Comparison of inhibition of N2 fixation and ureide accumulation under water deficit in four common bean genotypes of contrasting drought tolerance

Background and Aims

Drought is the principal constraint on world production of legume crops. There is considerable variability among genotypes in sensitivity of nitrogen fixation to drought, which has been related to accumulation of ureides in soybean. The aim of this study was to search for genotypic differences in drought sensitivity and ureide accumulation in common bean (Phaseolus vulgaris) germplasm that may be useful in the improvement of tolerance to water deficit in common bean.

Methods

Changes in response to water deficit of nitrogen fixation rates, ureide content and the expression and activity of key enzymes for ureide metabolism were measured in four P. vulgaris genotypes differing in drought tolerance.

Key Results

A variable degree of drought-induced nitrogen fixation inhibition was found among the bean genotypes. In addition to inhibition of nitrogen fixation, there was accumulation of ureides in stems and leaves of sensitive and tolerant genotypes, although this was higher in the leaves of the most sensitive ones. In contrast, there was no accumulation of ureides in the nodules or roots of stressed plants. In addition, the level of ureides in the most sensitive genotype increased after inhibition of nitrogen fixation, suggesting that ureides originate in vegetative tissues as a response to water stress, probably mediated by the induction of allantoinase.

Conclusions

Variability of drought-induced inhibition of nitrogen fixation among the P. vulgaris genotypes was accompanied by subsequent accumulation of ureides in stems and leaves, but not in nodules. The results indicate that shoot ureide accumulation after prolonged exposure to drought could not be the cause of inhibition of nitrogen fixation, as has been suggested in soybean. Instead, ureides seem to be produced as part of a general response to stress, and therefore higher accumulation might correspond to higher sensitivity to the stressful conditions.     

Biochemical characterisation of an allantoate-degrading enzyme from French bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis>): the requirement of phenylhydrazine

In tropical legumes like French bean (Phaseolus vulgaris) or soybean (Glycine max), most of the atmospheric nitrogen fixed in nodules 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 characterise the allantoate degradation step in Phaseolus vulgaris. The degradation of allantoin, allantoate and ureidoglycolate was determined “in vivo” using small pieces of chopped seedlings. With allantoate and ureidoglycolate as substrates, the determination of the reaction products required the addition of phenylhydrazine to the assay mixture. The protein associated with the allantoate degradation has been partially purified 22-fold by ultracentrifugation and batch separation with DEAE-Sephacel. This enzyme was specific for allantoate and could not use ureidoglycolate as substrate. The activity was completely dependent on phenylhydrazine, which acts as an activator at low concentrations and decreases the affinity of the enzyme for the substrate at higher concentrations. The optimal pH for the activity of the purified protein was 7.0 and the optimal temperature was 37°C. The activity was completely inhibited by EDTA and only manganese partially restored the activity. The level of activity was lower in extracts obtained from leaves and fruits of French bean grown with nitrate than in plants actively fixing nitrogen and, therefore, relying on ureides as nitrogen supply. This is the first time that an allantoate-degrading activity has been partially purified and characterised from a plant extract. The allosteric regulation of the enzyme suggests a critical role in the regulation of ureide degradation.     

Effects of drought on nitrogen fixation in soybean root nodules

Soybean plants [Glycine max (L.) Merr.] were grown in silica sand and were drought stressed for a 4 week period during reproductive development and without any mineral N supply in order to maximize demand for fixed nitrogen. A strain of Bradyrhizobium japonicum that forms large quantities of polysaccharide in nodules was used to determine whether or not the supply of reduced carbon to bacteroids limits nitrogenase activity. A depression of 30–40% in nitrogen content in leaves and pods of stressed plants indicated a marked decline in nitrogen fixation activity during the drought period. A 50% increase in the accumulation of bacterial polysaccharide in nodules accompanied this major decrease in nitrogen fixation activity and this result indicates that the negative impact of drought on nodules was not due to a depression of carbon supply to bacteroids. The drought treatment resulted in a statistically significant increase in N concentration in leaves and pods. Because N concentration and chlorophyll concentration in leaves were not depressed, there was no evidence of nitrogen deficiency in drought‐stressed plants, and this result indicates that the negative impact of drought on nodule function was not the cause of the depression of shoot growth. At the end of the drought period, the concentration of carbohydrates, amino nitrogen, and ureides was significantly increased in nodules on drought‐stressed plants. The overall results support the view that, under drought conditions, nitrogen fixation activity in nodules was depressed because demand for fixed N to support growth was lower.     

