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CROP PHYSIOLOGY & METABOLISM

Soybean Cultivar Differences in Ureides and the Relationship to Drought Tolerant Nitrogen Fixation and Manganese Nutrition

Univ.of Arkansas, Dep. of Crop, Soil, and Environmental Sciences, 276 Altheimer Drive, Fayetteville, AR 72704 USA

lpurcell{at}comp.uark.edu

	ABSTRACT

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results Discussion REFERENCES

 
Water deficit in soybean [Glycine max (L.) Merr.] results in the accumulation of the products of N₂ fixation (ureides) in shoots, and this may lead to feedback inhibition of N₂ fixation. Manganese is required for ureide degradation in leaves, and it was hypothesized that increased leaf Mn⁺² would alleviate ureide accumulation during drought, lessen feedback inhibition, and prolong N₂ fixation. In a growth chamber experiment, ureides supplied through roots decreased N₂ fixation in well-watered plants, and a soybean cultivar with demonstrated tolerance of N₂ fixation to water deficit (Jackson) had a lesser concentration of shoot ureides following exogenous ureide application than a cultivar sensitive to water deficit (KS4895). In a greenhouse experiment, N₂ fixation in Jackson under moderate water deficit was not different from the control in the absence or presence of soil-applied Mn⁺², whereas N₂ fixation in KS4895 was 30% of the control in the absence of soil-applied Mn⁺² and 111% of the control in the presence of soil-applied Mn⁺². Increased N₂ fixation in KS4895 with soil-applied Mn⁺² under water deficit was associated with decreased shoot ureide concentration. In field experiments, Jackson consistently had lesser shoot ureide concentrations than did KS4895, and enzymatic degradation of ureides was greater for Jackson on one date than for KS4895. We concluded that ureides inhibit N₂ fixation, that genetic variation in the ability to degrade ureides may be important in drought tolerance, and that increased leaf Mn⁺² concentration promotes ureide breakdown and prolongs N₂ fixation under water deficit.

Abbreviations: ARA, acetylene reduction activity • DAS, days after sowing • PAR, photosynthetically active radiation

	INTRODUCTION

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results Discussion REFERENCES

 
NITROGEN FIXATION PROVIDES an important advantage for the production of soybean compared with most grain crops in that soybean fixes the nitrogen required for its growth and for the production of high-protein seed. While the ability to fix N₂ is a major asset for soybean production, N₂ fixation is more sensitive to water deficit than are many other processes (reviewed by Serraj et al., 1999a), and this may restrict nitrogen supply and yield of soybean in many environments.

The cultivar Jackson was identified as having substantially greater drought tolerant N₂ fixation than eight other cultivars (Sall and Sinclair, 1991), and subsequently the superiority of Jackson for N₂ fixation under drought was confirmed in field studies (Serraj et al., 1997) and in controlled environments (Serraj and Sinclair, 1996; Purcell et al., 1997). Understanding the mechanism of drought tolerance in Jackson may provide insights into the identification of other germplasm with drought tolerance.

There are several differences between cultivars that are drought tolerant and drought sensitive for N₂ fixation that may be important. During water deficit, ureides accumulate in soybean shoots (deSilva et al., 1996; Serraj and Sinclair, 1996; Purcell et al., 1998; Serraj et al., 1999b). Ureides (allantoin and allantoate) are the products of N₂ fixation in soybean. Ureides are transported from the nodule to the shoot, where they are normally broken down (Winkler et al., 1987). The accumulation of ureides in response to water deficit is considerably less in the cultivar Jackson than in drought sensitive lines (Serraj and Sinclair, 1996; Purcell et al., 1998). Therefore, greater rates of N₂ fixation for Jackson during drought may be associated with an ability to maintain shoot ureides at lesser concentrations than those found in drought sensitive lines.

