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USDA-ARS, Agronomy Dep., Agronomy Physiology Lab., IFAS Building 350, 2005 SW 23rd Street, Univ. of Florida, P.O. Box110965, Gainesville, FL 32611-0965 USA
* Corresponding author (trsincl{at}gnv.ifas.ufl.edu)
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It has been reported that there are two pathways for the degradation of allantoic acid in soybean leaves. One, allantoate amidohydrolase (E.C. 3.5.3.9), is dependent on Mn as a cofactor (Winkler et al., 1987), while the other, allantoate amidinohydrolase (E.C. 3.5.3.4), is not dependent on Mn (Shelp and Ireland, 1985). The involvement of Mn in the response to water deficit and the regulation of ureide levels in leaves of some soybean cultivars has been documented in experiments in which increased Mn supply increased leaf ureide degradation rates and ameliorated N2 fixation sensitivity to water deficit (Purcell et al., 2000; Vadez et al., 2000).
Not all soybean cultivars, however, exhibit high sensitivity of N2 fixation to water deficit. The cultivar Jackson was discovered to have substantially greater tolerance of N2 fixation to soil drying than other soybean cultivars (Sall and Sinclair, 1991). The N2 fixation in Jackson was no more sensitive to drying soil than was transpiration. Several studies have confirmed the tolerance of N2 fixation in Jackson to water deficits and have shown that leaf ureide concentrations in Jackson are relatively low under water deficits (Serraj and Sinclair, 1996a; Purcell et al., 1997). The basis for the low ureide concentrations in Jackson, however, is still not resolved.
There are at least two possible hypotheses based on degradation of ureides in the leaves as water deficits develop to explain the low ureide concentrations in Jackson. One possibility is that the activity of allantoate amidohydrolase, which requires Mn as a cofactor, is sustained as a result of unusually high Mn concentrations in the leaves of Jackson. This assumes that there may be greater concentrations of unbound Mn in the leaves of Jackson than other cultivars. Higher Mn levels might support sustained allantoate amidohydrolase activity under water-deficit conditions. There are, in fact, preliminary reports that Jackson has leaf Mn concentrations greater than other cultivars (Purcell et al., 2000; Vadez and Sinclair, 2001b).
A second possibility is that Jackson may depend heavily on allantoate amidinohydrolase, which does not require Mn as a cofactor, as the main enzyme for ureide degradation in the leaves. Vadez and Sinclair (2001a) found that N2 fixation tolerance to water deficits among a limited number of cultivars was associated with ureide degradation that did not require Mn. They suggested that allantoate amidinohydrolase was likely active in the tolerant cultivars and this could be associated with the expression of N2 fixation drought tolerance. Their study did not include Jackson so it may be possible that the tolerance of Jackson is related to the presence of this alternate ureide degradation enzyme.
The objective of this study was to resolve these two competing hypotheses for explaining the N2 fixation tolerance of Jackson to water deficits. Experiments are presented that measured the leaf Mn concentration of Jackson under controlled and field conditions and the relationship of Mn concentrations to leaf ureide accumulation and degradation. These experiments included well-watered and water-deficit treatments. Additionally, data are presented that indicate the sensitivity of N2 fixation and ureide accumulation in Jackson to ureide added to the nutrient solutions of various Mn concentrations.
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MATERIALS AND METHODS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In Exp. 1, 20 plants each of Jackson and Biloxi were grown for 2 wk on the hydroponic nutrient solution containing an adequate Mn concentration of 6.6 µM Mn. Then, for the following 3 wk, plants were divided among nutrient solution treatments containing 0, 6.6, 26.4, or 52.8 µM Mn (8 plants on 6.6 µM Mn and 4 plants on each of the other concentrations). At 5 wk after transplanting, plants grown at 0, 26.4, 52.8 µM Mn, and half of the plants grown at 6.6 µM Mn were treated by replacing the nutrient solution with 400 mL of nutrient solution containing 5 mM allantoic acid and the respective Mn concentrations. Over the next 5 d, solution uptake by the plants was compensated by adding distilled water as required to keep the water level constant and, therefore, the allantoic acid concentration in the nutrient solution decreased during the experiment.
