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• The effects of N-availability and elevated atmospheric CO 2 partial pressure ( pCO2) on growth, allometry and N-metabolism of poplar plants are reported here.
• Poplar plants were grown hydroponically at deficient and sufficient N-supply under ambient and elevated pCO2. The N-fluxes within the plants were estimated by comparing the fate of newly acquired 15N-NO3− in plants either severely N-limited or with sufficient N-supply.
• At deficient N-supply, plants accumulated less biomass and exhibited an increased root : shoot ratio compared with sufficient N-supply; a larger fraction of newly acquired 15 N was allocated to the youngest leaves immediately after exchange of the nutrient solution. Increasing the external N-supply from deficient to sufficient shifted the site of nitrate reduction from roots to leaves.
• Elevated pCO2 increased total biomass and the root : shoot ratio at deficient N-supply, but had no effect at sufficient N-supply. Elevated pCO2 decreased rates of N-uptake in both treatments. Increased root : shoot ratio at deficient N-supply coincided with enhanced nitrate reduction in the root and elevated pCO2 also enhanced the allocation of newly acquired 15N to the youngest leaves. Root nitrate reduction as a possible factor controlling the root : shoot ratio and N-allocation is discussed.
The current increase of atmospheric CO2 partial pressure (pCO2) will enable C3 plants to perform photosynthesis at a higher rate, due to an increased carboxylation efficiency of Rubisco and, coincidentially, a decreased photorespiration at elevated pCO2 (Andrews & Lorimer, 1987; Stitt, 1991). However, after prolonged exposure, an acclimation of photosynthesis to elevated pCO2 often occurs (Drake et al., 1997). The extent of this down-regulation depends, for example, on the species examined, a variety of environmental factors and, in particular, on the availability of mineral N (Stitt & Krapp, 1999). Frequently, the increase of leaf C : N ratio at elevated pCO2 is not entirely due to an accumulation of nonstructural carbohydrates, especially starch, but also to a decrease of leaf protein-N, especially at deficient N-supply (Drake et al., 1997). Total leaf-N determines the capacity of photosynthesis in C3 plants (Evans, 1989). The distribution of leaf N between thylakoids, Rubisco or other Calvin-cycle enzymes may be important to optimize photosynthesis at the cellular level, responding to limitations of light, pCO2, or RuBP regeneration capacity (Murray et al., 2000). At the whole plant level, however, a deficient N-supply causes a reallocation of nitrogen from the leaves to promote root growth and, thus, to improve N-acquisition, hence resulting in an increased root : shoot ratio (Brouwer, 1983; Bloom et al., 1985; Field & Mooney, 1986). Thus, imbalances between photosynthetic C-fixation and N-uptake alter the root : shoot ratio, thereby indicating which process currently limits plant growth. Therefore, the root : shoot ratio has been proposed as an indicator for the plant's nutritional status (Baxter et al., 1997), which certainly is useful for herbaceous species.
In woody perennials, however, a large fraction of the shoot consists of nonphotosynthetic wood and bark-tissue with support and transport functions, which complicates our understanding of the functional equilibrium between shoot and root growth and, thus, photosynthesis and N-uptake (Cromer & Jarvis, 1990; Körner, 1994). For example, the stem of trees represents a large sink for carbohydrates which may also influence the acclimation response to elevated pCO2, since photosynthetic down-regulation is thought to be triggered by an exhaustion of sink-strength (Arp, 1991; Thomas & Strain, 1991; Moore et al., 1999). Moreover, trees are characterized by their ability to ‘mine’ for nutrients (Johnson et al., 1996). In woody species, root architecture and turnover of fine roots may be more important for the acquisition of nutrients than total root mass.
The rates of N-uptake in trees vary extensively at elevated pCO2. Responses range from a decrease (Rothstein et al., 2000), no response (BassiriRad et al., 1997) to an increase of N-uptake rates (BassiriRad et al., 1996). However, it is necessary to study both root architectural and physiological responses in order to assess the capacity for N-acquisition of trees, since there appears to be a ‘trade off’ between these two factors (Jackson et al., 1990). For example, elevated pCO2 increased root length production, but decreased rates of N uptake in Betula (Bauer & Berntson, 2001). In contrast, elevated pCO2 did not affect root architecture of Pinus, but significantly increased Vmax for NH4+ and NO3− uptake (Bauer & Berntson, 2001). In Populus tremuloides an increased turnover of fine roots was observed at elevated pCO2, especially at sufficient N-supply (Kubiske et al., 1998). In a different study, however, it was shown that the physiological capacity for N-uptake was decreased in Populus grown at elevated pCO2 (Rothstein et al., 2000).
The genus Populus is known to store large amounts of nitrogen in the leaves (Bradshaw et al., 2000). This trait is probably related to the periodically changing availability of mineral N in a natural habitat of Populus, the floodplain forest. In addition, the transport pool of nitrogen, cycling between xylem and phloem, may vary to a large extent. Thus, growth rates of woody species, and especially of Populus, may generally not correlate with N-uptake, like shown for herbaceous species (Millard, 1996; Kruse et al., 2002).