The increased shoot ureide concentration during drought (deSilva et al., 1996; Serraj and Sinclair, 1996; Purcell et al., 1998) may serve as a feedback signal to nodules to decrease N₂ fixation (Purcell and Sinclair, 1995). The inhibition of N₂ fixation that occurs following NO^-₃ application also results in increased leaf ureide concentration (Rice et al., 1990), and this increase in shoot ureide concentration has been associated with decreased activity of an enzyme that catabolizes allantoate, allantoate amidohydrolase (E.C. 3.5.3.9). Manganese is a required cofactor for allantoate amidohydrolase activity, and manganese is complexed under certain conditions (Lukaszewski et al.,1992), leading to increased shoot ureide concentration. There are no reports of the response of shoot ureide concentration to changes in manganese levels during water deficit.

A second factor that has been proposed as being important in conferring drought-tolerant N₂ fixation in soybean is the size of nodules (Purcell et al., 1997; Serraj et al., 1999a). The source of water for nodules is the phloem (Walsh et al., 1989) and large nodules generate a greater sink demand than do small nodules, resulting in preferential delivery of water to large nodules during drought relative to small nodules (Serraj et al., 1999a). Additionally, photosynthate is carried in the water so that sugars, which are needed to support N₂ fixation, are also delivered preferentially to large nodules.

We hypothesized that the decrease in N₂ fixation during water deficit was associated with ureide accumulation in shoots. Ureide accumulation during water deficit would, therefore, be differentially affected in cultivars differing in sensitivity of N₂ fixation to water deficit and by availability of manganese, which is required for ureide catabolism. Our objectives were to: (i) determine differences in ureide accumulation between drought-tolerant and drought-sensitive cultivars in response to exogenous ureide application and water deficit; (ii) evaluate the effect of manganese fertilization on lessening shoot ureide concentrations and prolonging N₂ fixation during water deficit; and (iii) explore and contrast nodule size responses to water deficit, shoot ureide accumulation, and manganese nutrition for cultivars differing in their sensitivities to water deficit.

	Materials and methods

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results Discussion REFERENCES

 
Growth Chamber Experiment
Seeds of the soybean cultivars Jackson and KS4895 were sown in 500-mL pots constructed of 5-cm diam polyvinylchloride pipe approximately 23 cm in length. Pots were sealed at the top and bottom by end caps, and a single plant was grown through a 1-cm-diam hole in the top end-cap. Fittings were inserted into the top and bottom end-caps to allow for nondestructive acetylene reduction measurements. The potting medium was a N-free peat-perlite-vermiculite mixture (LB2, Sungro Horticulture Inc., Bellevue, WA). At sowing, 200 mL of N-free nutrient solution (deSilva et al., 1996) was added, and the potting medium was inoculated with Bradyrhizobium japonicum (strain USDA 110). Growth chamber day/night temperatures were 25/23°C, and photosynthetically active radiation (PAR) at the top of plants was 300 ± 30 µmol m^-2 s^-1 on a 16-h photoperiod beginning at 0600 h. Plants were watered with deionized water as needed, and 100 mL of additional nutrient solution was added at 21 days after sowing (DAS).

Acetylene reduction measurements were begun 35 to 40 DAS when plants were at the V5 to V6 developmental stage (Fehr and Caviness, 1977). The morning of the first measurement (Day 1), leaves of two plants per replication of both Jackson and KS4895 were sprayed until runoff with a 5 mM solution of MnSO₄ containing 0.5 mL surfactant (Tween-20) per 100 mL of solution. Remaining plants were sprayed with the surfactant minus manganese. Plant stems were sealed with putty in the pots, and nitrogenase activity at 21 kPa O₂ was determined using a rapid, nondestructive, flow through, acetylene reduction technique (Bacanamwo and Purcell, 1999). Briefly, a 10 kPa C₂H₂ gas mixture (balance gases: 21 kPa O₂ and 69 kPa N₂ ) was passed through the inlet ports at the bottom of the pots at a flow rate of 200 mL min^-1 until acetylene reduction activity (ARA) was constant (8 min). Gas samples were collected from the exhaust port of the pots, and C₂H₄ concentration was determined by gas chromatography. Immediately after the gas samples were collected, C₂H₂ was removed from the gas stream, and pots were thoroughly flushed with air to remove residual C₂H₂.

