Journal of Experimental Botany, Vol. 53, No. 379, pp. 2351-2367,
December 1, 2002
© 2002 Oxford University Press
Elevated pCO2 favours nitrate reduction in the roots of wild-type tobacco (Nicotiana tabacum cv. Gat.) and significantly alters N-metabolism in transformants lacking functional nitrate reductase in the roots
Received 18 April 2002; Accepted 18 July 2002
1 Universität Freiburg, Institut für Forstbotanik und Baumphysiologie, Professur für Baumphysiologie, Georges-Köhler-Allee, Geb. 053/054, D-79085 Freiburg, Germany
2 Botanisches Institut der TU Braunschweig, Humboldtstr. 1, D-38106 Braunschweig, Germany
3 Universität Hohenheim, Institut für Pflanzenernährung, Fruwirthstr. 20, D-70593 Stuttgart, Germany
4 Universität Bayreuth, Fachgruppe Geowissenschaften, Abteilung Agrarökologie D-95440 Bayreuth, Germany
5 To whom correspondence should be sent. Fax: +49 761 203 8302. E-mail: here{at}uni-freiburg.de
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Abstract |
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The impact of elevated pCO2 on N-metabolism of hydroponically grown wild-type and transformed tobacco plants lacking root nitrate reduction was studied in order to elucidate the effects on (i) nitrate uptake, (ii) long-distance transport of N, (iii) nitrate reduction with emphasis on root-NR, and (iv) the allocation of N between the root and shoot. The findings were related to alterations of growth rates. At elevated pCO2 the wild type exhibited higher growth rates, which were accompanied by an increase of NO3–-uptake per plant, due to a higher root:shoot ratio. Furthermore, elevated pCO2 enhanced nitrate reduction in the roots of the wild type, resulting in enhanced xylem-loading of organic N (amino-N) to supply the shoot with sufficient nitrogen, and decreased phloem-transport of organic N in a basipetal direction. Transformed tobacco plants lacking root nitrate reduction were smaller than the wild type and exhibited lower growth rates. Nitrate uptake per plant was decreased in transformed plants as a consequence of an impeded root growth and, thus, a significantly decreased root:shoot ratio. Surprisingly, transformed plants showed an altered allocation of amino-N between the root and the shoot, with an increase of amino-N in the root and a substantial decrease of amino-N in the shoot. In transformed plants, xylem-loading of nitrate was increased and the roots were supplied with organic N via phloem transport. Elevated pCO2 increased shoot-NR, but only slightly affected the growth rates of transformed plants, whereas carbohydrates accumulated at elevated pCO2 as indicated by a significant increase of the C/N ratio in the leaves of transformed plants. Unexpectedly, the C/N balance and the functional equilibrium between root and shoot growth was disturbed dramatically by the loss of nitrate reduction in the root.
Key words: C:N balance, elevated pCO2, functional equilibrium, long-distance transport, N-uptake, nitrate reduction, root:shoot ratio.
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Introduction |
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During the last decade considerable attention has been paid to plant growth and metabolism under elevated pCO2, since atmospheric pCO2 is expected to double by the end of this century (Bowes, 1993; Saxe et al., 1998). Under these circumstances C3-plants may perform photosynthesis at a higher rate, because the carboxylation-efficiency of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) will be increased at the expense of a lower oxygenation rate of ribulose-1,5-bisphosphate, hence resulting in a lowered photorespiration (Stitt, 1991; Conroy and Hocking, 1993). Numerous studies have shown that elevated pCO2 initially stimulates carbohydrate-production and plant growth (Stitt and Krapp, 1999). However, this benefit ultimately vanished as the carboxylation capacity tended to decrease after prolonged exposure to elevated pCO2 (Makino et al., 1997; den Hertog et al., 1996). The acclimation of photosynthesis to elevated pCO2 seems to depend on several factors, for example, the species examined (Bazzaz and McConnaughay, 1992; Baker and Allen, 1993; Poorter, 1993), the developmental stage of the plants (Besford et al., 1990; Kelly et al., 1991; Coleman et al., 1993), the duration of CO2-exposure (Sionit and Kramer, 1986), the CO2-concentration applied (Allen et al, 1990), the temperature (Baker et al., 1993), the availability of water (Chaudhuri et al., 1990) and nutrients (Roberntz and Linder, 1999) or, more generally, down-regulation of photosynthesis depends on the sink-strength for carbohydrates (Herold, 1980; Stitt, 1991; Arp, 1991; Miller et al., 1997; Moore et al., 1999).
The capacity of CO2-assimilation and leaf-nitrogen concentation are tightly linked (Evans, 1989), and it is recognized that acclimation of photosynthesis is connected with the N-status of the plant (Stitt and Krapp, 1999). An increase of the C/N-ratio of leaf dry-matter is a common feature observed at elevated pCO2, mainly due to the accumulation of non-structural carbohydrates, especially starch (Sims et al., 1998), which leads to a ‘dilution’ of nitrogen in the dry matter (Stitt and Krapp, 1999). However, at elevated pCO2 the N-content of leaves is often decreased, even after correcting for increased starch contents (Poorter et al., 1997). Growth at elevated pCO2 appears to result in a decrease of the NO3–-contents of leaves (Hocking and Meyer, 1991), but after prolonged exposure the protein contents of leaves are also reduced by 15–20% (Peñuelas et al., 1997; Poorter et al., 1997). The decrease of total protein-N at elevated pCO2 corresponded to a decrease of Rubisco-N (Drake et al., 1997). These phenomena, observed at elevated pCO2, are more pronounced in plants grown at a deficient compared to a sufficient nitrogen supply (Petterson and MacDonald, 1994; Stitt and Krapp, 1999). The decrease of leaf-N concentrations suggest that N-uptake and -assimilation often fails to keep pace with photosynthesis and growth at elevated pCO2 (Stitt and Krapp, 1999). At elevated pCO2, N-uptake rates were reported to be decreased (Jackson and Reynolds, 1996; Makino et al., 1997), not affected (Bassirirad et al., 1997) or enhanced (Bassirirad et al., 1996; Makino et al., 1997). Such differences in the experimental findings might be explained, in part, by different N-application rates or by an exhaustion of the external N-supply followed by an initial acceleration of plant growth at elevated pCO2. Contradictory results have also been obtained for N-assimilation at elevated pCO2 (Stitt and Krapp, 1999). Although it may be assumed that nitrate reduction is enhanced concomitantly with photosynthesis and growth, until recently there was no consistent evidence for a stimulation of nitrate assimilation in elevated pCO2 (Stitt and Krapp, 1999). Geiger et al. (1998) showed that elevated pCO2 altered the diurnal rhythm of NR-activity in tobacco, because the decrease of NR-activity at the end of the light period was partly abolished at elevated pCO2. Thus, integrated over the entire day, elevated pCO2 appeared to enhance nitrate reduction in the leaves of tobacco plants.
