Journal of Experimental Botany, Vol. 52, No. 359, pp. 1251-1258,
June 1, 2001
© 2001 Oxford University Press
Original Papers |
Tobacco plants that lack expression of functional nitrate reductase in roots show changes in growth rates and metabolite accumulation
1 Botanisches Institut, Technische Universität Braunschweig, D-38106 Braunschweig, Germany
2 Institut für Pflanzenernährung, Universität Hohenheim, D-70593 Stuttgart, Germany
3 Institut für Forstbotanik und Baumphysiologie, Albert-Ludwigs-Universität, D-79110 Freiburg, Germany
4 Julius-von-Sachs-Institut für Biowissenschaften, Universität Würzburg, D-97082 Würzburg, Germany
Received 31 August 2000; Accepted 29 December 2000
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Abstract |
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When tobacco is provided with a high nitrate supply, only a small amount of the nitrate taken up by the roots is immediately assimilated inside the roots, while the majority is transported to the leaves where it is reduced to ammonium. To elucidate the importance of root nitrate assimilation, tobacco plants have been engineered that showed no detectable nitrate reductase activity in the roots. These plants expressed the nitrate reductase structural gene nia2 under control of the leaf-specific potato promoter ST-LS1 in the nitrate reductase-mutant Nia30 of Nicotiana tabacum. Homozygous T2-transformants grown in sand or hydroponics with 5.1 mM nitrate had approximately 55–70% of wild-type nitrate reductase acivity in leaves, but lacked nitrate reductase acivity in roots. These plants showed a retarded growth as compared with wild-type plants. The activation state of nitrate reductase was unchanged; however, diurnal variation of nitrate reductase acivity was not as pronounced as in wild-type plants. The transformants had higher levels of nitrate in the leaves and reduced amounts of glutamine both in leaves and roots, while roots showed higher levels of hexoses (3-fold) and sucrose (10-fold). It may be concluded that the loss of nitrate reductase acivity in the roots changes the allocation of reduced nitrogen compounds and sugars in the plant. These plants will be a useful tool for laboratories studying nitrate assimilation and its interactions with carbon metabolism.
Key words: Diurnal variation, leaf specific expression, nitrate reductase, tobacco, roots.
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Introduction |
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The assimilation of nitrate is a highly regulated process (Crawford, 1995
). Nitrate is taken up by the roots, but in many herbaceous plants only a small amount is reduced to ammonium within the roots. Plants provided with a high nitrate supply transport the majority of nitrate to the leaves where photosynthesis provides energy and C-skeletons for N-assimilation (Rufty, 1997). N- and C-assimilation are co-regulated in higher plants by reciprocal molecular and metabolic controls between both pathways (Kaiser, 1997; Koch, 1997; Ferrario et al., 1995). In particular, reversible protein phosphorylation of nitrate reductase (NR, EC 1.6.6.1) plays a key role in this regulatory network (Glaab and Kaiser, 1995; Huber et al., 1996; MacKintosh, 1997). The rate of nitrogen uptake and translocation to the shoot can determine the rate of shoot growth (see review by Rufty, 1997).
Following uptake into the root cells, nitrate is stored in vacuoles, reduced by NR, or loaded into the xylem. In the xylem, nitrate (mainly as KNO3) is transported with the transpiration stream primarily to source leaves where it can be stored in vacuoles until it is finally assimilated. The products of nitrate assimilation, the amino acids, are transferred via the phloem to the growth centres of the shoot, or transported back to the root through the phloem, for supporting their growth. Root growth and nitrate uptake are also dependent on a sustained flux of carbohydrates from the shoot. NR plays a key role in this interdependent regulatory network although the NR protein is present in excess in the cells, and only 20–70% of the NR molecules are active depending on the metabolic state of the cell (Kaiser and Huber, 1994). Studies with tobacco NR mutants and transgenic plants with altered expression of NR clearly showed that there is no direct correlation between plant growth and the nitrate reduction capacity of the plant, i.e. plants with lowered NR activity are phenotypically not different from wild-type plants (Vaucheret et al., 1990; Kleinhofs and Warner, 1990; Vincentz and Caboche, 1991; Dorbe et al., 1992; Wilkinson and Crawford, 1993; Foyer et al., 1994; Gojon et al., 1998). Only when NR activity is lower than 10% of the wild-type level is growth affected (Müller and Mendel, 1989; Foyer et al., 1994; Scheible et al., 1997a).