Following ARA measurements, at 1500 h, 100 mL of N-free nutrient solution plus 10 mM allantoin was added to half the pots that had been sprayed with MnSO₄ plus surfactant and half the pots that had been sprayed with surfactant only. To those plants not receiving the allantoin treatment, 100 mL of N-free nutrient solution was added.

On Days 2 to 4 of the experiment, plants received the same foliar spray treatments as described, and ARA measurements were made. After ARA measurements were made on Day 2, plants received a second application of the allantoin treatment. After ARA measurements on Day 4, plants were harvested and divided into roots, nodules, and shoots. Plant parts were dried at 70°C, weighed, and shoot tissue was ground to pass through a 1-mm screen. A 30 to 35 mg subsample was used to determine shoot ureide concentration using the colorimetric procedure of Young and Conway (1942) as described by deSilva et al. (1996).

The experiment was designed as a randomized complete block with a factorial arrangement of cultivars (Jackson and KS4895), foliar Mn⁺², and allantoin treatments for a total of eight treatment combinations. Four replications were run sequentially in the growth chamber. Data comparing responses of ARA over the 4 d of the experiment were first normalized on a per plant basis by dividing rates of subsequent days by rates on Day 1, and a second normalization was used to decrease day to day variability. The second normalization was the quotient of the first normalization (i.e., normalized for the initial rate of that plant on Day 1) and the mean rate for the same cultivar of the minus manganese, minus allantoin treatment for that day. Data were analyzed by analysis of variance, and means were separated by Fisher's least significance difference (LSD, P 0.05).

Greenhouse Experiment
Four seeds of either Jackson or KS4895 were sown in 1.9 L pots filled with the same potting medium used for the growth chamber experiment. Pots were saturated with deionized water, drained overnight and weighed. The following day, 500 mL of N-free nutrient solution, containing 9 µm MnCl₂, (deSilva et al., 1996) was added to half the pots. To the remaining pots, 500 mL of the same nutrient solution was added except this solution lacked Mn⁺². Pots were inoculated with B. japonicum (strain USDA 110), and plants were thinned to one per pot after emergence. Temperatures were 28 ± 3°C (day) and 22 ± 2°C (night), and metal halide lamps on a 16 h photoperiod provided, at plant height, approximately 300 µmol m^-2 s^-1 PAR above the level of the incident solar radiation. Plants were kept well watered by additions of deionized water.

At the V5 developmental stage, plants received an additional 500 mL of nutrient solution. After nutrient addition, pots were allowed to begin drying in increments to one of three assigned water levels: well-watered control (pot weight 70% of pot capacity); moderate water deficit (pot weight 35% of pot capacity); and severe water deficit (pot weight 30% of pot capacity). Similar moisture levels with this potting media were used previously to create three distinct levels of plant water availability (Purcell et al., 1997). The first step of drying was to allow pots to reach 70% of the pot capacity. Once all pots were dried to the same level on a given day, a lower drying step was targeted until pots reached their assigned treatment weights.

On the day all pots reached their treatment weight, one third of the plants were harvested (Day-1 harvest) and separated into shoots, roots, and nodules. The remaining pots were weighed daily, and water was added to maintain them at the target water-deficit pot weights. Additional harvests were made 7 (Day-7 harvest) and 14 d (Day-14 harvest) after the first harvest.

Nodules were counted, and plant parts were dried at 70°C, weighed, and ground to pass through a 1-mm sieve. Shoot ureide concentrations were analyzed as described previously. Shoot manganese concentrations were analyzed by digesting a 0.25-g subsample in 25 mL of concentrated nitric acid with peroxide. The digest was filtered and analyzed for manganese by inductively coupled plasma emission spectroscopy (Model D, Spectro Analytical, Fitchburg, MA) by the Soil Testing and Plant Analysis Laboratory at the University of Arkansas.

Field Experiments
Jackson and KS4895 were sown on 28 June 1997 at Fayetteville, AR, on a Captina silt loam (fine-silty, siliceous, mesic Typic Fragiudult) in 0.51 m rows at a density of 40 seeds m^-2. Soil pH was 5.7 and total manganese concentration in the top 15 cm of soil was 490 kg ha^-1. There were four rows per plot, and plot length was 6.1 m. The experiment was a randomized complete block design with six replications. There was a factorial arrangement of two cultivars and two levels of manganese spray treatment.