The central aim of this study was to characterize the effects of N-availability and elevated pCO2 on biomass accumulation and allometric relations of Populus, and to correlate the results to uptake, transport and partitioning of N between different organs. In particular, N-acquisition was studied and root architectural responses were separated from physiological responses. Second, we analysed whether elevated pCO2 and N-supply altered the storage of NO3− and organic N in different organs, the distribution of nitrate reduction between leaves and roots and, as a consequence, xylem transport of nitrate and organic N to the shoot and the redistribution of organic N via phloem transport.
Materials and Methods
Plant material and growth conditions
Our experiments were performed with hybrid poplar plants (Populus tremula × P. alba), INRA-clone no. 717–1-B4. Following micropropagation in vitro (Leple et al., 1992), poplar plants were transferred to hydroponic culture and placed into climate controlled glasshouses for a photoperiod of 16 h (250–300 µmol m−2 s−1 PAR at leaf level), at a temperature of 24°C, a relative air humidity of 75–80%, at ambient (36 Pa) and elevated pCO2 (75 ± 5 Pa). The plants were distributed among four growth chambers, two of them exposed to ambient and two to elevated pCO2. The plants were randomly rotated between the two chambers every week, and twice a week within each chamber.
In the present study we wanted to emphasise physiological responses (e.g. the capacity of nitrate uptake per unit of root f. wt) over root architectural responses (e.g. branching or lateral root elongation). Root architecture crucially depends on the heterogenous localization of available N in the rooting zone (Linkohr et al., 2002). In the present study the plants were grown hydroponically. Since the nutrients were homogenously dissolved in the solution, it was expected that alterations of N-uptake rates were more important than changes of root architecture. The 6.5 l-pots in which the plants were grown contained a well-aereated nutrient solution of the following composition: K+ 880 µM, Ca2+ 900 µM, Mg2+ 300 µM, PO 600 µM, SO 300 µM, FeEDTA 5 µM, H3BO3 10 µM, Mn2+ 2 µM, Zn2+ 0.5 µM, Cu2+ 0.2 µM, MoO42–0.02 µM. Buffering of the nutrient solution (pH 6.8 ± 0.1) was achieved by addition of phosphate as KH2PO4 and K2HPO4. Plants were grown for 8 wk in hydroponic culture and nutrient solutions were changed once a week. At the beginning of each week, plants had access either to 2.0 mM NO3− (= sufficient N-supply) or to 0.2 mM NO3− (= deficient N-supply). Because of N-uptake by the plants, the external N-concentration was expected to fluctuate within weekly intervals, especially at deficient N-supply and when the plants grew larger. Thus, there was no defined N-limitation like it may be achieved by the relative addition rate method (Ingestad, 1982). Since the precise impact of elevated pCO2 on growth rates of poplar plants and, thus, the putative N-addition rate under these conditions were not clear, we decided to grow the plants at the same initial N-concentration. It was expected that fluctuations of the external N-supply within weekly intervals would also cause fluctuations of the plant internal N-status. To study the fate of newly acquired N after exchange of the nutrient solution, 15N-NO3− was added to a final concentration of 0.2 and 2.0 mM NO3− with a 15N abundance of 10 and 20 atom% at sufficient and deficient N-supply, respectively. 15N abundance of the nutrient solution was lower at sufficient compared to deficient N-supply because of cost savings.
After 3 d of hydroponic growth, poplar plants were harvested 3–6 h after beginning of the light-period. Each plant was divided into mature and young leaves, stem, roots and apex. For this purpose, the stem was marked in a distance of every 10 cm. The leaves nearest to these marks were pooled (= ‘mature’ leaves). Around these marks, c. 1 cm-long pieces of the stem were cut and pooled (= ‘stem’). The three first leaves nearest to the apex with c. 2.0 ± 0.2 g total f. wt both at deficient and sufficient N-supply were combined to the fraction ‘young leaves’. The roots were separated into coarse and fine roots. Coarse roots were already lignified (d. wt: 5.1 ± 1.1% of f. wt). Fine roots had a diameter of 0.3–2.0 mm with a d. wt of 1.8 ± 0.1% of f. wt. We found hardly any visible organic residue in the nutrient solution. Since elevtated pCO2 did not change the coarse to fine root ratio, both fractions were combined. Part of the plant material was frozen in liquid nitrogen, and stored at −80°C until analysis. The other part was dried in an oven at 45°C for 3 d and stored at room temperature until analysis.
Transpiration and collection of xylem sap
Transpiration was calculated from the water loss of the 6.5 l-pots during the 3 d of 15N enrichment. For this purpose, water-loss was measured by weighing. Evaporational water-loss was determined from pots containing nutrient solution, but no plants, and was substracted from total water-loss during the time under study.
Xylem sap was collected by the modified method of Scholander et al. (1965) described by Rennenberg et al. (1996). The collection was started 3 h after the beginning of the lightperiod. The stem was cut with a razor blade above the root, the cut end was carefully rinsed with distilled water to avoid contamination with cellular compounds and, subsequently, the root was put into the pressure chamber. The pressure was slowly increased until the first drops appeared at the cut end. Aliquots of 50–75 µl of xylem sap were collected and stored at −80°C until analysis.
