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Published online 24 June 2005
Published in Crop Sci 45:1464-1468 (2005)
© 2005 Crop Science Society of America
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CROP PHYSIOLOGY & METABOLISM

Effects of Nitrogen and Calcium Supply on the Accumulation of Oxalate in Soybean Seeds

John G. Streeter*

Dep. of Horticulture and Crop Science, the Ohio State Univ./O.A.R.D.C., 1680 Madison Ave., Wooster, OH 44691

* Corresponding author (streeter.1{at}osu.edu)


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Soybean [Glycine max (L.) Merr.] seeds have sufficiently high oxalate concentration that they are not recommended for consumption by individuals prone to formation of kidney stones. One possible precursor of oxalate is glyoxylic acid and this acid is formed in soybean seeds as a product of ureide catabolism. A nodulating soybean variety was compared with its nonnodulating isoline and the lack of nodules resulted in significant decline in ureide supply to developing seeds. However, oxalate concentrations in the seeds of the isoline were not reduced, suggesting that oxalate is not formed via glyoxylate in soybean seeds. Because oxalate and Ca concentrations are often correlated, manipulation of Ca supply to soybean plants was also studied by increasing Ca supply during pod development by 3.5-fold relative to the level in the rooting medium. Surprisingly, there was no significant effect of Ca supply on Ca concentrations in leaves, pod walls, or seeds, indicating that Ca accumulation is stable in spite of Ca supply. The Ca treatment also did not significantly alter oxalate accumulation in developing seeds. In spite of this lack of treatment effect, the correlation of oxalate and Ca concentrations within sampling times was generally significant. Oxalate concentration was 2- to 3-fold higher in developing seeds than in mature seeds. It is concluded that (i) the correlation between Ca and oxalate in developing soybean seeds is probably related to unknown factors influencing oxalate accumulation and the subsequent binding of Ca by the oxalate accumulated, (ii) lowering oxalate in soybean seeds will most likely be achieved by selection of low-oxalate genotypes, and (iii) because oxalate concentrations are higher in immature soybean seeds than in mature seeds, those prone to formation of kidney stones should be cautious about consuming immature soybeans.

Abbreviations: CaOx, calcium oxalate • DAP, days after planting


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
OXALIC ACID is found in all plants and most of the compound is found as the calcium salt, which is essentially insoluble in water (Libert and Franceschi, 1987; Nakata, 2003). Calcium oxalate (CaOx) is the principle component of kidney stones (Mandel, 1996) and CaOx can be directly absorbed by the gut in spite of its insolubility (Hanes et al., 1999). For these reasons, the amount of CaOx in foods is of significant concern (Massey et al., 1993). The relative concentration of oxalate in many foods has been reported (Hönow and Hesse, 2002; Massey et al., 1993).

The content of oxalate in soybean seeds was reported to increase following fertilization to a peak at 16 d post-fertilization and then decline slightly to seed maturity (Ilarslan et al., 1997). The localization of oxalate deposits in various seed tissues has also been documented (Ilarslan et al., 2001). Recently, the concentration of insoluble oxalate in soybean seeds and soy foods was reported to be very high (Ilarslan et al., 1997; Massey et al., 2001). The latter authors concluded that "...no soybean or soyfood tested could be recommended for consumption by patients with a history of CaOx kidney stones." Also, "The amounts of total oxalate in soybean seeds, soy foods, and other common legume foods exceed current recommendations for oxalate consumption by individuals who have a history of CaOx kidney/urinary stones." An obvious question is what steps might be taken to lower the undesirable levels of CaOx in soybean seeds.

Studies on the biosynthesis of oxalate have come to different conclusions. In a 1987 review, Libert and Franceschi concluded that a major precursor of oxalate is glyoxylate. More recent studies suggest that oxalate is synthesized from ascorbate (Keates et al., 2000; reviewed in Nakata, 2003).

