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Nitrate reduction in leaves and roots of young pedunculate

oaks

(

Quercus robur

) growing on different nitrate

concentrations

Frank M. Thomas *, Christine Hilker

Uni6ersita¨t Go¨ttingen,Albrecht-6on-Haller-Institut fu¨r Pflanzenwissenschaften,Abt.O8kologie und O8kosystemforschung,

Untere Karspu¨le2,D-37073Go¨ttingen,Germany

Received 26 February 1999; received in revised form 29 July 1999; accepted 31 July 1999

Abstract

Against the background of high rates of nitrogen (N) input into forest ecosystems and, in part, high nitrate (NO3−)

concentrations in the soil solutions, NO3− reduction activity and N accumulation in leaves and roots of young

pedunculate oaks(Quercus robur) were investigated. Seedlings with unrestricted root growth, and 2-year-old saplings with cut root-stocks, were grown hydroponically at different forms and concentrations of N. The nitrate reductase activity (NRA) of the leaves and fine roots was measured in vivo without addition (NRAH2O) or with addition of

exogenous NO3−(NRAH2O) to the incubation assay. The amounts of reduced NO3

−_{as calculated with the NRA}

H2O

and NRAKNO3 were compared with the uptake of

15_NO 3

−_{, and with root-to-shoot translocation of NO}

− _as

determined by NO3− concentrations of the xylem sap and transpiration rates. Compared with the NRAH₂O, the

NRAKNO3was higher by a factor of approximately 10 in the current year’s fine roots, and by a factor of about 60

in the leaves. In only one case did increased NO3− concentrations of the nutrient solution result in an increase in

NRA. In some cases, NRA was diminished in the presence of ammonium (NH4+) in the root medium. The quantities

of reduced NO3−as calculated on the basis of NRAH2O agreed with the amounts of

15_{N accumulated in roots and}

leaves, and with the amounts of NO3−translocated from the roots to the shoots. The contribution of the leaves to the

total plant’s NO3−reduction as computed on the basis of NRAH2Owas 1 – 17% in the seedlings, but up to 86% in the

saplings; here, it correlated significantly with the leaf/root ratios on a fresh-weight basis. Compared with the leaf/root ratios, the form and concentration of the supplied N had a much lower impact on the share of the leaves in NO3−

reduction; and did not affect the foliar N concentrations. In the leaves as well as in the roots, the concentrations of soluble NO3−were very low (B0.5 mg NO3−N g dry weight−1). The results show that young pedunculate oaks have

Keywords: Ammonium; Leaf; Leaf/root ratio; 15_{Nitrogen; Nitrate; Nitrate translocation; Nitrate reductase activity; Nitrogen;}

Quercus robur; Root; Xylem sap

www.elsevier.com/locate/envexpbot

* Corresponding author. Tel.: +49-551-395724; fax+49-551-395701. E-mail address:[email protected] (F.M. Thomas)

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1. Introduction

Nearly all higher plants can use nitrate (NO3

−₎

as a source of nitrogen (N), and the majority of these species is capable of reducing NO3− _{in both}

roots and shoots (Runge, 1983). This is also true for many woody plants. The extent of NO3−

re-duction in roots and shoots is, inter alia, depen-dent upon the N form (solely NO3−_{; or both NO3}−

and ammonium (NH4+_{)), the amount of NO3}−

supplied, and the amount of radiation (cf. Blevins, 1989; Peuke and Kaiser, 1996). In the presence of NH4+

in the root medium of trees, NO3−

reduction can be suppressed (Bigg and Daniel, 1978; Titus and Kang, 1982; Yandow and Klein, 1986; Pieti-la¨inen and La¨hdesma¨ki, 1988). With increasing NO3

− _{concentration of the root medium, the}

con-tribution of the shoot to total N assimilation often increases, which has been explained by a saturation of the NO3− _{reduction process in the}

root (Titus and Kang, 1982; Gojon et al., 1991). In stands of pedunculate oak (Quercus robur) in northwestern Germany, the elevated N input due to atmospheric deposition of NH4+ _{and NO3}− _led

to high NO3− _{concentrations in the soil solution}

and to increased N concentrations in the leaves, but a substantial accumulation of NO3−

in the leaves was not observed (Thomas and Kiehne, 1995; Thomas and Bu¨ttner, 1998a,b). Although the oak is second only to the beech(Fagus syl6at

-ica)as the most important deciduous forest tree in Central Europe, investigations on the effects of the form and the quantity of the supplied N on the extent and partitioning of NO3− _reduction

under controlled conditions are scarce. Stadler et al. (1993) determined the distribution of NO3−

reduction in young pedunculate oaks. However, the study was conducted with exogenous NO3−

added to the in vivo nitrate reductase activity (NRA) assay. This is believed to indicate, at endogenous amounts of reductant, the potential capacity of the tissue to reduce nitrate rather than the actual reduction rates. The in situ activity is commonly considered to be most closely approxi-mated by an assay without exogenous NO3

−

(An-drews, 1986). The objectives of the present investigations were to test, in young pedunculate oaks under controlled conditions, whether an

in-crease of the NO3

− _{supply in the root medium}

leads to (1) an increase of the NRA in roots or leaves, (2) an increase of the contribution of the leaves to the total plant’s NO3

− _{reduction, or (3)}

to NO3−_{accumulation in the tissue, particularly at}

ample NO3− _{supply. The finding of reaction (1) or}

(3) would indicate that young oak trees exhibit a high affinity to NO3−_{in stands subject to elevated}

NO3−_{supply caused, for example, by atmospheric}

N deposition. To assess possible effects of the leaf/root ratio on the partitioning of the NO3−

reduction within the plants, the study was per-formed on seedlings with unrestricted root growth, and with 2-year-old saplings whose root-stocks had been cut back at the beginning of the cultivation, resulting in a low production of fine root biomass during the first 2 months of the investigation. In the saplings, effects of the pres-ence of NH4+ _{in the root medium on NRA were}

also studied.

In the temperate and boreal regions of the northern hemisphere, ectomycorrhizas play an im-portant role in the supply of forest trees with water and nutrients, particularly with respect to phosphorus and nitrogen. This is also true for the species of the Fagaceae (for example, Marschner, 1995). In the case of supraoptimal N supply, however, the number of mycorrhizas, related to root dry matter, decreases distinctly, due to changes in the root system (Kottke, 1995). A complete fertilization of pedunculate oak seedlings resulted in a very low extent of mycor-rhization, caused by a large reduction in the num-ber of finest roots (Herrmann et al., 1992). In those plants, it is improbable that mycorrhizas exert a great influence on nutrient relations. Since the present study aimed to reveal possible changes in NO3− _{metabolism under continuously high}

NO3− _{supply, non-mycorrhizal plants were used.}

The pedunculate oak belongs to the deciduous tree species with a rhythmic growth pattern. In the seedlings, the shoot exhibits an enhanced growth during about 2 weeks in spring and early summer, and, eventually, a small additional growth peak in late summer. Apart from these growth phases, the growth rate is only low (Hoff-mann, 1972; Alatou et al., 1989; Thomas, 1991). To test the capability of the pedunculate oak for

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NO3

− _{uptake and NO}

− _{reduction also in periods}

of relatively low shoot growth rates, the investiga-tions were carried out between the growth phases. The measurements of NRA were performed in vivo with and without addition of NO3− _{to the}

assay. To confirm the results of the NRA mea-surements, they were compared with data ob-tained by15_{N tracer studies, and by estimations of} root-to-shoot allocation of NO3− _{on the basis of}

transpiration and xylem sap concentration.