The role of nitrate reduction in the roots of herbaceous plants is not fully understood. It may constitute only 10% of the total nitrate reduction capacity of the plant, depending on plant type and N-nutrition (Gojon et al., 1998). Recently it was suggested that nitrate might serve not only as nutrient but also as signal for regulating N- and C-metabolism (Scheible et al., 1997b). In order to elucidate the role of root nitrate reduction, the consequences of a complete loss of this enzymatic activity in the roots on metabolism and plant development were studied. For this purpose tobacco plants were constructed without detectable NR activity in the roots. In this report, the first analyses of these transformed plants are presented and it is shown that the loss of nitrate reductase activity in the roots changes the allocation of reduced nitrogen compounds and sugars when NR is present only in the leaves.
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Materials and methods |
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Plasmid construction
All restriction endonuclease and ligase reactions (Sambrook et al., 1989
) were performed using the buffer conditions recommended by their respective manufactureres. The intermediate plasmid constructions were made in the pRT-series (Toepfer et al., 1988). Two binary vectors were constructed using the HindIII site of the pGA472 (An et al., 1987). The first vector contained the CaMV-35S promoter driving the nitrate reductase cDNA (nia2) of Nicotiana tabacum (Vaucheret et al., 1989; Vincentz and Caboche, 1991) followed by the polyA-tail from CaMV-35S. In the second vector, the 35S-promoter was replaced by the ST-LS1 promoter from Solanum tuberosum (Stockhaus et al., 1987) kindly provided by Dr Uwe Sonnewald, Gatersleben. For transformation, the Agrobacterium tumefaciens strain C58C1/pGV2260 was used.
Plant material and transformation
Experiments were performed with Nicotiana tabacum cv. Gatersleben (GAT). The nitrate reductase-deficient mutant Nia30 (Müller, 1983) derived from this wild type was used as recipient in transformation studies. The plants were grown in vitro on MS medium (Murashige and Skoog, 1962) supplemented with ammonium succinate in the case of the Nia30 mutants (Vaucheret et al., 1990). Leaf disc transformation and shoot regeneration were performed as described previously (Horsch et al., 1985). Regenerated plantlets were maintained on medium containing 50 mg l-1 kanamycin and screened by Southern blotting of genomic DNA to verify the integration of the foreign genes. The primary transformants were transferred into soil in controlled environment chambers (Hereaus-Vötsch, HPS 1500, Balingen, Germany) with 14 h daily light periods to allow self-fertilization.
Cultivation of plants and determination of relative growth rate (RGR)
Seeds of wild-type N. tabacum cv. GAT and the transformants were germinated on soil in a day/night-regime of 14/10 h, 25/20 °C, a relative humidity of 80% and 350–400 µE m-2s-1 PAR. After 3 weeks the plants were transferred to sand-filled 0.5 l pots or to 3.0 l pots containing a well-aerated nutrient solution of the following composition: 2.0 mM K+, 2.5 mM Ca2+, 1.2 mM Mg2+, 0.5 mM 
, 1.2 mM 
, 10 µM FeEDTA, 10 µM H3BO3, 0.5 µM Mn2+, 0.5 µM Zn2+, 0.1 µM Cu2+, and 0.07 µM 
. Nutrient solutions were changed three times a week. Sand-cultured plants were supplied daily with the complete nutrient solution containing 5 mM ammonium nitrate. During the first week of hydroponic growth, NO3- (3.4 mM) plus 
(1.7 mM) were added to the nutrient solution. Subsequently, 
(5.1 mM) was the sole nitrogen source of the plants for both the hydroponically and the sand-grown plants.
