Plant Physiol. (1982) 69, 171-1780032-0889/82/69/017 1/08/$00.50/0

Ion Balance, Uptake, and Transport Processes in N2-Fixing andNitrate- and Urea-Dependent Soybean Plants'

Received for publication March 6, 1981 and in revised form September 16, 1981

DANIEL W. ISRAEL AND WILLIAM A. JACKSONUnited States Department ofAgriculture, Agricultural Research Service, Department of Soil Science (D. W. L),and Department of Soil Science (W. A. J.), North Carolina State University, Raleigh, North Carolina 27650

ABSTRACT

The objective of this study was to examine the influence of N2 fixationand N03--N and urea-N assimilation on ion balance, uptake, and transportprocesses in soybean (Glycine max L. Merr.).

Inoculated plants were grown in Perlite supplied daily with nutrientsolutions which contained zero-N, 10 and 20 millimolar N03--N, and 10and 20 milimolar urea-N, and they were sampled 41, 76, and 151 daysafter transplanting. Total uptake of inorganic cations and anions wasdetermined by analysis of tissue for K , Ca2+, Mg2+, Na , total N fromN03-, total S, H2PO-, and Cl1. Diferences in total inorganic cations(C) and inorganic anions (A) in plant tissue were used to estimate totalcarboxylate content.

Internal OH- generation resulting from excess cation uptake (net H+excretion) by the roots accounted for more than 89% of the carboxylateaccumulation in Nr and urea-fed plants, while OH- generation resultingfrom s042- reduction accounted for less than 11%. Shoots contained over89% of the total plant carboxylate content. Malate balanced about 75% ofthe excess inorganic cationic charge of the xylem sap; ailantoate andaspartate balanced most of the remaining charge. These results indicatethat carboxylates (primarily malate) are synthesized in roots of Nr andurea-fed plants and transported to the shoots in the xylem to maintaincharge balance. The high malate concentration resulted in the C/N weightratio of xylem sap from N2-fed plants being >2.0, even though 83% of theN was transported as allantoin and ailantoic acid which have a C/N ratioof 1.0. The data emphasize that C and N content of N compounds shouldnot be the sole basis for calculating the C/N weight ratio of xylem sap.The C-to-A uptake ratio for plants supplied 10 millimolar N03- ranged

from 1.24 to 1.57 during development, indicating that internal OH- wasgenerated both by excess cation uptake and by N03- and S042- reduction.The C-to-A uptake ratio for 20 millimlar N03-fed plants ranged from0.86 to 0.96 during development, indicating a small net OH- efflux fromthe roots for support of excess anion uptake. On a seasonal basis, only15% of the OH- generated during N03- and SO42- reduction was asso-ciated with OH- efflux (excess anion uptake), while 85% was associatedwith carboxylate accumulation. Tle malate concentration in xylem sapfrom plants supplied 20 millimolar N03- was only one-third that of Nrand urea-fed plants; however, it did balance 75% of the excess inorganiccationic charge. Potassium, recycling to accommodate excess anion uptakeby 20 millimlar N03-fed plants, was calculated to involve at most 17% ofthe total K+ absorbed during the 41- to 76-day growth interval.

It has been shown that total inorganic cations (C) exceed total

1 Cooperative investigation of the United States Department of Agri-culture, Agricultural Research Service, and the North Carolina Agricul-tural Research Service, Raleigh, NC. Paper No. 6816 of the Journal Seriesof the North Carolina Agricultural Research Service at Raleigh.

inorganic anions (A) in the tissue of a number of nonleguminousspecies (3, 14, 17). Reduction of N03-, and s042-, as well asgreater uptake of inorganic cations than of inorganic anions,contributes to the accumulation of excess inorganic cations (C-A)and to the generation ofOH- within the tissue (17, 23). Cytoplas-mic acidity is maintained by transfer of the negative chargeassociated with the OH- to carboxylates, ie. nonvolatile organicacids and uronic residues of pectic substances (5, 9, 17, 23).For some species assimilating N03 , essentially equal amounts

of inorganic cations and inorganic anions were absorbed, theacidity of the ambient medium remained relatively constant, andthe quantity of carboxylates (C-A) in the tissue was equal to thetotal quantity of reduced N and organic S in the tissue (3, 16, 17).Thus, in these plants, the sole fate ofOH- generated by NO3 ands042- reduction was to support carboxylate accumulation. Forother species supplied NO3-, more inorganic anions than inorganiccations were absorbed, the pH of the ambient medium increased,and the quantity of carboxylates (C-A) in the tissue was less thanthe quantity of reduced N and organic S (4, 6, 15). In theseinstances, two fates are indicated for the OH- generated duringN03- and s042- reduction: exchange to the ambient medium todrive excess anion uptake; and consumption in carboxylate syn-thesis.For plants assimilating NH4' and urea, in the absence of the

readily permeable N03 anion, inorganic cation uptake exceedsinorganic anion uptake substantially (3, 12, 17), with a resultingacidification of the rooting medium. The quantity of carboxylate(C-A) in tissue of plants supplied NH4+ was only a small fractionof that observed for N03 -supplied plants and was much less thanthe quantity of reduced N and organic S (3, 12, 17). Most of theOH- generated during excess cation uptake is thought to beneutralized by the H+ produced during subsequent assimilation ofNH4' (17, 23). The quantity of carboxylates in tissue of tomatoplants assimilating urea was intermediate between that of plantssupplied N03 and plants supplied NH4' (17). Inasmuch asassimilation of urea does not alter the pH of the plant cellcytoplasm, the quantity ofcarboxylates measured in the tissue wasessentially equal to the quantity of excess cation uptake and ofS042- reduced (17).When leguminous plants are receiving most of their nitrogen

from N2 fixation, a sizeable excess cation uptake should occurwith a concomitant acidification of the root rhizosphere andaccumulation of tissue carboxylates (13, 22). In addition, suchplants must adjust their root metabolism to accommodate chargebalance in the upward-flowing xylem fluid. The paucity of infor-mation with leguminous plants led us to examine the various ionicbalance adjustments which occur in soybean plants obtaining theirnitrogen from N2 fixation, N03, or urea. A previously proposedconceptual model (13) was used in interpreting the results.

