Plant Physiol. (1988) 86, 166-1750032-0889/88/86/0166/10/$01 .00/0

Nitrogen Uptake by Wheat Seedlings, Interactive Effects of FourNitrogen Sources: NO3-, NO2-, NH4', and Urea'

Received for publication May 18, 1987 and in revised form September 14, 1987

RICHARD S. CRIDDLE, MICHAEL R. WARD, AND RAY C. HUFFAKER*Department ofBiochemistry and Biophysics (R.S.C.), Plant Growth Laboratory and Department ofAgronomy and Range Science (M.R.W., R.C.H.), University ofCalifornia, Davis, CA 95616

ABSTRACI

The net influx (uptake) rates of NO3-, NH4, N02-, and urea intoroots of wheat (Triticum aestivum cv Yecora Rojo) seedlings fromcomplete nutrient solutions containing all four compounds were monitoredsimultaneously. Although urea uptake was too slow to monitor, itspresence had major inhibitory effects on the uptake of each of the othercompounds. Rates of NO3-, NH4+, and N02- uptake depended in acomplex fashion on the concentration of afl four N compounds. Equationswere developed which describe the uptake rates ofeach ofthe compounds,and of total N, as functions of concentrations of all N sources. Contourplots of the results show the interactions over the range of concentrationsemployed. The coefficients of these equations provide quantitative valuesfor evaluating primary and interactive effects of each compound on Nuptake.

It is well known that plants are capable of assimilating NO3-and NH4' into amino N. In addition, plants can also assimilateNO2- with great efficiency (1, 2) and urea has long been used asa source ofN for plant growth (13). Much work has been donecharacterizing NO3- and NH4' uptake but few publications dealwith uptake ofNO2- or urea. Substrate inducibility of the trans-porters for NO3- (1 1, 16), NH4' (1 1) and NO2- (11, 17) has beenreported and their kinetics have been described (see Goyal andHuffaker [11 ] for summary of Km values found for variousplants). The uptake of urea is not well characterized with bothpassive (25) and active (14) uptake reported, respectively, forcorn and bean roots. Several investigators found that the uptakeof urea was very slow in comparison to NO- uptake by cornroot segments (20), and soybean seedlings (30).Much work has been done to determine the effect ofNH4+ on

the uptake ofNO3- by a wide variety of plants. Many investiga-tors have reported that NH4' decreases NO- uptake (5, 22, 26,27) (see Ref. 16 for review), whereas others found little effect(24, 28). Deane-Drummond and Glass (8) showed that NH-+(from 100 to 500 Mm) inhibited net NO- uptake by barleyseedlings at a NO- concentration of 10 gM but not at a concen-tration of 100 gM. Genotypic variation of NH4 inhibition ofNO- uptake has been shown (3, 26). Several investigators havereported that NO3- had no effect on NH4I uptake (5, 16). Weshow that NO3- has multiple effects on NH4V uptake dependingupon the relative concentrations employed. The differences de-scribed for interactive effects of the N species on each others

' This work was supported in part by a grant from the United StatesNational Aeronautics and Space Agency (NASA NCC-2-99).

I Abbreviation: OPA, o-phthaldialdehyde.

uptake can be largely attributed to the differences in relativeconcentrations of each source employed in the studies.

Relatively few publications are available describing the inter-actions of NO- and NO2-, NH4' and NO2-, or the effects ofurea on the uptake of any of these N species. Hentschel (14)found that NH4' inhibited urea uptake by bush bean. Jackson etal. (17) showed that NO3- and NO2- decreased each other'suptake in wheat seedlings, moderately and severely, respectively.They also found that NH4' affected NO2- uptake much less thanNO3- uptake.

Studies ofinteractive effects ofpaired N sources have generallyemployed long-term analyses using multiple sets ofplants. Whilesuch studies provided useful information, it is difficult to controlthe many factors affecting uptake, to analyze reversibility ofchanges in N uptake rates, and to minimize inter-plant variabil-ities which complicate interpretations. Also, most reports haveexamined effects of one N source on the uptake of another in aone-variable-at-a-time fashion. Such an approach can not yielda quantitative description of interactive effects.We have used a multivariable factorial design experimental

method to simultaneously test the main effects and interactionsofNO3-, NH4', NO2-, and urea on the individual rates ofuptakeand on the total rate ofN uptake using one set of wheat plants.Equations have been developed which describe the uptake ratesfor the N species from complete nutrient solutions containingvariable amounts of each. We show that strong and unexpectedinteractions among N sources occur. Concentration dependentenhancement, inhibition, or in some conditions lack of interac-tive effects help explain some of the variable results reported inthe literature. Three factors controlling relative and absoluterates of N uptake into the roots are illustrated: (a) direct com-petition between sources, (b) regulation oftotal uptake rates fromall available sources, and (c) a system for the general depressionofN uptake from all sources by molecules such as urea.

MATERIALS AND METHODSPlant Material. Wheat (Triticum aestivum cv Yecora Rojo)

seedlings were grown hydroponically. Seeds were surface-steri-lized in sodium hypochlorite (1% v/v) for 10 min, rinsed withdistilled water, and germinated at 25°C in aerated deionizedwater in the dark. After 24 h, the germinated seeds were spreadon a layer of cheesecloth supported on a stainless steel screensuspended about 1 cm above the surface of 5 L of aerated 0.2mM CaSO4 solution, and placed in the dark at 25°C. After 7 d,the seedlings were transferred to aerated one-quarter strengthHoagland solution (15) (pH 5.8) lacking N and placed in acontrolled environment growth chamber. In the N-free Hoaglandsolution, KH2PO4, K2HPO4, CaSO4, and Ca(H2P04)2 were sub-stituted for KNO3 and Ca(N03)2. To minimize the effect ofrhythms, the seedlings were transferred to continuous light for 3d at 25°C and 60 to 65% RH. The photon flux density at the

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UPTAKE OF NO3-, NH4+, NO2-, AND UREA

seedling canopy was 400 ,E m-2 s-' and was supplied by incan-descent and cool-white fluorescent lamps. After 2 d in continuouslight, the seedlings were transferred to one-quarter strength mod-ified Hoagland solution containing 1 mM KNO3, 1 mM KNO2,0.5 mm (NH4)2SO4, and 1 mM urea for 1 d prior to the uptakeexperiment to induce the uptake systems (1 1).

