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
3
.
1
. Growth and biomass In early August, the seedlings grown on the
highest NO
3 −
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 leafroot 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 NH
4 +
and NO
3 −
. 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 NH
4 +
concentrations, compared with the treatment with 2 mM N; thus, the leafroot ratios
had increased. In NO
3 −
-grown plants, the root biomass remained at a low level. Combined with
an increased leaf biomass, this resulted in higher leafroot ratios than in saplings grown on NH
4 +
+ NO
3 −
. In general, in the saplings, the leaf biomass was higher in plants grown on NH
4 +
+
Fig. 1. a Biomass of leaves and fine roots diameter 5 2 mm of pedunculate oak seedlings, grown on different NO
3 −
con- centrations, in early August and early September. b Amounts
of NO
3 −
reduced in these compartments during a 3-day period as calculated from the nitrate reductase activities measured
without the addition of NO
3 −
to the assay NRA
H
2
O
. For the leaves, all flushes were combined. NO
3 −
treatments marked with different letters differ significantly.
Fig. 2. a Biomass of fine roots diameter 5 2 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 NO
3 −
reduction as calculated from NRA
H
2
O
. 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 NO
3 −
concentration 2 mM NO
3 −
versus 4 mM [NH
4 +
+ NO
3 −
], and 4 mM NO
3 −
versus 8 mM [NH
4 +
+ NO
3 −
]; this test was performed only for the contribu- tion of the leaves to the NO
3 −
reduction.
NO
3 −
than in plants with NO
3 −
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 NH
4 +
+ NO
3 −
, the foliar CN ratios decreased, in tendency, with
increasing N concentration of the substrate. This tendency was less distinct in plants grown with
only NO
3 −
, 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 NH
4 +
+ NO
3 −
. 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 NO
3 −
concentration was below the threshold of detection B 0.5 mg NO
3 −
N g DW
− 1
. The foliar NO
3 −
concentrations determined after Kamphake et al. 1967 were
between 0.03 and 0.07 mg NO
3 −
N g DW
− 1
. Thus, the fraction of soluble NO
3 −
N in total N of the leaves was negligible.
3
.
3
. Nitrate reductase acti6ity Neither NRA
H
2
O
nor NRA
KN0
3
differed among the leaves of different flushes; thus, a mean value
was calculated for the entire leaf compartment of a single plant. In every case, NRA
KNO
3
was higher than NRA
H
2
O
measured in the same plant com- partment Table 1. On average, NRA
KNO
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 NO
3 −
concentrations of the nutrient solution, but only for NRA
H
2
O
in early August, differences were significant. In the
leaves of the seedlings, NRA
KNO
3
decreased, in tendency, with increasing NO
3 −
concentrations;
Table 1 Nitrate reductase activity nmol NO
2 −
g FW
− 1
h
− 1
without NO
3 −
NRA
H
2
O
and with NO
3 −
added to the assay NRA
KNO
3
, measured in fine roots and leaves average values of all flushes of seedlings and 2-year-old saplings of Q. robur grown on different
N forms and NO
3 −
concentrations, and harvested on different dates of the vegetation period
a
Early July Plants, compartments, N form, N concentration
Early August Early September
NRA
H
2
O
NRA
KNO
3
NRA
H
2
O
NRA
KNO
3
NRA
KNO
3
NRA
H
2
O
Seedlings, NO
3
−
Fine roots 27 9 10 a
298 9 98 38 9 19
297 9 56 1 mM
n.d. n.d.
n.d. n.d.
38 9 11 a 2 mM
321 9 77 45 9 31
400 9 128 408 9 90
33 9 13 382 9 92
84 9 11 b 4 mM
n.d. n.d.
n.d. n.d.
95 9 14 b 8 mM
480 9 50 460 9 118
42 9 10 Leaves
821 9 163 9 9 6
623 9 162 n.d.
n.d. 1 mM
13 9 5 751 9 218
9 9 7 11 9 9
546 9 240 n.d.