Update on ureide degradation in legumes

Warm season N2-fixing legumes move fixed N from the nodules to the aerial portions of the plant primarily in the form of ureides, allantoin and allantoate, oxidation products of purines synthesized de novo in the nodule. Ureides are also products of purine turnover in senescing tissues, such as seedling cotyledons. A combination of biochemical and molecular approaches in both crop and model species has shed new light on the metabolic pathways involved in both the synthesis and degradation of allantoin. Improved understanding of ureide biochemistry includes two 'additional' enzymatic steps in the conversion of uric acid to allantoin in the nodule and the mechanism of allantoin and allantoate breakdown in leaf tissue. Ureide accumulation and metabolism in leaves have also been implicated in the feedback inhibition of N2-fixation under water limitation. Sensitivity to water deficit differs among soybean cultivars. Manganese supplementation has been shown to modify relative susceptibility or tolerance to this process in a cultivar-dependent manner. A discussion of the potential roles for ureides and manganese in the feedback inhibition of N2-fixation under water limitation is presented. The existing data are examined in relation to potential changes in both aerial carbon and nitrogen supply under water deficit.     

Effects of drought stress on legume symbiotic nitrogen fixation: physiological mechanisms

Drought stress is one of the major factors affecting nitrogen fixation by legume-rhizobium symbiosis. Several mechanisms have been previously reported to be involved in the physiological response of symbiotic nitrogen fixation to drought stress, i.e. carbon shortage and nodule carbon metabolism, oxygen limitation, and feedback regulation by the accumulation of N fixation products. The carbon shortage hypothesis was previously investigated by studying the combined effects of CO2 enrichment and water deficits on nodulation and N2 fixation in soybean. Under drought, in a genotype with drought tolerant N2 fixation, approximately four times the amount of 14C was allocated to nodules compared to a drought sensitive genotype. It was found that an important effect of CO2 enrichment of soybean under drought was an enhancement of photo assimilation, an increased partitioning of carbon to nodules, whose main effect was to sustain nodule growth, which helped sustain N2 rates under soil water deficits. The interaction of nodule permeability to O2 and drought stress with N2 fixation was examined in soybean nodules and led to the overall conclusion that O2 limitation seems to be involved only in the initial stages of water deficit stresses in decreasing nodule activity. The involvement of ureides in the drought response of N2 fixation was initially suspected by an increased ureide concentration in shoots and nodules under drought leading to a negative feedback response between ureides and nodule activity. Direct evidence for inhibition of nitrogenase activity by its products, ureides and amides, supported this hypothesis. The overall conclusion was that all three physiological mechanisms are important in understanding the regulation of N2 fixation and its response of to soil drying.     

Developmental effects on ureide levels are mediated by tissue-specific regulation of allantoinase in Phaseolus vulgaris L

The ureides allantoin and allantoate are key molecules in the transport and storage of nitrogen in ureide legumes. In shoots and leaves from Phaseolus vulgaris plants using symbiotically fixed nitrogen as the sole nitrogen source, ureide levels were roughly equivalent to those of nitrate-supported plants during the whole vegetative stage, but they exhibited a sudden increase at the onset of flowering. This rise in the level of ureides, mainly in the form of allantoate, was accompanied by increases in allantoinase gene expression and enzyme activity, consistent with developmental regulation of ureide levels mainly through the tissue-specific induction of allantoate synthesis catalysed by allantoinase. Moreover, surprisingly high levels of ureides were also found in non-nodulated plants fertilized with nitrate, at both early and late developmental stages. The results suggest that remobilized N from lower leaves is probably involved in the sharp rise in ureides in shoots and leaves during early pod filling in N(2)-fixing plants and in the significant amounts of ureides observed in non-nodulated plants.     

