Incorporation of 15N into allantoin in nodulated soybean plants supplied with 15N2

Incorporation of ¹⁵N into allantoin and allantoic acid in noduleswas higher than that in roots. This confirms that nodules produceallantoin. The ¹⁵N concentration in allantoin was slightly higherthan that in allantoic acid, suggesting that allantoin decomposedto allantoic acid. Allantoin and allantoic acid in nodules weretranslocated rapidly to roots. (Received August 25, 1976; )     

Effects of exogenous nitrogen-compounds on the concentrations of allantoin and various constituents in several organs of soybean plants

The effects of nitrogen compounds supplied to culture solutionson the concentrations of allantoin and various constituentsin several organs of soybean plants A62-1 (nodulating variety)were studied to elucidate the symbiotic relation. A62-1 plantsbearing well developed nodules accumulated a large quantityof allantoin in the upper stems, roots, developing leaves, podsand maturing seeds in the reproductive stage. However, the additionof N lowered the allantoin accumulation without changing thesoluble Kjeldahl-N concentration in any organs of the host plants.Also addition of N increased the amino-N concentration in upperstems and roots in contrast with the lack of change in developingleaves, pods and maturing seeds. The decrease of allantoin accumulationwas parallel with the weakened formation of nodules. The additionof N also scarcely affected the concentrations of reducing sugarand sugar in the upper stems. There were few differences inthe concentrations of allantoin, amino-N and soluble Kjeldahl-Namong nodules attached to the A62-1 plants grown in variousamounts of nitrate. Statistical calculations showed that the allantoin concentrationin A62-1 plants was correlated negatively with N applicationand positively with nodule weights. Significant levels of bothcorrelation coefficients were attained in the reproductive stage.Thevariation in allantoin concentration in A62-1 plants was notcorrelated with that in the sugar/soluble Kjeldahl-N ratio andthe reducing sugar/soluble Kjeldahl-N ratio. A large quantity of nitrate added to the nutrient solutionsof the A62-2 (non-nodulating variety) plants elicited only asmall amount of allantoin accumulation in the upper stems, witha consequent increase in the concentrations of amino-N and solubleKjeldahl-N and a decrease in the concentrations of sugar andreducing sugar. (Received August 25, 1976; )     

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.     

Allantoin and allantoic acid titre in the faeces and tissues of the developing larva of the moth, Orthaga exvinacea Hampson

Allantoin and allantoic acid are investigated in the faeces and tissues of the developing sixth instar larva of the moth, Orthaga exvinacea. The nitrogen excreted as allantoin and allantoic acid is compared with nitrogen excreted as uric acid and ammonia. The larva excretes 2.35–5.14 μmol/g allantoin and 0.74–1.34 μmol/g allantoic acid which account for 0.83 to 2.39% and 0.23 to 0.53%, respectively, of the excreted total nitrogen. Allantoin and allantoic acid are found to be minor nitrogenous end-products of the larva. Allantoin and allantoic acid are also present in the haemolymph and fat body of the larva in varying concentrations. The level of allantoin in the haemolymph shows a negative correlation with the allantoin concentration of faeces and fat body. The allantoin is found to be stored in the fat body at a low level. The results of the present study also indicate the coexistence of uric acid storage and uricolysis.     