The ureides, allantoin and allantoic acid, were first identified as important nitrogenous compounds in soybean in 1970 by Ishizuka. Subsequently, it was shown that ureides are probably synthesized in root nodules (Fujihara and Yamaguchi, 1978). Enzymatic mechanisms for the biosynthesis in nodules were worked out (reviewed in Schubert, 1986) and the presence of large quantities of ureides in stem exudate during seed development confirmed the importance of these compounds in the nitrogen nutrition of nodulated soybean plants (Streeter, 1979). Ureides are delivered to pods (Matsumoto et al., 1977) before being broken down in the seed coat for transport of N to inner tissues of the developing seed (Winkler et al., 1988a). This degradation pathway in seed coats results in the formation of glyoxylate plus ammonium (Winkler et al., 1988a, 1988b). It has been suggested that oxalate accumulation in soybean seeds might be the result of ureide catabolism (Ilarslan et al., 1997). Thus, if glyoxylate is a precursor of oxalate in soybean, one strategy for lowering oxalate concentration in seeds might be to lower ureide transport to seeds.

A naturally occurring single locus recessive, nonnodulating mutant of soybean was reported by Williams and Lynch in 1954. Other nonnodulating genotypes have subsequently been isolated and one study showed that, in fact, the nonnodulating mutants contained much lower concentration of ureide in developing pods (Matsumoto et al., 1977). When provided with sufficient mineral N, the nonnodulating isolines were indistinguishable from the nodulated plants in terms of seed yield and protein concentration in seeds (Weber, 1966). A nonnodulating isoline of the soybean variety ‘Harosoy’ was employed in studies reported here.

Another possible strategy for lowering oxalate concentration in seeds would be to manipulate calcium concentration supplied to the plants. However, although several studies have reported a correlation between oxalate accumulation and Ca concentration (Massey et al., 2001; Mazen et al., 2003), it is not clear that Ca accumulation controls or even influences oxalate accumulation (McConn and Nakata, 2002). Also, although field soils in Ohio contain highly variable concentrations of calcium (Streeter et al., 1994), the extent to which it is possible to manipulate the Ca concentration in soil-grown soybean plants is not clear.

The objectives in these studies were to determine the influence of N supply (fixed versus combined N) on oxalate concentrations in soybean seeds and to determine whether it is possible to manipulate Ca concentration in seeds and, thereby, to influence oxalate accumulation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plant Growth
There were two greenhouse experiments. For the first, seeds of cv. Harosoy and the nonnodulating mutant of Harosoy soybeans were obtained from Dr. Randy Nelson at the USDA soybean germplasm collection at Univeristy of Illinois (rlnelson{at}uiuc.edu). The mutant, L65-1274 ("L65") does not form root nodules with any known species of rhizobia. For each genotype, five seeds were sown in 10 ceramic pots containing approximately 9 L of silica sand. Pots were autoclaved before use and seeds were surface sterilized and then inoculated with Bradyrhizobium japonicum strain USDA 110 after placement in the sand. (Commercial inoculant would be an acceptable substitute.) Seed quality was poor and germination averaged only about 60%, so that, for the six pots of each genotype, there were approximately three plants per pot.

After emergence, plants were supplied with a N-free nutrient solution (Streeter, 1989). L65 plants also received 100 mL of 5 mM NaNO3 daily during the week. However, L65 plants began to turn slightly yellow after about 4 wk and the N supplement was changed to 20 mM NH4NO3. L65 plants quickly became green and this supplemental N was continued for the remainder of the experiment. Harosoy plants, not supplied with any combined N, remained dark green throughout the experiment. At 90 d after planting, about 20 pods were removed from 20 different plants of each genotype, combined, frozen at –80°C, and freeze-dried. Dried pods were divided into pod walls and seeds, which were ground in a Wiley mill. At plant maturity, another 20 pods were combined, divided, and ground as above.

In the second experiment, soybean seeds, cv. Flint, were planted in pots containing 1.1 kg of a mixture of 250 g kg–1 local soil, 250 g kg–1 peat, and 500 g kg–1 Promix RX (Premier Horticulture, Riviere-du-Loup, Canada). This mixture was chosen because it provides very vigorous plants and contains only a small amount of water-soluble Ca, namely 155 mg Ca kg–1 of soil mix. Soil mixtures were not autoclaved, but seeds were inoculated with B. japonicum to ensure good nodulation. After emergence, plants were thinned to one per pot. Plants received daily irrigation with a commercial water-soluble fertilizer (Peters 20-20-20, Scotts Consumer Service, Marysville, OH) containing 20 mg each of N, P2O5, and K2O per liter. The amounts of Ca, Mg, and minor nutrients in this fertilizer are very small (<<0.05%).