Analysis of tissue C, N and 15N contents
Total nitrogen and total carbon were estimated in dried plant material with a C/N 2500 Element Analyser (CE Instruments, Milan, Italy) using Dumas combustion. In this instrument samples are energetically oxidised yielding a gas mixture in which CO2 and N2 are detected by a thermoconductivity detector. Subsequently, 15N contents of the plant material were determined by an isotope ratio mass spectrometer (IRMS, Finnigan MAT GmbH, Bremen, Germany), coupled in series with the C/N analyser. Nitrate uptake of poplar plants was calculated from the enrichment of 15N in different plant organs, summing up to the 15N accumulation per plant.
Extraction and analysis of amino-compounds
Amino compounds were extracted from plant material as described by Kruse et al. (2002). For analysis of amino compounds in xylem sap aliquots were diluted 20-fold with lithium citrate buffer. Samples were injected into an automated amino-acid analyser (Biochrom, Pharmacia LKB, Freiburg, Germany) using a system of five lithium citrate buffers and a gradient of pH 2.8–3.55. The amino compounds separated were subjected to postcolumn derivatisation with ninhydrin. The absorption of the aminoninhydrin derivates was measured at 440 and 570 nm.
Extraction and determination of nitrate
Nitrate was extracted from frozen plant material as decribed by Kruse et al. (2002). Anions were separated on a IonPac® column (AS9-Sc 250 × 4 mm; Dionex, Idstein, Germany) with a solution containing 1.8 mM Na2CO3 and 1.7 mM NaHCO3 at a flow rate of 1.0 ml min−1. Nitrate was detected with a conductivity detector module (CDM, Dionex, Idstein, Germany).
Extraction and analysis of carbohydrates
Soluble sugars were extracted from frozen plant material as described by Kruse et al. (2002). Sucrose, glucose and fructose were separated on an anion exchange column (CarboPac PA 1, 4 × 250 mm, Dionex, Idstein, Germany) with an isocratic NaOH system. Separation was carried out with 36 mM NaOH free of carbohydrate within 37 min at a flow rate of 1 ml min−1. Sugars were detected by pulsed amperometry.
Analyses of different parameters were performed in five independent replicates. ANOVA was performed with SPSS 11.0 for windows (SPSS Inc., USA), using a univariate linear model with N-supply and pCO2 as fixed factors. Comparisons among means were conducted using Student's t-test.
Biometrical data and transpiration
Biomass and root : shoot ratio of poplar plants grown for 8 wk in hydroponic culture were dependent on both N-supply and pCO2 (Fig. 1). On average, the increase from deficient (0.2 mM NO3−) to sufficient (2.0 mM NO3−) N-supply increased plant f. wt and decreased the root : shoot ratio by approx. 100%. Elevated pCO2 significantly increased the biomass and root : shoot ratio of poplar plants grown at deficient, but not at sufficient N-supply. The shift from deficient to sufficient N-supply substantially increased total dry mass of leaves, but significantly decreased total dry mass of the roots, irrespective of pCO2 (compare Fig. 2a and Fig. 2b). Increasing the N-supply altered the accumulation of dry mass, expressed as percentage of f. wt, in different organs of poplar plants (Fig. 2c,d). At deficient N-supply, most of the dry mass accumulated in the stem (23.5%). Relative dry mass was lower in mature and young leaves (22%). At sufficient N-supply an inverse pattern was observed, with a relative dry mass of approx. 17% in the stem and approx. 21% in young leaves. Elevated pCO2 increased relative dry mass of each tissue above-ground. This increase was, however, more pronounced at deficient N-supply (Fig. 2c,d). Elevated pCO2 did not affect total leaf area, irrespective of the N-supply (data not shown). Thus, specific leaf area (SLA, cm2 g−1 d. wt) was significantly decreased at elevated pCO2, especially at deficient N-supply.
Rates of transpiration were dependent on both N-supply and pCO2 (Fig. 3). At deficient N-supply, transpiration rates were decreased at elevated rather than ambient pCO2. At sufficient N-supply, transpiration rates decreased at ambient pCO2 rather than deficient N-supply, but were hardly affected by elevated pCO2. Thus, at sufficient N-supply transpiration rates were similar at ambient and elevated pCO2.
N-contents in root- and shoot-tissues
The increase of the N-supply from 0.2 mM to 2.0 mM NO3− led to a significant decrease of the C : N ratio in all tissues examined (Fig. 4, column 1). This decrease was, with the exception of root tissue, generally connected with increased N-contents in the dry matter of the tissues analysed (column 2). The C : N ratio of roots decreased by a factor of 4 by increasing the N-supply (Fig. 4(1a)), but N-contents only increased by a factor of 3 (Fig. 4(2a)). Consequently, at sufficient N-supply the decreased C : N ratio of the roots was not entirely due to increased N-contents, but also to a decreased allocation of C to the roots. At deficient N-supply the C : N ratio and N-content of the drymass were similar in mature and young leaves. In contrast, at sufficient N-supply the C : N ratio of young leaves was lower than in mature leaves due to increased N-contents in young leaves (compare Fig. 4(2c) and Fig. 4(2d)).
Elevated pCO2 significantly increased the C : N ratio of roots at deficient N-supply and in leaves, irrespective of the N-supply. The increase of C : N ratio was most pronounced in mature leaves, especially at deficient N-supply. The impact of elevated pCO2 on the C : N ratio became smaller in young leaves and vanished in the apex. Generally, the increased C : N ratio at elevated pCO2 was the result of decreased N-contents of the dry matter. However, with the exception of mature leaves grown at deficient N-supply (Fig. 4(3c)), N-contents of the freshmass were hardly affected by elevated pCO2. Thus, at elevated pCO2 decreased N-contents of the dry matter were a consequence of dilution by C accumulation.