2. Materials and methods

2.1. Plant culti6ation

At the end of February, acorns of pedunculate oak (Q. robur L.; provenance forest district Duingerwald, growth district Weserbergland, Lower Saxony, northern Germany), derived from the Forest Seed Centre in Oerrel (Lower Saxony), were sown in quartz sand. In mid-May, when the shoots were about 15 cm high, five seedlings each were transferred into 12-l culture vessels, accord-ing to Ahr (cf. Baumeister and Ernst, 1978), which contained tap water. To prevent anaerobio-sis, the water was constantly aerated. After a few days, the tap water was replaced with nutrient solution containing 1 mM NO3

− _{as the N source.}

The combination of other nutrients was slightly modified after Thomas and Gehlen (1997). For the duration of the entire experiment, the nutrient solutions were replaced with fresh solutions at weekly intervals. The pH (5.2) was adjusted 3 – 4 days after replacement. The plants were cultivated in a greenhouse at 20°C during the day (6.00 – 20.00 h) and 15°C at night with 60910% relative humidity. During daytime, additional light was given (Osram HQJE, 400 W) with a photon flux density of 120 – 200mmol m−2s−1at the level of

the central part of the shoot.

At the end of April, 2-year-old saplings of pedunculate oak, which had been obtained from a tree nursery (provenance lowlands of northwest-ern Germany), were taken into cultivation. The root-stocks were cut back by approximately one-third of their length. The preparation and condi-tioning of the plants for the culture in nutrient

solution were then carried out as described previ-ously (Thomas and Gehlen, 1997). At the end of May, when the buds began to open, the plants were supplied with nutrient solutions containing 1 mM N, provided in the form of either solely NO3−

or 0.5 mM NH4

+0.5 mM NO3

−_{. Light,}

tempera-ture and relative humidity were the same as for the seedlings.

During June, the N concentrations of the nutri-ent solutions of seedlings and saplings were suc-cessively increased until the desired final concentrations were reached. In the case of the seedlings, these were 1, 2, 4 and 8 mM NO3

−_{, and}

in the saplings, 2, 4 and 8 mM N, given in the form of only NO3

−_{, or as equimolar NH}

+NO3

−

. The concentrations of other macronutrients (ex-cept for P) were also increased to give constant ratios to N. For 1 day prior to the experiments, the plants were placed into tap water (NO3−

con-centration B0.1 mM) to increase their NO3

−

up-take capacity and to rinse NO3− _{from the roots.}

At the beginning of the experiments, Mikropur (Katadyn, Wallisellen, Switzerland) was added to the solutions to suppress microbial activity.

2.2. N-labelling experiment

In early August and early September, six seedlings, which were between two growth phases of the shoot (leaf expansion had ceased, and new buds had not yet opened; Alatou et al., 1989), were selected from each NO3

− _{treatment. These}

plants were placed into fresh nutrient solutions. In each three plants per NO3

− _{treatment, NO}

− _was

given, in the respective concentrations of 1, 2, 4 or 8 mM, in the form of Ca(15

NO3)2 with 10.8 atom% 15_{N. The other three plants of each NO}

−

treatment were supplied with unlabelled NO3−_.

After 3 days, the plants were harvested, and the fresh weight of leaves and fine roots (diameter 52 mm) was determined. The material was dried, pulverized and analyzed for total N and15_{N with} a C – N-analyzer (NA 1500; Carlo Erba, Rodano/

Milan, Italy) coupled to an isotope mass spec-trometer (MAT 251; Finnigan, Bremen, Germany). The N standard was acetanilide. From the differences in the 15_{N concentrations between} labelled and unlabelled plants, the 15_N accumula-tion for the experimental period was calculated.

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2.3. Determination of NO3 −

translocation from roots to shoot

In early August and early September, when the 2-year-old saplings had developed an appreciable mass of the current year’s fine roots, three plants, which were between two shoot growth phases (see earlier), were selected from each N-form treat-ment. The plants were placed into fresh solutions. On one of the two subsequent days, the transpira-tion of three leaves per plant was measured with a steady-state porometer (LI-1600; LI-COR, Lin-coln, NE, USA). A previous study had shown that the gas exchange is relatively constant during the light period in pedunculate oak under green-house conditions (Thomas, 1991). All flushes were considered. After 2 days, the plants were har-vested. The fresh weights of leaves and fine roots were measured. The leaves were subdivided into, at maximum, three size classes per plant. The fresh weight was determined from one representa-tive leaf per size class, and the leaf area was measured with a Digital Image Analysing System (Delta-T Devices, Burwell, UK). From the rela-tion of leaf area to fresh weight and the total leaf biomass, the total leaf area of the plant was calculated. The amount of water transpired dur-ing the 2-day experimental period was computed using the total leaf area and the transpiration rate.

After harvesting the leaves, the shoot was cut from the roots, and xylem sap was extracted from the shoot with a Scholander pressure chamber according to Berger et al. (1994). After the re-moval of the bark from the cut end of the shoot for a length of about 2.5 cm, a pressure of 0.2 – 0.3 MPa was applied for 10 min. The exuded xylem sap was collected with a pipette, and frozen at

−18°C until analysis. From each plant, 0.1 – 0.5 ml xylem sap were collected.

The NO3−

concentrations of the xylem sap were measured with high-performance liquid chro-matography (HPLC). After thawing, the samples were centrifuged (5000×g; 10 min), filtered through Oasis HLB cartridges (Waters, Milford, MA, USA), and analyzed with UV-HPLC at 210 nm after separation with an anion exchange column filled with Partisil-10 SAX (Whatman,

Maidstone, Kent, UK), according to Thayer and Huffaker (1980). The eluent was 30 mM KH2PO4 in distilled water (pH 3.0). From the NO3

−

con-centration of the xylem sap, the amount of NO3

−

translocated from the roots to the shoot within 2 days was calculated by means of the measured transpiration rate, the duration of the daily light period (14 h), and the leaf area (see earlier).

