Nodule Metabolism Research Articles

Can genotypes of soybean (Glycine max) selected for nitrate tolerance provide good “models” for studying the mechanism of nitrate inhibition of nitrogenase activity?

In soybeans (Glycine max L. Merr.), high levels of soil nitrate inhibit N2 fixation, and nitrate‐tolerant symbioses have been identified within a chemically mutagenized line of cv. Bragg denoted nts382 and within the line K466, a genotype representative of a number of Korean soybean cultivars. The genotypes nts382 and K466 were examined to see if they could be used as a model system for studying the mechanism responsible for the short‐term (i.e. 3‐day) inhibition of specific nitrogenase activity, especially the mechanism behind the greater O2 limitation of nodule metabolism that is characteristic of nitrate inhibition of N2 fixation in soybean. In nts382, total nitrogenase activity (TNA = H2 production in Ar:O2) was inhibited to a lesser degree (48% of control) relative to Bragg (30% of control), and the nitrate‐treated symbioses showed less of an O2 limitation of nodule metabolism in nts382 than in Bragg. However, the relative proportion of O2 limitation to the total nitrate inhibition was similar (40 and 41%) in nts382 and Bragg, respectively. Therefore, the nts382 symbioses may be useful in elucidating the general mechanism for down‐regulation of nitrogenase activity in soybean, but would not be a useful model system for studying the control of O2‐limited metabolism following nitrate exposure. The effects of nitrate on TNA and on the degree of O2 limitation of nodule metabolism were the same in K466 and a reference cultivar Maple Arrow. Consequently, the tolerance of K466 to nitrate reported previously was attributed to the ability of this symbiosis to maintain nodule biomass in the presence of nitrate, not to any ability to maintain specific nitrogenase activity in the presence of nitrate.

Immunogold localization of nodule‐enhanced phosphoenolpyruvate carboxylase in alfalfa

ABSTRACTPhosphoenolpyruvate carboxylase (PEPC; EC 4‐1‐1‐31) plays a paramount role in providing carbon for synthesis of malate and aspartate in alfalfa (Medicago sativa L.) root nodules. PEPC protein and activity levels are highly enhanced in N2‐fixing alfalfa nodules. To ascertain the relationship between the cellular location of PEPC and root nodule metabolism, enzyme localization was evaluated by immunogold cytochemistry using alfalfa nodule PEPC antibodies. Gold labelling patterns in effective nodules showed that PEPC is a cytosolic enzyme and is distributed relatively equally in infected and uninfected cells of the nodule symbiotic zone. A high amount of labelling was also observed in pericycle cells of the nodule vascular system. Labelling was also detected within inner cortical cells, but the density was reduced by 60%. When Lotus corniculatus was transformed with a chimeric gene consisting of the 5′‐upstream region of the PEPC gene fused to β‐glucuronidase (GUS), GUS staining in nodules was consistent with immunogold localization patterns. The occurrence of PEPC in both infected and uninfected cells of the symbiotic zone of effective nodules coupled to the reduced amounts in ineffective nodules suggests a direct role for this enzyme in supporting N2‐fixation. PEPC localization in the uninfected, interstitial cells of the symbiotic zone indicates that these cells may also have a role in nodule carbon metabolism. Moreover, the association of PEPC with the nodule vascular system implies a role for the enzyme in the transport of assimilates to and from the shoot.

Content of different phytohormones and indole acetic acid metabolism in root nodules of Derris scandens BENTH

AbstractRoot nodules of Derris scandens BENTH., an economic leguminous climbing shrub, contain high amount of IAA, gibberellin like (GA), cytokinin like (CK) and abscisic acid like (ABA) substances. A tryptophan pool present in the nodule may serve as a source of IAA production. Metabolism of IAA in the nodules was evidenced by the presence of IAA metabolising enzymes, IAA oxidase and peroxidase. The symbiont from the nodules, a Rhizobium sp., produced high amount of IAA in culture when supplemented with tryptophan. For IAA production the bacteria preferred L‐tryptophan to DL‐ or D‐isomers of tryptophan.