MATERIALS AND METHODS

Plant Culture. Soybean (Glycine max L. Merr.) plants werecultured in an unshaded greenhouse from May through mid-

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ISRAEL AND JACKSON

October of 1977. During daylight h, ambient temperatures werekept below 37°C with evaporative cooling. Night temperature wasallowed to equilibrate with outdoor temperature by leaving ventsopen. The RH ranged from 40 to 50o at midday and from 80 to90%o at night.

Seeds of 'Ransom' soybeans were germinated, and all seedlingswere inoculated with Rhizobium japonicum strain USDA 3 Ilbl 10and transplanted (two per pot) into 25.4-cm-diameter pots con-tamiing Perlite, as described previously (19, 20). Twelve pots ofinoculated seedlings were exposed to each of five nutrient solutiontreatments and arranged in a randomized complete block designwith treatments and sampling dates (at 41, 76, and 151 days aftertransplanting) randomized within blocks. Seedlings were thinnedto one per pot 2 weeks after transplanting.

Nutrient solutions containing N were prepared by adding 10and 20 mim KN03 and 10 and 20 mm urea-N to the N-free nutrientsolution previously described by McClure and Israel (19). Theinitial pH of the treatment solutions ranged from 6.6 to 6.8. TheK+ concentration of all solutions, including the N-free solution,was made equal to that of the 20 mm KNO3 solution by addingappropriate amounts of K2S04. From day 1 to day 9, the appro-priate nutrient solution (250 ml) was applied twice daily at 0900and 1500 h. From the 9th day to the 8th week, pots were flushedthoroughly twice daily (0900 and 1500 h) with tap water (1.5 1/flush), and, after the second flushing, appropriate nutrient solution(400 ml) was applied. From the 8th week to final harvest, appro-priate nutrient solution (400 ml) was applied after both themorning and afternoon flushings. From the 10th week to lateSeptember, pots were also flushed with 1.5 L tap water at noon tomeet the high transpirational demand of the plants. Inasmuch asthe pH of the root rhizospheres decreased after application of N-free and urea solutions and increased after application of N03solutions, the repeated flushing and application of nutrient solu-tions were used to prevent long-term exposure of roots to a low orhigh pH. From the water-holding capacity of the Perlite and thevolume of nutrient solution added, the concentrations of nutrientsin the solution phase of the growth medium shortly after additionwere estimated to be about one-fifth the concentrations in theapplied nutrient solutions. As the plants grew, pots were spread tominimize canopy interference. All plants produced branches atthe cotyledonary, primary, and lower three or four trifoliolatenodes on the main stem.Measurement of Tissue Dry Weight and Chemical Composition.

At 41- and 76-day samplings, plants were separated into leaflets(including the petiolules), stems plus petioles, roots, and nodules.At the 151-day sampling, seeds and pod walls were also separated.Plant material was dried at 65°C for 72 h, weighed, ground topass through a 1.0-mm screen, and mixed for 4 h before subsam-ples were taken for chemical analyses.The total N content of tissue samples (100 mg) was determined

by a Kjeldahl procedure that included a salicylic acid predigestionstep to convert N03 to NH4' and that employed a zirconium-copper catalyst (19). After extracting 500 mg tissue in 50 ml H20,nitrate-N was determined by a manual modification of the methodof Lowe and Hamilton (18). Reduced nitrogen in tissue wascalculated by subtracting the N03-N content from the total Ncontent. Ammonium-N content of tissue from 76-day-old plantswas determined, so that ash alkalinity values could be corrected.Samples of dry tissue (1 g) were shaken for 2 h in 15 ml redistilledH20 and then allowed to stand overnight. After filtration anddilution to 25 ml, NH3-N in 2.5-ml aliquots was diffused into 0.5N HCI and quantified as described previously (20), with theexceptions that 2.0 ml 25 mm sodium tetraborate (pH 10) was usedto alkalinize the aliquots, and flasks were shaken gently at 50°Cfor 48 h to obtain quantitative recovery of NH3.

Although nodulation was inhibited by the application of N03solutions, N03--fed plants formed a few nodules that were active

in N2 fixation. Thus, the total N and reduced N cotitents of tissueof N03 -supplied plants were derived both from NO03 uptakeand assimilation and from N2 fixation. The contribution of N03uptake and assimilation to total N and reduced N contents wasestimated using the relationship between relative ureide contentof xylem sap and relative N input from N2 fixation, determinedby McClure et al. (20). The relative dependence ofNO3 -suppliedplants on N2 fixation was estimated as follows: percentage of total(i.e. reduced) N input from N2 fixation = (percentage of total sapN as ureide-N + 0.41) + 0.84 (20). Reduced-N per plant from N2fixation was calculated as the product of relative N2 fixation andtotal plant N. Thus, reduced-N from nitrate uptake and assimila-tion = total plant N - (nitrate-N + reduced-N from N2 fixation).The relative ureide content values used in these calculations weredetermined on xylem sap collected from 76-day-old plants. It wasassumed that the relative dependence of plants on the two Nsources was constant throughout development. Because the rela-tive contribution of N03 uptake and assimilation to total Nsupply of plants may have decreased with age (20), its contributionto total N contents may have been underestimated for 41-day-oldplants and overestimated for 151-day-old plants. Possible errorsassociated with these calculations would not be of such magnitudeas to alter interpretation of the data.

