Journal of Experimental Botany, Vol. 51, No. 349, pp. 1459-1465,
August 2000
© 2000 Oxford University Press
Ureide degradation pathways in intact soybean leaves1
USDA-ARS, Agronomy Department, Agronomy Physiology Laboratory, IFAS Building 350, University of Florida, PO Box 110965, Gainesville, FL 32611–0965, USA
Received 11 February 2000; Accepted 3 April 2000
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Abstract |
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Ureides dramatically accumulate in shoots of N2-fixing soybean (Glycine max L. Merr.) under water deficit and this accumulation is higher in cultivars that have N2 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 N2 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 N2 fixation activity.
Key words: Ureide degradation, allantoate amidohydrolase, boric acid, water deficit.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Ureides, allantoin and allantoic acid, are the major nitrogenous compounds synthesized in soybean nodules and are transported via the xylem to the shoot where they are degraded. Shoot ureide concentrations are usually about 5–10 µmol g-1 dry weight. Ureide concentrations in the shoot accumulate dramatically, however, when plants are subjected to water deficit (Serraj and Sinclair, 1996
), even though at the same time nodule activity is very much decreased. The accumulation of ureides in the shoot might result from a decreased ureide degradation under water deficit, and could be linked to a feedback inhibition of nitrogenase activity (Parsons et al., 1993; Serraj et al., 1999). Such a sequence might also be important in explaining the great sensitivity in soybean of nitrogen fixation to water deficit compared to other physiological processes (Durand et al., 1987; Sinclair et al., 1987). Since cultivars with tolerant nitrogen fixation accumulate less ureides in the shoot under water deficit than sensitive ones (Serraj and Sinclair, 1996; Purcell et al., 1996), ureide degradation is potentially important in understanding the nitrogen fixation response to water deficit in soybean.
There are two initial steps in the degradation of ureides. Allantoin is first degraded into allantoic acid by allantoinase (EC 3.5.2.5.). The subsequent step of allantoic acid degradation in soybean has been attributed to one of two enzymatic pathways: allantoate amidinohydrolase (EC 3.5.3.4) (Shelp et al., 1984), and allantoate amidohydrolase (EC 3.5.3.9) (Winkler et al., 1987; Stebbins and Polacco, 1995). This latter enzyme was found to be manganese (Mn) dependent (Winkler et al., 1987; Lukaszewski et al., 1992). Consistent with the presence of the allantoate amidohydrolase pathway, it was found that increased Mn availability in the leaves of the soybean cultivar Biloxi resulted in enhanced rates of ureide degradation (Vadez et al., 2000). Increased levels of leaf Mn were also found in their study to result in lower leaf ureide concentrations and less of a decrease in ARA when the plants were fed ureide.
The relative importance of the two putative pathways for ureide degradation is unresolved, although it is striking that evidence provided for each pathway was gathered on different soybean genotypes (the cultivar Maple Arrow for allantoate amidinohydrolase and cultivar Williams for allantoate amidohydrolase). Therefore, it is possible that the existence of the pathways may differ among genotypes, or even both pathways may coexist in some soybean genotypes. Resolution of the controversy concerning the two pathways has been difficult because the methods provided thus far to measure allantoic degradation in leaves are either in vitro aimed at isolating allantoate amidohydrolase (Winkler et al., 1985), or in vivo based on by-products of the allantoate amidinohydrolase pathway (Shelp and Ireland, 1985). Furthermore, in any eventual efforts to compare large numbers of genotypes these methods are not readily applicable for processing a large number of samples.
The objective of this research was to compare the degradation pathways of ureides in the soybean cultivars Williams and Maple Arrow. A key basis for this comparison required measurements of in situ degradation rates of ureides in response to experimental treatments. An analytical technique was developed that involved feeding detached leaves a solution of allantoic acid, removing the leaf tissue from the allantoic source, and then monitoring the subsequent decline in the level of ureide in the leaves. A key in detecting the two degradation pathways was a comparison of the in situ degradation rate in response to variation in Mn availability.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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In situ ureide degradation methodology
Leaves harvested from the soybean cultivar Biloxi were used in developing the in situ ureide degradation protocol. The plants were grown in well-watered conditions in 2 l plastic pots in the greenhouse, with day/night temperatures of about 28/20 °C and a photoperiod close to 14 h. The pots were filled with a 2 : 1 mixture (v/v) of potting soil (Vitagreen, Clermont, FL) and a vegetable plug mix (WR Grace & Co., Cambridge, MA). Each pot contained about 2 kg of soil and was inoculated with a commercial preparation of Bradyrhizobium japonicum (Nitragin, Milwaukee, WI). Three seeds per pot were initially seeded and thinned to two per pot after plant emergence. Leaves were collected for the in situ ureide degradation test when the plants were 5–12 weeks old and still in vegetative development. Several sets of plants were grown to provide the required leaves.