Beginning 38 d after planting, plants were debranched twice per week to limit pod formation to the main stem and give a similar pod load among plants. Beginning 46 d after planting, each pot (plant) received a 100 mL supplement of either 5 mM CaCl2 or MgCl2 for 5 d per week. Ca or Mg supplements were continued for 6 wk, a total of 30 d, during which a total of about 600 mg of Ca was supplied per pot. Thus, the total amount of Ca supplied during the treatment period was approximately 3.5 times the water soluble Ca in the soil mix. The time when treatments were initiated corresponded to about midflowering and just before pod development. The Ca treatment was intended to increase Ca supply during reproductive development and the Mg treatment was a control to provide an equal amount of salt to each pot. The design was completely random. These plants were grown during the summer months without supplemental light and were dark green, and disease and insect free throughout the growth period. Plants were sampled beginning 67 d after planting and continuing to maturity when all leaves had dropped and pods were brown. There were five replicates (plants) per treatment and sampling time. Samples were frozen and freeze-dried and divided into plant parts as described above.

Extraction and Analysis of Plant Tissues
Oxalate Analysis
Various extraction methods were tried and a method was developed that is based on the procedure of Nakata and McConn (2003). Portions of dry, ground samples of 100 mg were weighed into Corex centrifuge tubes. Four milliliters of 50 mM HCl and a small amount (approx. 100 mg wet weight) of Dowex 50 ion exchange resin in the H+ form were added to each tube. Tubes were covered with Parafilm (American Can Co., Greenwich, CT), placed at a 45° angle in a test tube rack and shaken at 55°C and 270 rpm in an orbital shaker for 3 h. Tubes were turned upright and the solids were allowed to settle for a few minutes before sampling the cloudy liquid layer. (Tubes can be centrifuged, but achievement of a clear supernatant is difficult.) A 0.50-mL aliquot was transferred to a clean 9-mL test tube and 125 µL of 200 mM NaOH was added to neutralize the extract. (pH 7 is the target, but precise adjustment of pH is not necessary because the solution has very little buffering capacity and the analysis is done in buffer.)

All reagents were obtained from Sigma Chemical Co. St. Louis, MO. Oxalate analysis was done in screw-cap 2.0-mL microfuge tubes in duplicate mixtures. To 100 µL of sample or standard was added 1.7 mL of a solution containing 3.2 mM 3-(dimethylamino) benzoic acid (DMAB) and 0.22 mM 3-methyl-2-benzothiazolinone (MBTH) in 50 mM citrate buffer, pH 3.8. (This solution may not be completely clear, but precipitate is removed at a later step. Also, this reagent will develop color in storage over time and should be mixed fresh every few days.) To this mixture was added 200 µL of a solution containing 4.0 mg of oxalate oxidase plus 6.0 mg of peroxidase per 8.0 mL of citrate buffer. (This second reagent should be mixed by inversion and not vortexed; again the solution may not be completely clear.) A standard and blank were included with every batch of samples. Tubes were capped, mixed gently by inversion and incubated for 1 to 1.5 h at 30°C. Mixtures were microfuged at about 11000 g for 10 min and absorbance of the clear supernatant was measured at 590 nm. A standard of 25 nmole of oxalate (50 µL of a 0.50 mM solution) should give a net absorbance of about 0.40. In general, samples were extracted and analyzed once, but replicate analyses of the same dry ground tissue gave very similar results.

