Influence of nitrate on uptake of ammonium by nitrogen-depleted soybean: is the effect located in roots or shoots?

Journal of Experimental Botany, Vol. 45, No. 280, pp. 1575-1584, November 1994Journal ofExperimentalBotany

Influence of nitrate on uptake of ammonium bynitrogen-depleted soybean: is the effect located inroots or shoots?

Carole H. Saravitz1, Sylvain Chaillou2'4, Joanne Musset3, C. David Raper Jr.1 andJean-Francois Morot-Gaudry2

1 Department of Soil Science, Box 7619, North Carolina State University, Raleigh, NC 27695-7619, USA2 Laboratoire du Metabolisme, INRA, route de St Cyr, F-78026 Versailles Cedex, France3 Laboratoire de Physiologie Vegetale, Institut National Agronomique Paris-Grignon, 16 Rue Claude-Bernard,F-75231 Paris Cedex, France

Received 8 April 1994; Accepted 20 July 1994

Abstract

In non-nodulated soybean [Glycine max (L.) Merrill cv.Ransom] plants that were subjected to 15 d of nitrogendeprivation in flowing hydroponic culture, concentra-tions of nitrogen declined to 1.0 and 1.4mmol Ng 1

dry weight in shoots and roots, respectively, and theconcentration of soluble amino acids (determined asprimary amines) declined to 40//mol g 1 dry weight inboth shoots and roots. In one experiment, nitrogenwas resupplied for 10 d to one set of nitrogen-depletedplants as 1.0 mol m"3 NH4

+ to the whole root system,to a second set as 0.5 mol m 3 NH4

+ plus 0.5 mol m 3

NO3~ to the whole root system, and to a third set as1.0 mol m 3 NH4

+ to one-half of a split-root system and1.0 mol m"3 NO3"~ to the other half. In a second experi-ment, 1.0 mol m~3 of nitrogen was resupplied for 4 dto whole root systems in NH4

+ : NO3 ratios of 1:0, 9 : 1 ,and 1:1. Nutrient solutions were maintained at pH 6.0.

When NH^ was resupplied in combination withNO3" to the whole root system in Experiment I, cumulat-ive uptake of NH4

+ for the 10 d of resupply was abouttwice as great as when NH4

+ was resupplied alone.Also, about twice as much NH4

+ as NO3~ was taken upwhen both ions were resupplied to the whole rootsystem. When NH4

+ and NO3" were resupplied to separ-ate halves of a split-root system, however, cumulativeuptake of NH4

+ was about half that of NO3". The uptakeof NH4

+, which is inhibited in nitrogen-depleted plants,thus is facilitated by the presence of exogenous NO3",and the stimulating effect of NO3 on uptake of NH4

+

appears to be confined to processes within root tis-sues. In Experiment II, resupply of nitrogen as bothNH4

+ and NO3 in a ratio of either 1:1 or 9:1 enhancedthe uptake of NH4

+. The enhancement of NH4+ uptake

was 1.8-fold greater when the NH4+: NO3-resupply

ratio was 1:1 than when it was 9 : 1 ; however, only 1.3times as much NO3 was taken up by plants resuppliedwith the 1 :1 exogenous ratio. The effect of NO3~ onenhancement of uptake of NH4

+ apparently involvesmore than net uptake of NO3 itself and perhaps entailsan effect of NO3~ uptake on maintenance of K+ availab-ility within the plant. The concentration of K+ in plantsdeclined slightly during nitrogen deprivation and con-tinued to decline following resupply of nitrogen. Thegreatest decline in K+ concentration occurred whennitrogen was resupplied as NH4

+ alone. It is proposedthat decreased availability of K+ within the NH4

+-resup-plied plants inhibited NH4

+ uptake through restrictedtransfer of amino acids from the root symplasm intothe xylem.

Key words: Ammonium, Glycine max, nitrate, nitrogen-nutrition, nitrogen stress, split-root cultures.

Introduction

In previous experiments when we have controlled acidityin flowing solution culture at a pH near 6.0, we haveshown that under growth-chamber conditions NH^ andNOf are utilized with equal effectiveness as sole nitrogen

4 To whom all correspondence should be addressed. Fax: +33 1 30 83 30 96.

© Oxford University Press 1994

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1576 Saravitz et al.

sources by nitrogen-replete soybean (Henry and Raper,1989; Raper et al., 1991; Rufty et al., 1983; Vessey et al.,1990). Nitrogen concentrations of these plants typicallyexceed 40mgg~1 dry weight. More recently it wasobserved that, following depletion of the nitrogen concen-tration in soybean to about 20mgg~' dry weight, NH^was less readily utilized than NO " as a sole nitrogensource (Rideout et al., 1994). However, when thesenitrogen-depleted plants were resupplied with NH^ plusNO3~, their capacity for utilization of NH^ was restored.Similar responses have been observed for tobacco (unpub-lished data). The mechanism of this response is notcertain.

