Interactions of NH 4 + and L-glutamate with N0 3 transport processes of non-mycorrhizal Fagus sylvatica roots

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1 Journal of Experimental Botany, Vol. 48, No. 312, pp , July 1997 Journal of Experimental Botany Interactions of NH 4 + and L-glutamate with N0 3 transport processes of non-mycorrhizal Fagus sylvatica roots Jiirgen Kreuzwieser 1, Cornelia Herschbach 1, Ineke Stulen 2, Peter Wiersema 3, Willem Vaalburg 4 and Heinz Rennenberg 1 ' 5 1 Albert-Ludwigs-UniversMt Freiburg, Institut for Forstbotanik und Baumphysiologie, Professur for Baumphysiologie, Am Flughafen 17, D Freiburg i. Br., Germany 2 Department of Plant Biology, Biological Center, University of Groningen, 9750 AA Haren, The Netherlands 3 Isotope Laboratory, University of Groningen, 9750 AA Haren, The Netherlands 4 PET Center, University Hospital, 9713 EZ Groningen, The Netherlands Received 5 November 1996; Accepted 10 March 1997 Abstract The processes of NO 3 uptake and transport and the effects of NH + 4 or L-glutamate on these processes were investigated with excised non-mycorrhizal beech [Fagus sylvatica L.) roots. NO 3 ~ net uptake followed uniphasic Michaelis-Menten kinetics in a concentration range of 10//M to 1 mm with an apparent K m of 9.2 ftm and a V mmx of 366 nmol g~ 1 FW h" 1. NH + 4, when present in excess to NO 3, or 10 mm L-glutamate inhibited the net uptake of N0 3 ~. Apparently, part of NO 3 taken up was loaded into the xylem. Relative xylem loading of N0 3 ranged from 3.2 ±1.6 to 6.4 ±5.1% of NO 3 " net uptake. It was not affected by treatment with NH + 4 or L-glutamate. 1B N/ 13 N double labelling experiments showed that N0 3 efflux from roots increased with increasing influx of N0 3 " and, therefore, declined if influx was reduced by NH + 4 or L-glutamate exposure. From these results it is concluded that N0 3 net uptake by non-mycorrhizal beech roots is reduced by NH + 4 or L-glutamate at the level of influx and not at the level of efflux. Key words: Nitrate transport, net uptake, influx, efflux, ammonium, Fagus, Fagaceae. Introduction N is available to forest trees in the soil mainly as NO 3 ~ and NH^. The actual amount and relative proportion of 8 To whom correspondence should be addressed. Fax: Abbreviation: Bis-tris propane, 1,3-bis[tris(hydroxymethyl)methyl-amino]-propane. these ions in forest soils are highly dependent on soil processes such as immobilization, mineralization, nitrification, and denitrification, and environmental factors such as temperature, soil moisture, and N deposition (Haynes and Goh, 1978). NO 3 " concentrations in natural soils may vary by 3-4 orders of magnitude (Jackson and Caldwell, 1993). In forest stands, for example, Marschner et al. (1991) determined NO 3 ~ concentrations from c. 100 ftm to c. mm in the soil solution. This variation may partially be due to the patchiness of landscapes resulting in a close vicinity of many forests in central Europe to intensively used agricultural land. These forests are exposed to high loads of N from wet and dry deposition, predominantly as NH4/NH3 (G6ttlein and Kreutzer, 1991), and as a consequence, to excessive amounts of NH^ N in the soil. In many herbaceous plants NH^ strongly retards net uptake of NO3" (Lee and Drew, 1989). Similar results have been obtained in laboratory experiments (Marschner et al., 1991; Kamminga-van Wijk and Prins, 1993) or field studies (Rennenberg et al, 1996) with conifers. However, Stadler et al. (1993) described a positive effect of NH on NO 3 ~ uptake of deciduous trees, namely ash and oak. NO 3 ~ uptake of higher plants can proceed as a symport with protons (Glass and Siddiqi, 1995) or an antiport with OH" orhco 3 ~ (Touraineef a/., 1994). It is regulated by the N demand for growth (Clement et al., 1978) and/or by the N status of the whole plant (Touraine et al., 1994; Glass and Siddiqi, 1995). Since the rate of Oxford University Press 1997

