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Environmental and Experimental Botany 66 (2009) 94–99

Contents lists available at ScienceDirect

Environmental and Experimental Botany

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nteractive influence of phosphorus and iron on nitrogen fixation by soybean

ladimir Rotarua, Thomas R. Sinclairb,∗

Institute of Plant Physiology, Moldavian Academy of Sciences, 26/1 Padurii str., MD 2002, Chishinev, MoldaviaAgronomy Physiology Laboratory, P.O. Box 110965, University of Florida, Gainesville, FL 32611-0965, USA

r t i c l e i n f o

rticle history:eceived 9 November 2008ccepted 4 December 2008

eywords:ronhosphorusitrogen fixationodulesoybean

a b s t r a c t

Adequate supplies of phosphorus (P) and iron (Fe) to legumes have been shown to be crucial in obtaininghigh nitrogen fixation rates and growth. These responses are anticipated as a result of the high require-ment for P in energy transfer processes in the nodule and for Fe as a constituent of nitrogenase andleghemoglobin. However, little attention has been given to documenting the response of nitrogen fixa-tion rates resulting from concentrations of P and Fe that actually exist in nodules. In particular, an openquestion is whether there is an interaction between nodule P and Fe concentrations that maximize nitro-gen fixation activity. This study was designed to induce various concentrations of P and Fe in the noduleand to measure the resultant nitrogen accumulation and nitrogen fixation rates. Plant nitrogen accumu-lation was linearly correlated with both nodule P and Fe concentration, and with total plant nitrogenfixation rate as measured by acetylene reduction rate. Therefore, total nitrogen fixation rate was alsocorrelated with nodule P and Fe concentrations, but a higher linear correlation was obtained for Fe as

compared to P concentration. Surprisingly, nodule ureide concentration, which is generally assumed to bea positive index of nitrogen fixation rate, was negatively correlated with nodule P and Fe concentrations.These results indicated that higher concentrations of P and Fe in the nodules not only stimulated highernitrogen fixation rates, but were associated with an enhanced ability to export ureides from the nodules.Since there was a linear response to both P and Fe over the range of nodule concentrations induced inthese experiments, no evidence for optimum interactive concentrations of these two elements in the nodules was obtained.

. Introduction

Among environmental constraints, nutrient availability withinlants can be critical limitations on plant production (O’Hara et al.,988a; Sinclair, 1992; Sinclair and Vadez, 2002). Phosphorus (P) andron (Fe) deficiencies have been reported on a number of soils world

ide. Increased P supply from the soil has resulted in increasedhole-plant N concentration and whole-plant growth for several

eguminous species including soybean (Glycine max L. Merr.) (Israel,987). Low iron availability has been reported to result in low

concentration and accumulation for several legumes includinghaseolus vulgaris (Hemantaranjan, 1988), Arachis hypogaea (Terryt al., 1988; Tang et al., 1991), Lupinus albus (Tang et al., 2006),enus culinaris (Rai et al., 1984), and Cicer arietinum (Ohwaki andugahara, 1993).

Legumes appear to be especially vulnerable to P and Fe deficien-ies because of the elemental needs for supporting symbiotic N2xation. The consequences of P deficiency in soybean are directlyelated to declines in dry weight and nitrogen content in plants and

∗ Corresponding author. Tel.: +1 352 392 6180; fax: +1 352 392 6139.E-mail address: trsincl@ifas.ufl.edu (T.R. Sinclair).

098-8472/$ – see front matter © 2008 Elsevier B.V. All rights reserved.oi:10.1016/j.envexpbot.2008.12.001

© 2008 Elsevier B.V. All rights reserved.

to the reduction in nitrogen-metabolism activity (Gunawardena etal., 1992; Rufty et al., 1993; Sa and Israel, 1995). A major reason forthe sensitivity of legumes to P and Fe deficiency is the critical rolethese elements play in the growth and activity of nodules (O’Haraet al., 1988b, 1993; Freeden et al., 1989; Tang et al., 2001, 2006;Sinclair and Vadez, 2002).

Symbiotic N2 fixation has a higher P requirement for maximumactivity than growth supported by nitrate assimilation because ofthe high energy requirements in the reduction of atmospheric N2by the nitrogenase system. There appear to be several consequencesat the physiological level resulting from severe P deficiency onlegume nodules. In tissues of common bean (Phaseolus vulgaris L.),decreases in P concentration result in increased root-carbohydratecontent (Rychter and Randal, 1994), decreased total respirationrate (Wanke et al., 1998), and decreased root-ATP concentration(Rychter et al., 1992). Under P-limiting conditions, Jnshu and Israel(1994) found a decrease of sucrose and hexoses in nodules of soy-bean plants. Also, P deficit has been observed to result in a decreased

energy status in soybean (Glycine max Merr.) nodules (Sa and Israel,1991).

