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New Phytol. (1998), 138, 385399

Soluble N compounds in trees exposed tohigh loads of N: a comparison between theroots of Norway spruce (Picea abies) and

beech (Fagus sylvatica) trees grown underfield conditions

B A R T H U R G E L E R , S T E P H A N S C H N E I D E R , P A U L W E B E R ,

U L R I K E H A N E M A N N H E I N Z R E N N E N B E R G *

Albert-Ludwigs-UniversitaWt Freiburg, Institut fuWr Forstbotanik und Baumphysiologie,

Professur fuWr Baumphysiologie, Am Flughafen 17, D-79110 Freiburg i. Br., Germany

(Received 10 June 1997; accepted 13 October 1997)

During the growing session of 1995, the total soluble non-protein nitrogen (TSNN) composition and contents of

mycorrhizal fine roots, xylem sap and phloem exudates of roots from a coniferous ( Picea abies L.(Karst)) and a

deciduous (Fagus sylvatica L.) tree species were analysed at a field site (Ho$glwald, Germany) exposed to high

loads of N. In April, TSNN in fine roots of spruce and beech trees amounted to 16 mol N g" f. wt and

23n3 mol N g" f. wt, respectively. It decreased to 9n2 mol N g" f. wt and 18n1 mol N g" f. wt, respectively,

after bud break in June. The seasonal maximum of TSNN in fine roots of spruce was observed in July

(32n7 mol N g" f. wt) followed by a decline of c. 30% until the end of the growing season in September. TSNN

in fine roots of beech trees showed a further decline between June and July, when its seasonal minimum was

determined (15n6 mol N g"

f. wt), and increased to c. 29 mol N g"

f. wt until September. In spruce roots Glnand Arg were the most abundant TSNN compounds during the entire growing season. In roots of beech Asn

played an important role alongside Gln and Arg, especially in April, when it was the most abundant TSNN

compound. Other proteinogenic and non-proteinogenic N compounds comprised c. 2030% of TSNN. Nitrate

made up 1%, and ammonium 7% of TSNN in the fine roots of both species.

In April, TSNN in the xylem sap of roots of spruce and beech trees amounted to 3n4 and 8n6 mol N ml",

respectively. In roots of spruce trees xylem sap TSNN increased after bud break up to 12n7 mol N ml" in July.

At the end of the growing season TSNN had declined again to 3n9 mol N ml". TSNN in the root xylem sap of

beech trees decreased after bud break until July (2n4 mol N ml" in July) followed by a slight increase until

September (2n9 mol N ml"). Arg, Gln and Asp were the most abundant TSNN compounds in the xylem sap of

spruce trees contributing together c. 90% to TSNN. The same TSNN compounds prevailed in the root xylem

sap of beech trees in April and July, whereas in June and September Asp was replaced by Asn comprising 57%

of TSNN in June. In addition to the N compounds mentioned above, a number of other proteinogenic and non-

proteinogenic amino compounds were found in root xylem sap of both species. In either species, nitrate and

ammonium were present in small amounts, contributing 1% and 4% to TSNN, respectively. Apparently,inorganic N taken up by the mycorrhizal roots is mainly assimilated in root tissues or by the mycorrhiza and N

uptake by the roots is largely adapted to the assimilatory capacity of this organ.

In phloem exudates of spruce roots, TSNN amounted to 10n7 mol N g" f. wt in April, increased in June to

23n4 mol N g" f. wt and decreased again until September to a seasonal minimum of 4n8 mol N g" f. wt. In

contrast to spruce, TSNN content in phloem exudates of beech roots showed a seasonal maximum (c.

20 mol N g" f. wt) in April with a subsequent decrease in June after bud break (c. 2 mol N g" f. wt). A fourfold

increase in July was followed by a decrease in September, when TSNN in phloem exudates of beech roots

amounted to 4n3 mol N g" f. wt. Arg was the most abundant N compound in the phloem of roots from spruce

trees and made up c. 6085% of TSNN during the entire growing season. In beech trees the seasonal course of

* To whom correspondence should be addressed.E-mail: here!sun2.ruf.uni-freiburg.de


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386 A. Geler and others

TSNN correlated with the relative abundance of Arg. Arg comprised 69 and 57% of TSNN in April and July,

respectively, but contributed 20% in June and September. Besides Arg, other proteinogenic and non-

proteinogenic amino compounds could be detected in the phloem of both species. In addition, nitrate and

ammonium were present in considerable amounts.

From these results and a previous report on TSNN in above-ground parts of spruce and beech at the same site,

a whole-plant model for the cycling of TSNN in both species is proposed. Differences in the location of storage

pools are assumed to be responsible for the differences in the seasonal course of TSNN composition and contents

observed between the two tree species.

Key words: N composition, N cycling, amino acids, phloem exudate, xylem sap, nitrate, ammonium, roots.

Nitrogen supplies for higher plants in terrestrial

ecosystems are mainly met by the uptake of am-

monium and\or nitrate by the roots (Glass & Siddiqi,

1995). Total amounts in the soil and the availability

of both N forms for the plant depend on soil

properties, microbial processes in the soil, climatic

factors, and the N input into the ecosystem (Haynes

& Goh, 1978; Fangmeier et al., 1994; Bredemeier,

Dorenbusch & Murach, 1995). Ammonium plays amore important role in soils of forest ecosystems as

compared with agricultural soils, where good aer-

ation and neutral pH favour nitrification processes

and cause nitrate to prevail (Glass & Siddiqi, 1995).

In addition to root absorption, N can also be taken

up by above-ground parts of plants either in a

gaseous form, mainly as NH$

and NO#

(Nussbaum

et al., 1993; Pearson & Stewart, 1993; Weber et al.,

1995), or in a dissolved form as ammonium and

nitrate (Brumme, Leimcke & Matzner, 1992; Burk-

hardt & Eiden, 1994).