As in Shelp and Ireland (Shelp and Ireland, 1985), the two most recent fully expanded leaves were taken by detaching the petiole with a surgical blade at the stem. The petioles of the harvested leaves were placed in cold distilled water in the dark and transported from the greenhouse to the laboratory. Then, the base of the petiole was recut under water and each leaf was placed under artificial light in a 60 ml test tube containing the allantoic acid feeding solution. The artificial ligh (>500 µmol m-2 s-1) was provided by a Sun-Brella (Environmental Growth Chambers, Chagrin Falls, OH) which consisted of a multi-vapour metal halide lamp (General Electric No E-37) and a high pressure sodium lamp (General Electric No E-18) set in a water-cooled reflector housing. Test tubes were weighed before and after the feeding period to quantify leaf transpiration during the feeding.
The in situ method for estimating the rate of ureide degradation in leaves involved first feeding allantoic acid to detached soybean leaves for 12 h. It was found that feeding leaves of 5, 7.5 or 10 mM allantoic acid had no significant effect on the subsequent rate of degradation. Supplying leaves with 20 mM resulted in leaf burning and a degradation rate that was not significantly different from zero (P<0.05). Since increased variability in degradation rate was obtained when supplying 10 mM allantoic acid, a feeding solution of 7.5 mM allantoic acid was used in most experiments.
At the end of the feeding period, the leaves were incubated in distilled water under light. During this incubation period, the ureide concentration was measured in 1.6 cm diameter leaf discs at successive intervals to determine the rate of ureide loss. An initial test was done on the influence of the irradiance during the incubation period on degradation rate. Exposure of leaves to 1000 µmol m-2 s-1 resulted in rapid degradation rates. At an exposure of about 500 µmol m-2 s-1 the degradation rate was decreased to about half. An exposure of 500 µmol m-2 s-1 was used in all subsequent tests to ensure a linear degradation over at least an 8 h period.
Another important issue was the potential variability in ureide concentration among the 1.6 cm diameter leaf disc samples as a result of the position within the leaf from which the disc was harvested. Variability in leaf ureide concentration was found to be small within each blade of the trifoliolate leaf, but larger differences in ureide concentration existed among the blades. To eliminate this source of variation in the degradation data, a sample at each time period consisted of three discs with one disc harvested from each blade. This usually allowed six samples to be harvested during the incubation period.
Each three-disc sample was weighed to determine sample fresh weight and remnant ureide was immediately extracted in 2 ml vials with 1 ml of 0.2 M NaOH in a boiling water bath for 30 min. Samples were centrifuged and stored at 4 °C in the refrigerator until measurement. Remnant ureide was measured using the colorimetric method from Trijbel and Vogel (Trijbel and Vogel, 1966) on a 0.2–0.3 ml subsample of the supernatant. Leaf disc ureide concentrations were regressed as a linear function of time where the slope and the intercept corresponded to the ureide degradation rate and the initial ureide concentration, respectively. Slopes and intercepts were calculated for each individual leaf with Sigma Plot statistical software options and differences between mean slopes of treatments were determined by t-test.
A potential problem associated with sampling discs from intact leaves over time during the degradation period was the transfer of ureide from the petiole to the leaf blade. An approach to eliminate the petiole during the degradation period was to sample all leaf discs at the end of the feeding period and allow degradation to proceed in the discs. At various times during the degradation period, degradation in disc samples was stopped and the ureide concentration measured. Sets of three disc samples from each leaf, usually six, were immediately collected following the ureide-feeding period and incubated either by floating them on water in Petri dishes with one sample per dish, or by simply placing them in the 2 ml vials used for ureide extraction with one sample per vial. The Petri dishes contained 5 ml distilled water and the vials contained 0.1 ml distilled water to prevent dessication of samples. The leaf discs were incubated over different periods of time under artificial light (>500 µmol m-2 s-1) and were sequentially harvested at time 0, 1, 2, 3, 4, and 5 h after collecting the leaf discs. The water was also sampled from the Petri dishes to estimate possible ureide leakage from the leaf discs. The degradation rate obtained by incubating leaf discs was compared to that obtained by incubating intact leaves.