Iron deficiency results in decreased nodule number and massin peanuts (O’Hara et al., 1988b; Tang et al., 1991), French bean(Hemantaranjan and Gard, 1986) and lupines (Tang et al., 1990a,b,

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991). Symbiotic N2 fixation was shown to have a high require-ent for iron in lupine (Tang et al., 2006) because Fe is an essential

omponent of nitrogenase, leghemoglobin, and ferrodoxins (Evansnd Rossel, 1971). Acetylene reduction activity was found to bencreased with enhanced Fe iron nutrition for peanuts (O’Harat al., 1993), mungbean (Chahal and Chahal, 1988), and lentilRai et al., 1982). Decreased concentration of leghemoglobin dueo Fe deficiency has been reported in nodules of common beanHemantaranjan, 1988), although Fe deficiency has been reported toave a much greater influence on bacteroid number than on leghe-oglobin level (Kaczor et al., 1994). However, there is a crucial need

or heme in the synthesis of leghemoglobin in vivo, which makesp to 20% of the plant protein mass in the nodule cytosol of soybeanAppleby, 1984). Iron deficiency results in decreased nodule leghe-

oglobin content and nitrogenase activity leading to low N contentn shoots of several legumes (Tang et al., 1992), although soybeanas not included in their study. Maximum nitrogenase activity haseen found to be associated with high Fe concentration in nodulesf lentil (Rai et al., 1984).

In spite of the fact that soybean is by far the most economi-ally important grain legume world wide, there are only a limitedumber of studies that have examined directly N2 fixation responsef soybean to a range of P and Fe treatments. Response to P ande in soybean is of increasing concern as soybean production isxpanded in new areas where the price of fertilizer inhibits themount of nutrients that can be applied. Further, there appears toe no studies that have explored the possibility that there maye an interaction within soybean plants to varying supplies ofand Fe. Investigations on the interaction of P and Fe on plant

ehavior are often confounded because the results are expressedn the basis of the concentrations of the elements to which thelants were exposed. Since the interactions between P and Fe inhe soil solution can be complex (e.g. Romera et al., 1992), theesponse to the levels of P and Fe actually accumulated in the planteed to be considered in assessing plant activities such as nitrogenxation.

Therefore, the overall objective was to examine variousttributes of N2 fixation of soybean in response to various com-inations of P and Fe in nodules. In particular, the question wassked whether there existed a unique combination of P and Fe con-entration in nodules that resulted in especially high levels of Nccumulation and N2 fixation activity. Previously, we have reportedhe growth response to these treatments (Rotaru and Sinclair, inress). In this paper, we present results on N accumulation, noduleass, N2 fixation (acetylene reduction activity), and ureide accu-ulation obtained in two experiments in which soybean plantsere subjected to a range of P and Fe treatments to assess possible

nteractions between the two elements.

. Materials and methods

Soybean (cv. Biloxi) plants were grown from seed in PVC pots10 cm diameter, 40 cm height) containing 4 L silica sand. Beforehe experiments were initiated white quartz sand was washedith hydrochloric acid to remove residual nutrients. Seeds were

urface sterilized with ethanol (1 min) and calcium hypochlorite5 min) and then washed several times with deionized water.he seeds were inoculated at sowing with Bradyrhizobium japon-

cum and sown three seeds per pot. Each pot was thinned tone plant per pot 1 week after emergence. The plants wererown in a greenhouse in which temperature was maintained at

pproximately 28 ◦C by day and 24 ◦C by night under natural lightonditions.

