Uptake capacity of amino acids by ten grasses and forbs in
relation to soil acidity and nitrogen availability
U. Falkengren-Grerup *, K.F. Ma˚nsson, M.O. Olsson
Department of Ecology,Plant Ecology,Lund Uni6ersity,Ecology Building,S-223 62Lund,Sweden
Received 12 October 1999; received in revised form 6 July 2000; accepted 6 July 2000
Abstract
Uptake capacity of organic nitrogen was studied in solution experiments on eight grasses and two forbs growing in acid soils with relatively high nitrogen mineralisation in southern Sweden. Uptake of a mixture of amino acids (alanine, glutamine, glycine), that varied between 1.6 and 6.3mmol g−1dw root h−1, could not be explained by soil
data from the species’ field distributions (pH, total carbon and nitrogen, potential net mineralisation of ammonium and nitrate). The ratio between organic and inorganic nitrogen (methylamine) uptake wasB0.05 for the forbs, higher for the grasses with a maximum of 1.42 forDeschampsia flexuosa. The ratio was negatively correlated with measures related to soil acidity (Ellenberg’sR-value, soil nitrate and total carbon) but not, as hypothesised, with the total amount of mineralised nitrogen. The total demand on nitrogen by all components of the ecosystem would probably have described the extent to which competition among and between plants and microbes induced nitrogen limitation. In a methodological study two grasses were exposed to pH 3.8, 4.5 and 6.0 and to 50, 100 and 250mmol l−1of three
amino acids. Uptake was also compared between intact plants and excised roots. The treatment response varied considerably between the species which stresses the importance of studying intact plants at field-relevant pH and concentrations. © 2000 Elsevier Science B.V. All rights reserved.
Keywords:Alanine;Deschampsia flexuosa;Elymus caninus; Excised roots; Glutamine; Glycine; Soil pH
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1. Introduction
There is a growing interest in the capacity of plants to take up organic nitrogen and of the significance it has for the plants’ nitrogen nutri-tion (Stribley and Read, 1980; Chapin et al., 1993;
Kielland, 1994, 1997; Raab et al., 1996, 1999). Early reports usually reflected the physiological aspect, investigating concentration dependence of the amino acid uptake and the uptake systems (King, 1976; Soldal and Nissen, 1978) whereas the ecological significance of a range of species and geographical areas has come into focus in several later papers. Species studied have been predomi-nantly the non-mycorrhizal sedges (Chapin et al., 1993; Kielland, 1994; Raab et al., 1996) and woody, ericoid and ectomycorrhizial species
* Corresponding author. Tel.:+46-46-2224408; fax: + 46-46-2224423.
E-mail address:[email protected] (U. Falkengren-Grerup).
(Stribley and Read, 1980; Bajwa and Read, 1985; Abuzinadah and Read, 1989; Kielland, 1994; Na¨sholm et al., 1998). Plants with ectomycorrhiza and ericoid mycorrhiza, growing in soils with low mineralisation of inorganic nitrogen, have often been considered to be especially efficient in using organic nitrogen (Abuzinadah and Read, 1986). Organic nitrogen uptake has rarely been consid-ered in arbuscular mycorrhizal species and then usually in cultivated plant species (Schobert, et al., 1988; Chapin et al., 1993; Jones and Dar-rah, 1994). The uptake supplied by the fungi itself is usually low compared to the total demand by the plants (George et al., 1995).
There is a large temporal and spatial variation in availability of amino acids in soils and a rapid turnover into other organic or inorganic com-pounds. Soil solution concentrations in organic soils in temperate and arctic regions are com-monly 5 – 15 mmol l−1 of amino acids such as
alanine, aspartic acid, glutamic acid and glycine and analysed amino acids may add up to 100 mmol l−1 (Abuarghub and Read, 1988; Chapin
et al., 1993; Kielland, 1994; Raab et al., 1996, 1999). A high seasonal variation in total amino acids is exemplified in the study on Cyperaceae
species in four alpine meadows, being between 13 and 158 mmol l−1 (Raab et al., 1999). Some
results indicate that higher concentrations are found in soil solutions in highly organic soils (Raab et al., 1996), which the few results on brown soils, as compared with podsols, also show when the amounts per g soil are similar (Abuar-ghub and Read, 1988).
