JXB Advance Access originally published online on March 12, 2004
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Journal of Experimental Botany, Vol. 55, No. 398, pp. 867-877, April 1, 2004
© 2004 Oxford University Press
Cell and Molecular Biology, Biochemistry and Molecular Physiology |
Soybean cultivars ‘Williams 82’ and ‘Maple Arrow’ produce both urea and ammonia during ureide degradation
Received 12 November 2003; Accepted 20 January 2004
Department of Biochemistry and Interdisciplinary Plant Group, 117 Schweitzer Hall, University ofMissouri–Columbia, Columbia, MO 65211, USA
* To whom correspondence should be addressed. Fax: +1 573 882 5635. E-mail: polaccoj{at}missouri.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
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 14CO2 or [14C]urea from 14C-labelled ureides in the presence of various inhibitors. 14CO2 evolution from [2,7-14C]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 14CO2 and [14C]urea from [2,7-14C] (ureido carbon labelled) allantoin, not previously reported in either ‘Williams 82’ or in ‘Maple Arrow’. In situ leaf degradation of 14C-labelled allantoin confirmed that both urea and CO2/NH3 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.
Key words: Allantoate, allantoin, amidinohydrolase, amidohydrolase, nitrogen metabolism, N-fixation, urea, urease, ureide.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Following nitrogen fixation in symbiotic soybean plants the majority of xylem-borne nitrogen is delivered to the shoot as the ureides allantoin and allantoate (McClure and Israel, 1979; Thomas and Schrader, 1981). Once in the leaf tissue allantoin and allantoate are ultimately broken down to NH3, CO2, and glyoxylate (Winkler et al., 1988a). The mechanism of this conversion is still under debate. Two different enzymes have been proposed to hydrolyse allantoate, allantoate amidohydrolase (EC 3.5.3.9 [EC] ) (Winkler et al., 1985), and allantoate amidinohydrolase (EC 3.5.3.4 [EC] ) (Shelp and Ireland, 1985). The amidohydrolase releases NH3 and CO2 directly from allantoate, whereas the amidinohydrolase produces urea, which can then be converted to NH3 and CO2 by the nickel metalloenzyme urease (EC 3.5.1.5 [EC] ).
Soybean cultivar ‘Williams 82’ was shown to liberate NH3 and CO2 directly from allantoate, without a requirement for urease (Winkler et al., 1985), suggesting that it utilized an amidohydrolase route (Reactions 2 and 4, Fig. 1; Route D, Table 1). However, it was reported that cv. ‘Maple Arrow’ utilized the amidinohydrolase reaction (Reactions 1 and 3, Fig. 1; Route A, Table 1), since the urease inhibitor acetohydroxamate (AHA) blocked all 14CO2 release from [2-14C]allantoin in sliced leaf tissue (Shelp and Ireland, 1985). The conversion of ureidoglycolate to glyoxylate has not received as much attention in soybean, though a ureidoglycolate amidohydrolase activity was reported in developing seedcoats (Winkler et al., 1988b). However, both a ureidoglycolate amidohydrolase (EC 3.5.3.19 [EC] ) (Wells and Lees, 1991) and a ureidoglycolate urea-lyase (EC 4.3.2.3 [EC] ) (Muñoz et al., 2001) have been purified from other leguminous species.
|
|
It has been suggested that both allantoate amidohydrolase and amidinohydrolase are present in the soybean germplasm and that different cultivars may contain one pathway exclusively, or both (Vadez and Sinclair, 2000). This difference is thought to have agronomic importance, as the amidinohydrolase is believed to continue to degrade allantoate under water-limiting conditions, preventing the build-up of ureides and subsequent inhibition of nitrogen fixation. Conversely, the amidohydrolase is believed to become inactive under water-deficit, ultimately contributing to the inhibition of nitrogen fixation (Vadez and Sinclair, 2000; Sinclair et al., 2003). Allantoate amidohydrolase has been shown to require manganese for activity in vitro (Winkler et al., 1987) and in planta (Lukaszewski et al., 1992) and manganese has been implicated in the tolerance of nitrogen fixation to drought (Purcell et al., 2000). It has been assumed that allantoate amidinohydrolase does not require manganese (Vadez and Sinclair, 2001) since exogenous manganese was not applied in the experiments performed with ‘Maple Arrow’ (Shelp and Ireland, 1985), but this has yet to be demonstrated biochemically. The response of these two cultivars to water deficit has been examined physiologically (Vadez and Sinclair, 2000, 2001) and differences have been attributed to separate pathways of ureide degradation operating during water stress. Still, no biochemical evidence has been presented for the exclusivity of one mode of ureide degradation over another in either cultivar. For that reason a choice was made to employ a side-by-side biochemical and physiological comparison of ureide degradation in ‘Williams 82’ and ‘Maple Arrow’ leaves.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Plant material and growth conditions
Seeds from soybean (Glycine max [L.] Merr.) cultivars ‘Williams 82' and ‘Maple Arrow’ were allowed to germinate for 24–48 h at 27 °C in distilled water in rolls of germination paper (Anchor Paper, St Paul, MN, USA), inoculated with Bradyrhizobium japonicum (USDA 3I1B143), and transferred to 8" pots containing a 1:1 (v:v) vermiculite:perlite mixture with a cheesecloth wick. Plants were grown under greenhouse conditions at 26/21 °C day/night with a 16 h photoperiod maintained above 650 µmol photons m–2 s–1 with supplemental lighting. Plants were grown under N2-fixing conditions by placing the vermiculite:perlite in containers of nitrogen-free nutrient solution composed of 2.0 mM CaCl2, 0.63 mM K2SO4, 0.5 mM MgSO4, 0.5 mM K2HPO4, 25 µM FeSO4, 2.3 µM H3BO3, and 1 µM each MnSO4, ZnSO4, Na2MoO4, NiCl2, CoCl2, and CuSO4. Plants grown in the presence of supplemental nitrogen were not inoculated and the nutrient solution was identical except for the addition of 5 mM NH4NO3. The vermiculite:perlite support was kept hydrated via cheesecloth wicks and plant roots were allowed to grow into the nutrient solution; plants were always kept well hydrated. At the conclusion of the experiment, plant root systems were examined and, in all cases, were well nodulated when grown without nitrogen. No nodules were found on plants grown in the presence of NH4NO3.