Once the seedlings had emerged, treatments with various nutri-nt solutions were initiated. The base nutrient solution (Drevon etl., 1988) contained 3.3 mM CaCl2, 2.0 mM MgSO4, 1 mM K2SO4,

xperimental Botany 66 (2009) 94–99 95

6 �M MnSO4, 4 �M H3BO3, 1.6 �M ZnSO4, 0.3 �M CoCl2, and 0.1 �MNa2MoO4 (Drevon et al., 1988). In addition, 2 mM of Ca(NO3) wasincluded to provide a N source for early plant growth on thenutrient-free sand. In Expt. 1, additions of KH2PO4 were made to thebase nutrient solution to achieve P concentration of either 2 mM or0.1 mM and of Fe EDTA to achieve Fe concentration of either 20 �Mor 1 �M. Based on the results of Expt. 1, it was decided to imposemore nutrient treatments at lower concentrations so in Expt. 2 thebase nutrient solution was diluted by half. Three P and three Fetreatments were applied in Expt. 2 of 0.50, 0.125 and 0.05 mM Pand 10.0, 2.5, and 0.5 �M Fe. These treatments were applied in allcombinations so there were a total of nine solution treatments inExpt. 2. Potassium sulfate was added to the low P solutions to equal-ize the K supply for all solutions. In all cases, nutrient solution pHwas adjusted to 6.4 using 0.1N NaOH.

Treatments were placed on a greenhouse bench in a completelyrandomized design. The nutrient solution for each treatment wasmade on each day of solution application by mixing into the basesolution the appropriate amount of P and Fe stock solutions. In Expt.1, 200 mL of nutrient solution was applied every second day to eachpot. On alternate days the pots were irrigated with 200 mL of dis-tilled water to help minimize the accumulation of salts in the sand.In Expt. 2, 200 mL of the nutrient treatments was added to the pots3 days per week and 200 mL of water was applied on the other 4days.

Five replicate plants were harvested from each solution treat-ment at 4, 5, and 6 weeks after sowing (WAS) in Expt. 1 and fourreplicate plants were harvested from each solution treatment at 4and 6 WAS in Expt. 2. The individual plants were separated intoleaves, stems plus petioles, and roots. Roots were carefully washedseveral times and the nodules separated for counting and weighing.All tissue was dried in an oven (75 ◦C, for 48 h) and then weighedto record dry mass. Total N, P, and Fe concentrations of each ofthe four tissue samples for each plant were determined. N con-centration was measured using the Kjeldahl method and P wasdetermined colorimetrically (Hambleton, 1977). For Fe determina-tion, 1 g of powdered plant material was dry-ashed at 550 ◦C inmuffle furnace prior to analysis. The ash was dissolved in waterfor Fe analysis using an atomic absorption spectrophotometer withan air–acetylene flame. Ureide analysis was also performed on dryplant material for leaves and nodules. The tissue samples wereextracted in 0.2 M NaOH of boiling water (30 min) and ureide con-centration was measured by colorimetric assay (Young and Conway,1942).

A continuous flow system was used to measure acetylene reduc-tion activity (ARA) of individual pots (Serraj and Sinclair, 1996) priorto the 6 WAS harvest. ARA measurements were made on four repli-cate pots from each treatment on 28 November and 1, 4, 7, and 11December in Expt. 1, and on 28, 29, 30, and 31 March in Expt. 2.Between 11:00 and 12:00 on the day of measurement, an acety-lene:air mixture (9:1) was flowed through the pots at 1 L min−1.After flowing the gas mixture for 15 min to allow for equilibration,two 1-cm3 samples were collected in syringes at the exit port ofeach pot. The collection of gas samples from all pots took approxi-mately 5 min, after which the pots were flushed with only air for atleast another 1 h to remove acetylene from the pots. The gas sam-ples were injected into a gas chromatograph equipped with a flameionization detector to measure ethylene production in each pot asan index of N2 fixation activity.

The highest and lowest ARA values obtained among the eightmeasurements on each day from a treatment were discarded. This

allowed outlier values resulting from poor sample collection or poorinjection into the gas chromatograph to be discarded in a uniformway. Hence, the ARA results presented for each treatment representthe mean of 30 measurements (6 measurements × 5 days) in Expt.1 and 24 measurements (6 measurements × 4 days) in Expt. 2.

9 l and Experimental Botany 66 (2009) 94–99

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. Results

The growth data for these plants in the various treatments wasresented by Rotaru and Sinclair (in press). There was a steady

ncrease in mass over the period from 4 to 6 WAS. Since the Noncentration was fairly stable across the various sampling datesRotaru and Sinclair, in press), the increase in accumulated N byhese plants (Fig. 1) was very similar to the increase over timen total plant mass. Those plants grown with high levels of P, inarticular, in both experiments exhibited the greatest amount ofccumulated N (Fig. 1). The difference among nutrient treatmentsas especially obvious at the 6 WAS harvest when N accumulationsith the high P treatments were more than two-fold that accumu-

ated in the lower P treatments in both experiments. At the lastarvest, there also appeared to have been differences within theigh P treatment in response to the Fe treatment. The combinationf high P and high Fe resulted in the greatest N accumulation inoth experiments.