Enzymes of purine catabolism in soybean plants

Remarkable formation and utilization of allantoin is observedin soybean (Glycine max variety A62-1). To study this, variousenzymes involved in purine catabolism (i.e., xanthine oxidase,uricase and allantoinase) were measured in different regionsof soybean plants during development. Uricase, which catalyzesthe direct formation of allantoin from uric acid, was studiedin detail. The activities of these three enzymes were highest in the rootnodules, indicating that the nodules are the major site of allantoinmetabolism. Radicles only showed appreciable activity about80 hr after the seeds were planted. Allantoinase activity wasdetected in all regions tested, showing that allantoin translocatedfrom the nodules can be metabolized in the roots, stem and leaves.In the nodules, xanthine oxidase was localized in the nuclearfraction, while uricase was mainly restricted to the mitochondrialfraction and allantoinase to the soluble fraction. Uricase was partially purified from the nodules and radicles,respectively. The pH optimum of enzyme from the nodules was9.5, whereas that of enzyme from the radicles was 7.0. The enzymefrom the nodules did not require a cofactor, while that fromthe radicles showed an absolute requirement for a cofactor,which was a low molecular substance easily separable from theapoprotein. Thus, the uricase in nodules differs in chemicalproperties from that in the host plant. The results are discussedin relation to change in the allantoin level in soybean tissues. (Received November 1, 1974; )     

A possible role of allantion and the influence of nodulation on its production in soybean plants

Summary To examine the influence of nodulation on the production of nitrogenous compounds, soybean plants (Glycine max var. Tamanishiki) were grown with or without N-fertilizer in the field, and the changes in amino-N and allantoin-N content in the different organs were determined throughout the growth period. In the stem allantoin-N markedly increased after the flowering period and then decreased during seed formation. Allantoin accumulated in the pod (up to 70 per cent of total alcohol soluble-N) during pod formation, while in the seed the main N-compounds were amino acids, the allantoin concentration being very low. In well-nodulated soybeans grown without N-fertilizer, allantoin content in every plant organ was always high compared to beans grown with N-fertilizer, but amino-N content was comparatively low.Another experiment, in which soybean plants were allowed to form nodules by growing on a N-free medium, and in which a fixed-N supply was then controlled by the addition of various levels of ammonium, was made in a greenhouse. When nodules were formed, the subsequent addition of high concentrations of ammonium caused the accumulation of allantoin as well as the accumulation of amides and arginine. A possible role for allantoin and some aspects of its production in soybeans are discussed. re]19760421     

Allantoin metabolism in soybean plants as influenced by grafts, a delayed inoculation with Rhizobium, and a late supply of nitrogen-compounds

Reciprocal grafts between A62-1 (nodulating variety) and A62-2(non-nodulating variety) of soybeans, delayed inoculation withRhizobium and a late supply of N-compounds to nodulated anddenodulated A62-1 plants were tested to study the regulationof allantoin production in soybeans. In the upper portions of stems of the A62-2 plants grafted ontoA62-1 plants, allantoin was accumulated in a significant quantity,but lower than the ungrafted intact A62-1 plants. The concentrationsof odier nitrogenous and sugar components were similar to thoseof the ungrafted A62-1 plants. On the other hand, in the upperstems of the A62-1 plants grafted onto A62-2 plants, littleallantoin was accumulated and the concentrations of variouscomponents were similar to those of the ungrafted intact A62-2plants. A62-1 and A62-2 plants not inoculated with Rhizobium showedapproximately the same levels of allantoin and of other componentswhen the same concentration of Ncompounds was supplied. Witha late inoculation, A62-1 plants showed a delayed accumulationof allantoin in accordance with the delayed development of nodules. When nodulated soygean plants were denodulated, the allantoinconcentration in the stems and roots rapidly decreased. Additionof ammonia, urea, or nitrate to the denodulated plants retardedthe decrease of allantoin concentration in the stems, but maintainedthe soluble Kjeldahl-N and amino-N concentrations at the samelevels as those in nodulated plants. In contrast, addition ofany one to nodulated plants did not increase the allantoin accumulation. (Received April 17, 1978; )     