2.4. Nitrate reductase acti6ity

In the seedlings and saplings used for the 15 N labelling or the NO3−

translocation experiments, the NRA of leaves and fine roots was measured according to Jaworski (1971). In addition to the saplings investigated in August and September, NRA was determined also in early July, when the saplings had developed only small biomasses of the current year’s fine roots, in plants treated in the same way as in the later investigation periods. In preliminary investigations, the NRA measure-ment had been optimized for the pedunculate oak with respect to the time course and the concentra-tions of buffer, propanol, NO3− _{and H}+ _{in the}

assay. In these studies, it was also found that, under greenhouse conditions, NRA is relatively constant during the light period, and that the NRA of the lignified coarse roots is very low compared with fine roots. During the respective experimental periods, between 9.00 and 10.00 h Central European time, 100 mg of leaf material (pieces of ca. 3 mm2_{size from the intercostal area} of the central leaf blade, which had been previ-ously rinsed with deionized water) and 50 mg of the current year’s fine roots (pieces of ca. 2 mm length from the apical root sections without the root tip, after rinsing with deionized water) were taken from the plants. Until the start of the incubation, the samples were kept on ice and protected from light to prevent a premature onset of NO3−

reduction. They were infiltrated for 10 min under vacuum with 5 ml assay medium, consisting of 0.2 M KH2PO4 (pH 7.5) and 1.5% 1-propanol. The NRAKNO₃(as a measure of NO3

−

reduction capacity at non-limiting NO3

−_{) was}

de-termined by adding 0.5 ml of 0.4 M KNO3to the assay; whereas, for the determination of the NRAH

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Keltjens and van Loenen, 1989) in parallel mea-surements, 0.5 ml deionized water was added in-stead. After vacuum infiltration, the samples were incubated at 30°C in the dark for 90 min. During that period, the NO2− _{production was linear with}

time, as had been determined before. At 30 and 90 min after the start of the incubation, 1 ml assay was added to a mixture of 1 ml of 1% sulfanil-amide in 3 M HCl, 1 ml aqueous 0.1%N -naph-thylethylene diamine dihydrochloride, and 1 ml deionized water. After 20 min of incubation in the dark, absorbance was measured at 540 nm. The NRA (nmol NO2−

g fresh weight (FW)−1 h−1

) was calculated from the difference between the values measured at 30 and 90 min. For leaves and fine roots of each plant, three parallel measure-ments of NRAH₂Oand NRAKNO₃were performed. All flushes developed were considered. From the measured activities, the amounts of NO3−_reduced

in the leaves and roots were calculated by means of the compartments’ biomasses and the length of the experimental periods. In the case of the leaves, only the duration of the daily light period (14 h) was considered, since NO3− _{is assimilated only in}

light in the leaves (Riens and Heldt, 1992). For the roots, NO3− _{reduction was computed on the}

basis of 24 h per day.

2.5. Nitrogen concentrations of the plants

In dried and powdered root and leaf material, the total N concentration was measured with a C – N-analyzer (NA 1500; Carlo Erba). All devel-oped flushes were considered. After aqueous ex-traction (45°C; 1 h) and centrifugation (2500×g; 15 min) of dried and powdered material, the concentrations of soluble NO3− _{in leaves and}

roots were routinely determined photometrically after nitration of salicylic acid (Cataldo et al., 1975). Under the selected conditions, the determi-nation threshold was ca. 0.5 mg NO3−

N g dry weight (DW)−1_{. In about 15% of the plants, the} foliar NO3

− _{concentration was additionally}

ana-lyzed photometrically by the more laborious, but also more sensitive, method of NO3

− _reduction

with hydrazine sulfate, and subsequent reaction of the generated NO2− _with _N_{-naphthylethylene}

di-amine dihydrochloride (Kamphake et al., 1967).

Here, the determination threshold was about 0.02 mg NO3

−

N g DW−1.

2.6. Statistics

The results are given as means with standard errors. Comparisons of two different treatments were performed with the Mann – Whitney Ranked Sum Test (U-Test). Comparisons of more than two treatments were carried out with the non-parametric Ranked Sum Test after Nemenyi (Sachs, 1984). The significance of the correlation coefficients calculated from linear regressions was tested against the distribution of t-values. The significance level was 5% (PB0.05).

3. Results

3.1. Growth and biomass

In early August, the seedlings grown on the highest NO3− _{concentration had produced a}

sig-nificantly greater leaf mass than the plants of the other treatments (Fig. 1a). This resulted in a significantly increased ratio of leaf to fine root biomass (0.51 on a fresh-weight basis; as opposed to 0.25 – 0.28 in the other treatments). In early September, the leaf/root ratios were 0.24 – 0.27, and no significant differences in these ratios or in biomass production between the treatments were found.

In early July, the biomass of the current year’s fine roots of the 2-year-old saplings was still small (Fig. 2a). In August, the root biomass had in-creased, particularly in the saplings grown with both NH4+ _{and NO3}−_{. Compared with the first}

investigation period, this led to diminished leaf/

root ratios (Fig. 2b). In September, root biomass had distinctly decreased in saplings grown on higher NH4+

concentrations, compared with the treatment with 2 mM N; thus, the leaf/root ratios had increased. In NO3

−_{-grown plants, the root}

biomass remained at a low level. Combined with an increased leaf biomass, this resulted in higher leaf/root ratios than in saplings grown on NH4

+NO3−_{. In general, in the saplings, the leaf}

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Fig. 1. (a) Biomass of leaves and fine roots (diameter52 mm) of pedunculate oak seedlings, grown on different NO3−

con-centrations, in early August and early September. (b) Amounts of NO3−reduced in these compartments during a 3-day period

as calculated from the nitrate reductase activities measured without the addition of NO3−to the assay (NRAH₂O). For the

leaves, all flushes were combined. NO3− treatments marked

with different letters differ significantly.

Fig. 2. (a) Biomass of fine roots (diameter 52 mm), and (b) ratios of leaf to fine root biomass of 2-year-old saplings of pedunculate oak, which were grown on nutrient solutions differing in the form and concentration of N, and harvested at the end of three investigation periods. (c) Contribution of the leaves to the plant’s NO3− reduction as calculated from

NRAH2O. Significant differences among the various

concentra-tions of the same N form are marked with different letters. *, Significantly different, within the same investigation period, in comparing plants grown with the same N concentration; c, significantly higher, within the same period, in comparing plants grown with the same NO3−concentration (2 mM NO3−

versus 4 mM [NH4++NO3−], and 4 mM NO3−versus 8 mM

[NH4++NO3−]; this test was performed only for the

contribu-tion of the leaves to the NO3−reduction).

NO3

− _{than in plants with NO}

− _{as the only N}

source (data not shown), but an increase in the nutrient solutions’ N concentrations did not result in an enhanced production of leaf or root biomass.