Studies on organic nitrogen uptake by wild plants have been based largely on species from infertile and highly organic soils where nitrogen mineralisation rates are low (Chapin et al., 1993; Kielland, 1994, 1997; Na¨sholm et al., 1998;Raab et al., 1999). In a study by Kielland (1994) on arctic soils glycine, aspartic acid and glutamic acid accounted for as much as 80% of the total nitrogen uptake in Ledum palustre, while the same amino acids accounted for only 10% in
Eriophorum angustifolium. Organic nitrogen up-take has rarely been studied in more fertile soils where mineralisation rates are relatively high. Ni-trogen is often the limiting nutrient even in these
soils, as stated for example for temperate decidu-ous forest soils of varying soil fertility (Tamm, 1991).
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
Prunella6ulgarisL. 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 MoO4, 5 H3BO4, 10
phosphate (Na2HPO4), 500 Cl and 200 SO4, 250
NH4NO3and 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 studiesa
Total biomass
Plant species Plant age after sowing (weeks) Root Shoot Root:shoot ratio
Methodological study
0.390.1 11.193.7
D.flexuosa Grass 3 2.490.1 8.693.3
15.095.9 0.490.1 10.894.8
3.791.3 5
Grass E.caninus
Main study
Grass 7 1.690.4 1.590.3 3.190.6
A.capillaris 1.090.2
Grass 7 2.190.4
A.6inealis 2.090.4 4.090.7 1.190.2
2.290.4
D.cespitosa Grass 7 3.090.6 5.290.9 0.790.2
Grass 6 2.090.2 3.890.8 5.891.0 0.590.1
D.flexuosa
Grass 5 5.991.3 10.792.6 16.893.8 0.590.1 E.caninus
0.590.1 7.491.3
4.991.0 2.590.5
F.gigantea Grass 7
F.o6ina Grass 7 2.490.5 2.890.6 5.190.9 0.990.1
Forb 7 3.190.5
G.aparine 5.690.6 8.790.8 0.690.2
P.nemoralis Grass 7 2.690.5 3.690.5 6.290.9 0.790.1
Forb 7 3.290.2 4.590.3 7.790.5 0.790.1
P.6ulgaris
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 B0.5 mm. The temperature in the green-house was set to 16°C night/20°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 14C-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 grassesD.flexuosaandE.caninuswere 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 250mmol 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.caninusleaves 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 CaCl2 l
−1of
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 CaCl2 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 of14C-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 CaCl2 l−
1
to maintain the membrane integrity (Epstein, 1961). The solutions of methylamine and amino acids contained 1.65 kBq l−1. Alanine (14CH
had three labelled C-atoms, glycine two (H2N 14
CH2 14CO
2H) and methylamine hydrochloride one
(14CH
3NH2.HCl).
2.6. Radioisotope measurement
The roots and shoots were separately com-busted in a Packard sample oxidiser, model 307. The14
CO2 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 usingSPSS, 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.caninushad 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 250mmol l−1of 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
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−1dw root h−1)a
Treatment D.flexuosa E.caninus
Intact plant Excised roots Ratio Intact plant Excised roots Ratio intact:excised intact:excised
Glycine
2.590.3
50 2.490.2 1.1 10.790.8 7.290.9 1.5
2.790.2
100 3.790.4 1.6 14.190.5 8.190.6 1.7
3.890.1 1.5 21.590.3
5.390.6 8.690.8
250 2.5
Glutamine
2.590.1 3.3 9.590.9
7.891.7 7.391.0
50 1.3
5.990.7
100 2.090.2 3.2 13.090.3 8.590.8 1.5
2.290.4 3.2 15.691.4 11.490.7
6.691.0 1.4
250
Alanine
1.590.2 2.1 8.190.4 7.790.7 1.1
50 3.290.7
1.790.1 2.5 8.990.3
4.290.2 8.690.4
100 1.0
250 6.391.1 2.390.1 2.7 14.490.6 13.690.5 1.1
Methylamine
3.590.5 1.1 30.892.7
50 3.990.4 22.693.1 1.3
5.391.2 0.9 42.092.7
4.990.6 36.194.8
100 1.2
11.591.0
250 11.891.2 1.0 59.995.4 47.794.1 1.3
aMeans9S.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 forE.
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 ofD.
flexuosa. The largest effect onE. 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 11%in 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 forD.flexuosathat had a significantly higher transport at 250 mmol l−1than 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),
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 forD. flexuosa and at pH 4.5 and 6.0 forE.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. flexuosaand E. caninusplants at three pH-levels in solutions of 100 mmol l−1 methylamine or a mixture of three amino acids as nitrogen source (means9S.E., n=5)a
aUptake 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. flexuosaand pH 6.0, 4.5\pH 3.8 forE. caninus.