The role of sucrose synthase in the response of soybean nodules to drought

Experiments were carried out to investigate the effects of drought stress on enzymatic activities related to carbon and nitrogen metabolism in soybean nodules. Gradual drought stress, imposed by withholding water nutrients, resulted in declines in the water potential of leaves and nodules consistent with a significant decline in N2 fixation. However, the amounts of nitrogenase components 1 and 2 were virtually unaffected by drought stress. Similarly, no significant changes could be detected in aspartate aminotransferase, phosphoenolpyruvate carboxylase, glutamine synthetase or alkaline invertase activities throughout the experiment. In contrast, sucrose synthase (SS), one of the enzymes involved in sucrose metabolism in legume nodules, declined dramatically in activity and in content within a few days of withholding water. Coincident with this decline in SS activity were significant increases in the nodule contents of sucrose, total free amino acids and ureides. The amounts of proline, however, did not increase until some days later. It is suggested that SS may play a key role in the regulation of nodule carbon metabolism and, therefore, of nitrogen fixation under drought stress conditions.

Effects of validamycin A, a potent trehalase inhibitor, and phytohormones on trehalose metabolism in roots and root nodules of soybean and cowpea

Trehalose, a common microbial disaccharide, has been reported to be toxic to plants, and plant trehalase has therefore been hypothesized to function as a detoxifying enzyme. To test this, aseptically grown soybean (Glycine max L. Merr.) plantlets were supplied with trehalose. The plants accumulated trehalose only when validamycin A, a potent trehalase inhibitor, was added as well. Under these conditions, they accumulated trehalose to up to 8% of the dry weight in their primary leaves without any detectable impairment of growth or health. We have previously shown that in soybean nodules, trehalose is generated by the symbiotic bacteria, and trehalase is strongly induced. However, direct exposure of plants to trehalose did not affect their trehalase activity, whereas a treatment with auxin strongly increased it, indicating that the enzyme level is regulated by hormones rather than by its substrate. Addition of validamycin A to nodules caused an increase in the amount of trehalose and a decrease in the sucrose and starch pools, but nitrogen fixation was not affected. Similar results were obtained with cowpea (Vigna unguiculata L.) plantlets and nodules. These results indicate that plant trehalase is functional in metabolizing trehalose from exogenous and endogenous sources, even though the disaccharide has no obvious toxic effects.

Drought Stress, Permeability to O2 Diffusion, and the Respiratory Kinetics of Soybean Root Nodules.

In legume nodules, treatments such as detopping or nitrate fertilization inhibit nodule metabolism and N2 fixation by decreasing the nodule's permeability to O2 diffusion, thereby decreasing the infected cell O2 concentration (Oi) and increasing the degree to which nodule metabolism is limited by O2 availability. In the present study we used nodule oximetry to assess and compare the role of O2 limitation in soybean (Glycine max L. Merr) nodules inhibited by either drought or detopping. Compared to detopping, drought caused only minor decreases in Oi, and when the external O2 concentration was increased to raise Oi, the infected cell respiration rate in the drought-stressed plants was not stimulated as much as it was in the nodules of the detopped plants. Unlike those in detopped plants, nodules exposed to moderate drought stress displayed an O2-sufficient respiration rate that was significantly lower than that in control nodules. Despite possible side effects of oximetry in altering nodule metabolism, these results provided direct evidence that, compared to detopping, O2 limitation plays a minor role in the inhibition of nodule metabolism during drought stress and changes in nodule permeability are the effect, not the cause, of a drought-induced inhibition of nodule metabolism and the O2-suffiecient rate of respiration.