Boric acid and asparagine (Asn) are inhibitors of allantoate amidohydrolase, probably because Mn is complexed by these two compounds and is less available as a co-factor for the enzyme (Winkler et al., 1987). It was investigated whether the in situ methodology was suitable to evaluate the response of these inhibitors of allantoate amidohydrolase as a potential approach for assessing the presence or absence of a Mn-dependent pathway for ureide degradation using the cultivar Biloxi. A range of boric acid concentrations from 0.5 to 5 mM was studied. Five replicate leaves were fed, for 13 h, a 7.5 mM allantoic acid solution that also contained one of the following boric acid concentrations: 0, 0.5, 1.0, 3.0, or 5.0 mM. Six leaf disc samples per leaf were collected after feeding and placed in 2 ml vials for 0, 1, 2, 3, 4 or 5 h before ureide extraction. Another experiment was carried out similarly with Asn concentrations of 10, 20 or 30 mM. Four replicate leaves were fed, for 13 h, a 7.5 mM allantoic acid solution containing each of the Asn concentrations. Four leaf disc samples were collected and incubated in 2 ml vials under artificial light for 0, 3, 6 or 8 h before ureide extraction.
Degradation in cultivars Williams and Maple Arrow
Leaf test:
The possibility of discriminating between the pathways identified in the cultivars Williams and Maple Arrow was tested using the in situ technique with leaves harvested from soil-grown plants as described previously. Of particular interest was the use of inhibitors to evaluate for the presence or absence of the Mn-dependent allantoate amidohydrolase (EC 3.5.3.9) for ureide degradation in the cultivar Williams (Lukaszewski et al., 1992) as compared to the cultivar Maple Arrow, which was reported to degrade allantoic acid through allantoate amidinohydrolase (EC 3.5.3.4) (Shelp and Ireland, 1985). Leaves harvested from each cultivar were fed a 7.5 mM allantoic acid solution, containing either 5 mM boric acid, 30 mM Asn or neither of these two compounds, under artificial light for 13 h. Then four leaf disc samples per leaf were collected and incubated in 2 ml vials and ncubated for 0, 3.5, 6.5, and 9.5 h after collecting the samples.
Plant test:
An experiment was undertaken with hydroponically grown plants of Williams and Maple Arrow to determine if Mn treatments could be applied to the whole plant and allow a differentiation among the two cultivars. Seeds of each cultivar were inoculated with bacteria (Nitragin, Milwaukee, WI) and germinated in soil. The seedlings were transplanted 1 week after sowing to 1 l Erlenmeyer flasks containing a nutrient solution with the following concentrations of macro and micro-elements: CaCl2 (3.3 mM), MgSO4 (2.05 mM), K2SO4 (1.25 mM), KH2PO4 (0.35 mM), H3BO3 (4 µM), ZnSO4 (1.55 µM), CuSO4 (1.55 µM), NaMoO4 (0.12 µM), FeEDTA (40 µM) (Kalia and Drevon, 1985). The nutrient solution for the first 2 weeks after transplanting contained 1 mM urea. Thereafter, the hydroponic solution was replaced each week and no urea applied. Manganese nutrition was altered so that half of the plants of each genotype were grown without Mn in the first 2 weeks after transplanting and thereafter grown on 0.33 µM Mn concentration in the nutrient solution, which has been shown to be deficient for growth of the soybean cultivar Bragg (Ohki, 1976). The other half of the plants received normal Mn nutrition, i.e. 6.6 µM. The pH of the solution was maintained close to 7.0 by adding 0.2 g l-1 CaCO3. Air was continuously bubbled through the solution at a flow rate of 2 l min-1 to keep the solution fully aerated (Serraj and Sinclair, 1996). The volume of nutrient solution in each flask was maintained at c. 500 ml so that most of the nodules were above the nutrient solution.
At 4 weeks after transplanting, 400 ml of a nutrient solution containing 3 mM allantoic acid was applied to the root system of half of the plants grown at each Mn concentration. The other half of the plants were not exposed to allantoic acid. There were four replicate plants in each treatment. Flasks were weighed at the initiation of the treatments and daily transpiration losses, about 150–200 ml d-1 regardless of treatment, were replaced with distilled water from day 2 after treatment. Leaf disc samples were collected for ureide measurement at 0, 1, 2, 3, and 4 d after treatment. The samples consisted of three 1.6 cm diameter leaf discs obtained by taking a single disc from each blade of the most recently fully expanded leaf. The entire plant was harvested at 5 d after treatment, separated into leaves, stem, root, and nodules, and oven-dried. Ureide concentrations in leaves and nodules were measured as described previously. Manganese levels in leaves and nodules were also measured following ashing, using atomic absorption spectrophotometry.