PvUPS1, an allantoin transporter in nodulated roots of French bean

Nodulated legumes receive their nitrogen via nitrogen-fixing rhizobia, which exist in a symbiotic relationship with the root system. In tropical legumes like French bean (Phaseolus vulgaris) or soybean (Glycine max), most of the fixed nitrogen is used for synthesis of the ureides allantoin and allantoic acid, the major long-distance transport forms of organic nitrogen in these species. The purpose of this investigation was to identify a ureide transporter that would allow us to further characterize the mechanisms regulating ureide partitioning in legume roots. A putative allantoin transporter (PvUPS1) was isolated from nodulated roots of French bean and was functionally characterized in an allantoin transport-deficient yeast mutant showing that PvUPS1 transports allantoin but also binds its precursors xanthine and uric acid. In beans, PvUPS1 was expressed throughout the plant body, with strongest expression in nodulated roots, source leaves, pods, and seed coats. In roots, PvUPS1 expression was dependent on the status of nodulation, with highest expression in nodules and roots of nodulated plants compared with non-nodulated roots supplied with ammonium nitrate or allantoin. In situ RNA hybridization localized PvUPS1 to the nodule endodermis and the endodermis and phloem of the nodule vasculature. These results strengthen our prediction that in bean nodules, PvUPS1 is involved in delivery of allantoin to the vascular bundle and loading into the nodule phloem.     

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.

     

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.

     

Ontogenetic Changes in the Level of Ureides and Enzymes of their Metabolism in Various Plant Parts of Pigeonpea (Cajanus cajan)

Levels of allantoin and allantoic acid in shoots, roots, nodulesand leaves of pigeonpea plant, in general, followed the patternof acetylene reduction in nodules, increasing progressivelyfrom 15 days after sowing (DAS) and attaining peaks at 75 DASand 60 DAS, respectively, except in shoots where their contentsevinced maximum values at pod-setting (90 DAS). Activity ofGS in nodules and shoots reached a maximum at 60 DAS and 75DAS, respectively. However, in leaves and roots, the enzymeshowed a biphasic behaviour with peaks at days 60 and 105 inleaves and at days 75 and 105 in roots. GDH activity in nodulespeaked at 60 DAS, whereas, in leaves and roots, the maximumactivity was observed at flowering (75 DAS). Uricase was presentonly in nodules with peak activity at flowering. Allantoinaseactivity again peaked at flowering, where nodules had maximumactivity followed by leaves, roots and shoots. Urease couldbe detected in all the organs with maximum activity at 60 DASin leaves followed by roots and nodules. Except uricase, allthe enzymes reported above were also present in reproductivestructures. Compared to GS, GDH was more active both in flowerbuds and developing pods. Seeds, compared to podwalls, containedhigher activities of GDH, allantoinase and urease at day 105.Only allantoin could be detected in seeds and podwalls at day105. Key words: Cajanus cajan, Allantoin, Allantoic acid, Nitrogenase, Glutamine synthetase, Glutamate dehydrogenase, Uricase, Allantoinase, Urease, Development     

Allantoic Acid Synthesis in Soybean Root Nodule Cytosol via Xanthine Dehydrogenase

Allantoin and allantoic acid are the major forms of nitrogen transported from soybean nodules to other parts of the plant. Neither the pathway or the site of ureide synthesis has been demonstrated in root nodules.     

Soybean ureide transporters play a critical role in nodule development,function and nitrogen export

Legumes can access atmospheric nitrogen through a symbiotic relationship with nitrogen‐fixing bacteroids that reside in root nodules. In soybean, the products of fixation are the ureides allantoin and allantoic acid, which are also the dominant long‐distance transport forms of nitrogen from nodules to the shoot. Movement of nitrogen assimilates out of the nodules occurs via the nodule vasculature; however, the molecular mechanisms for ureide export and the importance of nitrogen transport processes for nodule physiology have not been resolved. Here, we demonstrate the function of two soybean proteins – GmUPS1‐1 (XP_003516366) and GmUPS1‐2 (XP_003518768) – in allantoin and allantoic acid transport out of the nodule. Localization studies revealed the presence of both transporters in the plasma membrane, and expression in nodule cortex cells and vascular endodermis. Functional analysis in soybean showed that repression of GmUPS1‐1 and GmUPS1‐2 in nodules leads to an accumulation of ureides and decreased nitrogen partitioning to roots and shoot. It was further demonstrated that nodule development, nitrogen fixation and nodule metabolism were negatively affected in RNAi UPS1 plants. Together, we conclude that export of ureides from nodules is mediated by UPS1 proteins, and that activity of the transporters is not only essential for shoot nitrogen supply but also for nodule development and function.     