ARA was measured every afternoon on Day -1, 0, 1, 2, 3, 4, 5, and 6 after the initiation of the ureide treatment by introducing acetylene into the air flow into each flask. The acetylene concentration in the air flow was at 10% and 10 min were allowed for equilibration. Gas samples were immediately collected after the equilibration from the exhaust of each flask and analyzed for ethylene concentration with a gas chromatograph equipped with a flame ionization detector (Model GC-8A, Shimadzu Corp., Columbia, MD). The exposure time of the plants to acetylene was less than 15 min and this short-exposure technique allows measurements of ARA over a number of days without any adverse effects (Vadez and Sinclair, 2000). The ARA for the individual plants treated with ureide were normalized against the mean ARA of those plants grown on 6.6 µM Mn but not treated with allantoic acid. This normalization was done to eliminate variations resulting from changes in the greenhouse environment or plants during the experiment. The leaves were harvested from each plant immediately after the last ARA measurement, dried for 2 d in an oven at 60°C, and then ground for Mn determination.
Experiment 2 was a repeat of Exp. 1 except that intensive measurements of leaf ureide concentrations and degradation rates were made during the period of allantoic acid addition to the nutrient solution. Biloxi and Jackson plants were grown for 4 wk on a hydroponic nutrient solution containing 0.33, 6.6, or 26.4 µM Mn. Eight plants per genotype were grown on 0.33 and 6.6 µM Mn and four plants per cultivar were grown on 26.4 µM Mn. Half of the plants grown on 0.33 and 6.6 µM Mn were treated with ureide at 4 wk after transplanting, by again replacing the nutrient solution with 400 mL of the same solution with the respective Mn nutrition plus 5 mM allantoic acid. ARA was measured every afternoon on Day -1, 0, 1, 2, 3, 4, and 5 after the initiation of the ureide treatment and normalized against the mean of the untreated plants. Leaf disk samples, consisting of a 1.6-cm leaf disk obtained from each of the three blades of the most recently expanded trifoliolate leaf, were harvested for immediate ureide extraction at 0, 1, 2, 3, and 4 d after the initiation of the ureide treatment. No leaf disks were sampled on plants grown with 26.4 µM Mn. In addition, a leaf ureide degradation measurement (see description below) was carried out on plants grown with 0.33, 6.6, and 26.4 µM Mn that were not treated with ureide. Finally, leaves were harvested immediately after the final leaf disk sampling, dried for 2 d in an oven at 60°C, and then ground for Mn determination.
The procedures for the ureide degradation test were described in detail by Vadez and Sinclair (2000). The top-most fully expanded leaves were harvested by detaching the petiole at the stem. The petiole was placed in a test tube, one leaf per tube, containing either 5 mM allantoic acid or 5 mM allantoic acid + 50 µM MnSO4, and incubated under a combination of sodium and metal halide lamps (
500 µmol m-2 s-1, Sun-brella, Environmental Growth Chambers, Chagrin, OH) for a 12-h period. Following the incubation, six leaf samples, each consisting of three 1.6-cm diameter leaf disks collected from each of the blades, were obtained from each trifoliolate leaf. The samples were incubated in 2-mL extraction vials under the same light conditions as were the intact leaves. At regular intervals during this second incubation (0, 1.5, 3, 4.5, 6, and 7.5 h), the ureide degradation was stopped in individual vials by adding 0.2 M NaOH and placing the vial in a boiling water bath for 30 min. This was also the first step in the ureide extraction procedure (see below). Ureide concentration of successive leaf samples was regressed over time to obtain an estimate of the ureide degradation rate (µmol h-1 g-1 FW).
Soil Tests
Leaf Mn concentrations under two watering treatments were compared among Jackson, Biloxi, and three genotypes (PI374163, PI423886, and PI507039) that had been previously identified as having the non-Mn-dependent enzyme for ureide degradation (Valdez and Sinclair, 2001a). Soybean plants were grown in 10-cm diameter and 30-cm high polyvinylchloride pots. The pots were filled with about 1.2 kg of soil that was a 2:1 mixture (v/v) of potting soil (Vitagreen, Clermont, FL) and a vegetable plug mix (W.R. Grace & Co., Cambridge, MA). Each pot was inoculated with a commercial preparation of Bradyrhizobium japonicum (Lipha Tech, Milwaukee, WI). The potting mixture had already been used to grow other soybean plants and was low in Mn. The pots were grown under the same greenhouse conditions as the hydroponic experiments.
Two seeds per pot were sown and thinned after emergence leaving the most vigorous plant in each pot. Plants were grown under well-watered conditions for 5 wk before initiating a dry-down experiment. The dehydration cycle lasted 11 d until transpiration rates of the water-deficit plants was below 10% of that of the well-watered plants. In this dry-down experiment, five water-deficit and four well-watered plants per genotype were used. At the end of the dry down, leaves were harvested, dried for 2 d in an oven at 60°C, and then ground for Mn determination.
