Journal of Experimental Botany, Vol. 54, No. 386, pp. 1389±1397, May 2003
DOI: 10.1093/jxb/erg146
RESEARCH PAPER
Bidirectional exchange of amino compounds between
phloem and xylem during long-distance transport in
Norway spruce trees (Picea abies [L.] Karst)
Arthur Geûler, Paul Weber, Stephan Schneider and Heinz Rennenberg1
Institut fuÈr Forstbotanik und Baumphysiologie, Professur fuÈr Baumphysiologie, Albert-UniversitaÈt Freiburg,
Georges-KoÈhler-Allee Geb 053/054, D-79110 Freiburg i. Br., Germany
Received 21 October 2002; Accepted 20 January 2003
Abstract
Introduction
14
In forest ecosystems the nitrogen demand of woody plants
is mainly met by the uptake of nitrate, ammonium and/or
amino acids from the soil (Glass and Siddiqi, 1995; Ohlund
and Nasholm, 2001). The rates of uptake of these
compounds largely depend on climatic conditions and
the properties of the forest soil (Marschner et al., 1991;
Geûler et al., 1998a). In particular, the share of ammonium
versus nitrate availability is of signi®cance (Downs et al.,
1993), because ammonium uptake by the roots of trees is
often preferential to the uptake of nitrate (Marschner et al.,
1991; Geûler et al., 1998a). In addition to nitrogen uptake
from the soil, gaseous nitrogen compounds can be taken up
by leaves/needles from the atmosphere, in particular NOx
(Wellburn, 1990; Nussbaum et al., 1993; Rennenberg and
Geûler, 1999; Geûler et al., 2000, 2002) and NH3 (PeÂrezSoba et al., 1994; Fangmeier et al., 1994; Geûler et al.,
2000, 2002). Solubilized nitrogen may also be taken up by
leaves/needles and bark from rainwater, fog or dew as
ammonium and nitrate (Brumme et al., 1992; Burkhardt
and Eiden, 1994; Rennenberg and Geûler, 1999).
In many woody plants nitrate reduction and ammonium
assimilation are thought to occur mainly in the roots
associated with ectomycorrhizal fungi (Martin and Botton,
1993; Martin and Lorillou, 1997). Irrespective of the
origin, organic nitrogen compounds synthesized in the
roots have to be allocated to other tissues (e.g. needles/
leaves, stem meristems, reproductive structures, etc.) that
require nitrogen for growth and development. In addition
to the use of external nitrogen sources, the actual nitrogen
demand of a given tissue can also be met by mobilization
of nitrogen compounds from internal sources (Millard,
1996). In spring, when soil temperatures and, as a
C-Gln, 14C-Asp, 15N-Gln, and 15N-Asp were fed to
cut tips of 2- or 3-year-old needles of spruce
twigs, still attached to the tree. After incubation,
distribution of the radiolabel and 15N enrichment
was studied in needles, bark and wood tissues of
girdled twigs and untreated controls. Analysis of
the twig tissues showed that between 22% and
26% of the total amount of the tracers applied had
been taken up. Since export out of the application
segment and distribution between needles, bark
and wood was comparable for 14C and 15N tracer,
it was concluded that, mainly the amino compounds that had been fed were subject to longdistance transport within the plant and supplied
the new sprout with nitrogen. Asp was exported to
a greater extent to developing needles compared
with Gln. This difference in export between the
two amino compounds applied may be explained
by the different pool sizes of Gln and Asp in
xylem and phloem or differences in xylem and
phloem loading. Girdling of the stem showed that
the transport of reduced nitrogen compounds from
older needle generations to current-year needles
proceeded in both xylem and phloem. In addition,
an intensive bidirectional exchange of Gln and Asp
between xylem and phloem was observed during
long-distance transport.
Key words: Amino acids, girdling, long-distance transport,
Picea abies, phloem±xylem exchange, spruce.
1
To whom correspondence should be addressed. Fax: +49 761 203 8302. E-mail: [email protected]
Journal of Experimental Botany, Vol. 54, No. 386, ã Society for Experimental Biology 2003; all rights reserved
1390 Geûler et al.
consequence, rates of nitrogen uptake from the soil are
generally low (Geûler et al., 1998a), mobilization of
nitrogen compounds from storage tissues is of particular
signi®cance for growth and development.