Degradation and utilization of exogenous allantoin by intact soybean root

Allantoin is produced by soybean [ Glycine max (L.) Merr. cv. Harper] nodules during nitrogen fixation. Decomposed nodules, therefore, may release allantoin into the surrounding soil. If the released allantoin were to be taken up by the plant without degradation, it is possible that the exogenous allantoin might repress subsequent nodulation. Using a hydroponic growth system, degradation of exogenous allantoin by soybean root was studied. In the presence of intact soybean root exogenous allantoin was rapidly degraded, yielding ca 2 mmol of urea per mmol of allantoin. Hydrolysis of urea to ammonia proceeded very slowly. Instead, the urea seemed to be taken up by the intact soybean root. The enzyme(s) required for the production of urea from exogenous allantoin could not be detected in the aqueous rooting medium. Therefore, these enzymes seem to be attached to the exterior surface of the intact soybean root. This study shows that exogenous allantoin can be readily degraded and assimilated by the growing soybean plant.     

Allantoin and Allantoic Acid in Tissues and Stem Exudate from Field-grown Soybean Plants

Samples of stem exudate and plant tissue collected from field-grown soybean (Glycine max [L.] Merr.) plants were analyzed for allantoin and allantoic acid. Nitrogen in nitrate plus amino acids exceeded ureide N concentration in stem exudate prior to flowering. During all of reproductive development (from about 40 days after planting until maturity), ureide N concentration was two to six times greater than amino acid plus nitrate N concentration. Allantoin and allantoic acid, not asparagine, are the principal forms of nitrogen transported from nodulated roots to shoots of the soybean plant. During pod and seed development ureide N comprised as high as 2.3, 37.7, and 15.8% of total N in leaf blades, stems + petioles, and fruits, respectively. The concentration of ureide in stems and fruits declined to nearly zero at maturity.     

Immunoaffinity purification and comparison of allantoinases from soybean root nodules and cotyledons.

Allantoinase (allantoin amidohydrolase, EC 3.5.2.5) catalyzes the conversion of allantoin to allantoic acid in the final step of ureide biogenesis. We have purified allantoinase more than 4000-fold by immunoaffinity chromatography from root nodules and cotyledons of soybean (Glycine max [L] Merr.). We characterized and compared properties of the enzyme from the two sources. Seed and nodule allantoinases had 80% identity in the first 24 amino acid residues of the N terminus. Two-dimensional gel electrophoresis of the purified enzymes showed that multiple forms were present in each. Allantoinases from nodules and cotyledons had very low affinity for allantoin with a Km for allantoin of 17.3 mM in cotyledons and 24.4 mM in nodules. Both had activity in a broad range of pH values from 6.5 to 7.5. In addition, purified allantoinase from both sources was very heat stable. Enzyme activity was stable after 1 h at 70 degrees C, decreased gradually with heating to 85 degrees C, and was lost at 90 to 95 degrees C. Although these studies have revealed some differences between allantoinases in seeds and nodules, the differences were not reflected in key enzyme properties. The immunoaffinity approach enabled purification of allantoinase from soybean root nodules and simplified its purification from cotyledons, thereby allowing characterization and comparison of the enzyme from the two sources.     

Allantoin and Allantoic Acid in the Nitrogen Economy of the Cowpea (Vigna unguiculata [L.] Walp.)

The ureides, allantoin and allantoic acid, represented major fractions of the soluble nitrogen pool of nodulated plants of cowpea (Vigna unguiculata [L.] Walp. cv. Caloona) throughout vegetative and reproductive growth. Stem and petioles were the principal sites of ureide accumulation, especially in early fruiting.

