bags with a mesh size of 0.2 mm roots of the intact plants. The synthetic bags could not be
used for the excised roots since the air bubbles that formed when the root bags were submerged
in the aerated solution lifted the bags to the surface. In the experiment with intact plants the
synthetic bags were inserted into slits in non- transparent polyethylene discs with shoot and
root at separate sides of the disc. The discs were 10 mm thick and, like the 1000 ml beakers, had a
diameter of 100 mm . The polyethylene discs were used to protect the shoots from contamination of
14
C-solution. The roots were allowed to equili- brate for 30 min in 0.5 mmol CaCl
2
l
− 1
to a solution temperature of 20°C that was used
throughout the experiment. After temperature equilibration the roots were placed for 30 min in
continuously aerated, well mixed, freshly prepared experimental solutions of
14
C-labelled amino acids or methylamine. The roots were rinsed for 2 min
in 1 mmol KCl l
− 1
to remove any possible sur- plus of labelled substrate and then put in 0.1 l
paper bags to be dried at 67°C for 24 h and thereafter weighed. The holding solution, the ex-
perimental solution and the rinsing solution all contained 0.5 mmol CaCl
2
l
− 1
to maintain the membrane integrity Epstein, 1961. The solutions
of methylamine and amino acids contained 1.65 kBq l
− 1
. Alanine
14
CH
3 14
CH[NH
2
]
14
CO
2
H and glutamine H
2
NCO[
14
CH
2
]
14
CH[NH
2
]
14
CO
2
H had three labelled C-atoms, glycine two H
2
N
14
CH
2 14
CO
2
H and methylamine hydrochloride one
14
CH
3
NH
2 .
HCl.
2
.
6
. Radioisotope measurement The roots and shoots were separately com-
busted in a Packard sample oxidiser, model 307. The
14
CO
2
was collected in a scintillation cocktail containing 6 ml Carbosorb and 13 ml Permafluor
and analysed by liquid scintillation.
2
.
7
. Statistical analysis Two-way ANOVA followed by Tukey’s test
was used to assess species, treatment and species x treatment effects on uptake. Testing for correla-
tion between soil characteristics and uptake of amino acids and methylamine was done using
linear correlation. Differences in uptake between intact plants and excised roots was tested by
ANCOVA. All statistical analysis were performed using
SPSS
, version 8.0, using logarithmic transfor- mation to obtain normality for the dependent
variable in Fig. 1.
3. Results
3
.
1
. Methodological study
3
.
1
.
1
. Uptake of amino acids and methylamine E. caninus and D. flexuosa were similar in size
and had a root:shoot ratio of 0.3 to 0.4 and a biomass of 11 – 15 mg Table 1. The uptake of
amino acids and methylamine differed consider- ably, however, between the two species. E. caninus
had an uptake several times higher than D. flexuosa, especially pronounced in the methyl-
amine uptake four to nine times higher and the amino acid uptake two to four times higher
Table 2. The uptake of the three different amino acids also differed between the species. Whereas
E. caninus had a similar uptake of all amino acids D. flexuosa took up glutamine an acid amino
acid about double that of glycine and alanine neutral amino acids. Although uptake generally
increased with concentration of amino acids and methylamine it was not directly proportional to
concentration. The uptake rate decreased some- what between 100 and 250 mmol l
− 1
of the amino acids as compared to the lower concentrations.
Only glutamine uptake by D. flexuosa was not concentration-dependent which was probably due
to the high variation in uptake in the lowest concentration. The uptake capacity of methy-
lamine was about three times higher than that of amino acids for E. caninus whereas it was about
the same for D. flexuosa, or somewhat higher at the highest concentration.
3
.
1
.
2
. Uptake in intact plants and excised roots The difference in uptake between excised roots
and intact plants depended to a large extent upon plant species, nitrogen source and concentration.