Ureide Analysis
To 100 mg of ground tissue in a 2.0-mL capped microfuge tube was added 1.6 mL of 75% (v/v) ethanol. After vortexing several times over a few hours, the tubes were centrifuged and the supernatant transferred to a 5-mL vial and dried under a stream of air at 35°C. The pellet was resuspended in 1 mL of 75% ethanol and, after additional vortexing and centrifugation, the second supernatant was added to the first. The extraction process was then repeated again, for a total of three extractions. After the combined ethanol extract was dried, solids were dissolved in 2.0 mL of pure water and a few drops of chloroform were added as a preservative. Portions of the aqueous layer were analyzed. The procedure for ureide analysis was as described by Young and Conway (1942) with minor modifications (Streeter, 1979). Only total ureide (sum of allantoin and allantoic acid) was analyzed.

Other Analyses
Mineral element concentration was determined in ashed dry samples by inductively coupled plasma emission spectroscopy (Streeter et al., 1994). Ascorbate concentration in seeds was determined by AOAC method 967.21 (AOAC, 2000). Dry ground tissue samples (200 mg) were suspended in 5.0 mL of a solution containing metaphosphoric acid and acetic acid and shaken at 30°C for 1 h. Ascorbic acid concentration in a 3.5-mL sample of the supernatant was determined by titration with a solution containing dichloroindophenol.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
In the first experiment, a nodulated soybean variety (Harosoy) was compared with its nonnodulating isoline L65. The ureide concentration in pod walls from immature plants of Harosoy was about 100-fold greater than in pod walls of the nonnodulated plants (Table 1). This confirmed that large amounts of ureides were being generated in nodules and were transported to developing pods. The amount of ureide in seeds was much less than the amount in pod walls; this would be predicted on the basis of the fact that ureide is broken down in the seed coat (Winkler et al., 1988a, 1988b). At maturity, seeds of the nodulated variety contained about four times as much ureide as seeds of the nonnodulated isoline, but, in both plant genotypes, the concentration of ureide was quite small.


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  		Table 1. Ureide and oxalate concentrations in immature and mature pod walls and seeds from nodulated and nonnodulated soybean plants.{dagger}

 
There was a substantial amount of oxalate in pod walls and seeds of both plant types at 90 DAP, when pods were immature (Table 1). The difference in plant types was significant, but, when both pod wall and seed oxalate concentrations are considered together, there was little difference between the two genotypes. Differences between genotypes here may relate to the fact that, because of N deficiency early in plant growth of L65, reproductive development was slightly delayed. At maturity, L65 seeds contained about 30% greater oxalate concentration and, in this case, stage of development was not involved.

In the second experiment, Ca supplied to each plant during reproductive development was about 3.5 times the amount of calcium in the rooting medium. In spite of this large supplement of Ca, there was no statistically significant effect of the Ca treatment relative to the control. Reproductive development of plants in the second experiment is documented in Table 2. At the first sampling time, pods were small, and there was very little seed dry weight. Only 17 d later, seed weight per plant had reached nearly 50% of maximum dry weight. At the third sampling, pods had achieved maximum weight but were still green. Calcium concentration in leaves and pod walls increased during reproductive development, but Ca concentration in seeds was essentially invariant (Table 2).


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  		Table 2. Changes in calcium and oxalate concentrations during soybean seed development.{dagger}

 
Between 67 DAP and 126 DAP, there was about a threefold decline in oxalate concentration in seeds. There was no significant effect of Ca supply on oxalate concentration in seeds. In spite of the lack of apparent treatment effects, there was variation among plants at each harvest in oxalate and Ca concentrations. Correlation analysis of these variations within sampling times showed a statistically significant positive relationship between Ca and oxalate concentration among individual plants, except the last sampling at 126 DAP. Oxalate concentration was not significantly correlated with the concentration of other elements at any sampling time (data not shown).