Growth was measured as the increase in fresh or dry weigth (DW) between five harvest occasions between 4–8 weeks after sowing. Dry weight was determined after oven-drying at 80 °C for 14 h. RGR for single plants was calculated according to: RGR=ln (DW final plant/DW seedling)/days of growth.
Extraction and detection of mRNA and protein
Plants were harvested, shoot and root biomass were determined, and the tissues were frozen in liquid nitrogen and stored at -80 °C. Tissues were extracted by grinding to a fine powder in a small mortar and pestle cooled in liquid nitrogen.
For RNA-analysis, 1 g powder was transferred to a precooled 15 ml centrifuge tube and total RNA was extracted (Verwoerd et al., 1989). Gel electrophoresis was performed in a formamide-formaldehyde-agarose-gel loaded with 40 µg of total RNA. The RNA was blotted to a charged membrane (Hybond-N+, Amersham-Pharmacia-Biotech, Freiburg, Germany) and fixed by UV-crosslinking followed by hybridization with a fluorescein-dUTP-labelled-DNA-probe. Bands of NR mRNA were visualized with an anti-fluorescin antibody labelled with alkaline phosphatase. Hybridization and detection were performed with CDP-Star Detection System according to the manufacturer (Amersham-Pharmacia-Biotech).
Protein from 20 mg tissue was extracted and loaded (Scheible et al., 1997b) on SDS-PAGE consisting of 7.5% acrylamide (Laemmli, 1970). The proteins were electroblotted to Hybond-PVDF (Amersham-Pharmacia-Biotech) and immuno-detected with NR-peptide antibodies (Scheible et al., 1997b) kindly provided by Dr Mark Stitt (Heidelberg). The NR bands were visualized using the ECL-Plus-System (Amersham-Pharmacia-Biotech).
Determination of NR activity
Nitrate reductase activity was determined (Scheible et al., 1997a). The activation state of NR is given by the ratio of its activity in extraction and reaction buffers with either 10 mM EDTA or with 10 mM MgCl2 (Kaiser and Spill, 1991).
Analysis of amino-compounds and tissue nitrate concentration
Amino compounds were extracted from plant material by a modification of the method described earlier (Gessler et al., 1998a). Roots and the first two adult plus the youngest leaves were ground with mortar and pestel, respectively. Aliquots of 0.1 g of the frozen powder were homogenized in 0.2 ml of 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 (v/v)). The homogenate was incubated for 30 min at 4 °C. Subsequently, water-soluble metabolites were extracted twice with 0.6 ml double-distilled water. The aqueous phases were combined and freeze-dried (Alpha 2–4, Christ, Osterode, Germany). The dried material was dissolved in 1 ml lithium citrate buffer (0.2 mM, pH 2.2) for amino acid analysis. Amino compounds were separated and subsequently detected after post-column derivatization with ninhydrin by an automated amino acid analyser (Biochrom, Pharmacia LKB, Freiburg, Germany). Analyses of amino compounds and nitrate in roots/shoots of the wild type and the transformant at day and night were performed in 5–6 independent replicates each. For statistical analysis data were subjected to ANOVA (Microcal Origin, Version 3.5, Microcal Software, Inc.)
Tissue nitrate was determined according to the method described previously (Gessler et al., 1998b) using a conductivity detector module (CDM, Dionex).