Experimental Design. Experiments simultaneously measuredthe uptake of four N sources (NO3-, NH4', NO2-, and urea)from solutions containing mixtures of all four compounds usinga randomized 24 factorial design (4). The concentrations of eachvaried over the range 0.2 to 0.6 mm, which are within the rangeof mechanism 1 for uptake of N03 , NH4', and NO2- (11).Replicate measurements were made at each concentration. Cen-ter point measurements with each N source at 0.4 mm werereplicated eight times at intervals spread over the entire durationof the experiment to allow examination of time dependentchanges occurring during the 8 hr required to measure all theuptake rates.An important part of the experimental design was to ensure

that all determinations of N uptake were run in duplicate, onone group of plants, and with samples and their replicates testedin randomized order. As a result, uptake measurements at anygiven concentration of an individual N compound were carriedout following exposure of the plant roots to both higher andlower levels of all four of the test N sources. No concentrationdependent irreversibility in uptake or major time lag in the plantsadjusting to new solution conditions or steady state rates to Nuptake were seen in these studies.N Uptake. A single set of 20 preinduced seedlings weighing 6

g was rinsed for 1 min with one-quarter strength modifiedHoagland solution containing one of 16 different combinationsof 0.2 or 0.6 mN KNO3, KNO2, (NH4)2SO4, and urea. Theseedlings were then transferred to 40 ml of the same solutionand placed in a mini growth chamber at 25C, 65% RH and aphoton flux density of 400 ,uE m-2 s-'. Depletion of each Nsource from the aerated nutrient solution was determined byassay of samples separated using an automated HPLC system(10) by sampling of solutions every 2 min. Sampling was contin-ued for 8 to 12 min. Rates of net influx (uptake) by seedlingswere determined from the depletion curves. After each uptakeassay the seedlings were rinsed for 1 min in 100 ml of the uptakesolution containing the next selected combination of the four Nsources and then transferred to 40 ml of the same solution foranother 8 to 12 min period. The process was repeated through16 different randomly ordered combinations of the four Nsources and their replicates.

Nitrogen Analyses. Urea, N02 , N03 , and NH4' were deter-mined sequentially using the automatic HPLC method of Goyaland Huffaker (10). Urea was determined spectrophotometricallyat 190 nm. Nitrite and N03 were determined spectrophotomet-rically at 221 nm following separation by HPLC on a WhatmanPartisil-10-SAX anion exchange column (29) using a seconddetector placed in series with the urea detector. Ammonium wasdetermined fluorimetrically by post-column derivation withOPA2 (21). The OPA reagent contained 1 g OPA dissolved in240 ml methanol, 120 ml saturated Na2B4O7, pH 12.0, and 1 ml,3-mercaptoethanol per liter. The sensitivity for detection of eachof the four N species was similar.

RESULTSSteady state uptake rates for each N species were achieved

after 2 to 4 min (Fig. 1). The results of duplicate measurementsfor uptake rates of NO3-, NH4', and N02 are included in TableI. Urea uptake was, in all cases studied, too low to be measured(<1 nmol/min). Experimental order for each of the rate analysesand its duplicate are also shown. The pooled standard deviationsfor measurements of uptake rates of each N compound are

151

12(

1-1

E;r4

9'

6(

30-

I I I0 2 4 6 8

TIME (min)FIG. 1. Time course of net uptake of NO3- (A), NH4' (0), and N02-

(U), each at 0.2 mm in the presence of 0.2 mm urea (see "Materials andMethods" for procedures).

summarized in Table I. Measured values of uptake rates at thecenter point (with concentrations of each ofthe N sources at 0.4mm and values predicted by the regression equation) were usedto estimate the data fit to linear equations relating rates ofuptaketo concentrations of the four N sources. The experimentallydetermined values measuring curvature (or deviation from line-arity) are presented along with estimates of minimum significantcurvature (P95%) calculated from observed pooled standard de-viations. In each case, the minimum significant curvature isgreater than observed values of curvature. Linear regressionequations, therefore, adequately described the data obtained overthe entire concentration range tested.A time dependent decrease of center point values occurred

during the eight hours required for measuring all the uptake rates(Fig. 2). Since all of the uptake rates decreased linearly, a timedependent correction factor was applied. These corrected valuesfor uptake rates are presented in Table I.

Table II summarizes the coefficients obtained for the equationsdescribing uptake rates for N03 , NH4', N02 and total uptakeas functions ofthe concentrations ofeach N species. Substitutionof appropriate concentrations of each nutrient into these equa-tions allows prediction of uptake rates of each individual com-ponent or the total rate ofN uptake. These equations hold overthe entire concentration range of 0.2 to 0.6 mm for each com-ponent. The values of the coefficients in Table II demonstrateimportant main effects as well as interactive effects of each ofthe N compounds upon the uptake rates of the others. Themagnitude of the coefficients indicate the degree of the depend-ence of uptake on each of the corresponding factors. The sign ofthe coefficient indicates whether the effect is stimulatory orinhibitory.Graphic representations of the solutions to the regression

equations via contour diagrams are used in Figures 3 and 4 toillustrate the rates ofuptake oftheN species and their interactionsover the entire range of concentrations employed. The degree ofcurvature of the contours shows the importance of interactiveeffects in determining uptake rates. Large curvature indicatesstrong interactive effects. There are major changes in shapes ofthe contour lines as concentrations of the N sources are varied.Consideration of the contour plots allows a visualization ofwhat

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Plant Physiol. Vol. 86, 1988

Table I. Rates ofNitrogen Compound Uptake into Wheat Roots at Test Concentrations ofFour NitrogenSources in Nutrient Solution