Physiology of the legume nodule and its response to stress

A common mechanism is sought for the effect of environmental variables (e.g. light, defoliation, temperature, N: nitrate, ammonium or organic) on nodule metabolism. A decrease in nodule cortical permeability has been suggested to enforce a primary limitation of O 2 supply to the infected zone during periods of environmental stress, but the mechanism by which cortical permeability is altered is controversial. It is proposed that the water requirement of xylem export from the nodule achieves a balance with water delivery to the nodule in the phloem, such that a change in either export or import will affect the turgor of cells surrounding the nodule vascular bundles. Cortical cell turgor may also be influenced by the balance of solutes between the apoplast and symplast. Stress events involving variations of phloem supply or decrease in rate of N 2 fixation (e.g. C 2H 2 assay) will result in changes in the soluble sugar and nitrogenous solute content of the cortex, with implication to cortical cell turgor. Change in cortical cell turgor may affect the turgor gradient driving phloem supply into the nodule in a form of turgor homeostat, and coincidentally may affect cortical cell shape or the intercellular water content and so alter the permeability of the nodule cortex to gases. It is proposed this regulation of nodule permeability is localized in the vicinity of the vascular bundles. Nodule anatomy and physiology is, however, heterogeneous. For example, nodule cortical anatomy is variable between species. Thus, the response of N 2 fixation to the effect of an environmental variable is species specific. The response will also reflect the duration and severity of stress, the plant growth history and will be complicated by effects on nodulation and anatomical adaptation in the longer term.

Effects of Temperature on Infected Cell O2 Concentration and Adenylate Levels in Attached Soybean Nodules.

To assess the role of O2 in the regulation of nodule metabolism following a decrease or an increase in temperature, the fractional oxygenation of leghemoglobin (FOL) was measured in soybean (Glycine max L. Merr.) nodules during rapid and gradual changes in temperature from 20[deg]C to either 15 or 25[deg]C. The affinity of leghemoglobin for O2 was also measured at each temperature and the values were used to calculate the infected cell O2 concentration (Oi). After nodules were transferred to 15[deg]C, FOL and Oi increased and adenylate energy charge (AEC = [ATP + 0.5ADP]/[ATP + ADP + AMP]) increased from 0.70 to 0.78. The temperature increase was associated with a decrease in FOL and Oi. We concluded that changes in nodule temperature alter the respiratory demand of the nodules for O2, resulting in a change in Oi and a shift in the balance between ATP consumption and ATP production within the nodule tissue.

The Role of Oxygen in the Regulation of Nitrogenase Activity in Drought-Stressed Soybean Nodules.

The aim of this study was to investigate the mechanism of nitrogenase inhibition in drought-stressed soybean (Glycine max L.) nodules to determine whether this stress was similar to other inhibitory treatments (e.g. detopping) known to cause an O2 limitation of nodule metabolism. Nodulated soybean plants were either detopped or subjected to mild, moderate, or severe drought stress by growth in different media and by withholding water for different periods. All treatments caused a decline in nitrogenase activity, and in the drought-stressed nodules, the decline was correlated with more negative nodule water potentials. Increases in rhizosphere O2 concentration stimulated nitrogenase activity much more in detopped plants than in drought-stressed plants, reflecting a greater degree of O2 limitation with the detopped treatment than with the drought-stressed treatment. These results indicated that drought stress differs from many other inhibitory treatments, such as detopping, in that its primary cause is not a decrease in nodule permeability and a greater O2 limitation of nodule metabolism. Rather, drought stress seems to cause a decrease in the maximum O2-sufficient rate of nodule respiration or nitrogenase activity, and the changes in nodule permeability reported to occur in drought-stressed nodules may be a response to elevated O2 concentrations in the infected cell that may occur as nodule respiration declines.

The relationship between nodule adenylates and the regulation of nitrogenase activity by O2 in soybean