The consequences of the Mn treatments on N2 fixation activity in each cultivar were measured by acetylene reduction activity (ARA). The ARA was done by introducing 10% acetylene into the air bubbled into the hydroponic flasks. After allowing 10 min for equilibration, a gas sample was collected at the exhaust of the flask and ethylene concentration was measured. After collecting the gas sample, the acetylene flow into the flasks was discontinued and the normal flow of air through the hydroponic flask flushed the acetylene from the system. ARA measurements were made on days -1, 0, 1, 2, 3, 4, and 5 relative to the ureide treatment. The data within each cultivar and Mn treatment were normalized relative to the mean ARA values obtained on days -1 and 0.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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In situ ureide degradation methodology
Detached leaves absorbed between 6 and 7 ml of allantoic acid solution over a 12 h feeding period under the artificial light. Leaves also remained turgid and looked healthy throughout the experiment. As expected, the absorption of allantoic acid solution was closely related to leaf size and the average amount of solution absorbed was 22.8±1.4 ml g-1 leaf dry weight after 12 h. Feeding ureide to detached leaves resulted in a significant increase in leaf ureide concentration over time (Fig. 1
).
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) (µmol) and increase in leaf ureide concentration (•) (µmol g-1 FW) as a function of time upon ureide feeding with a 5 mM allantoic acid solution. Data were fitted to the following quadratic equations (P<0.05):An important question in the development of the in situ technique for ureide degradation was whether harvested leaf discs only could be incubated following ureide feeding, or must the leaves remain intact during the incubation period. A constant ureide degradation over a 5 h period was found for incubated discs, with no significant difference in the degradation rate between discs incubated in Petri dishes and those incubated in extraction vials (Fig. 2
). Further, a comparison of the degradation rates between discs placed in extraction vials and intact leaves resulted in no statistical difference (P<0.05). Therefore, all subsequent degradation measurements were made by harvesting all leaf discs immediately after ureide feeding and allowing the discs to incubate in extraction vials.
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). Regression equations were: Petri dish: X=17.0±1.8–(0.80±0.14)t; 2 ml vials: X=15.8±2.3–(0.77±0.13)t. Slope of ureide leaks in Petri dish incubation solution as a function of time was not significantly different from zero. Data are means (±SE) of five replicate leaves.The use of inhibitors of the Mn-dependent pathway of ureide degradation was explored as a potential methodology to evaluate for the presence or absence of this pathway. The addition of boric acid to the solution fed to leaves at a concentration of 3 mM and less had either no or only a partial inhibitory effect on ureide degradation in the cultivar Biloxi (Table 1
). By contrast, there was a total inhibition with 5 mM. This inhibitory effect of feeding 5 mM boric acid was confirmed in other experiments (data not shown). Boric acid concentrations as high as 40 mM were tested, but this concentration induced severe burning on leaves (data not shown) while 5 mM did not result in any visible effect on the leaves.
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A similar trial was done by feeding Asn as a potential inhibitor of ureide degradation. There was no effect of 10 or 20 mM Asn on the rate of ureide degradation and there may be partial inhibition of ureide degradation with 30 mM Asn, although degradation rates with 30 mM Asn were not significantly different from the degradation rate in control leaves (Table 1
).
Degradation in cultivars Williams and Maple Arrow
The presence or absence of the Mn-dependent allantoate amidohydrolase for ureide degradation was tested on leaves from the cultivars Williams and Maple Arrow. Ureide degradation rate in Williams was decreased to less than half by the 30 mM Asn treatment and was not significantly different from zero in leaves that were treated with 5 mM boric acid (Table 2). These responses in Williams to the inhibitors of allantoate amidohydrolase are consistent with the conclusion of Lukaszewski et al. that this enzyme is dominate in ureide degradation in this cultivar (Lukaszewski et al., 1992). On the other hand, the rate of ureide degradation for Maple Arrow was not significantly affected by the application of either 5 mM boric acid or 30 mM Asn in the allantoic acid feeding solution. This contrasting response in Maple Arrow is consistent with the conclusion of Shelp and Ireland that allantoate amidinohydrolase is the main ureide degradation enzyme in this cultivar (Shelp and Ireland, 1985).
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The growth of plants on hydroponic solution with two levels of Mn resulted in substantial differences in the Mn concentration between treatments of each cultivar (Table 3
). In spite of the large differences in leaf Mn concentration between treatments, there was very little difference in leaf ureide levels between treatments within each cultivar (Table 3). There was, however, a substantial difference between the two cultivars as Maple Arrow had much larger ureide levels than did Williams.