Concentrations Rate of Uptakea bExp. Order

NO3- NH4+ NO2- Urea NO3- NH4+ NO2- TotalmM nmol/g x min

17, 35 0.2 0.2 0.2 0.2 16.9 22.8 19.1 58.815, 39 0.6 0.2 0.2 0.2 24.1 31.3 16.4 71.816, 25 0.2 0.6 0.2 0.2 17.5 41.2 18.1 76.84, 37 0.6 0.6 0.2 0.2 21.2 36.7 12.7 70.69, 32 0.2 0.2 0.6 0.2 12.6 24.5 35.4 72.511,28 0.6 0.2 0.6 0.2 21.5 22.1 23.1 66.710, 36 0.2 0.6 0.6 0.2 19.3 33.5 35.8 88.618, 30 0.6 0.6 0.6 0.2 18.1 33.2 29.2 80.519, 22 0.2 0.2 0.2 0.6 16.7 26.0 18.5 61.213, 34 0.6 0.2 0.2 0.6 20.4 27.5 12.4 60.312, 26 0.2 0.6 0.2 0.6 16.5 31.2 15.3 63.02, 23 0.6 0.6 0.2 0.6 19.0 32.6 14.4 66.03, 31 0.2 0.2 0.6 0.6 12.2 20.4 29.9 62.56, 33 0.6 0.2 0.6 0.6 19.6 23.9 23.6 67.15, 21 0.2 0.6 0.6 0.6 11.0 22.6 30.8 64.47, 27 0.6 0.6 0.6 0.6 23.8 29.6 22.8 76.2

1, 8, 14, 20, 24, 29, 38, 40 0.4 0.4 0.4 0.4 20.2c 29.4c 21.9c 71.5c

Pooled standard deviation (Sp) 2.32 2.94 2.25Curvature -2.00 -1.01 -0.16Minimum significant curvature (P95%) 2.11 2.14 2.04

' Average of two determinations. b Urea uptake too low to be measured (<1 nmol/min).of eight determinations.

'Average

32_

-1

' 24

E -

0

a 16-

EsC48-4-

ie--1

ILI---I.

41 _ s _ 0_~ ~ ~ 1 A

Al . "Il A

I I I I0 10 20 30 40

EXPERIMENTAL ORDER

FIG. 2. Time dependence of net uptake rates of NO3- (A), NH4+ (0),and N02- (U) through the duration ofthe multifactoral experiment. Thevalues represent eight repeat determinations at the midpoint concentra-tion levels (0.4 mM) for all N sources.

effect one N species has on uptake of another.N03- Uptake. A. Low Urea (0.2 mM). The effects of NH4+

and NO2- on NO3- uptake at low urea are presented in the seriesof contour plots in Figure 3A. Here, the values for NO3- uptakeare represented in the NH4+:NO2 plane as contour lines con-necting regions.with identical rates. In Figure 3A. 1, where theNO3- level is 0.2 mm, and starting from the lower left hand

corner (where NO2- and NH4' are also low), increasing NH4+along the x-axis crosses only one contour line (17.2 nmol/g xmin). Uptake rates thus remained essentially constant along thisaxis; hence, at low NO07, increasing NH4 had little effect onNO3- uptake. In contrast, starting from this same point andincreasing the concentration of NO2- along the y axis crosses sixregularly spaced contour lines, showing a decrease of N03uptake rate from about 17 to 13 nmol/g x min. Increasingambient NH4+ along with N02 removed the inhibition and highlevels of both NO2- and NH4+ gave the maximum observed rate(18.5 Amol/g x min). The same trends were seen in Figure 3A.2at 0.3 mm NO3-. At 0.4 mm NO3-, the above effects of NH4'and NO2- began to change. At 0.5 and 0.6 mM N03 , NH,+, andNO2- independently decreased NO3- uptake (Fig. 3A.4,5). Thechanging curvature of the contours shows that the interactiveeffects influencing NO3- uptake were lage when ambient N03was low (Fig. 3A. 1), but much smaller when NO3- was high (Fig.3A.5).Taking a three-dimensional look down through the five planes

of contours shows how all three variables progressively interactto influence NO3- uptake (Fig. 3A. 1-5). The effects ofincreasingNHI+ changes from having a small positive influence at lowNO3- and N02- (but a large enhancing effect at high NO2-) tohaving little effect at intermediate NH4' levels (0.4 mM NO3-),and to an increasingly inhibitory effect at 0.5 and 0.6 mm NO3-.Thus, NH4+ overcame the inhibition by NO2- and had littleeffect on NO3- uptake at low NO3- while at higher ambientNO3- both NH4+ and N02- independently inhibited NO3- up-take.

Effect of Urea on N03- Uptake. One additional method ofdata manipulation to aid in visualizing interactive effects is tosubstitute fixed values for the concentrations of two of thevariables into the regression equations, leaving only two variablesto deal with conceptually at a time. This allows coefficients ofthe equation to be interpreted more simply with just the maineffects and two-way interactive effects to consider. In Table IIIA

168 CRIDDLE ET AL.

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UPTAKE OF NO3-, NH+, NO2-, AND UREA

Table II. Coefficients in Equationsfor Rates of Uptake ofIndividual Nitrogenous Compounds andfor TotalNitrogen Uptakefrom Nutrient Mixtures Containing Nitrate, Ammonia, Nitrite, and Urea

Variables 1, 2, 3, and 4 correspond to nitrate, ammonia, nitrite, and urea, respectively. General equation:rate = C0 + CQ(NO3) + C2(NH4) + C3(NO2) + C4(urea) + C3(NO3XNH4) + C6(NO3XNO2) + C7(NO3Xurea) +Cg(NH4XNO2) + C9(NH4Xurea) + Clo(NO2Xurea) + C1l (NO3XNH4XNO2) + C12(NO3XNH4Xurea) + C13(NO3XNO2Xurea) + C14(NH4XNO2) + C,5(NO3XNH4XNO2Xurea).