2 1432 Kreuzwieser et al. net uptake of NO 3 is the balance between NO 3 influx and efflux processes, decreased net uptake of NO3" in the presence of NH^ may be mediated either by inhibition of NO 3 ~ influx or by stimulation of NO 3 " efflux (Aslam et al., 1994). The present study with non-mycorrhizal roots of young beech (Fagus sylvatica L.) trees was performed to address this question. For this purpose, experiments were carried out in which influx and efflux of NO^ were analysed in roots exposed to radioactive ( 13 NO 3 ~) and nonradioactive (''NO^) tracers. Non-mycorrhizal roots were chosen because they represent a relative simple experimental system and are thought to contribute considerably to NO 3 " uptake of trees (Marschner et al., 1991). Materials and methods Plant material For germination, beech nuts (Fagus sylvatica L.) were shelled, surface-sterilized in a suspension containing 5g I" 1 of the fungicide Polyram-Combi (BASF, Ludwigshafen, Germany), 100 mg I" 1 tetracycline and lml I" 1 Tween 80, and were transferred to plastic pots (18 cm diameter). An autoclaved mixture of perlite and blond peat (2:1, v/v) served as the substrate. Growing seedlings were supplied twice a week with a modified 1/5 MS medium (Murashige and Skoog, 1962) containing mm NO 3 " and mm NH.J". The plants were grown in a climate chamber (HPS 1500, Heraeus VStsch, Hanau, Germany) for weeks under long day conditions (16 h light) at day and night temperatures of 20 C and 15 C and relative humidities of 70% and 80%. Photosynthetic photon flux density (PPFD) during growth was kept at 140 to m~ 2 s" 1 at the top of the plants. This PPFD was chosen since young beech trees are adapted to growth in forest understoreys; as a consequence, young beech trees are shade-tolerant and highly sensitive to full sunlight (Ellenberg, 1986). The PPFD provided enabled photosynthesis to proceed at c. 70% of the rate at light saturation (Fig. 1) as determined with a LCA4 (ADC, Hoddeston, UK). Under these conditions, addition of sucrose did not increase NO 3 "~ uptake rates by the roots (Table 1). As stated by Butcher and Street (1964) excised roots of higher plants are able to take up sucrose and to use it for growth as a C-source. Apparently, NO^ uptake was not limited by C and energy availability. For preincubation the substrate was carefully removed from P.A.R. ( imol m s ) Fig. 1. Light dependency of the rate of photosynthesis of young beech trees. Beech trees were grown for weeks in climate controlled chambers under long day conditions with a 16 h photopenod. The photosynthetic active radiation amounted to ftmol m~ 2 s~' Photosynthetic rate was determined at the light intensities indicated using a LCA4 (ADC, Hoddeston, UK). the roots and the trees were exposed for 72 h to different, aerated 1/5 MS media as required for the experiments. Uptake and transport experiments with intact plants Trees were preincubated in 1/5 MS media containing either + mm NO 3 - without NH 4 or mm NO 3 " plus mm NH^. These N concentrations were the same as supplied during the growth on solid substrate. After preincubation the plants were transferred into Erlenmeyer flasks containing 50 ml aerated transport medium, consisting of 5.0 mm bis-tris propane (ph 6) and mm CaCl 2, supplemented with 0.1 mm NO 3 ~ and 0.0 or mm NH^. This NO3" concentration was chosen in order to determine net rates of NOf uptake of the inducible, high affinity transport system rather than the constitutive low affinity transport system active at high external NOf concentrations. The incubation lasted for 6 h. The ph remained constant during this period. Net uptake rate was calculated from the Table 1. Net uptake rate of NO 3 by excised roots and intact beech tree seedlings Seedlings were preincubated for 72 h in liquid 1/5 MS media containing either mm NO 3 " or mm NO 3 "/0 5 mm NH^. Excised roots were incubated in root chambers and exposed to 0.1 mm "NO 3 with or without mm NH< + in the uptake compartment. After 6 h of incubation roots were harvested and 15 N was analysed. Intact plants were incubated in 50 ml aerated media of the same composition used for excised roots and were harvested after 6 h Fresh weight of the fine roots was determined and net uptake was calculated from the depletion of external NO 3 " in the media. Data shown are means (±SD) of the number of independent experiments indicated. Statistical analysis was performed using LSD under ANOVA. Different indices show significant differences at P-^0.05. Nitrogen supply (mm) during Net uptake rate of NO 3 (mmol g" 1 FWh"" 1 ) Preincubation Incubation Excised roots Intact plant NO 3 " NH 4 + NO; NH; Sucrose +1% Sucrose ±102a(n = 4) 27±8b(n = 4) 225±44a(n = 5) n.d. 230±127a(n = 9) 33±42b(n = 7)

