Journal of Experimental Botany, Vol. 52, No. 359, pp. 1283-1289,
June 1, 2001
© 2001 Oxford University Press
Original Papers |
Diurnal changes in nitrogen assimilation of tobacco roots
1 Institut für Botanik, Technische Universität Darmstadt, Schnittspahnstr. 10, D-64287 Darmstadt, Germany
2 Plant Nutrition Laboratory, Department of Agricultural Sciences, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark
Received 26 July 2000; Accepted 25 January 2001
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Abstract |
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To gain an insight into the diurnal changes of nitrogen assimilation in roots the in vitro activities of cytosolic and plasma membrane-bound nitrate reductase (EC 1.6.6.1), nitrite reductase (EC 1.7.7.1) and cytosolic and plastidic glutamine synthetase (EC 6.3.1.2) were studied. Simultaneously, changes in the contents of total protein, nitrate, nitrite, and ammonium were followed. Roots of intact tobacco plants (Nicotiana tabacum cv. Samsun) were extracted every 3 h during a diurnal cycle. Nitrate reductase, nitrite reductase and glutamine synthetase were active throughout the day–night cycle. Two temporarily distinct peaks of nitrate reductase were detected: during the day a peak of soluble nitrate reductase in the cytosol, in the dark phase a peak of plasma membrane-bound nitrate reductase in the apoplast. The total activities of nitrate reduction were similar by day and night. High activities of nitrite reductase prevented the accumulation of toxic amounts of nitrite throughout the entire diurnal cycle. The resulting ammonium was assimilated by cytosolic glutamine synthetase whose two activity peaks, one in the light period and one in the dark, closely followed those of nitrate reductase. The contribution of plastidic glutamine synthetase was negligible. These results strongly indicate that nitrate assimilation in roots takes place at similar rates day and night and is thus differently regulated from that in leaves.
Key words: Diurnal cycle, nitrate reductase forms, glutamine synthetase, nitrate assimilation, roots.
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Introduction |
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The enzymes that catalyse the major steps of inorganic nitrogen assimilation are regulated diurnally in shoots of higher plants. In this case, the energy for uptake and reduction of nitrate and for assimilation of ammonium as well as the supply of carbon skeletons for incorporation of ammonium into amino acids are provided by photosynthesis. Consequently, the highest activities of the nitrate and ammonium assimilating enzymes in leaves are observed during the light phase. A phytochrome-mediated, light-induced expression of the genes of nitrate reductase (NR) and nitrite reductase (NIR) (Rajasekhar et al., 1988
; Schuster and Mohr, 1990; Becker et al., 1992b) as well as of chloroplastic glutamine synthetase (GS2; Becker et al., 1992a; Lam et al., 1995) has been demonstrated in etiolated leaves. In tobacco, however, GS2 is not light-inducible (Becker et al., 1992a) and in tomato gene expression of cytosolic GS (GS1) in leaves is also unaffected by light (Migge et al., 1996). Furthermore, cytosolic NR (cNR) is regulated by post-transcriptional mechanisms. Its activity is down-regulated in the dark by phosphorylation and binding of 14-3-3 proteins (Kaiser and Huber, 1994). GS2 from tobacco leaves is also a phosphoprotein which binds 14-3-3 proteins but, in contrast to their inhibiting effect on NR, they keep octameric GS2 active (Riedel et al., 2001). GS1 from leaves of oilseed rape is phosphorylated and activated by binding of 14-3-3 proteins (Finnemann and Schjørring, 2000).
Little is known about the diurnal regulation of these enzymes in roots. As they also depend on energy and carbon skeletons, which ultimately are derived from photosynthesis, it seems likely that their activities are also highest during the day. This view is confirmed by the observation that uptake (Delhon et al., 1995) and reduction (Glaab and Kaiser, 1993) of nitrate by roots follow a diurnal time-course with the highest rates during the day. In tobacco, maximum nia transcript levels in roots were also observed in the light, but activity of cytosolic NR (cNR) changed diurnally only under low-nitrate conditions (Scheible et al., 1997). Post-translational regulation of cNR from roots is reported to occur via similar mechanisms as in the leaves, but with a lower sensitivity for Mg2+(Merlo et al., 1995). The modulation of NiR activity seems less affected by light than that of cNR; furthermore, activity and protein of NiR from roots is more stable in darkness than NiR from leaves (Li and Oaks, 1995) pointing to a considerable capacity for nitrite reduction in the roots and in darkness. Also assimilation of the resulting 
has been reported to occur in darkness in roots (Aarnes et al., 1995; Knight and Weissman, 1982). This indicates that, in contrast to the leaf, a considerable assimilation of nitrite and ammonium may occur in darkness in the roots. This raised the question as to whether nitrate reduction may also be higher in darkness in roots than previously assumed. So far only cNR has been examined in this context, but the additional presence of a plasma membrane-bound NR (PM-NR) in roots has recently been demonstrated (Stöhr, 1999). To study its activities in light and darkness and its possible implication on root nitrogen metabolism roots were harvested every 3 h during a diurnal cycle and activities of key enzymes were measured (both types of NR, NiR, cytosolic (GS1) and plastidic (GS2) glutamine synthetase) and key metabolites (
).
