUS ROOT NODULES

ABSTRACT Activity of glutamine synthetase in the plant fraction of Phaseolus root nodules was approximately 14-fold higher than in the roots, when synthetase activities (per g fresh wt) were compared. Chromatography of the plant nodule enzyme on DEAE-Sephacel revealed the presence of three forms of the enzyme. These forms could also be separated by starch gel electrophoresis but not by gel filtration, suggesting that the three forms in the nodule differ in charge but not in molecular weight. Evidence is presented showing that the thud form of glutamine synthetase in the nodule is identical to the single form of the enzyme found in roots. Moreover, from measurements of the nodule activities it appears that the increase in glutamine synthetase during nodulation can be accounted for entirely by the production of the two new (nodule-specific) forms.

Birdsfoot trefoil (Lotus corniculatus) root nodules: morphogenesis and the effect of forage harvest on structure and function

Nodule structure, nodule enzymes of ammonia assimilation, nitrogenase-dependent acetylene reduction, and nodule soluble protein were studied during vegetative regrowth of detopped birdsfoot trefoil (Lotus corniculatus L.) seedlings grown in the glasshouse. Nodules senesced rapidly for a period of 14 days following shoot removal, but then pink nodule populations increased as shoot regrowth occurred. The structural sequence of senescence was similar in nodules whether the result of either aging or shoot removal. Membranes surrounding bacteroids showed degenerative changes as bacteroids senesced. Bacteroids aggregated within the nodule cells and ultimately disappeared in senescent cells. Infection threads and bacteria inside infection threads did not disintegrate.Nodule senescence as a result of shoot removal was accompanied by a marked decline in acetylene reduction, nodule soluble protein, nodule host plant glutamine synthetase (GS), and glutamate synthase (GOGAT). Nodule enzyme activity, soluble protein, and acetylene reduction activity recovered to initial values as shoot regrowth occurred and pink nodule populations increased. Nodule host plant glutamate dehydrogenase (GDH) did not change after shoot removal.This study shows that birdsfoot trefoil nodules respond to shoot removal by an increased senescence. Recovery of nodule function is associated with the formation of a new nodule population. The data also indicate that host plant GS and GOGAT function to assimilate fixed N. Spherical nodules with determinant growth may be less efficient than elongate nodules with indeterminant growth in nodule maintenance and function.

Root and Nodule Enzymes of Ammonia Assimilation in Two Plant-Conditioned Symbiotically Ineffective Genotypes of Alfalfa (Medicago sativa L.)

Biochemical and physiological parameters associated with nitrogen metabolism were measured in nodules and roots of glasshouse-grown clones of two symbiotically ineffective alfalfa (Medicago sativa L.) genotypes supplied with either NO(3) (-) or NH(4) (+). Significant differences were observed between genotypes for nodule soluble protein concentrations and glutamine synthetase (GS) and glutamate synthase (GOGAT) specific activities, both in untreated controls and in response to applied N. Nodule soluble protein of both genotypes declined in response to applied N, while nodule GS, GOGAT, and glutamate dehydrogenase (GDH) specific activities either decreased or remained relatively constant. In contrast, no genotype differences were observed in roots for soluble protein concentrations and GS, GOGAT, and GDH specific activities, either in untreated controls or in response to applied N. Root soluble protein levels and GS and GOGAT specific activities of N-treated plants increased 2- to 4-fold within 4 days and then decreased between days 13 and 24. Root GDH specific activity of NH(4) (+)-treated plants increased steadily throughout the experiment and was 50 times greater than root GS or GOGAT specific activities by day 24.Enzymological data indicate that nodules of these ineffective alfalfa genotypes are uniquely differentiated plant organs. Decreasing or constant plant GS and GOGAT specific activities in ineffective nodules in response to applied N suggest that factors in addition to N supply are involved in the induction of high levels of plant ammonia-assimilating enzymes in nodules. Genotype differences observed for nodule enzyme specific activities support the concept that ineffectiveness may be expressed in different ways within the nodule. Senescence was evident in ineffective nodules of N-treated plants of both genotypes, indicating that nodule senescence induced by applied N may not be closely linked to symbiotic effectiveness in alfalfa. Data for ammonia-assimilating enzymes in roots suggest the GS/GOGAT pathway operates only at low levels of soil N and that GDH functions to detoxify high levels of soil NH(4) (+).