Soybean nodulation and N2 fixation response to drought under carbon dioxide enrichment

The combined effects of carbon dioxide (CO₂) enrichment and water deficits on nodulation and N₂ fixation were analysed in soybean [Glycine max (L.) Merr.]. Two short-term experiments were conducted in greenhouses with plants subjected to soil drying, while exposed to CO₂ atmospheres of either 360 or 700 μmol CO₂ mol^–1. Under drought-stressed conditions, elevated [CO₂] resulted in a delay in the decrease in N₂ fixation rates associated with drying of the soil used in these experiments. The elevated [CO₂] also allowed the plants under drought to sustain significant increases in nodule number and mass relative to those under ambient [CO₂]. The total non-structural carbohydrate (TNC) concentration was lower in the shoots of the plants exposed to drought; however, plants under elevated CO₂ had much higher TNC levels than those under ambient CO₂. For both [CO₂] treatments, drought stress induced a substantial accumulation of TNC in the nodules that paralleled N₂ fixation decline, which indicates that nodule activity under drought may not be carbon limited. Under drought stress, ureide concentration increased in all plant tissues. However, exposure to elevated [CO₂] resulted in substantially less drought-induced ureide accumulation in leaf and petiole tissues. A strong negative correlation was found between ureide accumulation and TNC levels in the leaves. This relationship, together with the large effect of elevated [CO₂] on the decrease of ureide accumulation in the leaves, indicated the importance of ureide breakdown in the response of N₂ fixation to drought and of feedback inhibition by ureides on nodule activity. It is concluded that an important effect of CO₂ enrichment on soybean under drought conditions is an enhancement of photoassimilation, an increased partitioning of carbon to nodules and a decrease of leaf ureide levels, which is associated with sustained nodule growth and N₂ rates under soil water deficits. We suggest that future [CO₂] increases are likely to benefit soybean production by increasing the drought tolerance of N₂ fixation.     

Asparagine and ureide accumulation in nodules and shoots as feedback inhibitors of N2 fixation in soybean

Inhibition of N₂ fixation under water deficits has been hypothesized to result from N feedback within the plant involving ureides and/or asparagine (Asn). This study was undertaken to investigate ureide and Asn accumulation in shoots and nodules in response to treatments inhibiting nodule activity (acetylene reduction assay, ARA) such as Asn, ureide, or polyethylene glycol application to the nutrient solution of plants, boric acid on leaves, and imposition of a water deficit. ARA inhibition and nodule concentration of Asn and ureide were correlated to the ureide treatment applied (3–15 m M applied in the nutrient solution). Supplying Asn (3–9 m M applied in the nutrient solution) also induced an increase in nodule Asn and ureide concentration, which was associated with ARA inhibition. Spraying boric acid on leaves also inhibited ARA in parallel to an increase in shoot ureide and nodule Asn concentration while nodule ureide remained unchanged. By contrast, polyethylene glycol (PEG) inhibited ARA in parallel to an increase in nodule Asn and ureide concentration while shoot ureide was unchanged. The decline in ARA in response to water deficit was associated with an increase in nodule ureide, Asn and aspartate (Asp), although the increases in Asn and Asp were less than for ureides. Altogether, the results of these experiments indicated that Asn cannot be the only compound involved in the feedback inhibition of ARA. Instead ureide and Asn are probably both involved, either directly by accumulation of products that fail to be exported, or by feedback from the shoot due to an N -compound supply that exceeds shoot requirements.     

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.     

Reduced carbon availability to bacteroids and elevated ureides in nodules, but not in shoots, are involved in the nitrogen fixation response to early drought in soybean

Nitrogen fixation (NF) in soybean (Glycine max L. Merr.) is highly sensitive to soil drying. This sensitivity has been related to an accumulation of nitrogen compounds, either in shoots or in nodules, and a nodular carbon flux shortage under drought. To assess the relative importance of carbon and nitrogen status on NF regulation, the responses to the early stages of drought were monitored with two soybean cultivars with known contrasting tolerance to drought. In the sensitive cultivar ('Biloxi'), NF inhibition occurred earlier and was more dramatic than in the tolerant cultivar ('Jackson'). The carbon flux to bacteroids was also more affected in 'Biloxi' than in 'Jackson', due to an earlier inhibition of sucrose synthase activity and a larger decrease of malate concentration in the former. Drought provoked ureide accumulation in nodules of both cultivars, but this accumulation was higher and occurred earlier in 'Biloxi'. However, at this early stage of drought, there was no accumulation of ureides in the leaves of either cultivar. These results indicate that a combination of both reduced carbon flux and nitrogen accumulation in nodules, but not in shoots, is involved in the inhibition of NF in soybean under early drought.     

