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; )     

Probable site of allantoin formation in nodulating soybean plants

Nodulated soybean plants contain high concentration of allantoin in all parts. Excision of nodules from the roots brought about a marked decrease in allantoin. To examine the function of nodules in allantoin production, nodulated and nodule-detached soybeans were fed with ¹⁵NH₃ for 1 week. High abundance of ¹⁵N was found in the amino acid-N fraction of both plants. In the root and stem of the nodulated plants, ca 80% of the nitrogen in this fraction was derived from the NH₃ added in the medium. Excess ¹⁵N was detected also in allantoin-N fraction, but the ¹⁵N content was very low in contrast to that in amino acid-N fraction. The site involved in the allantoin formation and the possible significance of its synthesis are discussed in relation to symbiotic nitrogen fixation.     

Ineffective utilization of nitrate by soybean during pod fill

The relative effectiveness of nitrate, allantoin, or nitrate plus allantoin as sources of nitrogen for the indeterminate soybean plant [ Glycine max (L.) Merr cv. Harper] was studied throughout vegetative and reproductive growth. All plants were provided with 3.0 m M nitrogen and were grown hydroponically in growth chambers. During vegetative and early reproductive growth, plants given nitrate or nitrate plus allantoin grew faster than plants provided allantoin only. However, during pod fill, plants provided with allantoin or allantoin plus nitrate gained weight more rapidly than plants receiving just nitrate. More importantly, at maturity plants that had been provided with allantoin or allantoin plus nitrate during pod fill were 30% heavier in total dry weight, 50% higher in nitrogen content, and 50% higher in seed yield than plants that had received just nitrate. At full bloom, all plants were inoculated with the same culture of Bradyrhizobium japonicum , and twice each week throughout pod fill each plant was assayed for nitrogen fixation (acetylene reduction). Correlation coefficients obtained by linear regression analysis show a strong positive correlation between the measured rate of nitrogen fixation and maximum plant fresh weight (r = 0.83), total plant nitrogen (r = 0.81), or seed yield (r = 0.76). The fact that nitrogen fixation during pod fill stimulates plant growth and seed yield, coupled with the facts that nitrate blocks nodulation and is not used efficiently during pod fill by the soybean plant, may explain why seed yield of field-grown soybeans usually does not respond to added fertilizer nitrogen. Thus, it is suggested that enhanced nitrogen fixation may be the key factor in improving soybean seed yield.     

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; )     

Distribution and change in the contents of allantoin and allantoic acid in developing nodulating and non-nodulatng soybean plants

Distribution and change in contents of allantoin¹ in each organof nodulating variety, A62-1, and non-nodulating variety, A62-2,of soybean plants were measured over the growth period, andthe physiological significance of allantoin in soybean plantsis discussed. Allantoin in the cotyledons of both varieties increased andthen decreased in the germination stage. The allantoin levelin stems, roots and nodules of A62-1 was raised with the growthand attained a maximum at the green pod stage and then decreased.On the other hand, those organs of A62-2 accumulated littleallantoin over the growth period. The allantoin level in thestems of A62-1 was the highest compared with other organs. Inthe leaves of A62-1, the level was higher in the developingleaves than lower mature leaves. The level decreased just beforethe end of leaf development and became trace in the lower fullydeveloped leaves. The allantoin level in the pods of A62-1 duringthe young stage was fairly high; whereas that of A62-2 was lowbut significant, and then decreased with maturing. The dry seedsin both varieties showed low levels. Allantoin was concluded to be accumulated in roots and stemsof developing soybean plants bearing nodules and then decreasedin the stage of seed formation. ¹ In this article the sum of allantoin and allantoic acid ismeasured. Therefore, the expression "allantoin" in the textand abstract includes allantoic acid. (Received August 19, 1976; )     

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.     

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.

     

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.     

寄主植物与温度对大豆食心虫滞育期间糖类和脂质含量的影响

【目的】本文探究了大豆食心虫Leguminirora glycinioorella(Mats.)Obraztsov进入滞育以后体内糖类、脂质和水分含量的变化,以及不同寄主植物和滞育诱导期温度对大豆食心虫体内糖类、脂质和水分含量的影响。【方法】通过蒽酮硫酸法和氯仿甲醇分离法测定了滞育前后、以大豆Glycine max(L.)Merr和野大豆Glycine soja Sieb.et Zucc.两种植物为寄主以及不同滞育诱导温度下的大豆食心虫体内脂质、水分、总糖、糖原和海藻糖的含量,结果利用SPSS17.0数据分析软件进行分析。【结果】进入滞育阶段的大豆食心虫体内脂质、总糖、海藻糖和糖原含量显著增加,含水量显著下降。以大豆为寄主的大豆食心虫在滞育阶段体内水分含量、总糖和糖原含量较野大豆寄主的高,海藻糖含量较野大豆的低,差异均显著。不同滞育诱导温度下的大豆食心虫体内生化物质含量基本一致。【结论】滞育前后大豆食心虫体内各种生化物质变化明显,以栽培大豆为寄主和以野大豆为寄主的大豆食心虫体内生化物质含量不同,而滞育诱导温度对大豆食心虫体内生化物质含量影响不大。     