Table 3
Uptake of amino acids and methylamine in shoots as a percentage of the total uptake in roots and shootsa
Treatment D.flexuosa E.caninus
Shoot% total 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. flexuosaand methylamine\glycine\
glutamine, alanine forE.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.
6ulgarishad 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 175.2mmol g−1dw root h−1.
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 acidsa
Methylamine Ratio
aDifferences 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. Means9S.E.; n=5 for D. flexuosaandE.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 (R2=
0.158, PB0.001) and pH and the C/N ratio (R2=0.070, PB0.001) whereas pH is positively
related to the nitrification rate in the soil (R2=
0.635). No correlation is found between pH and nitrogen mineralisation (R2=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 mmol 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
NO3
aThe 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 (NH4), nitrate (NO3) or total nitrogen (NH4+NO3). Means9S.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.
bpH-data calculated from Tyler (1996) representing the province of Ska˚ne, Sweden,n=22. cn.a., not available.
on ignition (LOI). This measurement can also be divided into the amount that was mineralised to ammonium (4 – 65mmol g−1LOI) 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
4.1. Methodological study
The hypothesis, that species are specific in their amino acid uptake and that the uptake is depen-dent on concentration of these nitrogen sources and on pH, was supported by our results. There
were differences between the two species in all these aspects, and it may well be that the variety in responses among species, even from similar habitats, is much higher than exemplified by our two grass species, i.e. D.flexuosa and E. caninus. The concentration-dependent uptake, that was found to different extents in our study, has been studied earlier (e.g. Soldal and Nissen, 1978; Kielland, 1994) but the pH-dependence is little studied, which is surprising since we show that it was important for the interpretation of the results (see next paragraph). The pH-dependent uptake of amino acids may depend on which ionic form it takes. The amino acid is cationised below its isoelectric point, anionised above it and is in a neutral dipolar form around this point. The isoelectric point is at pH 6.0 for alanine and glycine and at pH 5.7 for glutamine. The amino acids may exist mainly as cations at the tested pH-levels 3.8 and 4.5 and as neutral or anion forms at pH 6.0. Uptake cannot, however, be predicted from the isoelectric point as shown by
Hordeum 6ulgare that had uptake optima at pH
3 – 5 and 5 for the two neutral amino acids me-thionine and proline (Soldal and Nissen, 1978). It has been suggested that amino acids are taken up by plant roots as zwitterions (Soldal and Nissen, 1978; Borstlap et al., 1986) or as anions (Chapin et al., 1993). As plants seem to contain multiple sets of amino acid transport proteins that are specific and/or general in their transport of amino acids (Fischer et al., 1998) it is impossible, at the present stage of knowledge, to predict the uptake patterns of single species.
We can calculate the ‘fault’ that we introduce by making the wrong assumptions of the most relevant pH and concentration of amino acids in the soil solution. The calculation illustrates the various impact of pH and type and concentration of amino acids that species would experience given that the laboratory conditions are relevant for soils and adult plants with mycorrhiza. If the field-relevant pH was 3.8 and D. flexuosa (or another similar species) was tested in a non-ad-justed solution of a neutral amino acid or methy-lamine then it would be exposed to a pH-value of nearly 6. Uptake of amino acids by D. flexuosa
by \300% and uptake of methylamine would be underestimated by 25%. The ratio between amino acid and methylamine uptake would change from 3.3 to 0.7. Making the same assumptions for E.
caninusmeans that uptake of methylamine would be overestimated by 200% whereas uptake of amino acids would be correctly estimated. Here the uptake ratio would change by 0.1 only. The corresponding fault for testing the wrong concen-tration would be, if 50mmol l−1was the
field-rel-evant concentration and 250mmol l−1
was tested, an overestimation by 150 – 300% depending on which amino acid is taken up and 200 – 300% for methylamine. The ratio between amino acid and methylamine uptake by intact plants would hardly change forE. caninusbut would increase by 50% (alanine, glycine) and 350% (glutamine) for D.
flexuosa. The misinterpretation would be consid-erable if both pH and concentration faults were present in a test.
