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Journal of Experimental Botany, Vol. 54, No. 386, pp. 1389-1397, May 1, 2003
© 2003 Oxford University Press

Bidirectional exchange of amino compounds between phloem and xylem during long-distance transport in Norway spruce trees (Picea abies [L.] Karst)

Received 21 October 2002; Accepted 20 January 2003

Arthur Geßler, Paul Weber, Stephan Schneider and Heinz Rennenberg1,

Institut für Forstbotanik und Baumphysiologie, Professur für Baumphysiologie, Albert-Universität Freiburg, Georges-Köhler-Allee Geb 053/054, D-79110 Freiburg i. Br., Germany

1 To whom correspondence should be addressed. Fax: +49 761 203 8302. E-mail: Heinz.rennenberg{at}ctp.uni-freiburg.de


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
14C-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 long- distance 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.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
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; Geßler et al., 1998a). In particular, the share of ammonium versus nitrate availability is of significance (Downs et al., 1993), because ammonium uptake by the roots of trees is often preferential to the uptake of nitrate (Marschner et al., 1991; Geßler 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 Geßler, 1999; Geßler et al., 2000, 2002) and NH3 (Pérez-Soba et al., 1994; Fangmeier et al., 1994; Geßler 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 Geßler, 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 consequence, rates of nitrogen uptake from the soil are generally low (Geßler et al., 1998a), mobilization of nitrogen compounds from storage tissues is of particular significance 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 (Geßler 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; Geßler 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 (Geßler et al., 1998b; Weber et al., 1998). From this finding it was hypothesized that these amino acids undergo a bidirectional phloem/xylem exchange during long-distance transport (Geßler 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
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
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 field 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 17±1 °C and 55±10% RH during illumination with 300±20 µmol m–2 s–1 PAR, and 13±1 °C and 70±15% 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 influence 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 14C 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 ({alpha}-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 µl 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, specific activity of 244.0 mCi mmol–1) or 14C-Asp (440–480 nmol ml–1 H2O in an ethanol/water mixture of 2:98, specific 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 ({alpha}-amino 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 artificial light (Osram Powerstar HQI-T 400W/DH, Osram, Germany) of about 500–620 µmol 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.

14C- and 15N-analysis
Analysis of 14C in plant sections was carried out by a modification 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 µl of the supernatants were transferred into scintillation vials (Mini Poly Q-Vials, Beckmann Instruments, München, 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 fluid (OptiPhase, Wallace, Turku, Finland) and the radioactivity was determined by liquid scintillation counting (LSC, Beckman LS 7500, Beckman, München, 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 µl isopropanol, transferred into scintillation vials (20 ml Mini Poly Q-Vials, Beckmann Instruments, München, Germany), and bleached twice with 300 µl 30 Vol% H2O2 for 1 d at RT each. Pellets of needle extracts were bleached with 200 µl 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 fluid (OptiPhase, Wallace, Turku, Finland) was added to all samples. Radioactivity was determined by LSC (Beckman LS 7500, Beckman, München, Germany) at efficiencies 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 79.4±18.0%.

For determination of 15N abundance (atom%) and total N, samples of the oven-dried plant material were ground with a ball mill into a fine 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 Conflo 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 15N enrichment determined in all plant tissues analysed, excluding the fed needles, and was set as 100%. It was subdivided into (1) the 14C 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, 14C-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. Significance 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 (Zöfel, 1988).


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Uptake of the labelled Gln and Asp
Individual needles of spruce trees were fed 14C-Gln, 14C-Asp, 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 (90±5%) in all twig sections analysed, whereas a minor portion of radioactivity was determined within the acid-insoluble fraction (10±5%). 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).


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  		Table 1. Absorption time, total uptake, and recovery of 14C- and 15N-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.
 
Export and distribution of 14C and 15N 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.


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  		Table 2. Export of 14C- and 15N-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 defined 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).
 
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, 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 significantly 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).



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  		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 figure shows the absolute amounts of radiolabel in the twig segments analysed in needles (white bars), bark (black bars), and wood (hatched bars). As about 90±5% 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.

 


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  		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 figure shows the absolute amounts of radiolabel in the twig segments analysed in needles (white bars), bark (black bars), and wood (hatched bars). As about 90±5% 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.

 
Effect of girdling on export and distribution of 14C and 15N tracer
Girdling treatment significantly 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 14C 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 14C-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 15N 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 approach (Figs 1, 3). Accumulation of radiolabel in the first twig segment apical to the application site was neither observed for Gln nor for Asp (Figs 3, 4).



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  		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 figure shows the absolute amounts of radiolabel in the twig segments analysed in needles (white bars), bark (black bars), and wood (hatched bars). As about 90±5% 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.

 


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  		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 figure shows the absolute amounts of radiolabel in the twig segments analysed in needles (white bars), bark (black bars), and wood (hatched bars). As about 90±5% 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.

 
Girdling treatment enforced loading of the fed tracer into the xylem before the girdle. As a consequence, in the first 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 radio label 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.


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  		Table 3. Distribution of 14C and 15N between wood, bark and needles beyond the girdle in spruce subjected to the girdling treatment 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).
 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
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 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 14C-labelled Asp and Gln amounted to 4.6±1.6 nmol (i.e. 24–26% of the tracer applied) or 4.1±1.8 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 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; Geßler et al., 1998a). As a consequence the nitrogen demand has to be met by mobilization from older needle generations. Since 90±5% 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 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 {alpha}-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 significant portion of the 14C-tracer fed was found in the bark tissue close to the application site (Figs 1, 2). This finding 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 -{gamma}-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-fifth 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 five times higher than Asp (Schneider et al., 1996; Weber et al., 1998), the difference in transport between these compounds may reflect 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 (Geßler 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 finding 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; Geßler 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 (Geßler 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 findings 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 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 {gamma}-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 field (Schupp et al., 1992; Geßler et al., 1998b; Köstner 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, Jürgen Kreuzwieser, and Cristian Cojocariu for providing the plant material used in the present study. Financial support by the Bundesministerium für Bildung und Forschung (BMBF, contract no. BEO/51–0339614) and by the ‘Projekt Europäisches Forschungszentrum für Maßnahmen zur Luftreinhaltung’ (PEF, contract no. PEF 1.93.002) is gratefully acknowledged.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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