Changes in nodule mass within each experiment closely paral-eled plant N accumulation (Fig. 2). Indeed, the correlation betweenlant N accumulation and nodule mass (Fig. 3) was very high withinach experiment (r2 = 0.98 in Expt. 1 and r2 = 0.99 in Expt. 2). Ofourse, it is not possible to resolve from these results the cause andhe effect between these two variables. It is clear, however, that thelope of the relationship between N accumulation and nodule massas quite different between the two experiments. The change in N

ccumulation was much greater per unit change in nodule mass inxpt. 1 than in Expt. 2. That is, these results imply that specific activ-ty of nodules in Expt. 1 was substantially greater than in Expt. 2.t is unknown whether this difference between experiments mighte a result of using nutrient solutions with lower basal concen-rations for all nutrients in Expt. 2 as compared to Expt. 1. Thereas, however, no significant correlation between nodule mass and

ither nodule P or Fe concentration across experiments (data nothown).

Combining the two experiments, the amount of N accumulationt 6 WAS was positively correlated both P and Fe concentrationsn the nodules (Fig. 4). The level of correlation of element concen-ration with N accumulation was essentially the same for P and Fe.

ig. 1. Plant nitrogen accumulation for each nutrient solution treatment at the var-ous sampling dates in each of the two experiments. Standard error is indicated forach datum where the value is greater than the size of the symbol.

Fig. 2. Nodule mass for each nutrient solution treatment at the various samplingdates in each of the two experiments. Standard error is indicated for each datumwhere the value is greater than the size of the symbol.

Therefore, it is not surprising that the correlation between P and Feconcentration of the nodules was high (r2 = 0.77, p < 0.001). Increasein the concentration of these two elements signified an increasedcapability for N2 fixation per unit nodule mass, since nodule massitself was not correlated with the element concentrations of thenodules.

ARA offers a relative index of the N2 fixation capacity of theplants. Expressed on a plant basis, ARA varied substantially amongnutrient treatments (Fig. 5). Within both experiments, the combi-nation of high P and high Fe resulted in the greatest ARA valuesamong all treatments. In contrast, low P and low Fe resulted in lowARA values.

There was a significant (r2 = 0.55, p < 0.01) trend of N accu-mulation being positively correlated with increased ARA (Fig. 6)across all treatments in both experiments. The same linear responseappeared to represent the data from both experiments. On the otherhand, there was no correlation between accumulated N and specific

ARA (data not shown). It appears that nodule mass overwhelmedthe variability in specific ARA to result in the correlation of totalplant ARA with N accumulation.

Considering the positive correlation of N accumulation withnodule P and Fe concentrations, it is not surprising that there

Fig. 3. Plant nitrogen accumulation at the harvest 6 weeks after sowing for eachnutrient solution treatment plotted against nodule mass.

V. Rotaru, T.R. Sinclair / Environmental and Experimental Botany 66 (2009) 94–99 97

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Fig. 6. Plant nitrogen accumulation at the harvest 6 weeks after sowing for eachnutrient solution treatment plotted against in situ acetylene reduction rate (ARA)measured prior to harvest for each of the nutrient solution treatments. Linear regres-sion included the results from the two experiments combined.

ig. 4. Plant nitrogen accumulation at the harvest 6 weeks after sowing for eachutrient solution treatment plotted against nodule phosphorus concentration andgainst nodule iron concentration. Linear regressions included the results from thewo experiments combined.

as also a positive correlation between ARA and nodule P

nd Fe concentrations (Fig. 7). Combining the results from thewo experiments, the correlation between ARA and Fe (r2 = 0.63,< 0.01) was somewhat higher than between P and ARA (r2 = 0.50,< 0.01).

ig. 5. In situ acetylene reduction rate (ARA) for plants prior to the harvest 6 weeksfter sowing for each of the nutrient solution treatments. Those treatments indicatedith different letters were found to be significantly different using Newman–Keulsultiple comparison test (p < 0.05).

Fig. 7. In situ acetylene reduction rate (ARA) for plants prior to the harvest 6 weeksafter sowing for each of the nutrient solution treatments plotted against nodulephosphorus concentration and against nodule iron concentration. Linear regressionsincluded the results from the two experiments combined.

