Ureide Synthesis in a Cell-Free System from Cowpea (Vigna unguiculata [L.] Walp.) Nodules : STUDIES WITH O(2), pH, AND PURINE METABOLITES

The effect of O₂ and pH on the in vitro synthesis of ¹⁴C-labeled ureides from [8-¹⁴C]hypoxanthine in a cell-free system from cowpea nodules was investigated. Under conditions which suppressed uricase (EC 1.7.3.3) activity, namely low O₂ concentrations and low pH, ureide synthesis was inhibited and the ¹⁴C label incorporated into uric acid was increased. Conversely, conditions which increased uricase activity, namely high O₂ concentrations and high pH, also stimulated ureide synthesis, and the ¹⁴C label was incorporated principally into allantoin. The overall response of the system to O₂ concentration and pH indicated that the per cent distribution of total ¹⁴C label incorporated into uric acid was inversely related to that into allantoin. In the present study there was evidence that uricase (EC 1.7.3.3) controlled the in vitro rate of ureide synthesis in the cell-free system. Adenine and guanine inhibited xanthine dehydrogenase (EC 1.2.1.37) and as a consequence ureide synthesis from [8-¹⁴C]hypoxanthine was also inhibited.     

The Assimilation of Ureides in Shoot Tissues of Soybeans : 1. CHANGES IN ALLANTOINASE ACTIVITY AND UREIDE CONTENTS OF LEAVES AND FRUITS

The ureides, allantoin and allantoic acid, are major forms of N transported from nodules to shoots in soybeans (Merr.). Little is known about the occurrence, localization, or properties of the enzymes involved in the assimilation of ureides in shoot tissues. We have examined the capacity of the shoot tissues to assimilate allantoin via allantoinase (EC 3.5.2.5) during leaf and fruit development in nodulated soybeans. Specific activity of allantoinase in leaves peaked during pod formation and early seed filling. In developing fruits allantoinase activity in the seeds was 2 to 4 times that in the pods when expressed on a fresh weight or organ basis. In seeds, the embryos contained the highest specific allantoinase activity. Stems and petioles also had appreciable allantoinase activity. With development, peaks in the amounts of allantoic acid, but not allantoin, were measured in both leaves and fruits suggesting that the assimilation of allantoic acid may be a limiting factor in ureide assimilation. Highest amounts of ureides were measured in the pith and xylem of stem tissues and in developing pod walls.     

Metabolism and translocation of allantoin in ureide-producing grain legumes

Transfer of the nitrogen and carbon of allantoin to amino acids and protein of leaflets, stems and petioles, apices, peduncles, pods, and seeds of detached shoots of nodulated cowpea (Vigna unguiculata L. Walp. cv. Caloona) plants was demonstrated following supply of [2-¹⁴C], [1,3-¹⁵N]allantoin in the transpiration stream. Throughout vegetative and reproductive growth all plant organs showed significant ureolytic activity and readily metabolized [2-¹⁴C]allantoin to ¹⁴CO₂. A metabolic pathway for ureide nitrogen utilization via allantoic acid, urea, and ammonia was indicated. Levels of ureolytic activity in extracts from leaves and roots of nodulated cowpea were consistently maintained at higher levels than in non-nodulated, NO₃⁻ grown plants.

[¹⁴C]Ureides were recovered in extracts of aphids (Aphis craccivora and Macrosiphum euphorbieae) feeding at different sites on cowpea plants supplied with [2-¹⁴C]allantoin through the transpiration stream or to the upper surface of single leaflets. The data indicated that the ureides were effectively transferred from xylem or leaf mesophyll to phloem, and then translocated in phloem to fruits, apices, and roots.

     

Biosynthesis of Ureides from Purines in a Cell-free System from Nodule Extracts of Cowpea [Vigna unguiculata (L) Walp.