By contrast to shoot-NR, the impact of elevated pCO2 on the nitrate reduction in roots has rather been neglected in recent years, probably because in herbaceous species the bulk of the nitrate taken up is reduced in the leaves (Andrews, 1986) and, thus, shoot-NR appeared to be more important than root-NR with respect to N-assimilation. Fonseca et al. (1997) observed an initial increase in nitrate reductase transcript level and a distinctly enhanced NR activity in roots of Plantago major following CO2-enrichment, which were, however, reversed after a few days of exposure. In young tobacco seedlings, elevated pCO2 distinctly increased Gln and Ala contents especially in the roots (Geiger et al., 1998), indicating an increased root-NR. However, it was shown recently that elevated pCO2 has a rather variable effect on root-NR-activity in nitrate-grown tobacco plants, depending on the type of cultivation and the time of the day (Matt et al., 2001). During the day, root-NR-activity increased in sand-grown and decreased in hydroponically grown plants, both having access to high nitrate. Furthermore, the effect of elevated pCO2 appears to interact with daytime, because at elevated pCO2 root-NR-activity was generally increased at night compared to the light-period (Matt et al., 2001).
The present experiments were performed with tobacco plants grown in hydroponic culture, supplied with a nitrogen concentration at a super-optimal level (5 mM NO3–, Matt et al., 2001), in order to avoid N-limitation, which may superimpose the ‘direct’ effects of elevated pCO2 on the N-metabolism of plants (Stitt and Krapp, 1999). The intention was first to investigate whether elevated pCO2 increases the growth rates of wild-type tobacco grown at a sufficient N-supply, decreases leaf-N or alters the allocation of nitrogen at the whole plant level. In a modelling approach the aim was to identify mechanisms which govern N allocation at ambient and elevated pCO2, including N-uptake, nitrate reduction and its partitioning between the root and the shoot, and xylem-loading of nitrate and organic N. In situ fluxes of N were integrated over the period of an entire day to avoid interference with diurnal rhythms. Second, the aim was to clarify if elevated pCO2 increases N-uptake and nitrate reduction, when plants are grown at sufficient N-supply. Finally, the definite importance of root-NR at elevated pCO2 was evaluated. For this purpose transformed tobacco plants lacking root-NR were employed, which exhibited similar shoot NR-activity to the wild type (Hänsch et al., 2001).
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Plant material
The present experiments were performed with wild-type Nicotiana tabacum cv. ‘Gatersleben’, and the transformant nia30/LNR (LNR, Leaf Nitrate Reductase). This transformant lacks nitrate reductase activity in the roots, but contains 75–100% of wild-type nitrate reductase activity in the leaves. The construction and basic features of nitrogen metabolism of this transformant as compared to the wild type have previously been reported (Hänsch et al., 2001). From several transgenic lines exhibiting similar features (Hänsch et al., 2001) the line nia30/LNR-H was chosen for the present experiments.
Tobacco seeds were germinated on the growth medium described by Scheible et al. (1997) and were grown for 3 weeks in a climate-controlled growth chamber (Hereaus-Vötsch, HPS 1500, Balingen, Germany) at a day/night regime of 12/12 h, at 25/20 °C, a relative air humidity of 80–90% and a photosynthetic active radiation (PAR) of 250 µE m–2s–1 at leaf level. After 3 weeks the plants were transferred to 2.8 l pots containing well-aerated nutrient solution of the following composition: K+ 2.0 mM, Ca2+ 2.5 mM, Mg2+ 1.2 mM, H2PO-4 0.5 mM, SO42– 1.2 mM, FeEDTA 10 µM, H3BO3 10 µM, Mn2+ 0.5 µM, Zn2+ 0.5 µM, Cu2+ 0.1 µM, MoO42– 0.07 µM. After transfer the growth conditions were changed to a day/night regime of 14/10 h at 25/20 °C, a relative air humidity of 80% and 350–400 µE m–2s–1 PAR. During the first week of hydroponic growth NO3– (3.4 mM) plus NH+4 (1.7 mM) were added to the nutrient solution. Subsequently, half of the plants were exposed to 35 Pa CO2, the other half to 70 Pa CO2, and NO3– (5.1 mM) was the sole nitrogen-source for the growth of the plants. Nutrient solutions were changed three times a week. Plants were rotated within the chambers after each exchange to minimize chamber effects. After 3 weeks of hydroponic growth, about half of the plants were harvested 3 h after the beginning of the light period, the other half 3 h after the beginning of the dark period. Some plants were left in the growth chambers for the determination of growth rates and transpiration. Shoot and root biomass were determined; 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 then stored until analysis.
Growth rate and transpiration
The accumulation of plant biomass was determined on the day of the experiment during an entire day/night cycle (24 h) and expressed as the relative growth rate on a fresh weight basis. Transpiration was calculated from the water loss of the 2.5 l pots during the 14 h light and 10 h dark period, respectively. 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.
Collection of xylem sap and phloem exudates
Xylem sap was collected by a modification of the method of Scholander et al. (1965) described by Rennenberg et al. (1996) within the light and dark periods, starting the collection 3 h after the beginning of the light or dark period, respectively. The stem was cut with a sharp blade above the root, which was put into a pressure chamber. Subsequently, the pressure was slowly increased until the first drops appeared at the cut end. Aliquots of 50–75 µl of the xylem sap were collected and stored at –80 °C until analysis. Phloem exudates were collected by the EDTA-technique desribed by Rennenberg and Thoene (1987). For this purpose, the roots of the tobacco plants were cut off and the shoot was placed at 100% relative air humidity into vials containing a solution with 20 mM EDTA and 0.15 µM chloramphenicol. Plants were allowed to exude for 2 h.