Ureide is the metabolic product of N2 fixation in soybean nod-ules that is exported to the shoot. In spite of the positive correlationsbetween ARA and nodule P and Fe, the accumulation of nodule ure-ide concentration had a strikingly negative correlation with noduleP and Fe concentrations (Fig. 8). That is, high P and Fe concentrationsin the nodules that resulted in increased ARA and plant N accumu-lation were associated with the lowest concentrations of N2 fixationproduct in the nodules. These results indicated that high elemen-tal concentrations in the nodules were associated with an enhancedability of the nodules to export ureides to the plant shoot. There was,however, no correlation between leaf ureide concentration and leafP or Fe concentration (data not shown).

4. Discussion

The positive response of N accumulation and N2 fixation activ-ity to increased supply of P and Fe has been documented in several

98 V. Rotaru, T.R. Sinclair / Environmental and E

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ig. 8. Nodule ureide concentration at the harvest 6 weeks after sowing for eachutrient solution treatment plotted against nodule phosphorus concentration andgainst nodule iron concentration. Linear regressions included the results from thewo experiments combined.

rain legumes including soybean. The results obtained in this studyith various combinations of P and Fe confirmed this overall trend.

n these studies with soybean, the range of P and Fe supplied inutrient solution treatments resulted in combinations of P ande concentration in nodules that were positively correlated withncreased N2 fixation (Fig. 7) and N accumulation (Fig. 4).

The unique aspect of this study was an investigation into theossibility that there might be an interaction of P and Fe that wouldause the response in N2 fixation activity to be especially high. Theesults of this study clearly did not identify any combination of Pnd Fe that resulted in synergistic responses resulting in any greatlynhanced N2 fixation trait. The nodule concentrations of these twolements changed in concert in response to the nutrient solutionreatments such that there were positive linear responses in plant

accumulation and ARA to both nodule P and Fe concentration.he linear response extended to the accumulation of ureides in theodules, but in this case the correlations were negative (Fig. 8).

Total N accumulation by the plant was also closely linked to nod-le mass, although the relationship was different between the twoxperiments (Fig. 3). Given the correlation of N accumulation withodule P and Fe concentration and with nodule mass, it was surpris-

ng that there was no linear correlation between either nodule P ore concentration, and nodule mass. These results may imply that tolarge extent nodule mass may be a consequence of plant growth

ather than altered nodule mass resulting in changed shoot growth.lthough the correlation between N accumulation and nodule mass

s high, this may reflect a tight regulation that keeps the growth ofodule mass compatible with growth in the plant shoot.

The major factor associated with N2 fixation factor that may behe basis for variation in N accumulation among treatments is the

otal N2 fixation activity of the nodules irrespective of mass (Fig. 6).he total activity of the nodules, in turn, was correlated with noduleand Fe concentration (Fig. 7). ARA was especially well correlatedith nodule Fe concentration, which may reflect the essentiality

f Fe in the structure of both nitrogenase and leghemoglobin in

xperimental Botany 66 (2009) 94–99

nodules. Nevertheless, these results show that both elements areneeded in high concentration in the nodules to achieve high N2fixation rates.

An interesting result from this study was the significant nega-tive correlation between nodule ureide concentration and nodule Pand Fe concentrations. Ureides are formed in the non-infected cellsin the nodules, and ureides are the main products transferred fromthe nodules to the plant shoot in soybean. The original assump-tion was that high levels of ureide concentration in nodules wouldbe associated with high N2 fixation rates (Herridge and People,1990). In fact, just the opposite response was obtained in this study(Fig. 8). These results imply that high concentrations of P and Fe inthe nodules alter the nodule physiology so that overall throughputof nitrogen is increased such that the accumulation of ureides isactually decreased. The active loading of ureides into the xylem forexport would require P to support this energy-dependent process.The role of Fe is less clear. These large differences in nodule ureideconcentrations with P and Fe levels may offer an important clue ininvestigating the key processes that influence nodule N2 fixationactivity.

Overall, the results from these experiments failed to identifyany significant interaction in P and Fe concentration in nodules inregards to N2 fixation activity and plant N accumulation. The resultsshowed that concentrations of P and Fe in the nodules were closelycorrelated. Increased concentrations of both P and Fe were asso-ciated with high level of nodule activity. The correlation betweennodule N2 fixation activity with both nodule P and Fe concentra-tion indicates the importance of these elements in key aspects ofthe physiology of N2 fixation in soybean nodules.

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