According to the ANCOVA-test of nitrogen
Fig. 1. Linear correlation between the logarithmic ratio between uptake of amino acids and methylamine and variables characterising the soil: R-values, pH 0.2 M KCl, total soil carbon C, the molar ratio between total amount of C and nitrogen
N, net potential N mineralisation mmol g
− 1
loss on ignition as ammonium NH
4
, nitrate NO
3
or total nitrogen total N. See Table 6 for details.
Table 2 Uptake by excised roots or intact plants in three concentrations mmol l
− 1
of amino acids and methylamine. Uptake was calculated as the total amount measured in the excised roots or in the whole intact plant mmol g
− 1
dw root h
− 1
a
Treatment E. caninus
D. flexuosa Intact plant
Excised roots Ratio
Intact plant Excised roots
Ratio intact:excised intact:excised
Glycine 2.5 9 0.3
50 1.1
2.4 9 0.2 10.7 9 0.8
7.2 9 0.9 1.5
2.7 9 0.2 100
1.6 3.7 9 0.4
14.1 9 0.5 8.1 9 0.6
1.7 3.8 9 0.1
1.5 21.5 9 0.3
5.3 9 0.6 8.6 9 0.8
250 2.5
Glutamine 2.5 9 0.1
3.3 9.5 9 0.9
7.8 9 1.7 7.3 9 1.0
50 1.3
5.9 9 0.7 100
2.0 9 0.2 3.2
13.0 9 0.3 8.5 9 0.8
1.5 2.2 9 0.4
3.2 15.6 9 1.4
11.4 9 0.7 6.6 9 1.0
1.4 250
Alanine 1.5 9 0.2
2.1 8.1 9 0.4
7.7 9 0.7 1.1
50 3.2 9 0.7
1.7 9 0.1 2.5
8.9 9 0.3 4.2 9 0.2
8.6 9 0.4 100
1.0 250
2.3 9 0.1 6.3 9 1.1
2.7 14.4 9 0.6
13.6 9 0.5 1.1
Methylamine 3.5 9 0.5
1.1 30.8 9 2.7
50 22.6 9 3.1
3.9 9 0.4 1.3
5.3 9 1.2 0.9
42.0 9 2.7 4.9 9 0.6
36.1 9 4.8 100
1.2 11.5 9 1.0
250 1.0
11.8 9 1.2 59.9 9 5.4
47.7 9 4.1 1.3
a
Means 9 S.E. n = 4–5. Differences between uptake by intact plants and excised roots were found according to an ANCOVA test for glycine and glutamine PB0.001, both species, alanine PB0.001, D. flexuosa, methylamine PB0.05, E. caninus while no
significant differences were found for methylamine D. flexuosa and alanine E. caninus.
source both species had a higher uptake of glycine and glutamine in intact plants as compared to
excised roots whereas uptake of alanine was higher only for D. flexuosa and of methylamine
for E. caninus Table 2. The effects of excision were in the following order: glutamine \ ala-
nine \ glycine \ methylamine for D. flexuosa and glycine \ glutamine, methylamine \ alanine for E.
caninus. The greatest effects were the glutamine uptake, more than three times higher by intact
plants compared to excised roots of D. flexuosa, and the alanine uptake, two to three times higher
in intact plants as compared to excised roots of D. flexuosa. The largest effect on E. caninus was the
uptake of glycine, about two times higher by intact plants than by excised roots.
3
.
1
.
3
. Transport of amino acids and methylamine to the shoot
It could be assumed that different rates of internal transport of amino acids and methy-
lamine explained the uptake rate with or without a shoot. However, all amino acids were trans-
ported to the shoot in relatively similar amounts, being on average 33 of total uptake in D.
flexuosa and 11in E. caninus Table 3. Signifi- cantly less methylamine was transported to the
shoot, being 5 and 1 for the two species. The transport to the shoot was dependent on the
treatment concentrations only for D. flexuosa that had a significantly higher transport at 250 mmol
l
− 1
than at lower concentrations P = 0.034, two- way ANOVA.