In spruce, older needle generations are thought to be the
main internal nitrogen source in spring (Millard, 1994).
From these needles nitrogen is allocated to tissues with
nitrogen demand. Apparently, the distribution of organic
nitrogen is achieved at the whole plant level by a cycling
pool that includes long-distance transport in both xylem
and phloem (Marschner et al., 1997), but also metabolic
interconversion in the roots and the leaves (Geûler et al.,
1998b).
Gln and Asp are the predominant amino compounds in
the xylem and the phloem of spruce trees (Schneider et al.,
1996; Weber et al., 1998; Geûler et al., 1998b). In
particular in spring, the amount of Gln and Asp in xylem
and phloem change with tree height, i.e. during transport
through the trunk (Geûler et al., 1998b; Weber et al., 1998).
From this ®nding it was hypothesized that these amino
acids undergo a bidirectional phloem/xylem exchange
during long-distance transport (Geûler et al., 1998b). In
order to test this hypothesis, needles of spruce twigs were
fed 14C-Gln, 14C-Asp, 15N-Gln, and 15N-Asp during the
mobilization of stored nitrogen compounds. The labelling
of C and N was applied to test whether both the C and N of
the amino compounds fed were subject to the bidirectional
phloem/xylem exchange. Girdling was applied to enforce
phloem-to-xylem exchange experimentally.
Materials and methods
Plant material
The present experiments were performed with 6±8-year-old spruce
trees (Picea abies [L.] Karst.) grown in polyethylene pots in a
commercial soil mixture. The pots were dug into the soil outdoors at
a ®eld site close to the Institute of Forest Botany and Tree
Physiology at the University of Freiburg. During the summer, trees
were watered once a week with tap water. About 4±6 weeks prior to
the experiments the trees were transferred to controlled environmental conditions (12/12 h light/dark cycle with 1761 °C and
55610% RH during illumination with 300620 mmol m±2 s±1 PAR,
and 1361 °C and 70615% RH during darkness). For the experiments, lateral sprouts were excised from a 3- or 4-year-old twig of
each tree with the exception of the terminal current year sprouts. The
cut planes were covered with vaseline. This procedure did not
visually in¯uence the vitality of the treated twigs (Schneider et al.,
1994).
Feeding with 14C- and 15N-labelled amino acids
One day prior to feeding labelled amino compounds, the trees were
transferred to the laboratory to allow adaptation to indoor conditions.
Room temperature was kept at 18±22 °C. The twigs which were used
for the feeding experiments were girdled close to the stem in all the
trees to avoid contamination of the whole tree with radiolabel in the
14
C feeding experiment. Twigs of one part of the trees were girdled a
second time by peeling about 2 cm of the bark, 4±6 cm apical to the
application site of the tracer to block phloem transport in the apical
direction. Twigs that were not subjected to this second girdling
procedure were used as a control. In initial experiments it was
ensured that girdling close to the stem did not affect total uptake,
export out of the fed needles, and long-distance transport of Asp and
Gln during the time of exposure to the labelled amino compounds
(data not shown).
Application of 14C-Gln, 14C-Asp, 15N-Gln (a-amino group
labelled), and 15N-Asp was carried out via two 2- or 3-year-old
needles as described by Schupp et al. (1992). For this purpose the
tips of two needles were cut with a razor blade. The remaining stump
of each needle was immediately immersed into 20 ml of the
incubation solutions. For the 14C feeding treatment the solution
consisted of distilled water containing 0.1 mCi ml±1 of 14C-Gln
(410±460 nmol ml±1 H2O, speci®c activity of 244.0 mCi mmol±1) or
14
C-Asp (440±480 nmol ml±1 H2O in an ethanol/water mixture of
2:98, speci®c activity of 207.2 mCi mmol±1) (Du Pont, Bad
Homburg, Germany), respectively. The total amount of 14C-Gln
and 14C-Asp fed per needle was 8.2 nmol and 9.7 nmol, respectively.