For analysis of seed nitrogen, whole pods were collected and dried at 70 °C for 2 d. Seeds were then removed from the pods and dried to constant weight at 70 °C. Seeds were weighed, ground to powder in a household coffee grinder, frozen at –80 °C, and lyophilized to remove any residual moisture. Leaf tissue was collected, frozen, lyophilized, and powdered. Total nitrogen content in the lyophilized seed samples was determined by the Kjeldahl method at the University of Missouri Agriculture Experiment Station Chemical Laboratories. Urea was determined as in Stebbins et al. (1991). Total ureide was determined by the method of Vogels and van der Drift (1970) and leaf protein levels were measured by the method of Lowry et al. (1951)
Synthesis of 14C-labelled ureides
[2,7-14C]allantoate was synthesized by condensing [14C]urea (Moravek, Brea, CA, USA) on glyoxylate, utilizing a method kindly provided by Dr DG Blevins. [2,7-14C]allantoin was synthesized by reacting [8-14C]uric acid (Moravek) with uricase (Sigma-Aldrich, St Louis, MO, USA). The end-product of uricase action, [7-14C]allantoin, racemizes, eventually resulting in an equal mixture of [2-14C]-labelled and [7-14C]-labelled molecules (Kahn and Tipton, 2000). Significantly, since allantoate is symmetrical around the 4 and 5 (glyoxylate) carbons, containing a urea moiety on each side, the release of both urea moieties and conversion to CO2 and NH3 from a single [2,7-14C]allantoin or [2,7-14C]allantoate molecule should result in a 4:1 NH3:14CO2 ratio, as the label resides in either the 2 or 7 carbon (but not both). Labelled allantoin and allantoate were purified by HPLC using a Shodex Asahipak GS-320 HQ column (Shodex, Showa Denko KK, Kawasaki, Japan) eluted with 5 mM NaH2PO4 (pH 4.4) monitored by UV detection and quantified by liquid scintillation counting. Immediately prior to use in assays both compounds were treated with Type IX Jack bean urease (Sigma–Aldrich) to remove any [14C]urea accumulated by non-enzymatic breakdown during storage.
In vitro assay of 14CO2 evolution from ammonium sulphate-precipitated protein extract
A 25–50% saturated (NH4)2SO4 protein fraction was generated from fully expanded third and fourth trifoliate leaves of both ‘Maple Arrow’ and ‘Williams 82’ plants using the method of Lukaszewski et al. (1992) in 50 mM Tricine (pH 8.75), 14 mM ß-mercaptoethanol, and 1 mM MnSO4. Enzyme assays were performed by the addition of 250 µl of extract to 250 µl of the same buffer containing 20 mM [2,7-14C]potassium allantoate (10 MBq mmol–1). Assays were incubated for 30 min at 30 °C and stopped by the addition of 1 ml 1 M H2SO4. 14CO2 evolved by the addition of the acid was trapped on glass filter paper (Whatman GFC, Whatman Inc., Clifton, NJ, USA) saturated with 9 M monoethanolamine and quantified by liquid scintillation counting. Urease activity in the same extracts was determined the same way except that 10 mM [14C]urea (5 MBq mmol–1) replaced the [2,7-14C]allantoate.
Preparation of leaf homogenates
Homogenates were made of fourth trifoliate leaves, harvested after full expansion of the fifth trifoliate, such that each plant was still in vegetative development. In both cultivars, expansion of the fifth trifoliate leaf preceded flowering. Fresh leaves were extracted in a chilled mortar in 20 vol. (total) chilled Buffer A (50 mM Tricine, pH 8.75, and 1 mM MnSO4) and a small amount of washed sea sand. Homogenates were collected in a single 50 ml conical tube on ice and were vortexed thoroughly prior to transferring 1 ml aliquots to individual 16x100 mm test tubes on ice. Samples were transferred using a 5 ml pipettor equipped with a truncated tip so that both buffer-soluble and -insoluble components of the homogenate were transferred. Inhibition of urease was accomplished by adjusting samples to 100 µM phenylphosphorodiamidate (PPD) and incubating on ice for 10 min.
Measurement of CO2 and NH3 production from leaf homogenates
Measurement of ammonia from allantoin was performed using an adaptation of the procedure described by Meyer-Bothling and Polacco (1987) for the production of ammonia from urea. Assays were initiated by the addition of 1 ml of Buffer A containing 20 mM allantoin (final allantoin concentration 10 mM) to leaf homogenates which were then capped with serum vial stoppers. Samples were incubated for 2 h at 30 °C in a shaking water bath in the dark and terminated by placing the samples in a watery ice bath followed by the addition of 4 ml saturated K2CO3. Immediately following addition of the K2CO3, a 2 ml collection tube containing 200 µl 0.1 M H2SO4 was floated on the mixture and the tubes resealed. Standard curves containing 0–2000 µmol NH4Cl in 2 ml distilled water were generated with each assay. Microdiffusion of ammonia from the alkalinized homogenate to the sulphuric acid was allowed to proceed overnight at room temperature with gentle agitation. An aliquot of the acid solution was analysed for ammonia using the Berthelot reaction as described by Meyer-Bothling and Polacco (1987). Control samples for endogenous ammonia levels and non-enzymatic production of ammonia from allantoin were also performed.