3
.
1
.
4
. pH-dependent uptake of amino acids and methylamine
In the above experiments the three concentra- tions of amino acids and methylamine caused
various pH-levels. Average n = 3 pH for 50, 100 and 250 mmol l
− 1
was 5.5, 5.9 and 5.7 glycine, 4.2, 4.0 and 3.8 glutamine, 5.6, 5.7 and 6.0
alanine and 5.9, 5.8 and 5.9 methylamine. This
shows that methylamine and the neutral amino acids glycine and alanine had a pH of around 1.5
unit higher than the acid glutamine. In this exper- iment we adjusted pH to 3.8, 4.5 and 6.0. It is
evident that pH had an effect on uptake, even more important as it differed between the species
and thus influenced the inter-species comparison Table 4. The ANOVA-test showed that uptake
was highest at pH 6.0 for D. flexuosa and at pH 4.5 and 6.0 for E. caninus. Uptake of amino acids
was more pH-dependent than uptake of methy- lamine for D. flexuosa and vice versa for E.
caninus.
The methodological study clearly showed that uptake depends on choice of amino acid, its con-
centration and pH of the solution. The impact of excision varies between species which means that
uptake of some amino acids may be largely under-
Table 4 Uptake by intact D. flexuosa and E. caninus plants at three
pH-levels in solutions of 100 mmol l
− 1
methylamine or a mixture of three amino acids as nitrogen source means 9 S.E.,
n = 5
a
E. caninus D. flexuosa
pH Amino acids
3.8 2.2 9 0.5
4.1 9 0.3 5.2 9 0.2
3.4 9 0.7 4.5
4.2 9 0.2 6.0
7.6 9 0.4 3.8
3.1 9 0.2 16.5 9 0.7
Methylamine 4.5
2.3 9 0.1 26.0 9 1.9
6.0 2.3 9 0.3
27.2 9 2.9
a
Uptake was calculated as the total amount in roots and shoots per gram root mmol g
− 1
dw root h
− 1
. Differences were analysed with two-way ANOVA showing that pH, nitro-
gen source, pH×nitrogen source had significant PB0.005 effects on uptake. Tukey’s tests of pH showed that pH 6.0\
pH 4.5, 3.8 for D. flexuosa and pH 6.0, 4.5\pH 3.8 for E. caninus.
Table 3 Uptake of amino acids and methylamine in shoots as a
percentage of the total uptake in roots and shoots
a
Treatment E. caninus
D. flexuosa Shoot total
Shoot total Glycine
29.1 9 6.8 50
17.5 9 2.1 16.9 9 1.0
100 46.3 9 2.4
45.1 9 7.0 250
15.9 9 2.5 Glutamine
35.8 9 8.8 50
10.1 9 1.2 15.9 9 6.8
100 10.9 9 1.3
31.8 9 7.9 250
6.9 9 1.0 Alanine
50 8.6 9 1.0
35.5 9 4.2 43.0 9 2.8
100 9.3 9 2.0
250 49.8 9 4.2
10.7 9 1.5 Methylamine
1.9 9 0.9 3.7 9 0.9
50 3.7 9 1.7
100 0.6 9 0.1
250 7.7 9 1.7
0.7 9 0.3
a
Means 9 S.E., n = 5. A two-way ANOVA showed an ef- fect of compound amino acids and methylamine; P = 0.000
but not of concentration or compound×concentration P\ 0.05.
Tukey’s tests
showed: methylamine\glutamine\
glycine, alanine for D. flexuosa and methylamine\glycine\ glutamine, alanine for E. caninus.
estimated for excised roots as compared to uptake by intact plants. In the main study we therefore
chose to expose intact plants to a mixture of amino acids at a concentration and pH that
would be close to field conditions.
3
.
2
. Main study
3
.
2
.