In the 15N feeding experiments the cut needles were incubated into
aqueous solutions containing 2000 nmol ml±1 15N-Asp (98%
Cambridge Isotope Laboratories, Andover, USA) or 15N-Gln (aamino group 98% labelled, Cambridge Isotope Laboratories,
Andover, USA). Hence, 40 nmol of the 15N labelled amino acids
were fed per needle. During experiments trees were exposed to
arti®cial light (Osram Powerstar HQI-T 400W/DH, Osram,
Germany) of about 500±620 mmol m±2 s±1 PAR measured at fed
twig level with a quantum sensor (Li-Cor, Model Li-185B,
Nebraska, USA). Feeding experiments were started between 09.00
h and 10.00 h.
Harvest
When the amino acid solution applied to the needles was removed
completely (between 190±310 min), experiments were terminated
by cutting the two fed needles from the twig. The cut needles were
rinsed twice with 1 ml distilled water to remove adhering
radioactivity or 15N-label of the incubation solutions. Then the
treated twig was excised from the stem and divided into sections of
2±6 cm length. Each section was subdivided into needles, bark and
wood, weighed and frozen in liquid nitrogen. Harvested plant
material was stored at ±24 °C until further analysis.
14
C- and 15N-analysis
Analysis of 14C in plant sections was carried out by a modi®cation of
the method described by Schupp et al. (1992) and Blaschke et al.
(1996). Frozen samples were ground with a mortar and pestle in
liquid nitrogen. Acid-soluble compounds were extracted in aliquots
of 50±150 mg of the frozen powder with 1 ml 0.1 N HCl and 50 mg
insoluble polyvinylpyrrolidone (PVP, Sigma Chemical Co.,
Deisenhofen, Germany) on a rotary shaker for 30 min at 4 °C.
After centrifugation for 15 min at 6000 g (Microfuge B, Beckman,
Germany) 400 ml of the supernatants were transferred into
scintillation vials (Mini Poly Q-Vials, Beckmann Instruments,
MuÈnchen, Germany). With the remaining pellets the extraction
procedure was repeated twice with 1.2 ml 0.1 N HCl. Then the
combined supernatants were mixed with 10 ml scintillation ¯uid
(OptiPhase, Wallace, Turku, Finland) and the radioactivity was
determined by liquid scintillation counting (LSC, Beckman LS
7500, Beckman, MuÈnchen, Germany).
The remaining pellets were used to determine the amount of
radioactivity incorporated into acid-insoluble compounds. For this
purpose pellets of bark and wood were digested for 1 d at 40 °C with
1 ml tissue solubilizer (Soluene 350, Packard, The Netherlands).
After drying, samples were resolved in 200 ml isopropanol,
transferred into scintillation vials (20 ml Mini Poly Q-Vials,
Beckmann Instruments, MuÈnchen, Germany), and bleached twice
with 300 ml 30 Vol% H2O2 for 1 d at RT each. Pellets of needle
Phloem±xylem exchange of amino acids 1391
Table 1. Absorption time, total uptake, and recovery of
14
C- and
15
N-labelled amino acids applied to needles of spruce twigs
Amino acids were fed to 3- or 4-year-old spruce twigs via the cut tips of two 2- or 3-year-old needles. After incubation, twigs were harvested
and divided into sections of about 2±6 cm length. Each section was subdivided into needles, bark and wood, weighed and frozen in liquid
nitrogen. The total uptake was calculated from radioactivity or 15N enrichment determined in all plant tissues analysed (excluding the fed
needles). Recovery is expressed as radioactivity in all plant tissues analysed (including the fed needles) as a percentage of the total amount of
tracer applied.
Fed amino acid
Number of
experiments (n)
Absorption time
(min)
14
6
6
6
6
282628
212625
272628
278616
C-Gln
C-Asp
15
N-Gln
15
N-Asp
14
a
aa
b
a
a
Total uptake
(nmol)
4.161.8
4.661.6
21.267.6
20.066.9
Recovery (% of the
radiolabel applied)
a
a
a
a
78.9619.4
81.2611.6
73.0623.9
68.5625.2
a
a
a
a
Different letters (a, b) indicate signi®cant differences at P <0.05.
extracts were bleached with 200 ml 30 Vol% H2O2 during drying for
4 d at 40 °C, and were subsequently digested with 1 ml tissue
solubilizer (Soluene 350, Packard, The Netherlands) for 1 d at RT.