Several possibilities exist for a direct role of NO3~ infacilitating utilization of NH^ by nitrogen-depletedplants. One possibility is that NO3~", either directly orindirectly, may promote assimilation of NH^. In radish,which is particularly sensitive to NH^" nutrition, assimila-tion of NH^" within the root is promoted by simultaneoussupply of exogenous NO^ (Goyal et al., 1982; Ota andYamamoto, 1989). Such a mechanism appears unlikelyin the case of nitrogen-depleted soybean, however, sinceNH.J" resupplied without NO3~" apparently was assimilatedrapidly in the roots and abnormal concentrations of freeNH41" were observed in neither shoots nor roots (Rideoutet al., 1994). Another possibility is that NO3~", eitherdirectly or indirectly, may promote synthesis of an ammo-nium transporter in nitrogen-depleted soybean. Entry ofNH^ and NO3" into roots involves separate transporters(Goyal and Huffaker, 1986). For nitrogen-depletedsoybean, however, the more repressed uptake of NH.J",relative to NO3~, apparently involved enhanced efflux ofexcessively absorbed NH^ rather than restriction of itsentry into roots (Rideout et al., 1994). Furthermore,most of the NO3~ absorbed by soybean is reduced in theleaves (Rufty et al., 1982Z>), and transport of NO3~ fromroot to shoot appeared to be less inhibited in nitrogen-depleted soybean than transport of amino acids fromroots of NH^-resupplied plants.

It also is possible that the effect of NO^ in facilitatingutilization of NH/ by nitrogen-depleted plants is indirectand occurs in the shoot. Resupply of nitrogen-depletedplants with NH/ plus NO3~ led to more rapid recoveryof photosynthetic activity than resupply with NH^ alone(Rideout et al., 1994). It is thus possible that NO3", whichcan be taken up and transported to shoots of nitrogen-depleted plants, indirectly facilitates utilization of NHîn nitrogen-depleted plants by stimulating recovery ofphotosynthetic activity in the shoot and restoring thecapacity for resupply of carbohydrates to roots.

The objective of this experiment was to establish as afirst step in determining the processes involved whetherthe stimulation of NH/ utilization by NO3" in nitrogen-depleted plants is localized within the root absorbing

^ or whether it is expressed through the shoot. The

experimental approach involved resupply of NH^ andNO3~ to separate halves of the split-root system of nitro-gen-depleted soybean plants. Since NO3~ presumably isnot transported in the phloem (Hocking, 1980), a stimula-tion of NH^ uptake by NO3~ would be indirect.Conversely, if NH^ uptake by one axis is not stimulatedby uptake of NO3~~ by the other axis, the effect of NO3~would be confined to processes within root tissues exposedto both ions.

Materials and methods

Plant material and growth conditions

After 3 d in a dark germination chamber at 25 °C and 98%relative humidity, soybean [Glycine max (L.) Merrill cv.Ransom] seedlings with radicle lengths between 8 and 12 cmwere placed in each of three continuous-flow, hydroponicculture units (Vessey et al., 1988) located within a controlled-environment room of the phytotron at North Carolina StateUniversity (Raleigh, NC, USA). During a 20 d pretreatmentperiod from transplanting until emergence of the fourthtrifoliolate leaf, roots in all hydroponic units received a completenutrient solution containing (in mol m~3) NO3"", 1.0; H2PO^",0.25; K + , 1.25; SO2,", 0.5; Ca2 + , 0.25; Mg2 + , 0.25; (in mmolm"3) B, 19; Mn, 3.7; Cl, 7.2; Zn, 0.3; Cu, 0.13; Mo, 0.05; and10 mmol m~3 Fe(II) as 300 Fe-Sequestrene (CIBA-GeigyCorp.). Half of the solution in each whole-root unit and all thesolution in each split-root unit was replaced with fresh solutionon days 9 and 16 of pretreatment, and equal parts of Ca(NO3)2and Mg(NO3)2 were added to adjust concentrations to 1.0 molm~3 NO3". Roots were not inoculated with rhizobia, and nonodules were observed during the experiment. Temperature ofthe nutrient solutions was maintained at 24 °C, and pH wasmaintained at 6.0 by automated additions of 0.01 M H2SO4 orCa(OH)2. Day/night aerial temperatures were 26/22 °C during9h day and 15 h night periods. A photosynthetic photon fluxdensity of 700/xmol m~2s~' and a photomorphogenic irradi-ance of 12 W m~2 were provided by a combination of cool-white fluorescent and incandescent lamps. Midway through the15 h night period, a 3h interruption with incandescent lamps(photosynthetic photon flux density of 70/xmol m~2s~' and aphotomorphogenic irradiance of 11 W m~2) were used torepress floral development. Atmospheric CO2 in the growthroom was maintained at 400 cm3 m"3.

Experiments

Two experiments were conducted. In Experiment I for split-root culture, one of the hydroponic units had two separate rootcompartments for each plant and each root compartmentcontained 60 dm3 of solution; the other two hydroponic unitswere designed for whole-root culture and contained 200 dm3 ofnutrient solution. Initially, 48 plants were placed in eachhydroponic unit, and after 2 d tap roots of plants in all threeunits were excised below the upper 6 or 7 lateral branches. At6 d after transplanting, all laterals present at the initial pruning,except four of similar length, were removed. Laterals initiatedafter the original pruning were removed at 6 and 9 d aftertransplanting. In the split-root unit, all roots remained in onecompartment until after pruning when the roots were separatedat 9 d after transplanting with two laterals in each half-rootcompartment. No new primary laterals were initiated from thetap root after 9 d in the whole-root or split-root systems. In

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Experiment II for determination of effects of NH^ : N 0 3 ratioon uptake of NHf, all three hydroponic units were configuredfor whole-root systems, and roots of the 48 plants initiallyplaced in each unit were not pruned.