In herbaceous plants N assimilation is thought to

take place mainly in the shoots (Gojon et al., 1991),

whereas in woody plants N assimilation in mycorr-

hizal fine roots (Gebauer & Stadler, 1990; Gojon,

Plassard & Bussi, 1994) is of significance. The

distribution of N assimilation between the roots and

the shoot of trees might depend on the species

(Truax et al., 1994), the N supply in the soil, the

capacity of root nitrate reductase in relation to N

uptake (Gojon et al., 1994), and atmospheric uptake

of NOx

and NH$

(Wellburn, 1990; Thoene et al.,

1991, Pe!rez-Soba et al., 1994). The actual N demand

of trees can also be met by mobilization of N

compounds from storage tissues (Millard & Proe,1992). Especially in spring during bud break, N

uptake from the soil is low as a consequence of low

soil temperatures (Geler et al., 1998). Under these

conditions N mobilized from storage tissues con-

tributes the major part of the N supply to newly

developing leaves (Millard, 1989; Millard & Proe,

1992; Millard, 1994). In deciduous trees the woody

tissues and the bark are the main sites of overwinter

storage, whereas remobilized N in coniferous trees

originates mainly from last years needles (Millard,

1996). Translocation of N compounds from the

tissues of uptake and storage to the tissues of demand

occurs via xylem and\or phloem transport. Xylem

transport is largely controlled by the direction of the

transpiration stream, whereas phloem transport takes

place from source to sink, and can be independent of

the relative positions of the tissues of supply and the

tissues of demand (Pate, 1975). In addition, N

compounds as well as other nutrients are known to

be exchanged between the two transport systems of

plants (Biddulph, 1956; Pate, 1975; Cooper &

Clarkson, 1989; Da Silva & Shelp, 1990; Schneider,

Schatten & Rennenberg, 1994; Pate & Jeschke,1995).

In order to adapt the uptake and mobilization of N

to the different seasonal needs of N-consuming

tissues, a signal indicating the N status of the plant

is required (Muller, Touraine & Rennenberg, 1996).

For herbaceous plants a well established model

(Imsande & Touraine, 1994) proposes that the

transport of products of N assimilation from the

shoot to the roots in the phloem acts as such a signal.

The enrichment of the phloem with amino com-

pounds is thought to repress nitrate uptake of the

roots and diminishes the rate of nitrate reduction.

Experiments on the interaction between atmospheric

and pedospheric N supply in spruce (Muller et al.,

1996) and on the influence of root-fed amino acids on

nitrate uptake in beech (Kreuzwieser et al., 1997)

indicate that this mechanism might also play a role in

the regulation of N uptake by trees. A pool of amino

acids circulating from the roots to the shoot and vice

versa would allow plants to remove N compounds

from this pool as required at the sites of demand,

whereas remaining N compounds can serve as a

signal indicating the N status of the tree (Cooper &

Clarkson, 1989).

The aim of the present study was to characterizethe N composition of roots of adult spruce and beech

trees grown in the field under conditions of high N

input. For this purpose N composition and contents

of phloem exudates and of xylem sap from roots as

well as of extracts of fine roots were analysed. Beech

(F. sylvatica) was chosen as the most important tree

in natural forests in Central Europe and Norway

spruce (P. abies) as one of the dominant conifers in

economically used forests. Combined with the

results of a previous study (Schneider et al., 1996)

characterizing the N status of above-ground parts of

beech and spruce trees at the same field location, a


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Soluble N compounds in roots of spruce and beech trees 387

model of N allocation at the whole tree level is

developed, and possible regulatory functions of a

circulating pool of amino compounds under con-

ditions of high N input are discussed.

Site characteristics

Studies were performed at the field site Ho$glwald

located 50 km west-north-west of Munich (Ger-

many) (longitude: 11m 10h E; latitude: 48m 30h N) in

the prealpine region at 540 m above sea level. Annual

average air temperature is 8 mC, and annual pre-

cipitation is 800 mm. The soil is an acidic podsolic

para brown earth (hapludalf, US Soil Taxonomy).

In 1770 the natural vegetation, a Luzulo Fagetum

typicum, was replaced by Norway spruce trees (Picea

abies (L.) Karst). In 1995 the second generation of

spruce had reached an age of 88 yr. In a small area,

beech trees (Fagus sylvatica L.) were planted afterthe first generation of spruce; in 1995 these trees had

reached an age of 95 yr. The field site is exposed to

high inputs of ammonium and nitrate caused by

intensive agriculture in close proximity (Go$ttlein &

Kreutzer, 1991). During the present investigation

throughfall within the spruce area contained 10 kg

NO$-N and 20 kg NH

%+j-N ha" yr" ; and within

the beech area the values are 7 and 9 kg ha" yr",

respectively. Nitrogen concentrations in the rainfall

amounted to 5 and 7 kg NO$-N and NH

%+-N,

respectively (Rothe, 1997).

Plant material

Plant material was collected during four field trials in

1995, these being performed in (I) April (before\

during bud break), (II) June (after bud break) (III)

July, and (IV) September at the end of the growing

season. Fine roots were dug out from soil at depths

of 010 cm, washed with double-demineralized

water to remove adhering soil particles, immediately

frozen in liquid N#, and stored at k80 mC. The fine

roots of both species were found to be mycorrhizal

by visual inspection. Roots of c. 1n0 cm diameter

were used for the collection of xylem sap and phloem

exudates.

Xylem sap collection

Xylem sap of roots was collected by the modification

of the procedure of Scholander etal. (1965) described

by Schneider et al. (1996). Roots were cut to a length

of 3050 cm immediately after harvest. Bark and

cambium were removed at a length of 30 mm from

the cut end to prevent contamination of the xylem

sap with cellular constituents. The cut end without

bark and cambium was rinsed thoroughly with

double-distilled water. Each root was fitted into a

pressure chamber (Soilmoisture, Santa Barbara,

USA) with 10 mm of the cut end protruding.

Subsequently, the pressure in the chamber was

raised at a rate of 0n10n2 MPa min". During this

procedure the cut end was observed with a dissecting

microscope withi10 magnification. The pressure at

which xylem sap first appeared was regarded as the

actual root water potential, being 1n4p1n2 and1n7p0n7 MPa for beech and spruce, respectively.

The first protruding solution was discarded in order

to avoid contamination. Subsequently, the pressure

was raised to 0n6 MPa over root water potential, and

was kept constant for 2 min. The xylem sap was

collected, immediately frozen in liquid N#, and

stored at k80 mC until analysis. Contamination with

cellular components was checked by measuring

luminometrically the ATP contents of the xylem sap

(Schupp, 1991; Rennenberg, Schneider & Weber,

1996). Measured ATP contents were in the same

range (0n5%) as those determined for the twigs of

both species (Schneider et al., 1996).