Uptake of NO3–
The determination of NO3–-uptake was performed 3 h after the beginning of the light and dark periods with excised roots according to the method described by Kreuzwieser et al. (1997), using 15N-NO3– as a tracer. Uptake of 15NO3– was assayed after transfer of excised roots to 300 ml beakers, containing the complete nutrient solution (pH: 7.0; atom% 15N: 20%). After 1 h the incubation was stopped, the fresh weight of the roots was determined and, subsequently, roots were dried. After measurement of dry weight, the roots were ground to a fine powder using a mill (Retsch, MM 2000, Haan, Germany). Total N and 15N contents of the ground samples were determined with an automated on-line CN analyser (NC 2500, CE Instruments, Milan, Italy) coupled with an isotope mass spectrometer (Finnigan MAT GmbH, Bremen, Germany).
Analysis of tissue-N and -C content
Total nitrogen and total carbon was estimated in dried plant material with an NCS 2500 Element Analyser (CE Instruments, Milan, Italy) using Dumas combustion. In this instrument the sample is oxidized yielding a gas mixture in which CO2 and N2 are detected by a thermoconductivity detector.
Extraction and analysis of amino compounds
Amino compounds were extracted from plant material by a modification of the method described by Geßler et al. (1998). Roots and and half of each leaf were ground with mortar and pestle. Plant material was homogenized in a buffer containing 20 mM HEPES (pH 7.0), 5 mM EGTA, 10 mM NaF, and 1.2 ml of chloroform:methanol (1.5:3.5, by vol). Water-soluble metabolites were extracted with double-distilled water and the aqueous phases were combined and freeze-dried (Alpha 2-4, Christ, Osterode, Germany). Amino compounds were also analysed in xylem sap and phloem exudates. For this purpose, the xylem sap collected was diluted with lithium citrate buffer. Phloem exudates were adjusted with 5 N HCl to pH 2.2.
An aliquot of each sample was injected into an automated amino-acid analyser (Biochrom, Pharmacia LKB, Freiburg, Germany) using a system of five lithium citrate buffers and a gradient from pH 2.8 to 3.55. The amino compounds separated were subjected to post-column derivatization with ninhydrin and, subsequently, measured at 440 and 570 nm.
Extraction and determination of nitrate
Aliquots of 20 mg of the homogenized plant material were added to 1 ml of double-distilled water. Samples were shaken for at least 2 h with 50 mg of PVPP (Sigma Chemie, Deisenhofen, Germany) to remove phenolic compounds. The homogenates were centrifuged for 10 min at 4 °C and 16 000 g. Aliquots of 300 µl of the clear supernatants were injected into an ion chromatograph (DX 100; Dionex, Idstein, Germany). Anions were seperated on a IonPac® column (AS9-Sc 250x4 mm; Dionex, Idstein, Germany) with a solution containing 1.8 mM Na2CO3 plus 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).
Data analysis
Different parameters of the N-metabolism were analysed in wild-type and transformed tobacco plants by day and night in 5–6 independent replicates each. For statistical analysis, data were subjected to ANOVA (SPSS 5.0 for windows, SPSS Inc., USA).
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Growth parameters and transpiration
Biomass accumulation was analysed in wild-type tobacco and the transformant nia30/LNR grown for 3 weeks at ambient and elevated pCO2. Elevated pCO2 increased the biomass by 80% and 45% in wild-type and transformed plants, respectively (Table 1). On the final day of cultivation, the growth rates of wild-type tobacco were higher at elevated than at ambient pCO2, whereas the growth-rates of the transformants were similar, irrespective of the pCO2. Elevated pCO2 enhanced the root/shoot-ratio of wild-type tobacco, whereas the distribution of the biomass between roots and the shoot was not affected by the pCO2 in transformed plants.
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On the day of analysis, the transpiration of whole tobacco wild-type plants was significantly enhanced at elevated pCO2 compared with controls grown at ambient pCO2 due to the enhanced biomass of wild-type plants grown at elevated pCO2 (Fig. 1A). However, elevated pCO2 decreased the rates of transpiration for both wild-type and transformed tobacco plants during the day. The transition from light to dark resulted in a decrease of transpiration rates by 75% (Fig. 1B).
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N-contents in leaf and root tissue
In the light, elevated pCO2 increased the dry weight and the C/N-ratio of the leaves of wild-type tobacco, whereas the N-content of the dry mass was decreased compared with controls at ambient pCO2. By contrast, elevated pCO2 had no effect on the N-content when expressed on a fresh weight basis (Table 2). In the night, the dry weight and the C/N-ratio of wild-type leaves were decreased compared to the day, whereas the N-content of the dry mass was enhanced. Statistical analysis of dry weight, C/N-ratio and N-content of dry mass showed a strong positive correlation between dry weight and the C/N-ratio of leaves, and a negative correlation between dry weight and the N-content of leaf dry mass (Fig. 2). From the change in these parameters between day and night, a decrease of the N-content in leaf fresh mass of approximately 50 µmol g–1 FW was calculated. By contrast to daytime, elevated pCO2 did not influence the dry weight and C/N-ratio of leaves from the wild type during the night.
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The dry weight and C/N-ratio of leaves from transformed plants were significantly increased, and the N-content of leaf dry mass significantly lowered compared to wild-type plants. Still, the N-contents in the leaf fresh mass of wild-type and transformed plants were similar. The effect of elevated pCO2 on dry weight, C/N-ratio and N-content in the dry mass of leaf tissue was more pronounced for the transformed than for wild-type plants. This finding was indicated by a significant interactive effect of transformation and CO2-concentration on the parameters mentioned (not shown). In the light period, elevated pCO2 led to a strong increase of the C/N-ratio and a decrease of N in the dry matter of leaves from transformed plants compared to controls at ambient pCO2. The differences observed during the day were maintained at night.