TSNN (total soluble nonprotein nitrogen), NO3− and total amino-N in different tissues of poplar plants
In all plants studied, increasing the N-supply from deficient to sufficient enhanced TSNN contents in roots and mature leaves, mainly due to elevated NO3−-contents (Fig. 5). In young leaves and apices, TSNN contents were substantially higher at sufficient N-supply because of increased amino-N contents. Amino-N contents of the apex were increased 10-fold compared to plants grown at deficient N-supply (Fig. 5(3d)).
Elevated pCO2 significantly decreased NO3− contents of roots at deficient, but not at sufficient N-supply (Fig. 5(2a)) and led to decreased NO3− contents in mature leaves, young leaves and the apex. In contrast, amino-N contents were significantly decreased by elevated pCO2 in young leaves only, but were similar at ambient and elevated pCO2 in all other tissues examined (Fig. 5).
Individual amino-compounds and NH4+ in different tissues of poplar plants
All of the nitrogen that has been reduced by nitrate reductase and nitrite reductase is assimilated in the GS/GOGAT pathway, the primary acceptor of reduced nitrogen being Gln (Heldt, 1996). However, NH4+ and Gln contents are also dependent on rates of photorespiration, whereas the pool of Glu is tightly regulated and rather constant (Stitt & Krapp, 1999). Thus, a shift of the Glu : Gln ratio points at alterations of photorespiration in relation to primary N-assimilation.
Increasing the N-supply from deficient to sufficient only slightly increased Gln- and Glu contents of the roots and did not affect the Glu : Gln ratio (Fig. 6). By contrast, it significantly increased Gln- and Glu-contents and decreased the Glu : Gln ratio in leaves. Large amounts of Gln and also Asn accumulated in the apex as a consequence of increased N-supply (not shown).
Elevated pCO2 did not affect Gln-, Glu- and NH4+ contents of the roots. In leaves, elevated pCO2 decreased Gln-, but hardly affected Glu-contents, hence resulting in a significantly increased Glu : Gln ratio (Fig. 6). Ammonium contents of leaves were also decreased at elevated pCO2, especially at sufficient N-supply. By contrast, Gln-, Glu- and NH4+ contents of the apex were similar at ambient and elevated pCO2 (not shown).
Uptake of 15N-NO3− and recovery of 15N in different organs of poplar plants
NO3− uptake was determined after the exchange of the nutrient solution and addition of 15N-NO3− to a final concentration of 0.2 and 2.0 mM NO3−, respectively. Note that N-uptake was related to total root mass, comprising both coarse and fine roots. Although the coarse to fine root ratio was constant among the treatments, rates of N-uptake were systematically underestimated, because coarse roots do not significantly contribute to N-uptake.
At ambient pCO2 and deficient N-supply, the rates of nitrate uptake amounted to 300 µmol NO3− g−1 root f. wt h−1, calculated as mean uptake during 3 d of exposure to 15N- NO3−. At sufficient N-supply, rates of N-uptake were increased by approx. 60% compared to deficient N-supply (Fig. 7a).
Elevated pCO2 significantly decreased rates of N-uptake, irrespective of the N-supply. However, at deficient N-supply decreased rates of N-uptake were overcompensated by an enhanced root : shoot ratio, hence resulting in about the same total N-uptake per plant (Fig. 7b).
With the exception of roots of poplar plants grown at deficient N-supply, elevated pCO2 did not significantly affect N-partitioning in any other plant organ (Fig. 8, column 1). The enhanced N-supply significantly increased total N-accumulation in each plant organ. This increase was most pronounced in the stem, irrespective of pCO2 (Fig. 8(1d)). However, since total N-accumulation predominantly reflects different plant size at different N-supply and pCO2, 15N-accumulation was also expressed on the basis of the f. wt of each plant organ (Fig. 8, column 2). The increase of N-supply from 0.2 mM to 2.0 mM NO3− significantly increased the N-accumulation per unit of fresh weight of root tissue. Surprisingly, it decreased N-accumulation in mature leaves, young leaves and apices. This decrease was most pronounced in young leaves of poplar plants (Fig. 8(2b)).
With the exception of young leaves, elevated pCO2 did not affect N-accumulation in any other tissue. In young leaves, however, 15N-accumulation was increased at elevated pCO2, irrespective of the N-supply (Fig. 8(2b)). Apparently, the allocation of 15N to the youngest leaves was higher at deficient compared to sufficient N-supply at the expense of the roots, and higher at elevated compared to ambient pCO2 (Fig. 9).
N-compounds in xylem sap and bark tissue
Xylem sap was collected 3 d after the beginning of the 15N-labeling. At deficient N-supply and ambient pCO2, TSNN concentration in xylem sap of poplar plants grown at ambient pCO2 amounted to 0.61 µmol ml−1 (Table 1). Nitrate made up 25% while reduced N, including NH4+ and amino-N, contributed approx. 75% of TSNN concentration (Table 1). At sufficient N-supply, TSNN contents of xylem sap were increased by a factor of approx. 13, and consisted to approx. 50% of each NO3− and amino-N.
