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.     

Two indirect methods for detecting ureide synthesis by nodulated legumes

Two methods were developed for the detection of altered ureide metabolism in legume nodules. Both techniques are based on the positive correlation between the presence of high xanthine dehydrogenase (EC 1.2.1.37) specific activity in nodules and the ability of those nodules to produce the ureides, allantoin and allantoic acid. In the first method, nodulated legumes are treated for 2 weeks with a soil drench of allopurinol. After allopurinol treatment, leaves of N₂-fed, ureide-producing legumes, soybean, cowpea, and lima bean, became very chlorotic. Leaves of KNO₃⁻ or NH₄Cl-fed ureide-producing legumes were unaffected by the allopurinol treatment. Leaves of the amide-producing legumes, alfalfa, clover, peak, and lupin, were unaffected by the allopurinol treatment with N₂, KNO₃, or NH₄Cl as nitrogen source. These experiments showed that long-term allopurinol treatments are useful in differentiating between ureide- and amide-producing legumes when effectively nodulated. A second method was developed for the rapid, qualitative estimation of xanthine dehydrogenase activity in legume nodules. This method utilizes pterin, an alternate substrate for xanthine dehydrogenase. Xanthine dehydrogenase hydroxylates pterin in the presence of NAD⁺ to produce isoxanthopterin. When exposed to long wave ultraviolet light (365 nanometers), isoxanthopterin emits blue fluorescence. When nodules of ureide-producing legumes were sliced in half and placed in microtiter plate wells containing NAD⁺ and pterin, isoxanthopterin was observed after 6 hours of incubation at room temperature. Allopurinol prevented isoxanthopterin production. When slices of amide-producing legume nodules were placed in wells with pterin and NAD⁺, no blue fluorescence was observed. The production of NADH by xanthine dehydrogenase does not interfere with the fluorescence of isoxanthopterin. These observations agree with the high specific activity of xanthine dehydrogenase in nodules of ureide-producing legumes and the low activity measured in amide-producing nodules. The wild soybean, Glycine soja Sieb. and Zucc., was examined for ureide synthesis. Stems of wild soybean plants had a high ureide abundance with N₂ as sole nitrogen source when nodulated with either Rhizobium fredii or Bradyrhizobium japonicum. Ureide abundance declined when nitrate or ammonium was added to the nutrient solution. Nodule slices of these plants produced isoxanthopterin when incubated with pterin. Nodule crude extracts of G. soja had high levels of xanthine dehydrogenase activity. Both Glycine max and G. soja plants were found to produce ureides when plants were inoculated with fast-growing R. fredii. The two methods described here can be used to discriminate ureide producers from amide producers as well as detect nitrogen-fixing legumes which have altered ureide metabolism. A nodulated legume that lacks xanthine dehydrogenase activity as demonstrated by the pterin assay cannot produce ureides since ureide synthesis has been shown to require xanthine dehydrogenase activity both in vivo and in vitro. A nodulated legume that remains green during allopurinol treatment also lacks ureide synthesis since the leaves of ureide-producing legumes are very chlorotic following allopurinol treatment.     

Pathways of Nitrogen Assimilation in Cowpea Nodules Studied using N(2) and Allopurinol

In the presence of 0.5 millimolar allopurinol (4-hydroxypyrazolo [3,4-d]pyrimidine), an inhibitor of NAD:xanthine oxidoreductase (EC 1.2.3.2), intact attached nodules of cowpea (Vigna unguiculata L. Walp. cv Vita 3) formed [¹⁵N]xanthine from ¹⁵N₂ at rates equivalent to those of ureide synthesis, confirming the direct assimilation of fixed nitrogen into purines. Xanthine accumulated in nodules and was exported in increasing amounts in xylem of allopurinol-treated plants. Other intermediates of purine oxidation, de novo purine synthesis, and ammonia assimilation did not increase and, over the time course of experiments (4 hours), allopurinol had no effect on nitrogenase (EC 1.7.99.2) activity. Negligible ¹⁵N-labeling of asparagine from ¹⁵N₂ was observed, suggesting that the significant pool (up to 14 micromoles per gram of nodule fresh weight) of this amide in cowpea nodules was not formed directly from fixation but may have accumulated as a consequence of phloem delivery.     