2 mM n.d.
4 mM 507 9 160
7 9 3 13 9 6
n.d. 660 9 91
n.d. 733 9 137
12 9 4 n.d.
10 9 2 n.d.
8 mM 410 9 133
Saplings, NO
3
−
Fine roots 28 9 6
538 9 173 33 9 14 271 9 36
2 mM 21 9 2
297 9 49 14 9 5
276 9 125 19 9 2 547 9 51
4 mM 21 9 13 286 9 2
447 9 148 31 9 8 812 9 442
8 mM 25 9 10
28 9 16 458 9 218 Leaves
661 9 190 a 17 9 3
5 9 3 625 9 182
532 9 76 13 9 5
2 mM 20 9 2
492 9 152 11 9 3
592 9 61 4 mM
12 9 1 596 9 14 a
14 9 4 11 9 1
368 9 171 b 514 9 107 10 9 4
686 9 115 8 mM
Saplings, NO
3
−
+ NH
4
+
Fine roots 225 9 54
26 9 10 8 9 4
199 9 41 a 127 9 53
14 9 2 2 mM
4 mM 18 9 13 b
16 9 1 224 9 45
28 9 8 177 9 57
20 9 1 29 9 6
467 9 66 12 9 1
65 9 15 b 212 9 51
20 9 20 8 mM
Leaves 8 9 2
224 9 51 10 9 5
2 mM 600 9 114
7 9 5 639 9 153
2 9 1 4 mM
500 9 155 233 9 145
6 9 5 550 9 71
8 9 4 11 9 6
8 mM 22 9 21
494 9 158 300 9 89
10 9 1 353 9 71
a
n.d., Not determined; , significantly lower in comparison with plants grown with the same NO
3 −
concentration 2 mM NO
3 −
versus 4 mM [NH
4 +
+ NO
3 −
], and 4 mM NO
3 −
versus 8 mM [NH
4 +
+ NO
3 −
]. N-concentration treatments marked with different letters a and b differ significantly.
Fig. 3. Amounts of
15
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 NO
3 −
reduced in the respective com- partments during the same time as calculated from the
NRA
H
2
O
. The regression was calculated for both compart- ments. The broken line indicates a 1:1 relation between mea-
sured
15
N accumulation and calculated NO
3 −
reduction.
ences were significant Table 1. In contrast, NRA
H
2
O
was hardly affected by the presence of NH
4 +
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
15
N accumulation
For the seedlings, the amount of NO
3 −
reduced in the roots or leaves as calculated on the basis of
NRA
H
2
O
was plotted against the amount of
15
N accumulated in the same compartments during
the 3-day investigation period Fig. 3. Since the foliar NO
3 −
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; P B 0.0001 was also obtained when the
15
N accumulation was plotted against the NO
3 −
reduction computed on the basis of NRA
KNO
3
. However, the amounts of NO
3 −
calculated this way were much higher than the corresponding amounts of accumulated
15
N, due to the fact that NRA
KNO
3
of leaves and roots was much higher than NRA
H
2
O
of these compart- ments cf. Table 1.
3
.