Analysis of 14CO2 evolution was performed as above, but [2,7-14C] allantoin (10 MBq mmol–1) was included in the reaction mixture. After incubation reactions were stopped by addition of 0.5 ml 5 M H2SO4 and allowed to diffuse overnight. 14CO2 was trapped on alkaline glass filter paper saturated with 9 M monoethanolamine and quantified by liquid scintillation counting. Control reactions for non-enzymatic evolution of 14CO2 were also performed.
In situ leaf allantoin degradation
The petiole of the fourth trifoliate leaf from nodulated, N-fixing ‘Williams 82’ and eu3-e1/eu3-e1 urease-negative soybean (Stebbins et al., 1991) was cut at the stem, then recut under distilled water before transfer to a solution of [2,7-14C]allantoin in distilled water. Leaves were incubated for 6 h under a sodium halide lamp (700–800 µmol photons m–2 s–1) before the entire trifoliate was extracted in 10 mM TRIS-Cl (pH 7.5). Extracts were spun briefly in a clinical centrifuge to pellet cell debris and aliquots (1.2 ml) were split, in duplicate, into 16x100 mM test tubes. Jack bean urease (Sigma) was added to half the samples, all tubes were capped, and were then incubated for 90 min at 37 °C. 14CO2 was trapped on alkaline filter paper as described above, after the addition of 0.5 ml 5 M H2SO4, and measured by liquid scintillation counting.
Urease assays
Measurement of urease activity from leaf discs of ‘Williams 82’ and ‘Maple Arrow’ plants grown with and without 50 µM PPD in the nutrient solution was performed as described by Zonia et al. (1995).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Inhibition of [2,7-14C]allantoate and [14C]urea degradation in vitro
In cell-free extracts of soybean leaves, allantoate amidohydrolase activity cannot be detected by the colorimetric analysis of glyoxylate derivatives (Vogels and van der Drift, 1970), although steady-state ureide levels may be determined (Lukaszewski et al., 1992). This may be due to active consumption of glyoxylate during incubation of the samples. However, allantoate amidohydrolase activity was detected in (NH4)2SO4-fractionated leaf protein samples (Winkler et al., 1988b; Lukaszewski et al., 1992) or by the use of radiolabelled ureide precursors (Winkler et al., 1987). A 25–50% saturated (NH4)2SO4 leaf protein fraction from both ‘Maple Arrow’ and ‘Williams 82’ was assayed for the ability to degrade [2,7-14C]allantoate (ureido carbon label) (Fig. 2A) and [14C]urea (Fig. 2B) in vitro, as determined by the evolution of 14CO2. Both allantoate amidohydrolase and urease were found in the 25–50% saturated (NH4)2SO4 fraction.
|
In the presence of three potential inhibitors (Fig. 2) allantoate degradation was measured. AHA at 50 mM, the concentration employed by Shelp and Ireland (1985), inhibited 14CO2 evolution from both allantoate and urea in both cultivars (Fig. 2A). Phenylphosphorodiamidate (PPD), a specific urease inhibitor (Liao and Raines, 1985), inhibited 14CO2 release from urea, but not from allantoate. This suggests that the observed allantoate degradation is via allantoate amidohydrolase in both cultivars, releasing CO2 directly, since inhibition of urease with PPD did not cause a decrease in CO2 evolution from allantoate. Since AHA inhibited both allantoate degradation and urease activity (Fig. 2), its use as a urease inhibitor is unsuitable for studies of ureide degradation. AHA may inhibit allantoate degradation by chelating divalent cations (Von Anderegg et al., 1963; Winkler et al., 1987), in agreement with EDTA inhibition of 14CO2 release from allantoate in both cultivars (Fig. 2A). Allantoate amidohydrolase in soybean was shown to require manganese (Winkler et al., 1987; Lukaszewski et al., 1992). The allantoate amidinohydrolase of Chlamydomonas reinhardtii also requires manganese (Piedras et al., 2000), but there are no data on this enzyme’s metal requirements in higher plants. The allantoate degrading activities of both soybean cultivars responded in the same manner to the three inhibitors, AHA, PPD, and EDTA. Clearly, ‘Williams 82’ and Maple Arrow’ are both able to evolve 14CO2 directly from allantoate without a urea intermediate.
Evolution of CO2 and NH3 from allantoin in leaf homogenates
It could be argued that the 25–50% saturated ammonium sulphate fraction did not contain amidinohydrolase activity. Therefore, an attempt was made to employ a modification of the intact leaf tissue assay used by Shelp and Ireland (1985) using unfractionated leaf homogenates to examine complete [2,7-14C]allantoin degradation in each cultivar. As outlined by Shelp and Ireland (1985), the relative inhibition of both 14CO2 and NH3 production from [2,7-14C]allantoin, under conditions where urease is inhibited, can be predicted as a function of the allantoin degradative pathways (Table 1). Since it was determined that AHA is not urease-specific (Fig. 2), 50 µM PPD was used, which reduced urease activity >95% (Fig. 3).