O2 regulation and O2‐limitation of nitrogenase activity in root nodules of pea and lupin

The gas exchange characteristics of intact attached nodulated roots of pea (Pisum sativum cv. Finale X) and lupin (Lupinus albus cv. Ultra) were studied under a number of environmental conditions to determine whether or not the nodules regulate resistance to oxygen diffusion. Nitrogenase activity (H₂ evolution) in both species was inhibited by an increase in rhizosphere pO₂ from 20% to 30%, but recovered within 30 min without a significant increase in nodulated root respiration (CO₂ evolution). These data suggest that the nodules possess a variable barrier to O₂ diffusion. Also, nitrogenase activity in both species declined when the roots were either exposed to an atmosphere of Ar:O₂ or when the shoots of the plants were excised. These declines could be reversed by elevating rhizosphere pO₂, indicating that the inhibition of nitrogenase activity resulted from an increase in gas diffusion resistance and consequent O₂-limitation of nitrogenase-linked respiration. These results indicate that nodules of pea and lupin regulate their internal O₂ concentration in a manner similar to nodules of soybean, despite the distinct morphological and biochemical differences that exist between the nodules of the 3 species. Experiments in which total nitrogenase activity (TNA = H₂ production in Ar:O₂) in pea and lupin nodules was monitored while rhizosphere pO₂ was increased gradually to 100%, showed that the resistance of the nodules to O₂ diffusion maintains nitrogenase activity at about 80% of its potential activity (PNA) under normal atmospheric conditions. The O₂-limitation coefficient of nitrogenase (OLC_N= TNA/PNA) declined significantly with prolonged exposure to Ar:O₂ or with shoot excision. Together, these results indicate a significant degree of O₂-limitation of nitrogenase activity in pea and lupin nodules, and that yields may be increased by realizing full potential activity.     

Nitrogen Nutrition and Xylem Transport of Nitrogen in Ureide-producing Grain Legumes

Xylem sap composition was examined in nodulated and nonnodulated cowpea (Vigna unguiculata [L.] Walp.) plants receiving a range of levels of NO₃ and in eight other ureide-forming legumes utilizing NO₃ or N₂ as sole source of nitrogen. A ¹⁵N dilution technique determined the proportions of plant nitrogen derived from N₂ in the nodulated cowpeas fed NO₃. Xylem sap composition of NO₃-fed, nodulated cowpea varied predictably with the relative extents to which N₂ and NO₃ were being utilized. The ratios of asparagine to glutamine (N/N) and of NO₃ to ureide (N/N) in xylem sap increased with increasing dependence on NO₃ whereas per cent of xylem nitrogen as ureide and the ratio of ureide plus glutamine to asparagine plus NO₃ (N/N) in xylem sap increased with increasing dependence on N₂ fixation. The amounts of NO₃ and ureides stored in leaflets, stems plus petioles, and roots of cowpea varied in a complex manner with level of NO₃ and the presence or absence of N₂ fixation. All species showed higher proportions of organic nitrogen as ureide and several-fold lower ratios of asparagine to glutamine in their xylem sap when relying on N₂ than when utilizing NO₃. In nodulated (minus nitrate) cowpea and mung bean (Vigna radiata [L.] Wilczek) the percentage of xylem nitrogen as ureide remained constant during growth but the ratio of asparagine to glutamine varied considerably. The biochemical significance of the above differences in xylem sap composition was discussed.     