Nitrogen deprivation culture

At the fourth trifoliolate leaf stage, plants for both experimentswere thinned to 40 plants per hydroponic unit and exposed fora 15 d nitrogen-deprivation period to a minus-nitrogen solution(containing the same concentrations as the complete solutionof all other nutrients except SO4~, which was increased to1.0 mol m~3) to which 200 fimol NOf per plant were addeddaily as equal parts of Ca(NO3)2 and Mg(NO3)2. This dailyaddition of NO3", supplied at 5 h into the day period inExperiment I and in hourly increments in Experiment II, wasestimated from previous experiments as sufficient to provide anitrogen stress (Tolley-Henry and Raper, 1986; Rideout et al.,1994) while maintaining induction of the NO3" transport system.Concentration of NO^ was monitored immediately prior toaddition of the NO^, utilizing a Model 21 lOi Dionex IonChromatograph with an AS4A anion separator column, andall but trace amounts added on the preceding day had beenabsorbed. Every 3 or 4 d, half of the solution in each whole-root unit and all the solution in each split-root unit wasreplaced with fresh solution to maintain other nutrients within10% of initial concentrations (Vessey et al., 1988). Otherconditions during the depletion period were the same as duringthe pretreatment period.

Nitrogen resupply culture

At the end of the 15 d period of nitrogen deprivation, nitrogenwas resupplied for 10 d in Experiment I as 1.0 mol m~3 NH.J"in one whole-root unit, as 0.5 mol m~3 NH.J" plus 0.5 mol m~3

NO3" in the second whole-root unit, and as 1.0 mol m~3 NHîn one half-root unit of the split-root system and 1.0 mol m" 3

NO3" in the other half-root unit. In Experiment II, nitrogenwas resupplied for 4d in one hydroponic unit as 1.0 mol m" 3

NH^", in the second unit as 0.9 mol m" 3 NH4+ plus 0.1 mol

m" 3 NOf, and in the third unit as 0.5 mol m" 3 NH^ plus0.5 mol m~3 NO3"\ Except for SO4~, which was used as thevariable ion, concentrations of other nutrients were the sameas for the pretreatment and depletion solutions. Daily duringthe 10 d treatment period, deionized water was added to eachof the root units at 4 h into the day period to return thevolumes to 200 dm3 for the whole-root units and 60 dm3 forthe half-root units. Concentrations of NH^ and NO^ weremeasured 15min later by ion chromatography using an AS4Acolumn and pulsed amperametric detector for NO^ and apostcolumn reaction with o-phthalaldehyde and fluorescencedetection for NH^ (Goyal et al., 1988). Appropriate amountsof Ca(NO3)2 plus Mg(NO3)2 or (NH4)2SO4 then were addedto return NH^ and NOf in the solutions to 1.0 mol m~3. Halfof the nutrient solution in each whole-root unit and all thesolution in each half-root unit was replaced with fresh solutionevery 3 or 4 d. Other conditions were the same as during thepretreatment and depletion periods.

Plant sampling and net CO2 exchange rates

Immediately prior to initiation of the nitrogen-deprivationperiod and at 4, 9, and 15 d into the deprivation period, fourplants were harvested from each hydroponic unit at 5.5 h intothe day period. These sample periods are referred to as —15,— 11, — 6, and 0 d after nitrogen resupply. Harvests of fourplants from each unit also were taken at + 1 , + 3 , +6, +8,and + 10 d of nitrogen resupply in Experiment I and at 0 and

Influence of nitrate on ammonium uptake 1577

+ 4d in Experiment II. Each plant was separated into leaves,stems (plus petioles), and roots. For the split-root unit, the twohalves of the root system (each half consisted of two primarylaterals) were harvested separately. Leaf area was measuredphotometrically with a Li-cor LI-3000 area meter. The plantparts were placed immediately in a freezer at — 22 °C and laterfreeze-dried, weighed, ground to pass a 1 mm screen, and storedin sealed vials until analysed.

Accumulation of dry matter in shoots (leaves plus stems)and roots in Experiment I was described by regression modelsas a function of days at treatment. Separate equations werederived for the nitrogen deprivation and resupply periods. Dryweights of shoots (leaves plus stems) and total roots withineach harvest were statistically analysed by a one-way analysisof variance. Differences in dry weight of halves of the split-rootsystem were analysed by a two-tailed standard Student's t test.For the deprivation period, there were no significant differencesfor dry weight among the three hydroponic units or betweenthe halves of the split-root system; thus, these data for eachharvest were pooled and fitted to a single linear regressionequation. For the resupply period, accumulation of dry matterin shoots and roots was described for each hydroponic unit inExperiment I as a linear model of the natural logarithm of dryweights. Within the hour preceding destructive harvests, netCO2-exchange rates of the middle leaflet of the second, fifth,eighth, and thereafter the youngest expanded mainstem trifolio-late leaves of the four sample plants from each hydroponic unitin Experiment I were determined using a clamp-on Plexiglascuvette and an Anarad AR-500R infra-red gas analyser in thedifferential mode (Tolley and Raper, 1985).