The synthesis of ¹⁴C-labeled xanthine/hypoxanthine, uric acid, allantoin, allantoic acid, and urea from [8-¹⁴C]guanine or [8-¹⁴C]hypoxanthine, but not from [8-¹⁴C]adenine, was demonstrated in a cell-free extract from N₂-fixing nodules of cowpea (Walp.). The ¹⁴C recovered in the acid/neutral fraction was present predominantly in uric acid and allantoin (88-97%), with less than 10% of the ¹⁴C in allantoic acid and urea. Time courses of labeling in the cell-free system suggested the sequence of synthesis from guanine to be uric acid, allantoin, and allantoic acid. Ureide synthesis was confined to soluble extracts from the bacteroid-containing tissue, was stimulated by pyridine nucleotides and intermediates of the pathways of aerobic oxidation of ureides, but was completely inhibited by allopurinol, a potent inhibitor of xanthine dehydrogenase (EC 1.2.1.37). The data indicated a purine-based pathway for ureide synthesis by cowpea nodules, and this suggestion is discussed.     

Effect of oxygen pressure on synthesis and export of nitrogenous solutes by nodules of cowpea

Nodules of cowpea plants (Vigna unguiculata (L.) Walp. cv. Vita 3 :Bradyrhizobium CB756) cultured for periods of 23 d with their root systems maintained in atmospheres containing a range of partial pressures of O₂ (pO₂; 1–80%, v/v, in N₂) formed and exported ureides (allantoin and allantoic acid) as the major products of fixation at all pO₂ tested. In sub-ambient pO₂ (1 and 2.5%) nodules contained specific activities of uricase (urate: O₂ oxidoreductase; EC 1.7.3.3) and allantoinase (allantoin hydrolyase; EC 3.5.2.5) as much as sevenfold higher than in those from air. On a cell basis, uninfected cells in nodules from 1% O₂ contained around five times the level of uricase. Except for NAD: glutamate synthase (EC 1.4.1.14), which was reduced in sub-ambient O₂, the activities of other enzymes of ureide synthesis were relatively unaffected by pO₂. Short-term effects of pO₂ on assimilation of fixed nitrogen were measured in nodules of air-grown plants exposed to subambient pO₂ (1, 2.5 or 5%, v/v in N₂) and¹⁵N₂. Despite a fall in total¹⁵N₂ fixation, ureide synthesis and export was maintained at a high level except in 1% O₂ where formation was halved. The data indicate that in addition to the structural and diffusional adaptations of cowpea nodules which allow the balance between O₂ supply and demand to be maintained over a wide range of pO₂, nodules also show evidence of biochemical adaptations which maintain and enhance normal pathways for the assimilation of fixed nitrogen. This work was supported by a grant from the Australian Research Council (to C.A.A.) and an Australian Development Assistance Bureau postgraduate fellowship (to F.D.D.).     

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.     

Purine synthesis and catabolism in soybean seedlings : the biogenesis of ureides

The ureides, allantoin and allantoic acid, are the major nitrogenous substances transported within the xylem of N₂-fixing soybeans (Glycine max L. Merr. cv Amsoy 71). The ureides accumulated in the cotyledons, roots and shoots of soybean seedlings inoculated with Rhizobium or grown in the presence of 10 millimolar nitrate. The patterns of activity for uricase and allantoinase, enzymes involved in ureide synthesis, were positively correlated with the accumulation of ureides in the roots and cotyledons. Allopurinol and azaserine inhibited ureide production in 3-day-old cotyledons while no inhibition was observed in the roots. Incubation of 4-day-old seedlings with [¹⁴C]serine indicated that in the cotyledons ureides arose via de novo synthesis of purines. The source of ureides in both 3- and 4-day-old roots was probably the cotyledons. The inhibition of ureide accumulation by allopurinol but not azaserine in 8-day-old cotyledons suggested that ureides in these older cotyledons arose via nucleotide breakdown. Incubation of 8-day-old plants with [¹⁴C]serine suggested that the roots had acquired the capability to synthesize ureides via de novo synthesis of purines. These data indicate that both de novo purine synthesis and nucleotide breakdown are involved in the production of ureides in young soybean seedlings.     