3 depletion of NO 3 in the media by analysis of 0.1 ml ahquots, which were taken in hourly intervals. NOf was measured by a HPLC system consisting of a Dionex GP40 Pump (Dionex, Idstein, Germany), a Sykam LCA-A03 polystyrole-column (lofim, 4.6x125 mm, Sykam GmbH, Gilching, Germany), a UV-Vis-spectrophotometric detector (210 nm; Shimadzu SPP-6AV, Shimadzu, Duisburg, Germany) and a Shimadzu C-R3A integrator. A solution containing 50 mm NaCl was used as eluent at a flow rate of 1 ml min" 1. NO^~ was detected c. 7 min after injection. Net uptake and transport experiments with excised roots For transport experiments with excised roots, the modification of the root chamber of Pitman (1971) described by Herschbach and Rennenberg (1991) was used. Root chambers were divided into three compartments by Plexiglas bars. Roots of preincubated trees were excised and incubated in the root chamber with the tips placed within the uptake compartment. Vaseline was used as a seal between the roots and the Plexiglas dividers. This compartment contained 200 ml transport medium (see above). The buffer compartment, containing 30 ml transport medium plus % agar (high gel-strength Agar Agar, Serva, Heidelberg, Germany), prevented contamination of the exudation compartment by solutes leaking out of the uptake compartment. The exudation compartment contained the cut ends of the roots in 15 ml transport medium. Incubation was started by adding 15 NO;f and NH.J" from stock solutions into the uptake compartment to obtain final concentrations of IOJIM to mm 15 NO 3 ~ (99 atoms% 15 N) and 0 or mm NH + 4. After 115 min 13 NO 3 " (c. 0.3 GBq per chamber) was added for 300 s. Subsequently, the roots were washed three times for 45 s with transport medium lacking NO3". Appreciable amounts of radioactivity were not detected in the final washing. Roots were cut and removed separately from the chamber compartments for tracer analysis. Tracer analysis For 15 N-analysis, the samples were oven-dried, milled, redried at 80 C for 24 h and placed in cleaned tin cups. The cups were loaded onto the automatic sample change of a Roboprep combustion system and the gaseous N 2 produced was analysed in a mass spectrometer (Europe, Crewe, UK). The 13 NO3~ was produced in the Scanditronix MC-17V cyclotron of the Positron Emission Tomography Center of the Groningen University Hospital by the 16 O(p,a) 13 N nuclear reaction using water as target material (Vaalburg and Paans, 1983). Radioactivity was determined by counting the 11 kev annihilation peak for 40 s using a sodium iodide scintillation crystal. The data obtained were corrected for background, decay and 18 F contamination (Ter Steege, 1996). Extraction ofxylem sap Xylem sap was extracted from the shoots of young beech trees by a modification of the protocol described by Scholander et al. (1965). Trees were cut at the epicotyl. Bark and cambium were removed for a length of 30 mm from the cut end. To reduce contamination by cellular compounds, the cut end was rinsed with distilled water. The trees were fitted into the pressure chamber and xylem sap was extracted by the procedure described by Rennenberg et al. (1996). The xylem sap collected was immediately frozen in liquid N 2 and stored at 80 C until analysis. NO^ in the xylem sap was analysed by HPLC analysis as described above. As a control of symplastic contamination, the ATP content of the extracted xylem sap was determined luminometncally with an ATP Bioluminescence Assay Kit HS Nitrate transport of beech 1433 II (Boehringer Mannheim, Germany). Symplastic contamination was found to be negligible ( %) based on the calculation provided by Schupp (1991). Data analysis From the 15 N data, net uptake of NO 3 ~, xylem loading and relative xylem loading of NO-,"", i.e. the portion of NO 3 ~ taken up that was loaded into the xylem, were calculated as previously described (Herschbach and Rennenberg, 1991). Total influx of NO;f was derived from ^NO^ uptake experiments. The efflux of NO 3 ~ was calculated as the difference between the net uptake rate of NO 3 ~ measured with 15 NO;f and the influx measured with 13 NO3~. Significant differences of independent experiments were determined by LSD under ANOVA and are indicated in the tables and figures. Results and discussion Net uptake rate of A/O 3 ~ Net uptake rate of NO^ was studied with intact plants and with excised roots of beech (Table 1). At 0.1 mm NO3", the rates of uptake did not differ significantly between these approaches. Addition of mm NH^ reduced net uptake of NO3" in both experimental systems by 85-90% (Table 1). Excised roots can grow on sucrose