After digestion, bleaching was repeated once as described above for
bark and wood samples. An aliquot of 15 ml scintillation ¯uid
(OptiPhase, Wallace, Turku, Finland) was added to all samples.
Radioactivity was determined by LSC (Beckman LS 7500,
Beckman, MuÈnchen, Germany) at ef®ciencies higher than 65% for
acid-insoluble compounds and higher than 91% for acid-soluble
compounds with correction for quenching. Recovery of the radioactivity applied amounted to 79.4618.0%.
For determination of 15N abundance (atom%) and total N, samples
of the oven-dried plant material were ground with a ball mill into a
®ne homogenous powder. 1±2 mg samples were transferred into tin
capsules (Type A; Thermo Quest, Egelsbach, Germany) and injected
into an elemental analyser (NA 2500; CE Instruments, Milan, Italy)
coupled by a Con¯o II-Interface (Finnigan MAT GmbH, Bremen,
Germany) to an isotope ratio mass spectrometer (Delta Plus;
Finnigan MAT GmbH, Bremen, Germany). 15N enrichment in the
tissues of the 15N-Gln or 15N-Asp fed plants was calculated on the
basis of the natural 15N abundance of wood, bark and needles from
untreated control plants (for a detailed description see Fotelli et al.,
2002).
Data analysis
Total uptake of the tracers was calculated as the sum of radiolabel or
15
N enrichment determined in all plant tissues analysed, excluding
the fed needles, and was set as 100%. It was subdivided into (1) the
14
C or 15N tracer remaining in the application segment (without the
fed needles) and the label exported to (2) basal and (3) apical tissues.
In girdling experiments, export into apical tissues was subdivided
into segments before and beyond the girdle. Application of 14C-Gln,
14
C-Asp, 15N-Gln, and 15N-Asp was carried out in six independent
experiments, with six different trees each. Half of the experiments
were performed with girdled twigs, the other half with non-girdled
twigs as a control. Similar results were observed in each of the three
replicate treatments. Signi®cance of differences in total uptake,
export, and recovery between the different tracers applied as well as
between girdled twigs and non-girdled controls (n=3) were analysed
by Student's t-test (ZoÈfel, 1988).
Results
Uptake of the labelled Gln and Asp
Individual needles of spruce trees were fed 14C-Gln, 14CAsp, 15N-Gln, and 15N-Asp. Twigs fed 14C-Asp absorbed
the solutions applied about 30% faster as compared to
twigs of trees supplied with the other tracer substances.
This difference in incubation time may be attributed to the
application of 14C-Asp as an ethanol/water mixture.
In the 14C feeding experiments the portion of radioactivity found in the acid-soluble fraction was dominant
(9065%) in all twig sections analysed, whereas a minor
portion of radioactivity was determined within the acidinsoluble fraction (1065%). Apparently, the major part of
the tracer substances applied was integrated into the
mobile pool of organic nitrogen compounds in the tissues
analysed and was not subjected to de novo protein
synthesis. Recovery of the different tracers applied ranged
between 68.5% and 78.9% (Table 1).
Export and distribution of
14
C and
15
N tracer
The tracer compounds taken up from the applied solution
were transported via the phloem out of the immersed
needles to the application segment. From this segment 14C
and 15N were further allocated partly to basal, partly to
apical tissue segments (Table 2). When Gln was applied,
more than 80% of 14C and 15N taken up remained in tissues
of the application segment. Only a minor portion of the 14C
and 15N tracer was exported to basal (2.1% (14C); 4.4%
(15N)) and apical (9.5% (14C); 12.3% (15N)) tissue
segments. By contrast, most of the 15N and 14C from
labelled Asp was exported to apical (58.2% (14C); 47.3%
(15N)) and basal (8.2% (14C); 12.5% (15N)) tissue segments, and only a minor portion remained in the tissues of
the application segment. Apparently, apical tissues were a
stronger sink for C and N derived from Asp and Gln than
basal tissues, and Asp was exported out of the fed needles
and allocated within the trees in preference to Gln.