Plots were furrow irrigated as needed until 28 July 1997, when they were allowed to begin drying. Both cultivars at this time were in late vegetative growth. On 29 July 1997 (Day 1 of the treatment period), plots were sprayed with 200 L ha^-1 of either 5 mM MnSO₄ containing 5 mL surfactant (Tween-20) per L solution, or the surfactant solution alone. Additional foliar sprays were applied on Days 5, 13, and 21 of the treatment period. On Days 15, 17, 18, and 19 there were 6, 5, 8, and 3 mm of rain, respectively.

Six of the uppermost fully expanded leaves were chosen at random from the two center rows of a plot on Days 4, 7, 10, 15, and 19. Leaflets were separated from petioles, dried, finely cut and analyzed for ureide concentration, as described previously.

In 1998, the same cultivars were sown on 7 July in a field adjacent to the 1997 experiment. Soil type, seeding rate, row spacing, and plot size were similar to the 1997 experiment, except that the field had been limed to pH 6.7 in the fall of 1997 to decrease availability of manganese. There were four replications and irrigation treatments were included as main plots. Sub-plots were a factorial combination of cultivar and manganese spray treatments.

All treatments in the 1998 experiment were furrow irrigated as needed to maintain well-watered conditions until 18 August, at which time irrigation was withheld from the drought treatment. After 18 August, irrigated plots were supplied with water by a drip irrigation system approximately every second or third day. Spray-treatment rates, plant sampling and processing, and chemical analyses were similar to the 1997 field experiment. Foliar spray treatments were begun on 21 August (Day 1 of the treatment period) and were repeated on Days 8 and 19. There was 2 mm of rain on Day 1 and on Day 17.

Allantoate amidohydrolase activity was assayed in 1998 on Days 6 and 11 of the measurement period. The procedure was similar to the method described by Lukaszewski et al. (1992). Six leaflets from upper, fully expanded leaves were sampled at 1400 h, placed on ice, and transported to the laboratory. Leaves were ground in 10 mL of ice-cold buffer (pH 8.75), composed of tricine (50 mM) and 2-beta-mercaptoethanol (2-ME, 14 mM). Extracts were filtered through cheese cloth, and 1.5 mL of the extract was centrifuged at 1300 x g for 3 min. A 750-µL volume of extract was treated with (NH₄)₂SO₄ to 50% saturation. After centrifuging for an additional 3 min, samples were frozen at -20°C for analysis.

Samples were thawed on ice, the supernatant was discarded, and the pellet was resuspended in 0.5 mL of tricine–2ME buffer (pH 8.75). A 240-µL aliquot of the resuspended extract was placed in test tubes with 300 µL of 20 mM potassium allantoate and diluted with water for a total reaction volume of 560 µL. The reaction mixture was incubated in a water bath at 25°C, and the reaction was stopped after 0.5 and 3.5 h by placing test tubes in a 95°C water bath. Protein concentration of the enzyme reaction was determined on a 50-µL aliquot of the reaction mixture (Lowry et al., 1951). Allantoate amidohydrolase-dependent formation of glyoxylate between the 0.5 h and 3.5 h incubation times was quantified on 500 µL of the reaction mixtures by colorimetric procedures (Vogels and Van der Drift, 1970).

	Results

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results Discussion REFERENCES

 
Growth Chamber Experiments
Application of allantoin to soybean roots decreased relative ARA greatly (Fig. 1) and this response was similar for both cultivars. The rapid decrease in ARA in response to allantoin indicates the inhibitory nature of ureides on nodule activity. Similar effects of ureides were reported in hydroponic systems (Serraj et al., 1999b).