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Materials and methods |
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Plant material and growth conditions
Tobacco seeds (Nicotiana tabacum L. cv. Samsun; received from Genbank, Gatersleben, Germany) were germinated for 5 d with 0.5 mM CaSO4, then transferred to sand culture in a greenhouse and supplied with 2.5 mM Ca(NO3)2 in a modified Hoagland's nutrient solution (Stöhr, 1999
) (light/dark rhythm 15 h/9 h and 28/22 °C). From 3 weeks onwards the seedlings were supplied with 10 mM nitrate. Nine weeks after germination roots were harvested at 3 h intervals during the diurnal cycle. At each sampling point roots from three different plants were analysed.
Preparation of extracts for measuring cNR and PM-NR activities
Root tissue was pulverized in a mortar under liquid nitrogen and homogenized with buffers for the preparation of soluble extracts or plasma membranes (Stöhr and Ullrich, 1997). The homogenized tissue was then sucked through glass filters and pre-centrifuged in the cold for 20 min at 104 g. The resulting supernatant was centrifuged for 1.5 h at 5x104 g. Activity of cNR was measured in the new supernatant, whereas the microsomal fraction was used for purification of plasma membranes as described earlier (Stöhr and Ullrich, 1997). Activities of the two nitrate reductase forms were assayed by the formation of nitrite as described previously (Stöhr and Ullrich, 1997).
Preparation of extracts for measuring GS1 and GS2 activities
Root plastids were isolated in a Percoll gradient immediately upon harvesting and additionally purified by washing the plastid fraction of the gradient (Mäck, 1998). Washing reduced the yield by c. 30%, but it was applied routinely to ensure high-quality of plastids. Purified plastids were osmotically shocked in 5 mM imidazole-HCl (pH 7.5) with 0.1% ß-mercaptoethanol. The supernatant of a 104 g centrifugation (15 min) was used for the determination of soluble plastid protein and for activity assays of GS2 and marker enzymes. Marker enzymes were measured as described earlier (Borchert et al., 1993). Their recovery in the plastid fraction as a percentage of total root homogenate was as follows: plastid marker nitrite reductase 30%, cytosol marker alcohol dehydrogenase 0.4%, mitochondria marker citrate synthase 4–5%. The 30% recovery of the plastid marker indicates a contamination of the plastids by cytosolic material of c. 1% and by mitochondrial material of c. 14%. Thus, contamination with cytosolic GS1 could be ignored. The mitochondrial contamination could also be tolerated because it is not likely that GS is located in root mitochondria but absent in leaf mitochondria (Wallsgrove et al., 1980; Nishimura et al., 1982). Plastid intactness was measured as described previously (Borchert et al., 1993); the latency of ADP-glucose pyrophosphorylase was determined in the presence of 0.33 M sorbitol±0.1% Triton X-100. Intactness was 70–80%.
For assaying total root GS activity, an aliquot of the roots was immediately frozen with liquid N2 and stored at -80 °C for 2 d. The frozen roots were ground in a mortar with 50 mM imidazole-HCl (pH 7.5) containing 1 mM MgSO4 and 5 mM glutamate.
GS activities were measured as synthetase reaction (biosynthetic assay) by formation of 
-glutamylhydroxamate (O'Neal and Joy, 1973) in a reaction mixture as described previously (Mäck, 1998). The different preparation methods did not allow a quantitative comparison of GS1 and GS2 activity. The former was expressed on a total soluble protein basis whereas the latter had to be based on soluble plastid protein. For a rough estimation, however, both activities were based on fresh weight; this calculation revealed that c. 1% of total GS activity was due to GS2. Thus, GS1 activity corresponded to total GS activity.
Determination of inorganic N-compounds and protein
For determination of nitrate and nitrite, ground frozen tissue was extracted with hot water, deproteinated by heating in a water bath at 100 °C for 10 min and then ultrafiltered (Nanosep TM, Pall Filtron). Nitrate was analysed by HPLC on a Partisil 10 SAX column (Whatman) as described previously (Thayer and Huffaker, 1980). Nitrite was determined spectrophotometrically upon diazotation and forming of azo dye (Snell and Snell, 1957). For determination of ammonium (Bergmeyer and Beutler, 1990), ground tissue was extracted with 5% TCA and centrifuged; the neutralized supernatant was used for enzymatic (GDH) analysis. To control ammonium contamination in the solutions or by atmospheric entry parallel analysis without plant tissue were performed. Total protein was extracted with SDS and ß-mercaptoethanol (Nejidat et al., 1997). Protein of all fractions was quantified by the method described previously using BSA as standard (Bradford, 1976).