Table 1. N-compounds in the xylem sap of poplar plants grown at ambient and elevated CO 2 with access to suboptimal (0.2 mM NO 3− ) and optimal N-supply (2.0 mM NO 3− )
µmol ml−1 xylem sap
After 8 wk of growth in hydroponic culture, xylem sap was collected with the pressure-chamber technique. The stem was cut with a razor blade above the root, the cut end was carefully rinsed with distilled water to avoid contaminations and, subsequently, the root was put into the pressure chamber. The pressure was slowly increased until the first drops appeared at the cut end. Aliquots of 50–75 µl xylem sap were collected and stored at −80°C until analysis as described in Fig. 5. Data shown are means ± SD of 5 independent replicates.
0.2 mM NO3−
0.61 ± 0.14
0.18 ± 0.03
0.05 ± 0.02
0.38 ± 0.14
36 Pa CO2
0.2 mM NO3−
0.46 ± 0.05
0.02 ± 0.01
0.05 ± 0.02
0.40 ± 0.06
75 Pa CO2
2.0 mM NO3−
7.99 ± 3.07
3.76 ± 1.70
0.09 ± 0.03
4.16 ± 2.59
36 Pa CO2
2.0 mM NO3−
6.82 ± 2.28
3.03 ± 1.24
0.07 ± 0.02
3.72 ± 1.66
75 Pa CO2
Elevated pCO2 decreased TSNN contents in the xylem sap at deficient N-supply, due to a significant decrease of NO3− contents (Table 1). Only traces of NO3− were present in the xylem sap of poplar plants grown at elevated pCO2 and deficient N-supply. By contrast, when plants were grown at sufficient N-supply, elevated pCO2 only slightly decreased TSNN contents of the xylem sap, and hardly affected the distribution between organic N and NO3−.
Bark tissue was removed at the root : shoot interface, above the root collar, and analysed for amino-N as an estimate of phloem-mobile N translocated to the roots. At deficient N-supply and ambient pCO2, amino-N contents amounted to 5 µmol g−1 f. wt. Amino-N contents increased 10-fold at sufficient compared to deficient N-supply (Fig. 10). Elevated pCO2 significantly decreased amino-N in bark tissue both at sufficient and deficient N-supply.
Model calculations of plant internal N-cycling
From the results of the present study, N-accumulation and -fluxes were calculated for the final 3-d period of 15N-tracer period (Fig. 11). For the sake of simplicity, mature and young leaves plus apices were combined and treated as a single fraction ‘leaves’. Further, it was assumed that the contribution of stem tissue to nitrate reduction was negligible (for a detailed description of the model calculations see Appendix).
At deficient N-supply and ambient pCO2 only 10% of the nitrate taken up was loaded into the xylem and transported to the shoot, while the bulk was already reduced in the root (Fig. 11a). Since the accumulation of organic N in the root was lower than nitrate reduction, the surplus, which amounted to 130 µmol N plant−1 day−1, was loaded into the xylem. However, xylem transport of organic N determined experimentally amounted to only 65 µmol N plant−1 day−1, suggesting that either xylem transport of organic N was under- or nitrate reduction in the root was overestimated by the model calculations. In the shoot, accumulation of organic N exceeded the amount which could be provided by nitrate reduction. Thus, the shoot was dependent on xylem unloading of organic N originating from the root. Accumulation of organic N in the shoot could have been met exactly by xylem transport of organic N originating from the root plus nitrate reduction in the shoot. As a consequence it has to be assumed that reallocation of organic N in the phloem is low under these conditions.
Poplar plants grown at deficient N-supply and elevated pCO2 exhibited increased NO3− uptake compared to controls at ambient pCO2 (Fig. 11c), due to an increased root : shoot ratio (Fig. 1, see above). At elevated pCO2, poplar plants did not accumulate any NO3− in the root, and xylem transport and accumulation of NO3− in the shoot was negligible. Thus, almost all nitrate taken up appears to be reduced in the root of poplar plants grown at 0.2 mM NO3− and elevated pCO2. Accumulation of organic N in the leaves was similar at ambient and elevated pCO2, but xylem transport of amino-N originating from the root was definitely not sufficient to provide the leaves with organic N, indicating that xylem transport of nitrogen was underestimated at deficient N-supply by the model calculations.
At sufficient N-supply and ambient pCO2 plants took up 550 µmol NO3− per day (Fig. 11d). Xylem transport of NO3− accounted for 775 µmol per plant and day. Since transport of NO3− within the plant is thought to occur predominantly in the xylem, and recyling of large amounts of NO3− via phloem transport is unlikely (Marschner, 1995), xylem transport of NO3− was probably overestimated at sufficient N-supply. Only a minor fraction of the NO3− arriving in the leaves accumulated; the majority was reduced at this site. Obviously, the site of nitrate reduction shifted from the roots towards the leaves at enhanced N-supply. Nitrate reduction exceeded the accumulation of organic N in the leaves by far and, thus, large amounts of amino-N were loaded into the phloem. Thus, the cycling pool of amino-N appears to be increased substantially at sufficient compared to deficient N-supply. Furthermore, at sufficient N-supply the roots of poplar plants act as a sink for organic N originating from the shoot, whereas the opposite was found at deficient N-supply.