|
PPD decreased 14CO2 and NH3 evolution from [2,7-14C]allantoin in both ‘Maple Arrow’ and ‘Williams 82’ leaf homogenates (Fig. 4). Values in Fig. 4 show relative inhibition in samples treated with PPD compared with their untreated controls. Absolute values are expressed in Table 2. In both cultivars, the observed 14CO2 evolution most closely resembled the theoretical 50% predicted by Table 1, routes B or C (61.3±8.9% for ‘Maple Arrow’ and 48.6±8.9% for ‘Williams 82’), suggesting the involvement of one ammonia and one urea liberating reaction. Evolution of ammonia was more consistent between samples and between cultivars, but showed only 25–30% inhibition in each cultivar (73.0±.6% for ‘Maple Arrow’ and 72.9±1.4% for ‘Williams 82’, Fig. 4B). The decrease in ammonia evolution in the absence of urease activity again suggests the involvement of ureo-lytic activity, consistent with routes B or C (Table 1). Increased NH3:14CO2 ratios in the presence of PPD (Table 2) suggest an additional source of NH3, perhaps from glyoxylate reacting with intermediates and enzymes of photorespiration.
|
|
Allantoin breakdown in intact leaves
To confirm that production of both 14CO2 and [14C]urea occurs in situ and to demonstrate that the inhibitor PPD is not altering the mechanism of breakdown, [2,7-14C]allantoin was fed through the petiole to leaves of ‘Williams 82’ and urease-negative eu3-e1/eu3-e1 plants. After 6 h of feeding under supplemental lighting the entire trifoliate leaf was extracted with aqueous buffer and assayed for the presence of soluble 14CO2 with and without the addition of jack bean urease. The experiment was carried out with supplemental lighting in order to allow the soluble 14CO2 produced to be assimilated through photosynthesis. Based on the experiments of Fig. 4, the prediction was that in ‘Williams 82’ leaves, endogenous urease should metabolize any [14C]urea produced and the addition of exogenous urease to the extracts should have little, if any, effect on soluble 14CO2 levels. By contrast, in the urease-negative mutants, urea should accumulate as a product of ureide degradation and should be released as soluble 14CO2 only after treatment with exogenous urease. This assertion proved to be correct. Addition of jack bean urease to extracts of ‘Williams 82’ trifoliate leaves had no effect on 14CO2 evolution. However, incubation with jack bean urease caused eu3-e1/eu3-e1 extracts to release ten times the soluble 14CO2 upon acidification (Fig. 5). It is notable that, in the untreated samples, eu3-e1/eu3-e1 leaves had residual soluble 14CO2 values that were approximately half of those of ‘Williams 82’ (calculated on the basis of total soluble counts extracted), which is strikingly similar to the inhibition of 14CO2 release by PPD in Fig. 4.
|
Whole plant urease inhibition
It was tested whether the ureo-lytic activity observed in leaf homogenates predominated in one of the varieties in planta. A prediction is that, as dependence on urea production from ureides increases, the role of urease becomes more critical in plants acquiring nitrogen exclusively from fixation. A cultivar relying solely on urea-N from fixation should be severely impacted by the inhibition of urease. Nodulated ‘Maple Arrow’ and ‘Williams 82’ plants were grown without added nitrogen in the presence and absence of 50 µM PPD in the nutrient solution. Both cultivars were also grown with 5 mM NH4NO3 in the nutrient solution with and without PPD treatment. Total protein, ureide, and urea levels were determined in individual trifoliate leaves sampled at the fourth trifoliate stage (as before) and during mid-pod fill. As a crude measure of nitrogen availability, PPD had little or no effect on total protein in either the +N or –N treatments (Fig. 6A, B), although in the fourth trifoliate leaves supplementation with NH4NO3 appeared to allow the young leaves to accumulate more protein compared with N-fixing plants (Fig. 6A).
|
The effect of PPD on urea accumulation was most evident during mid-pod fill (Fig. 6F). In the presence of PPD, urea accumulated in both the +N and –N treatments in both cultivars, but twice as much urea accumulated in older leaves under N-fixation in both cultivars (Fig. 6F). This provides additional evidence that urea is indeed a product of ureide degradation. Plants grown in the presence of PPD developed necrotic leaf tips, a phenotype exhibited by soybean genotypes which lack the ubiquitous urease by mutation in either the leaf urease structural gene or in one of the urease activation accessory factors (Stebbins et al., 1991). However, other than the leaf tip burn, there were no observable differences between the PPD-treated and control plants during any stage of development.
Under N-fixing conditions ‘Maple Arrow’ accumulated 3–4-fold more total ureide compared with ‘Williams 82’. In both cultivars ureide accumulation under fixation appeared independent of PPD treatment (Fig. 6C). Interestingly, when supplemented with NH4NO3, both cultivars increased total ureide levels in young leaf tissue when treated with PPD. This effect is apparently unrelated to N-fixation, since all plant root systems were examined at the termination of the experiment and only those plants which were grown in the absence of a nitrogen source showed nodule development. At mid-pod fill, total ureide levels in leaf tissue were substantially lower, but again, unaffected by PPD in either cultivar under either nitrogen regime (Fig. 6D).
Total seed nitrogen was determined for mature seeds harvested from both cultivars. All plants accumulated between 6.1% and 6.5% nitrogen (g N 100 g–1 DW). In both cultivars nitrogen deposition in the seed was unaffected by either nitrogen source or inhibition of urease, contrary to what would be expected if either cultivar were nitrogen-limited, because it degraded ureides solely through a urea intermediate.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Allantoate degradation in soybean
Two pathways for utilization of allantoate by soybean have been proposed based on previous studies on the two cultivars used in this work. An allantoate amidohydrolase activity was demonstrated in ‘Williams 82’, both in vitro (Winkler et al., 1985) and in intact leaf tissue (Winkler et al., 1987), releasing CO2 and two NH3 directly from allantoate, without a urea intermediate. Evidence for an allantoate amidinohydrolase was presented by Shelp and Ireland (1985) in the cultivar ‘Maple Arrow’. Surprisingly, there has not been a side-by-side biochemical characterization of allantoate degradation in these two cultivars. This is unfortunate as the differences in ureide degradation observed in these two cultivars have become somewhat anchored in the literature and have been the basis for ascribing differences in response of nitrogen fixation and ureide degradation to drought (Sinclair et al., 2003).