3.2. Nitrogen concentrations of the plants

The mean foliar N concentrations of the vari-ous treatments were 17.5 – 30.0 mg N g DW−1 _in the seedlings and 19.3 – 27.5 mg N g DW−1_{in the} saplings. In none of the age classes did the foliar N concentrations significantly differ among the various N compositions of the nutrient solutions. In the saplings grown with NH4+₊_NO3−_{, the}

foliar C/N ratios decreased, in tendency, with increasing N concentration of the substrate. This tendency was less distinct in plants grown with only NO3−_{, and absent in the seedlings (data not}

shown).

In the roots of the seedlings, the N concentra-tions were 16.8 – 25.6 mg N g DW−1_{, and in those} of the saplings, 18.5 – 43.0 mg N g DW−1_{. The} seedlings grown with the highest N supply exhib-ited the highest root N concentrations. In the saplings, an increase in root N concentrations with increasing N supply was only observed in the plants nourished with NH4+

+NO3−

. Distinctly increased root N concentrations were only found in plants with reduced root biomass.

In all leaf and root samples analyzed after Cataldo et al. (1975), the NO3

−_{concentration was}

below the threshold of detection (B0.5 mg NO3

−

N g DW−1). The foliar NO3− concentrations determined after Kamphake et al. (1967) were

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between 0.03 and 0.07 mg NO3

−

N g DW−1. Thus, the fraction of soluble NO3

−

N in total N of the leaves was negligible.

3.3. Nitrate reductase acti6ity Neither NRAH

2O nor NRAKN03differed among

the leaves of different flushes; thus, a mean value was calculated for the entire leaf compartment of a single plant. In every case, NRAKNO

3was higher

than NRAH₂O measured in the same plant com-partment (Table 1). On average, NRAKNO

3 was

higher by a factor of about 10 in fine roots, and by a factor of ca. 60 in the leaves.

In the roots of the seedlings, NRA tended to increase with increasing NO3

− _{concentrations of}

the nutrient solution, but only for NRAH

2O in

early August, differences were significant. In the leaves of the seedlings, NRAKNO₃ decreased, in tendency, with increasing NO3− _{concentrations;}

Table 1

Nitrate reductase activity (nmol NO2−g FW−1 h−1) without NO−3 (NRAH₂O) and with NO3−added to the assay (NRAKNO₃),

measured in fine roots and leaves (average values of all flushes) of seedlings and 2-year-old saplings ofQ.roburgrown on different N forms and NO3−concentrations, and harvested on different dates of the vegetation perioda

Early July

Plants, compartments, N form, N concentration Early August Early September

NRAH2ONRAKNO3 NRAH2O NRAKNO3 NRAH2O NRAKNO3

Seedlings,NO−3

Fine roots

27910 (a) 298998 38919 297956

1 mM n.d. n.d.

n.d. n.d. 38911 (a)

2 mM 321977 45931 4009128

408990 33913

382992 84911 (b)

4 mM n.d. n.d.

n.d. n.d. 95914 (b)

8 mM 480950 42910 4609118

Leaves

8219163 996 6239162 n.d. n.d.

1 mM 1395

7519218

997 1199 5469240

n.d.

2 mM n.d.

4 mM n.d. n.d. 1396 660991 793 5079160

7339137 1294

n.d. 1092

n.d.

8 mM 4109133

Saplings,NO−3

Fine roots

2896 5389173 33914 271936

2 mM 2192 297949

1495 2769125 1992 547951

4 mM 21913 28692

4479148 3198 8129442

8 mM 28916 4589218 25910

Leaves

6619190 (a) 1793 6259182 593

532976 1395

2 mM

2092 4929152

1193 592961

4 mM 1291 596914 (a)

1494

1191 5149107 1094 6869115 3689171 (b) 8 mM

Saplings,NO−3+NH+4

Fine roots

225954

26910 127953 1492 894 199941 (a) 2 mM

4 mM 2091 177957 2898 224945* 1691 18913 b*

2996 467966

1291 65915 b*

212951 20920

8 mM Leaves

892 224951 1095

2 mM 6009114 795 6399153

291*

4 mM 894 5009155 550971 695 2339145*

1196

8 mM 353971 22921 4949158 1091 300989*

a_{n.d., Not determined; *, significantly lower in comparison with plants grown with the same NO} 3

−_{concentration (2 mM NO}

−

versus 4 mM [NH4++NO3−], and 4 mM NO−3 versus 8 mM [NH4++NO3−]). N-concentration treatments marked with different

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Fig. 3. Amounts of15_{N, accumulated in fine roots and leaves}

of pedunculate oak seedlings, resulting from the uptake of

15_NO 3

− _{from the nutrient solution during 3 days, plotted}

against the amounts of NO3−reduced in the respective

com-partments during the same time as calculated from the NRAH₂O. The regression was calculated for both

compart-ments. The broken line indicates a 1:1 relation between mea-sured15_{N accumulation and calculated NO}

−_reduction.

ences were significant (Table 1). In contrast, NRAH₂O was hardly affected by the presence of NH4

+ _{in the nutrient solution, and a significant}

difference between the N-form treatments was found in only one case.

3.4. Correlation between NO−3 reduction and 15N accumulation

For the seedlings, the amount of NO3− _reduced

in the roots or leaves as calculated on the basis of NRAH

2O was plotted against the amount of

15 N accumulated in the same compartments during the 3-day investigation period (Fig. 3). Since the foliar NO3

− _{reduction was rather low, the}

respec-tive values were pooled with the data obtained from the roots in calculating the regression. The correlation between both parameters was signifi-cant. Deviations from the 1:1 ratio occurred, but both parameters were of the same magnitude. A significant correlation (r=0.76; PB0.0001) was also obtained when the 15_{N accumulation was} plotted against the NO3− _{reduction computed on}

the basis of NRAKNO

3. However, the amounts of

NO3− _{calculated this way were much higher than}

the corresponding amounts of accumulated 15 N, due to the fact that NRAKNO

3of leaves and roots

was much higher than NRAH₂Oof these compart-ments (cf. Table 1).

3.5. Correlation between NO−3 reduction and NO−3 translocated in the xylem

The average NO3− _{concentration of the xylem}

sap obtained from the saplings was 96918 mM

and did not exhibit distinct differences between plants grown on different forms and concentra-tions of N. The mean transpiration rate was 0.5590.07 mmol H2O m−2

s−1

. Generally, no differences between the N treatments were found, but in September, the saplings grown on 8 mM (NO3

−

+NH4

+_{) showed diminished transpiration}

rates compared with all other treatments. The amount of NO3

− _{translocated from the roots to}

the shoot as computed from the NO3

−

concentra-tions of the xylem sap, the transpiration rates, and the leaf area, correlated significantly with the amount of reduced NO3−_{, calculated on the basis}

however, differences were not significant (Table 1).

In the fine roots of the 2-year-old saplings grown on NO3

−_{, no distinct responses of NRA to}

increasing NO3

− _{concentrations of the substrate}

were found. This was also true for the fine root NRAH₂O of saplings supplied with NH4

+NO3

−_.