1
. Uptake of amino acids and methylamine In this study we tested uptake of methylamine
and a mixture of alanine, glutamine and glycine at 100 mmol l
− 1
and pH was set to 4.5. The ten species had a biomass of 3 – 17 mg and root:shoot
ratio of 0.5 – 1.1 Table 1. All species were able to take up amino acids, their capacities ranging from
1.6 to 6.3 mmol g
− 1
dw root h
− 1
Table 5. P. 6
ulgaris had a lower uptake than all other species and D. flexuosa a significantly lower uptake than
six of the other species. The variation in uptake of methylamine was larger than of amino acids,
ranging from 2.4 to 175.2 mmol g
− 1
dw root h
− 1
. Here the two forbs had the largest uptake,
whereas D. flexuosa had also the lowest uptake of this nitrogen source. There was no correlation
between uptake of amino acids and the soil data in Table 6 P \ 0.30.
Table 5 Uptake mmol g
− 1
dw root h
− 1
of amino acids and methy- lamine, and their ratio, by the ten studied species shown in
order of increasing uptake of amino acids
a
Methylamine Ratio
Plant species Amino acids
70.1 9 9.4
b
P. 6ulgaris 0.02
1.6 9 0.2
d
2.4 9 0.2
h
D. flexuosa 1.42
3.4 9 0.7
c
11.8 9 1.2
fg
4.3 9 0.2
b
0.36 A. 6inealis
26.0 9 1.9
cd
E. caninus 0.17
4.5 9 0.6
abc
22.9 9 2.3
de
4.8 9 0.2
abc
0.21 F. gigantea
40.3 9 2.7
c
D. cespitosa 0.12
4.9 9 0.1
ab
25.2 9 3.1
de
5.8 9 0.2
ab
0.23 P. nemoralis
13.8 9 1.2
fg
0.43 F. o6ina
6.0 9 0.3
ab
175.2 9 23.6
a
6.1 9 0.3
a
0.03 G. aparine
21.3 9 2.2
def
0.30 A. capillaris
6.3 9 0.4
a a
Differences between species and treatments were tested on logarithmic values by two-way ANOVA followed by Tukey’s
test. Species, treatment and species×treatment effects were significant PB0.000. Differences within treatments P50.05
are denoted by different letters. Means 9 S.E.; n = 5 for D. flexuosa and E. caninus, for all other species 9–10.
berg, 1992 or as soil analyses from deciduous forest sites in Sweden where the species occur
Table 6. Although the soils are all relatively acid, their variation according to different soil
variables, shown below, are of significance for the species distribution. Acid forest soils often have a
high organic matter content, a low nitrification rate and a low nitrogen mineralisation rate, then
changing as pH rises. Calculated for all sites n = 194, Table 6, there is a significant negative
correlation between pH and total carbon R
2
= 0.158, P B 0.001 and pH and the CN ratio
R
2
= 0.070, P B 0.001 whereas pH is positively
related to the nitrification rate in the soil R
2
= 0.635. No correlation is found between pH and
nitrogen mineralisation R
2
= 0.001, P = 0.666.
The pH-values for the species’ distributions in southern Sweden, where the seeds were collected,
varied between 3.6 and 4.3. This is a relatively small range, as compared to the whole of Sweden,
which relates to soil acidification that has taken place during recent decades. Total C and N varied
between 16 and 21. The potential net nitrogen mineralisation
was 104 – 129
m mol
g
− 1
loss
3
.
2
.
2
. Relation between uptake of amino acids and soil characteristics
Soil data are available for the species studied either as an index of soil acidity R-value; Ellen-
Table 6 The index for soil acidity R-value according to Ellenberg 1992 and soil characteristics for the plants’ distributions in the province
of Ska˚ne, southern Sweden, calculated from Falkengren-Grerup et al. 1998
a
NO
3
NH
4
C:N N
C pH
R-value Total N
3.62 9 0.06
b
3 A. 6inealis
n.a. n.a.
c
n.a. n.a.
n.a. n.a.