To test a urea intermediate, PPD was employed as a more avid and specific urease inhibitor than AHA. Although both PPD and AHA inhibited urease, only AHA inhibited CO2 release from [2,7-14C]allantoate, in both soybean cultivars (Fig. 2). This is in agreement with previous reports (Winkler et al., 1985; Lukaszewski et al., 1992) that AHA inhibited allantoate amidohydrolase, while PPD had no effect. To be consistent with Shelp and Ireland (1985) AHA was used at 50 mM. It is proposed that, at this concentration, AHA inhibition is mediated by its chelation of Mn2+, in agreement with the observed EDTA inhibition of 14CO2 release from [2,7-14C]allantoate. EDTA had no effect on urease (Fig. 2B), in which the nickel metallocentre is well-sheltered (Jabri et al., 1995; Goldraij et al., 2003).
Interestingly, Reddy et al. (1989) reported that AHA inhibits allantoinase of Vigna radiata competitively and not by chelating metal ions. The V. radiata allantoinase did not show a cation requirement. The authors suggest that AHA may have more than one inhibitory mechanism, in agreement with its role as a non-competitive urease inhibitor (Fishbein and Carbone, 1965). Given that AHA inhibits urease, allantoate amidohydrolase, and possibly allantoinase, the conclusion of Shelp and Ireland (1985) that ‘Maple Arrow’ degrades ureides solely through urea intermediates (Route A, Table 1) must be questioned. Based on their observed complete AHA-mediated inhibition of 14CO2 release from [2,7-14C]allantoin, Shelp and Ireland (1985) concluded that both ureido groups of allantoate are released as urea. It is argued here that a more specific urease inhibitor, one that did not inhibit the allantoinase and allantoate amidohydrolase, would have allowed significant CO2 evolution if either allantoate (Fig. 2) or allantoin (Fig. 4) were employed as a substrate.
Under the assay conditions used in this work, with and without PPD, ‘Maple and Arrow’ and ‘Williams 82’ show no significant difference in the patterns of CO2 and NH3 release, arguing against radically different ureide degradation pathways. However, in both cultivars there is evidence for the operation of both an amidohydrolase and an amidinohydrolase (or urea-lyase), which, it is postulated, employs a ureidoglycolate substrate.
Potential routes of ureide degradation in leaf homogenates
In the total conversion of allantoin to glyoxylate involving one ammonia and one urea liberating step, PPD will inhibit 50% of 14CO2 release, regardless of urea production from allantoate or ureidoglycolate (Routes B, C, Table 1). To approximate the Shelp and Ireland (1985) leaf tissue experiment, unfractionated leaf homogenates were incubated with [2,7-14C]allantoin. 14CO2 evolution was inhibited approximately 50% by PPD. In the same leaf homogenates PPD inhibited neither 0% or 100% of NH3 release, again suggesting routes B or C (Table 1). However, PPD inhibited NH3 evolution by only 25–30% compared with its near 50% inhibition of 14CO2 production (Fig. 4). The NH3:14CO2 ratio increase in leaf homogenates incubated with PPD (Table 2) is consistent with an allantoin-dependent increase in NH3 production. Complete release of ureido nitrogen produces glyoxylate. It is suggested here that glyoxylate stimulates NH3 production from endogenous stores via the photorespiratory pathway. This is consistent with the observations of Winkler et al. (1987) that label from [4,5-14C]allantoin (labelled in the ‘glyoxylate’ carbons) was recovered in glyoxylate, glycine, and serine, all photorespiratory intermediates. Preliminary results indicate that glyoxylate incubated with unfractionated leaf homogenates may stimulate ammonia production (CD Todd, unpublished results).
A single pathway for ureide degradation in plants has not been identified. Osuji and Ory (1987) suggested that, in the tubers of Dioscorea cayensis (yam) and Ipomoea batatas (sweet potato), allantoate amidohydrolase is responsible for catabolic degradation of allantoate and allantoate amidinohydrolase may be involved in anabolic ureide production. Ureidoglycolate amidohydrolase was purified from developing Phaseolus vulgaris and shown to be a manganese-requiring enzyme. Interestingly, the enzyme was found to be associated with peroxisomes (Wells and Lees, 1991), suggesting a potential spatial link to photorespiration. A ureidoglycolate urea-lyase purified from chickpea (Cicer arietinum [L.]) was also shown to be activated by manganese (Muñoz et al., 2001). Unfortunately, neither allantoate amidohydrolase nor allantoate amidinohydrolase has been purified from a plant source, however, a manganese-requiring allantoate amidinohydrolase has been purified from Chlamydomonas reinhardtii, a photosynthetic eukaryote (Piedras et al., 2000).
These results suggest sequential degradation of allantoate to glyoxylate via allantoate amidohydrolase and ureidoglycolate urea-lyase in both soybean cultivars examined (Route C, Table 1). In both ‘Williams 82’ and ‘Maple Arrow’ the presence of allantoate amidohydrolase in a 25–50% ammonium sulphate fraction (Fig. 2) and evolution of NH3, CO2, and urea from allantoin in a leaf homogenate (Fig. 4) were demonstrated. Atkins et al. (1982) also identified NH3, urea and CO2 as ureide breakdown products in cowpea (Vigna unguiculata).