In the first two investigation periods, the fine root NRAKNO₃ of these plants tended to increase with increasing N supply, but in September, a signifi-cant decrease with increasing N concentration was found. At this time, a significant decrease in NRAKNO

3 was also detected in the leaves of the

saplings grown on NO3−

, whereas this trend was insignificant in the leaves of the saplings supplied with NH4+₊_NO3−_{. Generally, from August to}

September, a decrease in foliar leaf NRAKNO

3was

observed in seedlings and saplings grown on high N concentrations (Table 1).

In the comparisons of saplings grown on NH4+

+NO3− _{with NO3}−_{-grown plants, statistical tests}

were performed with plants supplied with the same concentrations of NO3− _{(2 mM NO3}− _versus

4 mM [NH4+₊_NO3−_{], and 4 mM NO3}− _{versus 8}

mM [NH4+₊_NO3−_{]). Generally, NRAKNO}

3 was

higher in both leaves and roots of saplings grown with NO3− _{as the only N source. In August (fine}

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differ-of NRAH₂O (Fig. 4). In the range of higher amounts of translocated and reduced NO3

−_{, the}

calculated amounts of reduced NO3

− _{were lower}

than the corresponding amounts of translocated NO3−_{, but, as in the case of the} 15_{N labelling} experiment, the parameters were of the same mag-nitude. A significant correlation (r=0.58; PB 0.002) was also obtained when the translocated NO3− _{was plotted against the reduced NO3}− _as

computed on the basis of NRAKNO

3. However,

the values resulting from the calculation with the NRAKNO

3 data were much higher than the

corre-sponding amounts of translocated NO3−

, since the NRAKNO

3 in the leaves was much higher than

NRAH₂O (cf. Table 1).

3.6. Reduction of NO−3 in lea6es and roots The quantities of NO3− _{reduced in roots and}

leaves were calculated on the basis of NRAH

2O.

This could be done for three reasons. First, the amounts of soluble NO3

− _{accumulated in roots}

and leaves were negligible; thus, in the leaves of the seedlings, the15_{N almost completely consisted} of reduced N. Second, the quantities of reduced NO3− _{as computed on the basis of NRAH}

2O were

in accordance with the magnitude of accumulated 15_{N in the case of the seedlings, and third, also in}

accordance with the amounts of NO3

−

translo-cated from the roots to the shoots in the case of the saplings (Figs. 3 and 4).

In the seedlings, the roots contributed by far the largest fraction to NO3− _{reduction (Fig. 1b).}

Under the assumption that the quantity of NO3−

reduction in the stem and in the petioles was negligible, the seedlings’ leaves contributed 4 – 14% to the total NO3− _{reduction of the plant.}

Differences between the NO3−_{treatments were not}

found. However, with regard to the NO3−

reduc-tion per plant compartment determined on the basis of the NRAH

2O data from the experiment

performed in August, the amount of NO3−

re-duced in the roots was significantly higher in the 4 and 8 mM treatment, compared with the 1 and 2 mM treatment (Fig. 1b).

In comparison with the seedlings, much greater quantities of NO3− _{were reduced in the leaves of}

the saplings. Here, the contribution of the leaves to the whole plant’s NO3− _{reduction was}

particu-larly large in early July, when only small fine root biomasses had developed (Fig. 2a,c). It decreased from July to August; at this time, the biomasses of current year’s fine roots had distinctly in-creased. In September, more leaf biomass had been produced, and, in some plants, the genera-tion of new fine roots could not compensate for the loss of fine roots produced in early summer. This resulted in higher leaf/root biomass ratios and, accordingly, a greater contribution of the leaves to NO3

− _{reduction (Fig. 2b,c). In the}

saplings, the ratio of leaves to fine roots on a fresh-weight basis correlated significantly with the contribution of the leaves to the NO3− _reduction

of the total plant (r=0.43;PB0.002). Generally, the contribution of the leaves to the whole plant’s NO3− _{reduction was larger in the saplings grown}

with NO3− _{as the only N source; however, the}

differences to plants grown with NH4+₊_NO3−

were insignificant in most cases.

A completely different pattern emerged when the contribution of the leaves to the capacity of the plant’s NO3

− _{reduction (on the basis of the}

NRAKNO₃) was calculated. In the seedlings, the share of the leaves was about 20 – 50% (decreasing from August to September); and in the saplings, about 50 – 90%. In none of the age classes were

Fig. 4. Amounts of NO3−translocated from the roots to the

shoot of 2-year-old saplings of pedunculate oak during 2 days as calculated from the NO3−concentrations of the xylem sap

and from the transpiration rates, plotted against the amounts of NO3− reduced in the leaves during the same time as

computed from the foliar NRAH₂O. The broken line indicates

a 1:1 relation between NO3−translocation and NO3−

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differences in the foliar contributions among the N treatments found.

4. Discussion

4.1. Biomass, and nitrate reductase acti6ity of

roots and lea6es

Like most terrestrial plants (Runge, 1983), the pedunculate oak produces the largest biomass when supplied with both N forms. The impair-ment of root growth in plants cultivated over prolonged periods with high NH4+

concentrations (Fig. 2a) was also found in a previous study on pedunculate oak (Thomas and Gehlen, 1997) as well as in an investigation on Douglas fir (Olst-hoorn et al., 1991). This may be due to an in-creased demand of carbon skeletons for NH4+

assimilation, which results in reduced contents of carbohydrates in the roots (Marschner, 1995). However, detailed mechanisms of this NH4+

-in-duced reaction are still unclear (Bloom, 1997). Ammonium-induced alterations of biomass pro-duction and metabolism may also be responsible for the distinct reduction in fine-root NRA and transpiration rates of saplings grown on high NH4+ _{concentrations and harvested in September}

(cf. Gerenda´s et al., 1997).

Compared with woody pioneer species which tend to have high leaf nitrate reductase activities (Smirnoff et al., 1984; Smirnoff and Stewart, 1985), late-successional species such as peduncu-late oak generally exhibit low foliar NRA. The range of the foliar NRAKNO

3(0.2 – 0.8mmol NO2

−

g FW−1_{) was approximately the same as} deter-mined in field-grown pedunculate oaks (Al Gharbi and Hipkin, 1984; Gebauer and Schulze, 1997). Root NRAH

2O was normally higher than

leaf NRAH

2O(Table 1). In contrast, the NRAKNO3

per unit of fresh weight was generally higher in leaves than in roots; but, on a dry-weight basis, these differences would vanish or even reverse in most cases, since the water content of fine roots (ca. 90%) is considerably higher than that of leaves (about 50%). However, the ratios of NRAKNO

3 to NRAH2O in the respective

compart-ments show that the leaves have the potential to

increase their NO3

− _{reduction rates to a greater}

extent than the roots. These findings are in accor-dance with the minute concentrations of NO3

−

present in the leaves: in the case of increased NO3− _{fluxes from the roots to the leaves, the}

leaves are able to readily reduce the NO3−_{, thereby}

avoiding its accumulation. In adult peach-trees, it was recently shown that NO3− _{supplied to the}

leaves through the xylem induces a noticeable level of in situ NRA, even in the absence of tissue-accumulated NO3− _{(Bussi et al., 1997).} 4.2. Correlations between nitrate reductase acti6ity, and uptake and translocation of nitrate

The good correlations between the amounts of reduced NO3

−_{calculated on the basis of NRA}

H₂O, on the one hand, and the amounts of accumulated 15_{N in roots and leaves (Fig. 3) or the amounts of} NO3− _{translocated from the roots to the shoot}

(Fig. 4), on the other, provide evidence that NRAH

2O also gives a much closer approximation

to the in situ NO3− _{reduction than NRAKNO}

3 in

pedunculate oak. In contrast, NRAKNO

3 is rather

a measure of the capacity of NO3− _reduction.