Sugar determination
Leaf samples (0.5 g FW) were ground in small mortars in liquid nitrogen. The frozen powder was suspended in 5 ml deionized water, and grinding continued until thawing. Subsequently, samples were boiled for 3 min in a heating block and cooled immediately on ice. After removal of insoluble material by centrifugation (16000 g, 20 min, 2 °C), the supernatant was used after suitable dilution to separate and quantify sugars by isocratic ion chromotography with PAD (pulsed amerometric detection) (4500 i, Carbopac P1 column plus precolumn, 0.12 M NaOH as eluent, Dionex, Idstein, Germany). Mixtures containing sorbitol, glucose, fructose, sucrose, and maltose (0.1 mM each) were used as standards every fifth sample. Occasionally, extracts were passed through mixed-bed ion exchange columns before sugar analysis in order to check for ionic contamination which might give a signal on the PAD. However, in all cases, analysed contaminations were negligible.
Statistics
Biomass and nitrate reductase activity values were obtained from 3 leaves per plant using 3–5 plants per line. The results are given as the mean values for each population with the SE=
n/
, where n is the SD. If not pointed out otherwise, for biochemical analyses the youngest fully expanded three leaves (7 and 15 cm in length) were pooled. The values given for these analyses represent the means of three plants, and each experiment was repeated independently three times. Northern and Western analyses were done with a mix of ground powder from these plants.
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Results |
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Construction of tobacco plants without NR activity in the roots
For engineering tobacco plants without detectable NR activity in the roots, tobacco NR-cDNA was introduced into the widely used tobacco NR-mutant Nia30. This mutant is a homozygous double mutant in the NR loci nia1 nia2 that was purified by backcrossing to the wild type (Müller, 1983
). It lacks detectable NR activity in leaves and roots and is unable to grow in soil. Cultured in vitro, Nia30 plants are able to use ammonium-succinate as the nitrogen source but cannot grow on nitrate (Müller, 1983). For transformation, the nia2-cDNA (Vaucheret et al., 1989) was under the control of the potato ST-LS1 promoter that drives expression of the genes only in green tissues (Stockhaus et al., 1987). As a control, Nia30 mutants were transformed with NR-cDNA under the control of the constitutive CaMV-35S promoter. T0-plants that exhibited NR activity in the leaves were subjected to Southern blot analysis of genomic DNA using a nptII- and a nia2-(Moco-domain only) radiolabelled probe, respectively. Three plants (H, P and U) with a one copy-integration of the transgene were chosen to produce homozygous T2-seeds. Untransformed Nia30-mutants were unable to grow in hydroponic culture or sand with nitrate as the sole nitrogen source and died. Among the 35S-NR transformants, plants were identified that showed NR activities similar to the wild type both in leaves and roots. Compared to the wild type, the three LNR plants (LNR=leaf nitrate reductase) had NR activities between 25% and 70% in the leaves, and under no circumstances were NR activity detectable in the roots.
LNR plants show retarded growth
LNR plants were considerably smaller than the wild type of the same age while 35S-NR plants grew as fast as wild-type plants. Figure 1A shows the phenotype of homozygous T2-plants of the LNR type, i.e. retarded growth with smaller leaves as compared with a wild-type plant of the same age. Eight weeks after germination, LNR plants had accumulated less than half the biomass compared with the wild type (Fig. 1B). Between 4 and 8 weeks of cultivation the RGR were not different (LNR: 0.112 and wild type: 0.119). Given enough time LNR plants developed into plants of similar size as the wild type (not shown) with a flowering time of 83.2±5.3 d (69.4±4.1 d in wild type). The retarded growth of LNR plants made it necessary to measure NR activities not only in leaves of the same plant age but also in leaves of a given developmental stage and size when comparing LNR plants with wild-type plants. Seven-week-old wild-type plants correspond in developmental stage and size to LNR plants of an age between 8 and 9 weeks. NR activities were therefore determined separately for the first four fully expanded leaves at regular intervals between 7 and 10 weeks after sowing. Figure 2 shows that NR activities stayed comparable over a period of 7–9 weeks for both wild-type and LNR plants. The slower growth of the LNR plants was surprising because it was previously shown that tobacco plants with considerably reduced NR activity exhibited no growth retardation (Foyer et al., 1994). When Nia30 mutants were transformed with 35S-NR cDNA they showed NR activity in both leaves and roots and grew as fast as wild-type plants. Thus the slower growth of the LNR plants might be attributable to the loss of root NR activity. Therefore LNR lines H and U were chosen for further analyses and were compared with wild-type plants and, where indicated, also with Nia30 transformed with 35S-NR cDNA as the positive control.