Effects of Allopurinol [4-Hydroxypyrazolo(3,4-d)Pyrimidine] on the Metabolism of Allantoin in Soybean Plants

Some studies on the effects of xanthine oxidase inhibitor allopurinol [4-hydroxypyrazolo(3,4-d)pyrimidine] on allantoin metabolism of soybean plants (Glycine max cv. Tamanishiki) are reported. Soybean seedlings, aseptically germinated for 96 hours on agar containing 1 millimolar allopurinol, contained only slight amounts of allantoin, allantoic acid, and urea as compared with controls. Analysis of purines and pyrimidines of the allopurinol-treated seedlings showed marked accumulation of xanthine both in the cotyledons and seedling axes. No hypoxanthine accumulation was found. Xanthine accumulation due to allopurinol treatment was relatively low after the cotyledons had fallen. For nodulated plants, allopurinol caused a significant drop in allantoin (+allantoic acid) in the stems and nodules, accompanied by a striking accumulation of xanthine in the nodules. The xanthine concentration in the nodules far exceeded that in the germinated seedlings. Allopurinol at a concentration of 50 micromolar strongly inhibited xanthine oxidase prepared from soybean nodules.

The results suggested that the main pathway of allantoin formation in soybean plants was through purine decomposition, via xanthine-uric acid. It was specially noted that a very active purine-decomposing system existed in soybean nodules.

     

Asparagine and boric Acid cause allantoate accumulation in soybean leaves by inhibiting manganese-dependent allantoate amidohydrolase

Our previous work demonstrated substantial accumulation of allantoate in leaf tissue of nodulated soybeans (Glycine max L. Merr., cv Williams) in response to nitrogen fertilization. Research was continued to determine the effect of nitrate and asparagine on ureide assimilation in soybean leaves. Stem infusion of asparagine into ureide-transporting soybeans resulted in a significant increase in allantoate concentration in leaf tissue. Accumulation of allantoate was also observed when asparagine was supplied in the presence of allopurinol, an inhibitor of xanthine dehydrogenase in the pathway of ureide biosynthesis. In vitro, asparagine was found to have an inhibitory effect on the activity of allantoate amidohydrolase, a Mn²⁺-dependent enzyme catalyzing allantoate breakdown in soybean leaves. The inhibition was partially overcome by supplemental Mn²⁺ in enzyme assays. Another inhibitor of allantoate amidohydrolase, boric acid, applied foliarly on field-grown nodulated soybeans, caused up to a 10-fold increase in allantoate content of leaf tissue. Accumulation of allantoate in response to boric acid was either eliminated or greatly reduced in plants presprayed with Mn²⁺. We conclude that elevated levels of allantoate in leaves of ureide-transporting soybeans fertilized with ammonium nitrate result from inhibition of allantoate degradation by asparagine and that Mn²⁺ is a critical factor in this inhibition. Furthermore, our studies with asparagine and boric acid indicate that availability of Mn²⁺ has a direct effect on ureide catabolism in soybean.     

The involvement of aspartate aminotransferases in ammonium assimilation in lupin nodules

Aspartate aminotransferase (AAT) activity has been detected in the plant and bacteroid fractions of lupin nodules, and in free-living Rhizobium lupini. Two electrophoretically distinct forms of AAT were detected in the plant fraction of the nodule and a third form in the bacteroid fraction. AAT activity increased in the plant fraction during nodule development and this increase may be due to an increase in the activity of one of the AAT forms in this fraction. The single form of AAT detected in the bacteroid fraction had the same electrophoretic mobility as that detected in free-living R. lupini. The nodulated roots of lupins, grown in a media supplemented with nitrate and ammonium, had a 3- and 4-fold lower activity of AAT and nitrogenase activity respectively, compared to the nodulated roots of plants grown in the absence of added nitrogen. A role for the plant AAT in ammonium assimilation in lupin nodules is proposed.     