For both Gln and Asp feeding treatment, the newly
developing needles were the main sink for the 14C tracer
exported out of the application segment in an apical
direction (Figs 1, 2). The relative portion of the radiolabel
exported in an apical direction, found in the new developing needles, was higher for Asp (up to 50% of apical
export) compared with Gln feeding (up to 20% of apical
export). Irrespective of the 14C-amino compound applied,
1392 Geûler et al.
Table 2. Export of
14
C- and
15
N-labelled Asp and Gln taken up by the fed needles
After incubation the whole twig was harvested and analysed for radiolabel as described in Table 1. Radioactivity or 15N enrichment in all twig
fractions (with the exception of the fed needles) was at set at 100% (total uptake). Radioactivity or 15N enrichment determined in the application
segment was de®ned as the non-allocated portion of total tracer amount taken up. Radioactivity or 15N enrichment in the basal and apical
segments was attributed to long-distance transport (export). In girdling experiments, transport of 14C and 15N into apical direction was subdivided
into the portion up to and beyond the girdle. Experiments were performed in three independent replicates (n=3).
Tracer
applied
Non-girdled
controls
Gln
Asp
Girdled twigs
Gln
Asp
Tracer remaining in
the application
segments (% of
total uptake)
Export of tracer to
basal segments (%
of total uptake)
Export of tracer to
apical segments
(% of total uptake)
Apical export of tracer
into segments up to the
girdle (% of total
uptake)
Apical export of tracer
into segments beyond
the girdle (% of total
uptake)
14
14
14
14
14
C
15
N
C
15
N
15
C
N
88.462.1
a; aa
33.664.7
b; a
83.369.8
a; a
40.2610.3
b; a
2.162.8
a; a
8.268.0
a; a
4.463.2
a; a
12.567.2
a; a
9.563.5
a; a
58.2612.7
b; a
12.364.2
a; a
47.363.3
b; b
93.863.2
a; aa
76.5612.7
a; a
88.769.68
a; a
84.867.98
a; a
1.962.3
a; a
4.564.2
a; a
3.262.2
a; a
7.163.6
b; a
4.364.5
a; a
19.0612.9
a; a
8.165.5
a; a
8.164.5
a; b
C
2.964.4
a; a
4.265.2
a; a
15
N
4.363.1
a; a
2.163.2
a; a
a
Different letters indicate signi®cant differences (1) between the different amino acids fed (a, b), and (2) among
at P <0.05.
radiolabel was determined in wood, bark and needles of all
twig segments apical to the application site (Figs 1, 2).
With the exception of the young sprouts, the highest
portion of radiolabel was found in bark tissues. In the
experiments shown in Figs 1 and 2, the relative contribution of transport of radiolabel from Asp to basal tissue
(2.5% of total transport, i.e. 23.5 pmol Asp) was signi®cantly lower compared with apical transport (67.2% of
total transport, i.e. 621.8 pmol Asp), but was still higher
than basal transport of radiolabel from Gln (0.3% of total
transport, i.e. 3.9 pmol Gln).
Effect of girdling on export and distribution of
15
N tracer
14
C and
Girdling treatment signi®cantly increased the remaining
portion of 14C and 15N in the application segment of the
twig in the Asp treatment (Table 2) and reduced export of
14
C and 15N out of the application segment compared with
non-girdled controls. The transport of tracer to apical
segments was reduced, while the absolute amount of
14
C-Asp transported in a basal direction was increased as a
consequence of girdling (Fig. 4). The new developing
sprouts of girdled twigs no longer showed high enrichment
of the 14C tracer applied (Figs 2, 4). In the Gln incubation
treatments girdling enhanced the remaining portion of 14C
and 15N only slightly (Table 2). Thus, the export of 14C and
15
N to apical segments was also only slightly reduced
compared with non-girdled controls (Table 2; Figs 1, 3).
By contrast with Asp, the new developing needles of the
terminal sprouts remained the main sink (up to 39% of
apical export) of the 14C exported in the 14C-Gln feeding
14
C and
C
1.460.8
a; a
14.8611.8
b; a
15
N
3.863.2
a; a
6.063.2
a; a
15
N feeding experiments (a, b)
approach (Figs 1, 3). Accumulation of radiolabel in the
®rst twig segment apical to the application site was neither
observed for Gln nor for Asp (Figs 3, 4).