We also hypothesised that uptake by excised roots is lower than by intact plants and that this relation varies between species. The value of labo-ratory experiments with excised roots would thereby decrease. The hypothesis was partly confi-rmed. The excision effect on D. flexuosa was substantial for most of the amino acids but not for methylamine. It was also found forE.caninus
for two of the amino acids and for methylamine, though not quite as strong. Thus, comparing spe-cies in amino acid and methylamine uptake would be uncertain with excised roots as the presence – absence of a shoot seems to give highly variable results. Depending on the concentrations used, the effects of excision between plants and nitrogen sources were more or less pronounced. For exam-ple, using 50 mmol l−1, either intact plants or
excised roots could be chosen and still give the same differences between glycine and methy-lamine uptake for E. caninus. If, on the other hand, 250 mmol l−1
was used the uptake of glycine would be 2.5 times higher than the methy-lamine uptake when using intact plants as com-pared to the excised roots.
The root:shoot biomass may explain some of the variation in uptake patterns of the two spe-cies, e.g. that intact plants of D. flexuosa had a higher uptake of glutamine and alanine than
ex-cised roots whereas excision had little effect onE.
caninus. E. caninus had a higher root:shoot ratio which implies that the shoots are either a weaker sink for xylem solutes or a stronger source for carbohydrates than shoots of D. flexuosa. The fact that the strength of the shoot differs between nitrogen sources complicates the use of excised roots to estimate uptake of inorganic organic nitrogen.
4.2. Main study
The results from this study show that herba-ceous plants from a range of different habitats can take up amino acids. Previous studies have demonstrated uptake of organic nitrogen by spe-cies from soils with low nitrogen mineralisation rates (Stribley and Read, 1980; Abuzinadah and Read, 1986; Chapin et al., 1993; Kielland, 1994; Na¨sholm et al., 1998). Our hypothesis was in line with these studies, stating that the absolute up-take of amino acids should be negatively related to availability of inorganic nitrogen in the soil even on the regional scale used in this study. The results did not support our hypothesis. This may be exemplified by G. aparine, as compared to D.
flexuosa, where the former species grows in soils with comparatively higher nitrogen availability and lower acidity. Comparing our results to ear-lier studies, our species took up amounts of amino acids as high as species growing on soils with low nitrogen mineralisation: 0.3 – 6.9 mmol g−1 dw
root h−1 was taken up by ten arctic species
rela-tively high pH (Brunet et al., 1998), had an up-take of amino acids of 6.1 and 4.5mmol.g−1 dw
root.h−1 respectively, values that lie in the higher
range of those reported for the arctic plants (Kielland 1994). Plant uptake of amino acids may be of importance also in more nitrogen rich soils as there is a higher demand by the mass and numbers of plants and soil organisms in these ecosystems (Kaye and Hart, 1997).
Our last hypothesis stated that the ratio be-tween organic and inorganic nitrogen uptake should be low for all species. The forbs re-sponded with the assumed low uptake ratio, whereas the grasses had a higher uptake, espe-cially D. flexuosa that had a ratio \1. This species responded similarly in a field experiment in north Sweden where it took up as much as 60% as organic nitrogen (Na¨sholm et al., 1998). We also hypothesised that the relative uptake of amino acids should be negatively related to nitro-gen mineralisation in the soil but this hypothesis was rejected. There was, however, a negative cor-relation with soil acidity and nitrification and a positive correlation with total carbon content. Several studies have claimed that uptake of or-ganic nitrogen is most important in oror-ganic soils where mineralisation of nitrogen is low (Kielland, 1994; Raab et al., 1996). Interestingly, our species also responded to the varying but relatively low carbon content in the mineral soils. The uptake ratio between organic and inorganic nitrogen was the most powerful variable in understanding the species’ physiological capacity to use various ni-trogen sources in relation to their soil ecology.
The missing correlation between uptake of or-ganic to inoror-ganic nitrogen and potential net ni-trogen mineralisation is probably not explained solely by differences between laboratory condi-tions and field condicondi-tions as other soil character-istics were related to the uptake. It is most likely that several soil characteristics are involved, in-cluding acidity, availability of different nitrogen forms and the demand on nitrogen by the whole ecosystem. The uptake by mycorrhiza may also be considerable but is still little known (Johansen et al., 1993; Ma¨der et al., 2000). Field studies must therefore be performed to further explain the advantage of uptake of organic nitrogen
un-der natural conditions when both inorganic and organic nitrogen may also be supplied by mycor-rhizal fungi.
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