Nodulated soybeans (Glycine max L. Merr, cv. Maple Arrow) were exposed to various physiological and environmental treatments to determine the relationship between nodule adenylate pools and the degree of O2 limitation of nitrogenase. Adenylate energy charge (AEC = [ATP + 0.5 ADP]/[ATP + ADP + AMP]) and ATP/ADP ratios declined under conditions of decreased (10%) external pO2 but increased in nodules exposed to elevated (30%) external pO2. Nitrogenase activity was inhibited by both pO2 treatments, but recovered towards initial levels within 45 min. AEC also returned to initial levels during this period. To account for these and related data in the literature, it was hypothesized that 1) legume nodules regulate infected cell O2 concentration (Oi) to maintain adenylate pools at levels which limit respiratory metabolism: 2) treatments which decrease Oi alter the adenylate pools and further limit nodule metabolism; 3) treatments which increase Oi to levels in excess of a narrow range alter the adenylate pools and activate biochemical pathways which are not conducive to nitrogenase activity. In a preliminary test of these hypotheses, changes in AEC and ATP/ADP ratio were studied in nodules in which nitrogenase activity was inhibited by stem girdling, nitrate fertilization and exposure to an Ar:O2 atmosphere. All three treatments caused an increased O2 limitation of nodule respiration and nitrogenase activity. However, decreases in AEC were observed only in the stem girdling and nitrate fertilization treatment: Ar:O2 exposure had no effect on whole nodule AEC. While this result challenged the hypotheses suggesting a central role for adenylates in the regulation of O2‐limited metabolism, it was noted that the Ar:O2 treatment would differ from the other treatments in that it would have a specific effect on the ATP demands for NH3 assimilation in the plant fraction. Since AEC and ATP/ADP ratio would be affected by both the rate of ATP synthesis (potentially an O2‐limited process) and the demand for ATP, changes in these parameters in the whole nodule may not be a reliable indicator of adenylate‐mediated O2 limitation. Futher studies are needed to examine in vivo changes in adenylate pools in the plant and bacteroid fractions in nodules which vary in their degree of O2‐limited metabolism.

Composition and Distribution of Adenylates in Soybean (Glycine max L.) Nodule Tissue.

Adenylates (ATP, ADP, and AMP) may play a central role in the regulation of the O2-limited C and N metabolism of soybean nodules. To be able to interpret measurements of adenylate levels in whole nodules and to appreciate the significance of observed changes in adenylates associated with changes in O2-limited metabolism, methods were developed for measuring in vivo levels of adenylate pools in the cortex, plant central zone, and bacteroid fractions of soybean (Glycine max L. Merr cv Maple Arrow x Bradyrhizobium japonicum strain USDA 16) nodules. Intact nodulated roots were either frozen in situ by flushing with prechilled Freon-113(-156[deg]C) or by rapidly (<1 s) uprooting plants and plunging them into liquid N2. The adenylate energy charge (AEC = [ATP + 0.5 x ADP]/[ATP + ADP + AMP]) of whole-nodule tissue (0.65 [plus or minus] 0.01, n = 4) was low compared to that of subtending roots (0.80 [plus or minus] 0.03, n = 4), a finding indicative of hypoxic metabolism in nodules. The cortex and central zone tissues were dissected apart in lyophilized nodules, and AEC values were 0.84 [plus or minus] 0.04 and 0.61 [plus or minus] 0.03, respectively. Although the total adenylate pool in the lyophilized nodules was only 41% of that measured in hydrated tissues, the AEC values were similar, and the lyophilized nodules were assumed to provide useful material for assessing adenylate distribution. The nodule cortex contained 4.4% of whole-nodule adenylates, with 95.6% being located in the central zone. Aqueous fractionation of bacteroids from the plant fraction of whole nodules and the use of marker enzymes or compounds to correct for recovery of bacteroids and cross-contamination of the bacteroid and plant fractions resulted in estimates that 36.2% of the total adenylate pool was in bacteroids, and 59.4% was in the plant fraction of the central zone. These are the first quantitative assessments of adenylate distribution in the plant and bacteroid fractions of legume nodules. These estimates were combined with theoretical calculations of rates of ATP consumption in the cortex (9.5 nmol g-1 fresh weight of nodule s-1), plant central zone (38 nmol g-1 fresh weight of nodule s-1), and bacteroids (62 nmol g-1 fresh weight of nodule s-1) of soybean nodules to estimate the time constants for turnover of the total adenylate pool and the ATP pool within each nodule fraction. The low values for time constant (1.6-5.8 s for total adenylate, 0.9-2.5 s for ATP only) in each fraction reflect the high metabolic activity of soybean nodules and provide a background for further studies of the role of adenylates in O2-limited nodule metabolism.