Urease-negative eu3-e1/eu3-e1 ‘Williams’ demonstrated that urea is a product of allantoin degradation in situ (Fig. 5). Yet even in the complete absence of urease activity some 14CO2 evolution was detected, confirming the presence of an amidohydrolase, although the majority of 14CO2 produced directly was probably assimilated through photosynthesis. It is suggested that the 10-fold increase in 14CO2 evolution by added jack bean urease accounts for approximately half of the total 14CO2 produced, representing the release of urea from ureidoglycolate. 14CO2 released in the absence of added urease is probably generated directly and is that not assimilated through photosynthesis. The genetic block in urease production in eu3-e1/eu3-e1 confirmed that PPD-derived data were not an artefact. While urea-generating activities in both cultivars deserve further investigation, however, the main objective here was to assess whether ‘Maple Arrow’ and ‘Williams 82’ differ significantly in the ureide degradation pathway. It was concluded that they do not.
Physiological responses to urease inhibition
When leaf urease activity was inhibited by 50 µM PPD in the nutrient solution both ‘Maple Arrow’ and ‘Williams 82’ accumulated significant amounts of urea in the mature leaves (Fig. 6F). ‘Williams 82’ accumulated more urea than ‘Maple Arrow’ under N-fixation, contrary to the prediction based on exclusive ureide degradation through urea in ‘Maple Arrow’. ‘Maple Arrow’ (group 00) and ‘Williams 82’ (group III) are in different soybean maturity groups (USDA, ARS. 2003. National Genetic Resources Program. Germplasm Resources Information Network – (GRIN) [Online Database] National Germplasm Resources Laboratory, Beltsville, MD, USA. http://www.ars-grin.gov/npgs.), and under the greenhouse conditions mid-pod fill leaves of ‘Williams 82’ were 6-weeks-older than those of ‘Maple Arrow’, possibly allowing for greater total urea accumulation. Significantly, however, urea levels in both cultivars doubled when grown under N-fixing conditions (Fig. 6F), providing physiological evidence that ureide degradation contributes to urea production. This agrees with the accumulation pattern in the urease negative eu3-e1/eu3-e1 mutant, which accumulated 5–8 times more urea under nitrogen-fixing conditions (Stebbins and Polacco, 1995). Urea accumulation under continuous 50 µM PPD treatment was not as dramatic, possibly due to incomplete inhibition of urease in the older leaves However, the PPD effects, combined with in situ [14C]-urea production in eu3-e1/eu3-e1 leaf tissue (Fig. 5) and in vitro data (Figs 2, 4) lead the authors to conclude that both ammonia and urea are products of ureide degradation in both cultivars.
PPD did not impair the accumulation of seed nitrogen. The 6.1–6.5 % N observed for ‘Maple Arrow’ and 6.2–6.5 % N for ‘Williams 82’, values are similar to the 6.2 g % N observed by Stebbins and Polacco (1995) in the eu3-e1/eu3-e1 mutant of ‘Williams 82’. Similarly, urea levels in the seeds of PPD treated ‘Williams 82’ and ‘Maple Arrow’ approximated those found in seeds of eu4/eu4; eu1-sun/eu1-sun (leaf urease and seed urease structural genes, respectively) urease-negative double mutant (Stebbins et al., 1991). No difference were observed in form or growth between plants grown in nutrient solution alone or in the presence of PPD, apart from a PPD-induced leaf-tip burn, also observed in eu3-e1/eu3-e1 or eu4/eu4 plants (Stebbins et al., 1991). eu3-e1/eu3-e1 callus was incapable of growth with urea as a sole nitrogen source, but was able to utilize allantoin (Stebbins and Polacco, 1995), suggesting that urea is not the sole nitrogenous compound liberated from ureides. In this study, if either cultivar degraded ureides solely via urea, inhibition of urease should have had drastic negative effects, but like the eu3-e1/eu3-e1 mutant (Stebbins and Polacco, 1995), neither ‘Williams 82’ nor ‘Maple Arrow’ showed a negative response, providing further physiological evidence that fixed nitrogen can be liberated as ammonia.
Ureide degradation, manganese and drought
Both Purcell and Sinclair have proposed that the sensitivity of N-fixation to water-deficit in soybean is related to the nature of the ureide degradative pathway, the more tolerant genotype employing one with much reduced or no Mn requirement. This assertion was examined, focusing on ‘Maple Arrow’ and ‘Williams 82’, the two varieties which formed much of the basis for their conclusions. Firstly, as discussed above, no differences were seen in ureide degradation under well-watered conditions. Secondly, corroborations of unique pathways (Vadez and Sinclair, 2000) could be explained by differences in Mn movement and availability. Thirdly, it appears unlikely that the narrow germplasm base of North American soybean could harbour varieties with enzymatically distinct ureide degradative pathways. Finally, the assumption of no Mn requirement for the proposed urea-generating activities of ‘Maple Arrow’ is counter to the Mn requirements of allantoate amidinohydrolase of Chlamydomonas reinhardtii (Piedras et al., 2000) and ureidoglycolate urea-lyase of chickpea (Muñoz et al., 2001).