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). Values presented are for LNR H plants, LNR U plants gave similar values. The increase of fresh and dry weight of hydroponically grown plants was measured over a period of 8 weeks starting from seed germination.
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Correlation of nitrate reductase protein-level and enzyme-activity
Homozygous T2-generation plants of the LNR type and the wild type were analysed for NR activity and for the amount of NR protein expressed in the leaves and roots. Figure 3A shows that NR activity correlates with the amount of NR-protein detected by NR-specific antibodies. LNR plants had approximately half the NR activity of the wild type in their leaves which directly correlated with the amount of NR protein detected immunologically. Further, the activation state of NR was analysed according to Kaiser and Spill (Kaiser and Spill, 1991). Figure 3B shows that the percentage of active NR in the leaves of both wild-type and LNR plants ranged from 60% to 80%, whereas the activation state of the root NR was much lower in the wild type.
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Diurnal variation of NR expression
Expression of NR was characterized by a typical diurnal variation of the level of mRNA, protein and enzyme activity (Fig. 4). For generating LNR plants the potato ST-LS1 promoter was used that is expressed only in photosynthetically active, green tissues (Stockhaus et al., 1989). This promoter is known to increase transcription about 2-fold when the green tissues are exposed to light (Stockhaus et al., 1987), but nothing is known about its behaviour during a complete day–night cycle. Therefore, NR expression was analysed in LNR plants and it was compared to the wild type. Figure 4 also shows that the LNR type also exhibited diurnal variation of NR expression on the level of enzyme activity, reflecting the strength of the ST-LS1 promoter in dark and light; however, the amplitude of this variation was not so pronounced as in the wild type. The level of NR-mRNA in wild-type leaves showed the expected diurnal variation with a maximum during the night, while the amount of NR protein was decreased at night. Also in wild-type roots, the amount of NR protein was lower at the end of the day and during the night. In LNR leaves, however, constitutive NR-mRNA levels were measured over the whole period. LNR roots showed a NR-mRNA pattern similar to the wild type. The trace amounts of NR protein in LNR roots originated from the mutated and unstable NR protein of mutant line Nia30 that was used as recipient for the transformation experiments.
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Soluble nitrogen compounds in the shoot and roots
In the leaves, total soluble non-protein nitrogen (TSNN) contents were substantially higher in the LNR transformants as compared to the wild type and the 35S-NR plants (Table 1). Enhanced TSNN levels in the leaves of LNR plants were attributed to an enhanced content that overcompensated the strongly reduced levels of amino-N. Most remarkably, glutamine levels in the leaves of the LNR transformants were over 95% reduced as compared to the wild type and to the 35S-NR plants (Table 1). and glutamate levels of the leaves were higher in the wild type and the 35S-NR plants as compared to the LNR type.
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In the roots, TSNN contents were slightly lower as compared to the leaves (Table 1
). Both, TSNN and contents were similar in the roots of the wild type, 35S-NR plants and the LNR plants. Still, the glutamine levels in the roots of the LNR transformants were over 90% reduced as compared to wild type and 35S-NR plants, which is also reflected in the lower levels of amino-N in LNR roots. Total protein contents in wild type and LNR were also examined. They were very similar in leaves and roots of wild-type, LNR and 35S-NR plants, with a trend towards higher values in LNR roots (data not shown).
Hexoses and sucrose in the shoot and roots at day and night
The contents of glucose, fructose and sucrose of the leaves did not show differences between LNR plants and the wild type (Table 2 ). As expected, the sugar content of the leaves increased during the day. In the roots of LNR plants, however, a strong accumulation of sucrose did occur with levels 8–10 times higher than in the wild type (Table 2). Hexose levels in the LNR roots were 2–3 times higher as compared to the wild type, but did not increase during the day. Malic acid concentrations in roots of LNR were 5–10 times higher than in the wild type, and cation concentrations were also increased (data not shown).