Girdling treatment enforced loading of the fed tracer
into the xylem before the girdle. As a consequence, in the
®rst twig segments beyond the girdle, relatively higher
labelling was found in wood tissue than in bark and leaves
(Figs 3, 4). Still less label was found in these tissues in
absolute terms than in non-girdled controls (Figs 1, 2).
Irrespective of feeding 14C-Gln or 14C-Asp, radiolabel
was observed in all twig tissues analysed apical to the
girdle, including the bark (Figs 3, 4). Apparently,
radiolabel transported in the xylem was redistributed into
the phloem beyond the girdle. Table 3 shows that this
redistribution was comparable between 14C and 15N,
independent of the amino acid applied. 14C and 15N tracer
from Asp that had passed the girdle, was mainly
determined in wood tissues (apical segment 3 in Fig. 4;
Table 3), whereas most of the tracer from Gln was found in
needles of the new developing sprouts (Fig. 3; Table 3).
Differences in the relative distribution patterns of radiolabel between wood, bark and needles in the apical
segments beyond the girdle (Figs 3, 4) compared with
apical segments of non-girdled controls (Figs 1, 2) is
supposed to be caused by the fact that phloem transport in
an apical direction has been prevented by girdling.
Discussion
In the present study 14C- and 15N-labelled Gln or Asp were
fed to 2- or 3-year-old needles of spruce trees. The feeding
Phloem±xylem exchange of amino acids 1393
Fig. 1. The distribution of the total amount of radiolabel along a non-girdled twig of spruce fed 14C-Gln. Application of radiolabel was carried out
via the cut tips of two 2- or 3-year-old needles. After incubation, the twig was excised from the stem and divided into segments of 2±6 cm length.
Each twig segment was subdivided into needles, bark, and wood, weighed and frozen in liquid nitrogen. For the preparation of 14C analysis,
samples were ground and extracted with 0.1 N HCl. Radiolabel was determined in the soluble and insoluble fraction separately by liquid
scintilation counting (LSC). The ®gure shows the absolute amounts of radiolabel in the twig segments analysed in needles (white bars), bark
(black bars), and wood (hatched bars). As about 9065% of the radiolabel was found in the soluble fraction, data given in the graph are the sum of
both fractions. Data given are taken from one out of three experiments with similar results from each.
Fig. 2. The distribution of the total amount of radiolabel along a non-girdled twig of spruce fed 14C-Asp. For details of the application of
radiolabel and analysis of the samples see the legend to Fig. 1. The ®gure shows the absolute amounts of radiolabel in the twig segments analysed
in needles (white bars), bark (black bars), and wood (hatched bars). As about 9065% of the radiolabel was found in the soluble fraction, data
given in the graph are the sum of both fractions. Data given are taken from one out of three experiments with similar results from each.
technique applied was previously used in experiments with
sulphur compounds (Schupp et al., 1992; Schneider et al.,
1994; Blaschke et al., 1996). The total uptake of 14Clabelled Asp and Gln amounted to 4.661.6 nmol (i.e. 24±
26% of the tracer applied) or 4.161.8 nmol (i.e. 22±25% of
the tracer applied), respectively. Comparable relative
uptake rates (c. 25%) were observed for the 15N compounds applied in the present study even though c. 4-fold
higher concentrations were used in the incubation solutions. Hence, the relative uptake rates were in the same
range as determined for glutathione (Schupp et al., 1992:
max. 13%; Schneider et al., 1994: max. 16%; Blaschke
1394 Geûler et al.
Fig. 3. The distribution of the total amount of radiolabel along a girdled twig of spruce fed 14C-Gln. For details of the application of radiolabel
and analysis of the samples see legend to Fig. 1. In addition, the twig fed radiolabel was girdled in order to enforce the exchange of Gln from
phloem to xylem. The ®gure shows the absolute amounts of radiolabel in the twig segments analysed in needles (white bars), bark (black bars),
and wood (hatched bars). As about 9065% of the radiolabel was found in the soluble fraction, data given in the graph are the sum of both
fractions. Data given are taken from one out of three experiments with similar results from each.