For determination of Ca2+, Mg2+, K+, Na+, and H2PO4-, theash from 1.25 g of dry tissue was dissolved in 3 ml 4.0 N HCI.Calcium and Mg2+ were determined by atomic absorption, andK+ and Na+ were determined by flame photometry. Phosphoruswas determined spectrophotometrically at 470 nm after reactionwith ammonium molybdate-vanadate. Chloride was determinedwith an automatic chloride titrator.

For total S measurements, 0.25-g samples of dry tissue wereashed in the presence of 5.4 meq Mg(NO3)2. The ash was dissolvedin 3 ml 4.0 N HCI and determined turbidometrically (27). OrganicS content of tissue was calculated from the organic N contentusing the relation: organic S = organic N x 0.05 meq organic S/meq organic N. Inasmuch as the NH4+ concentration in tissue of76-day-old plants was small (<5%) relative to the total reduced Nconcentration, reduced N was taken to be organic N. The ratio oforganic S to organic N is the average determined for tissues of anumber of leguminous plants supplied adequate sulfur (7).

Sulfate-S was calculated as the difference between total S andorganic S. Occasionally the calculated organic S of tissue samplesexpected to have low SO4-S content was greater than the measuredtotal S. In these cases, organic S was taken to be equal to total S.Inasmuch as total S content of the tissue was small in relation tonitrogen and total cation contents, errors associated with thoseestimates had a small impact on the ion balance calculations.Ash alkalinity of tissue samples from 76-day-old plants was

determined by ashing 0.5 g of dry material in the presence of 0.5meq NaOH at 550°C for 3 h, dissolving the ash in 10 or 20 ml 0.1N HCI, and titrating the excess acid to pH 5 with 0.1 N NaOH,using methyl red-methylene blue as an indicator. Ash alkalinityvalues were corrected for NO3 and NH4 contents of the tissue,and, thus, should be a measure of total carboxylate content (17).Ash alkalinity of tissue from 76-day-old plants was used to verifythat the previously described measurements of inorganic cationsand anions accounted for the major positively and negativelycharged inorganic substances. Excess cation content (C-A) wasused as the measure of total carboxylate content of tissue from 41-and 15 1-day-old plants.

Collection and Analysis of Xylem Sap. Sap from 76-day-oldplants was collected between 1000 and 1200 h for time periods upto 20 min and kept on ice until transferred to a freezer (- 18°C)for storage, as described previously (19, 20). After appropriatedilution, K+, Na+, Ca2+, Mg2+, s042-, Cl-, H2PO4-, and N03-contents of the sap were quantified using procedures described fortissue analyses. Amino acids and ureides (allantoin and allantoic

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ION BALANCE, UPTAKE, AND TRANSPORT PROCESSES

acid) were separated and quantified using an amino acid analyzer(19). Total N was determined by a summation ofN in amino acid,ureide, and N03 fractions, which has been shown to account foressentially all of the N in the sap (19). Malate was determined bythe malate dehydrogenase method (11). Total carbon was calcu-lated by summation of carbon associated with amino acid, ureides,and malate.

RESULTS

Yield and Cation-Anion Uptake. The growth of N2-fed plantswas restricted relative to that of plants supplied combined N(Table I), because they experienced a transient N stress as evi-denced by reversible yellowing of leaves between the time thatcotyledonary N reserves were depleted (2.5 to 3 weeks) and thetime that nodules became capable of supplying adequate N forgrowth (approximately 4 weeks). For N2-fixing and urea-depend-ent plants, cation uptake exceeded anion uptake 4- to 6-fold

throughout the developmental cycle, indicating a considerable netH+ efflux from the roots. The cation-to-anion uptake ratio rangedfrom 1.27 to 1.57 for plants supplied 10 mm N03, indicating a

greatly reduced net H+ efflux as NO03 uptake contributed to totalanion uptake. The ratio ranged from 0.86 to 0.96 for plantssupplied 20 mm N03, indicating a relatively small net OH effluxfrom the roots, as N03 uptake and assimilation accounted formost (82%) of the whole plant N input.

Sulfate and H2PO4 were the major anions absorbed by N2-fixing and urea-dependent plants. Nitrate was the major anionabsorbed by N03 -dependent plants, but its uptake did not mark-edly affect the uptake of S042 or H2PO4 .

Potassium plus Ca2+ uptake comprised more than 80o of totalcation uptake by plants exposed to the different N sources. Re-gardless of the N source, K+ uptake exceeded Ca2+ uptake, butthe ratio of K+ to Ca2+ uptake was lower for N03--dependentplants than it was for N2- and urea-dependent plants. With all Nsources, the ratio of K+ to Ca2e uptake declined as plants ap-

Table I. Influence ofAge andN Source on Growth and Inorganic Cation and Anion Uptake by Whole SoybeanPlants

Values are means of four replicates.

Time Cations

Treatments after DryTrans- weight K Ca Mg Na Totalplant

d giplant meqiplantZero-N (N2 fixation) 41 9.5 10.6 3.8 2.0 0.3 16.7

76 139.3 114.0 48.0 24.0 2.0 188.0151 446.0 317.0 164.0 94.0 5.0 580.0

N03-N, 10 mim 41 22.4 22.9 12.3 6.1 0.7 42.076 234.2 200.0 98.0 45.0 4.0 347.0151 633.0 491.0 285.0 143.0 7.0 926.0

N03-N, 20 mM 41 24.5 24.8 15.7 7.8 0.7 49.076 243.9 204.0 116.0 54.0 4.0 378.0151 607.0 492.0 343.0 172.0 6.0 1,013.0

Urea-N, 10 mM 41 18.9 20.0 8.5 4.5 0.6 33.676 205.3 173.0 62.0 36.0 3.0 274.0151 543.0 361.0 183.0 112.0 5.0 661.0

