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.     

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.     

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 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.     

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.     

Regulation of allantoate transport in wild-type and mutant strains of Saccharomyces cerevisiae.

Accumulation of intracellular allantoin and allantoate is mediated by two distinct active transport systems in Saccharomyces cerevisiae. Allantoin transport (DAL4 gene) is inducible, while allantoate uptake is constitutive (it occurs at full levels in the absence of any allantoate-related compounds from the culture medium). Both systems appear to be sensitive to nitrogen catabolite repression, feedback inhibition, and trans-inhibition. Mutants (dal5) that lack allantoate transport have been isolated. These strains also exhibit a 60% loss of allantoin transport capability. Conversely, dal4 mutants previously described are unable to transport allantoin and exhibit a 50% loss of allantoate transport. We interpret the pleiotropic behavior of the dal4 and dal5 mutations as deriving from a functional interaction between elements of the two transport systems.     

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.     

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.     

Urease Is Not Essential for Ureide Degradation in Soybean

The hypothesis that soybean (Glycine max L. [Merrill]) catabolizes ureides to urea to a physiologically significant extent was tested and rejected. Urease-negative (eu3-e1/eu3-e1) plants were supported by fixed N2 or by 2 mM NH4NO3, so that xylem-borne nitrogen contained predominantly ureides (allantoin and allantoic acid) or amide amino acids, respectively. Seed nitrogen yield was equal on either nitrogen regime, although 35-d-old fixing plants accumulated about 6 times more leaf urea. In callus, lack of an active urease reduced growth on either arginine or allantoin as the sole nitrogen source, but the reduction was greater on arginine (73%) than on allantoin (39%). Furthermore, urease-negative cells accumulated 17 times more urea than urease-positive cells on arginine; for allantoin the ratio was 1.8. Urease-negative callus accumulated urea at 3% the rate of seedlings. To test whether urea accumulating in urease-negative seedlings was derived from ureides, seeds were first allowed to imbibe in 1 mM allopurinol, an inhibitor of ureide formation. Seedling ureides were decreased by 90%, but urea levels were unchanged. Thus, ureides are poor precursors of urea, which was confirmed in seedlings that converted no more than 5% of seed-absorbed [14C-ureido]allantoate to [14C]urea, whereas 40 to 70% of [14C-guanido]arginine was recovered as [14C]urea.     

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.     

Effect of water stress on the enzymes of nitrogen metabolism in mung bean (Vigna radiata Wilczeck) nodules

Abstract. Water stress created by withholding irrigation in mung bean resulted in decreased leaf water potential and nodule moisture content. Decreased leaf water potential was associated with decreased activity of nitrogenase, glutamine synthetase (GS), asparagine synthetase (AS), aspartate amino transferase (AAT), xanthine dehydrogenase (XDH) and uricase. However, the activity of glutamate dehydrogenase increased three-fold under severe stress. The activity of allantoinase and allantoicase was not affected by moderate stress but decreased under severe stress. The in vitro production of allantoic acid from allantoin and uric acid in the cytosol fraction decreased more than its production from xanthine and hypoxanthine. The production of NADH also decreased under stress.
During recovery from severe stress, the activity of XDH and uricase further decreased, whilst that of allantoinase and allantoicase increased compared to the control. This corresponded with the higher content of ureides during recovery. The recovery in other enzymes was not complete although leaf water potential and nodule moisture content recovered fully within 24 h.     

The biochemistry of nitrogen mobilization: purine ring catabolism

The enzymatic route of purine ring catabolism has recently been completed by the discovery of several novel enzymes identified through comparative genome analyses. Here, we review these recent discoveries and present an overview of purine ring catabolism in plants. Xanthine is oxidized to urate in the cytosol, followed by three enzymatic steps taking place in the peroxisome and four reactions in the endoplasmic reticulum releasing the four ring nitrogen as ammonia. Although the main physiological function of purine degradation might lie in the remobilization of nitrogen resources, it has also emerged that catabolic intermediates, the ureides allantoin and allantoate, are likely to be involved in protecting plants against abiotic stress. Conserved alternative splicing mediating the peroxisomal as well as cytosolic localization of allantoin synthase potentially links purine ring catabolism to brassinosteroid signaling.     

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.

     

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.     

The influence of phosphorus deficiency on growth and nitrogen fixation of white clover plants

The effects of P deficiency on growth, N(2)-fixation and photosynthesis in white clover (Trifolium repens L.) plants were investigated using three contrasting relative addition rates of P, or following abrupt withdrawal of the P supply. Responses to a constant below-optimum P supply rate consisted of a decline in N(2)-fixation per unit root weight and a small reduction in the efficiency with which electrons were allocated to the reduction of N(2) in nodules. Abrupt removal of P arrested nodule growth and caused a substantial decline in nitrogenase activity per unit root weight, but not per unit nodule mass. Similarly, the rate of photosynthesis per unit leaf area was unaffected by abrupt P removal, whereas CO(2) acquisition for the plant as a whole decreased due to a decline in total leaf area, leaf area per unit leaf weight and utilization of incoming radiation. These changes followed the decline in tissue P concentrations. The ratio between CO(2)-fixation and N(2)-fixation was maintained under short-term P deprivation but increased under long-term low P supply, indicating a regulatory inhibition of nodule activity following morphological and growth adjustments. It is concluded that N(2)-fixation did not limit the growth of clover plants experiencing P deficiency. A low P status induced changes in the relative growth of roots, nodules and shoots rather than changes in N and/or C uptake rates per unit mass or area of these organs.     

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.