In root tissue of wild-type and transformed plants, dry weight and N-content were similar for night and day. Elevated pCO2 did not affect these parameters either, whereas a significant impact of the transformation on the dry weight and N-content of the roots was found: a slight reduction of the N-content in the dry matter was compensated by an accumulation of dry matter in the root of transformed tobacco plants, resulting in an increased N-content per unit root fresh weight (Table 2).
TSNN, NO3–, NH4+, and total amino-N in leaf and root tissue
The fraction of low molecular weight N-compounds was analysed in more detail in leaves and roots during the day and at night (Table 3). The bulk of TSNN (total soluble non-protein nitrogen) in both leaves and roots consisted of NO3–, with generally higher contents in leaf compared to root tissue. After 2 weeks exposure to elevated pCO2 the TSNN contents in the leaves of wild-type plants were decreased by approximately 17% compared to controls at ambient pCO2, due to a reduction of the NO3–-contents by c. 30%. The amount of amino-N was enhanced at elevated pCO2 in leaves of the wild type during the day. The NH4+ contents in leaves of wild-type plants grown at elevated pCO2, which repesented only a small fraction of TSNN (<5%), were reduced during the day by 40% compared to controls at ambient pCO2. By contrast to daytime, elevated pCO2 had no effect on amino-N and NH4+ in leaves of wild-type plants during the night. The interactive effect of pCO2xlight on the parameters mentioned was statistically significant (not shown).
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Table 3 shows that the leaves of the transformant differ substantially from the wild type in all parameters analysed. At ambient pCO2 transformed plants accumulated more NO3– in their leaves than wild-type plants. This effect was less pronounced at elevated pCO2, which resulted in a significant interaction of pCO2xtransformation on the NO3–-content of the leaves (not shown). By contrast to the enhanced levels of NO3– in leaves of transformed plants, the amounts of amino-N and NH4+ were significantly reduced compared to wild-type plants. During the light, elevated pCO2 increased the amounts of amino-N in leaves of wild-type plants, which was not observed for transformed plants (significant interaction of pCO2xtransformation). The transition from light to dark led to a decrease in amino-N by 25–50% at ambient or elevated pCO2, respectively, and a reduction in NH4+ by 75% in leaves of wild-type plants, whereas amino-N and NH4+ in leaves of transformed plants were much less affected by light.
Exposure to different pCO2 and the change from day to night did not influence the TSNN, NO3–, NH4+, and amino-N contents of roots from wild-type and transformed tobacco plants. Still, roots of transformed plants differed significantly from wild-type plants in these parameters. Roots of the transformed plants exhibited higher NO3– contents, especially during the day and at ambient pCO2. During the day the NH4+-contents in the roots of transformed plants were generally lower compared to the wild type. Surprisingly, amino-N contents in roots of transformed plants, lacking functional NR activity in the root, were significantly higher than root amino-N contents of the wild type.
Nitrate uptake
After 2 weeks of growth in hydroponic culture with NO3– as the sole nitrogen source, nitrate net-uptake was measured during the day and at night (Fig. 3). At ambient pCO2, wild-type plants exhibited a rate of NO3–-uptake of 15 µmol N g–1 FW h–1. Rates of nitrate uptake of wild-type plants grown at elevated pCO2 were slightly, but not significantly decreased. During the day, the rates of NO3–-uptake of transformed plants were increased by approximately 20% compared to the wild type at ambient or elevated pCO2, respectively. The transition from light to darkness significantly decreased the rates of nitrate-uptake, especially in transformed plants grown at ambient pCO2. In wild-type plants, the rates of NO3–-uptake were decreased by 10% in the night, irrespective of the pCO2.
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TSNN, NO3–, NH4+, and amino-N in the xylem sap
On the day of analysis, xylem sap was collected during light and darkness and analysed for NO3–, NH4+ and amino-N (Table 4). The contents of TSNN summed to 41.3 µmol N ml–1 xylem sap from wild-type plants grown at ambient pCO2. Approximately 75% of TSNN was in NO3–-N, whereas NH4+ represented only a minor fraction of TSNN in the xylem sap. The remaining 25% of TSNN were found in amino-N, with Gln as the most abundant amino-compound (>60% of total amino-acids, not shown).
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TSNN-contents in the xylem sap of transformed plants were significantly enhanced compared to the wild type, both at ambient and elevated pCO2, because of enhanced NO3–-contents. At ambient pCO2, the amino-N content in the xylem sap of transformed plants was similar to the wild type. By contrast, elevated pCO2 led to a sharp decline of amino-N in the xylem sap of transformed plants, but not in the wild type. Analysis of variance of the data revealed a significant interaction between the pCO2 and the transformation (not shown). The composition of amino compounds in the xylem sap of the transformant differed significantly from the wild type. At ambient pCO2 transformed plants exhibited the highest amount of amino-N in the xylem sap, but still Gln-contents were significantly reduced compared to the wild type and, hence, the Gln/Glu-ratio was much lower. Elevated pCO2 further reduced the Gln/Glu-ratio in the xylem sap of transformed plants. With the exception of transformed plants at elevated pCO2 the transition from light to darkness caused a significant decrease of TSNN in the xylem sap of the plants, mediated especially by Gln. By contrast, NO3–-concentrations of the xylem sap were lower in wild-type plants by 10% at ambient and elevated pCO2, but were not affected in transformed plants by light.
N composition of phloem exudates
With the method applied, phloem exudates can only provide qualitative information. During day the most abundant amino compound in phloem exudates of all plants studied was Gln (Fig. 4). Gln made up 10% of total amino acids in leaves of transformed plants, but more than 30% of the amino compounds in phloem exudates. Relative abundance of Gln in phloem exudates collected by day was highest in wild-type plants grown at ambient pCO2, and was diminished in wild-type plants grown at elevated pCO2. This effect was also observed in transformed plants grown at elevated pCO2. By day, the amino acid composition of phloem exudates was similar for wild-type and transformed plants. The transition from day to night led to an overall decrease of Gln-abundance in phloem exudates compared to daytime. This decrease was more pronounced in transformed compared to wild-type plants. It was accompanied by an increase in Asp-abundance during the transition from light to darkness in wild type, but not in transformed plants, which instead exhibited an increase of Ser and other N-compounds.