Urea-N, 20 mM 41 20.0 20.9 9.0 5.1 0.6 35.676 235.1 182.0 75.0 40.0 3.0 300.0151 586.0 372.0 179.0 113.0 29.0 693.0

Time Anionsa Excess Cation/Treatments

after Cation AnionTrans- Nb S P Cl Total Uptakec Uptakeplant

d meqlplant ratio

Zero-N (N2 fixation) 41 1.9 1.3 3.2 13.5 5.2276 23.0 13.0 1.0 37.0 151.0 5.08151 72.0 53.0 125.0 455.0 4.64

N03-N, 10mM 41 21.4 3.1 2.3 26.8 15.2 1.5776 186.0 28.0 17.0 1.0 232.0 115.0 1.49151 608.0 77.0 59.0 744.0 182.0 1.24

N03-N, 20 mm 41 45.6 3.1 2.2 50.9 -1.9 0.9676 368.0 25.0 16.0 1.0 410.0 -32.0 0.92151 1,047.0 74.0 58.0 1,179.0 -166.0 0.86

Urea-N, 10 mM 41 3.4 1.9 5.3 28.3 6.3476 43.0 17.0 2.0 62.0 212.0 4.41151 100.0 56.0 156.0 505.0 4.24

Urea-N, 20 mM 41 3.4 1.8 5.2 30.4 6.8576 58.0 17.0 2.0 77.0 223.0 3.90151 109.0 58.0 167.0 526.0 4.15

a S considered divalent; P, monovalent.b N from N03- uptake.-_, Excess anion uptake.

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ISRAEL AND JACKSON

Table II. Effect on N-Source on Cation-Anion Balance of 76-Day-Old Ransom Soybean Plant TissuesValues represent the means of four replicates.

Dry CationsTreatments Plant Part CationWeight K Ca Mg Na Total

glplant meqlplantZero-N (N2 fixation) Leaflets 52.4 31.7 23.5 11.6 0.5 67.3

Stem + petiole 68.8 72.0 19.1 10.9 0.7 102.7Root 18.1 10.2 5.0 1.6 1.2 18.0

NO3, 10 mM Leaflets 76.5 44.5 45.2 19.7 0.9 110.3Stem + petiole 132.7 141.3 45.4 22.0 0.7 209.4Root 25.0 14.8 7.3 3.1 1.8 27.0

NO3, 20 mm Leaflets 81.1 48.4 54.2 22.2 0.9 125.7Stem + petiole 136.1 137.1 51.4 26.9 0.5 215.9Root 26.7 18.0 10.3 4.7 2.3 35.3

Urea, 10 mm Leaflets 71.7 45.5 26.8 16.3 0.4 89.0Stem + petiole 112.2 114.9 27.4 17.5 0.8 160.6Root 21.4 12.4 7.8 2.0 1.5 23.7

Urea, 20 mm Leaflets 84.3 54.2 35.0 17.4 0.7 107.3Stem + petiole 124.3 114.3 29.8 20.4 1.1 165.6Root 26.3 13.9 10.4 2.5 1.6 28.4

Anions

Treatments Plant PartNO3 SO4 H2PO4 Cl Cartexy- Totalate

meqlplantZero-N (N2 fixation) Leaflets 0 6.2 0.1 64.7 71.0

Stem + petiole 0.3 7.5 5.9 0.3 94.5 108.5Root 0.2 4.1 1.1 0.5 16.7 22.6

NO3, 10 mM Leaflets 1.3 7.8 0.2 100.8 110.1Stem + petiole 14.7 9.0 7.4 0.4 181.2 212.7Root 1.9 3.3 1.2 0.7 18.1 25.2

NO3, 20 mM Leaflets 2.8 7.8 0.1 118.1 128.8Stem + petiole 23.8 5.0 6.9 0.5 180.9 217.1Root 3.9 2.5 1.3 0.8 25.0 33.5

Urea, 10 mm Leaflets 0 2.3 7.8 0.2 79.1 89.4Stem + petiole 1.9 15.8 7.7 0.6 123.5 149.5Root 0.5 5.5 1.3 1.0 17.8 26.1

Urea, 20 mm Leaflets 0.2 6.4 8.3 0.2 89.9 105.0Stem + petiole 2.4 20.2 7.1 0.6 121.1 151.4Root 0.8 6.9 1.3 1.4 23.0 33.4

proached maturity.Cation-Anion Balance. In most instances, the sums of total

cations in tissues of 76-day-old plants (K+, Ca2+, Mg2+, and Na+)closely balanced (within 10o) the sums of total inorganic anions(NO3-, s042-, H2PO4-, and CL-) and total carboxylates, as deter-mined by ash alkalinity (Table II). Agreement was less precise inroots of N2- and 20 mm urea-fed plants, where balances werewithin 25% and 15%, respectively. Leaflet and stem plus petioletissues contained higher carboxylate concentrations than did roottissue (derived from information in Table II). With all N sources,carboxylates as a percentage of total anions were 68 to 74% inroot tissues, 80 to 87% in stem plus petiole tissue, and 86 to 92%in leaf tissue. Inasmuch as urea- and N03 -N stimulated plantgrowth, the total quantity of carboxylates in plants supplied theseN sources was greater than that in N2-fed plants.

