under laboratory conditions our results must be validated for soil conditions in the field and for
mature plants infected by arbuscular mycorrhiza. The following hypotheses are tested:
1. The capacity to take up amino acids varies between species and amino acids and depends
on concentration and pH in the solution. 2. Uptake is sink-source driven. Experiments
with excised roots, as compared to intact plants, underestimates uptake and to various
degrees in different species. 3. A species’ uptake capacity of amino acids is
negatively related to availability of inorganic nitrogen in the soil. This is reflected by varying
nitrogen mineralisation in soils of the species’ field distributions.
4. Uptake of amino acids relative to inorganic nitrogen is low for all studied species. The
importance of uptake of organic to inorganic nitrogen is highest in soils with low availability
of inorganic nitrogen.
2. Material and methods
2
.
1
. Plant material Eight grasses and two forbs were used in
the study: Agrostis capillaris L., Deschampsia cespitosa L. Beauv., Deschampsia flexuosa L.
Trin., Agrostis 6inealis Schreb., Elymus caninus L. L., Festuca gigantea L. Vill., Festuca o6ina
L., Poa nemoralis L., Galium aparine L. and Prunella 6ulgaris L. We chose species occurring on
soils with low to high nitrogen availability as estimated from field observations in Central Eu-
rope Ellenberg, 1992 and as measured in 600 sites in south Sweden Diekmann and Falkengren-
Grerup, 1998. The data on nitrogen and other soil chemical characteristics from southernmost
Sweden, from where the seeds were collected, are given in Table 6. The plants were grown from
seeds in \ 99 pure silica sand with the addition of a nutrient solution containing in mmol l
− 1
250 Ca, 200 K, 80 Mg, 200 Na, 5 Fe as citrate, 20 Mn, 2.0 Zn, 0.2 Cu, 0.1 MoO
4
, 5 H
3
BO
4
, 10 phosphate Na
2
HPO
4
, 500 Cl and 200 SO
4
, 250 NH
4
NO
3
and with pH set to 4.5 Falkengren-Gre- rup, 1998 Establishment of mycorrhiza in silica
sand is very slow according to own experience and highly unlikely in this study. The plants were used
in the experiments 3 – 7 weeks after sowing that was done repeatedly, using the same seed collec-
tions, between August and October to co-ordinate plant size with time of experiment. In spite of a
longer growth period for D. flexuosa in the main study than in the methodological study the
seedlings were smaller. The seedlings were chosen
Table 1 Biomass mg dry weight and the root:shoot ratio of plants used in the two studies
a
Total biomass Plant species
Plant age after sowing weeks Root
Root:shoot ratio Shoot
Methodological study 0.3 9 0.1
11.1 9 3.7 D. flexuosa
Grass 3
2.4 9 0.1 8.6 9 3.3
15.0 9 5.9 0.4 9 0.1
10.8 9 4.8 3.7 9 1.3
5 Grass
E. caninus Main study
Grass 7
1.6 9 0.4 1.5 9 0.3
3.1 9 0.6 A. capillaris
1.0 9 0.2 Grass
7 2.1 9 0.4
A. 6inealis 2.0 9 0.4
4.0 9 0.7 1.1 9 0.2
2.2 9 0.4 D. cespitosa
3.0 9 0.6 Grass
5.2 9 0.9 0.7 9 0.2
7 Grass
6 2.0 9 0.2
3.8 9 0.8 5.8 9 1.0
0.5 9 0.1 D. flexuosa
Grass 5
5.9 9 1.3 10.7 9 2.6
16.8 9 3.8 0.5 9 0.1
E. caninus 0.5 9 0.1
7.4 9 1.3 4.9 9 1.0
2.5 9 0.5 F. gigantea
7 Grass
F. o6ina 2.4 9 0.5
Grass 2.8 9 0.6
5.1 9 0.9 0.9 9 0.1
7 Forb
7 3.1 9 0.5
G. aparine 5.6 9 0.6
8.7 9 0.8 0.6 9 0.2
P. nemoralis 0.7 9 0.1
6.2 9 0.9 3.6 9 0.5
2.6 9 0.5 Grass
7 Forb
7 3.2 9 0.2
4.5 9 0.3 7.7 9 0.5
0.7 9 0.1 P. 6ulgaris
a
Means 9 S.E., n = 9–10 in the methodological study and 19–20 in the main study.
to be of similar root biomass in both experiments which resulted in a higher root:shoot ratio in the
main study Table 1. The diameter of all roots was B 0.5 mm. The temperature in the green-
house was set to 16°C night20°C day and with 16 h per day of radiation of 160 mmol m
− 2
s
− 1
at plant surface. Relative air humidity was set at
50.