The concentration of ascorbic acid in ground seeds from the second experiment was analyzed for the last three sampling times. The ascorbic acid concentrations (nmole per mg dry weight; mean ± SE) were 2.13 ± 0.41, 1.23 ± 0.23, and 0.35 ± 0.01 at 84, 98, and 126 DAP, respectively. Ascorbic acid in this concentration range is difficult to quantify accurately and this is reflected in the standard errors. The ascorbate concentrations were on the order of 10% of the oxalate concentrations and there was no effect of the Ca supplement on ascorbate. The concentration of ascorbic acid declined during reproductive development and this decline was significantly correlated with the decline in oxalate concentration over the three sampling times (r = 0.599, based on all observations).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The hypothesis underlying the first experiment was that, by depressing ureide supply to developing seeds, glyoxylate supplied to seeds would be lowered, thus reducing the oxalate concentration in seeds. The use of a nonnodulating isoline of soybean was very effective in lowering ureide supply to pods, but there was little or no effect of ureide supply on oxalate accumulation. In fact, oxalate concentration in mature L65 seeds was higher than that in Harosoy seeds (Table 1). Because there is strong evidence that ureide breakdown in soybean seed coats leads to glyoxylate formation (Winkler et al., 1988a, 1988b), the results reported here provide indirect evidence that oxalate accumulation in soybean seeds is not via glyoxylate.

Instead, oxalate synthesis is probably via ascorbic acid (see Nakata, 2003). Ascorbate concentration in seeds was about 10% of the concentration of oxalate and ascorbate concentration declined between 84 DAP and maturity. However, the concentration of ureide also declined during maturation of pods (Table 1), so that the ascorbate decline over time is not strong evidence for a precursor–product relationship. Additional studies with labeled compounds will be required, but it is likely that ascorbate is the precursor of oxalate in soybean seeds.

When Ca was supplied to plants during pod development at a level of 3.5 times the available Ca in the soil mix, there was no statistically significant effect on Ca concentration in vegetative or reproductive plant parts. This surprising result suggests that Ca content of soil-grown soybean plants is relatively difficult to manipulate. Note that, while Ca concentration in leaves and pod walls was increasing during pod development, the Ca concentration in seeds remained relatively low and was unchanged (Table 2). Again, this suggests that Ca in seeds would be difficult to manipulate. Although Ca concentration in seeds remained invariant during pod development, the concentration of oxalate declined three-fold. Also, the concentration of Ca in seeds on a molar basis was about 60 nmole per mg dry weight– two to six times the concentration of oxalate, depending on the stage of development. These facts lead to the suggestion that oxalate concentration was not dependent on Ca accumulation in seeds. In other words, Ca levels in seeds appear to have been above a threshold concentration required for oxalate accumulation.

However, within sampling times, variations in seed oxalate and Ca concentrations among individual plants were significantly correlated (Table 2). This result is in agreement with results reported by others (Massey et al., 2001; Mazen et al., 2003, McConn and Nakata, 2002). The latter studies indicated that oxalate accumulation is probably not the result of Ca accumulation and this is supported by the fact that Ca concentrations on a molar basis were several times greater than oxalate concentrations, in the studies reported here. Because of the affinity of this dicarboxylic acid for Ca, the insolubility of the Ca salt, and because the correlation coefficient was significant in three out of four data sets in these studies, it is doubtful that the oxalate–Ca correlation is spurious. If the size of the Ca pool has no influence on oxalate accumulation, it is possible that the oxalate level in the seeds influences the accumulation of at least a portion of the Ca in the seeds.

In conclusion, the use of nonnodulating cultivars of soybean is not likely to influence the undesirable accumulation of oxalate in soybean seeds and soyfoods. It is likely that oxalate synthesis in soybean seeds is via ascorbate, not glyoxylate. Also, the possibility of growing soybeans on low Ca soils, thereby reducing oxalate accumulation, does not appear to be a viable strategy. Possibly, the best approach to limiting oxalate accumulation will be via selection of lower oxalate genotypes (Massey et al., 2001). Also, because of the plant-to-plant variation in oxalate accumulation seen in the second experiment and given the presumed genetic purity of these plants, additional studies are justified to try to establish the reason for this random variation. Finally, although the trends in oxalate accumulation during seed development are not exactly the same as the trends previously reported (Ilarslan et al., 2001), much higher oxalate concentrations in immature seeds were clearly evident in both studies. This result provides a cautionary note concerning consumption of immature soybean seeds (e.g., edamame) by those prone to kidney stone formation.


   ACKNOWLEDGMENTS
 
I thank Jim Sonowski and Rocio Aviles-Nava for technical assistance. This work was supported by funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University.

Received for publication April 1, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 




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