Poplar plants grown at sufficient N-supply and elevated pCO2 exhibited similar biomass and root : shoot ratio compared to controls grown at ambient pCO2. Thus, accumulation and fluxes of N at ambient and elevated pCO2 can be compared directly (Fig. 11d,e). Uptake of NO3− was decreased at elevated pCO2. Elevated pCO2 also slightly decreased accumulation of NO3− and organic N in both the root and the leaves. Also acropetal xylem transport of NO3− and amino-N as well as basipetal phloem transport of organic N were decreased. However, as indicated by increased allocation of 15N to the youngest leaves (see above), acropetal phloem transport of organic N appeared to be increased at elevated pCO2, which is not resolved by the current model where all leaves are treated as a single fraction.
Soluble sugars in different organs of poplar plants
To assess the carbohydrate status of the plants, which may depend on both photosynthesis and N-assimilation, soluble sugars were determined in different organs (Fig. 12). Increasing the N-supply significantly decreased glucose and sucrose contents of the roots. Sucrose was only present at deficient, but was not detected at sufficient N-supply (Fig. 12(3a)). In mature and young leaves, glucose and fructose contents were significantly increased, but sucrose contents slightly decreased as a result of enhanced N-supply. Glucose contents of the apex were significantly increased at deficient compared to sufficient N-supply, even though glucose contents of young leaves were decreased at deficient compared to sufficient N-supply (Fig. 12(1c)). Furthermore, fructose and sucrose contents were decreased in the apex compared to young leaves of poplar plants with access to sufficient N-supply, but contents were similar in apex and young leaves at deficient N-supply.
Highest amounts of sucrose within poplar plants were observed in bark tissue. However, the bark contained only minor amounts of glucose and fructose (Fig. 12, row e). Sucrose contents were significantly higher at deficient compared to sufficient N-supply.
Elevated pCO2 significantly increased glucose and fructose contents of mature leaves, and also tended to increase sucrose contents (Fig. 12(3b)). By contrast, glucose and fructose contents of young leaves were similar at ambient and elevated pCO2, but sucrose contents tended to be decreased at elevated pCO2.
Elevated pCO2 increased the root : shoot ratio of poplar plants only at deficient N-supply, and consistently decreased the rates of nitrate uptake irrespective of the N-supply
In the present study poplar plants were grown hydroponically in order to emphasize alterations of physiological N-uptake over changes in root architecture, for example, lateral root density and elongation, as a consequence of different N-supply and pCO2. Thus, in hydroponic culture, it was considered reasonable to simply determine the root : shoot ratio as a reflection of the overall nutritional status of young poplar plants (Stitt & Schulze, 1994). Both the N-supply and pCO2 significantly affected biomass accumulation and the root : shoot ratio of poplar plants. With access to sufficient N, the plants grew larger and exhibited a decreased root : shoot ratio as compared to deficient N-supply. However, elevated pCO2 increased the root : shoot ratio only at deficient, but not at sufficient N-supply. As to the interactive effect of elevated pCO2 and N-supply on the root : shoot ratio, similar results were obtained by Curtis & Wang (1998) in a comprehensive literature analysis of recent studies. By contrast, several authors reported that elevated pCO2 predominantly increased total biomass at nonlimiting N-supply (Curtis & Wang, 1998; Murray et al., 2000; Maroco et al., 2002). This effect was not observed in the present study. Nitrogen was not limiting for plant growth at elevated pCO2 and sufficient N-supply, since total nitrogen contents per f. wt of mature leaves were not decreased compared to controls at ambient pCO2. Still, elevated pCO2 decreased the N content of leaves on a d. wt basis also at sufficient N-supply, not only because of a ‘C-dilution’ by increased amounts of soluble sugars (Fig. 12), but probably also due to increased starch contents in mature leaves (Drake et al., 1997).
Although N-acquisition was decreased at sufficient N-supply and elevated pCO2, due to lowered uptake rates rather than changes of the root : shoot ratio, total N-contents of the leaves were similar at ambient and elevated pCO2. Conse-quently, the pool of organic N circulating between xylem and phloem must have been decreased. Such a decrease was indeed predicted by model calculations (Fig. 11e) and was also confirmed by decreased contents of amino-N in bark tissue (Fig. 10). A decrease of nitrogen contents in the transport pool was also found for Quercus as a consequence of elevated pCO2 (Norby et al., 1986).
N-uptake rates at elevated pCO2 were decreased compared to controls at ambient pCO2. However, at deficient N-supply the decreased uptake rates were overcompensated by a significant increase of the root : shoot ratio. The enhanced root : shoot ratio at elevated pCO2 coincided with decreased N-contents of mature leaves, both on a f.- and d. wt basis. Apparently, in addition to an increased allocation of carbon to the roots, as indicated by enhanced sucrose contents in the bark tissue compared to sufficient N-supply, a second prerequisite to favour root growth may be an increased allocation of nitrogen to the roots at the expense of the leaves (Peuke, 2000; Walch-Liu et al., 2001). However, at elevated pCO2 the N-transport in basipetal direction appeared to be decreased rather than increased, irrespective of the N-supply.