as a C-source (Butcher and Street, 1964). Since addition of sucrose did not improve the rate of NO 3 ~ net uptake by excised roots C- and energy-supply seemed to be sufficient under the experimental conditions applied. It can therefore be assumed that experiments on NO 3 ~ fluxes with excised beech roots provide reliable information on NO3" uptake rates by intact plants. In earlier studies excised roots were successfully used to analyse SO^" uptake and xylem loading of beech (Kreuzwieser et al., 1996), oak (Seegmtiller et al., 1996) and tobacco (Herschbach and Rennenberg, 1991) using the same experimental system. Experiments with excised beech roots showed that 15 NO 3 ~ is not only taken up (Fig. 2A), but is also loaded into the xylem in considerable amounts (Fig. 2B). At a concentration of 1 mm, both processes showed linear correlation with incubation time, suggesting that a sufficient energy supply was maintained in the roots during the 6 h period of investigation. Net uptake of 1 mm NO 3 " (Fig. 2A) and xylem loading of NO 3 ~ (Fig. 2B) also increased linearly with time of incubation in the presence of mm NH 4 +. The rate of net uptake of NO 3 ~ was similar in the presence or absence of mm NH^ (Fig. 2A). Since inhibition of NO 3 ~ net uptake in the presence of mm NH^ was observed when excised beech roots were exposed to 0.1 mm NO 3 ~ (Table 1), it appears that a surplus of NOf over NH^ is able to prevent inhibition of NOf net uptake. Similar results were obtained by Marschner et al. (1991) in laboratory studies with Norway spruce. Xylem loading of NO3" appeared to be stimulated in the presence of

4 1434 Kreuzwieser et al. O _ c % i so T x r'" Y = x / 0.99S5 time (h) 1 Y x r' t- Y x r** Fig. 2. Relationship between uptake (A) and xylem loading (B) of 15 NOf with time of incubation. Beech trees were preincubated for 72 h in a liquid 1/5 MS medium containing either l.5mm NO 3 " ( ) or l.o mm NO 3 ""/ mm NH 4 + (A). Excised roots were incubated in root chambers and were exposed to l.o mm 15 NO 3 with (A) or without ( ) mm UHJ in the uptake compartment. At the times indicated roots were harvested and analysed for "N by mass spectroscopy. Data shown are means (±SD) of 2-9 independent experiments with 4-6 roots each. Significant differences of the slopes obtained by linear regression are indicated by different letters for each plot. (Fig2B), but due to the high scattering of the data of NH4 treatments the curves did not differ significantly. The rate of NO 3 ~ net uptake was dependent on the external concentration of NO 3 ". It showed uniphasic Michaelis-Menten kinetics in the range from 10 ^M to mm NO3" (Fig. 3A). In order to determine apparent K m and K ma, of NO 3 ~ net uptake, the graph shown in Fig. 3A was linearized according to Hanes (1932). An apparent K m of 9.2 MM and a V^ of 366 nmol g~ 1 FW h" 1 (c. 3^mol g" 1 DW h" 1 ) was calculated (Fig. 3B). Comparable data from other studies on NO 3 ~ transport of woody plants are scarce. Kamminga-van Wijk and Prins (1993) analysed the uptake kinetics of nonmycorrhizal roots of Douglas fir and found similar values for apparent K m (17^M) and K max (5fxmol g" 1 DW h" 1 ) as in the present study. V mmx of non-mycorrhizal maritime pine was in the same order of magnitude (550 nmol g" 1 FW h" 1 ; Plassard et al., 1994) as beech, whereas the apparent K m was distinctly higher (120 ^M). However, K m of barley plants ranged from 7 ^M (Aslam et al., 1992) to 110/xM (Rao and Rains, 1976). Net uptake of NO 3 ~ by non-mycorrhizal Douglas fir supplied with 700 nm NO 3 " amounted to 2 ^mol g" 1 DW h" 1 (Rygjewicz and Bledsoe, 1986). In a recent study Lajtha (1994) investigated uptake of various deciduous trees in the range from fim NO 3 ~. For beech roots the author found one order of magnitude higher rates of NO 3 " net uptake (c. 20,xmol g" 1 DW h" 1 ) than in the present study at all concentrations measured. Such high rates of NO 3 " net uptake are usually found for herbaceous plants. For example, Rao et al. (1993) calculated V^ values of 2-7 ^mol g" 1 FW h" 1 for different legumes and cereals and apparent K m -values of c. 100 ^M. The high rates of NOf uptake of Lajtha (1994) may be due to the experimental system used. This is assumed since the author did not obtain any concentration dependence of NO 3 " uptake, a phenomenon observed for almost all energy dependent processes in biology. The low rates of NO 3 " net uptake found