Manganese application alleviates the water deficit-induced decline of N2 fixation

Water deficit is a very serious constraint on N₂ fixation rates and grain yield of soybean (Glycine max Merr.). Ureides are transported from the nodules and they accumulate in the leaves during soil drying. This accumulation appears responsible for a feedback mechanism on nitrogen fixation, and it is hypothesized to result from a decreased ureide degradation in the leaf. One enzyme involved in the ureide degradation, allantoate amidohydrolase, is manganese (Mn) dependent. As Mn deficiency can occur in soils where soybean is grown, this deficiency may aggravate soybean sensitivity to water deficit. In situ ureide breakdown was measured by incubating soybean leaves in a 5 mol m ^{? 3} allantoic acid solution for 9 h before sampling leaf discs in which remnant ureide was measured over time. In situ ureide breakdown was dramatically decreased in leaves from plants grown without Mn. At the plant level, allantoic acid application in the nutrient solution of hydroponically grown soybean resulted in a higher accumulation of ureide in leaves and lower acetylene reduction activity (ARA) by plants grown with 0 mol m ^{? 3} Mn than those grown with 6·6 mol m ^{? 3} Mn. Those plants grown with 6·6 mol m ^{? 3} Mn in comparison with those grown with 52·8 mol m ^{? 3} Mn had, in turn, higher accumulated ureide and lower ARA. To determine if Mn level also influenced N₂ fixation sensitivity to water deficit, a dry‐down experiment was carried out by slowly dehydrating plants that were grown in soil under four different Mn nutritions. Plants receiving no Mn had the lowest leaf Mn concentration, 11·9 mg kg ^{? 1}, and had N₂ fixation more sensitive to water deficit than plants treated with Mn in which leaf Mn concentration was in the range of 21–33 mg kg ^{? 1}. The highest Mn treatments increased leaf Mn concentration to 37·5 mg kg ^{? 1} and above but did not delay the decline of ARA with soil drying, although these plants showed a significant increase in ARA under well‐watered conditions.     

Inhibition of N2 fixation in soybean is associated with elevated ureides and amino acids

Decreased N2 fixation in soybean (Glycine max) L. Merr. during water deficits has been associated with increases in ureides and free amino acids in plant tissues, indicating a potential feedback inhibition by these compounds in response to drought. We evaluated concentrations of ureides and amino acids in leaf and nodule tissue and the concurrent change in N2 fixation in response to exogenous ureides and soil-water treatments for the cultivars Jackson and KS4895. Exogenous ureides applied to the soil and water-deficit treatments inhibited N2 fixation by 85% to 90%. Mn fertilization increased the apparent catabolism of ureides in leaves and hastened the recovery of N2 fixation following exogenous ureide application for both cultivars. Ureides and total free amino acids in leaves and nodules increased during water deficits and coincided with a decline in N2 fixation for both cultivars. N2 fixation recovered to 74% to 90% of control levels 2 d after rewatering drought-stressed plants, but leaf ureides and total nodule amino acids remained elevated in KS4895. Asparagine accounted for 82% of the increase in nodule amino acids relative to well-watered plants at 2 d after rewatering. These results indicate that leaf ureides and nodule asparagine do not feedback inhibit N2 fixation. Compounds whose increase and decrease in concentration mirrored the decline and recovery of N2 fixation included nodule ureides, nodule aspartate, and several amino acids in leaves, indicating that these are potential candidate molecules for feedback inhibition of N2 fixation.     

Soybean cultivars 'Williams 82' and 'Maple Arrow' produce both urea and ammonia during ureide degradation

The ability of two soybean (Glycine max L. [Merrill]) cultivars, 'Williams 82' and 'Maple Arrow', which were reported to use different ureide degradation pathways, to degrade the ureides allantoin and allantoate was investigated. Protein fractions and total leaf homogenates from the fourth trifoliate leaves of both cultivars were examined for the ability to evolve either (14)CO(2) or [(14)C]urea from (14)C-labelled ureides in the presence of various inhibitors. (14)CO(2) evolution from [2,7-(14)C]allantoate was catalysed by 25-50% saturated ammonium sulphate fractions of both cultivars. This activity was inhibited by acetohydroxamate (AHA), which has been used to inhibit plant ureases, but not by phenylphosphorodiamidate (PPD), a more specific urease inhibitor. Thus, in both cultivars, allantoate may be metabolized by allantoate amidohydrolase. This activity was sensitive to EDTA, consistent with previous reports demonstrating that allantoate amidohydrolase requires manganese for full activity. Total leaf homogenates of both cultivars evolved both (14)CO(2) and [(14)C]urea from [2,7-(14)C] (ureido carbon labelled) allantoin, not previously reported in either 'Williams 82' or in 'Maple Arrow'. In situ leaf degradation of (14)C-labelled allantoin confirmed that both urea and CO(2)/NH(3) are direct products of ureide degradation. Growth of plants in the presence of PPD under fixing and non-fixing conditions caused urea accumulation in both cultivars, but did not have a significant impact on total seed nitrogen. Urea levels were higher in N-fixing plants of both cultivars. Contrary to previous reports, no significant biochemical difference was found in the ability of these two cultivars to degrade ureides under the conditions used.     