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Discussion |
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Nitrate reduction in the roots of herbacious plants like tobacco constitutes approximately one-tenth of the total nitrate reduction capacity of the plant (Gojon et al., 1998
). To study the consequences of the loss of this small percentage of NR in the roots for plant metabolism and development, tobacco plants were constructed without detectable NR activity in the roots. This might principally be achievable by an antisense approach using a strong and tightly expressing root-specific promoter. Antisense approaches are a good means for lowering the expression of a given gene, but only rarely reduce its expression tightly to zero. This problem was circumvented by using a completely NR-deficient mutant as recipient for the NR cDNA in sense under control of a leaf-specific promoter.
The potato ST-LS1 promoter chosen for these experiments is known to increase transcription about 2-fold when exposed to light and it is active only in green tissues containing photosynthetically active chloroplasts (Stockhaus et al., 1987). In the leaf, it is expressed only in the mesophyll cells and the stomata guard cells, but not in the epidermis (Stockhaus et al., 1989). The leaf mesophyll is also the main site of nitrate assimilation so that the ST-LS1-driven NR expression is appropriately placed. The ST-LS1 promoter turned out to be very tight in its organ specificity, i.e. no residual NR activity was ever detected in the roots of the LNR transformants. It permitted a diurnal expression pattern of NR activity, albeit not with the same amplitude as observed for the nia-promoter (Fig. 4). The ST-LS1 promoter, however is not nitrate inducible. As in wild-type plants, the amount of NR protein in LNR plants is decreased at night, and also the activation state of NR in wild-type and LNR plants was very similar (Fig. 4). NR-mRNA levels, however, stayed constant over the whole period in LNR plants. Here it has to be taken into account that mutant line Nia30 used as a recipient for the transformations experiments has point mutations in the two nia genes rendering its NR protein inactive and unstable, but the genes are still transcribed. Therefore the NR-mRNA expression pattern observed in LNR leaves is the sum of two transcriptions: one driven by the residual nia promoters and the other driven by the introduced ST-LS1 promoter.
The LNR transformants showed a slower growth than the wild type. This was surprising for several reasons: (1) In wild-type tobacco, root NR activity is approximately 10 times lower than leaf NR activity (Gojon et al., 1998) and hence its loss should not cause such a delay in growth. (2) Tobacco plants with lowered NR activity did not show growth retardations. Only when NR activity was lower than 10% of the wild-type level was growth affected (Müller and Mendel, 1989; Foyer et al., 1994; Scheible et al., 1997a; Gojon et al., 1998). (3) The latter plants with less than 10% of wild-type NR activity showed, however, actual 
reduction rates between 30–70% of those measured in wild-type plants (Gojon et al., 1998). These high rates, however, were also observed in detached leaves so that Gojon et al. came to the conclusion that in tobacco the contribution of the roots to total nitrate reduction is not high (Gojon et al., 1998). Further, the data of these authors demonstrate that there is a discrepancy between the potential nitrate reduction (as measured by NR activity) and the actual nitrate reduction, and this discrepancy has not been solved yet. LNR plants had approximately 50% of wild-type NR activity in the leaves which corresponded with the amount of NR protein detected immunologically. Taking into account the results of other authors (Gojon et al., 1998; Foyer et al., 1994), neither the amount of NR protein expressed in leaves nor the activity and the activation state of NR could account for the slower growth of LNR plants.
The loss NR activity in the roots of LNR plants had an effect on their development. One could argue that this phenotype is unrelated to NR but rather might be due to secondary mutations in the recipient plant Nia30. This possible intrepretation, however, can be ruled out because the control 35S-NR transformants that regained NR activity both in leaves and roots showed normal growth and metabolite values. The determination of NR activity in leaves over a period of 7–9 weeks after sowing gave rather steady values for single leaves of wild-type and LNR plants. Thus, it rules out the possibility that strongly changed NR activities that might occur during different developmental stages were the reason for the observed growth retardation.