View larger version (15K): [in this window] [in a new window]   Fig. 1 Acetylene reduction activity (ARA) following allantoin application to roots. Relative ARA was normalized for activity prior to treatment and for rates of the control treatment on a given day. Data were averaged over foliar manganese-spray treatments (n.s.) and soybean cultivars (n.s.). The * indicates that rates differed significantly within a day between control and allantoin-treated plants, as indicated by an

Greenhouse Experiment
Plant Dry Weight and Nodule Number
There were several general responses that were consistent over the three harvests: manganese addition tended to increase plant dry weight, water deficit decreased root and shoot dry weights, Jackson had greater shoot and root mass than did KS4895, and individual nodule mass was greater for Jackson than for KS4895 (Table 2) . The most important interactions by harvest are reported in the following paragraphs.

At the Day-7 harvest, there was a striking difference between Jackson and KS4895 in response of dry weight to water deficit that was similar for both manganese treatments (Table 2). For KS4895, dry weight of nodules, roots, and shoots and nodule number was significantly decreased by the severe water-deficit treatment compared with the control treatment, and dry weight of nodules and shoots and nodule number was decreased by the moderate water-deficit treatment. In contrast to KS4895, dry weight and nodule number of Jackson were not affected by water deficit with the exception of individual nodule mass, which was increased by the moderate and severe water-deficit treatments relative to the control. The main effect of manganese addition at the Day-7 harvest was to increase dry weight and nodule number for plants of the +Mn treatment relative to plants of the -Mn treatment.

After 14 d of the water-deficit treatments (Day-14 harvest), nodule number per plant and dry weight of nodules, roots, and shoots was generally decreased for plants of the water-deficit treatments compared with plants of the well-watered treatment, averaged over cultivars (Table 2). There was also a positive response to manganese addition for Jackson and KS4895 for nodule, root, and shoot dry weight. Averaged over water treatments, addition of manganese decreased nodule number per plant and increased individual nodule mass in Jackson, whereas addition of manganese to KS4895 increased nodule number per plant but had no effect on individual nodule mass.

Shoot Ureide and Manganese Concentrations
Shoot ureide concentration at the Day-1 and Day-7 harvests was greater in water-deficit treatments than control treatments (Fig. 2a) , which agrees with previous reports (de Silva et al., 1996; Purcell et al., 1998). At the Day-1 harvest, Jackson had a lesser concentration of ureides than did KS4895 with or without Mn (Fig. 2b), but shoot ureide concentration of KS4895 was greatly decreased in the +Mn treatment compared with the -Mn treatment.

View larger version (38K): [in this window] [in a new window]   Fig. 2 Shoot-ureide concentration response to water-deficit treatments (A.) and manganese treatment (B.) for cultivars Jackson (J) and KS4895 (KS). Within a harvest date, different letters above bars indicate that means were significantly different, as determined by an LSD (P = 0.05). In (A.), data were averaged over cultivar and manganese treatments (water x cultivar x manganese, n.s.), and in (B.), data were averaged over water treatment

Although there were fairly large effects of Mn treatment on plant dry weight (Table 2), there were relatively small effects of Mn treatment on shoot manganese concentration (Table 3) . At the Day-1 harvest, there was no difference in manganese concentration between Mn treatments for KS4895, and for Jackson, shoot manganese concentration for the +Mn treatment was less than that for the -Mn treatment. Similarly, at Day-7 and Day-14 harvests there was no effect of Mn treatment on manganese concentration. Water-deficit stress also affected manganese concentration, resulting in increased manganese concentration at the Day-1 harvest and decreased manganese concentration at the Day-7 and Day-14 harvests compared with plants of the control treatments. The concentration of manganese was from a subsample of the total shoot tissue. Manganese deficiency would result in remobilization of manganese from older leaves to younger leaves (Marschner, 1995), and differences in manganese concentration for different age leaves would not be accounted for in our measurements.

The ratio of ureide concentration to manganese at the Day-1 harvest revealed important cultivar differences in the ability to breakdown ureides (Table 3). Jackson had a lesser ratio of ureide to manganese than did KS4895 for both manganese treatments, whereas this ratio was increased for KS4895 in the -Mn treatment. The ureide to manganese ratio was also increased in plants of the moderate drought treatment relative to plants of the control treatment when averaged across cultivars and manganese treatments.