Polyamine metabolism in nodules of Vigna mungo during senescence

The polyamine (PA) concentration was greatest in the nodules at stage 2 (flowering) in Vigna mungo. The amount of putrescine (Put) was the highest and changed significantly with senescence, whereas the spermine (Spm) level was the least. During senescence, both arginine decarboxylase (ADC, EC 4.1.1.19) and ornithine decarboxylase (ODC, EC 4.1.1.17) were positively correlated with the endogenous PA titre, particularly in the late stages of development, while the catabolic enzyme, diamine oxidase (DAO, EC 1.4.3.6) increased with senescence, being highest (five-fold) in nodules at stage 5 (i.e. mature pod stage). In vivo labelling studies using l-[U- 14C]arginine with difluoromethylarginine (DFMA) and difluoromethylornithine (DFMO) indicated that both Put biosynthetic enzymes are involved during the process of senescence. Moreover, Spm promoted nitrogenase activity in vivo.

Control of nitrogen and carbon metabolism in root nodules

Because legume root nodules have high rates of carbon and nitrogen metabolism, they are ideal for the study of plant physiology, biochemistry and molecular biology. Many plant enzymes involved in carbon and nitrogen assimilation have enhanced activity and enzyme protein in nodules as compared to other plant organs. For all intents and purposes the interior of the root nodule is O2 limited. Both plant and bacterial components of effective root nodules have unique adaptive features for maximizing carbon and nitrogen metabolism in an O2‐limited environment. Plant glycolysis appears to be shunted to malic acid synthesis with further reductive synthesis to fumarate and succinate. Nodule bacteroids utilize these organic acids for the energy to fuel nitrogenase activity. Activities of the plant enzymes phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), malate dehydrogenase (MDH, EC 1.1.1.37) and aspartate aminotransferase (AAT, EC 2.6.1.1), which are very high in nodules, may mediate the flux of carbon between organic and amino acid pools. Dark CO2 fixation via nodule PEPC can provide up to 25% of the carbon needed for malate and aspartate synthesis. At least three of the plant proteins showing enhanced expression in root nodules are O2 regulated. Isolation of alfalfa cDNAs encoding PEPC, AAT, NADH‐glutamate synthase (NADH‐GOGAT, EC 1.4.1.14) and aldolase (EC 4.1.2.13) will offer new tools to assess molecular events controlling nodule carbon and nitrogen metabolism.

A metabolic connection between nitrogenase activity and the synthesis of ureides in nodulated soybean

Application of allopurinol (AP; 1H‐pyrazolo‐[3,5‐d]pyrimidine‐4‐o1) to intact nodulated roots of ureide‐forming legumes causes rapid inhibition of NAD:xanthine dehydrogenase (XDH: EC 1.2.1.37), cessation of ureide synthesis and, subsequently, severe nitrogen deficiency (Atkins et al. 1988. Plant Physiology 88: 1229–1234). Nitrogen deficiency is a result of inhibited nitrogenase (EC 1.7.99.2) activity. Using an open gas exchange system to measure H2 and CO2 evolution, short term effects of AP application were examined in a Hup− soybean symbiosis [Glycine max (L.) Merr. cv. Harosoy: USDA 16]. The onset of inhibition of nitrogenase was detected after ca 2 h exposure of the roots to AP. At the same time xanthine began to accumulate and ureide levels declined in nodules as a result of inhibition of XDH. The decline in H2 evolution following AP application was not due to altered electron allocation between N2 and H+ by nitrogenease but was coincident with increased gaseous diffusive resistance of nodules and a decline in intracellular oxygen concentration. A possible scheme for the intermediary metabolism of soybean nodules which might account for a direct connection between nitrogenase activity and ureide synthesis is proposed. The suggested mechanism envisages coupling production of reducing power by cytosolic enzymes of purine oxidation to synthesis of dicarboxylic acid substrates (malate and succinate) required for bacteroid respiration.