A/H4+ and NO3 uptake

Daily net uptake of NH^ and NOf per plant during the 10 dperiod of nitrogen resupply in Experiment I and the 4 d periodin Experiment II was determined from the daily ion chromato-graphic analyses of depletion from the solutions in eachhydroponic unit. Additionally, in Experiment I net uptake ofNH^ and NO^ was monitored at hourly intervals for the first3 d of resupply (Rideout et al., 1994). Daily and hourly specificnet uptake rates (uptake per unit root dry weight) inExperiment I were calculated as the quotient of the net uptakerate per plant divided by the dry weight of roots per plant aspredicted by the regression equations for the hydroponic unit.Cumulative uptake of NH^ and NO^ in both Experiments Iand II was derived by summation of their daily depletionfrom solution.

Tissue analyses

Composite samples of tissue for shoots (leaves plus stems) androots of the four plants from each harvest of each treatment inExperiment I were prepared by combining subsamples ofground tissue proportional to their dry weights. Total carbonand nitrogen in each composite sample were determined utilizinga Perkin Elmer 2400 CHN elemental analyser. After extractionwith water at 90°C for 2 h, NO3~" and K+ were determined byion chromatography. Soluble carbohydrates, starch, organicacids, soluble free primary amines (including NH^), and freeNH^ were determined as previously described (Chaillou et al.,1991, 1994; Rideout et al., 1994) after extraction with ethanoland water at 5°C. Starch in the residue was solubilized inDMSO and 8.0 kmol m~3 HC1 at 60 °C and hydrolysed intoglucose by amyloglucosidase (EC 3.2.1.3).

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Results

Uptake and accumulation of nitrogen following resupply(Experiment I)

When nitrogen was resupplied to nitrogen-depletedsoybean as NH<J~ alone to the whole root or to half of asplit-root system, cumulative uptake of NH^ during the10 d resupply period was restricted relative to that whennitrogen was resupplied as NH^ plus NO ~ to the wholeroot (Fig. 1A, B, C). The restricted uptake of NH^ whenwhole or half-root systems of nitrogen-depleted plantswere resupplied with NH^ alone was a consequence ofgenerally lower rates of both uptake per unit root weight(specific uptake rate) (Fig. ID, E, F) and of significantlydepressed root growth (Fig. 2F, G, H). The diminishedcapacity for uptake of NH.J" became more pronouncedduring the last 4 d of the resupply period, as both specific

RESUPPLYWhole RootNHJ +NO;

O NO, hall• NHjholfQ Combined ax«

0 5 10 0 5 10 0 5

DAYS AFTER NITROGEN RESUPPLY

Fig. 1. Cumulative uptake (A, B, C) and daily specific uptake rate (D,E, F) of NH4

+, NOf, and total nitrogen by roots of soybean following15 d of nitrogen deprivation when nitrogen was resupplied as 1.0 molm"3 NH^ to the whole root system (A, D), 0.5 mbl m~3 NH^ plus0.5 mol m~3 NO7 to the whole root system (B, E), or 1.0 mol m~3

NH^ to one half of a split-root system and 1.0 mol m~3 NOf to theother half (C, F). Experiment I.

uptake rate (Fig. IF) and mass (Fig. 2H) of NH4+-

resupplied roots declined in comparison to NO3"-resupplied roots.

Only net uptake of NH^ and NO3", rather than separ-ate influx and efflux components, was measured in thisexperiment. Monitoring of net uptake at hourly intervalsduring the first 3 d of resupply, however, revealed intervalsof both depletion (net influx into roots) and enrichment(net efflux from roots) in the exogenous solution(Table 1). The number of hourly intervals of solutionenrichment for NH^ was about twice that for NO3"regardless of whether NH^ was resupplied alone to wholeroot systems or halves of split-root systems or wasresupplied with NO3~ to whole root systems.

Concentrations of total nitrogen in the shoot and roots(Fig. 3) increased similarly among treatments since drymatter accumulation (Fig. 2) following nitrogen resupplywas approximately proportional to nitrogen accumula-tion. The slightly greater concentration of total nitrogenin the NO^-resupplied half than the NH^"-resupplied halfof the split-root system was attributable to accumulationof NO3". Concentrations of soluble primary amines in theshoot (Fig. 4B, C, D) also increased similarly amongtreatments. However, concentrations in roots of solubleprimary amines (Fig. 4F, G, H) and free NH^ (Fig. 4J,K, L) increased more rapidly and to a greater level inwhole or half-root systems that were resupplied withNH^ alone than in whole root systems that were resup-plied with NH.J" plus NOf or in halves of split-rootsystems that were resupplied with NO^. The increasedconcentrations of free NH^", however, were transient anddeclined within 2 or 3 d after resupply.

Concentration and uptake rate ofK+ (Experiment I)

The concentration of K+ within plants (Table 2), whichdeclined slightly during the nitrogen deprivation period,continued to decline following resupply of nitrogen.Declines in both average rate of K+ uptake and tissueconcentrations of K+ following resupply were related tonitrogen source, with the greatest declines occurring whennitrogen was resupplied to the whole root system asNH^ alone. This inhibition of NH^ on K+ uptake waslessened by resupply of exogenous NO3" in combinationwith NH^ either to the whole root system or to separatehalves of the split-root system.