Main Effects Two-Factor EffectsUptake Rate N03- NH4+ N02- Urea 1x2 1 x3 1 x4 2x3 2x4 3x4

Cl C2 C3 C4 Cs C6 C7 C8 C9 CIONO3- 2.83* 0.13 -0.91 -0.75 -0.60 0.67 0.48 0.63 0.03 0.13NH4- 1.24* 4.21* -2.16* -2.25* -0.76 -0.24 1.08* -0.68 -1.29* 0.21+

NO2- -2.98* 0.06 6.46* -1.41* 0.37 -1.09 0.37 0.80* -0.13 -0.69Total 1.09 4.42* 3.39* -4.41* -0.99* -0.66 1.93* 0.75 -1.3* -0.34

Three- and Four-Factor Interaction EffectsConstant

1x2x3 1 x2x4 1 x3x4 2x3x4 1x2x3x4 CIClI C12 C13 C14 C15

NO-3 0.02 1.12* 1.08* -0.05 0.83* 18.2NH+4 1.50* 0.61 0.59 -0.24 0.37 28.3NO-2 0.07 0.01 0.26 -0.59 -0.99* 22.3Total 1.59* 1.73* 1.93* -0.89* -0.53 68.9

* Coefficients significant at >90% level.

the regression equations for NO3- uptake are reduced to sets oftwo factor (NH4' and NO2-) equations at different fixed levelsof NO3- and urea. At low urea, NH4' (K2) inhibits (negativecoefficients) NO3- uptake when NO3- is high but enhances(positive coefficients) when NO3- is low. This pattern is invertedat high levels of urea. Increasing ambient urea also progressivelychanged the effect ofNO2- on NO3- uptake from negative at allincreasing NO3- concentrations, to positive at higher concentra-tions of NO3- (see coefficient K3, Table IIIA).

Considering the contours for NO3- uptake at high urea (Fig.4A) in conjunction with Figure 3A allows simultaneous evalua-tion of all four N species on NO3- uptake. At high urea, NH4Iovercame the inhibition of increasing ambient NO2- on N03uptake at high but not at low ambient NO3-. In contrast, at lowurea, NIH4 overcame the inhibition ofincreasing NO2- on NO3-uptake at low but not high ambient NO3-. Also, at high urea andlow N02-, NH4' had slight effect on NO3- uptake at any con-centration of ambient NO3- whereas at low urea, increasingambient NH4I and NO2- independently inhibited NO3- uptakeat high NO3-.NH4' Uptake: Low Urea (0.2 mM). The profiles in the contour

plots show that the interactive effects of NO3- and NO2- onNH4' uptake were again large at the lower concentrations ofNH4' and much smaller at the higher concentrations of NH4I.Scanning through the three-dimensional profile of NH4I up-

take (Fig. 3B. 1-5), the effect of NO3- on NH4' uptake changedprogressively from that ofenhancing NH4' uptake and removingthe inhibitory effect of increasing NO2- at low and intermediateconcentrations of NO2-, to having no effect, to more independ-ently inhibiting NHI4 uptake as ambient NH14 increased. Theeffect of NO2- changed from being small at low NH4' and lowNO3- to inhibiting NH,4 uptake strongly at both low and highconcentrations of NO3- at high ambient NIVW.

Effect of Urea on NH4L Uptake. The coefficients for the effectofNO3- on NH4I uptake became progressively more positive asambient urea increased (Table IIIB and Fig. 4B). Increasingambient urea caused a reversal of the effect of NO3- on NHIVuptake at high ambient NH4I. Increasing ambient NO3- signifi-cantly overcame the inhibitory effect of NO2- (Fig. 4B).NO2- Uptake: Low urea (0.2 mM). Interactive effects between

NO3- and NHIV on NO2- uptake were small (Fig. 3C. 1). N03

consistently inhibited NO2- uptake at all levels. NH4' had neg-ligible effects at low NO3-. At 0.5 mM N02-, NH4+ began toenhance NO2- uptake if NO3- was high, and at 0.6 mm theenhancement became significant. Increasing NHE4 progressivelyincreased NO2- uptake as NO3- increased above 0.4 mM.At high urea, interactions between NO3- and NH4' affecting

NO2- uptake were similar to those at low urea except at highN02 .Uptake of Total N from all Sources. The greatest total uptake

rate (88.6 nmol/g x min) from all sources occurred when ureawas low, N03 was low and NH4' and NO2- were both high(Table I). At this point, 40% of total N was taken up as NO2-,38% as NH4', and 22% as NO3- (Table IV). Other combinationsofthe N species in the uptake solutions also resulted in high totalrates of net influx, although the relative contribution of each tothe total uptake rate varied widely (Tables I and IV). When allN sources were at equal concentrations in the nutrient solutions,39 to 41% of the N uptake was as NH4', 30 to 32% as NO2-,and 28 to 29% as NO3-, irrespective of whether all were at thehigh or low range of concentrations. Generally, increased uptakeof either NO3- or NO2- caused a decrease in the proportion ofthe total uptake of the other. High urea in all cases decreasedtotal uptake; however, the proportion each species contributedto the total uptake was little changed. Solutions of the regressionequations at any selected values can be used to optimize totaluptake rates using the four N compounds.To illustrate the interactions among the various N sources on

total uptake, contour profiles are presented at fixed concentra-tions of NH,4, with low urea and with varying ambient N03and NO2- (Fig. 3D). Interactive effects were greatest when all Nsources are at low levels. Scanning through the profile for uptakeof total N at increasing concentrations of NH4I shows that atlow NHIV increasing concentrations of both NO3- and NO2- upto 0.5 mm increased uptake of total N. As ambient NH4' con-centration increased, increasing NO3- changed from increasingtotal N uptake to decreasing it, whereas increasing NO2- uni-formly increased uptake of total N. Increasing urea reversed theeffects of NO- on total N uptake as ambient NH4' increased.The effect of NO3- changed from strongly negative to stronglypositive (Table IIIC). The coefficients for NO2- showed a positiveeffect on total N uptake at all levels of ambient urea and NH4A.

169

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CRIDDLE ET AL. Plant Physiol. Vol. 86, 1988

A. NO; UPTAKE B. NH4+ UPTAKE

NO2 N020.6 1o3 .14A IliZ 10.5.

0.5. 24.8245O0. 26.7

CN3 28N0

3~~~29.

0.6 13II 17z0 17.6 "16

2029

0.2 I"'

0.6 134 17.8 162 15

]~~~~ lOB

30.5047-II

0.3 -§192

20.5 202 195 MS

4152

0.4

228220220.8Q224

0.6

0.5 17

o0.4

0.211

245 230 226

0.2 0.3 0.4 0.5 0.6

24.825c6

_ 26.427.2

- 2 8JD__~~~~~~ 2CD

30.3u0

32.7

27.0

28.2

208*29.5

30**

308

IA32.7

340

33,

335

34.7

35.