in the present study are not surprising when the relatively slow growth of beech compared to herbaceous plants is considered. Apparently, the uptake of NO 3 " is down regulated when NH^ is present in excess to NO 3 " (Fig. 3A). NH^ therefore may be preferred as a N source as compared to NO 3 ~~ under these conditions. Preferential net uptake of NH + 4 versus NO 3 " has also been described by Finlay et al. (1989) for mycorrhizal, and in a recent study by Ge/31er et al. (unpublished results), for non-mycorrhizal roots of beech trees. These results are consistent with data reported by Kronzucker et al. (1995, 1996) who found a significantly higher influx of NH^ than NO 3 ~ in spruce seedlings. Relative xylem loading of NOf ranged between and 6.4±5.1% of the NO 3 " taken up (Fig. 3C). It appears to increase with increasing NO 3 ~" concentrations until 100 im; at higher NO 3 " concentrations relative xylem loading seemed to decrease slightly. Due to the scattering of the data this effect was not significant. It may be assumed that the process of xylem loading is saturated at high NO 3 " concentrations and additionally incoming NO 3 "" is not loaded into the xylem. This assumption is partially supported by xylem loading of NO 3 " as influenced by NH + 4 (Fig. 3C). In the presence of NH + 4 a more distinct increase of relative xylem loading was

5 Nitrate transport of beech r '0.806 A ± H 1 1- A observed at external NO 3 concentrations from jxm; but xylem loading decreased at higher NOf concentrations that exhibited higher rates of NO^~ uptake, when NH^ was fed. These findings indicate that xylem loading of NO^~ was directly dependent on net uptake of external NOf. Results obtained with excised roots treated with mm NH^ support this conclusion. At NO 3 ~ concentrations from 10 to 100/xM, both net uptake (c. 90%; Fig. 3A) and xylem loading of NO 3 ~ (data not shown) were strongly inhibited. Relative xylem loading, therefore, was similar with and without NH^ (Fig. 3C). The present finding of xylem loading of NO^ (Fig. 2B; Fig. 3C) suggests the presence of NO3" in the xylem. To address this question, xylem sap of beech trees incubated with l.5mm NO 3 "" and l.omm NO 3 "/ mm NH 4 +, respectively, were investigated. As expected from the results of the transport experiments, the NO^ content found in the xylem sap was similar and amounted to A- A t A N0 3 cone. (mm) Mechanism of NO3 transport and the effect of A/H 4 + and L-glutamate While NO3 uptake is a regulated process, full extent of the control system is not known. NO 3 ~ itself (Siddiqi et al., 1989; King et al., 1993), NH 4 + (Siddiqi et al., 1989; King et al., 1993) or products of NH^ assimilation (Muller et al, 1995) may act as feedback inhibitors of NO^ net uptake. Free, soluble amino compounds also may regulate NO 3 "" net uptake (Muller et al., 1995; Imsande and Touraine, 1994; Touraine et al., 1994). In several studies amino acids (Muller et al., 1995; Muller and Touraine, 1992; Breteler and Arnozis, 1985) as well as NH 4 + (Chaillou al., 1994; Marschner et al., 1991; Lee and Drew, 1989; Scheromm and Plassard, 1988) exerted inhibitory effects on NO^ net uptake. L-gJutamate was chosen for the present study, because it is present in considerable amounts in the phloem sap of beech trees (Schneider et al., 1996) and, therefore, fulfils the general requirements to act as feedback inhibitor of NO 3 " uptake. Radiolabelled I3 NO 3 " in combination with ls NOf was used to obtain more information on the mechanism of NO^ influx and its interaction with NH^. Because of its 511 kev y-radiation arising from positron annihilation, 13 NC>3~ can easily be detected and allows incubation for Fig. 3. Kinetics of 15 NO 3 " uptake rate (A), Hanes plot of 15 NO 3 uptake rate (B) and relative xylem loading of 15 NO 3 " (C). Beech trees were preincubated for 72 h in a liquid 1/5 MS medium containing either mm NO 3 " ( ) or mm NO 3 "/0 5mM NH< + (A). Excised roots were incubated in root chambers and exposed to 10 JJM to mm 15 + NO 3 " with (A) or without ( ) mm NH 4 in the uptake compartment. After 6 h of incubation "N in the root segments and in the solution in the exudation compartment were determined. (C) Data from Fig. 3B were converted according to Hanes (1932) and linear regression was used to calculate A^, and V mul of NOf net uptake by roots incubated without NH^. Means (±SD) are from 4-16 independent experiments with 4-6 roots each.