Labeling studies using ¹⁴CO₂ and ¹⁵N₂ and incubation periods of 25 to 245 minutes indicated that synthesis of allantoin and allantoic acid in root nodules involved currently delivered photosynthate and recently fixed N, and that the ureides were exported from nodule to shoot via the xylem. From 60 to 80% of xylem-borne N consisted of ureides; the remainder was glutamine, asparagine, and amino acids. Allantoin predominated in the soluble N fraction of nodules and fruits, allantoin and allantoic acid were present in approximately equal proportions in xylem exudate, stems, and petioles.

Extracts of the plant tissue fraction of nitrogen-fixing cowpea nodules contained glutamate synthase (EC 2.6.1.53) and glutamine synthetase (EC 6.3.1.2), but little activity of glutamate dehydrogenase (EC 1.4.1.3). High levels of uricase (EC 1.7.3.3) and allantoinase (EC 3.5.2.5) were also detected. Allantoinase but little uricase was found in extracts of leaflets, pods, and seeds.

Balance sheets were constructed for production, storage, and utilization of ureide N during growth. Virtually all (average 92%) of the ureides exported from roots was metabolized on entering the shoot, the compounds being presumably used as N sources for protein synthesis.

     

Allantoin involved in species interactions with rice and other organisms in paddy soil

Allantoin (5-ureidohydantoin) plays an essential role in the assimilation, metabolism, transport, and storage of nitrogen in numerous higher plants, but its ecological implications are largely unknown. In this study allantoin was found in tissues of 11 rice (Oryza sativa) varieties tested, and its structure was characterised by X-ray diffraction analysis to confirm the fact that allantoin was actually obtained from the rice plants. Furthermore, the endogenous allantoin was exuded from the rice roots into the rhizosphere soils and had a great diversity of biological effects on associated weeds and microbes by soil interactions once released. However, allantoin levels in tissues or soils could not be distinguished between the allelopathic and non-allelopathic rice varieties. Field experiments showed that levels of allantoin released from rice varieties varied with their growth stages and reached the maximal levels at the stem elongation or panicle initiation to booting stages and then decreased dramatically. Allantoin could significantly stimulate the germination and growth of Echinochloa crus-galli and populations of soil bacteria and actinomycetes at selected test concentrations (30–500 μg/g), but had no effect on soil fungi. The half-life (t _1/2 ) of allantoin in autoclaved soil (20.2 ± 2.5 h, r ² = 0.95) was almost three-times longer than in non-autoclaved soil (7.3 ± 1.9 h, r ² = 0.92), indicating that rapid biodegradation or transformation of allantoin occurs in paddy soil. The results suggest that not only may allantoin play a role in the transport and storage of nitrogen in rice tissues but it may also participate in species interactions between rice and other organisms in paddy soil.     

Effect of Substrate Moisture and Potassium on Water Relations and C, N and K Distribution in Vigna Radiata

The Vigna radiata L. plants were grown in greenhouse at moisture content of sand (SMC) of 12.0 ± 0.5 %. At flower bud initiation stage, i.e. 45 - 50 d after sowing, the SMC was decreased to 3.5 ± 0.5 %, and the effects of applied potassium (0, 2.56 and 3.84 mmol dm-3) were studied. During water stress, K-fed plants maintained higher leaf water potential and relative water content (RWC) of leaves and nodules and lower osmotic potential as compared to untreated plants. The proline content was higher in nodules than in leaves showing their difference in degree of stress. A partial recovery was found after re-irrigation. When subjected to drought, carbon was accumulated in the leaves and declined in nodules and roots. K-fed plants showed higher C and N content in stem, roots and nodules than untreated plants. The content of K significantly increased in stem and nodules in K-fed plants, irrespective of SMC. Dry masses of different plant parts were also increased in K-fed plants. This revised version was published online in July 2006 with corrections to the Cover Date.