Carboxylate Accumulation, H+ and OH- Efflux, and NO- andSO42- Reduction. For N2- and urea-fed plants, internal OH-generated by H+ excretion during excess cation uptake was asso-ciated with more than 89% of the carboxylate accumulation atthree stages of growth; that generated by s042- reduction was lessthan 11% (Table III). For plants supplied 10 mm N03-, theproportion of total carboxylate accumulation associated with in-ternal OH- generated by excess cation uptake declined from 49%

to 22% as the plants aged, that associated with OH- generated byN03 reduction increased from 46% to 71%, and that associatedwith OH- generated by So42- reduction remained essentiallyconstant (Table III). In contrast, 85% ofthe OH- generated duringthe entire season by N03- and SQ42- reduction in plants supplied20 mm NO3- was associated with carboxylate accumulation, while15% of this OH- was effluxed to drive excess anion uptake (TableIII).At all sampling dates and for plants grown on all N sources,

shoot tissue contained more than 89%o ofthe total plant carboxylatecontent (Table IV). Stem plus petiole tissue contained a slightlygreater proportion of the carboxylate than the leaflet tissue. Atthe final harvest, reproductive tissue contained 37, 42, and 52% ofthe total carboxylate within N03 -, urea-, and N2-fed plants,respectively.

Ionic Composition of Xylem Sap. The exudation rate of N03-fed plants was higher than that of urea- and N2-fed plants (TableV). Nitrogen source had a relatively small effect on sap acidity, aspH values ranged from 6.07 to 6.26. Total cations were closelybalanced (within 10o) by total inorganic anions plus carboxylates.Carboxylates comprised more than 74% of the total anion chargein sap from N2- and urea-fed plants. Increasing the NO3 supplyfrom 10 to 20 mm resulted in N03 increasing from 25 to 54% of

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Table III. Influence ofAge andN Source on Carboxylate (C-A) Accumulation, HI Excretion (Excess CationUptake), and N03- and So42- Reduction by Whole Soybean Plants

Values represent means of four replicates.

Time

Treatments after H' Excretion Reduced N Organic S CarboxylateTrans- from NO3 Accumulationplant

d meqiplantZero-N (N2 fixation) 41 14 (93)8 1 (7)8 15

76 151(93) 11(7) 162151 455(90) 49(10) 504

N03-N, 10 mM 41 20 (49)b 19 (46)b 2 (5)b 4176 114 (38) 169 (57) 16 (5) 299151 182(22) 599(71) 60(7) 841

N03--N, 20 mM 41 0 41 2 43 (100)-76 -33 (9)` 337 17 321 (91)151 -166 (15) 1029 60 923 (85)

Urea-N, 10 mm 41 28 (93)8 2(7)a 3076 212 (91) 20 (9) 232151 505 (89) 65 (11) 570

Urea-N, 20 mm 41 30 (94)8 2 (6)8 3276 224 (91) 22 (9) 246151 526(90) 60(10) 586

'Values in parentheses for Nr and urea-dependent plants indicate percentage of total carboxylate accumu-lation attributed to excess cation uptake and to S042- reduction.

b Values in parentheses for 10 mm N03--dependent plants indicate percentage of total carboxylate accumu-lation attributed to excess cation uptake, to N03- reduction, and to S042- reduction.

'Values in parentheses for 20 mM NO3--dependent plants indicate percentage of total anion charge resultingfrom N03- and S042- reduction associated with OH- efflux (negative H+ excretion = excess anion uptake) andwith carboxylate accumulation.

the total anion charge, while the proportion due to carboxylatesdecreased from 60%o to 33%.

Potassium and Ca2" were the dominant cations in sap fromplants exposed to all N sources (Table VI). The K+ to Ca + ratiowas lower in sap from plants supplied 20 mm NO03 than in sap

from urea- and N2-fed plants, as well as those supplied 10 mmNO3-.The negative charge associated with malate, aspartate, and

allantoate closely balanced (within 10%o) the excess inorganiccation charge in the xylem sap (Table VI). Regressing the totalcarboxylate concentration against the excess cation concentrationin the xylem sap revealed a linear relationship, y = 1.0 lx + 0.312,with an R2 = 0.902, indicating that the most important carboxyl-ates in the xylem sap have been taken into account. Malatecomprised 76 to 85% of the carboxylate charge in xylem sap fromplants exposed to the different N treatments (Table VI). Theconcentration of malate in sap from N2-, 10 mm NO03-, and urea-

fed plants was 2 to 3 times that in sap from plants supplied 20 mmN03-.

Carbon-Nitrogen Balance of Xylem Sap. Because of the high

malate concentration, the carbon in sap from N2- and urea-fedplants was more than twice that in sap from plants supplied 20mm N03 (Table VII). Forty-eight%, 35%, and 17% of the carbonin sap from N2-fed plants was associated with malate, ureides, andamino acids, respectively. More of the carbon in sap from urea-

and 20mm nitrate-fed plants was present as amino acids (includingamides) than as malate. The carbon-to-nitrogen weight ratio ofsap from N2- and urea-fed plants was greater than 2.0, while thatof sap from plants supplied 20 mtm N03 was 1.29. The ureidescontained 83% of the total N in sap from N2-fed plants. The

importance of ureides as N transport forms in xylem sap declinedas plants were supplied increasing levels of N03 and urea (cf.Ref. 19).

DISCUSSION

A conceptual model has been proposed to account for the effectsof assimilation of different nitrogen sources on ion uptake, trans-location, and balance in plants (13). The model assumes that totalcation and total anion (inorganic plus organic) charges of planttissue must be equal (17) and that the pH of the cell cytoplasmmust be maintained within close limits (pH 7-8) (23).An electrogenic H+ transport to the ambient medium by a

reversible ATPase located in the plasmalemmae of the root cells(Fig. IA) is viewed as maintaining both an electrical and anacidity gradient across those membranes (10, 24, 25). Cations enterthe tissue in response to the resulting electrical potential gradient(Fig. 1B) or as an integral part of the pump operation. In additionto the alkalinizing effect in the root cytoplasm resulting from theH+ efflux process, cytoplasmic OH- generation occurs when N03is reduced(Fig. ID; Ref. 6). Sulfate reduction (not shown) alsogenerates OH- (6). Cytoplasmic OH- generated by any of theprocesses can either serve as counterion for the inward transportof anions (Fig. IC) or stimulate carboxylate synthesis (Fig. 1E;Refs. 5, 9). The latter prevents dramatic increases in the pH of thecell cytoplasm when generation ofOH- exceeds the inward move-ment of anions. Organic acid decarboxylation (Fig. IG; Ref. 5)can occur when rapid exchange of N03 with cytoplasmic OH(Fig. IC) or assimilation of NH+ (6, 17) causes a decrease incytoplasmic pH. Excess anion uptake by plants in which N03reduction occurs primarily in the shoot is viewed as being sus-