Fig. 4. The distribution of total amount of radiolabel along a girdled twig of spruce fed 14C-Asp. For details of the application of radiolabel and
analysis of the samples see legend to Fig. 1. The ®gure shows the absolute amounts of radiolabel in the twig segments analysed in needles (white
bars), bark (black bars), and wood (hatched bars). As about 9065% of the radiolabel was found in the soluble fraction, data given in the graph are
the sum of both fractions. Data given are taken from one out of three experiments with similar results from each.
et al., 1996: max. 37%). Incubation was performed during
illumination, since Smith and Cheema (1985) observed
that the uptake rate of the tracers applied increased in the
presence of sucrose.
Feeding was performed during the early development of
the current-year needles. During this time, the nitrogen
demand is relatively high, whereas nitrogen uptake from
the soil is supposed to be low (Millard, 1994; Geûler et al.,
1998a). As a consequence the nitrogen demand has to be
met by mobilization from older needle generations. Since
9065% of the radiolabel fed was found in the acid-soluble
fraction of all bark, wood and needle tissues analysed, the
tracer applied seems to be incorporated into the mobile
pool of organic nitrogen compounds mobilized from
storage pools in needle tissues.
Table 2 shows that the export of 14C label to the apical
and basal segments was comparable to the 15N transport.
Hence, there is clear evidence that the amino acids fed are
Phloem±xylem exchange of amino acids 1395
Table 3. Distribution of
treatment
14
C and
15
N between wood, bark and needles beyond the girdle in spruce subjected to the girdling
The total amount of apical export of 14C and 15N to segments beyond the girdle is set at 100% and the amount of 14C and 15N in the needles,
bark and wood of all segments beyond the girdle related to that total amount. Experiments were performed in three independent replicates (n=3).
Tracer
applied
Gln
Asp
a
Tracer in the needles (% of total
export apical export beyond the
girdle)
Tracer in the bark (% of total
export apical export beyond the
girdle)
Tracer in the wood (% of total
export apical export beyond the
girdle)
14
14
15
14
24.869.8 A
26.867.9 A
22.2617.6 A
32.6615.2 A
17.968.2 a
45.368.2 a
C
57.3612.2 aa
27.969.8 a
15
N
55.5618.7 a
30.3616.5 a
C
N
C
15
N
22.3614.9 a
37.1618.9 a
Different letters (a, A, a) indicate signi®cant differences between 14C and 15N application at P < 0.05.
the compounds that are transported within the plant.
However, especially in the case of Gln, metabolic
conversion taking place before or during transport cannot
totally be excluded. Since only the a-amino group of Gln
was labelled, it is also possible that Glu or a mixture of Gln
and Glu are exported out of the application segment.
A signi®cant portion of the 14C-tracer fed was found in
the bark tissue close to the application site (Figs 1, 2). This
®nding indicates that both amino compounds were
exported out of the needles by phloem transport. Similar
results were obtained by feeding 35S-cysteine (Blaschke
et al., 1996), 35S -g-glutamylcysteine (Blaschke et al.,
1996), and 35S-glutathione (Schupp et al., 1992; Schneider
et al., 1994; Blaschke et al., 1996). 14C tracer applied as
Asp was mainly transported to the new developing needles
of the current-year sprouts (Table 2; Fig. 2); the transport
of the tracer applied as Gln out of the application segment
was only one-®fth as compared to Asp (Table 2; Fig. 1).
Since the pool of Gln in the xylem as well as in the phloem
of spruce twigs is in general about four to ®ve times higher
than Asp (Schneider et al., 1996; Weber et al., 1998), the
difference in transport between these compounds may
re¯ect differences in pool sizes. Alternatively, a preferential uptake of Gln by bark parenchyma cells or a
preferential phloem loading of Asp could be responsible
for this phenomenon. The latter explanation is consistent
with a function of Asp in supporting current sprout growth
and a function of Gln in regulating nitrate uptake by the
roots (Geûler et al., 1998a, b, c). Strong differences in
phloem mobility and transfer between xylem and phloem
among different amino compounds have also been
observed for lupin (Atkins, 2000).