The effect of excision on O2 diffusion and metabolism in soybean nodules

Nitrogen‐fixing nodules of soybean [Glycine max (L.) Merr. cv. Maple Arrow inoculated with Bradyrhizobium japonicum USDA 16] were studied before and after excision from the root to determine the role the O2 regulation plays in the inhibition of nodule activity and the potential for using excised nodules nodules in studies of nodule metabolism. Relative nitrogenase (EC 1.7.99.2) activity (H2 evolution in N2:O2) and nodule respiration (CO2 evolution) were monitored first in intact nodulated roots and then in freshly excised nodules of the same plant to determine the time course of the decline in nodule metabolism. Folowing excision, nitrogenase activity and respiration declined rapidly in the first minute and then more gradually. After 40 min the rate of H2 evolution was only 14–28% of that in the intact plant. In some nodules activity declined steadily, and in others there was a partial recovery in activity ca 10 min after detachment. Infected cell O2 concentration (Oi), measured by a spectro‐photometric technique, also declined after nodule detachment with a time course similar to the declines in nitrogenase activity and respiration. Following excision, Oi levels declined rapidly from ca 21 nM in attached nodules to 8–12 nM at 4–10 min after excision and then more gradually to 2–3 nM O2 at 30–40 min after excision. These results show that the nodules' permeability to gas diffusion continued to be regulated for up to 40 min after detachement. At 40 min after detachment, when excised nodules displayed steady‐state rates of gas exchange, linear increases in pO2 from 20 to 100% at 4% min−1 resulted in recoveries of H2 and CO2 evolution, indicating that Oi limited nitrogenase activity durig this period, and that energy reserves were greatly in excess of the O2 available for respiration. When detached nodules were equilibrated for 12 h at 20, 30 and 50% O2, Oi values measured at supra‐ambient pO2 were greater than those at 20% O2 and were linked with a more rapid decline in nitrogenase activity. Also, increases in external pO2 (Oc) failed to stimulate nodule metabolism, suggesting that the nodules' energy reserves were no longer greatly in excess of their respiratory demands. It was concluded that soybean nodules could provide useful material for steady‐state studies of nodule metabolism between 40 and 240 min after detachment, but to attain metabolic rates equivalent to in vivo rates the nodules must be exposed to above‐ambient pO2.

The effect of excision on O2 diffusion and metabolism in soybean nodules

The effect of excision on O2 diffusion and metabolism in soybean nodules

Proline Accumulation, Nitrogenase (C2H2reducing) Activity and Activities of Enzymes related to Proline Metabolism in Drought-Stressed Soybean Nodules

We examined the effect of drought stress on proline accumulation, nitrogenase activity and activities of enzymes related to proline metabolism in soybean (Glycine max [L.] Merr.) nodules. Nitrogenase (C2H2 reducing) activity was inhibited 90% or more as a result of drought stress. This inhibition was substantially reversed after a 4 h recovery period. Pyrroline-5-carboxylate reductase activity in extracts of drought-stressed nodules from 25-d-old plants was 55% higher than in unstressed nodules, but the same activity in preparations from 55-d-old plants was similar to that of control plants. Extracts of recovering nodules on plants of both ages had activities near those of controls. Drought stress increased the activity of the pentose phosphate pathway by about 65% in extracts of nodules from 55-d-old plants, but there was no effect in extracts of nodules from younger plants (25-d-old). Proline dehydrogenase activity was 3.7 and 1.6 times higher in bacteroids isolated from nodules taken from 25- and 55-d-old stressed plants, respectively, than in comparable control bacteroids. This activity remained high in bacteroids from both sets of recovering nodules. The amount of proline in extracts from stressed nodules was 3- to 4-fold higher than in unstressed nodules, despite increased proline dehydrogenase activity and remained high in nodules collected 4 h after rewatering. This increase was observed in both cytoplasmic and bacteroid fractions. The possible physiological significance of these results is discussed.