Concentrations of soluble carbohydrates and organic acids(Experiment I)

Concentrations of soluble carbohydrates initially declinedin shoots after nitrogen resupply in all combinations;however, a subsequent increase after + 3 d was morerapid when NH^ was resupplied alone to the whole rootsystem (Fig. 5B, C, D). Concentrations of starch changedlittle in shoots or roots after resupply. Concentrations of

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Table 1. Effect of source of nitrogen on uptake of NHf and N0$ measured for 65 1 h intervals during the first 3 d of resupply

Net efflux intervals represent hourly periods for which an enrichment of the exogenous solution was measured, and net influx intervals representhourly periods for which a depletion from the solution was measured. Experiment I.

Nitrogensource

Net efflux intervals Net influx intervals

Number Specific rate(mmol h" 1 g"1 root DW)

Number Specific rate(mmol h" 1 g"1 root DW)

NH4+ NO3- NH4

+ NO3- NH4+ NO3- NH4

+ NO3-

Whole rootNH4

+

NH4++NO3-

Split rootNH4

+

NO3-

2119

18

13

10

-0 .073- 0 123

-0.061

-0.055

-0.054

4446

47

52

55

0.0890.181

0.100

0.046

0.182

15.0

12.0

9.0

6.0

"5. 3.0

0t 2.5

2.0

1.5

1.0

0.5

DEPRIVATION

SHOOT

(¥) ROOTS

What* RootNHJ

RESUPPLYWhokRootNH: + NO;

Split Root

«OOTS

) SHOOT

ROOTS

I SHOOT

) ROOTS

-15 - 1 0 - 5 0 0 5 10 0 5 10


10

Fig. 2. Accumulation of dry matter in shoots (A, B, C, D) and roots(E, F, G, H) of soybean during 15 d of nitrogen deprivation (A, E)and 10 d following resupply of nitrogen as 1.0 mol m" 3 NH^ to thewhole root system (B, F), 0.5 mol m~3 NH4

+ plus 0.5 mol m" 3 NO3~to the whole root system (C, G), or 1.0 mol m~3 NH^ to one half ofa split-root system and 1.0 mol m" 3 NO3~ to the other half (D, H).LSDs at / ) = 0.05 for dry weights of the shoots and total root systemon day 10 of resupply are 1.17 and 0.28 g, respectively. 'Indicatesdifference in dry weight of the two halves of the split-root system issignificant at ^ = 0.05 according to a two-tailed standard Student's (test. Experiment I.

I 2

"5

JL

DEPRIVATION

) SHOOT

) ROOTS

o -....rr.

RESUPPLYWhole Root Whok Root Split RootNHJ N H : + N O ;

JJ) SHOOT

(D ROOTS

(C) SHOOT

(BOOTS

SHOOT

H j ROOTS

total N. D NO] half

• NHJ haH

-15 - 1 0 - 5 0 0 5 10 0 5 10 0


10

Fig. 3. Concentrations of total and NO3 -nitrogen in shoots (A, B, C,D) and roots (E, F, G, H) of soybean during 15 d of nitrogendeprivation (A, E) and 10 d following resupply of nitrogen as 1.0 molm~3 NH4

+ to the whole root system (B, F), 0.5 mol ITT 3 NH4+ plus

0.5 mol m~3 NO3" to the whole root system (C, G), or 1.0 mol m~3

NH^ to one half of a split-root system and 1.0 mol m" 3 NOf to theother half (D, H) Experiment I.

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240

160

80

gj 0S 320

240

160

80

0

60

40

20

ft£ 0

DEPRIVATION

I SHOOT

ROOTS

I ROOTS

RESUPPLYW h o l . Root WhoU Root Split Root

NH; NH; + NOJ

SHOOT

rJ) ROOTS

(J) ROOTS

SHOOT ( D ) SHOOT

( 6 ) ROOTS

ROOTS

•<B> ROOTS

DNOjhdl• NHjholf

-15 - 1 0 - 5 0 0 5 10 0 5 10 0


10

Fig. 4. Concentrations of soluble primary amines in shoots (A, B, C,D) and roots (E, F, G, H) and of free NH4

+ in roots (I, J, K, L) ofsoybean during 15 d of nitrogen deprivation (A, E, I) and 10 dfollowing resupply of nitrogen as 1.0 mol m~3 NH^ to the whole rootsystem (B, F, J), 0.5 mol m"3 NH4

+ plus 0.5 mol m~3 NO3" to thewhole root system (C, G, K), or 1.0 mol m~3 NH4 to one half of asplit-root system and 1.0 mol m~3 NOJ" to the other half (D, H, L).Experiment I.

organic acids in shoots of the NH^-resupplied plantsdeclined more rapidly than in shoots of plants thatalso were resupplied with NO3~ (Fig. 6B, C, D).Concentrations in roots of soluble carbohydrates(Fig. 5F, G, H) and organic acids (Fig. 6F, G, H)declined similarly following resupply of NH^ and NH^plus NO3~ to whole root systems and NH^ and NO^~ toseparate halves of the split-root system.