The observations of Vadez and Sinclair (2001) that well-watered ‘Maple Arrow’ had 3-fold greater ureide levels than ‘Williams 82’ under high and low Mn treatments have been corroborated, in this case focusing on the fourth trifoliate leaves of plants with sufficient water and Mn (Fig. 6C). If ureide metabolism is indeed related to drought-tolerant fixation then perhaps the sum of ureide synthesis, transport, accumulation, and degradation bears on N-fixation efficiency during soil drying. Streeter (2003) observed that N-fixing soybean accumulated higher leaf and pod N under drought stress. This, plus the observation that drought-stressed nodules accumulated more of both reduced carbon and nitrogen, led him to conclude that down-regulation of fixation under drought-stress is due to reduced demand for fixed N in the shoot. This hypothesis could explain accumulation of ureides in shoot tissues under drought stress; those cultivars able to continue fixation would be those able to utilize the products (ureides) of N-fixation.
Carbon:nitrogen balance and the ability to assimilate ureide breakdown products may be the mechanism underlying down-regulation of nitrogenase activity. Complete degradation of allantoin to glyoxylate, 2 CO2 and 4 NH+4 requires four turns of the GS-GOGAT cycle, requiring four carbon skeletons (Fig. 7). So, a difference between ‘Williams 82’ and ‘Maple Arrow’ (or other sensitive versus tolerant varieties) could be the availability of molecules to accept the released NH+4, consistent with elevated CO2 preventing both the build-up of leaf ureides and inhibition of nitrogenase when allantoate is supplied in the nutrient solution (Serraj and Sinclair, 2003). These results are consistent with Streeter’s hypothesis that reduced nitrogen demand may be related to inhibition of fixation. In a recent study on N-stress under well-watered conditions it was observed that nodulated soybean show decreased Asn in the xylem sap and increased Asp (Lima and Sodek, 2003). Under water stress, if C-skeletons became limiting, Asn could be synthesized from Asp (Fig. 7). An increase in [Asn] may be a potential indicator of nitrogen status and may also directly inhibit ureide breakdown by chelating manganese (Lukaszewski et al., 1992). As far as is known, the relationship between ureide breakdown, carbon availability, and operation of the GS-GOGAT cycle has not been studied in drought-stressed soybean.
|
The work was done on well-watered plants and it can not be ruled out that alternate modes of ureide degradation during water stress or different developmental stages. The role leaf ureide accumulation may play in feedback inhibition of nitrogen fixation is not disputed. However, the interplay among ureide accumulation, ureide degradation, manganese, and water deficit may involve more than merely a question of whether allantoate amidohydrolase or allantoate amidinohydrolase is the enzyme responsible for allantoate breakdown. Perhaps a re-examination of all the existing data, without preconception of the operative pathway, may yield alternative hypotheses on the role manganese, ureides and, possibly, carbon, play in tolerance of nitrogen fixation to drought. Additional tolerant lines (Sinclair et al., 2000, 2003) will surely aid in determining the basis for the plant response to drought stress. The isolation of ureide catabolic genes is the current focus for determining whether the inhibition of ureide degradation plays a causal role in the sensitivity of N-fixation to water stress.
|
Acknowledgements |
|---|
This work was supported by funds from grant no. 2003-00779, NRI, CSREES, USDA, and the Monsanto-MU Plant Biotechnology Research Program. CDT was supported by a Natural Sciences and Engineering Research Council of Canada Post-Doctoral Fellowship and a University of Missouri Life Sciences Post-Doctoral Fellowship. The authors would like to thank Peter Tipton and Nicholas Power for use of and assistance with their HPLC equipment. We would also like to thank Dale Blevins and Krystina Lukaszewski for helpful discussion of this work.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Atkins CA, Pate JS, Ritchie A, Peoples MB. 1982. Metabolism and translocation of allantoin in ureide producing grain legumes. Plant Physiology 70, 476–482.
Fishbein WN, Carbone PP. 1965. Urease catalysis. II. Inhibition of the enzyme by hydroxyurea, hydroxylamine, and aceto hydroxamate. Journal of Biological Chemistry 240, 2407–2414.
Goldraij A, Beamer LJ, Polacco JC. 2003. Interallelic complementation at the ubiquitous urease locus of soybean. Plant Physiology 132, 1801–1810.
Jabri E, Carr MB, Hausinger RP, Karplus PA. 1995. The crystal structure of urease from Klebsiella aerogenes. Science 268, 998–1004.
Kahn K, Tipton PA. 2000. Kinetics and mechanism of allantoin racemization. Bioorganic Chemistry 27, 62–72.[CrossRef]
Liao CFH, Raines SG. 1985. Inhibition of soil urease activity by amido derivatives of phosphoric and triphosphoric acids. Plant and Soil 85, 149–152.[CrossRef]
Lima JD, Sodek L. 2003. N-stress alters aspartate and asparagine levels of xylem sap in soybean. Plant Science 165, 649–656.[CrossRef]
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. 1951. Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193, 265–275.
Lukaszewski KM, Blevins DG, Randall DG. 1992. Asparagine and boric acid cause allantoate accumulation in soybean leaves by inhibiting manganese-dependent allantoate amidohydrolase. Plant Physiology 99, 1670–1676.
McClure PR, Israel DW. 1979. Transport of nitrogen in the xylem of soybean plants. Plant Physiology 64, 411–416.
Meyer-Bothling LE, Polacco JC. 1987. Mutational analysis of the embryo-specific urease locus of soybean. Molecular and General Genetics 209, 439–444.[CrossRef]
Muñoz A, Piedras P, Aguilar M, Pineda M. 2001. Urea is a product of ureidoglycolate degradation in chickpea. Purification and characterization of the ureidoglycolate urea-lyase. Plant Physiology 125, 828–834.