Nitrogen fixation in several grain legume species with contrasting sensitivities to copper nutrition

The nitrogen fixation response to copper nutrition in faba bean, yellow lupin and soybean was studied. Copper nutrition significantly increased the pod yields of all tested grain legumes but faba bean gave the greatest Cu-use efficiency for pod and grain production. The accumulation of dry matter in vegetative parts, nodules, N and leghemoglobin concentration in nodules and nitrogen accumulation in the whole plants were increased by copper supply in faba bean and yellow lupin in contrast with soybean. Cu nutrition significantly increased the Cu concentrations in nodules of all cultivated plants. The differential sensitivity of N₂ fixation in tested grain legume species to copper nutrition could be connected with the level of phenols in nodules and depended on both the host plants and strains of rhizobia, which differ in their ability to produce catechol-like siderophores. Copper requirements by symbiotic N₂ fixation could also depend on the nature of phenols in nodules (presence of o-dihydroxyphenols or number of hydroxyls in molecule).     

Effect of soil moisture stress on legume-rhizobium symbiosis in soybeans

Summary In a pot culture experiment, the influence of soil moisture stress at different physiological stages of soybean, cv. Hark, on nodulation, symbiosis and nitrogen accumulation was studied. Moisture stress reduced leghemoglobin content of root nodules and nitrogen uptake by plants. It had no effect on number of bacteroids. Stress at mid bloom and rapid pod filling stages reduced yield and seed protein content. However, these parameters were not affected by stress at nodule initiation and early flowering stages, though, flower initiation and maturity of the plant were delayed. Moisture stress at any stage did not alter nitrogen status of roots.     

Effects of applied nitrogen upon aspects of nitrogen metabolism and transport in developing nodulated winged bean plants

Nodulated winged bean [Psophocarpus tetragonolobus (L.) DC., cv. UPS 122] were grown under constant environmental conditions and supplied with mineral nutrient solution in which nitrogen was absent or was present as nitrate (12 mg N week^-1 plant^-1). Nitrate treatment dramatically promoted plant growth, increased fruit weight 1.6 fold, was necessary for tuberisation and enhanced nodulation. The in vitro accumulation of ¹⁴C into asparagine and aspartate components of excised nodules supplied with exogenous ¹⁴CO₂ and [¹⁴C]-D-glucose was greater for nitrate-treated plants, whilst accumulation into ureides was reduced by nitrate treatment. Levels of amino acids in xylem sap were greater for plants supplied with a complete nutrient solution, than those grown without applied nitrate, particularly for asparagine, glutamine and proline. Xylem ureide levels were greater for plants grown in the absence of supplementary nitrate. Nitrogen accumulated in leaf, stem and petiole, and root nodule tissues for utilisation during fruit development; peak nitrogen levels and time of anthesis were retarded for plants grown without applied nitrate. The shoot ureide content increased during fruiting, coincident with decreases in the total nitrogen content, indicating that ureide pools are not utilised during the early reproductive phase. However ureide reserves, particularly allantoin, were utilised during the later stages of pod fill. Enzyme activity which metabolised asparagine was found throughout the plant and was identified as K⁺-dependent asparaginase (EC 3.5.1.1) and an aminotransferase. Apart from temporal differences in developmental profiles of enzyme activity, the activity of these enzymes and of allantoinase (EC 3.5.2.5) in developing tissues were similar for both treatments. The main differences were greater asparaginase and asparagine:pyruvate aminotransferase activities in root tissues and fruit of nitrate-supplied plants; allantoinase activity in the primary roots of plants grown without nitrate decreased during development, whilst activity in developing tubers (nitrate-supplied plants) increased.     

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.     

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; )     

Effect of Removal of Flower Buds, Open Flowers, Young Pods and Shoot Apex on Growth and Pod Set in Soybean

The complete removal of the reproductive structures once andshoot apices of soybeans (Glycine max L. Merrill) during earlyanthesis but before the rapid seed development stage significantlyincreased flowering and pod set in greenhouse and field grownplants. The treated plants had darker green leaves, shorterstems and petioles and retained their chlorophyll content longerthan control plants. Pod maturation was also delayed. Althoughdecapitation and the removal of reproductive structures increasedthe number of 3- or more-seeded pods in all varieties tested,seed weight per plant was not consistently increased. The possibleinvolvement of endogenous hormones in pod set and multi-loculepod production in soybeans is discussed. Key words: Glycine max (L.) Merr, Reproductive structures, Shoot apex, Growth, Flowering, Pod set, Multi-locule pods     

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.     