6 1436 Kreuzwieser et al. very short periods of time. This is a prerequisite to measure unidirectional fluxes. Incubation must be long enough to obtain an equilibrium of the apparent free space. To determine influx of NO 3 ~ in conditions where efflux can occur, observations have to be made well within the time it takes to fill the cytoplasmic pool because efflux of the tracer from a partially labelled pool will lead to an underestimation of influx (Clarkson et al., 1996). In the present study, detached roots were incubated for 5 min, which seemed to be an acceptable compromise between both constraints (Ter Steege, 1996; Muller et al., 1995). At low external NO 3 ~ concentrations (10/xM) there was no significant difference between NO 3 ~ influx and net uptake rate (Table 2). Therefore, no efflux of NO 3 ~ took place. At a supply of and 1 mm NO^, NO 3 ~ influx was times higher, respectively, than influx at 10 fim external NO^. Since net uptake at these NO^ concentrations ranged between 495 and 413nmol g" 1 FW h" 1, the NO3" efflux calculated amounted to 45 ±13 to % of NO 3 " influx, respectively (Table 2). Under these conditions NH^ inhibited the influx of NO3" to 60-70% as compared to treatments without NH^. Due to exposure to NH^" the net uptake at mm NO 3 ~ was decreased to c %, whereas NO^ net uptake at 1 mm NO3" remained unaffected. The efflux under these conditions amounted to % (Table 2). L-Glutamate like NH^ inhibited influx of NO^. Net uptake also appeared to be slightly reduced, but due to the large scatter of data this difference was not significant. Compared to treatments without reduced N, influx of NC>3~ was reduced up to 50% (Table 2), whereas efflux amounted to 57 ±21%. From these results it appears that reduced net uptake of NO^ in response to treatment with NH^ and L- glutamate is mainly caused by inhibition of the influx of NO 3 ~ (Table 2). Diminished NO 3 " net uptake mediated by decreased influx of NO^ has been reported in several studies (Muller et al., 1995; Ayling, 1993; Lee and Drew, 1989). The present work shows that efflux of NO 3 decreases if reduced N compounds are applied to the roots. Apparently, this effect is a consequence of reduced internal NO^ concentration due to inhibited influx of NO 3 ~. This assumption is supported by findings of Aslam et al. (1994); these authors showed that NO 3 " efflux is a function of internal NO 3 ~ concentration, as also assumed in the present study (Table 2). However, these and other authors (Chaillou et al., 1994; Deane-Drummond and Glass, 1983) suggested that enhanced efflux is responsible for changes in NO^ net uptake in the presence of NH^. This conclusion does not seem to be valid for beech, since reduced NO 3 ~ net uptake was mainly a consequence of decreased influx rather than enhanced efflux. Although feedback inhibitor effects of reduced N compounds on NO 3 ~ influx are a well-known phenomenon (Muller et al., 1995; Aslam et al., 1994; Chaillou et al., 1994; Ayling, 1993; Lee and Drew, 1989; Deane- Drummond and Glass, 1983), the molecular basis of this effect has not been established. On the one hand, ^ and L-glutamate may directly affect the activity of transporter proteins; on the other hand, an inhibition of the synthesis of the transport entities seems to be possible. Because of the experimental set-up used in the present study (co-incubation of reduced N compounds and NO 3 ~) also a competition between uptake systems cannot be excluded. Since studies of Schobert and Komor (1987) indicated that uptake of NO 3 ~ and organic N sources operate quite independently, this effect may be of minor significance. Besides direct effects of reduced N compounds it also may be assumed that inhibition of NO 3 ~ net uptake is mediated indirectly by an effect on assimilatory NO 3 ~" reduction. The products of assimilatory NO 3 ~ reduction, NH^ and, to a minor extent, the amino acid L-glutamate, both showed inhibitory effects on NO 3 ~ net uptake. This is a prerequisite to act as feedback inhibitor of NO^ net uptake. Together with the fact that both compounds are Table 2. Effect of NHf and L-glutamate on influx, net uptake and efflux of NO 3 Beech trees were preincubated for 72 h in liquid 1/5 MS media as indicated. The roots were excised and incubated in root chambers for 120 min with 13 NOi". 