402404 396 349 37*0 376

0.2 03 04 05 0.6

C. NO2 UPTAKENH4

1533

146i\\\\ 14?15.9

1X 1014 7 17.1 N03

21p5 .2X 1S

230 24.4

264 257 219 24Z235 223D 212

2 l2

32.7 34.4 352 36O 307 39F

20.2

26.5

0.2 0.3 0.4 0.5 0.6

D. TOTAL N UPTAKENO2

72) 7I067.0

p~~2605 67V 65 685 N03

flA 76 748 735 722 7X

6Sr 6X0 68) 69S

80.5 792 77.9 767 75.4 742

797

C9J 705

8A5 867 85.2

77.8769

70.6

0.2 Q3 0.4 0.5 0.6

mM

FIG. 3. Representation of uptake rates of N03- (A), NH14' (B), N02- (C), and total N (D) at a constant 0.2 mM urea as contour plots illustratingvariation with changing concentrations of pairs of variables. The top row ofcontour plots designated number 1, corresponds to 0.2 mm of the uptakenutrient. In turn, rows numbered 2, 3, 4, and 5 correspond to 0.3, 0.4, 0.5, and 0.6 mm of the uptake nutrient. The lines in each contour plotconnect regions of constant uptake rates and are calculated from the regression equation and coefficients from Table II. Values of the uptake ratesalong each contour are shown at the intersection of the lines with the axis. The series of plots in A (I1-5) show variation of NO3- uptake with NIH4and NO2- as the level of N03- in the nutrient solution increases from 0.2 mm to 0.6 mm in 0.1 mm increments. The series of plots in B (1-5) showvariations ofNH4' uptake with NO3- and NO2- as the level ofNH4' in the nutrient solution increases from 0.2 mM to 0.6 mM in 0.1 mm increments.The series of plots in C (1-5) show variations ofNO2- uptake with NO3- and NH4' as the level ofNO2- in the nutrient solutions increases from 0.2mm to 0.6 mm in 0. 1-mM increments. The series of plots in D (1-5) present corresponding rates of total N uptake as a function of NO3- and N02-as the NH4' concentration increases from 0.2 mm to 0.6 mM in 0.1-mM increments. Difference required for significance, 0.5, = 3.6 nmol/g x min.

DISCUSSION

An overall evaluation of the multiple interactions among theN sources shows several regulatory features influencing the netinflux of the N species tested: (a) competitive effects were seen;(b) urea had strong interactive effects on the uptake of the otherN sources, although it was not taken up at a measurable rate;and (c) the uptake of total N appeared to be under regulatorycontrol.Complex interactive kinetics ofthe different N sources on each

others uptake were found. Increasing N03 always inhibitedNO2- uptake, but the inhibition was greater at high than at lowconcentrations ofN02- (Fig. 3C. 1-5). NH4' did not affect N03-

uptake at low NO3- but inhibited uptake at high NO3- (Fig.3A.1-5). Similarly, NO2- had no effect on NH4' uptake at lowNH4' but inhibited NH4' uptake as ambient NHIV increased(Fig. 3B. 1-5). NO3- inhibited NH4V uptake only at high ambientNH4+, but the interaction was more complex. At low ambientNH4+, increasing NO3- greatly enhanced NH4+ uptake (Fig.3B. 1-5). This pattern of inhibition at the high concentrations ofsubstrate but not at the low is directly opposite to what wouldbe expected for competitive inhibition; hence, these are notstrictly competitive responses in the kinetic sense. NO2- inhibitedN03- uptake at all levels and the inhibition by NO2- was greateTat low N03- than at high NO3-. This appears to be a moretypical competition, but it should be noted that there is not a

170

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UPTAKE OF N03-, NH4+, NO2-, AND UREA

A. N140 UlVAKE

NO2; * .0

22.23.=12.24.

is.

is.:

MY4

C. No2 U[rAIKE

20.9 21.7 22.5 '22.2 24.0 24.8 1- 22 1 7

25.6 ~~~~~~~~~16.4

219.420.4

27.? 15.9

20.627.9

1 28.721.229.7

0.2 0.3 0.4 0.5 0.6 0.2 0.3 0.4 0.5 0.6

mU0.2 0.3 0.4 0.5 0.6

FIG. 4. Same as Figure 3, except at a constant 0.6 mm urea.

reciprocal competitive inhibition ofNO2- uptake by NO3-. Thesemultiple interactions substantiate and help explain the conffict-ing results reported in the literature concerning the effects ofdifferent N sources on each others net influx. The uptake ofN03 , NH4', and NO2- were stimulated, inhibited, or not af-fected by the otherN source(s) depending upon the concentrationof the N sources supplied.

Recent evidence shows that net uptake of NO3- could beinfluenced by an efflux component. We are unaware of anyreports on the efflux of either NH4 or N02-. Efflux ofNO3- byseveral different plant species has been reported (7, 18, 19, 23).Efflux was greater in wheat seedlings in the presence of ambientNO3- than in water; however, the rate of effiux was the same in0.25 and 1.0 mm NO3- solutions (18) which is within the con-centration range used in our study. Prolonged leakage of N03

may occur from root storage pools dependent upon the N statusof the roots (23).NH4' could exert an effect on net NO3- uptake by affecting

either influx or efflux. Reports are mixed in this regard. UsingC103- as a tracer for N03 , NH4' reportedly had no effect onNO3- influx but largely affected efflux (8). Later, it was reportedthat NH4R strongly inhibited influx ofNO3- by barley roots using'3N03- (9). Jackson et al. (18) reported that NH4' decreasedNO3- influx but had no effect on efflux in roots of wheat andcorn. Lee and Clarkson (19) found no effect ofNH4' on the rateof NO3- efflux from barley roots. They proposed that N03effiux was sensitive to NH4I only under nonsteady state condi-tions of extreme variations in NH4' and NO3- concentrations.We are unaware of any reports concerning the effects of N02-or urea on NO3- efflux.

0.6

0.5

1 0.4

0.3

0.2

0.6 14-14.14.1

0.5 is.;1S.is..2 0.4 'Sis.