After severe N-starvation the root : shoot ratio does not correlate with the allocation of newly aquired 15N
In a 13C and 15N double-labeling experiment, low N-supply increased the allocation of newly fixed carbon and nitrogen to the root of Quercus robur, whereas high N-supply tended to decrease allocation of new C to roots and increase allocation of new N to the shoot (Maillard et al., 2001). In the present experiment with Populus, however, just the opposite was observed for the allocation of newly acquired 15N. At deficient N-supply, the allocation of 15N was increased in mature leaves, but decreased in root tissue as compared to sufficient N-supply, irrespective of pCO2. To interpret these findings, it is necessary to consider that the nutrient solutions were changed once a week and the NO3− concentration was depleted, probably more at deficient and less at sufficient N-supply, by the time of 15N application. Obviously, the allocation of newly aquired 15N does not reflect the balance of biomass accumulation in the shoot and root during the time of 15N enrichment, but points at a more dynamic response.
Therefore, enhanced allocation of newly aquired 15N to mature leaves at deficient compared to sufficient N-supply may partly be a replenishment of previously depleted N-contents. However, at deficient N-supply the allocation of 15N to young, actively growing leaves was even more pronounced compared to sufficient N-supply. During the time of 15N enrichment, shoot growth appeared to be higher at deficient than at sufficient N-supply, although poplar plants still exhibited an increased root : shoot ratio at deficient N-supply. This assumption is supported by differential effects of the N-supply on the 15N and amino-N accumulation in the apex. The growth of the apex is dependend on the supply of amino-N by acropetal phloem transport, since the reduction of NO3− is neglectable in apical tissue, initially not connected to the transpiration stream (Bush, 1999). At sufficient N-supply, large amounts of amino-N, especially Asn and Gln, accumulated in the apex. Compared to deficient N-supply, however, the accumulation of newly aquired 15N decreased, indicating that at sufficient N-supply large amounts of the amino-N were present in the apex before the application of 15N. In contrast, at deficient N-supply 15N accumulation in the apex was slightly increased, whereas amino-N contents were substantially decreased compared to sufficient N supply. These findings suggest intensified N-metabolism, leading to enhanced protein synthesis, which in turn may accelerate the growth of apical tissue and, thus, promote shoot growth. How can we account for these results at deficient N-supply, which ought to emphasise root growth over shoot growth? Obviously, a larger fraction of the assimilated nitrogen taken up was invested into shoot growth, immediately after exchange of the nutrient solution. However, after depletion of NO3− in the nutrient solution at deficient N-supply, root growth was promoted, as indicated by the increased root : shoot ratio as a longterm indication of the nutrient status. In contrast, mechanisms which govern the allocation of newly aquired 15N appear to respond more rapidly and in a flexible manner to alterations of the external N-availability.
Discrepancies between actual measurements and model calculations of N-accumulation and fluxes
N-accumulation and fluxes in the present model calculations are flawed by two factors. First, at deficient N-supply the exchange of the nutrient solution appeared to affect 15N-partitioning differently from what may be expected under steady state conditions (e.g. at a constant external NO3−). After three days of 15N exposure, NO3− concentrations of the nutrient solution were decreased by 60% to 70% at deficient N-supply, but only by approx. 15% at sufficient N-supply. Thus, in the long term, allocation of newly acquired N between roots and leaves appeared to fluctuate to a higher extent at deficient compared to sufficient N-supply. Second, xylem sap was collected at a single time point at the end of the tracer-period, but transpirational water loss was calculated as the mean response of the entire 3-d period. At deficient N-supply, this led to a considerable underestimation of xylem transport of N-compounds, since the external N-concentration was depleted by the time of xylem sap collection. It cannot be excluded that after exchange of the nutrient solution, NO3− transport to the shoot and its subsequent reduction was initially higher than resolved by the present model calculations. By contrast, xylem transport of nitrogen was slightly overestimated at sufficient N-supply, probably because diurnal variations of N-concentrations in the xylem sap were not analysed.
Mechanisms governing N-allocation at the whole plant level and alterations observed at different N-supplies and pCO2
Despite the lack of precise flux calculations, the model estimates revealed the most important physiological alterations, which occur as a consequence of different external NO3− supply. Increasing the N-supply from deficient to sufficient caused the site of nitrate reduction to shift from the roots to the leaves. At deficient N-supply, elevated pCO2 further increased root nitrate reduction compared to controls at ambient pCO2. Although at the end of the 15N labeling period there was still some NO3− left in the nutrient solution, NO3− was almost absent in the xylem sap of plants grown at elevated pCO2, but made up 25% of TSNN at ambient pCO2. By contrast, amino-N contents of the xylem sap were slightly increased at elevated compared to ambient pCO2, indicating increased nitrate reduction in the roots at elevated pCO2. Recently, it was shown that the loss of root nitrate reduction dramatically disturbs the coordination of C- and N-metabolism of tobacco plants (Hänsch et al., 2001; Kruse et al., 2002). Root nitrate reduction appeared to be an indispensable factor for the regulation of the root : shoot ratio in tobacco. Because of the increased root nitrate reduction and root : shoot ratio in the present study, it may be concluded that root nitrate reduction triggers root growth also in poplar plants. The distribution of total nitrate reduction between the roots and the shoot depends on the species (Gebauer & Schulze, 1997; Bauer & Berntson, 2001) and, more importantly, on the nutrient supply, because limiting NO3− availability favours root- over shoot nitrate reduction (Andrews, 1986; Andrews et al., 1992). At elevated pCO2, the sensitivity of poplar plants to nitrogen limitation appears to be enhanced, resulting in an overproportional increase of root growth. At appropriate N-supply (e.g. immediately after exchange of the nutrient solution), total N-acquisition of poplar plants was higher at elevated than at ambient pCO2, although the rates of N-uptake were decreased. However, under these circumstances the nitrogen acquired was obviously not used for root growth, but predominatly allocated to the youngest leaves.