The ratio of ureide concentration to manganese concentration at the Day-7 harvest was greater for both of the water-deficit treatments compared with the control, and for the -Mn treatment relative to the +Mn treatment. At the Day-14 harvest, the ratio of ureide concentration to manganese concentration was also greater for KS4895 than for Jackson. An increased ureide:manganese concentration is consistent with decreased efficiency of ureide breakdown under manganese-limiting conditions.

Shoot Nitrogen Accumulation Rates
Shoot N accumulation rates for cultivars and Mn treatments within a water treatment were determined by covariate regression of shoot N against time, with cultivar and manganese treatment as covariates (Table 4) . Our previous work with this potting mixture had established that there was no available nitrogen, and N accumulation was due exclusively to N₂ fixation. For the severe water-deficit treatment and the control, there were no significant effects of cultivar, Mn treatment, or their interaction on the rates of N accumulation. Averaged over Mn treatments and cultivars, N accumulation rates were 1.9 mg N d^-1 for plants of the severe water-deficit treatment and 4.4 mg N d^-1 for plants of the control treatment.

Field Experiments
In general, ureide data for both years agreed with growth chamber and greenhouse experiments with Jackson having lesser concentrations of ureides in leaves and petioles than KS4895. In 1997, Mn spray treatment increased petiole ureide concentration for KS4895 3 d following the initial foliar application, but petiole ureide concentration of Jackson was unaffected by Mn treatment (Fig. 3a) . On Days 4, 7, 10, 15, and 19, Jackson always had a lesser petiole ureide concentration than did KS4895. Leaf ureide concentration was also greater for KS4895 than for Jackson throughout the measurement period, but there was no significant effect of Mn treatment on leaf ureide concentration (Fig. 3b).

View larger version (22K): [in this window] [in a new window]   Fig. 3 Petiole ureide concentration (A.) and leaf ureide concentration (B.) for Jackson and KS4895 during a drying cycle for a field experiment in 1997. Except where indicated, data were averaged over foliar manganese treatments (n.s.). Within a day, * indicates that ureide concentrations differed significantly between cultivars as determined by an F-test (P 0.05). LSD bar in panel A is for comparisons of means on Day 4

In 1997, manganese application increased leaf manganese concentration from 143 to 159 mg kg^-1, and in 1998, manganese application increased manganese concentration from 141 to 193 mg kg^-1 after two foliar applications (data not shown). Jackson had a greater concentration of leaf manganese than KS4895 in both 1997 and 1998. Although these effects of Mn treatment were significant, the concentrations were three to four times greater than those seen in the greenhouse experiment. These concentrations are relatively high but are considered nontoxic for soybean growth (Bell et al., 1995).

Allantoate amidohydrolase is a key enzyme in ureide breakdown, and this enzyme requires manganese as a cofactor. In 1998, we measured allantoate amidohydrolase activity on Days 8 and 15 of the measurement period. The data were variable, particularly on Day 8, which showed no significant differences between cultivars, Mn treatment, or irrigation treatment (Table 6) . On Day 15 of the measurement period, there was a significant cultivar x Mn treatment interaction. Allantoate amidohydrolase activity of the -Mn treatment of Jackson was greater than both the +Mn or -Mn treatments of KS4895.

	Discussion

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results Discussion REFERENCES

In the growth chamber, application of ureides to roots of plants under well-watered conditions clearly resulted in rapid and substantial decreases in ARA. Serraj et al. (1999b) found a similar decrease in ARA following a 10 mM allantoate application to roots of hydroponically grown soybean. Although results from our growth chamber experiment indicated that there were no differences in foliar manganese treatments or cultivars in the response of N₂ fixation to allantoin application, the high concentration of ureides applied may have overpowered any differences that would have been apparent at a lesser concentration. Nevertheless, Jackson, which has drought tolerant N₂ fixation (Serraj et al., 1997; Purcell et al., 1997), maintained shoot ureide concentrations less than those of KS4895, which has drought sensitive N₂ fixation (Purcell et al., 1997).