13 NOf was introduced during the last 5 min of this exposure. Roots were then washed in transport media lacking NOf, and analysed for 15 N and 13 N. 15 N-analysis was used to determine NOf net uptake rate, 13 N-analysis to determine NO 3 ~ influx. Data shown are means (±SD) of the number (n) of independent experiments with 4-6 roots each. Statistical analysis was performed using LSD under ANOVA. Different indices show significant differences within columns at P< Nitrogen supply (mm) during Preincubation Incubation NO 3 " influx (nmolg- 1 FWh 1 ) NO 3 net uptake (nmol g-'fwli ') NO 3 " efflux (% of influx) NO3- NH NO 3 - NH; L-GIU ±20f(/; = 6) 1084±204b(n = 6) 1092±631a(n = 6) 413±166d(n = 4) 495±128cd(n = 5) 792±205bc(n = 4) 139±53f(n = 5) 495±80cd(n = 5) 413±126d(n=16) 200±60e(n = 5) 395±186d(n=13) 272±62ed(n = 5) 0±9c(n = 6) 45±13b(n = 6) 78±lla(n = 6) 49±28b(n = 4) 53±13b(n = 5) 57±21ab(n = 4)

7 found in considerable amounts in the phloem of beech (Schneider et al., 1996) they may act as shoot-to-root signals to adapt NO^" uptake to the N requirement of beech trees. In the present study NH^ and L-glutamate were fed to the roots in the external medium. Further experiments with a more direct application of these compounds, e.g. by phloem feeding are required to explore the nature of the shoot-to-root signal regulating NO3" uptake by beech roots. In addition, the effect of mycorrhization an NO^" transport processes and its interaction with NH/ and amino compounds has to be considered. Acknowledgements The authors thank Dr P Delhon, Montpellier, for her support in 15 N-analysis. We are also grateful to the PET Center, Groningen, for providing 13 N. This study was financially supported by the Deutsche Forschungsgemeinschaft (DFG) under contract no. Re 515/4 and the BMBF under contract no. BEO References Aslam M, Travis RL, Huffaker RC Comparative kinetics and reciprocal inhibition of nitrate and nitrite uptake in roots of uninduced and induced barley (Hordeum vulgare L.) seedlings. Plant Physiology 99, Aslam M, Travis RL, Huffaker RC Stimulation of nitrate and nitrite efflux by ammonium in barley (Hordeum vulgare L.) seedlings. Plant Physiology 106, Ayling SM The effect of ammonium ion on membrane potential and anion flux in roots of barley and tomato. Plant, Cell and Environment 16, Breteler H, Amozis PA Effects of amino compounds on nitrate utilization by roots of dwarf bean. Phytochemistrv 24, Butcher DN, Street HE Excised root culture. Botanical Reviews 30, Chaillou S, Rideout JW, Raper CD, Morot-Gaudry J-F Responses of soybean to ammonium and nitrate supplied in combination to the whole root system or separately in a splitroot system. Plant Physiology 90, Clarkson DT, Gojon A, Saker LR, Wiersema PK, Purves JV, Tillard P, Anold GM, Paans AJM, Vaalburg W, Stulen I Nitrate and ammonium influxes in soybean (Glycine max) roots: direct comparison of 13 N and 15 N tracing. Plant, Cell and Environment 19, Clement CR, Hopper MJ, Jones LHP, Leaf EL The uptake of nitrate by Lolium perenne from flowing nutrient solution. II. Effect of light, defoliation and relationship of CO 2 flux. Journal of Experimental Botany 29, Deane-Dnimmond CE, Glass ADM Short term studies of nitrate uptake into barley plants using ion-specific electrodes and 36 C1O 3 ". II. Regulation of NO 3 " efflux by NH^. Plant Physiology 73, Ellenberg H Vegetation Mitteleuropas mil den Alpen. Verlag Eugen Ulmer, Stuttgart, Germany. Finlay RD, Ek H, Odham G, SaderstrSm B Uptake, translocation and assimilation of nitrogen from 15 N-labelled ammonium and nitrate sources by internal ectomycorrhizal systems of Fagus sylvaiica infected with Paxillus involutus. New Phytologist 113, Nitrate transport of beech 1437 Glass ADM, Siddiqi MY Nitrogen absorption by plant roots. In: HS Srivastava, RP Singh, eds. Nitrogen nutrition in higher plants. New Delhi, India: Associated Publishing Co., Gottlein A, Kreutzer K Der Standort H6glwald im Vergleich zu anderen flkologischen Fallstudien The H6gJwald experimental site as compared to other ecological field studies. Forstwissenschaftliches Centralblatt 39, Hanes CS Studies on plants amylases. I. The effect of starch concentration upon velocity of hydrolases by the amylases of germinated barley. Biochemical Journal 26, Haynes RJ, Goh KM Ammonium and nitrate nutrition of plants. Biological Reviews 53, Herschbach C, Rennenberg H Influence of glutathione (GSH) in sulphate influx, xylem loading and exudation in excised tobacco roots. Journal of Experimental Botany 42, Imsande H, Touraine B N-demand and the regulation of nitrate uptake. Plant Physiology 105, 3-7. Jackson RB, CaldweU