Effect of exogenous WH4+ : WO3" ratio on NH? and NO3"

uptake (Experiment II)

Soybean plants that had been deprived of nitrogen for15 d by a similar protocol as used in Experiment I wereresupplied for 4d with 1.0 mol m~3 nitrogen asNO3- ratios of either 1:0 (1.0 mol m~3 NH4

+), 9:1(0.9 mol m" 3 NH4

+ plus 0.1 mol m"3 NO3") or 1:1(0.5 mol m " 3 NH4

+ plus 0.5 mol m" 3 NO3~") The cumulat-

ive uptake of NH^ per plant by nitrogen-depleted plantsresupplied with 1:1 or 9:1 NH^ : NO3" ratios was greaterthan the NH4

+ per plant taken up by those resuppliedwith the 1:0 ratio (Table 3), and the enhancement ofNFL+ uptake was greater for the 1:1 ratio than for the9:1 ratio. While there was a non-significant effect ofnitrogen source on root growth during the 4 d of resupply(Table 3), the differences in increased mass of roots werenot sufficient in themselves to account for the differencesin NH^ uptake; thus, the enhancement of NH^ uptakewas due more to uptake per unit root weight than to adifference in root mass.

Discussion

Responses to nitrogen deprivation

During the 15 d period of nitrogen deprivation (indicatedas —15 to Od after nitrogen resupply), in which asuboptimum daily addition of 200 /xmol NO-T per plantwas supplied in the nutrient solution, the responses ofplants generally were as expected from previous observa-tions (Rideout et al, 1994). Concentrations of total andNO3" nitrogen (Fig. 3 A, E) and of soluble primary aminesand free NH^ (Fig. 4A, E, I) declined during nitrogendeprivation in both shoots and roots, and concentrationsof organic acids decreased slightly in shoots and increasednearly 2-fold in roots (Fig. 6A, E). Concentrations ofsoluble carbohydrates in shoot and roots increased duringthe first days of nitrogen deprivation and then decreased(Fig. 5A, E). The values at the end of nitrogen depriva-tion, however, were comparable to those obtained in theprevious experiment.

Inhibition ofNHf uptake following resupply as related toinitial root composition

Nitrogen-replete soybean plants can utilize NH^, NO3",or NH^ plus NO^ with equal effectiveness under growth-room conditions (Chaillou et al., 1994; Rufty et al, 1983;Vessey et al, 1990). For nitrogen-depleted soybean plants,the decreased concentrations of NO3~" (Fig. 3) and solubleamines (Fig. 4) and the increased concentrations of sol-uble carbohydrates (Fig. 5) and organic acids (Fig. 6) inroots for the present, as well as a previous (Rideout et al,1994), experiment might be expected to enhance uptakeof both NH4

+ and NO3" (Ivanko and Ingversen, 1971;Jackson and Volk, 1992; Lee et al, 1992; Lee and Rudge,1986; Talouizte et al, 1984; Teyker et al, 1988). As inthe previous experiment, however, uptake of NH^(Fig. 1) was restricted when it was resupplied alone tothe whole root system or to half of the split-root systemrelative to when it was resupplied in combination withNO3" to the whole root system.

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Table 2. Effect of source of nitrogen on concentration and mean uptake rate of K+

Mean uptake rate of K.+ is calculated from the accumulation of K+ in whole plants and mean root dry weights during deprivation and resupplyperiods. Experiment I.

Nitrogen source K+ uptake rate(mmol d"1 g"1 root DW)

K+ concentration (mmol g ' DW)

day - 1 5 day 0 day 10

Nitrogen deprivationNitrogen resupply

Whole rootNH4

+

NH4++NO3-

Split-root

0.41

0.100.300.27

1.08 0.89

0.890.890.89

0.630.760.77

150

120

Q 90

90

60

30

DEPRIVATION

SHOOT

DSoUbO Starch

(ROOTS

RESUPPLYWhol. Root WhoURoot Split RootNH1 NHj +NOj

(B) SHOOT

( F ) ROOTS

) SHOOT

I ROOTS

® SHOOT

) ROOTS

-15 - 1 0 - 5 0 0 5 10 0 5 10 0


10

Fig. 5. Concentrations of soluble sugars (glucose + fructose + sucrose)and total starch in shoots (A, B, C, D) and roots (E, F, G, H) ofsoybean during 15 d of nitrogen deprivation (A, E) and 10 d followingresupply of nitrogen as 1.0 mol m~3 NH^ to the whole root system (B,F) , 0 5 mol m~3 NH« plus 0.5 mol m~3 NO3 to the whole root system(C, G), or 1.0 mol m" 3 NH.J" to one half of a split-root system and1.0 mol m" 3 NO3" to the other half (D, H). Experiment I.

400

320

240

160

80

0400

320

240

160

80

0 •

DEPRIVATION

T 1 1 r

J) SHOOT

<D ROOTS

-15 -10 -5

RESUPPLYWhol* Root Whole Root Split RootNHJ NHJ+NOJ

l 1

(B) SHOOT

( ? ) ROOTS

( £ ) SHOOT

©ROOTS

(B ) SHOOT

$j) ROOTS

ONOjkolf• NHjholl

0 0 10 0 10 0 10


Fig. 6. Concentrations of organic acids (succinate + citrate + malate +malonate) in shoots (A, B, C, D) and roots (E, F, G, H) of soybeanduring 15 d of nitrogen deprivation (A, E) and 10 d following resupplyof nitrogen as 1.0mol m~3 NH^ to the whole root system (B, F),0.5 mol m~3 NH^ plus 0.5 mol m~3 NOf to the whole root system(C, G), or 1.0 mol m" 3 NH^ to one half of a split-root system and1.0 mol m" 3 NO3~ to the other half (D, H). Experiment I.