Nitrogen fixation of nodulation mutants of soybean as affected by nitrate

It was previously reported that three soybean (Glycine max [L.] Merr.) nodulation mutants (NOD1-3, NOD2-4, and NOD3-7) were partially tolerant to nitrate when nitrate was supplied simultaneously with inoculation at the time of transplanting. The current study evaluated the effect of short-term nitrate treatment on nitrogenase activity (C₂H₂ reduction per plant and per nodule weight) and on relative abundance of ureides when nitrate application was delayed until plants were 3 weeks old and nodules were fully developed. Nitrogenase activity of the mutants was similar to that of Williams after an initial 3-week growth period, prior to nitrate treatment. Application of 5 millimolar nitrate resulted in greater inhibition of nitrogenase activity in Williams than in the three mutants. NOD1-3 was most tolerant of nitrate among the mutants tested and showed the highest relative abundance of ureides. Although C₂H₂ reduction activity per plant for NOD1-3 was higher than for Williams in the presence of nitrate, C₂H₂ reduction activity per gram of nodules was lower for NOD1-3 than for Williams in the presence and absence of nitrate. Compared to Williams, NOD1-3 had higher nodule ureide concentration and had similar glutamine synthetase activity in nodule tissue, indicating its nodules have normal nitrogen assimilation pathways. Nitrate application resulted in ureide accumulation in nodule tissue as well as in all plant parts assayed. Unexpectedly, nitrate treatment also increased the rate of ureide degradative capacity of leaves in both NOD1-3 and Williams. The data confirmed that nitrogenase activity of the selected nodulation mutants was more, but still only partially, tolerant of nitrate compared with the Williams parent.     

Effects of Glycolate Pathway Intermediates on Glycine Decarboxylation and Serine Synthesis in Pea (Pisum sativum L.)

The study aimed to test the hypothesis that ammonia production by Rhizobium bacteroids provides not only a source of nitrogen for growth but has a central regulatory role in maintaining the metabolic activity and functional integrity of the legume nodule. Production of ammonia in intact, attached nodules was interrupted by short-term (up to 3 days) exposure of the nodulated root systems of cowpea (Vigna unguiculata L. Walp cv Vita 3: Rhizobium CB 756) and lupin (Lupinus albus L. cv Ultra: Rhizobium WU 425) to atmospheres of argon:oxygen (80:20; v/v). Treatment did not affect nodule growth, levels of plant cell and bacteroid protein, leghaemoglobin content, or nitrogenase (EC 1.7.99.2) activity (acetylene reduction) but severely reduced (by 90%) synthesis and export of the major nitrogenous solutes produced by the two symbioses (ureides in cowpea, amides in lupin). Glutamine synthetase (EC 6.3.1.2) and NAD:glutamate oxidoreductase (EC I.4.1.2) were more or less stable to Ar:O₂ treatment, but activities of the glutamine-utilizing enzymes, glutamate synthase (EC 2.6.1.53), asparagine synthetase (EC 6.3.5.4) (lupin only), and de novo purine synthesis (cowpea only), were all markedly reduced. Production and export of nitrogenous solutes by both symbioses resumed within 2 hours after transferring Ar:O₂-treated plants back to air. In each case the major exported product of fixation after transfer was initially glutamine, reflecting the relative stability of glutamine synthetase activity. Subsequently, glutamine declined and products of its assimilation became predominant consistent with resurgence of enzymes for the synthesis of asparagine in lupin and ureides in cowpea. Enzymes not directly involved with either ammonia or glutamine assimilation (purine synthesis, purine oxidation, and carbon metabolism of both bacteroids and plant cells) also showed transient changes in activity following interruption of N₂ supply. These data have been interpreted to indicate a far-reaching effect of the production of ammonia by bacteroids on a wide range of enzymes, possibly through control of protein turnover, rather than a highly specific effect of ammonia, or some product of its assimilation, on a few enzyme species.     