5
. Correlation between NO
3
−
reduction and NO
3
−
translocated in the xylem The average NO
3 −
concentration of the xylem sap obtained from the saplings was 96 9 18 mM
and did not exhibit distinct differences between plants grown on different forms and concentra-
tions of N. The mean transpiration rate was 0.55 9 0.07 mmol H
2
O m
− 2
s
− 1
. Generally, no differences between the N treatments were found,
but in September, the saplings grown on 8 mM NO
3 −
+ NH
4 +
showed diminished transpiration rates compared with all other treatments. The
amount of NO
3 −
translocated from the roots to the shoot as computed from the NO
3 −
concentra- tions of the xylem sap, the transpiration rates,
and the leaf area, correlated significantly with the amount of reduced NO
3 −
, calculated on the basis however, differences were not significant Table
1. In the fine roots of the 2-year-old saplings
grown on NO
3 −
, no distinct responses of NRA to increasing NO
3 −
concentrations of the substrate were found. This was also true for the fine root
NRA
H
2
O
of saplings supplied with NH
4 +
+ NO
3 −
. In the first two investigation periods, the fine root
NRA
KNO
3
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
NRA
KNO
3
was also detected in the leaves of the saplings grown on NO
3 −
, whereas this trend was insignificant in the leaves of the saplings supplied
with NH
4 +
+ NO
3 −
. Generally, from August to September, a decrease in foliar leaf NRA
KNO
3
was observed in seedlings and saplings grown on high
N concentrations Table 1. In the comparisons of saplings grown on NH
4 +
+ NO
3 −
with NO
3 −
-grown plants, statistical tests were performed with plants supplied with the
same concentrations of NO
3 −
2 mM NO
3 −
versus 4 mM [NH
4 +
+ NO
3 −
], and 4 mM NO
3 −
versus 8 mM [NH
4 +
+ NO
3 −
]. Generally, NRA
KNO
3
was higher in both leaves and roots of saplings grown
with NO
3 −
as the only N source. In August fine roots, 2 mM NO
3 −
and in September, the differ-
of NRA
H
2
O
Fig. 4. In the range of higher amounts of translocated and reduced NO
3 −
, the calculated amounts of reduced NO
3 −
were lower than the corresponding amounts of translocated
NO
3 −
, 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; P B 0.002 was also obtained when the translocated
NO
3 −
was plotted against the reduced NO
3 −
as computed on the basis of NRA
KNO
3
. However, the values resulting from the calculation with the
NRA
KNO
3
data were much higher than the corre- sponding amounts of translocated NO
3 −
, since the NRA
KNO
3
in the leaves was much higher than NRA
H
2
O
cf. Table 1.
3
.
6
. Reduction of NO
3
−
in lea6es and roots The quantities of NO
3 −
reduced in roots and leaves were calculated on the basis of NRA
H
2
O
. This could be done for three reasons. First, the
amounts of soluble NO
3 −
accumulated in roots and leaves were negligible; thus, in the leaves of
the seedlings, the
15
N almost completely consisted of reduced N. Second, the quantities of reduced
NO
3 −
as computed on the basis of NRA
H
2
O
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 NO
3 −
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 NO
3 −
reduction Fig. 1b. Under the assumption that the quantity of NO
3 −
reduction in the stem and in the petioles was negligible, the seedlings’ leaves contributed 4 –
14 to the total NO
3 −
reduction of the plant. Differences between the NO
3 −
treatments were not found. However, with regard to the NO
3 −
reduc- tion per plant compartment determined on the
basis of the NRA
H
2
O
data from the experiment performed in August, the amount of NO
3 −
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 NO
3 −
were reduced in the leaves of the saplings. Here, the contribution of the leaves
to the whole plant’s NO
3 −
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 leafroot biomass ratios
and, accordingly, a greater contribution of the leaves to NO
3 −
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 NO
3 −
reduction of the total plant r = 0.43; P B 0.002. Generally,
the contribution of the leaves to the whole plant’s NO
3 −
reduction was larger in the saplings grown with NO
3 −
as the only N source; however, the differences to plants grown with NH
4 +
+ NO
3 −
were insignificant in most cases. A completely different pattern emerged when
the contribution of the leaves to the capacity of the plant’s NO
3 −
reduction on the basis of the NRA
KNO
3
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 NO
3 −
translocated from the roots to the shoot of 2-year-old saplings of pedunculate oak during 2 days
as calculated from the NO
3 −
concentrations of the xylem sap and from the transpiration rates, plotted against the amounts
of NO
3 −
reduced in the leaves during the same time as computed from the foliar NRA
H
2
O
. The broken line indicates a 1:1 relation between NO
3 −
translocation and NO
3 −
reduc- tion.
differences in the foliar contributions among the N treatments found.
4. Discussion