Collection of phloem exudate

Phloem exudate was collected by the EDTA-

technique described by Rennenberg et al. (1996) and

Schneider et al. (1996). Small pieces of bark (c.

150 mg f. wt) were removed from roots with a

diameter of c. 1n0 cm and washed with double-

demineralized water. Subsequently, the bark pieces

were placed in 6-ml vials with 2 ml of exudation

solution containing 10 m EDTA and 0n015 m

chloramphenicol at pH 7n0 for 5 h. Phloem exudateswere frozen in liquid N

#and stored at k80 mC until

analysis. Previous studies (Schneider et al., 1996)

showed that contamination of phloem exudates of

beech and spruce with cellular constituents can be

neglected under the experimental conditions applied.

Extraction of N compounds from the fine roots

Nitrogen compounds were extracted from fine roots

as described by Winter, Lohaus & Heldt (1992).

Fine roots frozen in liquid N were ground with

mortar and pestle. Aliquots of 0n3 g of the frozenpowder were homogenized in 0n4 ml of buffer

containing 20 m HEPES (pH 7n0), 5 m EGTA,

10 m NaF and 2n5 ml of chloroform: methanol

(1n5 : 3n5, (v\v)). The homogenate was incubated for

30 min at 4 mC. Subsequently, water-soluble meta-

bolites were extracted twice with 3 ml of double-

distilled water. The aqueous phases were combined

and freeze-dried (Alpha 24, Christ, Osterode,

Germany). The dried material was dissolved in 1 ml

double-demineralized H#O or 1 ml of lithium citrate

buffer (0n2 , pH 2n2) for nitrate and amino acid

analysis, respectively.


4/15


Analysis of amino compounds

Samples of xylem sap and phloem exudate were

centrifuged at 16000 g and 4 mC for 5 min before

analysis. The pH values of the clear supernatants

were adjusted with 1 HCl to pH 2n2. Solutions of

the root extracts in lithium citrate buffer were

directly used for analysis of amino compounds. An

aliquot of 3570 l of each sample was injected intoan automated amino-acid analyser (Biochrom, Phar-

macia LKB, Freiburg, Germany). Amino com-

pounds were separated on a PEEK column (Ultrapac

8 Resin, Lithium 250i4n6 mm, Biochrom, Pharma-

cia, Freiburg, Germany) using a system of five

lithium citrate buffers causing a pH gradient from

2n80 to 3n55. The separated amino components and

ammonium were subjected to postcolumn deriva-

tisation with ninhydrin. The absorption of the

aminoninhydrin derivates was measured at 440 and

570 nm. Peaks were identified and quantified by

means of a standard solution (Sigma Chemie,

Deisenhofen, Germany) containing 39 amino com-pounds and ammonium. To estimate decomposition

of amino acids during the treatment, commercial

amino compounds were added during phloem exu-

dation and extraction of roots in control experiments.

Neither decomposition of the added amino com-

pounds, nor increase of ammonium contents were

measured in these experiments.

Determination of nitrate

Aliquots of 1 ml of each sample were shaken for at

least 2 h with 50 mg of PVPP (Sigma Chemie,

Deisenhofen, Germany) to remove phenolic com-pounds. The samples were centrifuged for 10 min at

4 mC and 16000 g. Aliquots of 250 l of the clear

supernatants were injected into an ion chromato-

graph (DX 100; Dionex, Idstein, Germany). Anions

were separated on a IonPac2 column (AS9-Sc

250i4 mm; Dionex, Idstein, Germany) eluted with

a solution containing 1n8 m Na#CO

$plus 1n7 m

NaHCO$

at a flow rate of 1n0 ml min". Nitrate in

freeze-dried fine root samples and xylem sap was

detected with a conductivity detector module (CDM,

Dionex, Idstein, Germany). The detection limit of

this method is 0n3 nmol ml". In phloem exudates

determination of nitrate contents was performedwith an UV-VIS detector (SPD-6AV; Shimadzu,

Duisburg, Germany ) at 210 nm as described by

Hayashi & Chino (1985). The detection limit of this

method is 0n3 nmol ml".

Data analysis

For phloem exudate analyses three to four roots each

of beech and spruce were sampled. Analyses of

amino compounds and nitrate of fine roots were

performed in three to five independent samples. The

data were subjected to (Microcal Origin,

version 3.5, Microcal Software, Inc.). For the

analyses of xylem sap two roots of each tree species

were sampled during each trial with the exception of

June 1995, when five roots were collected.

N composition and contents in the fine roots

The total soluble non-protein N (TSNN) contents

of fine roots of spruce (Fig. 1 a) amounted to

16n0 mol N g" f. wt in April, decreased to

9n2 mol N g" f. wt in June and reached the seasonal

maximum in July at 32n7 mol N g" f. wt. At the

end of the growing season in September TSNN

content had declined to 23n6 mol N g" f. wt. In

April, Gln and Arg were the most abundant amino

compounds in the fine roots of spruce and con-

tributed together 64% to TSNN (4n5 mol

N g" f. wt and 5n7 mol N g" f. wt, respectively).

Between April and June Gln contents decreased c.

fourfold, whereas Arg contents remained constant.In July, when TSNN in spruce roots reached

maximum amounts, Arg plus Gln made up 74% of

TSNN with 16n4 mol N g" f. wt for Arg and

7n8 mol g" f. wt for Gln. In September Arg and

Gln contents decreased to 10n4 mol N g" f. wt and

5n8 mol N g" f. wt, respectively. In addition to Arg

and Gln considerable amounts of Asp, Asn and Glu

could be detected during the entire growing season.

These compounds contributed together between 9

and 12n5% to TSNN. Several other proteinogenic

amino acids such as Thr, Val, Gly, Al, Pro, Ile, Leu,

His, Tyr, Lys and Trp were found in small amounts

of up to 0n2 mol N g" f. wt throughout the year.

Non-proteinogenic amino compounds, i.e. ornithine

(Orn), -amino butyric acid (GABA) and etha-

nolamine, comprised together 3% of TSNN.