The presented data are the means of three independent experiments performed with three different plants and three repetitions of each single measurement.
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Results |
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The highest activity of cNR in tobacco roots was found in the fourth hour of the light period (Fig. 1A
). Thereafter it declined to constantly low levels for the rest of the light period and in the dark to increase again before the onset of light. In contrast, PM-NR had only low but constant activity during the day (Fig. 1B) with either NADH or succinate as electron donor. Yet, with the onset of darkness PM-NR activity was strongly enhanced. This increase in activity was higher for the succinate-dependent (6.5 times) than for the NADH-dependent reaction (5 times). In both cases, maximum activity was reached after 1 h of darkness. In the following hours of darkness PM-NR activity was down-regulated and succ-PM-NR reached its minimum early in the light phase. NIR activity was one order of magnitude higher than cNR and PM-NR activities (Fig. 1C) and showed a time-course with the highest activity in darkness and the lowest in the early light phase.
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The ammonium assimilating enzyme, cytosolic GS1, was present with high activity in tobacco roots during the entire diurnal cycle (Fig. 2A
). Despite its high activity, it exhibited diurnal variations with two activity peaks, the first after 6 h of light and a second after 4 h of darkness. Contrasting to GS1, the activity of plastidic GS2 was much lower and showed no clear diurnal changes (Fig. 2B).
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The nitrate content of the roots also varied during the diurnal cycle (Fig. 3A
). Nitrate accumulation was highest in the middle of the light period. The nitrate content rapidly declined towards the end of the day, remained at an intermediate level during the night and reached its minimum at the onset of light. Nitrite could be detected only in small amounts with a slight increase in the morning hours (Fig. 3B). Ammonium levels in the root tissue were two orders of magnitude lower than those of nitrate (Fig. 3C). Despite this low amount, a diurnal change could be observed: a decrease of free ammonium during the early light period and an increase in darkness. The protein content of the roots showed no significant diurnal variation (Fig. 3D).
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Discussion |
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Roots of tobacco assimilate inorganic nitrogen day and night. Although it has been reported that nitrate uptake is reduced during darkness (Delhon et al., 1995
), the enzymes of the nitrate and ammonium assimilating pathway were highly active in tobacco roots at night.
Glutamine synthetase
GS1 activity was more than 200-fold the in vitro activity of cNR and 2-fold that of NIR during both day and night and thus was not limiting for ammonium assimilation in tobacco roots. The peak of GS1 activity in the light indicates that the ammonium assimilated in roots during the first half of the light period was used for protein synthesis. In the afternoon GS1 activity declined to lower rates; during the same period the ammonium content of the roots also reached its lowest values. This points to an increased translocation of ammonium to the shoot during the second half of the light period, in agreement with the report of Finnemann and Schjørring, who observed an enhancement of xylem translocation to the shoot of oilseed rape when gene expression of GS1 was repressed in roots (Finnemann and Schjørring, 1999). In darkness, GS1 activity reached a second peak, but the protein content (based on fresh weight) of the roots did not alter significantly. One explanation for this observation could be that the ammonium assimilated in darkness was not primarily used for protein synthesis but for nitrogen storage and, perhaps, nucleic acid synthesis. Recently, evidence was provided that GS1 localized in the phloem of tobacco roots is involved in the production of proline which is regarded as a storage compound for nitrogen (Brugière et al., 1999).
The function of GS2 in roots and its regulation are not clear yet. Although the reduction of nitrite to ammonium is considered to occur in root plastids (Lea et al., 1990), it was assumed that the key enzyme in root ammonium assimilation is GS1 (Hirel et al., 1993; Oliveira et al., 1997) and that GS2 is present only in the roots of legumes (Woodall and Forde, 1996; Vézina et al., 1987). Meanwhile, however, it became evident that GS2 mRNA and subunits (reviewed by Mäck, 1998) and even a catalytically active GS2 (Mäck, 1998) are also present in roots of non-legumes. In contrast to leaves, where chloroplast GS2 is the predominant isoenzyme in ammonium assimilation, the contribution of plastidic GS2 in the roots is very small. According to a rough estimation GS2 activity was only c. 1% of total root GS activity in tobacco roots, c. 11% in roots of sugarbeet (Mäck, 1998) and c. 20% in pea roots (Vézina and Langlois, 1989). GS2 activity in roots of tobacco and sugarbeet (Mäck, 1998) was much less affected by dark-light changes than GS1.