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-aminobutyrate, and Orn.Plant internal N cycling
On the basis of the data set collected, uptake, accumulation and plant internal N-cycling was calculated in the plants studied. For modelling the different fluxes of N through the plant it was assumed that (1) the root/shoot-ratio did not change significantly over the period of analysis (1 d) and (2) that the tissue-N concentrations did not change during the day of the study for reasons other than the diurnal fluctuations of tissue-N (for a detailed description of the model see Appendix). Figure 5a depicts the flow profile of N for the wild type grown at ambient pCO2. Only 4% of the nitrate taken up was already reduced in the root of these plants, while the major part (94% of the nitrate taken up by the plant) was transported with the transpiration stream via the xylem to the shoot (Table 5). At ambient pCO2 13% of the nitrate taken up accumulated in the shoot, while the rest, delivered by xylem transport, was reduced in the shoot. The bulk of reduced nitrogen accumulated in the shoot, for example, in proteins, nucleic acids, amino-N or other high- and low-molecular N-compounds. However, a fraction of 20% from the nitrate taken up was loaded as amino-N into the phloem and was transported in a basipetal direction, in order to supply the root with organic N. At ambient pCO2, the roots of wild-type plants were strongly dependent on phloem-borne amino-N, as 70% of the organic N needed for root growth originated from the shoot.
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At elevated pCO2 the total nitrate uptake of the wild type was enhanced compared to controls grown at ambient pCO2. This effect was predominantly due to an increase of plant biomass by a factor of 1.77 at elevated pCO2 (Fig. 5c). Still, uptake of NO3– was enhanced by 7% compared to controls, after differences in plant biomass were taken into account. This increase in NO3–-uptake coincided with the increase in relative growth rates by 9% at elevated pCO2 (see above). For wild-type plants grown at elevated pCO2 the decline of the rates of NO3–-uptake (µmol N g–1 FW h–1) by a factor of 0.83 (see above) was overcompensated by an increase of the root/shoot-ratio by a factor of 1.32 (see above) compared to controls at ambient pCO2.
At elevated pCO2, a distinctly higher fraction of the nitrate taken up was already reduced in the roots of wild-type plants, while xylem transport of nitrate declined by 27% compared to controls at ambient pCO2 (Table 5). In the shoot of the wild type grown at elevated pCO2, the accumulation and reduction of NO3– was decreased in relation to the controls at ambient pCO2, whereas the allocation of NO3– and organic N to the root increased at elevated pCO2. At elevated pCO2, more organic N accumulated in leaves of the wild type than could be provided by the reduction of NO3– in the shoot. Thus, the shoot was dependent on the supply of organic N via xylem transport. In relative terms, the accumulation of organic N in leaves of wild-type plants was the same at ambient and elevated pCO2, but the origin of the organic N in the shoot differed between CO2-supplies, because of a shift of NO3–-reduction towards the root at elevated pCO2.
Total nitrate uptake of transformed plants grown at ambient pCO2 was diminished compared to the wild type (Fig. 5b). Even after correction for differences in plant biomass, the N-uptake of transformed plants was decreased by 5% compared to the wild type, correlating with the decrease of growth rates of transformed plants by 5%. The root/shoot-ratio of transformed plants was decreased by a factor of 0.81 compared to the wild type and could not fully be compensated by an increase of NO3– uptake rates by a factor of 1.15.
Only a small fraction of the nitrate taken up accumulated in the roots of transformed plants. The bulk was transported via xylem transport to the shoot. At ambient pCO2, the amounts of nitrate which were reduced in the shoot of transformed plants were similar to the wild type, at least in relative terms (Table 5). However, transformed plants accumulated more NO3– and less organic N in the leaves than the wild type. The roots of transformed plants were totally dependent on organic N derived from the shoot. Thus, loading, basipetal transport and unloading of the phloem with amino-compounds was enhanced in transformed plants. Xylem transport of organic N, however, was similar in transformed and wild-type plants (Table 5).
At elevated pCO2, whole-plant uptake of nitrate by the transformants was enhanced by a factor of 1.4 compared to transformed plants at ambient pCO2. This effect corresponded to the increase of the biomass by 40% at elevated pCO2 (Fig. 5d). The relative growth rate at the day of analysis, the root/shoot-ratio and the NO3– uptake rate were similar for transformed plants, irrespective of the pCO2.
At elevated pCO2, 2% of the nitrate taken up by transformed plants accumulated in the root and 90% was transported to the shoot via xylem transport (Table 5). From the model calculations, 9% of the NO3– taken up were reduced in the root of transformed plants lacking NR activity in the root. For the model, mean values gained experimentally were used to calculate different N-fluxes. The nitrate reduction calculated for roots of transformed plants grown at elevated pCO2 may have been the consequence of the deviation of the parameters measured. Thus, the nitrate-reduction in roots calculated for transformed plants without root-NR activity was not taken into account.
At elevated pCO2, 11.5% of the nitrate taken up accumulated in the leaves of transformed plants, 10.5% less than in transformants grown at ambient pCO2 (Table 5). By contrast, shoot-NR and accumulation of organic N in the shoot of transformed plants was higher at elevated than at ambient pCO2. The roots of transformed plants were dependent on phloem-borne organic N, irrespective of the pCO2. Phloem-unloading of organic N in the roots of transformed plants was similar at 35 and 70 Pa CO2, and larger than in wild-type plants. Still, at elevated pCO2 the transport of organic N was diminished by 34% in the phloem and 21% in the xylem compared to transformants at ambient pCO2. Thus, elevated pCO2 resulted in a decrease of plant internal N-cycling for both transformed and wild-type plants, but in wild-type plants this decrease could partially be compensated for by an enhanced xylem loading of organic N in the root.