3.66 9 0.03 9.0 9 0.5
0.52 9 0.02 21 9 1
65 9 3 D. flexuosa
51 9 3 2
116 9 3 A. capillaris
3.77 9 0.03 4
6.9 9 0.3 0.45 9 0.02
19 9 1 50 9 4
69 9 3 119 9 3
F. o6ina 3
3.87 9 0.08 6.1 9 0.3
0.47 9 0.03 17 9 1
41 9 5 68 9 7
109 9 5 D. cespitosa
x
d
3.90 9 0.07 117 9 6
6.2 9 0.4 80 9 5
0.37 9 0.02 20 9 1
38 9 5 P. nemoralis
5 3.98 9 0.06
6.5 9 0.4 0.40 9 0.02
19 9 1 46 9 5
78 9 5 124 9 4
129 9 9 97 9 9
32 9 6 18 9 1
0.34 9 0.02 G. aparine
5.0 9 0.4 3.99 9 0.08
6 E. caninus
7 4.24 9 0.07
4.7 9 0.3 0.34 9 0.03
16 9 1 4 9 1
100 9 8 104 9 8
6 108 9 7
93 9 7 F. gigantea
15 9 5 4.27 9 0.12
20 9 1 0.26 9 0.02
4.4 9 0.4 P. 6ulgaris
n.a. n.a.
n.a. n.a.
n.a. n.a.
7 n.a.
3.87 9 0.03 7.7 9 0.4
0.46 9 0.02 All sites
19 9 0 51 9 3
69 9 3 119 9 3
a
The soil analyses, referring to 0–5 cm below litter layer in deciduous forests, are: pH 0.2 M KCl, total amount of carbon C, nitrogen N and their molar ratio, net potential nitrogen mineralisation mmol g
− 1
loss on ignition measured in a 15-week incubation experiment as ammonium NH
4
, nitrate NO
3
or total nitrogen NH
4
+ NO
3
. Means 9 S.E. for soil data are based on the number of sites where the species was present 6–121 out of a total of 194 sites. The species are ranked according to increasing
soil acidity.
b
pH-data calculated from Tyler 1996 representing the province of Ska˚ne, Sweden, n = 22.
c
n.a., not available.
d
R-value denoted as indifferent by Ellenberg 1992.
on ignition LOI. This measurement can also be divided into the amount that was mineralised to
ammonium 4 – 65 mmol g
− 1
LOI or was nitrified 51 – 100 mmol g
− 1
LOI. The two latter had a wider range than the total amount of nitrogen.
The ratio between amino acid and methy- lamine uptake capacity is used to eliminate the
variation in absolute amounts that are taken up by the different species and thereby to be able to
compare the importance of organic nitrogen among the species. The uptake ratio between
amino acids and methylamine ranged from 0.02 to 1.42 Table 5. We tested whether the capacity
to take up organic nitrogen was a plant charac- teristic that co-varied with any of our soil vari-
ables and if so could be considered to be selective for survival on soils with different nitrogen
status. The amount of nitrate in the soil ex- plained most of the variation, closely followed by
the Ellenberg’s R-value indicating soil acidity of the species’ distributions and total carbon con-
tent in the soil Fig. 1. pH-values for the species’ distribution in southern Sweden was, however,
not significantly correlated with the ratio between amino acid and methylamine uptake. This can
probably be explained by the low frequency n = 6 and 13 of the two species with the highest
pH-values as the correlation was significant P = 0.049 when a data set for the whole of Sweden
was used n = 93 and 30.
An interesting non-existent relationship is the missing correlation between the ratio between
amino acid to methylamine uptake and the total amount of mineralised nitrogen Fig. 1. In spite
of the high amounts of nitrogen produced in soils where the ten species grow, all species but G.
aparine and P. 6ulgaris could take up relatively high proportions as organic nitrogen.
4. Discussion