In 2 yr of field experiments, Jackson also had lesser concentrations of ureides in both leaves and petioles than did KS4895. In the greenhouse experiment, ureide concentration of Jackson was less than that of KS4895 at the Day-1 harvest, but these cultivar differences were not apparent at the Day-7 and Day-14 harvests. A mitigating factor that appeared to affect the cultivar response to ureides was manganese availability in shoots. At the Day-1 harvest, Jackson had a greater concentration of shoot manganese than did KS4895; however, at the Day-7 harvest, this relationship was reversed with KS4895 having a greater concentration of shoot manganese than Jackson.

The importance of manganese in continued N₂ fixation in response to water deficit was demonstrated in the greenhouse experiment. Nitrogen fixation for KS4895 under moderate water-deficit stress increased from 30% of the control in the absence of soil-applied manganese to 111% of the control in the presence of soil-applied manganese. For Jackson, N₂ fixation rate for the moderate-stress treatment was not different from the control, regardless of manganese treatment. The increased N₂ fixation rate of the moderate-stress treatment of the +Mn treatment of KS4895 was accompanied by decreased shoot ureide concentration at both the Day-1 and Day-7 harvests.

There were discrepancies in the response of ureides to additional manganese. In the growth chamber and field experiments, plants were well supplied with manganese from the soil solution, and these high concentrations of manganese may have been adequate for continued ureide breakdown, preventing a response to foliar MnSO₄ application. Foliar applications with Mn-EDTA also may be more effective than foliar MnSO₄ applications for increasing leaf manganese concentration and decreasing leaf ureides. Further research is required to evaluate the response of ureide concentration to different sources of manganese. Additional research is also required to determine the location of manganese and ureide pools within the plant and how supplemental manganese and drought may affect these pools.

Our working model of the role of manganese in ureide metabolism and nodule activity is presented in Fig. 4 . In the absence of sufficient manganese in leaves, allantoate amidohydrolase activity is decreased, leading to an accumulation of ureides in leaves. The water source for nodules is almost exclusively derived from the phloem (Walsh et al., 1989), and inhibition of ureide breakdown would be expected to increase ureide concentration in the phloem. Increased ureide delivery to nodules through the phloem would decrease N₂ fixation and lessen de novo ureide production in nodules. Increased manganese supply in leaves is envisioned as promoting ureide catabolism and preventing a feedback inhibition in nodules.

View larger version (61K): [in this window] [in a new window]   Fig. 4 Model of nitrogen fixation response to manganese and ureide accumulation during water deficit. In the absence of sufficient manganese, ureide breakdown is impaired, leading to an accumulation of leaf ureides. An increase in leaf ureides results in the export of ureides from leaves to nodules, along with water and sugars carried in phloem. Increased ureide concentration in nodules inhibits N2 fixation, lessens nodule respiration and sink strength, and decreases flux of water to nodules

Results from these experiments documented that the drought tolerance of N₂ fixation of Jackson relative to KS4895 was associated with a lesser tissue ureide concentration than for KS4895, a drought sensitive line. That N₂ fixation sensitivity to drought may be lessened by manganese availability in leaves of drought sensitive cultivars offers potential management options for increasing N₂ fixation for moderate water-deficit conditions. Manganese deficiencies in soybean are common in soils that are either highly leached, have a pH > 6.5, or are high in organic matter (Marschner, 1995). These soil conditions are relatively common, and an evaluation of shoot manganese management in drought-sensitive cultivars should be explored such that ureide concentrations are kept at a minimum, leading to prolonged N₂ fixation during a drought. A second possible research approach is to evaluate germplasm identified as tolerant to manganese deficiency (Graham et al., 1995), and to determine if these cultivars maintain low ureide concentrations and have drought tolerant N₂ fixation under conditions of limiting manganese.

	NOTES

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results Discussion REFERENCES

 
This paper is published with the approval of the director of the Arkansas Agric. Exp. Stn. (manuscript number 99081). Research supported in part by the United Soybean Board, project 8206.

Received for publication July 26, 1999.