TSNN contents in fine roots of beech trees

(Fig. 1 b) were 23n3 mol N g" f. wt in April and

subsequently showed a slight decline to 18n1

and 15n6 mol N g" f. wt in June and July, respec-

tively. At the end of the growing season in

September TSNN contents had increased to

28n8 mol N g" f. wt. Asn, Gln and Arg dominated

TSNN in fine roots of beech trees in April and

amounted to 7n4 mol N g"

f. wt, 6n8 mol g"

f. wt,and 4n5 mol N g" f. wt, respectively. Together

these amino compounds comprised 80% of TSNN.

The decline in TSNN between April and June was

mainly caused by a 4-fold decrease in the Asn

content. The contents of other TSNN compounds

remained constant between April and June. In July

a 2n5-fold decline in the Arg content was observed.

In September Arg, Asn and Gln contributed to-

gether 68% to TSNN. The increase of TSNN

between July and September was caused by an

increase in all of the major amino compounds

detected. In addition to the N compounds mentioned


5/15


NH

4+

NO

3

24018012060

30

20

10

April

(a)

April

(b)

25020015010050

20

10

350

300

150

100

50

350

300

250

200

150

100

50

00

September

J une J une

J uly J uly

September

T Pr

Np

Asp

Asn

Glu

Arg T P

rNp

Asp

Asn

Glu

Arg

NH

4+

NO

3

Gln

Gln

Ncompos

ition

(mo

lNg1

f.w

t)

Figure 1. Nitrogen composition and contents fine roots from adult spruce and beech trees. Fine roots of beechand spruce trees were dug out from depths 010 cm. The data shown are means of three independent rootsamples with two replicate analyses, each. Significant differences between spruce (8) (a) and beech () (b) areindicated at P0n05 (*) and P0n01 (**) T, Total; Pr, proteinogenic (rest); Np, non-proteinogenic.

above, between 16% and 26% of TSNN in the

beech roots was made up by the 17 other pro-

teinogenic amino acids throughout the year. The

non-proteinogenic amino compounds Orn, GABA

and ethanolamine contributed together 5 % t o

TSNN.


6/15


NO

3

NH

4+

NH

4+

NO

3

30

20

10

April

(a)

April

(b)

75

20

10

16014012010080

35

30

25

20

15

10

05

00

September

J une J une

J uly J uly

September

T Pr

Np

Asp

Asn

Arg T P

rNp

Asp

Asn

Arg

Gln

Gln

Ncompos

ition

(mo

lNg

1f

.wt)

40

45

30

20

10

05

15

25

60

45

40

60

80

Figure 2. Nitrogen composition and contents of the root xylem sap of adult spruce and beech trees. Xylem sapwas collected from roots with 1n0-cm diameter by a modification of the technique described by Scholander etal. (1965). For the trials conducted in April, July and September, data from two roots each of spruce ( a) andbeech (b) are shown separately. For the trial in June the data shown are meansp of five roots of each species.T, Total; Pr, proteinogenic (rest); Np, non-proteinogenic.


7/15


Nitrate made up 1% of TSNN between April

and September in roots of both tree species, whereas

the contribution of ammonium amounted to 2n37%

of TSNN in fine roots of spruce and 12n9% of

TSNN in fine roots of beech.

N composition and contents in the xylem sap of roots

Figure 2 shows the TSNN composition and contentsin root xylem sap of spruce and beech trees at

different times of the growing season. During the

trials in April, July and September, two roots of each

species were examined and are shown separately; for

the trial in June, the data shown represents

meansp of five independent samples from five

roots. In April, before bud break of both tree species,

mean concentrations of TSNN in the root xylem sap

were 3n4 mol N ml" and 8n6 mol N ml" for

spruce (Fig. 2 a) and beech (Fig. 2 b), respectively.

TSNN content in the root xylem sap of spruce trees

increased between April and July and reached

6n6 mol N ml" and 12n7 mol N ml" in June andJuly, respectively. At the end of the growing season

in September TSNN contents of root xylem sap had

declined to 3n9 mol N ml". TSNN in the xylem

sap of beech roots decreased after bud break to

4n2 mol N ml" in June. A seasonal minimum in the

TSNN content was reached in July with 2 n4 mol

N ml". Thereafter, TSNN slightly increased and

amounted to 2n9 mol N ml" in September. In June

of TSNN contents of the five roots analysed

amounted to 26% and 21% of the mean value for

spruce and beech, respectively.

In April and June Asp, Gln and Arg were the

predominant amino compounds in the xylem sap of

roots in spruce trees (Fig. 2 a) contributing together

more than 90% to TSNN. The increase of TSNN

between April and June was caused by an increase of

these three amino compounds. In July, when TSNN

reached its highest contents within the growing

season, Arg alone was 81% of total soluble N. Asp

and Gln contents were 0n5 mol N ml" and

0n3 mol N ml", respectively, comprising together

c. 6 % of TSNN. In September Arg content was c. 6-

fold lower than in July and reached c. 1n7 mol

N ml". At this time of the year Asp and Gln

contents amounted to 1n0 and 0n9 mol N ml"

,respectively.

In the xylem sap of the beech roots (Fig. 2 b) Asp,

Gln and Arg were the most abundant TSNN

compounds in April comprising together 96% of

TSNN. Concomitant with the decline of TSNN

between April and June, contents of these amino

compounds decreased 25-fold (Asp) and c. 5-fold

(Gln and Arg), respectively. With c. 57% of TSNN

Asn was the most abundant such compound in June,

but was not found in April. In July, when TSNN

content reached a seasonal minimum, Asn declined

from 2n3 mol N ml" in June to 0n02 mol N ml".

Contents of Gln and Arg remained unchanged,

whereas Asp increased c. 8-fold between June and

July. In September, Asn became again the most

abundant TSNN compound with 1n0 mol N ml",

whereas the Asp content declined to the value

observed in June. Contents of Gln and Arg showed

a slight increase in September and amounted to

0n7 mol N ml" and 0n6 mol N ml", respectively.

In addition to the amino compounds mentionedabove root xylem sap of both species contained other

proteinogenic and non-proteinogenic N compounds.

In spruce roots the proteinogenic amino acids Thr,

Ser, Glu, Gly, Ala, Ile, Leu, Tyr, Phe, Lys and His

contributed together c. 57% to TSNN. Proteino-

genic N compounds other than Asp, Asn, Gln and

Arg in root xylem sap of beech trees comprised

between 2n3% of TSNN in April and 10% in

September.