A field experiment subjecting plants to various soil Mn treatments, as described in detail by Vadez and Sinclair (2001b), was performed at the Green Acres Farm, University of Florida, Gainesville, FL. The soil type is classified as Kendrick fine sand (loamy, siliceous, hyperthermic arenic paleudult; USDA taxonomy). The objective of the trial was to study leaf Mn concentrations in response to Mn application (0 or 30 kg Mn ha-1) in interaction with watering treatments (water-deficit or well-watered condition). This experiment included Jackson plus two additional tolerant cultivars (PI374163 and PI423886) and two sensitive cultivars (Lee and Biloxi). Lee was included because its N2 fixation is sensitive to water deficit, and its growth is similar to that of Jackson (L.C. Purcell, University of Arkansas, personal communication).
The field was fertilized and treated with nematicide (Counter, American Cyanamid Co., Wayne, NJ) and herbicide (Trifluralin—
,,-trifluoro-2,6-dinitro-N,N-dipropyl-p-toluidine, United Horticultural Supply, Fremont, NE) on 24 and 25 March 1999. The field was low in Mn and fertilization with 80 kg K ha-1 and 40 kg P ha-1 added virtually no Mn. The field experiment was sown on 30 March 1999 with a push-planter. Manganese treatments were applied on 1 April 1999 in the form of manganese sulfate that was dissolved in water and applied with a portable backpack sprayer to each plot. The field was irrigated on 2 April 1999. A commercial inoculant (Urbana Laboratories, St. Joseph, MO) was surface applied followed by irrigation on 6 April 1999. The experimental design was a split plot with irrigated and nonirrigated treatments as the main blocks, and each block contained four replicate genotype x Mn treatments. Because of limited seed, each cultivar subplot was either six 6-m rows (Jackson and Lee), or four 6-m rows (PI374163, PI423886, and Biloxi). The rows were spaced 0.25 m apart with 0.10 m between plants within row.
The water treatment started on 11 May 1999, after which the well-watered plot continued to be irrigated regularly and the water-deficit treatment was irrigated with only small amounts of water (11 mm or less per application), to avoid severe stress, upon visible signs of leaf wilting. Because of the long daylength during this experiment, the plants were in vegetative development throughout the experiment. On 2 June, the top-most fully expanded leaves from three to four plants were harvested and combined to form a tissue sample from each water x Mn x genotype replicate subplot. The samples were dried for 3 d in an oven at 60°C, and then ground for Mn determination.
Chemical Measurements and Data Analysis
A colorimetric method was used (Trijbels and Vogel, 1966) to measure ureide levels. Ureides were extracted by boiling leaf samples in 1 mL of 0.2 M NaOH for 30 min. Samples were centrifuged, put into a refrigerator overnight, and 0.2 to 0.3 mL of supernatant used for ureide determination.
Mn was measured in leaf tissue samples by ashing a 1-g subsample overnight at 500°C in a furnace. The ashes were resuspended with 2 mL of concentrated hydrochloric acid and 20 mL of deionized water. The crucibles were put onto a hot plate and slowly boiled to dryness. The resuspension procedure was repeated and brought to a vigorous boil before reaching dryness and then being cooled to room temperature. This sample was brought to volume in 50-mL volumetric flasks, then filtered and a subsample taken for Mn analysis with an atomic absorption spectrophotometer (Model 3030B, Perkin-Elmer, Norwalk, CT).
Statistical comparisons among all cultivars were done by a Duncan multiple range test with a 0.05 level of significance. A linear regression was used to analyze the response of ARA to variations in leaf Mn concentration.
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RESULTS |
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Soil Tests
In the former experiments, plants were grown on nutrient solutions, and thereby, under well-watered conditions. An experiment using soil in pots was performed to investigate the response to water deficit and leaf Mn concentration of various cultivars. The leaf Mn concentrations obtained in this experiment were much less than generally observed in the hydroponic experiments. Under well-watered conditions, the leaf Mn concentration was not different between Biloxi and Jackson, while under water-deficit conditions, Jackson had a higher leaf Mn concentration than the sensitive Biloxi (Table 3). In each of the two watering treatments, two of three plant introductions had leaf Mn concentrations that were not different from Jackson. All three N2 fixation tolerant plant introductions had leaf Mn concentrations that were not significantly different from Biloxi.