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Table IV. Influence ofAge and N Source on Distribution of Carboxylates (C-A) in Soybean Plants

CarboxylatesTreatments Plant Part

41 Days 76 Days 151 Daysmeqlplant

Zero-N (N2 fixation) Leaflets 5.96 (41)a 61 (38) 109 (21)Stem + Petiole 7.32 (50) 89 (55) 122 (24)Pod wall 139 (28)Seed 120 (24)Shootb 13.28 (91) 150 (93) 490 (97)Root 1.40 (9) 12 (7) 14 (3)Whole plant 14.68 162 504

N03-N, 20 mm Leaflets 17.31 (42) 115 (36) 258 (28)Stem + petiole 19.55 (47) 180 (56) 300 (32)Pod Wall 202 (22)Seed 135 (15)Shootb 36.86 (89) 295 (92) 895 (97)Root 4.83 (11) 26 (8) 28 (3)Whole plant 41.69 321 923

Urea-N, 20 mm Leaflets 13.34 (42) 92 (38) 139 (24)Stem + petiole 15.60 (48) 135 (55) 177 (30)Pod wall 120 (20)Seed 131 (22)Shootb 28.94 (90) 227 (93) 567 (96)Root 3.32 (10) 19 (7) 21 (4)Whole plant 32.26 246 588

a Values in parentheses indicate percentage of whole plant carboxylates.b Shoot includes leaflets and stems + petioles at 41 and 76 days and these plus pod wall and seed at 151 days.

Table V. Influence of Nitrogen Source on Exudation rate, pH, and Ionic Composition of Xylem Sapfrom 76-Day-Old PlantsValues represent means of three replicates.

Ionic Composition of Xylem SapExuda-

tion Rate K Ca Mg Na Total cat- N S P Cl Carbox- Total

ions ylatesa anions

g min-'planC' ii~~~~~~~~~~~~,eq/mlplantr'

Zero-N (N2 fixation) 0.27 6.07 8.10 2.16 1.59 NDb 11.85 0.06 0.63 0.47 0.96 9.90 12.02N03-N, 10mM 0.39 6.26 7.03 1.82 1.11 ND 9.96 2.46 0.51 0.37 0.47 5.84 9.65N03-N, 20 mM 0.44 6.13 5.64 2.11 1.02 ND 8.77 4.57 0.38 0.35 0.42 2.79 8.51Urea-N, 10 mM 0.26 6.10 7.14 2.26 1.50 ND 10.90 0.30 0.6l 0.51 0.90 8.02 10.39Urea-N, 20 mM 0.29 6.23 7.03 2.04 1.43 ND 10.50 0.54 0.87 0.43 0.63 7.18 9.65

a Sum of microequivalents of malate, aspartic acid, and allantoic acid, assuming a net negative charge at pH of sap of -2, -1, and -1, respectively.b Not detected.

Table VI. Influence of Nitrogen Source on the Carboxylate Composition and Charge Balance of Xylem Sapfrom76-Day-Old Soybean Plants

Values represent means of three replicates.

Aspar- Allantoic Total Carboxylate (,ueq)Treatments Malate tate Acid Carbox- C-A C-A (,ueq)tate Acid ~ylate,ieq/ml sap ratio

Zero-N (N2 fixation) 7.55 0.12 2.23 9.90 9.85 1.01N03-N, 10 mM 4.83 0.25 0.76 5.84 6.15 0.95N03-N, 20 mM 2.37 0.15 0.28 2.80 3.03 0.92Urea-N, 10 mM 6.49 0.34 1.19 8.02 8.52 0.94Urea-N, 20 mM 5.83 0.36 1.00 7.19 8.03 0.89

tained by decarboxylation of carboxylates (Fig. 1G) which are N03- reduction (1, 2, 6, 8, 15). Transport of cations, organic andtransferred with accompanying cations (K+) in the phloem to the inorganic anions, and organic N forms into and out of the xylemroots (Fig. IF) following their synthesis in association with leaf (Fig. 1H), and vacuoles also must occur in a manner that allows

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ION BALANCE, UPTAKE, AND TRANSPORT PROCESSES

Table VII. Influence of Nitrogen Source on the C-N Balance of Xylem Sapfrom 76-Day-Old Soybean Plants

Sap N as

Treatments C/N Total C Malate Ureide .m° Total N . NH4+acids Ureide Amo N03-acids

ratio ,ug/ml % lg/ml %Zero-N (N2 fixa-

tion) 2.04 381 48 35 17 188 83 16 <1 <1NO3, 10mM 1.84 247 46 22 32 134 48 26 26 <1N03,20 mM 1.29 177 32 10 58 138 15 38 47 <1Urea, 10mM 2.27 397 39 19 42 174 49 48 3 <1Urea, 20 mM 2.17 421 33 16 51 193 39 57 4 <1

Table VIII. The Influence of Nitrogen Source on the Carbon Requirementfor Maintenance of Ionic Balance in76-Day-Old Soybean Plants

It was assumed that the carboxylate was malate and that dry matter contained 41% carbon. Values representmeans of four replicates.