Besides the proteinogenic components, several

non-proteinogenic amino compounds were found in

root xylem sap of both species, the most abundant

being Orn, GABA and ethanolamine. Together thesecompounds contributed 1%, and 7%, to xylem

sap TSNN in spruce and beech, respectively. In

root xylem sap of both species small amounts

of nitrate and ammonium could be detected.

Ammonium was present at 0n040n17 mol N ml"

(1%4% of TSNN), whereas nitrate accounted for

0n010n04 mol N ml" (0n11% of TSNN).

N composition and contents in phloem exudates of

roots

In April TSNN in root phloem exudates of adult

spruce trees was 10n7 mol N g" f. wt and increased

in June after bud break to 23n4 mol N g" f. wt (Fig.

3 a). In July TSNN was present in only half the

amount found in June, and there was a further

decline in September to 4n8 mol N g" f. wt. TSNN

in phloem exudates of beech roots (Fig. 3 b) showed

a different seasonal course, with maximum levels of

20n2 mol N g" f. wt in April, a decrease in June

(2n1 mol N g" f. wt), a slight increase in July

(8n9 mol N g" f. wt) and another decrease in Sep

tember (4n3 mol N g" f. wt). The most abundant

TSNN compound in phloem exudates of spruce

roots during the entire growing season was Arg,comprising c. 62% of TSNN in April and Sep-

tember, 85% in June and 59% in July. The changes

in TSNN contents during the growing season

correlated with changes in Arg contents. The 19

other proteinogenic amino compounds (most abun-

dant were Ala, Glu, Gln and Asp) contributed c.

10% in April and June and c. 30% in July and

September to TSNN. The non-proteinogenic

TSNN compounds Orn, GABA and ethanolamine

together made up between c. 2 and 12% of TSNN.

As in spruce roots, Arg was the most abundant

TSNN compound in phloem exudates of beech roots


8/15


NO

3

NH

4+

NO

3

NH

4+

30

20

10

April

(a)

April

(b)

20

10

1209060

30

15

00

September

J une J une

J uly J uly

September

Ncompos

ition

(mo

lNg

1f.w

t)

45

25

20

10

05

15

150

40

450

T Pr

Np

Asp

Asn

Arg

Gln

Ala

Glu T P

rNp

Asp

Asn

Arg

Gln

Ala

Glu

05

15

200

250

300

300

150

Figure 3. Nitrogen composition and contents of phloem exudates from roots of adult spruce and beech trees.The phloem exudate of bark pieces from the roots of Picea abies and Fagus sylvatica was extracted in 2 ml ofa solution containing 10 m EDTA and 0n015 m choramphenicol at pH 7n0 for 5 h. Subsequently, sampleswere subjected to automatic amino acid and anion analyses. The data shown are meansp of threeindependent root samples with two replicate analyses each. Significant differences between spruce (8) (a) andbeech () (b) are indicated at P0n05 (*) and P0n01 (**). T, Total; Pr, proteinogenic (rest); Np, non-proteinogenic.


9/15


in April. With a content of 14n0 mol N g" f. wt this

amino compound contributed 69 % to TSNN. In

contrast to spruce roots, Arg contents declined in

phloem exudates of beech roots to a very low level in

June (11% of TSNN), but still remained the most

abundant amino compound. In July Arg contents

increased 20-fold (57% of TSNN) and declined

again in September when it made up c. 15% of

TSNN. The variation of the TSNN contents in rootphloem exudates of beech during the growing season

was mainly attributed to a variation in Arg contents.

Other proteinogenic amino acids such as Ala, Glu,

Gln, Asn, Asp and Gly, contributed together

between 13% and 35% to TSNN. The three non-

proteinogenic amino compounds Orn, GABA and

ethanolamine together made up 5% of TSNN in

beech roots throughout the year.

The relative abundance of nitrate in phloem

exudates of spruce roots was high in April with

21n5% of TSNN, but low (c. 1% of TSNN) during

the rest of the growing season. Ammonium

amounted to up to 7n5% of TSNN in the phloemexudate of spruce roots. In phloem exudates of the

beech roots, relative ammonium contents showed a

strong seasonal oscillation, making up in April 2n1 %,

in June 39%, in July 5% and in September 21% of

TSNN. Nitrate contents in root phloem exudates of

beech amounted to c. 10% of TSNN in April, June

and July and to 41% in September.

TSNN in the roots of spruce and beech trees

In the present study the composition and contents of

TSNN in the xylem and phloem of roots as well as

in extracts of fine roots of a deciduous (F. sylvatica)

and a coniferous (P. abies) tree species grown in the

field under similar environmental conditions were

analysed. The field site Ho$glwald was chosen,

because it is exposed to high loads of N as a result of

its close proximity to intensive agriculture (Go$ttlein

& Kreutzer, 1991).

Differences in TSNN contents in beech and spruce in

spring might be due to differences in N mobilization

TSNN in root xylem from spruce trees showed

lowest seasonal contents immediately before bud-

break and increased until June. In the phloem of

spruce roots TSNN also increased from a low level

in April to a seasonal maximum in June. A totally

different situation was observed for beech which

showed a seasonal maximum of TSNN in the root

xylem and phloem before\during bud break with a

subsequent decrease. A seasonal course of N contents

similar to that in beech roots has previously been

observed in above-ground parts of beech and spruce

trees, with maximum N transport in xylem (Glavac

& Jochheim, 1993; Dambrine et al., 1995; Schneider

et al., 1996) and phloem (Schneider et al., 1996)

immediately before and during bud break. The N

supply of newly developing tissues of trees during

spring can at least partly be met by remobilization of

internal N sources from storage tissues (Millard &

Proe, 1992; Millard, 1994). During this period of

time N uptake by roots seems to be of minor

significance (Millard, 1996; Geler et al., 1997).Therefore, the differences in TSNN contents in root

xylem and phloem (Figs 2, 3) between spruce and

beech during the development of new leaf tissues

may be attributed to differences in N mobilization.

Since N is stored in conifers mainly in last years

needles (Nambiar & Fife, 1991; Millard & Proe,

1993; Millard, 1996), the roots might not contribute

as much to the transport of mobilized N to the new

developing leaves during bud break as in deciduous

trees where N is stored in the wood and bark of the

trunk and the roots (Millard, 1996). Therefore, the

high amount of TSNN in the transport systems of

underground parts of beech during bud break mightoriginate from N mobilized from storage tissues,

whereas the lower amounts in pruce indicate that

root-borne N is less important for new developing

needles.