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At sufficient N-supply, elevated pCO2 did not decrease leaf-N, and growth rates of wild-type tobacco were enhanced concomitantly with N-uptake per plant
In the current experiment, wild-type tobacco accumulated more biomass at elevated pCO2 and exhibited higher growth rates than controls grown at ambient pCO2. After prolonged exposure to elevated pCO2, there is often a gradual decline of photosynthesis and growth, usually accompanied by a decrease of protein-contents in the leaves (Poorter et al., 1997; Drake et al., 1997). In the present experiment, however, amounts of organic N in leaf tissue were similar at ambient and elevated pCO2. Judging from the increased growth rates and similar organic leaf-N concentrations at elevated pCO2 it appears unlikely that acclimation of photosynthesis had already occurred. As a logical consequence, N-metabolism must have been intensified at elevated pCO2 to provide the faster growing sinks with organic nitrogen. Intensification of N- metabolism at elevated pCO2 is indicated by the enhanced amounts of amino-N in the leaves of tobacco plants. Decreased NH4+ contents observed at elevated pCO2 were presumably due to lowered photorespiration, since only a small fraction of the NH4+ found in leaves originates from primary NO3– reduction, but instead has been released by photorespiration (Heldt, 1996). Although N-metabolism appeared to be intensified at elevated pCO2, the rates of N uptake (µmol g–1 FW h–1) were slightly decreased compared to controls at ambient pCO2. Nevertheless, the lowered rates of N-uptake were overcompensated by an increase in the root:shoot ratio, so that enhanced growth rates could be met by an increased N-uptake per plant. A shift of the root:shoot ratio has been proposed as a useful indicator of a deficient nitrogen supply at elevated pCO2 (Baxter et al., 1997). It has been stated that some of the characteristics of the photosynthetic acclimation to elevated pCO2 are similar to those with N-deficiency (Paul and Driscoll, 1997). For example, starch accumulates as a result of limited use of carbon skeletons in N-assimilation, which may hence lead to restricted growth and a subsequent drop of photosynthetic C-fixation. As a consequence, the source–sink balance of plants is altered and, under conditions of N-limitation, root-growth is favoured over shoot growth, which enables the plant to exploit the rooting medium more efficiently and to establish a new balance between C-fixation and N-acquisition (Brouwer, 1983; Field and Mooney, 1986).
At limiting N-supply, a further increase of the root:shoot ratio has been observed at elevated pCO2 (Walch-Liu et al., 2001), but this is unlikely to occur when plants are grown at sufficient N-supply (Curtis and Wang, 1998; Stitt and Krapp, 1999), as in the present experiment. Although it cannot be entirely excluded that the increase of the root:shoot ratio at elevated pCO2 was due to an inappropriate supply of nitrate to the external medium, such a scenario appears unlikely. The plants were grown in hydroponic culture with free access to 14 mmol of nitrate (5.1 mM, pot volume 2.8 l), the nitrate uptake of plants grown at elevated pCO2 was 3.6 mmol and, thus, the initial nitrate concentration was decreased by only 25%, which is still considered to be sufficient (Matt et al., 2001). Instead, the increase of the root:shoot ratio reflects the advanced development of wild-type plants grown at elevated pCO2. Subtle changes in the root:shoot ratio are a usual process in the development of plants (E Beck, personal communication). After the transition from a sink- to a source-leaf, the flow of assimilates in the phloem is redirected to the root, and may therefore affect root growth. Recently, it was shown that the root:shoot ratio, determined for tobacco plants of different size, oscillates around a given value (Walch-Liu et al., 2001). In the present experiment, wild-type plants grown at elevated pCO2 were significantly larger than controls, hence exhibiting an increased root:shoot ratio as a reflection of their advanced development. Nitrogen nutrition of plants is dependent on both the root:shoot ratio and the rates of N-uptake. The present results indicate that rates of N-uptake are low, when the root:shoot ratio is high as shown for wild-type plants grown at elevated pCO2. This relationship was reversed in transformed tobacco plants, which exhibited a low root:shoot ratio and significantly increased the rates of nitrate uptake, irrespective of the pCO2. Thus, part of the variability reported for the rates of N-uptake at elevated pCO2 (see Introduction for references) may be caused by developmental alterations of N-uptake.
Elevated pCO2 favours root-NR and xylem-loading of organic N
According to the model described, at elevated pCO2 a larger fraction of the NO3– taken up was reduced in the roots and the surplus was loaded into the xylem to provide the shoot with organic nitrogen. The distribution of NR between the root and the shoot varies greatly between species (Gojon et al., 1994), but is more dependent on the nitrate-supply (Andrews, 1986; Peuke, 2000). At a low external NO3– concentration, N-assimilation in the roots is enhanced, whereas translocation to the shoot and subsequent reduction becomes more important, when the external NO3– supply is increased. Again it is difficult to distinguish between a direct effect of elevated pCO2 or a putative N-limitation, resulting in an increased reduction of NO3– in the roots of wild-type plants. It is, however, possible that root-NR also varies with the plant developmental stage. Fonseca et al. (1997) observed an initial increase of nitrate reductase transcript level and a distinctly enhanced NR activity in roots of Plantago major following immediately after CO2-enrichment which was then reversed after 4 d of exposure. These observations are in good agreement with the finding that Gln and Glu-contents in the xylem sap of tobacco plants were significantly enhanced when xylem sap was collected shortly (3 d) after exposure to elevated pCO2 (unpublished results). In the present study, however, amino-N concentrations of the xylem sap were not different from those at ambient pCO2, although nitrate reduction in the roots was enhanced at elevated pCO2. At first glance this observation appears to contradict an enhanced root-NR. However, the existence of an internal N-pool, cycling between xylem and phloem is now well established (Marschner et al., 1997). At elevated pCO2, more organic N reaching the vein terminals via the xylem was taken up by the mesophyll and less of it was redistributed via phloem transport. This is in contrast to plants grown at ambient pCO2, where the majority of the NO-3 taken up was translocated to the shoot and subsequently reduced. The surplus of reduced N was loaded into the phloem to provide the root with reduced N.