The youngest leaves are exposed to full light and may therefore perform the highest rates of photosynthesis. In the youngest leaves, maximal rates of photosynthesis depend more crucially on total nitrogen contents than in mature leaves frequently restricted by self-shading (Takeuchi et al., 2001). At deficient N-supply, with low leaf N concentrations, as well as at elevated pCO2 the accelerated allocation of newly acquired nitrogen to the youngest leaves appears to optimize photosynthesis on a whole plant basis. Takeuchi et al. (2001) observed that elevated pCO2 increased maximum rates of photosynthesis only in the upper canopy of Populus tremuloides trees, but did not stimulate photosynthesis in the shade. Leaf developmental stage has been proposed an important factor for the acclimation response at elevated pCO2 (Miller et al., 1997) and it has been observed that C-assimilation was decreased in mature, but increased in young leaves (Kelly et al., 1991). Such differential response to elevated pCO2 may also be the consequence of a prefered allocation of nitrogen to the youngest leaves, as observed in the present study.
A role for root nitrate reduction to ‘sense’ external N-availability for the N-partitioning between roots and the shoot?
Several mechanisms that may control the root : shoot ratio and the distribution of nitrogen within the plant have been proposed, especially involving the effects of phytohormones (Beck, 1996; Munns & Cramer, 1996). Since root nitrate reduction affects the distribution of biomass between roots and the shoot of tobacco (Kruse et al., 2002), it may also be suggested that root nitrate reduction is important for the partitioning of nitrogen between different organs. For example, the preference of many woody species to reduce NO3− in the root appears to be a consequence of their low N-uptake capacity, preventing xylem loading of NO3− (Gojon et al., 1991). However, Populus, a rapidly growing pioneer species, exhibits a relatively high physiological upake capacity and the external N-supply appears to affect the extent of NO3− xylem loading directly (Siebrecht & Tischner, 1999). It seems that the external N-supply is ‘sensed’ by root nitrate reduction. At a certain threshhold value, which in the present study may have been achieved at deficient N-supply after exchange of the nutrient solution, this unknown signal triggers whether newly aquired N is predominantly allocated to the roots or to the shoot.
At sufficient N-supply, elevated pCO2 also increased the allocation of 15N to the youngest leaves. However, at sufficient N-supply growth was not affected by the pCO2. The reasons for the discrepancy between literature data revealing a distinct CO2 effect on growth especially at sufficient N-supply (Curtis & Wang, 1998) and the present findings are not clear. It was shown that genotypic variations of Populus are crucial for the response to elevated pCO2 (Wang et al., 2000). At sufficient N-supply, elevated pCO2 decreased NO3− acquisation and xylem transport, leading to decreased NO3− contents in mature leaves. Reduction of NO3−in situ and the concentrations of organic N in leaves, however, appeared to be hardly affected by the pCO2. The decrease of Gln, NH4+, Gly- and Ser contents in mature leaves rather point to decreased photorespiration at elevated pCO2, with a significant fraction of the protein-N not involved in the photosynthetic processes.
This study was financially supported by the Deutsche Forschungsgemeinschaft (Re: 515/5 and Po: 362/3). We are grateful to C. Kettner for maintenance of the plants.
For model calculations, the source-leaves, sink-leaves und the apex of poplar plants were combined to the fraction ‘leaf’. Accumulation and fluxes of nitrogen were determined as follows:
where (NO3−)r is the accumulation of NO3− in the root, [N]root the N-content of the root, [NO3−]root the NO3− content of the root and 15Nroot the 15N-accumulation in the root during one day.
(NO3−)l is the accumulation of NO3− in the leaves, [N]leaf the N-content of the leaves, [NO3−]leaf the NO3− content of the leaves and 15Nleaf the 15N-accumulation in the leaves during one day.
(Nred)r is the accumulation of reduced N in the root and [Nred]root the content of reduced N in the root.
(Nred)l is the accumulation of reduced N in the leaf and [Nred]leaf the content of reduced N in the leaf.
The following fluxes were determined experimentally:
( Eqn 5)
JuptNO3− is the uptake of nitrate during the period of enrichment, calculated from the sum of 15N accumulation in the different plant organs harvested.
JxNO3− = T × [NO3−]xyl(Eqn 6)
JxNO3− is the xylem transport of NO3− to the shoot, T is the transpiration of the plant (ml plant−1 day−1) and [NO3−]xyl the NO3− -content of the xylem.
JxNred = T × [Nred]xyl(Eqn 7)
JxNred is the xylem transport of reduced N to the shoot and [Nred]xyl the amounts of reduced N ml−1 xylem sap. From equations 1–7 the remaining fluxes can be calculated:
is the amount of nitrate reduced in the root.
is the amount of nitrate reduced in the leaves.
Jp load is the amount of reduced N, loaded into the phloem.
Jp unload is the amount of reduced N, unloaded from the phloem.
(N)s is the amount of N, accumulating in the stem.