N uptake and N mobilization expand the N pool in

the roots of spruce trees

The increase of TSNN in fine roots and root xylem

of spruce in summer might result from enhanced N

uptake and assimilation (Geler et al., 1998) in the

roots that might increase TSNN loading into the

xylem (Martin & Amraoui, 1989) and, as a conse-

quence, might expand the cycling N pool (Cooper &

Clarkson, 1989 ). Nitrogen mobilized from last

years needles, and N taken up by the roots and

transported to the twigs in summer might not only

be transferred to new needles (Millard & Proe,

1993), but might also be transported to the roots in

the phloem, especially if the supply of N in above

ground parts of spruce exceeds the actual demand of

the newly developing tissues. The observed increase

in TSNN contents in the phloem of beech roots in

July might be explained by the beginning of N

translocation to storage tissues of the roots thatmight also be responsible for the increase of TSNN

in the fine roots in September.

TSNN compounds in the xylem of beech and spruce

roots

From the predominant N compound in the xylem

sap plants can be divided into allantoin-, amide- and

citrulline-type species (Sauter, 1981). A previous

study that examined the TSNN composition and

contents of above ground parts of spruce and beech

trees at the same site confirmed the finding of Bollard


10/15


(1956) that both species transported predominantly

amides, i.e. Gln in the xylem sap of spruce and Asn

plus Gln in the xylem sap of beech trees (Schneider

et al., 1996). The present results show that such a

clear-cut classification is no longer possible, if root

xylem sap and its seasonal changes are considered.

As observed in the shoot, Gln plays an important

role in root xylem sap of spruce, but Arg pre-

dominates throughout the season and Asp contri-butes to TSNN in considerable amounts (Fig. 2 a).

In the xylem sap of beech roots TSNN composition

varies considerably during the seasons (Fig. 2 b). Arg

and Gln were always present, but Asp played an

important role in April and July, when Asn contents

were negligible; Asn was the most abundant TSNN

compound in June and September when Asp

contents were low. Since similar fluctuations be-

tween Asp and Asn were not observed in fine roots or

in root phloem exudates of beech, it may be

concluded that seasonal changes in xylem loading of

amino compounds in the roots are responsible for the

observations made.

N uptake seems to be adapted to the N reduction and

assimilation capacity of the roots

Besides organic N compounds only small amounts of

nitrate and ammonium were present in the root

xylem sap and in the fine roots of both species,

contributing together4% to TSNN. This finding

is in agreement with the observations of Dambrine et

al. (1995) for spruce and Martin & Amraoui (1989)

for beech xylem sap. Apparently, inorganic N uptake

by the mycorrhizal roots is well adapted to the

capacity for N reduction and assimilation in the roottissue and the hyphae. The absence of considerable

amounts of nitrate in fine roots and root xylem sap is

not surprising, since nitrate is not taken up by the

roots of either species at the Ho$glwald field site

during most of the growing season (Geler et al.,

1998). By contrast, Glavac & Jochheim (1993)

observed high nitrate contents in samples of xylem

sap from lower trunk sections of beech trees,

contributing c. 35% to total N transported in the

xylem. Experiments with peach seedlings showed

that the roots were the main sites of nitrate reduction,

but increasing nitrate availability and, as a conse-

quence, increased rates of nitrate uptake could resultin enhanced xylem transport of nitrate from the roots

to the shoot (Gojon et al., 1991, 1994). Differences in

the uptake of nitrate by the roots might have led to

the different nitrate contents in the xylem observed

by Glavac & Jochheim (1993) compared with the

present study.

TSNN compounds in the phloem of beech and spruce

roots

In the phloem of spruce trees Arg was the pre-

dominant TSSN compound during the entire grow-

ing season, but a wide spectrum of other proteino-

genic and non-proteinogenic amino compounds

could be detected. The TSNN contents of the

phloem exudates of the roots from beech trees

showed remarkable variations that could mainly be

attributed to a variation of Arg contents in the

phloem. Since Arg is thought to be a N-storage

compound (Flaig & Mohr, 1992; Gezelius &

Na$

sholm, 1993), high amounts of Arg in the phloemmight indicate remobilization of N (April) or

transport of N to the storage tissues (July). Arginine

accumulation might also indicate excess N nutrition,

and may be considered as a means by which plants

cope with excess ammonium in the soil (Na$sholm &

Ericson, 1989). High contents of ammonium and

nitrate observed in the phloem exudates of both

species might at least partly be due to diffusion of

these ions from the soil solution into the bark. It has

been suggested that both ions may be taken up across

the bark (Schulze, Gebauer & Katz, 1991). However,

a correlation between seasonal changes in nitrate and

ammonium contents in the soil solution (Geler etal., 1998) and nitrate and ammonium contents in

phloem exudates was not found in the present study

(data not shown). Since nitrate and ammonium were

also present in the phloem exudates of twigs from

both species at the same site (Schneider et al., 1996)

it cannot be ruled out that these inorganic N

compounds are also transported from the shoot to

the roots in the phloem in spruce and beech exposed

to high loads of N.

A cycling pool of amino acids might be involved in

the regulation of N uptake by the roots

Nitrogen uptake by plants is thought to be adapted

to N demand (Imsande & Touraine, 1994; Muller, et

al., 1996). A pool of amino acids cycling between

shoot and roots and vice versa might be involved in

this regulatory process. Nitrogen compounds re-

quired for growth of developing tissues can be taken

out of this pool, and remaining N might serve as an

indicator of the N status of the tree, regulating N

uptake (Cooper & Clarkson, 1989; Larsson et al.,

1991; Touraine, Clarkson & Muller, 1994). A

prerequisite for such a circulation within the tree is

the exchange of TSNN compounds between xylemand phloem as already observed for Gln in Populus

deltoides (Dickson, Vogelmann & Larson, 1985) and

for glutathione in spruce (Schneider et al, 1994).