0.3 27.17.:17.

0.2

0.6 1S.

0.5

3 0.4:E 0.3

0.2

0.6 2718.,

0.5

4 0.4 s.:

0.3

0.2

0.6

0.5

5 0.4

0.3

0.2

171

B. MC IWAKE4

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Plant Physiol. Vol. 86, 1988

Table II. Coefficientsfor Regression Equations Describing Uptake of(A) N03-, (B) NH,+, and (C) TotalNitrogen into Wheat Roots

The predicted values of uptake (Vi were obtained by solution of the equations of Table II as functions ofonly (A) NH4+ and N02- and (B) N03- and NO2- at various fixed levels of urea (U) and (A) N03- or (B)NH.+ (N). Coefficients in these equations are indicated by -K" to illustrate that they differ from the "C" valuesin Table II. However, subscripts were utilized which are consistent with their desgnated N sources and crosscoefficients in Table II.

Predicted Values of Uptake (1)

A. N03- uptakeEquation: =k+ K2NIL+ + K3NO2- + 4 NH*+NO2-

K4 K2 K3 K8 (Urea NO3-)16.80 +1.82 -0.63 +1.49 (0.2 0.2)17.78 +0.96 -0.83 +1.09 (0.2 0.3)18.95 +0.10 -1.04 +0.68 (0.2 0.4)20.10 -0.75 -1.25 +0.28 (0.2 0.5)21.30 -1.58 -1.45 -0.13 (0.2 0.6)16.07 +1.00 -1.10 +1.06 (0.3 0.2)17.28 +0.53 -1.04 +0.86 (0.3 0.3)18.58 +0.11 -0.97 +0.66 (0.3 0.4)19.86 -0.43 -0.90 -0.46 (0.3 0.5)21.21 -0.75 -0.84 +0.26 (0.3 0.6)15.37 +0.19 -1.58 +0.61 (0.4 0.2)16.79 +0.16 -1.29 +0.62 (0.4 0.3)18.20 +0.13 -0.91 +0.63 (0.4 0.4)19.61 -0.10 -0.53 +0.64 (0.4 0.5)21.03 +0.07 -0.24 +0.65 (0.4 0.6)14.61 -0.59 -2.06 +0.16 (0.5 0.2)16.38 -0.22 -1.45 +0.38 (0.5 0.3)17.92 +0.15 -0.85 +0.60 (0.5 0.4)19.36 +0.52 -0.25 +0.82 (0.5 0.5)20.85 +0.89 +0.36 +1.04 (0.5 0.6)13.91 -0.99 -2.54 +0.29 (0.6 0.2)15.93 -0.28 -1.62 +0.14 (0.6 0.3)17.61 +0.17 -0.79 +0.57 (0.6 0.4)19.05 +0.94 +0.03 +1.00 (0.6 0.5)20.64 +1.71 +0.96 +1.43 (0.6 0.6)

B. NH4+ UptakeEquation Y (rate) =K + K1 N03- +K3 NO2- +6 NO3- NO2-

Ko K1 K3 K6 (Urea,

25.15 +1.53 -1.93 -2.70 (0.2 0.2)27.89 +0.85 -2.15 -1.77 (0.2 0.3)30.65 +0.16 -2.37 -0.83 (0.2 0.4)33.40 -0.53 -2.59 +0.10 (0.2 0.5)36.15 -1.21 -2.82 +1.04 (0.2 0.6)24.67 +1.77 -1.70 -2.22 (0.3 0.2)27.11 +0.93 -1.99 -1.38 (0.3 0.3)29.53 +0.70 -2.27 -0.54 (0.3 0.4)31.95 +0.47 -2.55 +0.31 (0.3 0.5)34.39 -0.37 -2.83 +1.15 (0.3 0.6)24.19 +2.00 -1.48 -1.74 (0.4 0.2)26.30 +1.62 -1.82 0.99 (0.4 0.3)28.40 +1.24 -2.16 -0.24 (0.4 0.4)30.50 +0.86 -2.50 +0.51 (0.4 0.5)32.61 +0.48 -2.84 +1.26 (0.4 0.6)23.71 +2.24 -1.25 -1.26 (0.5 0.2)25.50 +2.01 -1.65 -0.60 (0.5 0.3)27.28 +1.78 -2.05 -0.06 (0.5 0.4)29.05 +1.55 -2.45 +0.72 (0.5 0.5)30.83 +1.32 -2.85 -1.37 (0.5 0.6)23.23 +2.48 -1.03 -0.78 (0.6 0.2)24.70 +2.40 -1.49 -0.21 (0.6 0.3)27.27 +2.32 -1.95 +0.36 (0.6 0.4)27.60 +2.24 -2.41 +0.92 (0.6 0.5)29.07 +2.16 -2.87 + 1.49 (0.6 0.6)

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UPTAKE OF NO3-, NH4+, NO2-, AND UREA

Table III. ContinuedC. Total "N" Uptake at fixed levels of urea + NH4+

Equation: YK=0 + K, N03- +3 NO2- + K6 NO3- N02K,K, K, ~~~~~~~~~~~~~~(Urea,Ko K, K3 K6 H+

67.51 +1.89 +2.09 -4.71 (0.2 0.2)70.41 -0.53 +2.90 -3.66 (0.2 0.3)73.31 -0.84 +3.73 -2.59 (0.2 0.4)76.21 -2.21 +4.56 -1.52 (0.2 0.5)79.11 -3.57 +5.37 -0.47 (0.2 0.6)65.99 +1.99 +3.37 -3.49 (0.3 0.2)68.55 +1.06 +2.96 -2.56 (0.3 0.3)71.10 +0.13 +3.56 -1.86 (0.3 0.4)73.66 -0.81 +4.16 -0.70 (0.3 0.5)76.21 -1.74 +4.76 +0.23 (0.3 0.6)64.48 +2.09 +2.64 -2.25 (0.4 0.2)66.69 +1.59 +3.02 -1.46 (0.4 0.3)68.90 +1.09 +3.39 -0.66 (0.4 0.4)71.11 +0.59 +3.77 +0.14 (0.4 0.5)73.32 +0.09 +4.14 +0.93 (0.4 0.6)62.97 +2.19 +2.92 -1.01 (0.5 0.2)64.84 +2.12 +3.07 -0.34 (0.5 0.3)66.70 +2.06 +3.22 +0.31 (0.5 0.4)68.57 +1.99 +3.37 +0.97 (0.5 0.5)70.43 +1.92 +3.53 +1.63 (0.5 0.6)61.40 +2.20 +3.19 +0.21 (0.6 0.2)62.97 +2.65 +3.13 +7.40 (0.6 0.3)64.49 +3.02 +3.05 +1.27 (0.6 0.4)60.01 +3.39 +2.98 +1.80 (0.6 0.5)67.53 +3.75 +2.91 +2.33 (0.6 0.6)