More detailed biochemical analyses of the LNR plants showed that they had striking changes in nitrogen and sugar content. A strong difference in total soluble nitrogen contents between wild-type Nicotiana tabacum cv. ‘Gatersleben’ and the transformants was observed. The glutamine content of the LNR plants was more than 90% reduced as compared to wild-type plants while 35S-NR transformants that do express root NR showed normal levels of glutamine. The glutamate contents in LNR plants were only moderately lowered (2–3-fold), in both leaves and roots. The decreased levels of glutamine and glutamate in the leaves of LNR plants could be explained with the reduced NR activity in the leaves of LNR plants. However, the following explanation should also be considered: because root NR activity was not measurable in the LNR transformants and NO3- was the sole source of nitrogen of the plants, the amino-N accumulated in the roots cannot originate from NO3- reduction and assimilation in the roots. Thus, the roots of the LNR plants constitute a larger sink for amino compounds than the roots of the wild type because down-regulation of NR activity in LNR roots creates an additional demand for reduced nitrogen and seems to interact with the cycling pool of amino compounds in the plants. Glutamine, as the major amino compound allocated from the leaves to the roots in the phloem of several species (Gessler et al., 1998a), has apparently to satisfy this additional demand which might explain the sharp decline of glutamine levels in the leaves of LNR plants that was not seen in the 35S-NR control transformants. Roots need reduced nitrogen in order to maintain an appropriate rate of growth, therefore glutamine as the major source for LNR roots is readily metabolized.
The lack of NR activity in the roots of the LNR transformants resulted in enhanced accumulation of in the leaves but not in the roots. This might be explained in two ways: either the lower NR activities in the leaves are the reason for the higher contents or/and accumulation in leaves is under control of root NR activity. 35S-NR control transformants expressing root NR do not accumulate high in the leaves. Scheible et al. suggested that higher concentration of nitrate in the shoot acts as a signal to regulate shoot–root allocation of nitrogen in tobacco (Scheible et al., 1997b).
In LNR roots, reduced nitrogen cannot be gained by any nitrate reduction in the root. As a consequence, all reduced nitrogen has to be allocated by phloem transport from the leaves to the root. Winter et al. showed that phloem transport of amino acids is mediated by a mass flow of sucrose driven by active phloem loading of sucrose (Winter et al., 1992). Thus, enhanced phloem loading of sucrose may be required to achieve an allocation of amino compounds sufficient to maintain appropriate root growth. This view is supported by the observation that not only the levels of nitrogen metabolites were changed in transformants lacking root NR activity, but also sugar metabolism was affected. Roots of LNR plants had sucrose levels 6–10 times higher than in the wild type. Also the hexose levels in the roots of the LNR transformants were 2–3 times higher than in the wild type, while the sugar levels in the leaves of LNR plants and the wild type were very similar. Roots of LNR grew smaller than roots of wild type with respect to biomass production. Apparently, the lack of nitrate reduction produces signals controlling root growth in a yet unknown manner. Accordingly, sugar accumulation in LNR roots may be considered as a general consequence of impaired growth. In addition, the high sucrose accumulation suggests, that sucrose translocation or unloading was a major point of direct or indirect control by nitrate reduction. Detailed analyses (J Kruse, unpublished results) of metabolite allocation in Nia30-transformants will further elucidate the interactions between nitrate reduction, carbon metabolism, allocation, and growth.
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Acknowledgments |
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We gratefully acknowledge the financial support of the Deutsche Forschungsgemeinschaft.
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Notes |
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5 To whom correspondence should be addressed. Fax: +49 531 391 8128. E-mail: r.mendel{at}tu\|[hyphen]\|bs.de
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References |
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