Therefore, a calculation of the portions of the different compartments to the plant’s total NO3−

reduction on the basis of foliar NRAKNO₃ will lead, at least in this species, to an overestimation of the contribution of the leaves (cf. Gebauer and Schulze, 1997).

Since the foliar NRAH₂O was rather low, the slope of the regression in Fig. 3 is determined by root NRAH

2O more strongly than by leaf

NRAH

2O. The finding that, in the range of higher

amounts of NO3− _{reduced, the computation of}

NO3− _{reduction on the basis of NRAH}

2O yields

larger results than the calculation on the basis of 15

N, can be easily explained by the translocation of reduced N compounds from the roots to the shoot. Within this range, the computation of NO3

−_{reduction on the basis of}15_{N would lead to} an underestimation of root NRA (cf. Fig. 3). For the whole range, however, the correlation between the15_{N values and the NO}

− _{reduction was rather}

good. Although the plants were, during the exper-imental periods, in an intermediate state between two growth phases, the shoot could have acted as

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a sink for N compounds reduced in the roots. To estimate the fraction of reduced N in the leaves, which was translocated from the roots to the shoot during the 3-day experimental period, the differences between foliar15_{N and foliar NRAH}

were calculated. This was feasible since: (1) the amounts of NO3− _{accumulated in the leaves were}

only very small; (2) the NO3− _{flux via the phloem}

is negligible (Peuke and Kaiser, 1996); and (3) the net phloem export of reduced N from non-senes-cent leaves to roots during few days represents only a very small fraction of the NO3− _assimilated

in the shoot, even when the leaves contribute a large part to the entire plant’s N assimilation (Gojon et al., 1991). According to the calculation, 51 – 86% of the foliar reduced N had been translo-cated from the roots to the shoots. This finding agrees well with results obtained from peach-tree seedlings grown on different NO3− _{concentrations}

(50 – 78%; Gojon et al., 1991). The portion of foliar reduced N originating from the roots in-creased with increasing NO3−_{concentration of the}

nutrient solution during the first, but not during the second, experimental period (data not shown); thus reflecting the increase in the amount of NO3−

reduced in the roots (Fig. 1b).

In the 2-year-old saplings, the finding that the amounts of NO3

− _{translocated from the roots to}

the leaves were somewhat higher than the corre-sponding amounts of NO3

− _{reduced in the leaves}

as calculated on the basis of the NRAH₂O(Fig. 4) could be due to a slow, but noticeable decrease of gas exchange rates from midday to evening under greenhouse conditions (Thomas, 1991). Thus, the computation of the NO3− _{flux by means of the}

transpiration rates may have resulted in an over-estimation. Nevertheless, the correlation between NRAH

2O and NO3

− _{flux is satisfying also in the}

saplings.

4.3. Effects of form and concentration of N on nitrate reduction

Increased NO3

− _{concentrations of the nutrient}

solution caused an increase of NRA in only one case (NRAH₂O in the roots of the seedlings at early August; Table 1). A stimulation of NRAKNO

3 by increasing NO3

−_{concentrations was}

also absent in Acer saccharum (Rothstein et al., 1996) and, in the case of foliar application of NO3

−_{-containing mist, in} _Q_. _robur _{(Pearson and}

Soares, 1995), but was found in other forest tree species (Picea abies; Peuke and Tischner, 1991;

Acer rubrum, Pinus strobus, Pinus rigida; Downs et al., 1993); whereas in Pinus syl6estris, an in-crease of substrate NO3− _{had opposite effects on}

NRA of roots and needles (Pietila¨inen and La¨hdesma¨ki, 1988). In spite of a lack in an in-crease of NRA, NO3− _{accumulation was found}

neither in the roots nor in the leaves. The same observations have been made in seedlings ofPinus syl6estris (Flaig and Mohr, 1992) and those of

Prunus persica(Gojon et al., 1991) grown on high NO3

− _{concentrations. A lack of NO}

−

accumula-tion in the tissue can be expected for periods with enhanced growth due to an increased demand of reduced N; but the same finding in periods with low growth rates points to an effective control of NO3− _{uptake in the pedunculate oak (cf. Imsande}

and Touraine 1994). Corresponding results were obtained in field and laboratory experiments with

Picea abies and F. syl6atica, which took up only minute amounts of NO3− _{from solutions}

contain-ing high NO3− _{concentrations (Geßler et al.,}

1998).

An inhibitory effect of NH4

+_{in the substrate on}

root or leaf NRA as was found, in some cases, in our study (Table 1) is a common observation in conifers (Bigg and Daniel, 1978; Yandow and Klein, 1986; Pietila¨inen and La¨hdesma¨ki, 1988; Peuke and Tischner, 1991) and has also been detected in some deciduous trees (e.g. Malus do

-mestica; Titus and Kang, 1982); but not in young plants of Fraxinus excelsiorand Q.robur (Stadler et al., 1993), possibly due to a different experi-mental design (shorter investigation period, N starvation before N treatment).

The findings that, at undisturbed root growth, the roots contributed by far the biggest part to NO3

− _{reduction, and that, at restricted root}

growth, the leaf/fine-root ratios correlated signifi-cantly with the portions of the leaves in NO3

−

reduction, demonstrate that the biomass distribu-tion between root and shoot plays a decisive role in the relative contribution of these compartments to NO3−_{assimilation. This may be also due to the}

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fact that the roots are not capable of compensat-ing a reduced biomass by increascompensat-ing their in situ NRA. Compared with the leaf/root ratio, the form and concentration of N had a much lower impact on the foliar contribution to NO3−

reduc-tion. In a previous study on 2-year-old F. excel

-sior and Q. robur grown on lower N concentrations (3 mM), effects of the N form and NO3− _{concentration on the NO3}−_reduction

capac-ities of roots and leaves were not found either (Stadler et al., 1993).