Carboxylate (C-A) Carbon in Carbon as Car-boxylate

CarboxylateTreatment Dry matter (C-A)

Shoot Whole Shoot e Shoot e Shootplant plant plant plant

meqlplant glplant %Zero-N (N2 fixation) 162 150 57.1 49.7 3.9 3.6 6.8 7.2N03-N, 10 mM 299 279 95.9 85.6 7.2 6.7 7.5 7.8N03-N, 20 mM 321 295 100.0 89.1 7.7 7.1 7.7 8.0Urea-N, 10 mM 232 213 84.2 75.4 5.5 5.1 6.6 6.8Urea-N, 20 mM 246 228 96.4 85.6 5.9 5.5 6.1 6.4

FIG. 1. A schematic illustrating interrelationships of processes associ-ated with maintenance of intracellular pH and ion balance in, and ionintake by, higher plants.

for maintenance of charge balance.Large amounts of carboxylates (C-A) were synthesized regard-

less of the N source (Table II), and more than 89% of thesecarboxylates accumulated in shoot tissue. However, the processesassociated with synthesis and distribution of carboxylates differedappreciably. In N2- and urea-fed plants, the large proportion oftotal plant carboxylate content in shoots (Table IV) and the high

levels of malate in xylem sap (Table VI) suggest that carboxylatesare synthesized in the root cells (Fig. IE) in response to OH-generation during H+ excretion (excess cation uptake) (Fig. IA).The anion change which balances excess cation uptake is presum-ably converted from the OH- form to carboxylate (malate) via thePEP carboxylase half of the intracellular pH stat system (Fig. IE;Ref. 5), thereby permitting maintenance of both ionic balance andpH within the cytoplasm of root cells (23). The malate andassociated cations are then deposited in the xylem (Fig. IH) fortransport to the shoot. Carboxylates remain in the shoot tissuebalanced by inorganic cations. Inasmuch as nodules are not likelyto have any significant involvement in uptake of inorganic cationsand anions from the rhizosphere (23), most ofthe malate observedin the xylem sap is probably synthesized in the root tissue.

Stumpf and Burns (26) reported that malonate was the predom-inate dicarboxylic acid in fresh nodule, root, and leaf tissue of N2-fixing soybeans. Although we did not measure the malonatecontent ofxylem sap, anion charge associated with malate, asparticacid, and allantoic acid was sufficient to largely balance excesscations (Table VI), implying that malonate was not a majorcarboxylate in the xylem. The predominance of malate in thexylem (Table VI) and ofmalonate in leaf tissue (26) would suggestmetabolism of malate upon delivery to the leaves. Apparently,malate that remains in root and nodule tissue would also bemetabolized to malonate.For N2-fixing plants, the C/N weight ratio of xylem sap was

2.04, even though 83% of the N was transported as ureides whichhave a C/N ratio of 1. This high C/N ratio resulted from thetransport of malate which contained almost 50o of the carbon insap (Table VII). It is evident that C/N ratios of the N compoundsin the xylem sap did not indicate the total upward carbon flow.Failure to consider the carbon in xylem organic acid anions ofN2-fixing plants may lead to significant errors in constructingoverall plant carbon balances.Most (85%) of the OH- formed during N03 and so42 reduc-

tion in either roots (Fig. ID) or shoots of plants supplied 20 mmN03 was neutralized by carboxylate (C-A) synthesis in eitherroots (Fig. IE) or shoots, and only a small amount (15%) wastransported (Fig. IC) to the ambient medium during excess anion(NO3) uptake (Table III). Nitrate reduction accounted for about95% of the cytoplasmic OH- generation, and s042- reductionaccounted for 5% (Table III). This partitioning of cytoplasmicanion charge (OH-) between carboxylate formation and efflux tothe ambient medium is similar to that reported for tomato plantssupplied adequate N03 (16). In 10 mm N03 -fed plants, excesscation uptake indicated that carboxylate accumulation was asso-ciated with OH- generated by net H+ excretion (Fig. IA) inaddition to that generated by N03- and so42- reduction in roots

177

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ISRAEL AND JACKSON

(Fig. ID) and shoots.During the 41- to 76-day growth interval, inorganic anion

uptake by 20 mm N03 -fed plants exceeded cation uptake by 32meq/plant, and total N03 and K+ uptake were 322 and 179meq/plant, respectively (Table I). This information can be usedto assess the extent of K+ recycling. Reduction of 10o (32 out of322 meq/plant) of the N03 which entered the plant in the rootswould have generated sufficient OH- to sustain the excess anionuptake (Fig. IC). On the other hand, if it is assumed that no NO3reduction occurred in the roots and that K+ is the only cationreadily transported in the phloem, about 17% (32 out of 179 meq)of K+ ions which entered the plant would have recycled withcarboxylate in the phloem (Fig. IF) to sustain the excess anionuptake. Thus, N03--fed soybean plants are similar to N03--fedtomato plants (16) in requiring relatively minimal K+ recycling.The requirement for ionic balance and intracellular pH main-

tenance by carboxylates involves the sequestration of energy inthe form of carbon (23). Calculations assuming that plant drymatter is 41% carbon and that malate or citrate is the organic acidin the tissue reveal that, regardless of the N source, 5 to 8% oftotal plant carbon accumulated is involved in maintenance of ionbalance and intracellular pH (Table VIII). Values of 4 to 6%would be obtained if malonate were the organic acid, as it is inyounger soybean tissue (26). While not great, this is energy whichpotentially could be used to support other growth processes.Whether alleviation of inorganic cation-anion imbalance wouldimprove growth and productivity would depend on whether car-bon supply is the factor limiting growth and whether the meansused to improve cation-anion balance involved a comparableenergy expenditure.The results of this greenhouse investigation can be used to make

predictions about the behavior of soybean plants in the field. Forexample, the C-A value for seed from N2-fixing plants was 0.68meq/g dry weight. A 3,000-kg per hectare crop of seed with thiscomposition would be associated with acidification of the soil,requiring 102 kg CaCO3 per hectare for neutralization. Productionof soybeans on low N soils, such as those of the SoutheasternCoastal Plain, is likely to cause substantial acidification whichmust be corrected by application of CaCO3. Implications foreffects of rhizosphere acidification on the nodulation process mayalso be advanced (13, 21).