MM The scale of nutrient heterogeneity around individual plants and its quantification with geostatics. Ecology 74, Kamminga-van Wijk C, Prins HBA The kinetics of NH^" and NO3" uptake by Douglas fir from single N-solutions containing both NH^" and NO 3 ". Plant and Soil 151, King BJ, Siddiqi MY, Ruth TJ, Warner RL, Glass ADM Feedback regulation of nitrate influx in barley roots by nitrate, nitrite, and ammonium. Plant Physiology 102, Kreuzwieser J, Herschbach C, Rennenberg H Sulfate uptake and xylem loading of non-mycorrhizal excised roots of young beech (Fagus sylvatica L.) trees. Plant Physiology and Biochemistry 34, Kronzucker HJ, Siddiqi MY, Glass ADM Kinetics of NO 3 " influx in spruce. Plant Physiology 109, Kronzucker HJ, Siddiqi MY, Glass ADM Kinetics of NH + 4 influx in spruce. Plant Physiology 110, Lajtfaa K Nutrient uptake in eastern deciduous tree seedlings. Plant and Soil 160, Lee RB, Drew MC Rapid, reversible inhibition of nitrate influx in barley by ammonium. Journal of Experimental Botany 40, Marschner H, Haussling M, George E Ammonium and nitrate uptake rates and rhizosphere ph in non-mycorrhizal roots of Norway spruce [Picea abies (L.) Karst.]. Trees 5, Muller B, Tillard P, Touraine B Nitrate fluxes in soybean seedling roots and their response to amino acids: an approach using 15 N. Plant, Cell and Environment 18, Muller B, Touraine B Inhibition of NO 3 ~ uptake by various phloem-translocated amino acids in soybean seedlings. Journal of Experimental Botany 43, Murashige T, Skoog F A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiologia Plantarum 15, Pitman MG Uptake and transport of ions in barley seedlings. I. Estimation of chloride fluxes in cells of excised roots. Australian Journal of Biological Science 24, Plassard C, Barry D, Eltrop L, Moussain D Nitrate uptake in maritime pine (Pinus pinaster) and the ectomycorrhizal fungus Hebeloma cylindrosporum: effect of ectomycorrhizal symbiosis. Canadian Journal of Botany 11, Rao KP, Rains DW Nitrate absorption by barley. I. Kinetics and energetics. Plant Physiology 57, Rao TP, Ito O, Matsuga R Differences in uptake kinetics

8 1438 Kreuzwieser et al. of ammonium and nitrate in legumes and cereals. Plant and Soil 154, Rennenberg H, Schneider S, Weber P Analysis of uptake and allocation of nitrogen and sulphur compounds by trees in the field. Journal of Experimental Botany 47, Ryglewicz PT, Bledsoe CS Effects of pre-treatment conditions on ammonium and nitrate uptake by Douglas fir seedlings. Tree Physiology 1, Scheromm P, Plassard C Nitrogen nutrition of nonmycorrhized maritime pine (Pinus pinaster) grown on nitrate or ammonium. Plant Physiology and Biochemistry 26, Schneider S, Ge/9ler A, Weber P, von Sengbusch D, Hanemann U and Rennenberg H Soluble N compounds in trees exposed to high loads of N: a comparison of spruce (Picea abies [L.] Karst) and beech (Fagus sylvatica L.) grown under field conditions. New Phytologist, 134, Schobert C, Komor E Amino acid uptake by Ricinus communis roots. Characterization and physiological significance. Plant, Cell and Environment 10, Scholander PF, Hammel T, Bradstreet ED, Hemmingsen EA Sap pressure in vascular plants. Science 148, Schupp R Untersuchungen zur Schwefelerndhrung der Fichte (Picea abies L.): Die Bedeutung der Sulfatassimilation und des Transportes von Thiolen. Frankfurt/Main: Wissenschaftsverlag Dr Wigbert Maraun, SeegmQIler S, Schulte M, Herschbach C, Rennenberg H Interactive effects of mycorrhization and elevated atmospheric CO 2 on sulphur nutrition of young pedunculate oak (Quercus robur L.) trees. Plant, Cell and Environment 19, Siddiqi MY, Glass ADM, Ruth TJ, Fernando M Studies of the regulation of nitrate influx by barley seedlings using 13 NO 3 ". Plant Physiology 90, Stadler J, Gebauer G, Schulze E-D The influence of ammonium on nitrate uptake and assimilation in 2-year-old ash and oak trees a tracer study with 15 N. Isotopenpraxis Environmental Health Studies 29, Ter Steege MW Regulation of nitrate uptake in a whole plant perspective. Changes in influx and efflux of nitrate in spinach. Thesis RUG, The Netherlands. Touraine B, Clarkson DT, Muller B Regulation of nitrate uptake at the whole plant level. In: Roy J, Gamier E, eds, A whole plant perspective on carbon-nitrogen interactions. The Hague: SPB Academic Publishing bv., Vaalburg W, Paans AMJ Short-lived positron emitting radionuclides. In: Helms F, Colombetti LG, eds. Radionuclides production II. Boca Raton: CRC Press,