4.4. Affinity of young pedunculate oaks to NO3 −

On the basis of the following findings, it can be concluded that young pedunculate oaks are not effective sinks for N in stands subjected to ele-vated NO3

− _{supply resulting from, for example,}

atmospheric N deposition: (1) increased NO3−

concentrations of the substrate stimulated the biomass production and the presumed in situ NO3− _{reduction in only one case (seedlings in}

early August); (2) a distinct increase in root N concentrations was only found when the root biomass was relatively low; (3) the foliar N con-centrations of the plants were unaffected; and (4) NO3−

did not accumulate in the tissues. The same conclusion was drawn for seedlings and adult trees ofA.saccharum(Rothstein et al. 1996), and might be a general feature of climax species of temperate deciduous forests. Within stands, it is also improbable that mycorrhizas associated with the pedunculate oak can act as an effective sink for N. The development of the extraradical mycelium as well as the mycorrhization of the roots is negatively affected by supraoptimal N availability (Wallenda and Kottke, 1998). Mycor-rhizas are able to accumulate N in their mantle hyphae (Kottke et al., 1995), but, under con-trolled conditions, the total amount of N accumu-lated in the plant was found to be the same in mycorrhizal and non-mycorrhizal seedlings at low or high N addition rate (Colpaert et al., 1996). Additionally, the affinity of the plants to soil NO3

− _{might be reduced at high rates of NH}

3 or NH4

+_{deposition caused by atmospheric pollution.}

Especially in the northwestern parts of Central Europe, the deposition of reduced N compounds

reaches high levels (cf. Lo¨vblad and Erisman, 1992). Evidence was provided that uptake of at-mospheric NH3by the shoot results in a reduction of NO3

− _{uptake from the root medium (Clement}

et al., 1997). Model calculations showed that the adverse effects of NH3 uptake by the leaves on NO3− _{uptake by the roots can be relevant in}

plants with low relative growth rates, like late-suc-cessional forest trees such as oak, which are sub-jected to high atmospheric NH3 concentrations (Stulen et al., 1998).

Acknowledgements

We thank Prof. Dr Rudolf Tischner and Dipl.-Biol. Sylke Siebrecht, Albrecht-von-Haller-Institut fu¨r Pflanzenwissenschaften, Universita¨t Go¨ttin-gen, for their valuable support in the analysis of xylem sap. The help of Dr August Reineking and Reinhard Langel, Isotopenlaboratorium fu¨r biolo-gische und medizinische Forschung, Universita¨t Go¨ttingen, in the measurement of 15_{N is greatly} appreciated.

References

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of NRAH₂O (Fig. 4). In the range of higher

amounts of translocated and reduced NO3

−_{, the} calculated amounts of reduced NO3

− _{were lower} than the corresponding amounts of translocated NO3−, but, as in the case of the 15N labelling

experiment, the parameters were of the same mag-nitude. A significant correlation (r=0.58; PB 0.002) was also obtained when the translocated NO3− was plotted against the reduced NO3− as

computed on the basis of NRAKNO₃. However,

the values resulting from the calculation with the NRAKNO₃ data were much higher than the

corre-sponding amounts of translocated NO3

−

, since the NRAKNO₃ in the leaves was much higher than

NRAH₂O (cf. Table 1).

3.6. Reduction of NO−3 in lea6es and roots The quantities of NO3− reduced in roots and

leaves were calculated on the basis of NRAH₂O.

This could be done for three reasons. First, the amounts of soluble NO3

− _{accumulated in roots} and leaves were negligible; thus, in the leaves of the seedlings, the15_{N almost completely consisted}

of reduced N. Second, the quantities of reduced NO3− as computed on the basis of NRAH₂O were

in accordance with the magnitude of accumulated

15_{N in the case of the seedlings, and third, also in}

accordance with the amounts of NO3

− translo-cated from the roots to the shoots in the case of the saplings (Figs. 3 and 4).

In the seedlings, the roots contributed by far the largest fraction to NO3− reduction (Fig. 1b).

Under the assumption that the quantity of NO3−

reduction in the stem and in the petioles was negligible, the seedlings’ leaves contributed 4 – 14% to the total NO3− reduction of the plant.

Differences between the NO3−treatments were not

found. However, with regard to the NO3−

reduc-tion per plant compartment determined on the basis of the NRAH₂O data from the experiment

performed in August, the amount of NO3

− re-duced in the roots was significantly higher in the 4 and 8 mM treatment, compared with the 1 and 2 mM treatment (Fig. 1b).

In comparison with the seedlings, much greater quantities of NO3− were reduced in the leaves of

the saplings. Here, the contribution of the leaves to the whole plant’s NO3− reduction was

− _{reduction (Fig. 2b,c). In the} saplings, the ratio of leaves to fine roots on a fresh-weight basis correlated significantly with the contribution of the leaves to the NO3− reduction

of the total plant (r=0.43;PB0.002). Generally, the contribution of the leaves to the whole plant’s NO3− reduction was larger in the saplings grown

with NO3− as the only N source; however, the

differences to plants grown with NH4++NO3−

were insignificant in most cases.

A completely different pattern emerged when the contribution of the leaves to the capacity of the plant’s NO3

− _{reduction (on the basis of the} NRAKNO₃) was calculated. In the seedlings, the

share of the leaves was about 20 – 50% (decreasing from August to September); and in the saplings, about 50 – 90%. In none of the age classes were Fig. 4. Amounts of NO3−translocated from the roots to the

shoot of 2-year-old saplings of pedunculate oak during 2 days as calculated from the NO3−concentrations of the xylem sap

and from the transpiration rates, plotted against the amounts of NO3− reduced in the leaves during the same time as

computed from the foliar NRAH₂O. The broken line indicates

a 1:1 relation between NO3−translocation and NO3−

(2)

differences in the foliar contributions among the N treatments found.

4. Discussion

4.1. Biomass, and nitrate reductase acti6ity of

roots and lea6es

assimilation, which results in reduced contents of carbohydrates in the roots (Marschner, 1995). However, detailed mechanisms of this NH4+

+ _{concentrations and harvested in September} (cf. Gerenda´s et al., 1997).

g FW−1_{) was approximately the same as}

deter-mined in field-grown pedunculate oaks (Al

Gharbi and Hipkin, 1984; Gebauer and Schulze, 1997). Root NRAH₂O was normally higher than

leaf NRAH₂O(Table 1). In contrast, the NRAKNO₃

compart-ments show that the leaves have the potential to

increase their NO3

− _{reduction rates to a greater} extent than the roots. These findings are in accor-dance with the minute concentrations of NO3

− present in the leaves: in the case of increased NO3− fluxes from the roots to the leaves, the

leaves are able to readily reduce the NO3−, thereby

avoiding its accumulation. In adult peach-trees, it was recently shown that NO3− supplied to the

leaves through the xylem induces a noticeable level of in situ NRA, even in the absence of tissue-accumulated NO3− (Bussi et al., 1997). 4.2. Correlations between nitrate reductase acti6ity, and uptake and translocation of nitrate

The good correlations between the amounts of reduced NO3

−_{calculated on the basis of NRA}

H₂O,

on the one hand, and the amounts of accumulated

15_{N in roots and leaves (Fig. 3) or the amounts of}

NO3− translocated from the roots to the shoot

(Fig. 4), on the other, provide evidence that NRAH₂O also gives a much closer approximation

to the in situ NO3− reduction than NRAKNO₃ in

pedunculate oak. In contrast, NRAKNO₃ is rather

a measure of the capacity of NO3− reduction.