Table IV. Fraction of Total Nitrogen Uptake as N03-, NH4+, and N02- at Selected Solution Compositions ofNitrogen Sourcesa

Percent of Total Uptake perNitrogen Solution Composition of Nitrogen Sources (NO3-/NH4+/NO2/Urea):Source

(0.2/0.2/0.2/0.2) (0.4/0.4/0.4/0.4) (0.6/0.6/0.6/0.6) (0.2/0.6/0.6/0.2) (0.6/0.6/0.6/0.2) (0.2/0.6/0.2/0.2)N03- 29 28 31 22 23 23NH4+ 39 41 39 38 41 54N02- 32 30 30 40 36 24

See Table I for relative uptake rates. b Solution composition at which maximum nitrogen uptake occurred. c,d Selected solutionconcentrations at which high levels of total rates of nitrogen uptake were observed.

Nitrate efflux rates are regulated by the concentration ofNO3-in the cytoplasm of root cells (6). The cytoplasmic NO3- pool is,in turn, determined by the rates ofN03- influx, NO3- reduction,net influx into the vacuole, and translocation of NO3- to theleaves where the major reduction occurs (1). Of the three Nspecies, an efflux component might have influenced net uptakeof NO3- the most. Barley roots of carbohydrate rich seedlingsreduce only about 20% of the net NO3- taken up (1), while theyreduce nearly 100% of the NO2 and assimilate over 95% of theNH4F taken up from 1 mm solutions (S Goyal, RC Huffaker,unpublished data). However, the concentration of N03 in thecytoplasm is further decreased by (a) movement into the vacuole,which is the major storage site for NO3- in barley leaf cells (12);and (b) translocation to leaves.The conditions ofour experiments were optimized to maintain

the cytoplasmic N pool size at low levels. The experiments wereconducted using carbohydrate rich seedlings under high lightintensity to facilitate N03 translocation (from roots to leaves)and reduction (1, 2), and also to maintain photosynthate supplyto roots at a high level to support N03 , N02 , and NHIVassimilation. The randomized replications of the rate studies,which were both step up and step down (from 0.2 to 0.6 mm andfrom 0.6 to 0.2 mM) were designed to show any effects, past 3 to

4 min, of a changing efflux rate on net uptake of the N speciesdue to changing cytoplasmic pool sizes. Our studies were allshort term; steady state uptake rates were established rapidly,most often after 3 min (1 min wash in next uptake solution plus2 min of measured net uptake), and never later than 5 min (1minwash in next uptake solution plus 4 min ofmeasured uptake)after changing to new solutions (Fig. 1). No net contribution ofan effect of pretreating plants with different N concentrationswas detected in the experiments.A second regulatory feature was noted in the effects of urea

on the combined interaction of the uptake of N03, NHM4, andNO2-. Even though urea itselfwas not taken up at a measureablerate it had strong interactive effects on the uptake of the others.Urea had generally inhibitory effects on uptake of all the otherN species (Note the large negative coefficient for urea, Table II).Once again, urea inhibition was greater when total available Nlevels were high, making an explanation based on competitiveinteractions untenable. High urea reversed the interactive effectsof NH4' and NO2- on N03 uptake (compare Figs. 3A.5, and4B.5), changed the interaction of NO3- and N02 on NH4Iuptake at high NH4' from nearly independent inhibition to N03overcoming the inhibition of NO2- (compare Figs. 3B.5 and4B.5), and nullified the interactive effects ofN03 and NH4' on

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Plant Physiol. Vol. 86, 1988

NO2- uptake (compare Figs. 3C.5 and 4C.5).Urea may be acting at the plasma membrane surface since its

uptake was not detected. Slow uptake of urea in comparison tothat of NO3- was shown for soybeans (30) and corn rootssegments (20). Conflicting views (passive [14] and active [25]uptake) concerning the mechanism of urea uptake have beenreported.The third class of regulatory effects appear linked to a general

upper limit upon the total rate of N uptake. As total N sourcelevels become higher, competitive inhibitory effects becomegreater. Thus, there appears to be some limit on the total uptakerate such that any increase in amount entering the cell from onesource is compensated for by a decrease in uptake of anothersource. Some property such as the N status of plants may be animportant determinant ofuptake when total available N sourcesare high.Are the effects of one N source upon the uptake rates of others

a property of the concentration of the N source in the substratesolution or of its uptake rate? The rates of uptake of each sourcealways increased with their increasing concentration in the sub-strate solution. But, since the uptake rate of any single N sourcemay either increase or decrease upon elevation of the substrateconcentration of one of the other N sources, it follows that theuptake rate of any single N species may also increase or decreasewith an elevated uptake rate of one of the other N sources. Thismakes it impossible to generally distinguish between rates ofuptake or concentration ofN species as the controlling factor. Apossible exception may be found at high concentrations of Nsources. Increasing levels ofone nutrient caused decreased uptakerates of the others. This suggests that competitive rates may bestrong determinants of relative uptake under these conditions.At low N levels, such as are commonly found in nature, regula-tion by relative uptake rates seems less likely. On the other hand,the reversible and rapid reestablishment of the interactive effectsof each N species on the others uptake rates as each was steppedup or stepped down in concentration shows either that internalconcentration effects were of little consequence or else that theywere established rapidly and were readily reversible.