Apical to the application site, tracer was found in wood,
bark and needle tissues (Figs 1, 2). However, the present
analyses neither distinguished wood parenchyma cells
from xylem vessels nor bark parenchyma cells from sieve
tubes. In order to prevent apical transport via the phloem, a
small ring of bark was peeled from the twig apical to the
application site. In twigs fed Gln (Fig. 3; Table 2) and Asp
(Fig. 4; Table 2) 14C and 15N tracer was determined in twig
tissues beyond the girdle segment. For long-distance
transport through the girdled segment it can be assumed
that Asp as well as Gln were loaded from the phloem into
the xylem. Such an exchange has been reported for
glutathione in spruce (Schneider et al., 1994) and beech
trees (Herschbach and Rennenberg, 1995). The present
experiments with non-girdled twigs show that a major
portion of tracer in tissues apical to the application site was
determined in bark tissues (Figs 1, 2). In girdled twigs, the
total amount of tracer in all twig segments apical to the
application site was found to be reduced and, in addition,
the dominance of tracer tended from bark to wood tissues
(Figs 3, 4). This ®nding indicates that mobilized nitrogen
compounds can be transported to the current-year sprouts
by both xylem and phloem transport as observed.
Apparently, the cycling and recycling model of mineral
nutrition in plants (Marschner et al., 1997; Geûler et al.,
1998c) that proposed transport in the xylem exclusively in
an apical direction and transport in the phloem exclusively
in a basal direction can not readily be transferred to trees.
The present results clearly show that nitrogen compounds
can also be allocated via the phloem in an apical direction.
This view is supported by the investigation of seasonal
changes in xylem sap and phloem exudate composition of
spruce (Geûler et al., 1998b; Weber et al., 1998).
Beyond the girdle, 14C and 15N tracer was not only
found in the wood, but also in bark tissues (Table 3; Figs 3,
4). As the transport of tracer in a basal direction was
negligible (Figs 1, 2), radiolabel and 15N enrichment
determined in bark tissues beyond the girdle must be a
result of reallocation of 14C- and 15N-labelled compounds
from the xylem to bark parenchyma cells or sieve tubes of
the phloem. Since the distribution between needles, bark
and wood was comparable for 14C and 15N (Table 3) it is
concluded that the amino compounds fed are subject to
such a reallocation. This is in contrast to ®ndings by Atkins
et al. (1980), who observed a high percentage of direct
phloem to xylem transfer in lupins without metabolic
interconversion for Gln but not for Asp. There the amino
group of transferred Asp was found to be utilized to form
other amino compounds.
As the existence of amino acid transporters in stem
phloem is indicated in other publications (Hirner et al.,
1998), an exchange of Gln and Asp between phloem and
1396 Geûler et al.
xylem and vice versa during long-distance transport in
spruce is likely. Such a bidirectional exchange was already
documented for sulphate and phosphate (Biddulph, 1956),
sodium and potassium (Jeschke et al., 1987), chloride,
calcium and magnesium (Jeschke and Pate, 1991) as well
as for glutathione, L-cysteine and g-glutamylcysteine
(Schneider et al., 1994; Herschbach and Rennenberg,
1995; Blaschke et al., 1996) and amino compounds
(Atkins et al., 1980; Atkins, 2000). It may explain the
vertical gradients of these compounds and the seasonal
changes of these gradients observed in the ®eld (Schupp
et al., 1992; Geûler et al., 1998b; KoÈstner et al., 1998;
Weber et al., 1998).
Acknowledgements
The authors thank C Herschbach and L Blaschke for their
introduction into methods applied in this study, and Karl Merz,
Siegrid Hagenguth, JuÈrgen Kreuzwieser, and Cristian Cojocariu for
providing the plant material used in the present study. Financial
support by the Bundesministerium fuÈr Bildung und Forschung
(BMBF, contract no. BEO/51±0339614) and by the `Projekt
EuropaÈisches
Forschungszentrum
fuÈr
Mabnahmen
zur
Luftreinhaltung' (PEF, contract no. PEF 1.93.002) is gratefully
acknowledged.
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