	REFERENCES

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results Discussion REFERENCES

 

• Bacanamwo M., Purcell L.C. Soybean dry matter and N accumulation responses to flooding stress, N sources and hypoxia. J. Exp. Bot. 1999;50:689-696.[Abstract/Free Full Text]
• Bell P.F., Hallmark W.B., Sabbe W.E., Dombek D.G. Diagnosing nutrient deficiencies in soybean using M-Dris and critical nutrient level procedures. Agron. J. 1995;87:859-865.[Abstract/Free Full Text]
• de Silva M., Purcell L.C., King C.A. Soybean petiole ureide response to water deficits and decreased transpiration. Crop Sci. 1996;36:611-616.[Abstract/Free Full Text]
• Fehr, W. R., and C.E. Caviness. 1977. Stages of soybean development. Spec. Report No. 80, Iowa State Univ. Coop. Ext. Ser., Ames, IA.
• Graham M.J., Nickell C.D., Hoeft R.G. Inheritance of tolerance to manganese deficiency in soybean. Crop Sci. 1995;35:1007-1010.[Abstract/Free Full Text]
• Lowry O.H., Rosebrough N.J., Farr A.L., Randall R.J. Protein measurement with the Folin phenol reagent. J. Bio. Chem. 1951;193:265-275.[Free Full Text]
• Lukaszewski K.M., Blevins D.G., Randall D.D. Asparagine and boric acid cause allantoate accumulation in soybean leaves by inhibiting manganese-dependent allantoate amidohydrolase. Plant Physiol. 1992;99:1670-1676.[Abstract/Free Full Text]
• Marschner H. Mineral nutrition of higher plants, second edition. New York: Academic Press, 1995.
• Purcell L.C., deSilva M., King C.A., Kim W.H. Biomass accumulation and allocation in soybean associated with genotypic differences in tolerance of nitrogen fixation to water deficits. Plant Soil 1997;196:101-113.
• Purcell L.C., Serraj R., de Silva M., Sinclair T.R., Bona S. Ureide concentration in field-grown soybean in response to drought and the relationship to nitrogen fixation. J. Plant Nutr. 1998;21:949-966.[ISI]
• Purcell L.C., Sinclair T.R. Nodule gas exchange and water potential response to rapid imposition of water deficit. Plant Cell Environ. 1995;18:179-187.
• Rice C.F., Lukaszewski K.M., Walker S., Blevins D.G., Winkler R.G., Randall D.D. Changes in ureide synthesis, transport and assimilation following ammonium nitrate fertilization of nodulated soybeans. J. Plant Nutr. 1990;13:1539-1553.
• Sall K., Sinclair T.R. Soybean genotypic differences in sensitivity of symbiotic nitrogen fixation to soil dehydration. Plant Soil. 1991;133:31-37.
• Serraj R., Bona S., Purcell L.C., Sinclair T.R. Nitrogen accumulation and nodule activity of field-grown `Jackson' soybean in response to water deficits. Field Crops Res. 1997;52:111-118.
• Serraj R., Sinclair T.R. Nitrogen fixation insensitivity to drought in soybean cultivar "Jackson". Crop Sci. 1996;36:961-968.[Abstract/Free Full Text]
• Serraj R., Sinclair T.R., Purcell L.C. Symbiotic N2 fixation response to drought. J. Exp. Bot. 1999;50:143-155 a.[Abstract/Free Full Text]
• Serraj R., Vadez V., Denison R.F., Sinclair T.R. Involvement of ureides in nitrogen fixation inhibition in soybean. Plant Physiol. 1999;119:289-296 b.[Abstract/Free Full Text]
• Vogels G.D., Van der Drift C. Differential analyses of glyoxylate derivatives. Ann. Biochem. 1970;33:143-157.
• Walsh K.B., McCully M.E., Canny M.J. Vascular transport and soybean nodule function: nodule xylem is a blind alley not a throughway. Plant Cell Environ. 1989;12:395-405.
• Winkler R.D., Blevins D.G., Polacco J.C., Randall D.D. Ureide catabolism in soybeans. II. Pathway of catabolism in intact leaf tissue. Plant Physiol. 1987;83:585-591.[Abstract/Free Full Text]
• Young E.G., Conway C.F. On the estimation of allantoin by the Rimini-Schryver reaction. J. Biol. Chem. 1942;142:839-853.[Free Full Text]

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