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In spite of the differences in leaf ureide levels, the ARA activity of Williams under both Mn treatments and Maple Arrow under the high Mn treatment had essentially identical ARA values (Table 3
). The ARA for Maple Arrow under low Mn had a lower ARA, which may be related to the lower ureide level in the leaves for this treatment, than when the cultivar was subjected to the high Mn treatment.
The feeding of ureide to the plants resulted in large increases in leaf ureide concentration and decreases in ARA (Figs 3, 4). Similar to the results of Vadez et al. (Vadez et al., 2000) with the cultivar Biloxi, the cultivar Williams tended to have greater normalized leaf ureide accumulation during ureide feeding for the 0.33 µM Mn treatment as compared to 6.6 µM Mn treatment, with a statistically significant difference after 4 d (Fig. 3). The difference in leaf ureide levels between Mn treatments was associated with the relative decrease in ARA during ureide feeding. The plants grown on 6.6 µM Mn had smaller decreases in ARA than those plants grown on 0.33 µM Mn (Fig. 3).
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In contrast to Williams, Maple Arrow had similar levels in leaf ureide accumulation and ARA decrease in response to the ureide feeding for both Mn treatments (Fig. 4
). That is, enhanced levels of Mn in the leaves did not decrease the accumulation of leaf ureide. The level of ureide accumulation and ARA decrease in Maple Arrow were similar to those of plants of Williams grown on the low Mn solution.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Significant ureide degradation in leaves was readily measured by feeding leaves allantoic acid, subsequently measuring remnant ureide concentration in leaf disc samples over time, and fitting the data to a linear regression line as a function of time. The degradation rates were in the range of 0.5–0.8 µmol g-1 FW h-1, similar to the values obtained using a complex vacuum infiltration technique (Shelp and Ireland, 1985
). These degradation rates were higher than those obtained involving in vivo incubation of ureide (Winkler et al., 1987; Atkins et al., 1982). The degradation rates reported for the in vitro method of Lukaszewski et al. were also much less than those obtained using the intact leaf technique (Lukaszewski et al., 1992).
Boric acid (5 mM) completely inhibited ureide degradation in Williams, which is reported to degrade allantoic acid through allantoate amidohydrolase (Table 2). By contrast, ureide degradation in Maple Arrow, which was reported to involve allantoate amidinohydrolase, showed no response to boric acid or Asn addition to the allantoic acid feeding solution. These leaf degradation experiments are consistent within each cultivar of the reported ureide degradation pathways. Hence, it appears that both pathways exist within the soybean germplasm.
Further evidence of the existence of different ureide degradation pathways in Williams and Maple Arrow was obtained by comparing intact plants grown on hydroponic solutions that varied in Mn levels. When these plants were fed ureide, the accumulation of ureide and response of ARA in Maple Arrow was not influenced by the Mn treatment (Fig. 4). This is consistent with a ureide pathway relying on allantoate amidinohydrolase, which does not require Mn as a cofactor. In contrast, ureide accumulation and ARA response in Williams during ureide feeding did differ between the Mn treatments (Fig. 3). This indicates the involvement of Mn as a cofactor in the degradation pathway relying on allantoate amidohydrolase in Williams.
In conclusion, the results from the newly developed leaf degradation technique and from intact plants grown hydroponically confirm that two pathways for ureide degradation exist within soybean. The basis for the conflicting conclusions concerning two differing degradation pathways for ureide reported in the literature appears to have resulted from the fact that the investigations were done on differing cultivars. Consequently, both pathways are extant within the soybean germplasm. Additional research is needed to elicit the relative advantage of each pathway for enhanced production of soybean, and to determine if selecting one pathway over another is advantageous under specific cropping conditions.
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Acknowledgments |
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We thank Dr J Harper for providing seed of cultivar Williams and Dr D Layzell for providing seed of cultivar Maple Arrow. This research was partially supported by United Soybean Board Project No. 8206P.
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Notes |
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1 Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the US Department of Agriculture and does not imply approval or the exclusion of other products that may also be suitable.
2 To whom correspondence should be addressed. Fax: +1 352 392 6139. E-mail: trsincl{at}gnv.ifas.ufl.edu
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References |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Atkins CA, Pate JS, Ritchie A, Peoples MB.1982. Metabolism and translocation of allantoin in ureide-producing grain legumes. Plant Physiology 70, 476–482.
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