In summary, the development ofequations describing the ratesof uptake of NO3-, NO2-, NH4', and urea from solutions con-taining all of these compounds, gives for the first time a quanti-tative description of the interactive effects among these com-pounds across a selected part of System I absorption for NO3-,NH4+, and NO2-. The kinetics for urea are not known and itsuptake was too slow to measure in these studies. Contour plotsof the resulting simultaneously determined uptake rates showedthe range of responses across the concentrations employed. Thewidely fluctuating changes in the contour curves as the concen-trations are varied show the strong interactions of the N com-pounds. Hence, the effects of any N source upon uptake ofanother into wheat seedlings are a complex function of theirconcentrations in the nutrient solution.

APPENDIXThe N uptake data were used to generate regression equations

describing dependence of N uptake rates for each compound,and of total N, upon the concentrations of all of the N sources.The resulting equations have the form:Rate of uptake = C0+ CI(NO3) + C2(NH4) + C3(NO2) + C4(urea)+ CA(NO3)(NH4) + C6(NO3XNO2) + C7(NO3)(urea) +C8(NH4)(NO2) + C9(NH4Xurea) + C,O(NO2Xurea) +C, ,(NO3)(NH4)(NO2) + CI2(NO3XNH4Xurea) +C13(NO3XNO2)(urea) + C14(NH4)(NO2Xurea +C,s(N03)(NH4XNO2Xurea)where: (NO3), (NH4+), (NO2-), and (urea) are the concentrationsof these compounds in the nutrient solutions and the values of

C, through C15 are the coefficients which relate the effects of thecorrespondingN source concentrations to the uptake rates. C0 isa constant, corresponding to the value of uptake at the midpointof the range of concentrations predicted by the equations foreach factor tested.The rates of uptake of any of the N sources in a nutrient

solution with selected concentration can be calculated by appro-priate substitution of coefficients and concentrations into theappropriate regression equation. To allow ready comparisons ofrelative values of the coefficients contributing to uptake, theconcentrations are entered in a scaled form. The regressionequations as presented are solved by entering values scaled from-1 to +1 for concentrations of each N source. These valuescorrespond to actual concentrations of 0.2 to 0.6 mm. A simpleconversion nomogram relating coded and actual concentrationvalues is presented.

Conc. of nitrogen compound (mM): 0.2 0.3 0.4 0.5 0.6Value used in regression equation: -1 -0.5 0 +0.5 +1.0Numerical values to be used in solving the regression equations

at any selection N source concentrations can be obtained byentering the nomogram at the actual concentration of N com-pound of interest and obtaining the corresponding coded valuefor use in the calculation.Contour plots of N uptake were generated using the above

regression equations and the "Minitab" software program.

Acknowledgment-The authors thank Lucia Lojewski for skillful technical asss-istance in preparing the figures.

LITERATURE CI'TED

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2. ASLAM M, RC HUFFAKER, CC DELWICHE 1981 Reduction of nitrate and nitritein barley leaves in darkness. In: JM Lyons, RC Valentine, DA Phillips, DWRains, RC Huffaker, eds. Genetic Engineering of Symbiotic Nitrogen Fixa-tion and Conservation of Fixed Nitrogen. Plenum Press, New York

3. BLOOM AJ, J FINAZZO 1986 The influence of ammonium and chloride onpotassium and nitrate absorption by barley roots depends on time ofexposureand cultivar. Plant Physiol 81: 67-69

4. Box GEP, WG HUNTER, HS HUNTER 1978 Statistics for Experimentors. JohnWiley & Sons, New York

5. BRETELER H, M SIEGERIST 1984 Effect of ammonium on nitrate utilization byroots of dwarf bean. Plant Physiol 75: 1099-1103

6. CLARKSON DT 1985 Factors affecting mineral nutrient acquisition by plants.Annu Rev Plant Physiol 36: 77-115

7. DEANE-DRUMMOND CE, ADM GLASS 1983 Short term studies ofnitrate uptakeinto barley plants using ion-specific electrodes and "C103-. I. Control of netuptake by NO3- efflux. Plant Physiol 73: 100-104

8. DEANE-DRUMMOND CE, ADM GLASS 1983 Short term studies ofnitrate uptakeinto barley plants using ion-specific electrodes and '3C03 -. II. Regulation ofNO3- efflux by NH4'. Plant Physiol 73: 105-110.

9. GLAss ADM, RG THOMPSON, L BORDELEAU 1985 Regulation ofNO3 influxin barley. Studies using "3NO3-. Plant Physiol 77: 379-381

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12. GRANSTEDT RC, RC HUFFAKER 1982 Identification of the leaf vacuole as amajor nitrate storage pool. Plant Physiol 70: 410-413

13. HARPER JE 1984 Uptake of organic nitrogen forms by roots and leaves. In RDHauch, ed. Nitrogen in Crop Production. American Society of Agronomy,Madison, WI, pp 165-170

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UPTAKE OF N03-, NIH4+, N02-, AND UREA

seedlings as influenced by nitrate and ammonium. Physiol Plant 32: 108-114

18. JACKSON WA, KD KWIK, RJ VOLK, RG BUTZ 1976 Nitrate influx and effluxby intact wheat seedlings: effects of prior nitrate nutrition. Planta 132: 149-156

19. LEE RB, DT CLARKSON 1986 Nitrogen-13 studies of nitrate fluxes in barleyroots. I. Compartmental analysis from measurements of '3N efflux. J ExpBot 37: 1753-1767

20. LIN W, JC KAUER 1985 Peptide alcohols as promoters of nitrate and ammo-nium ion uptake in plants. Plant Physiol 77: 403-406

21. LINDROTH P, K MOPPER 1979 High performance liquid chromatographydetermination of subpicomole amounts of amino acids by precolumn deri-vation with o-phthaldialdehyde. Anal Chem 51:1667-1674

22. LYCKLAMA JC 1963 The absorption of ammonium and nitrate by perennialryegrass. Acta Bot Neerl 12: 361-423

23. MAcKowN CT, WA JACKSON, RJ VOLK 1982 Restricted nitrate influx andreduction in corn seedlings exposed to ammonium. Plant Physiol 69: 353-359

24. OAKS A, I STULEN, IL BOESEL 1979 Influence of amino acids and ammoniumon nitrate reduction in corn seedlings. Can J Bot 57: 1824-1829

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