Various studies have been performed to examine the

influence of phloem-translocated amino acids on

nitrate uptake by roots (Cooper & Clarkson, 1989;

Muller & Touraine, 1992; Muller, Tillard &

Touraine, 1995). Experiments on the effect of amino

compounds on nitrate uptake by roots of beech trees

showed that Asp, Gln and Glu had the capability to

reduce net uptake of nitrate (Geler et al., 1998;

Kreuzwieser et al., 1997). The present study shows


11/15


NH4+

NH4+

NOxNHy

ArgGlnGluAsp

previousyearsneedles

Arg

currentyearsneedles

GlnArg

GlnGlnAsp(Arg)

Arg

Arg

Arg

Gln

Arg

GlnAsp

Xylemof

twigs

Phloemof

twigs

stem stem

roots roots

ArgGln

fine roots

during N mobilization after N mobilization(NO3

)(NO3

)

Figure 4. Allocation and cycling of TSNN compounds inspruce trees. The compounds shown made up at least 5 %of TSNN. Bold printed compounds comprised more than50%, compounds in italics more than 15% of TSNN.

that these amino compounds are present in root

phloem (Fig. 3 b) and in the fine roots of beech trees

(Fig. 1 b), and, therefore, might contribute to the

regulation of nitrate uptake in the field. In spruce

seedlings, an enhanced phloem transport of reduced

N compounds caused by NO#

fumigation led to a

reduction of net uptake of N by the roots (Muller, et

al., 1996). Enrichment of roots with Glu, Gaba, Gln

and Asn that are all transported in the root phloem

(Fig. 3 a) and all contribute to TSNN of fine root

tissues of spruce (Fig. 1 a) at the Ho$glwald site,

significantly reduced net uptake of nitrate by roots of

1-yr-old spruce seedlings (Geler et al., 1997).

A whole-plant model for allocation and cycling of

TSNN in spruce and beech trees exposed to high

loads of N

Bringing in the results of a previous study (Schneider

et al. 1996) investigating the TSNN composition

and contents of above ground parts of beech and

spruce trees at the same field location, we now

propose the following models of TSNN allocation

and cycling at the whole-tree level for spruce and

beech.

TSNN cycling in spruce

In spruce trees, Arg and\or Gln are the most

abundant TSNN compounds in all plant sections

analysed during the whole growing season. In the

roots Arg and Gln might originate from both

synthesis from inorganic N taken up from the soil,

and phloem transport from the leaves to the roots,

since both TSNN compounds could be detected inthe twig as well as in root phloem (Fig. 4). In spring

during bud break, N uptake by roots is supposed to

be negligible compared with mobilization of N from

storage tissues (Millard, 1996; Geler et al., 1998).

The uptake of inorganic N and subsequent as-

similation of Asn and Arg in the roots might be

important after N pools of the storage tissues are

exhausted.

Arg is cycled within the roots of spruce during spring

Arginine, Gln and in addition Asp are loaded into

the root xylem during the whole growing season. Inspring, Gln and Asp, but not Arg, were present in

the xylem of twigs. This finding indicates that Arg

loaded into the root xylem is subsequently removed

from the xylem in the root and\or stem region and

might serve as N source for stem growth. Since an

enrichment of the phloem with Arg from the twigs to

the roots was observed, Arg unloaded from the

xylem might also be reloaded into the phloem and,

thus, might circulate within the trunk and the roots

of spruce trees. In September during N storage Arg

was not only present in the xylem of roots but also

in that of twigs. Arginine found in the twig xylem

might originate either from Arg loaded into the root

xylem, but no longer removed in the root or stem

region during transport, or from Arg transported in

phloem.

Arginine is the most important TSNN compound in

the needles

Previous years needles contain Arg throughout the

growing season. Since these organs cannot be

considered sinks of phloem transported amino

compounds, the Arg found in previous years needles

might originate from (1) Gln and Asp transported inthe xylem and\or (2) assimilation of inorganic N

compounds taken up from the atmosphere. It might

represent part of the N storage pool mobilized

during growth of current years needles (Nambiar &

Fife, 1991; Flaig & Mohr, 1992; Gezelius &

Na$sholm, 1993). During initial development of the

current years needles Arg, the predominant TSNN

compound, might originate from three sources: (1)

phloem transport of Arg from the previous years

needles, as previously also observed for glutathione

in spruce trees (Schneider et al., 1994; Blaschke et

al., 1996), (2) transformation of Gln allocated to the


12/15


current years needles in xylem and phloem, and (3)

assimilation of inorganic N taken up from the

atmosphere. Since trees at the field site studied are

exposed to a high atmospheric N input (Go$ttlein &

Kreutzer, 1991) uptake of NHy

and NOx

might be

significant (Brumme et al., 1992; Nussbaum et al.,

1993; Pearson & Stewart, 1993; Weber et al., 1995 ;

Muller et al., 1996) and Arg accumulation in the

needles might indicate excess N supply (Na$

sholm &Ericson, 1989).

TSNN in the phloem of spruce might originate from

the leaves or from xylem to phloem exchange

TSNN allocated to, or produced in the needles and

exceeding the immediate demand for growth or

storage is supposed to be transported in the phloem

to the roots. The predominant TSNN compounds

transported in the phloem during the entire growing

season are Arg and Gln. Especially in spring and

early summer, gradients in the relative abundance of

both compounds between twig and root phloem wereevident (Fig. 4). Arg is enriched in the phloem of the

root compared with that of the twig. This might be

a result of (1) xylem to phloem exchange of Arg in

the root or stem region, or (2) the consequence of a

slow phloem unloading in the root region compared

with the phloem loading of Arg in the shoot.

Glutamine is enriched in the twig compared with

the root phloem and, therefore, might be exchanged

from phloem to xylem in the twig or stem region.

The enrichment of Gln from root to shoot xylem in

spring and early summer is consistent with this

assumption. Alternatively, phloem loading of Gln

might be low compared with phloem unloading in

the roots. Glutamine transported to the roots might

regulate nitrate uptake, since experiments with

spruce seedlings showed that enrichment of this

amino compound correlated with the reduction of

root uptake of nitrate (Geler et al., 1998). Regu-

lation of nitrate uptake has to be assumed, since adult

spruce trees at the Ho$glwald site did not take up

nitrate during the entire growing season (Geler et

al., 1998).

TSNN cycling in beech

Nitrogen cycling and allocation in beech are more

complex and they change during the growing season.

Therefore, it is necessary to discuss them separately

for the time of N remobilization in spring, and for

the phase of N storage at the end of the growing

season.