Osuji GO, Ory RL. 1987. Regulation of allantoin and allantoic acid degradation in the yam and sweet potato. Journal of Agricultural and Food Chemistry 35, 219–223.[CrossRef]
Piedras P, Muñoz A, Aguilar M, Pineda M. 2000. Allantoate amidinohydrolase (allantoicase) from Chlamydomonas rein hardtii: its purification and catalytic and molecular characteriza tion. Archives of Biochemistry and Biophysics 378, 340–348.[CrossRef][Web of Science][Medline]
Purcell LC, King CA, Ball RA, 2000. Soybean cultivar differences in ureides and the relationship to drought-tolerant nitrogen fixation and manganese nutrition. Crop Science 40, 1062–1070.
Reddy RS, Rao NV, Sastry KS. 1989. Acetohydroxamate, a competitive inhibitor of allantoinase of Vigna radiata. Phyto chemistry 28, 47–49.
Serraj R, Sinclair TR. 2003. Evidence that carbon dioxide enrichment alleviates ureide-induced decline of nodule nitro genase activity. Annals of Botany 91, 85–89.
Shelp BJ, Ireland RJ. 1985. Ureide metabolism in leaves of nitrogen-fixing soybean plants. Plant Physiology 77, 779–783.
Sinclair TR, Purcell LC, Vadez V, Serraj R, King CA, Nelson R. 2000. Identification of soybean genotypes with N2 fixation tolerance to water deficits. Crop Science 40, 1803–1809.
Sinclair TR, Vadez V, Chenu K. 2003. Ureide accumulation in response to Mn nutrition by eight soybean genotypes with N2 fixation tolerance to soil drying. Crop Science 43, 592–597.
Stebbins N, Holland MA, Cianzio SR, Polacco JC. 1991. Genetic tests of the roles of the embryonic ureases of soybean. Plant Physiology 97, 1004–1010.
Stebbins NE, Polacco JC. 1995. Urease is not essential for ureide degradation in soybean. Plant Physiology 109, 169–175.[Abstract]
Streeter JG. 2003. Effects of drought on nitrogen fixation in soybean root nodules. Plant, Cell and Environment 26, 1199–1204.[CrossRef]
Thomas RJ, Schrader LE. 1981. Assimilation of ureides in shoot tissues of soybeans. Plant Physiology 67, 973–976.
Vadez V, Sinclair TR. 2000. Ureide degradation pathways in intact soybean leaves. Journal of Experimental Botany 51, 1459–1465.
Vadez V, Sinclair TR. 2001. Leaf ureide degradation and N2 fixation tolerance to water deficit in soybean. Journal of Experimental Botany 52, 153–159.
Von Anderegg G, Epplattenier FL, Schartzenbach G. 1963. Hydroxamatkomplexe. II. Dir Anwendanzder pH method. Helvetica Chimica Acta 46, 1400–1408.[CrossRef]
Vogels GD, van der Drift C. 1970. Differential analysis of glyoxylate derivatives. Analytical Biochemistry 33, 143–157.[CrossRef][Web of Science][Medline]
Wells XE, Lees EM. 1991. Ureidoglycolate amidohydrolase from developing French bean fruits (Phaseolus vulgaris [L.]). Archives of Biochemistry and Biophysics 287, 151–159.[CrossRef][Web of Science][Medline]
Winkler RG, Blevins DG, Polacco JC, Randall DD. 1987. Ureide catabolism of soybeans. II. Pathway of catabolism in intact leaf tissue. Plant Physiology 83, 585–591.
Winkler RG, Blevins DG, Polacco JC, Randall DD. 1988a. Ureide catabolism in nitrogen-fixing legumes. Trends in Biochemical Sciences 13, 97–100.[CrossRef][Web of Science][Medline]
Winkler RG, Blevins DG, Randall DD. 1988b. Ureide catabolism in soybeans. III. Ureidoglycolate amidohydrolase and allantoate amidohydrolase are activities of an allantoate degrading complex. Plant Physiology 86, 1084–1088.
Winkler RG, Polacco JC, Blevins DG, Randall DD. 1985. Enzymatic degradation of allantoate in developing soybeans. Plant Physiology 79, 787–793.
Zonia LE, Stebbins NE, Polacco JC. 1995. Essential role of urease in germination of nitrogen-limited Arabidopsis thaliana seeds. Plant Physiology 107, 1097–1103.[Abstract]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  A. K. Werner, I. A. Sparkes, T. Romeis, and C.-P. Witte Identification, Biochemical Characterization, and Subcellular Localization of Allantoate Amidohydrolases from Arabidopsis and Soybean Plant Physiology, February 1, 2008; 146(2): 418 - 430. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Ladrera, D. Marino, E. Larrainzar, E. M. Gonzalez, and C. Arrese-Igor 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 Plant Physiology, October 1, 2007; 145(2): 539 - 546. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. D. Todd, P. A. Tipton, D. G. Blevins, P. Piedras, M. Pineda, and J. C. Polacco Update on ureide degradation in legumes J. Exp. Bot., January 1, 2006; 57(1): 5 - 12. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C.-P. Witte, M. G. Rosso, and T. Romeis Identification of Three Urease Accessory Proteins That Are Required for Urease Activation in Arabidopsis Plant Physiology, November 1, 2005; 139(3): 1155 - 1162. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. L. IZAGUIRRE-MAYORAL and T. R. SINCLAIR Soybean Genotypic Difference in Growth, Nutrient Accumulation and Ultrastructure in Response to Manganese and Iron Supply in Solution Culture Ann. Bot., July 1, 2005; 96(1): 149 - 158. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. A. King and L. C. Purcell Inhibition of N2 Fixation in Soybean Is Associated with Elevated Ureides and Amino Acids Plant Physiology, April 1, 2005; 137(4): 1389 - 1396. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||