Inhibition ofNHf uptake as related to changes in rootcomposition

Following nitrogen resupply, concentrations of solublecarbohydrates (Fig. 5) and organic acids (Fig. 6) rapidlydeclined in roots of all treatments with no apparent effectson the differential uptake of NH^ in the presence or

absence of exogenous supply of NO3 (Fig. 1). It ispossible that the restricted uptake of NH^ by rootsresupplied with NH " alone was a consequence of themore rapid accumulation within the roots, relative toroots resupplied with NH^ plus NO3~~, of soluble aminoacids or free NH^ (Fig. 4) following nitrogen resupply(Lee et cil., 1992; Ullrich el ai, 1984). The concentration

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Table 3. Effect of ratio of NHf : NO^ in resupply of 1.0 mol Nw " 3 on cumulative uptake ofNHf and NO^ and on accumulationof dry matter during 4 d of resupply following 15 d of nitrogenstress

Dry weights at the time of nitrogen resupply (day 0) were 4.60 g forthe shoot and 1 18 g for the roots. Experiment II.

ExogenousNH4

+: NO3-ratio

1:09 11 1

LSD(0 05)

Cumulative uptake(mmol per plant)

N H ;

1.602.614.67

NO3"

1.742.18

Total N

1.604.356.85

Dry matter increase(g per plant)

Shoot

1.010.801.09

ns

Roots

0.390.200.49

ns

of free NH4+, however, declined between the first and

third day after resupply, and uptake rates of NH^(Fig. ID, E, F) were not consistently enhanced upon thedecline in free NH^. While the contents of soluble primaryamines in NH^-resupplied roots throughout the 10 dresupply period remained elevated, relative to NH^ plusNO^~ resupplied whole root systems or NO3~ resuppliedsplit-root systems, their concentrations were less than halfthe 550 to 600 /xmol g"1 DW that are observed in rootsof non-stressed NH^-fed soybean with no apparentrestriction of NH4

+ uptake (Chaillou et al., 1991, 1994).Asparagine probably accounts for most of the increasedconcentration of soluble primary amines in roots of bothnon-stressed NH^-fed and nitrogen-depleted NH^"-resup-plied soybeans (Chaillou et al., 1991, 1994; Rideout et al.,1994). Thus, while remaining a possibility, it seemsunlikely for this experiment that the persistent restrictionof NH^ uptake when resupplied to nitrogen-depletedplants in the absence of NO3~ can be attributed directlyto the accumulation of soluble primary amines within thewhole root system.

Does inhibition of A/H4+ uptake involve restricted entry of

A/H4+ into roots?

Net NH^ uptake during short-term experiments involvesboth inward and outward movement across theplasmalemma of root cells (Jackson et al., 1993; Morganand Jackson, 1988). If NH^ enters the root symplasmbut amino acids are not loaded into the xylem fortranslocation to the shoot, feedback effects from accumu-lation of free NH^ and/or amino acids could account forthe limited net uptake of exogenous NH^ by enhancingefflux of endogenous N H / from the root. During theinitial 3 d of nitrogen resupply, the frequency of hourlyintervals of solution enrichment for NH^ was about twicethat for NO3~ (Table 1) whether NH^ was resuppliedalone to either whole root systems or halves of split-rootsystems or was supplied in combination with NO3~ towhole root systems. Together with the increased concen-

trations of free NH^ during these first 3 d after resupply(Fig. 4J, K, L), this apparent enhancement in efflux ofNH^, whether the effluxed NH^ represented turnaroundof non-assimilated NH^ or NH^ endogenously regener-ated following assimilation, suggests the possibility thatthe restriction in net uptake of NH^ by nitrogen-depletedplants involves a stimulation of efflux rather than aninhibition of influx (Jackson et al., 1993).

The possible involvement of enhanced efflux in long-term regulation of NH^ uptake by nitrogen-depletedplants, however, is contrary to the conclusion of Lee(1993) that uptake of NH4

+, as well as of NO3", iscontrolled primarily by changes in influx. Uptake rateswere determined by Lee (1993) only at the beginning ofthe seventh hour following resupply of nitrogen to defi-cient barley plants. Our observations in both a previous(Rideout et al., 1994) and the present (data not shown)experiments, which extended for 3 d following resupply,indicate that the frequency of hourly intervals of netefflux increased beyond the first 4-6 h of resupply.

Does the effect of A/O3~ in enhanced uptake of /VH4+ directly

involve the NO3 ion?