Involvement of Ureides in Nitrogen Fixation Inhibition in Soybean

The sensitivity of N₂ fixation to drought stress in soybean (Glycine max Merr.) has been shown to be associated with high ureide accumulation in the shoots, which has led to the hypothesis that N₂ fixation during drought is decreased by a feedback mechanism. The ureide feedback hypothesis was tested directly by measuring the effect of 10 mm ureide applied by stem infusion or in the nutrient solution of hydroponically grown plants on acetylene reduction activity (ARA). An almost complete inhibition of ARA was observed within 4 to 7 d after treatment, accompanied by an increase in ureide concentration in the shoot but not in the nodules. The inhibition of ARA resulting from ureide treatments was dependent on the concentration of applied ureide. Urea also inhibited ARA but asparagine resulted in the greatest inhibition of nodule activity. Because ureides did not accumulate in the nodule upon ureide treatment, it was concluded that they were not directly inhibitory to the nodules but that their influence mediated through a derivative compound, with asparagine being a potential candidate. Ureide treatment resulted in a continual decrease in nodule permeability to O₂ simultaneous with the inhibition of nitrogenase activity during a 5-d treatment period, although it was not clear whether the latter phenomenon was a consequence or a cause of the decrease in the nodule permeability to O₂.The physiological basis of N₂ fixation inhibition by water deficits in legume nodules is not clearly understood. A potential physiological basis for this water-deficit sensitivity may be that drought stress decreases the P_o (Weisz et al., 1985), as has been shown with other stresses such as temperature, salinity, or nitrate (Hunt and Layzell, 1993; Serraj et al., 1994; Denison and Harter, 1995). The role of O₂ limitation in the response of nitrogenase activity to drought stress has been discussed extensively (Diaz del Castillo and Layzell, 1995; Purcell and Sinclair, 1995; Serraj and Sinclair, 1996b; Serraj et al., 1999). However, the mechanisms by which drought affects P_o have not been elucidated. It is not clear whether drought stress has a direct effect on P_o, or whether the decrease in P_o is a consequence of a decrease in nodule activity.An alternative explanation for the decrease in nitrogenase activity under drought could be a feedback mechanism involving the accumulation of N compounds. Pate et al. (1969) proposed that lower rates of water movement out of the nodule during drought stress may restrict export of products of N₂ fixation, and the accumulation of these products would inhibit nitrogenase activity. Others have suggested that N₂ fixation in legumes might be regulated by a feedback mechanism involving N metabolism and the pool of reduced N in the plant (Silsbury et al., 1986; Parsons et al., 1993; Hartwig et al., 1994). Oti-Boateng and Silsbury (1993) reported an inhibition of nitrogenase activity in fava bean after plant uptake of Asn or Gln.Soybean (Glycine max Merr.) usually exports more than 80% of the N compounds out of the nodules in the form of the ureides Aln and Alac. They are transported in the xylem to the shoots, where they are catabolized (Winkler et al., 1987). High accumulation of petiole ureides has been measured during the inhibition of N₂ fixation by drought in both controlled (de Silva et al., 1996; Serraj and Sinclair, 1996a) and field (Purcell et al., 1998) environments. Furthermore, in a comparison of grain legume species, Sinclair and Serraj (1995) reported that those species exporting ureides from the nodules had N₂ fixation that was drought sensitive. Those species that exported little or no ureide had N₂ fixation that was relatively drought tolerant.An important possibility is that the accumulation of ureides in soybean nodules under soil-water deficits might trigger a feedback mechanism that results in decreased N₂ fixation activity (Sinclair and Serraj, 1995; Serraj et al., 1999). This paper reports a series of experiments to investigate the hypothesis of a ureide feedback inhibition of N₂ fixation in soybean. First, ureide levels were measured in plant tissue (nodules, roots, and shoots) upon the imposition of water deficits to confirm that ureide levels increased in the nodules themselves, and not just in the shoot. Second, the influence of ureides on nodule activity was examined by introducing ureides, along with other compounds, into soybean plants. These experiments were designed to examine the time course of the response and to determine the concentration response. Third, data were collected to determine if P_o and the response of N₂ fixation to pO₂ were also sensitive to introduced ureides.     