It has been proposed that the pool of amino acids circulating between the xylem and the phloem represents a key element in the adaptation of N-uptake to the N-demand of the plant, for example, that remobilization of nitrogen from the shoot signals a decreasing demand and, thus, rates of N-uptake are down-regulated (Cooper and Clarkson, 1989; Imsande and Touraine, 1994). In the present study, an overall decrease of organic N circulating between the xylem and the phloem was observed at elevated pCO2. Also, at elevated pCO2 the abundance of Gln, which has been shown to play an important regulatory role for N- uptake in beech (Geßler et al., 1998), was diminished in phloem exudates. Still, the rates of nitrate uptake were rather decreased than increased at elevated pCO2. It appears, as if rates of N-uptake were affected by additional factors. However, there may also be a different explaination. As outlined above, the root:shoot ratio depends on the developmental stage of the plants. With the development of new leaves, the root:shoot ratio decreases, and total C-fixation increases as well as the growth rate. The growth rate of tobacco is just as variable as the root:shoot ratio and changes rapidly (E Komor, personal communication). In the present experiment, the decreased redistribution of organic N via phloem transport indicates that sink-strength of the shoot for organic nitrogen increases, probably preceding a further enhancement of growth rates. Consequently, up-regulation of nitrate uptake rates may just lag behind these alterations.
Taken together, alterations of growth rates appear to be mediated by flexible changes in the root:shoot ratio, rates of N-uptake and plant internal cycling of organic N between the xylem and phloem. There are indications in the literature that NR-activity in the roots is also very variable (Fonseca et al., 1997; Matt et al., 2001). Recently, plants have become available, which totally lack root-NR, but exhibit similar NR-activity in the shoot, as compared to the wild type (Hänsch et al., 2001). These transformants were used as a tool in order to assess the importance of root-NR for the N-nutrition of tobacco plants.
The loss of root-NR significantly alters plant growth, N-fluxes within the plant and allocation of nitrogen at the whole plant level. Elevated pCO2 increases shoot-NR in transformed plants, but only slightly affects growth rates
The basic features of the transformant nia30/LNR (LNR=Leaf Nitrate Reductase) lacking functional NR activity in the root have been described previously (Hänsch et al., 2001). At ambient pCO2, the growth of transformed plants was retarded compared to the wild type. Given enough time, transformed plants reached the same size as the wild type, but flowering was retarded by approximately 2 weeks (Hänsch et al., 2001). This is surprising, since it was shown that growth is only inhibited when NR activity is decreased by more than 90% compared to the wild type (Dorbe et al., 1992) and since tobacco has a much higher NR activity in the shoot than in the root (Hänsch et al., 2001). The roots of transformed tobacco plants are totally dependent on organic N originating from the shoot, and the results of the current experiments indicate that Gln in particular is translocated to, and metabolized in root tissue. By contrast with the wild type, all of the nitrate taken up by transformed plants was transported via the xylem to the shoot. It is presently under discussion whether NO3– also functions as a signal for plant growth (Crawford, 1995). Recently, Scheible et al. (1997) found that the root:shoot ratio correlates with an accumulation of nitrate in the shoot. This is in agreement with the present observation that the root:shoot ratio was decreased and NO3– contents of leaves substantially increased, when nia30/LNR was grown at ambient pCO2. At elevated pCO2, however, leaf nitrate contents of the transformed plants were similar to the wildtype, but the root:shoot ratio was still significantly decreased. It has been hypothesized by Stitt and Krapp (1999) that the root:shoot ratio depends more crucially on the influx of NO3–, rather than its accumulation in leaf tissue. From the results of the present experiment with transformed plants it may also be considered that the root:shoot ratio is under the control of xylem loading of NO3–. Such a mode of regulation is also confirmed by the recent finding that the xylem sap of transformed plants, exuded as a consequence of root pressure, contained 30% more nitrate than the wild type, irrespective of the pCO2 (J Kruse, C Engels, and H Rennenberg, unpublished results).
The impact of elevated pCO2 on plant size on the day of study was less pronounced than for the wild type and the current growth rates did not differ significantly from transformed plants grown at ambient pCO2. However, the reduction of nitrate in the shoot of transformed plants was increased at elevated pCO2. This may indicate a transition to increased growth rates, although the current growth rates were similar at ambient and elevated pCO2. Still, integrated over the entire growth period, elevated pCO2 preferentially enhanced the growth rates of wild-type plants, since its effect on biomass accumulation was higher in the wild type than in transformed plants.
On the day of analysis, tobacco wild type grown at elevated pCO2 was dependent on root-NR to provide the shoot with reduced N, but this was not possible in nia30/LNR. In transformed plants grown at elevated pCO2 the sink strength of organic N was high in both roots and the shoot and, consequently, the cycling-pool of organic N within the plant was substantially decreased. However, the nitrogen unloaded from the phloem was not invested into further root growth, but accumulated instead. Scheible et al. (1997) also found that roots of tobacco mutants with very low NR activity (1–3% compared to the wild type) contained more proteins and amino acids, but the amounts of soluble sugars decreased. As shown recently, the roots of transformed plants used in the present experiment also exhibited higher amounts of sugars (Hänsch et al., 2001). Thus, the impeded root growth of transformed plants was not the result of a deficiency of building blocks, for example, sugars and amino compounds. Rather, the loss of root-NR appears to influence root growth in an as yet unknown manner.