It is evident from results of this and previous (Chaillouet al., 1994; Rideout et al., 1994) experiments that,regardless of the mechanism of inhibition of NH^ uptake,the capacity for roots of nitrogen-depleted plants toutilize NH^ as a nitrogen source is enhanced by theavailability of exogenous NO3~ (Fig. 1A, B). The questionaddressed in the split-root configuration is whether theeffect of NO3~ occurs within the root or whether it isintegrated through NO3" translocated to and assimilatedwithin the shoot. When NH^ and NO^ are supplied toseparate halves of a split-root system of nitrogen-repletesoybean, the specific uptake rate of NH^ is equal to orexceeds that of NO^, although growth of the NH^-fedroots is less than that of the NOf-fed roots (Chaillouet al., 1994). The uptake of NH^ when resupplied to halfof the split-root system of nitrogen-depleted soybean,however, was less than uptake of NO3~ by the other halfof the root system (Fig. 1C), especially during the last4 d of the resupply period as both specific uptake rate(Fig. IF) and mass (Fig. 2H) of the NH^-resuppliedportion declined in comparison to the NO3"-resuppliedportion. During these last 4 d, photosynthetic capacity ofthe shoot for the split-root plants had recovered to anextent comparable to that of plants resupplied with

^ plus NO3"~ and to an extent greater than that of^-resupplied plants (data not shown). [Net CO2

exchange rates for the several individual leaves of plantsresupplied with NH^ plus NO3~ and with N H / alonewere comparable during both nitrogen-deprivation andresupply periods to those reported for correspondingleaves in the analogous treatments of the previous experi-

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ment (Rideout et al., 1994).] These results suggest thatthe effect of NO^ on enhancing NH^ uptake, as occurredfor whole root systems resupplied with NH^ plus NO3~(Fig. IB, E), was not expressed through enhanced avail-ability of either photosynthates or assimilated nitrogenouscompounds from the common shoot of the split-rootsystem.

Since NO^ can be translocated through the phloemonly in negligible quantities (Hocking, 1980) and NO3"did not appear in detectable quantities in the NH^-resupplied half of the split-root system (Fig. 3H), NO3~presumably was unavailable to the NH^ -resupplied por-tion of the root. For nitrogen-depleted soybean, the roleof NO3~ in enhancing NH^ uptake thus appears to beconfined to availability of NO3~~ itself or to a product ofNO3" reduction and assimilation within the absorbingroot. The latter possibility seems unlikely. Only a smallproportion of absorbed NO^ usually is reduced in rootsof soybean (Rufty et al., 19826), and as the expectedproducts of NC>3~ reduction and assimilation, amino acids(Fig. 4F, G, H) and organic acids (Fig. 6F, G, H) in theNH^"-resupplied portion of the roots are either higherthan or comparable to concentrations in the NO^-resup-plied portion of split-root systems and in whole rootsystems resupplied with NH^ plus NO3~.

The ratio of NH^ to NO^ in the exogenous solutionaltered the extent to which NO3~ enhanced the uptake ofNH^ by nitrogen-depleted soybean. The enhancement ofNH4

+ uptake was 1.8-fold greater when the NH4+ :NO3~-

resupply ratio was 1:1 than when it was 9:1 (Table 3).Interestingly, only 1.3 times as much NO^ was taken upby plants resupplied with the 1:1 exogenous ratio ofNH^ to NO3~~ as by plants resupplied with the 9:1 ratio(Table 3); thus, the effect of NO3~ on enhancement ofNH^ uptake by nitrogen-depleted soybean appears toinvolve more than net uptake of NO^ itself. Furthermore,since 200 /^mol NO3~ per plant were supplied during theperiod of nitrogen deprivation and roots contained about30 ^mol NOf g"1 DW at the time of resupply, the effectof NO3~ on enhancement of NH^ uptake possibly involvesmore than the presence of NO3" within the root.

Does the effect of A/O3~ involve maintenance ofK+

availability?

The effect of NO^ uptake on enhancement of NHûptake by roots of nitrogen-depleted plants may bethrough maintenance of K+ availability within plants.Uptake of K + , which is competitively inhibited by NH^(Rufty et al., 1982a), is enhanced by NO3" (Triplett et al.,1980). Similar responses of K+ uptake to NH^ andNO3~ resupply occurred in this experiment (Table 2). Asthe predominant inorganic cation involved in cation-anion balance, K+ is essential for maintenance of thecation-anion balance, which in turn is necessary for


transfer of ions, presumably including amino acids, outof root cytoplasm and their translocation through thexylem (Anderson and Collins, 1969; Triplett et al., 1980).Decreased availability of K+ within the NH^1"-resuppliedplants (Table 2) possibly inhibited NH^ uptake by nitro-gen-depleted plants through restricted transfer of aminoacids from the root cytoplasm into the xylem. Since K +

is extensively recycled within plants, the lowered concen-tration of K + during nitrogen deprivation may be thecritical parameter in restricted uptake of NH^ by nitro-gen-depleted plants rather than the concurrent K + uptakeper se. A higher concentration of K+ in nitrogen-repleteplants at the time of transfer from NO3~ to NH^ nutritionafter several weeks of growth could explain why NHûptake was not inhibited in nitrogen-replete plants ofprevious experiments (Chaillou et al., 1991, 1994; Raperet al., 1991; Vessey et al., 1990).

Acknowledgements

We thank Emory York, Madeleine Provot, Sylvie Wuilleme,Audra McRae, Chad Winslow, Laura KJemm, and LionelFlutto for their excellent technical assistance and the staff ofthe Southeastern Plant Environment Laboratory at NorthCarolina State University for assistance in the use of thephytotron facilities. This research was funded in part byNational Aeronautics and Space Administration grant NCC2-101 and by the North Carolina Agricultural Research Service.Trade names are given as part of the exact experimentalconditions and do not imply endorsement by the North CarolinaAgricultural Research Service of the product named norcriticism of similar ones not mentioned.

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Influence of nitrate on uptake of ammonium by nitrogen-depleted soybean: is the effect located in roots or shoots?