Metabolism and translocation of fixed nitrogen in the nodulated legume

Summary Nitrogen (N₂) fixed by Rhizobium bacteroids in the legume nodule is excreted as ammonia to the surrounding host cell where it is efficiently assimilated into the amide group of glutamine. Generally glutamine is a minor exported solute of nitrogen, being further metabolised to asparagine in temperate species and to the ureides, allantoin and allantoic acid in tropical species. These solutes serve as the principal translocated forms of nitrogen in xylem. Compartmentalisation of the pathways of nitrogen metabolism and the role of ammonia in regulation of their activity is examined in nodules of both asparagine-forming (Lupinus albus L.) and ureide-forming (Vigna unguiculata L. Walp) symbioses.     

Enzymes of ureide synthesis in pea and soybean

Soybean (Glycine max) and pea (Pisum sativum) differ in the transport of fixed nitrogen from nodules to shoots. The dominant nitrogen transport compounds for soybean are ureides, while amides dominate in pea. A possible enzymic basis for this difference was examined.

The level of enzymes involved in the formation of the ureides allantoin and allantoic acid from inosine 5′-monophosphate (IMP) was compared in different tissues of pea and soybean. Two enzymes, 5′-nucleotidase and uricase, from soybean nodules were found to be 50- and 25-fold higher, respectively, than the level found in pea nodules. Other purine catabolizing enzymes (purine nucleosidase, xanthine dehydrogenase, and allantoinase) were found to be at the same level in the two species. From comparison of enzyme activities in nodules with those from roots, stems, and leaves, two enzymes were found to be nodule specific, namely uricase and xanthine dehydrogenase. The level of enzymes found in the bacteroids indicated no significant contribution of Rhizobium japonicum purine catabolism in the overall formation of ureides in the soybean nodule. The presence in the nodules of purine nucleosidase and ribokinase activities makes a recirculation of the ribose moiety possible. In concert with phosphoribosylpyrophosphate synthetase, ribose becomes available for a new round of purine de novo synthesis, and thereby ureide formation.

     

Nitrogen nutrition and the development of biochemical functions associated with nitrogen fixation and ammonia assimilation of nodules on cowpea seedlings

During early development (up to 18 d after sowing) of nodules of an effective cowpea symbiosis (Vigna unguiculata (L.) Walp cv. Vita 3: Rhizobium strain CB756), rapidly increasing nitrogenase (EC 1.7.99.2) activity and leghaemoglobin content were accompanied by rapid increases in activities of glutamine synthetase (EC 6.3.1.2), glutamate synthase (EC 2.6.1.53), enzymes of denovo purine synthesis (forming inosine monophosphate) xanthine oxidoreductase (EC 1.2.3.2), urate oxidase (EC 1.7.3.3), phosphoenolpyruvate carboxylase (EC 4.1.1.31) and led to increased export of ureides (allantoin and allantoic acid) to the shoot of the host plant in the xylem. Culturing plants with the nodulated root systems maintained in the absence of N₂ (in 80 Ar: 20 O₂, v/v) had little effect on the rates of induction and increase in nitrogenase activity and leghaemoglobin content but, in the absence of N₂ fixation and consequent ammonia production by bacteroids, there was no stimulation of activity of enzymes of ammonia assimilation or of the synthesis of purines or ureides. Addition of NO ₃ ^- (0.1–0.2 mM) relieved host-plant nitrogen deficiency caused by the Ar: O₂ treatment but failed to increase levels of enzymes of N metabolism in either the bacteroid or the plant-cell fractions of the nodule. Premature senescence in Ar: O₂-grown nodules occurred at 18–20 d after sowing, and resulted in reduced levels of nitrogenase activity and leghaemoglobin but increased the activity of hydroxybutyrate oxidoreductase (EC 1.1.1.30).     

Enzymes of Purine Biosynthesis and Catabolism in Glycine max: I. COMPARISON OF ACTIVITIES WITH N(2) FIXATION AND COMPOSITION OF XYLEM EXUDATE DURING NODULE DEVELOPMENT