Effects of gradual increases in o(2) concentration on nodule activity in soybean

The objectives of this study were to determine whether attached nodules of soybean (Glycine max L. Merr.) could adjust to gradual increases in rhizosphere pO₂ without nitrogenase inhibition and to determine whether the nitrogenase activity of the nodules is limited by pO₂ under ambient conditions. A computer-controlled gas blending apparatus was used to produce linear increases (ramps) in pO₂ around attached nodulated roots of soybean plants in an open gas exchange system. Nitrogenase activity (H₂ production in N₂:O₂ and Ar:O₂) and respiration (CO₂ evolution) were monitored continuously as pO₂ was ramped from 20 to 30 kilopascals over periods of 0, 5, 10, 15, and 30 minutes. The 0, 5, and 10 minute ramps caused inhibitions of nitrogenase and respiration rates followed by recoveries of these rates to their initial values within 30 minutes. Distinct oscillations in nitrogenase activity and respiration were observed during the recovery period, and the possible basis for these oscillations is discussed. The 15 and 30 minute ramps did not inhibit nitrogenase activity, suggesting that such inhibition is not a factor in the regulation of nodule diffusion resistance. During the 30 minute ramp, a stimulation of nitrogenase activity was observed, indicating that an O₂-based limitation to nitrogenase activity occurs in soybean nodules under ambient conditions.     

Alterations in Apoplastic and Total Solute Concentrations in Soybean Nodules Resulting from Treatments Known to Affect Gas Diffusion

Ureide concentration in the cortical apoplast of soybean (Glycinemax(L.) Merr.) nodules increases rapidly in response to noduleexcision. The objective here was to determine if changes inapoplastic ureide may be related to the control of resistanceto gas diffusion which is thought to be localized in the nodulecortex. Following decapitation of shoots, nitrogenase activity(acetylene reduction) and ureide concentration in total noduleextracts declined over a period of several hours. Apoplasticureide concentration relative to total nodule ureide was elevatedunder these conditions, but the treatment effect was small comparedto non-decapitated controls. Decapitation also caused a significantdecline in the concentrations of sucrose, glucose, and D-pinitolin nodules. However, the decline in carbohydrates was similarin the nodule cortex and the nodule as a whole, suggesting thatthe carbohydrate changes are not related to a cortex-localizedmechanism. Non-invasive treatments involving increases or decreasesin oxygen concentration supplied to nodulated roots caused rapiddecreases in respiration of nodulated roots and in ureide concentrationin total nodule extracts, but did not cause major changes inapoplastic ureide concentrations. The combined results indicatethat apoplastic ureide is probably not involved in the regulationof resistance to gas diffusion. The rapid decline in noduleureide concentrations in response to changing oxygen supplydocuments the sensitivity of ureide synthesis and/or transportto alterations in nodule respiration and/or nitrogenase activity Key words: Glycine max, Pisum sativum, ureide, carbohydrates     

Nitrate Inhibition of Legume Nodule Growth and Activity : II. Short Term Studies with High Nitrate Supply

Soybean plants (Glycine max [L.] Merr) were grown in sand culture with 2 millimolar nitrate for 37 days and then supplied with 15 millimolar nitrate for 7 days. Control plants received 2 millimolar nitrate and 13 millimolar chloride and, after the 7-day treatment period, all plants were supplied with nil nitrate. The temporary treatment with high nitrate inhibited nitrogenase (acetylene reduction) activity by 80% whether or not Rhizobium japonicum bacteroids had nitrate reductase (NR) activity. The pattern of nitrite accumulation in nodules formed by NR⁺ rhizobia was inversely related to the decrease and recovery of nitrogenase activity. However, nitrite concentration in nodules formed by NR⁻ rhizobia appeared to be too low to explain the inhibition of nitrogenase. Carbohydrate composition was similar in control nodules and nodules receiving 15 millimolar nitrate suggesting that the inhibition of nitrogenase by nitrate was not related to the availability of carbohydrate.