Vascular transport and soybean nodule function. III: Implications of a continual phloem supply of carbon and water

Abstract. In soybean, stores of carbon within the leaf have been demonstrated to support nodule metabolism under both photosynthetic and non‐photosynthetic conditions. Indeed, a net depletion of nodule starch is observed only under conditions of suboptimal rates of nodule metabolism. Therefore, maximal rates of nodule metabolism are associated with a continual supply of phloem sap to the nodule, delivering water, carbon and other solutes. A restriction of phloem supply to the nodule may result in changes in turgor between the apoplast of the export pathway and the symplast of the nodule. This change may cause the observed decrease in the permeability to gases and to the rate of product export from nodules deprived of a phloem supply. It is suggested that nodule metabolism is homeostatically regulated in terms of internal O2 levels by the delivery of phloem water and solutes.

Metabolite Regulation of Partially Purified Soybean Nodule Phosphoenolpyruvate Carboxylase

Phosphoenolpyruvate carboxylase (PEPC) was purified 40-fold from soybean (Glycine max L. Merr.) nodules to a specific activity of 5.2 units per milligram per protein and an estimated purity of 28%. Native and subunit molecular masses were determined to be 440 and 100 kilodaltons, respectively, indicating that the enzyme is a homotetramer. The response of enzyme activity to phosphoenolpyruvate (PEP) concentration and to various effectors was influenced by assay pH and glycerol addition to the assay. At pH 7 in the absence of glycerol, the K(m) (PEP) was about twofold greater than at pH 7 in the presence of glycerol or at pH 8. At pH 7 or pH 8 the K(m) (MgPEP) was found to be significantly lower than the respective K(m) (PEP) values. Glucose-6-phosphate, fructose-6-phosphate, glucose-1-phosphate, and dihydroxyacetone phosphate activated PEPC at pH 7 in the absence of glycerol, but had no effect under the other assay conditions. Malate, aspartate, glutamate, citrate, and 2-oxoglutarate were potent inhibitors of PEPC at pH 7 in the absence of glycerol, but their effectiveness was decreased by raising the pH to 8 and/or by adding glycerol. In contrast, 3-phosphoglycerate and 2-phosphoglycerate were less effective inhibitors at pH 7 in the absence of glycerol than under the other assay conditions. Inorganic phosphate (up to 20 millimolar) was an activator at pH 7 in the absence of glycerol but an inhibitor under the other assay conditions. The possible significance of metabolite regulation of PEPC is discussed in relation to the proposed functions of this enzyme in legume nodule metabolism.

Nitrate metabolism in roots and nodules ofVicia fabain response to exogenous nitrate

Activities of nitrate reduction enzymes, nitrate reductase activity (NRA) and nitrite reductase activity (NiRA) from roots and nodules of 5 mutant genotypes and one commercial cultivar (Alameda) of faba bean (Vicia fabaL. var. minor) grown in the presence of N2alone or with additional NO−3in the medium have been studied. A naturally occurring mutant (VFM109) with impaired ability to reduce nitrate in its nodules is described. All the other cultivars ofV. fabashowed nodule NRA, although the range was very wide, from almost negligible (VFM72) up to 2 μmol h−1(g FW)−1. This activity was entirely of plant origin. Root NRA also ranged widely accross cultivars. However, the level of activity expressed as well as the response of NRA to nitrate followed a pattern opposite to that observed in nodules. Roots and nodules of all cultivars showed very high rates of NiRA, respectively 50 and 150‐fold higher than NRA, thus precluding accumulation of nitrite in these tissues. Root enzymes were significantly stimulated by nitrate while negative (NRA) or little effect (NiRA) was found for nodules. Nitrate and nitrite reduction are carried out by inducible enzymes in roots ofV. fabaand by constitutive enzymes in nodules, indicating that there may be different forms of these enzymes in each tissue. Differences in the plant genotype were a major cause of the variability in nitrate and nitrite reduction by nodulated root systems ofV. faba.