NO
3 −
uptake and NO
3 −
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 NO
3 −
to the assay. To confirm the results of the NRA mea-
surements, they were compared with data ob- tained by
15
N tracer studies, and by estimations of root-to-shoot allocation of NO
3 −
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 NO
3 −
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 60 9 10 relative humidity. During daytime, additional light was
given Osram HQJE, 400 W with a photon flux density of 120 – 200 mmol m
− 2
s
− 1
at 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 NO
3 −
or 0.5 mM NH
4 +
+ 0.5 mM NO
3 −
. 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 NO
3 −
, and in the saplings, 2, 4 and 8 mM N, given in the
form of only NO
3 −
, or as equimolar NH
4 +
+ NO
3 −
. 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 NO
3 −
con- centration B 0.1 mM to increase their NO
3 −
up- take capacity and to rinse NO
3 −
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 NO
3 −
treatment. These plants were placed into fresh nutrient solutions. In
each three plants per NO
3 −
treatment, NO
3 −
was given, in the respective concentrations of 1, 2, 4 or
8 mM, in the form of Ca
15
NO
3 2
with 10.8 atom
15
N. The other three plants of each NO
3 −
treatment were supplied with unlabelled NO
3 −
. After 3 days, the plants were harvested, and the
fresh weight of leaves and fine roots diameter 5
2 mm was determined. The material was dried, pulverized and analyzed for total N and
15
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.
2
.
3
. Determination of NO
3
−
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 NO
3 −
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 KH
2
PO
4
in distilled water pH 3.0. From the NO
3 −
con- centration of the xylem sap, the amount of NO
3 −
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 NO
3 −
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, NO
3 −
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 mm
2
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 NO
3 −
reduction. They were infiltrated for 10 min under vacuum with 5 ml assay medium,
consisting of 0.2 M KH
2
PO
4
pH 7.5 and 1.5 1-propanol. The NRA
KNO
3
as a measure of NO
3 −
reduction capacity at non-limiting NO
3 −
was de- termined by adding 0.5 ml of 0.4 M KNO
3
to the assay; whereas, for the determination of the
NRA
H
3
O
as a measure of the actual NRA;
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 NO
2 −
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 NO
2 −
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 NRA
H
2
O
and NRA
KNO
3
were performed. All flushes developed were considered. From the
measured activities, the amounts of NO
3 −
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 NO
3 −
is assimilated only in light in the leaves Riens and Heldt, 1992. For
the roots, NO
3 −
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 NO
3 −
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 NO
3 −
N g dry weight DW
− 1
. In about 15 of the plants, the foliar NO
3 −
concentration was additionally ana- lyzed photometrically by the more laborious, but
also more sensitive, method of NO
3 −
reduction with hydrazine sulfate, and subsequent reaction of
the generated NO
2 −
with N-naphthylethylene di- amine dihydrochloride Kamphake et al., 1967.
Here, the determination threshold was about 0.02 mg NO
3 −
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 P B 0.05.
3. Results