Nodules on plants treated with 15 millimolar nitrate contained higher concentrations of amino N and, especially, ureide N than control nodules and, after withdrawal of nitrate, reduced N content of treated and control nodules returned to similar levels. The accumulation of N₂ fixation products in nodules in response to high nitrate treatment was observed with three R. japonicum strains, two NR⁺ and one NR⁻. The high nitrate treatment did not affect the allantoate/allantoin ratio or the proportion of amino N or ureide N in bacteroids (4%) and cytosol (96%).

     

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.     

Nitrogenase (C2H2) Activities of Isolated Peanut and Cowpea Bacteroids at Optimal Oxygen Availability and Comparison with Whole Nodule Activities

Bacteroids, formed by the same strain of Rhizobium, were isolatedanaerobically from peanut and cowpea root nodules and theirC₂H₂ reduction activities were measured. Measurements were startedin a pure N₂ atmosphere followed by stepwise addition of smallamounts of O₂. The procedures may have general application andare described in detail. With increasing O₂ level, a gradualincrease in nitrogenase activity was observed which reacheda peak, presumably at the optimum availability of O₂ to bacteroids,and then declined. The maximum activity attained by isolatedbacteroids of cowpea was much higher than that obtained frommeasurement of activities of intact nodules and their bacteroidcontent, whereas for peanut the two were nearly equal. The resultsindicated that intranodular conditions are probably responsiblefor the difference in nitrogenase activities of peanut and cowpeanodules rather than the unique morphological modification ofpeanut bacteroids. Key words: Root nodules, Peanut, Cowpea, Bacteroids, Nitrogenase activity     

Biochemical characterization of some pyrazolopyrimidine-based inhibitors of xanthine oxidase

Inhibition of xanthine oxidase-catalyzed conversion of xanthine to uric acid by various pyrazolopyrimidine-based inhibitors (allopurinol derivatives) was evaluated and compared with the standard inhibitor allopurinol. Three compounds out of the seven compounds used in the study were found to be reasonably good inhibitors of xanthine oxidase (XO). 4-Amino-6-mercaptopyrazolo-3,4-d-pyrimidine was found to be the most potent inhibitor of XO (IC₅₀=0.600±0.009 µM). 4-Mercapto-1H-pyrazolo-3,4-d-pyrimidine (IC₅₀=1.326±0.013 µM) and 4-amino-6-hydroxypyrazolo-3,4-d-pyrimidine (IC₅₀=1.564±0.065 µM) also showed inhibitory activity comparable to that of allopurinol (IC₅₀ = 0.776 ± 0.012 µM). All three compounds showed competitive type of inhibition with comparable K_i values. Induction of the electron transfer reaction catalyzed by XO in the presence of these compounds monitored as reduction of 2,6-dichlorophe nolindophenol (DCPIP) revealed that electron transfer by 4-amino-6-mercaptopyrazolo-3,4-d-pyrimidine is comparable to that obtained by allopurinol or xanthine. However, 4-mercapto-1H-pyrazolo-3,4-d-pyrimidine and 4-amino-6-hydroxypyrazolo-3,4-d-pyrimidine did not show DCPIP reduction. On the other hand, enzymatic reduction of cytochrome c in the presence of the three compounds was found to be insignificant and much less in comparison to allopurinol and xanthine. Therefore, both 4-amino-6-hydroxypyrazolo-3,4-d-pyrimidine and 4-mercapto-1H-pyrazolo-3,4-d-pyrimidine displayed the inhibitory property and also did not produce XO-mediated reactive oxygen species (ROS). Since 4-mercapto-1H-pyrazolo-3,4-d-pyrimidine was found to have some toxicity, the effect of 4-amino-6-hydroxypyrazolo-3,4-d-pyrimidine on the enzymatic formation of uric acid and ROS was investigated and it was found that this compound inhibited enzymatic generation of both uric acid and ROS. It can be noted that the standard inhibitor, allopurinol, inhibits uric acid formation but produces ROS.     