Early-season growth of beech depends on N mobilized

from storage sites

The N demand of deciduous trees in spring is met

almost completely by N compounds mobilized from

the storage tissues in the stem and the woody tissues

of roots (Millard, 1996). Arginine, Gln and Asn

found in the fine roots of beech (Fig. 5 a) in spring

can originate from stored soluble-N or from break-

down of storage protein. Nitrogen mobilization in

the roots results in xylem loading of Arg, Gln and

Asp. However, Asp could not be detected in the

xylem of twigs and Asn was present instead. The

presence of Asn in the twig xylem of beech might beexplained by its loading into the xylem from storage

tissues in the stem or by transfer of Asn from twig

phloem into the xylem. Since Asn also contributes

significantly to TSNN in the leaves (24% of TSNN

in April), a circulating pool of Asn in the upper part

of the tree is suggested. Another explanation for the

differences in Asp and Asn contents between xylem

sap of roots and twigs is the transformation of Asp

into Asn in the parenchymatous tissues of wood and

bark in the stem. Aspartic acid might be removed

from the xylem in the stem region and loaded with an

amide group originating from N storage compounds.

Asparagine produced might be loaded into the xylemto be transported to the leaves. Thus, during times of

N mobilization, Asp and Asn play an important role

for N distribution within beech trees. A similar role

of amino acids and corresponding amides for N

translocation and distribution has been observed in

herbaceous plants (Pate, Wallace & van Die, 1964;

Pate, Layzell & Atkins, 1979).

Nitrogen by developing leaves might be met by

phloem and xylem transport

In spring Asn is the most abundant TSNN com-

pound in the developing leaves. It might originate

from different sources: immediately before and at

the beginning of bud break a major part of N

consumed by the developing leaves has to be

transported in the phloem (Da Silva & Shelp, 1990).

The high abundance of Arg in the phloem of twigs

during spring might therefore represent remobiliza-

tion of stored N transported into an acropetal

direction. When leaves are unfolded and xylem sap

flow is driven by transpiration, N transport in the

xylem might contribute considerably to the N

demand of the developing foliage (Moreno & Garcia-

Martinez, 1983; Sauter & van Cleve, 1992). Arginineand Gln are present in the phloem and xylem of roots

and twigs as well as in the tissues of fine roots and

leaves. It may be assumed that these compounds are

subjected to an intensive exchange between phloem

and xylem as well as efficient plant internal cycling in

xylem and phloem.

TSNN in autumn might partly originate from N

uptake by the roots of beech trees

In autumn during N storage, Gln is the most

abundant TSNN compound in the roots. Since


13/15


NH4+

(NO3) (NH4

+)

Arg, Glu, Gln,Arg, Asp

leaves

Gln,Arg

AsnArgGlnArg

Arg, Gln Asn

ArgGln

ArgGln

Asp

Xylemof

twigs

Phloemof

twigs

stem stem

roots roots

ArgGlnAsnfine roots

(NO3) (NH4

+)

NOxNHy

Arg, Gln, Glu, Aspleaves

ArgGlnAsn

Arg

Arg

Arg

GlnAsn

Xylemof

twigs

Phloemof

twigs

stem stem

roots roots

GlnArgAsnfine roots

(a) Spring

AsnGlnArg

AsnAsp

Asp

Arg,Gln

stored Nbreakdown

NH2

soil

(b) Autumn

ArgGlnAsn

storage tissues

Gln,Asn

Arg, Gln,

Asn

Arg

GlnAsn

soil

Figure 5. Allocation and cycling of TSNN compounds in beech trees, (a) during N mobilization in spring and(b) during N storage at the end of the growing season. The compounds shown made up at least 5% of TSNN.Bold printed compounds comprised more than 50%, compounds in italics more than 15% of TSNN.

significant ammonium uptake was observed at the

Ho$glwald site in summer and autumn (Geler et

al., 1998) at least a part of the TSNN found in the

root might originate from ammonium uptake and

assimilation. In contrast to the situation in spring,

Asn is loaded into the xylem of the roots, whereas the

abundance of Asp in the root xylem is low.

Enrichment of Arg from root to shoot xylem was

observed and might be explained by phloem to

xylem exchange in the twigs or the stem region. The

relative abundance of Gln and Asn in the xylem sap

decreased from the roots to the twigs. This decrease

might indicate removal of these compounds from the

xylem for N storage in the stem.

High Arg contents of beech leaves in autumn might

result from N remobilization and uptake of

atmospheric N

Arg contents (21% of TSNN) as well as TSNN

contents of the leaves increased in September,

indicating the beginning of remobilization of N from

the foliage into storage tissues due to the onset of

senescence (Co# te! & Dawson, 1986; Millard, 1996).

Besides Arg, other N compounds as Gln, Glu and

Asp contributed to the TSNN pool of the leaves

(Fig. 5 b). Apparently, Arg, Gln and Asn are

transported in the phloem to the roots. Since the

relative Arg content decreased from the shoot (52%

of TSNN) to the roots (15% of TSNN), Arg seems

to be removed from the phloem most likely for

storage in the parenchyma cells in the stem wood.

Since Arg is enriched in the xylem of the twigs

compared with the roots, at least a part of that

removed from the phloem in the stem might be

reloaded into the xylem, and it therefore seems to be

cycled in the above-ground parts of the beech trees.

High Arg contents observed in foliage and phloem in

autumn might also partly originate from uptake of

NOx

and NHy

by the leaves. Gln is present in all

tissues and transport systems during the entire

growing season and therefore shows the charac-teristics of an N compound circulating between

shoot and roots. Thus, Gln might serve as a signal

compound adapting N uptake of roots to the N

demand of the plant. This assumption is consistent

with the observation that Gln but not Asn or Arg

inhibits nitrate uptake by the roots of beech

seedlings, and that beech trees at the Ho$glwald site

did not show nitrate uptake during most of the

growing season (Geler et al., 1998).

Further studies are required to compare the

present models for spruce and beech trees subjected

to high atmospheric N input with TSNN allocation


14/15


and cycling in trees growing at low N supply. Such

a comparison might provide additional information

on the consequences of high N input for N allocation

and cycling.

This investigation is part of the Ho$glwald ecosystem

study and was financially supported by the Bundesministerfu$r Bildung, Wissenschaft, Forschung und Technologie

(BMBF) under contract no. BEO 51 0339614.

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compus solubil n in arborii expusi la sarcini mari _o comparatie intre radacinile molidului din...

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