The timing and rate of phytic Acid accumulation in developing soybean seeds

The time-course of phosphorus (P) accumulation in the phytic acid fraction of developing soybean (Glycine max [L.] Merr. cv `Williams 79') seeds as well as the relation of phytic acid P to total P content were determined. Phytic acid was detected early in embryogenesis in field-grown soybeans and accumulated in a linear fashion throughout most of seed development. Although the observed rates of accumulation ranged from 18.7 micrograms phytic acid P per seed per day in pods positioned low on the plant to 33.6 micrograms in pods positioned high on the plant, the final concentrations were the same in all cases. Nearly all of the P translocated to developing seeds was incorporated into phytic acid from the third week after flowering until physiological maturity, with the sum of nonphytic acid P compounds remaining constant. Phytic acid accumulation was also linear throughout development when soybean plants were grown in solutions having nutrient P levels that ranged from severely limiting (2.0 milligrams P per liter) to excess (50 milligrams P per liter). However, there was a pronounced effect on rate of accumulation, which ranged from 7.2 micrograms phytic acid per seed per day with limiting nutrient P to 44.7 micrograms with excess P. The change in level of phytic acid accounted for most of the alteration in total seed P that was caused by altering the P status of the plants. These results support the view that phytic acid synthesis is involved in P homeostasis of the developing soybean seed.     

Spatial relationships between uninfected and infected cells in root nodules of soybean

In soybean (Glycine max (L.) Merr.) the uninfected cells of the root nodule are responsible for the final steps in ureide production from recently fixed nitrogen. Stereological methods and an original quantitative method were used to investigate the organization of these cells and their spatial relationships to infected cells in the central region of nodules of soybean inoculated with Rhizobium japonicum strain USDA 3I1B110 and grown with and without nitrogen (as nitrate) in the nutrient medium. The volume occupied by the uninfected tissue was 21% of the total volume of the central infected region for nodules of plants grown without nitrate, and 31% for nodules of plants grown with nitrate. Despite their low relative volume, the uninfected cells outnumbered the much larger infected cells in nodules of plants grown both without and with nitrate. The surface density of the interface between the ininfected and infected tissue in the infected region was similar for nodules in both cases also, the total range being from 24 to 26 mm²/mm³. In nodules of plants grown without nitrate, all sampled infected cells were found to be in contact with at least one uninfected cell. The study demonstrates that although the uninfected tissue in soybean nodules occupies a relatively small volume, it is organized so as to produce a large surface area for interaction with the infected tissue.     

Genotypic Difference in Molybdenum Accumulation Affects N2-fixation in Tropical Phaseolus vulgar is L.

Differences in uptake, seed storage and distribution of molybdenumwithin plants of two genotypes of Phaseolus vulgaris L. wereobserved with and without external sources of molybdenum ina glasshouse experiment. Differences in the amount of totalplant Mo translocated into seed appeared to be under genotypiccontrol, with a consistently larger proportion of total plantmolybdenum being accumulated in seed by Kabanima compared toBaseka. The uptake efficiency (total amounts accumulated withtime) did not differ between the two genotypes, but the allocationof Mo to plant parts did. Kabanima had larger nodule dry weight(30 to 40%), and nodule molybdenum content up to three timesgreater than Baseka with concurrent higher rates of N_2- fixation,particularly when plants were grown in the absence of an externalsource of Mo. Kabanima was also more efficient in translocatingMo in roots, nodules and pod walls to the seeds, particularlywhen Mo was deficient (43% of total plant Mo allocated to seedsin Kabanima compared to 31% in Baseka). Large amounts of molybdenumwere accumulated in the pod walls of Baseka plants (11 µgplant^-1) but this was not translocated to the seed.     

Spectral quality during pod development modulates soybean seed fatty acid desaturation

High-density cropping of soybeans results in considerable mutual shading. Consequently, pods mature under a range of light conditions, with those lower in the canopy exposed to drastically altered spectral quality as well as lower irradiance. The influence of spectral quality on reproductive development and seed quality was investigated in soybeans raised to physiological maturity under either broad spectrum or blue-deficient light sources. The absence of blue light had a large influence on vegetative morphology, but the timing of reproductive events was not affected. Total seed yield per plant, dry matter per seed, per cent protein and per cent oil were similar for all treatments. However, seeds harvested from plants matured under broad spectrum illumination contained high levels of oleic acid (18:1) and low linoleic acid (18:2) compared to seeds from plants grown under blue-deficient conditions. In addition to the spectral quality effect, there was a smaller effect of pod position. Seeds from pods lower in the canopy contained less 18:1 and more 18:2 than seeds that matured closer to the top of the canopy. Considering both spectral quality and pod position, the ratio of 18:1 to 18:2 varied four-fold between 0·35 and 1·43, indicative of a possible photoregulatory step in fatty acid desaturation. The spectral effects are consistent with the participation of a photomorphogenetic photoreceptor in the control of fatty acid metabolism during seed maturation and triglyceride accumulation.