Nitrogenase Activity and Amounts of Nitrogenase Proteins in a Frankia-Alnus incana Symbiosis Subjected to Darkness

Effects of prolonged darkness on nitrogenase activity in vivo, nitrogenase activity in vitro, and the amounts of nitrogenase proteins were studied in symbiotic Frankia. Plants of Alnus incana (L.) Moench in symbiosis with a local source of Frankia were grown for 9 to 10 weeks in an 18/6 hour light/darkness cycle. After 12 hours of a light period, the plants were exposed to darkness for up to 40 hours. Nitrogenase activity (acetylene reduction activity) of intact plants was measured repeatedly. Frankia vesicle clusters were prepared from the nodules with an anaerobic homogenization and filtration technique and were used for measurements of in vitro nitrogenase activity and for measurements of the amounts of nitrogenase proteins on Western blots. Antisera made against dinitrogenase reductase (Fe-protein) of Rhodospirillum rubrum and against dinitrogenase (MoFe-protein) of Azotobacter vinelandii were used. Western blots were made transparent and nitrogenase proteins were quantified spectrophotometrically. Nitrogenase activity both in vivo and in vitro decreased after about 23 hours of darkness and continued to decrease to about 25% and 16% of initial activity, respectively, after 40 hours. The amount of Fe-protein and MoFe-protein in Frankia of the same plants decreased to 60% and 35%, respectively, after 40 hours of darkness. Loss of nitrogenase activity thus appeared to be largely explained by loss of MoFe-protein.     

A functional relationship between leghaemoglobin and nitrogenase based on novel measurements of the two proteins in legume root nodules

A combination of physiological and structural measurements made on nodulated cowpea and soybean plants cultured with roots in different pO(2) permitted the expression of data in various ways. Values of leghemoglobin concentration and nitrogenase activity from the two legumes were expressed conventionally either on a per plant or per gram nodule fresh weight basis, and where microscopy was done, on the basis of nitrogenase-containing, N(2)-fixing units (i.e. per bacteroid, per infected cell, or per gram infected tissue). In both legumes, acetylene reduction, N fixed and ureide content expressed on the basis of whole plants or per nitrogenase-containing units were very significantly correlated with values of leghaemoglobin concentrations expressed in a similar manner. The use of mathematical correlations in this study involving leghaemoglobin concentrations and various indices of N(2) fixation indicated a strong functional relationship between the two proteins in symbiotic legumes. These findings confirm previous suggestions that leghaemoglobin and the nitrogenase complex are two proteins closely associated with N(2)-fixing efficiency in legume root nodules.     

Intercellular nodule localization and nodule specificity of xanthine dehydrogenase in soybean

The distribution of xanthine dehydrogenase throughout the soybean plant as well as the intercellular localization of xanthine dehydrogenase within soybean nodules was determined. Polyclonal antibodies against purified xanthine dehydrogenase were prepared and used in an enzymelinked immunosorbent assay to determine whether xanthine dehydrogenase is a nodule-specific protein. This immunological assay showed that xanthine dehydrogenase is present in far greater concentration in the nodule than in any other plant organ. Immunodiffusion tests showed that anti-soybean nodule xanthine dehydrogenase would cross-react with nodule crude extracts from the ureide producers, soybean, cowpea, and lima bean, but would not cross-react with those of the amide producers, alfalfa and lupine. A crude extract from pea nodules cross-reacted slightly with anti-soybean xanthine dehydrogenase. Anti-soybean xanthine dehydrogenase did not cross-react with buttermilk xanthine oxidase either by enzyme-linked immunosorbent assay or by immunodiffusion test.

Fresh nodule sections from the ureide-producers, soybean, cowpea, and lima bean, all stained positively for xanthine dehydrogenase. The substrate-dependent stain was inhibited by allopurinol and was observed only in the infected nodule cells of these species. Nodules from the amideproducers, alfalfa and white lupine, did not stain for xanthine dehydrogenase.

     

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.