2
.
2
. Amino acids and methylamine The
14
C-labelled amino acids alanine, glycine and glutamine were used in our experiments,
partly to be able to compare with other studies where they were frequently used but mainly as
they are commonly found in a range of different soils Abuarghub and Read, 1988; Stevenson,
1994. Methylamine hydrochloride was used as an ammonium analogue Hackette et al., 1970; Roon
et al., 1975; Kleiner, 1985; Franco et al., 1987; Ritchie, 1987; Kielland, 1994; Kosola and Bloom,
1994. The choice of concentrations was based on field concentrations reported in earlier studies
Kielland, 1994; Raab et al., 1996.
2
.
3
. Methodological study The two grasses D. flexuosa and E. caninus were
tested on uptake by excised roots and intact plants of each of the three amino acids alanine,
glutamine and glycine, and uptake of methy- lamine at concentrations of 50, 100 and 250 mmol
l
− 1
. The effects of pH were tested at 3.8, 4.5 and 6.0 in 100 mmol l
− 1
of a mixture of the three amino acids and methylamine. The two species
have a wide distribution and represent different morphologies and growth rates. D. flexuosa has
tightly inrolled leaves 0.5 mm wide and
E. caninus leaves are flat, rather thin, up to 13 mm wide Hubbard, 1980. The two grasses represent
species growing at the extreme ends of the soil pH found in deciduous forests in southern Swe-
den Table 6. D. flexuosa tolerates more acid soils than E. caninus and has a lower biomass
Mossberg and Stenberg, 1992. Plant age after sowing was 3 D. flexuosa and 5 weeks E. cani-
nus. The experiment is described in Section 2.5.
2
.
4
. Main study Intact plants of the eight grasses and two forbs
identified above were exposed to a mixture of alanine, glutamine and glycine and to a methy-
lamine solution. The intermediate nitrogen con- centration used in the methodological study was
chosen 100 mmol l
− 1
; 33.3 mmol l
− 1
of each amino acid. pH was adjusted to 4.5 by addition
of HCl. This pH lies within the physiological range of all species Falkengren-Grerup and
Tyler, 1993. Plant age after sowing was 5 E. caninus
, 6 D. flexuosa and 7 weeks all other species. The same root biomass was sought here
as in the methodological study. The experiment is further described in Section 2.5.
Uptake of amino acids and the ratio between amino acids and methylamine were related to soil
data from a subsample of 600 deciduous forests in total in south Sweden Falkengren-Grerup et al.,
1998. The subsample consists of sites where the studied species was present, usually only for the
province of Ska˚ne from where the seeds were taken. The soil was sampled from 5 cm below the
litter layer, that represents the main rooting hori- zon in many forest soils but roots may penetrate
to 10 – 20 cm depending on soil type. We used data on pH 0.2 M KCl, net potential nitrogen
mineralisation 15 weeks laboratory incubation on soil was sampled in June – August performed in
darkness at 18°C and 45 – 60 of water holding capacity, total carbon and nitrogen content mea-
sured on soils sampled in June to August. We also used the indirect measure of soil acidity R-value
of the species’ field distributions as estimated by Ellenberg 1992.
2
.
5
. Experimental procedure The entire root system was used in the experi-
ments with the intact plants as well as with the excised roots. The root biomass ranged between 2
and 6 mg dw per replicate Table 1. Plants with damaged or senescent roots were discarded. The
experimental procedure follows Kielland 1994. The roots were rinsed in a 0.5 mmol CaCl
2
l
− 1
of 20°C and then enclosed in cotton cloth bags with
a mesh size of 1 mm excised roots or synthetic
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