Acknowledgments-The authors thank Ms. Virginia Flynt and Ms. Peggy Long-mire for excellent technical assistance.

LITERATURE CITED

1. BEN-ZIONi A, Y VAADIA, SH LIPs 1970 Correlations between nitrate reduction,protein synthesis and malate accumulation. Physiol Plant 23: 1039-47

2. BEN-ZIONI A, Y VAADIA, SH Lips 1971 Nitrate uptake by roots as regulated bynitrate reduction products of the shoots. Physiol Plant 24: 288-290

3. BRETELER H 1973 A comparison between ammonium and nitrate nutrition of

young sugar beet plants grown in nutrient solutions of constant acidity. I.Production of dry matter, ionic balance and chemical composition. Neth JAgric Sci 21: 227-244

4. BRETELER H, AL SMIT 1974 Effect of ammonium nutrition on uptake andmetabolism of nitrate in wheat. Neth J Agric Sci 22: 73-81

5. DAVIEs DD 1973 Metabolic control in higher plants. In BV Milborrow, ed,Biosynthesis and Its Control in Plants. Academic Press, New York, pp 1-20

6. DUKSHOORN W 1962 Metabolic regulation of the alkaline effect of nitrateutilization in plants. Nature 194: 165-167

7. DIJKSHOORN W, AL VAN WIJK 1967 The sulphur requirements of plants asevidenced by the sulphur-nitrogen ratio in the organic matter: a review ofpublished data. Plant Soil 26: 129-157

8. DIJKSHOORN W, DJ LATHWELL, CT DE WIT 1968 Temporal changes in carbox-ylate content of ryegrass with stepwise changes in nutrition. Plant Soil 29: 369-390

9. HIATT AJ 1967 Relationship of cell sap pH to organic acid change during ionuptake. Plant Physiol 42: 294-298

10. HODGES TK 1973 Ion absorption by plant roots. Adv Agron 25: 163-20711. HOHORST HJ 1965 Malate determination with malic dehydrogenase and DPN.

In HU Bergmeyer, ed, Methods of Enzymatic Analysis. Academic Press, NewYork, pp 328-332

12. HOUBA VFG, F VAN EGMOND, EM WITriCH 1971 Changes in production ororganic nitrogen and carboxylates (C-A) in young sugar-beet plants grown innutrient solutions of different nitrogen composition. Neth J Agric Sci 19: 39-47

13. IsRAEL DW, WA JACKSON 1978 The influence of nitrogen nutrition on ion uptakeand translocation by leguminous plants. In CS Andrew, EJ Kamprath, eds,Mineral Nutrition of Legumes in Tropical and Subtropical Soils. Common-wealth Scientific and Industrial Research Organization, Australia, pp 1I13-129

14. KiRKBY EA 1969 Ion uptake and ionic balance in plants in relation to the formof nitrogen nutrition. In IH Rorison, ed, Ecological Aspects of the MineralNutrition of Plants. Blackwell Oxford, pp 215-235

15. KIuRBY EA, MJ ARMSTRONG 1980 Nitrate uptake by roots as regulated by nitrateassimilation in the shoot of castor oil plants. Plant Physiol 65: 286-290

16. KiRxuBY EA, AH KNIGHT 1977 Influence of the level of nitrate nutrition on ionuptake and assimilation, organic acid accumulation, and cation-anion balancein whole tomato plants. Plant Physiol 60: 349-353

17. KIRKBY EA, K MENGEL 1967 Ionic balance in different tissues of the tomatoplant in relation to nitrate, urea and ammonium nutrition. Plant Physiol 42: 6-14

18. LowE RH, JL HAMILTON 1967 Rapid method for determination of nitrate inplant and soil extracts. J Agric Food Chem 15: 359-361

19. MCCLURE PR, DW IsRAEL 1979 Transport of nitrogen in the xylem of soybeanplants. Plant Physiol 64: 411-416

20. MCCLURE PR, DW ISRAEL, RJ VOLK 1980 Evaluation of the relative ureidecontent of xylem sap as an indicator of N2 fixation in soybeans. Greenhousestudies. Plant Physiol 66: 720-725

21. MUNNS DN 1978 Soil acidity and nodulation. In CS Andrew, EJ Kamprath, eds,Mineral Nutrition of Legumes in Tropical and Subtropical Soils. Common-wealth Scientific and Industrial Research Organization, Australia, pp 247-263

22. NYATSANGA T, WH PIERRE 1973 Effect of nitrogen fixation by legumes on soilacidity. Agron J 65: 936-940

23. RAvEN JA, FA SMITH 1976 Nitrogen assimilation and transport in vascular landplants in relation to intracellular pH regulation. New Phytol 76: 415-423

24. SMITH FA 1970 The mechanism of chloride transport in Characean cells. NewPhytol 69: 903-917

25. SMITH FA 1973 The internal control of nitrate uptake into barley roots withdiffering salt content. New Phytol 72: 769-782

26. STUMPF DK, RH BuRRis 1979 A micromethod for the purification and quanti-fication of organic acids of the tricarboxylic acid cycle in plant tissues. AnalBiochem 95: 311-315

27. TABATABAI MA, JM BREMNER 1970 A simple method of determining sulfur inplant materials. Agron J 62: 805-806

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