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DISCUSSION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Tolerant genotype Jackson was able to maintain higher leaf Mn concentration than Biloxi when grown on hydroponic solutions containing between 6.6 to 26.4 µM Mn (Tables 1 and 2). On solutions with very low Mn availability (0 and 0.33 µM Mn), however, Jackson had Mn concentrations not different from Biloxi. This result may have developed because there was simply too little available Mn to allow Jackson to express superior accumulation. At the other extreme, high Mn availability in the nutrient solution (52.8 µM Mn), Jackson also had leaf Mn concentrations similar to Biloxi (Table 1), which appears to have resulted from very high uptake by Biloxi under these conditions (Table 1).
The greater leaf Mn concentration of Jackson under moderate Mn availability was supported in pot experiments done with soil. Even though the pot experiment resulted in low leaf Mn concentrations, Jackson tended to have leaf Mn concentrations greater than the other cultivars (Table 3). The difference between Jackson and other cultivars was clearly observed in the field experiment in leaves collected from well-watered plots (Table 4). These results are consistent with the field experiments of Purcell et al. (2000) in which leaf Mn concentrations of Jackson were greater than KS4895. No statistical difference in leaf Mn concentration among cultivars other than Jackson was observed from plants in the well-watered treatment, which is consistent with the report of Parker et al. (1981) showing little genotypic variation in leaf Mn when grown on sufficient Mn.
The fact that Jackson generally had higher leaf Mn concentrations than other cultivars may indicate a possibility for increased capacity for ureide degradation and improved N2 fixation tolerance to water deficit. That is, higher leaf Mn concentrations in Jackson could hypothetically be beneficial in maintaining higher rates of ureide degradation because of the need for Mn as a cofactor by allantoate amidohydrolase (Winkler et al., 1987). In fact, Jackson had rates of ureide degradation that were not different from, and even lower under 26.4 µM Mn, than those in Biloxi (Table 2). Especially crucial was that Jackson showed no changes in the rate of ureide degradation with increased Mn supply to the nutrient solution, whereas the ureide degradation rate doubled in Biloxi upon supplemental Mn nutrition. This occurred even though the leaf Mn concentration of Jackson was not different from Biloxi under deficient Mn nutrition. The differences between Jackson and Biloxi in response of ureide degradation rates to leaf Mn concentration is consistent with the accumulation of ureide in leaves when ureide was added to the nutrient solution (Fig. 3). Jackson exhibited no difference in ureide accumulation between the two concentrations of Mn in the nutrient solution on which the plants were grown in contrast to the ureide concentration differences between Mn treatments found in Biloxi. Therefore these experiments failed to indicate an increased Mn degradation in Jackson as a result of higher leaf Mn concentrations.
The stability of leaf ureide degradation with varying leaf Mn concentrations in Jackson, in contrast to Biloxi, was also reflected in the recovery of ARA following the addition of ureide to the nutrient solution. Jackson showed substantial recovery of ARA following ureide treatment even with 0 µM Mn in the nutrient solution (Fig. 1). Combining all data from Exp. 1 and 2 showed that Jackson had a pattern of recovery from ureide treatment that was unaffected by leaf Mn concentration, unlike Biloxi (Fig. 2). These results indicate that ureide degradation in the leaves of Jackson may be predominantly by allantoate amidinohydrolase, which is not dependent on Mn. This conclusion is consistent with the evidence for the presence of allantoate amidinohydrolase in other cultivars with drought-tolerant N2 fixation (Vadez and Sinclair, 2001a). The observed greater accumulation of Mn in leaves of Jackson appears not to be directly related to ureide degradation and N2 fixation tolerance to water deficit.
In conclusion, these experiments confirmed that under most conditions Jackson can accumulate higher concentrations of Mn in the leaves than other tested cultivars. The hypothesis was rejected, however, that Jackson's advantage of drought-tolerant N2 fixation was a result of increased leaf Mn concentration which allowed sustained Mn-dependent catabolism of ureides in leaves. Indeed, the results of these studies indicate that ureide catabolism in Jackson is likely dependent on allantoate amidinohydrolase, which does not require Mn as a cofactor. This conclusion is consistent with the observation of Vadez and Sinclair (2001a) that cultivars with N2 fixation tolerant of water deficit tend to rely on the allantoate amidinohydrolase pathway for ureide degradation.
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ACKNOWLEDGMENTS |
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Received for publication March 15, 2001.
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REFERENCES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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