Therefore, a calculation of the portions of the different compartments to the plant’s total NO3

− reduction on the basis of foliar NRAKNO₃ will

lead, at least in this species, to an overestimation of the contribution of the leaves (cf. Gebauer and Schulze, 1997).

Since the foliar NRAH₂O was rather low, the

slope of the regression in Fig. 3 is determined by

root NRAH₂O more strongly than by leaf

NRAH₂O. The finding that, in the range of higher

amounts of NO3− reduced, the computation of

NO3− reduction on the basis of NRAH₂O yields

larger results than the calculation on the basis of

N, can be easily explained by the translocation of reduced N compounds from the roots to the shoot. Within this range, the computation of NO3

−_{reduction on the basis of}15_{N would lead to}

an underestimation of root NRA (cf. Fig. 3). For the whole range, however, the correlation between the15_{N values and the NO}

− _{reduction was rather} good. Although the plants were, during the exper-imental periods, in an intermediate state between two growth phases, the shoot could have acted as

(3)

H₂O

were calculated. This was feasible since: (1) the amounts of NO3− accumulated in the leaves were

only very small; (2) the NO3− flux via the phloem

is negligible (Peuke and Kaiser, 1996); and (3) the net phloem export of reduced N from non-senes-cent leaves to roots during few days represents only a very small fraction of the NO3− assimilated

(50 – 78%; Gojon et al., 1991). The portion of foliar reduced N originating from the roots in-creased with increasing NO3−concentration of the

nutrient solution during the first, but not during the second, experimental period (data not shown); thus reflecting the increase in the amount of NO3−

reduced in the roots (Fig. 1b).

In the 2-year-old saplings, the finding that the amounts of NO3

− _{translocated from the roots to} the leaves were somewhat higher than the corre-sponding amounts of NO3

− _{reduced in the leaves} as calculated on the basis of the NRAH₂O(Fig. 4)

could be due to a slow, but noticeable decrease of gas exchange rates from midday to evening under greenhouse conditions (Thomas, 1991). Thus, the computation of the NO3− flux by means of the

transpiration rates may have resulted in an over-estimation. Nevertheless, the correlation between NRAH₂O and NO3− flux is satisfying also in the

saplings.

4.3. Effects of form and concentration of N on nitrate reduction

Increased NO3

− _{concentrations of the nutrient} solution caused an increase of NRA in only one case (NRAH₂O in the roots of the seedlings at

early August; Table 1). A stimulation of

NRAKNO₃ by increasing NO3−concentrations was

also absent in Acer saccharum (Rothstein et al., 1996) and, in the case of foliar application of NO3

−_{-containing mist, in} _Q_. _robur _{(Pearson and} Soares, 1995), but was found in other forest tree species (Picea abies; Peuke and Tischner, 1991;

Acer rubrum, Pinus strobus, Pinus rigida; Downs et al., 1993); whereas in Pinus syl6estris, an in-crease of substrate NO3− had opposite effects on

NRA of roots and needles (Pietila¨inen and La¨hdesma¨ki, 1988). In spite of a lack in an in-crease of NRA, NO3− accumulation was found

neither in the roots nor in the leaves. The same observations have been made in seedlings ofPinus syl6estris (Flaig and Mohr, 1992) and those of

Prunus persica(Gojon et al., 1991) grown on high NO3

− _{concentrations. A lack of NO}

− accumula-tion in the tissue can be expected for periods with enhanced growth due to an increased demand of reduced N; but the same finding in periods with low growth rates points to an effective control of NO3− uptake in the pedunculate oak (cf. Imsande

and Touraine 1994). Corresponding results were obtained in field and laboratory experiments with

Picea abies and F. syl6atica, which took up only minute amounts of NO3− from solutions

contain-ing high NO3− concentrations (Geßler et al.,

1998).

An inhibitory effect of NH4

+_{in the substrate on} root or leaf NRA as was found, in some cases, in our study (Table 1) is a common observation in conifers (Bigg and Daniel, 1978; Yandow and Klein, 1986; Pietila¨inen and La¨hdesma¨ki, 1988; Peuke and Tischner, 1991) and has also been detected in some deciduous trees (e.g. Malus do

The findings that, at undisturbed root growth, the roots contributed by far the biggest part to NO3

− _{reduction, and that, at restricted root} growth, the leaf/fine-root ratios correlated signifi-cantly with the portions of the leaves in NO3

− reduction, demonstrate that the biomass distribu-tion between root and shoot plays a decisive role in the relative contribution of these compartments to NO3−assimilation. This may be also due to the

(4)

reduc-tion. In a previous study on 2-year-old F. excel

-sior and Q. robur grown on lower N concentrations (3 mM), effects of the N form and NO3− concentration on the NO3−reduction

capac-ities of roots and leaves were not found either (Stadler et al., 1993).

4.4. Affinity of young pedunculate oaks to NO3 −

On the basis of the following findings, it can be concluded that young pedunculate oaks are not effective sinks for N in stands subjected to ele-vated NO3

− _{supply resulting from, for example,} atmospheric N deposition: (1) increased NO3−

concentrations of the substrate stimulated the biomass production and the presumed in situ NO3− reduction in only one case (seedlings in

early August); (2) a distinct increase in root N concentrations was only found when the root biomass was relatively low; (3) the foliar N con-centrations of the plants were unaffected; and (4) NO3

−

− _{might be reduced at high rates of NH}

3 or

NH4

+_{deposition caused by atmospheric pollution.} Especially in the northwestern parts of Central Europe, the deposition of reduced N compounds

reaches high levels (cf. Lo¨vblad and Erisman, 1992). Evidence was provided that uptake of at-mospheric NH3by the shoot results in a reduction

of NO3

− _{uptake from the root medium (Clement} et al., 1997). Model calculations showed that the adverse effects of NH3 uptake by the leaves on

NO3− uptake by the roots can be relevant in

plants with low relative growth rates, like late-suc-cessional forest trees such as oak, which are sub-jected to high atmospheric NH3 concentrations

(Stulen et al., 1998).

Acknowledgements

appreciated.

References

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Stadler, J., Gebauer, G., Schulze, E.-D., 1993. The influence of ammonium on nitrate uptake and assimilation in 2-year-old ash and oak trees — a tracer study with15_{N. Isotopenpraxis}

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Frank M. Thomas *, Christine Hilker

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