During the period examined from 12 to 63 days after planting, the ureides, allantoin and allantoic acid, were the predominant nitrogenous solutes in the xylem exudate of soybeans (Glycine max [L.]) growing solely on symbiotically fixed nitrogen, accounting for approximately 60% and greater than 95% of the total nitrogen in the xylem exudate before and after the onset of active nitrogen fixation, respectively. For plants between 18 and 49 days of age, the apparent rate of ureide export estimated from concentrations of ureides in xylem exudate collected over a period of one hour was closely related to the rate of nitrogen fixation estimated from measurements of C₂H₂ reduction by nodulated root systems. After this time, the apparent rate of ureide export per plant continued to increase, reaching a maximum value at day 63 of 12 micromoles per plant per hour, even though the rate of C₂H₂ reduction per plant declined approximately four-fold. The most probable pathway for the biosynthesis of ureides involves the catabolism of purines. The levels of phosphoribosylpyrophosphate (PRPP) synthetase, which catalyzes the formation of the PRPP required for purine synthesis, increased in parallel with the rates of nitrogen fixation (C₂H₂) from day 18 reaching a maximum value of 13.9 micromoles per plant per hour at day 49, and then both activities declined rapidly. During the period of active nitrogen fixation the ratio of PRPP synthesis estimated from measurements of PRPP synthetase activity in cell-free extracts to the apparent rate of ureide export was between 1 and 2. The activities of the enzymes of purine catabolism, xanthine dehydrogenase, uricase, and allantoinase, increased in parallel with the increases in nodule mass and the export of ureides with maximum activities of 13, 119, and 79 micromoles per plant per hour, corresponding with apparent rates of ureide export in the range of 9.5 to 11.9 micromoles per plant per hour. These results demonstrate that there is a close association between nitrogen fixation, PRPP synthetase activity, and ureide export in soybeans and support the proposal that recently-fixed nitrogen is utilized in the de novo synthesis of purines which are subsequently catabolized to produce the ureides.     

Appearance of purine-catabolizing enzymes in fix and fix root nodules on soybean and effect of oxygen on the expression of the enzymes in callus tissue

The appearance of enzymes involved in the formation of ureides, allantoin, and allantoic acid, from inosine 5′-monophosphate was analyzed in developing root nodules of soybean (Glycine max). Concomitant with development of effective nodules, a substantial increase in specific activities of the enzymes 5′-nucleotidase (35-fold), purine nucleosidase (10-fold), xanthine dehydrogenase (25-fold), and uricase (200-fold), over root levels was observed. The specific activity of allantoinase remained constant during nodule development. With ineffective nodules the activities were generally lower than in effective nodules; however, the activities of 5′-nucleotidase and allantoinase were 2-fold higher in ineffective nodules unable to synthesize leghemoglobin than in effective nodules. Since the expression of uricase has been shown to be regulated by oxygen (K Larsen, BU Jochimsen 1986 EMBO J 5: 15-19), the expression of the remaining enzymes in the purine catabolic pathway were tested in response to variations in O₂ concentration in sterile soybean callus tissue. Purine nucleosidase responded to this treatment, exhibiting a 4-fold increase in activity around 2% O₂. 5′-Nucleotidase, xanthine dehydrogenase, and allantoinase remained unaffected by variations in the O₂ concentration. Hence, the expression of two enzymes involved in ureide formation, purine nucleosidase and uricase, has been demonstrated to be influenced by O₂ concentration.     

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.     

Transport of nitrogen in the xylem of soybean plants

Experiments were conducted to characterize the distribution of N compounds in the xylem sap of nodulated and nonnodulated soybean plants through development and to determine the effects of exogenous N on the distribution of N compounds in the xylem. Xylem sap was collected from nodulated and nonnodulated greenhouse-grown soybean plants (Glycine max [L.] Merr. “Ransom”) from the vegetative phase to the pod-filling phase. The sum of the nitrogen in the amino acid, nitrate, ureide (allantoic acid and allantoin), and ammonium fractions of the sap from both types of plants agreed closely with total N as assayed by a Kjeldahl technique. Sap from nodulated plants supplied with N-free nutrient solution contained seasonal averages of 78 and 20% of the total N as ureide-N and amino acid-N, respectively. Sap from nonnodulated plants supplied with a 20 millimolar KNO₃ nutrient solution contained seasonal averages of 6, 36, and 58% of total N as ureide-N, amino acid-N, and nitrate-N, respectively. Allantoic acid was the predominant ureide in the xylem sap and asparagine was the predominant amino acid. When well nodulated plants were supplied with 20 millimolar KNO₃, beginning at 65 days, C₂H₂ reduction (N₂ fixation) decreased relative to nontreated plants and there was a concomitant decrease in the ureide content of the sap. A positive correlation (r = 0.89) was found between the ureide levels in xylem sap and nodule dry weights when either exogenous nitrate-N or urea-N was supplied at 10 and 20 millimolar concentrations to inoculated plants. The results demonstrate that ureides play a dominant role in N transport in nodulated soybeans and that the synthesis of ureides is largely dependent upon nodulation and N₂ fixation.     