N2Fixation Response to Drought in Common Bean (Phaseolus vulgarisL.)

Nitrogen fixation activity in common bean is generally thoughtto be low and sensitive to soil drying and, consequently, droughtcan have important negative effects on N accumulation and yieldpotential. The objectives of this research were to examine theresponse of N₂fixation to drought stress in common bean, andto test the hypothesis that drought sensitivity of N₂fixationin common bean is linked to ureide levels in the plants. Twoglasshouse experiments were conducted to compare the responsesof leaf transpiration and acetylene reduction activity (ARA)to soil water contents. ARA decrease during soil dehydrationwas found to lag behind the decline in transpiration. This indicatesthat ARA is relatively less sensitive to soil dehydration comparedto leaf gas exchange. Further, in comparing two cultivars therewas no consistent difference in the relative response of ARAand transpiration to soil drying. The ureide concentrationsmeasured in common bean plants were low, ranging from 0.1 to1.0 mmol l^-1in xylem sap exudates. Ureide concentrations inthe sap exudate varied significantly among the two genotypeseven though there was no difference in ARA response to drought.It was concluded that in common bean, the lower sensitivityof N₂fixation to drought compared to leaf gas exchange couldbe related to low ureide concentrations in petioles and xylemsap.Copyright 1998 Annals of Botany Company Phaseolus vulgaris,nitrogen fixation, drought stress, nodules, ureides.     

Ureide degradation pathways in intact soybean leaves

Ureides dramatically accumulate in shoots of N(2)-fixing soybean (Glycine max L. Merr.) under water deficit and this accumulation is higher in cultivars that have N(2) fixation that is sensitive to water deficit. One possible explanation is that ureide accumulation is associated with a feedback inhibition of nitrogenase activity. A critical factor involved in ureide accumulation is likely to be the rate of ureide degradation in the leaves. There exists, however, a controversy concerning the pathway of allantoic acid degradation in soybean. Allantoate amidinohydrolase was reported to be the pathway of degradation in studies using the cultivar Maple Arrow and allantoate amidohydrolase was the pathway that existed in the cultivar Williams. This investigation was undertaken to resolve the existence of these two pathways. An in situ technique was developed to examine the response of ureide degradation in leaf tissue to various treatments. In addition, the response of ureide accumulation and N(2) fixation activity was measured for intact plants in response to treatments that differentially influenced the two degradation pathways. The results from these studies confirmed that Maple Arrow and Williams degraded allantoic acid by different pathways as originally reported. The existence of two degradation pathways within the soybean germplasm opens the possibility of modifying ureide degradation to minimize the influence of soil water deficits on N(2) fixation activity.     

Functional specialization of one copy of glutamine phosphoribosyl pyrophosphate amidotransferase in ureide production from symbiotically fixed nitrogen in Phaseolus vulgaris

Purines are essential molecules formed in a highly regulated pathway in all organisms. In tropical legumes, the nitrogen fixed in the nodules is used to generate ureides through the oxidation of de novo synthesized purines. Glutamine phosphoribosyl pyrophosphate amidotransferase (PRAT) catalyses the first committed step of de novo purine synthesis. In Phaseolus vulgaris there are three genes coding for PRAT. The three full‐length sequences, which are intron‐less genes, were cloned, and their expression levels were determined under conditions that affect the synthesis of purines. One of the three genes, PvPRAT3, is highly expressed in nodules and protein amount and enzymatic activity in these tissues correlate with nitrogen fixation activity. Inhibition of PvPRAT3 gene expression by RNAi‐silencing and subsequent metabolomic analysis of the transformed roots shows that PvPRAT3 is essential for the synthesis of ureides in P. vulgaris nodules.