At elevated pCO2 the C/N-balance of tobacco plants was significantly altered by the loss of root-NR
Unexpectedly, the loss of root-NR not only led to an enhancement of organic N in the roots, irrespective of the pCO2, but at elevated pCO2 it also significantly increased the C/N ratio of leaf tissue. The C/N ratio varies according to the availability of resources, especially nitrogen. With inadequate N, the amino acid content of leaves falls, less protein is synthesized and growth is decreased generally more than photosynthesis, so carbohydrates accumulate and the C/N ratio is high (Lawlor, 2002). Frequently, elevated pCO2 also increases the C/N ratio of leaves (Paul and Driscoll, 1997). In wild-type tobacco, elevated pCO2 increased the C/N ratio only during the light period, whereas it was similar at ambient and elevated pCO2 at night, indicating an enhanced remobilization of starch during the dark period (see also Grimmer and Komor, 1999). The transient accumulation of starch seems to buffer the increased C-fixation at elevated pCO2 and is an important prerequisite for enhanced growth rates at elevated pCO2 (Ludewig et al., 1998). If the C/N ratio is used as a measure for the balance between C-fixation and growth, it may be concluded that the growth of transformed plants was inhibited to a greater extent than photosynthesis. However, a putative N-limitation, for example, as a consequence of exhaustion of NO3– in the external medium, was obviously not the cause for the increased C/N ratio. Obviously, the loss of root-NR not only dramatically disturbs the equilibrium between root and shoot growth, but also alters the C/N balance of leaves. According to the ‘functional equilibrium hypothesis’ root growth is limited by the supply of photosynthate from the shoot and the shoot growth by the supply of minerals from the root (Brouwer, 1983; Jackson, 1993). However, as sugars accumulate in roots of nia30/LNR (Hänsch et al., 2001), their impeded root growth cannot be explained by a lack of photosynthate. Sink-strength may be determined by metabolism, probably under control of phytohormones, such as cytokinins (Beck, 1993), which are also responsible for the remobilization of starch to support the growth of sink-tissues (Fetene and Beck, 1993). Still, the significance of root-NR, or the alteration of the metabolite profile induced by the loss of it, for the interaction with such signals has to be investigated.
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Conclusion |
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TopAbstractIntroductionMaterials and methodsResultsDisccussionConclusionAppendixReferences |
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At elevated pCO2, enhanced growth rates of wild-type tobacco coincided with increased N-uptake, mediated by a higher root:shoot ratio. Nitrate reduction was increased, preferentially in the roots of the wild type as well as xylem loading of amino-N to supply the shoot with organic N. The diminished redistribution of organic N in a basipetal direction via phloem transport suggests an increasing demand for shoot organic N to maintain enhanced growth rates at elevated pCO2. However, the results indicate that these alterations observed at elevated pCO2 may only represent a snapshot, taken at a given stage of plant development. It is suggested that alterations of the growth rate are co-ordinated by flexible responses in the rates of N-uptake, the root:shoot ratio, the distribution of nitrate reduction between the root and the shoot and plant internal cycling of organic N, in order to adjust the N-uptake to C-fixation, or vice versa. Unexpectedly, harmonized physiological responses are disturbed dramatically by the loss of root-NR. It significantly decreased growth and N-uptake, due to a low root:shoot ratio, increased xylem-loading of NO3– and, at ambient pCO2, led to an accumulation of NO3– in the leaves. Intriguingly, root growth was impeded, although organic N accumulated. Elevated pCO2 increased shoot-NR of transformed plants. Calculated over the entire growth period, it also increased growth rates, even though to a lesser extent than in the wild type. At elevated pCO2, a significantly increased C/N ratio in the leaves of transformed plants indicate that the remobilization of photosynthate is also dependent on root-NR.
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Acknowledgement |
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This research was supported by the Deutsche Forschungsgemeinschaft (RE 515/5).
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Appendix |
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Calculation of plant internal N-cycling
Over the period under study (1 d) the N-uptake and incorporation into different N forms were detemined as follows:
where (NO3–)r is the incorporation of NO3– into the root, RGRDexp is the plant’s growth rate at the day of the experiment, FWroot the root fresh weight, and [NO3–]r,day [NO3–]r,night the amount of nitrate g–1 root FW measured during the light and dark periods, respectively.
where (NO3–)s,day is the incorporation of NO3– into the shoot, FWshoot the shoot fresh weight, and [NO3–]s,day [NO3–]s,night the nitrate concentration of the shoot measured during the light and dark periods, respectively.
where [Nred]r is the incorporation of reduced N into the root and [Nred]r,day, [Nred]r,night the amount of organic N g–1 root FW measured during the light and dark periods, respectively.
where (Nred)s is the incorporation of reduced N into the shoot and [Nred]s,day [Nred]s,night is the concentration of organic N in the shoots measured during the light and dark periods, respectively.
The following fluxes of N were calculated:
where Jupt NO3– is the uptake of nitrate on the day of the experiment, [Upspec]day, [Upspec]night the specific uptake of nitrate in µmol g–1 FW h–1, measured at day and night. With the addition of the integral in equation (5) the growth of the root during the day of study (t=1 d) was taken into account.
JxNO3–=Tdayx[NO-3]xyl,day+Tnightx[NO-3]xyl,night(6)
where JxNO3– is the xylem transport of NO3– to the shoot, Tday, Tnight is the plant’s transpiration (ml plant–1) during the day and at night and [NO-3]xyl,,day [NO-3]xyl,night is the amount of NO-3 ml–1 xylem sap during the day and at night.
JxNred=Tdayx[Nred]xyl,day+Tnightx[Nred]xyl,night(7)
where JxNred is the xylem transport of reduced N to the shoot and [Nred]xyl,day [Nred]xyl,night is the amount of Nred ml–1 xylem sap during the day and at night.
From equations 1–7, the remaining flows within the plant can be calculated:
Jrred=JUptNO-3–JxNO-3–(NO-3)r(8)
where Jrred is the amount of nitrate reduced in the root;
Jsred=JxNO-3–(NO-3)s(9)
where Jsred is the amount of nitrate reduced in the shoot;
Jp,load=Jsred–(Nred)s(10)
where Jp,load is the amount of reduced N loaded into the phloem;
Jp,unload=(Nred)r–Jrred(11)
where Jp,unload is the amount of reduced N unloaded from the phloem;
JpNred=JxNred+Jp,load(12)
where JpNred is the amount of reduced N transported via the phloem from the shoot to the root.
Assumptions
For modelling the different fluxes of N through the plant, it is assumed that (1) the root/shoot ratio and (2) the tissue-N concentration did not change significantly during the day of study for reasons other than diurnal fluctuations (e.g. remobilization of leaf-N due to external N-limitation).
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  M. ANDREWS, J. A. RAVEN, P. J. LEA, and J. I. SPRENT A Role for Shoot Protein in Shoot-Root Dry Matter Allocation in Higher Plants Ann. Bot., January 1, 2006; 97(1): 3 - 10. [Abstract] [Full Text] [PDF]
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