Effect of nitrogen source on ureides in soybean

In field-grown soybeans (Glycine max L. Merr. cv Harosoy), the percentage of N in the xylem as ureides increased with increasing N₂ fixation. During a 9-week collection period, the ureide content varied from 9.0 to 69.2% of the xylary N. Between 9 and 11 weeks (early pod fill), there was a good correlation (r = 0.93) between C₂H₂ reduction and the per cent N in xylem as ureides. The per cent N as ureides, however, does not always indicate the reliance of the plant on symbiotic N₂ fixation. This ureide content also depended on the level of NO₃⁻ available to the roots. Non-nodulated soybeans given from 0 to 200 kilogram N per hectare produced xylem sap which averaged from 31.8% to 9.0% N, respectively, in the xylem as ureides over the 9-week period.

Feeding of ¹⁵N₂, ¹⁵NH₄, or ¹⁵NO₃ to greenhouse-grown soybeans indicated substantial differences in the initial distribution of N by the xylem stream, but the ultimate distribution of N between plant parts and grain did not vary with available N or percentage of xylary N as ureides. Amino acids, not ureides, were the major source of N in the phloem. The soybeans maintained a similar composition in phloem irrespective of the xylem sap constituents, with N derived from N₂, NH₄, or NO₃ being equally accessible to the phloem stream.

     

Effect of NO3 on N2 fixation and nitrogenous solutes of xylem in two nodulated West African geocarpic legumes,Kersting's bean (Macrotyloma geocarpum L.) and Bambara groundnut (Vigna subterranea L.)

Nodulation, nitrogen (N₂) fixation and xylem sap composition were examined in sand cultured plants of Bambara groundnut (Vigna subterranea L.) and Kersting's bean (Macrotyloma geocarpum L.) inoculated with Bradyrhizobium strain CB756 and supplied via the roots for a 4 week period from the third week onwards with different levels of (¹⁵N)-nitrate (0–15 mM). The separate contributions of nitrate and N₂ to plant nitrogen were measured by isotope dilution. Increasing levels of nitrate inhibited nodule growth (measured as dry matter or nodule N) of both species parallel with decreased dependence on symbiotically-fixed N. Specific nodule activity (N₂ fixed g nodule dry⁻¹ d⁻¹ of nodules) was reduced progressively with time in V. subterranea at higher (5 or 15 mM) levels of NO₃, but this was not so for M. geocarpum. Root xylem bleeding sap of both species showed ureides (allantoin and allantoic acid) as predominant (>90%) solutes of nitrogen when plants were relying solely on atmospheric N. Levels of ureide and glutamine decreased and those of asparagine and nitrate in xylem increased with increasing level of applied nitrate. Relative levels of xylem ureide-N were positively correlated (R²=0.842 for M. geocarpum and 0.556 for V. subterranea), and the ratio of asparagine to glutamine in xylem exudate negatively correlated (R²=0.955 for M. geocarpum and 0.736 for V. subterranea) with plant reliance on nitrogen fixation. The data indicate that xylem sap analyses might be useful for indirect field assays of nitrogen fixation by the species and that Kersting's bean might offer some potential as a symbiosis in which N₂ fixation is relatively tolerant of soil N.     

Nitrogen Fixation and Allantoin Formation in Soybean Plants

The activity of nitrogenase and the concentration of ammonia and allantoin (+ allantoic acid) in root nodules were measured throughout the growth period of soybean plants. Nitrogenase activity measured by acetylene reduction increased with plant growth and reached a maximum level at the flowering period. The level of ammonia and allantoin concentration in nodules was parallel with increased nitrogenase activity. At the late reproductive stage (pod-forming period), nitrogenase activity showed a marked decrease, but the ammonia and allantoin in the nodules remained at a constant level. Detached nodules from 56 day-old soybean plants were exposed to ¹⁵N₂ gas, and the distribution of ¹⁵N among nitrogen compounds was investigated. Enrichment of ¹⁵N in allantoin and allantoic acid reached a fairly high level after 90 min of nitrogen fixation; ca. 22% of ¹⁵N in acid-soluble nitrogen compounds was incorporated into allantoin + allantoic acid. In contrast, enrichment of ¹⁵N in amide nitrogen was relatively low. No significant ¹⁵N was detected in the RNA fraction. The data suggested that ureide formation in nitrogen-fixing root nodules did not take place through the breakdown of nucleic acids, but directly associated with the assimilating system of biologically fixed nitrogen.     

Translocation of nitrogen and expression of nodule-specific uricase (nodulin-35) in Robinia pseudoacacia

Uricase (urate oxidase, EC 1.7.3.3) activity and nodule-specific uricase II (nodulin-35) were detected in the nodules from a number of legume: Rhizobium symbioses ( Vigna unguiculata (L.) Walp., Phaseolus vulgaris L., and Kennedia coccinea Vent.) in the Phaseoleae, as well as in those of Robinia pseudoacacia L. which belongs to the tribe Robineae. Neither uricase activity nor nodulin-35 was detected in nodules from Lupinus angustifolius L., an amide-forming symbiosis of the tribe Genisteae. Nodules of R. pseudoacacia also showed high levels of allantoinase (EC 3.5.2.5) activity but activity of enzymes earlier in the pathway of ureide synthesis (xanthine dehydrogenase, EC 1.2.1.37; inosine monophosphate dehydrogenase, EC 1.2.1.14; and xanthosine nucleosidase, EC 3.2.2.1) could not be detected. Analysis of transport fluids (xylem, phloem and nodule exudates) from R. pseudoacacia found that asparagine, and, to a lesser extent, glutamine were the major translocated nitrogenous solutes. Ureides accounted for, at most, 2.6% of the N in transport fluids (tracheal xylem sap) and in nodule exudate, 0.1%. In common with nodules of the ureide-forming symbioses, those of R. pseudoacacia contained a high proportion of uninfected interstitial cells (53.7 ± 2.3%) in the central N₂-fixing tissue whereas in L. angustifolius only 2.5 ± 0.4% of cells in this tissue were uninfected. These data have been interpreted to indicate that expression of nodule-specific uricase is related to the differentiation of uninfected interstitial cells in nodules and not to the synthesis of ureides.     

Ureide biogenesis and the enzymes of ammonia assimilation and ureide biosynthesis in nitrogen fixing pigeonpea (Cajanus cajan) nodules

Allantoic acid production from IMP, XMP, inosine, xanthosine, hypoxanthine, xanthine, uric acid and allantoin was investigated by incubating each of these substrates withCajanus cajan cytosol and bacteroid fractions separately in the presence and absence of NAD+ and allopurinol. Allantoic acid synthesis by bacteroid fraction could only be observed with uric acid and allantoin as substrates. Addition of NAD⁺ or allopurinol to the reaction mixtures had no effect. However, with cytosol fraction, allantoic acid was produced by each of these substrates, with maximum rate with allantoin. With NAD⁺ or with allopurinol, allantoic acid was produced only with uric acid and allantoin as substrates. NADH production with cytosol fraction could again be observed with all the substrates. Except with uric acid and allantoin, allopurinol completely inhibited NADH formation. Regardless of the presence or absence of allopurinol, none of the substrates exhibited significant activity with bacteroid fraction. Based on the activities of glutamine synthetase, glutamate synthase, glutamate dehydrogenase, aspartate aminotransferase, asparagine synthetase, nucleotidase, nucleosidase, xanthine de-hydrogenase, uricase and allantoinase and their intracellular localisation in various nodule fractions, a probable pathway for the biogenesis of ureides in pigeonpea nodules has been proposed     

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.