Nitrite reductase
In tobacco, four genes coding for NIR have been identified, but only nir-2 and nir-4 are predominantly expressed in roots (Kronenberger et al., 1993) which indicates a different transcriptional regulation in roots and leaves. This indication is confirmed by the results of Duncanson et al. who showed that the synthesis of NIR in green leaves strongly depends on both nitrate and light whereas in roots NIR is synthesized in the dark and requires only nitrate (Duncanson et al., 1992). The authors observed an increase of NIR activity in darkness in barley roots which was due to the nitrate-induced synthesis of NIR molecules. As in barley, NIR activity in tobacco roots was highest in the dark. At that time the nitrate content had reached a medium level and was obviously sufficiently high for NIR induction. NIR activity was lowest at the beginning of the light period which resulted in a small accumulation of nitrite. In general, however, NIR activity was one order of magnitude higher than cNR activity and thus the accumulation of toxic amounts of nitrite (Pécsváradi and Zsoldos, 1996) was clearly prevented. The bulk of the resulting ammonium was obviously not assimilated in the plastids, but primarily in the cytosol by GS1 (as discussed above). The decrease of free ammonium concentrations during the early light period and the increase in darkness are consistent with the low and high activity of NIR. Ammonium increased in the dark, although GS1 activity was very high. This indicates that ammonium was not easily accessible to cytosolic GS1 in the night; an accumulation in the vacuole (Roberts and Pang, 1992) seems likely.
Cytosolic and plasma membrane-bound nitrate reductases
In contrast to GS and NIR, cNR was only partially active during the night. Since NR was extracted in the presence of EDTA, those molecules of cNR which might have been inactivated in vivo by phosphorylation and binding of 14-3-3 protein also became active in vitro and were thus included in activity assays (Kaiser and Huber, 1994). In agreement with recent reports (Glaab and Kaiser, 1993) cNR of tobacco roots was down-regulated upon darkness similar to that of leaves. Post-translational down-regulation of cNR in roots is assumed to be caused by a decrease of malate rather than by the direct mediation of photoreceptors (Kaiser, 1997).
Thus, the time-course of cNR activity was opposite to that of PM-NR. According to present knowledge succinate-dependent PM-NR is only present in roots (Stöhr and Ullrich, 1997). Generally, PM-NR activity was lower with NADH than with succinate as electron donor. Only at the onset of light was this reversed pointing to a specific post-translational regulation of the protein. The activity values, as presented in the figures, were based on PM-protein. The same result was found by calculating on a fresh weight base (not shown). However, as the preparation method (two-phase partitioning) leads to purification of right-side-out PM vesicles (Larsson et al., 1994), this can only be a rough estimation. Taking the entire population of vesicles into account, PM-NR activity in vivo might be twice as high. At its maximum value, PM-NR activity was estimated between 270 nmol h-1 g-1 FW (right-side-out vesicles) and 540 nmol h-1 g-1 FW assuming that 50% of the prepared vesicles were orientated inside-out. Recently, growing parameters of tobacco plants were compared with the organic nitrogen content of roots and shoots (Stöhr, 1999). In the present paper, these data were used to estimate the amount of nitrate necessary to enable plant growth during the main vegetative growth phase and it was compared with NR activity of the entire root system of a single plant over a 24 h cycle (Table 1). Provided the in vitro cNR activity reflects the situation in vivo, root cNR activity would be sufficient to cover the nitrogen demand of the roots. Surprisingly, the estimated PM-NR activity even exceeds cNR during the entire diurnal cycle. Since this additional nitrate reducing activity is obviously not needed to maintain root growth, the surplus of reduced nitrogen might be translocated to the shoot (Oji et al., 1989). It has been suggested that roots may be responsible for nitrate reduction during the night and PM-NR might be the nitrate reductase concerned. This nitrate assimilation in the dark may meet the demand of the root itself to maintain root growth during night (Ferris and Taylor, 1994), whereas during the day the nitrate reduced by root cNR may be partly translocated to other organs.
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Conclusion |
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These data suggest two independent nitrate assimilating phases in roots, one during the day and a second during the dark period. Nitrate reduction in roots is catalysed in the cytosol by cNR during the day, but in the apoplast by PM-NR during the night. High activities of NIR and GS1 ensure that the resulting nitrite is not accumulated, but further assimilated into organic N-compounds both during the day and night.
Conflicting data exist on nitrate reduction in darkness. When calculated from cytosolic nitrate reductase activity, nitrate reduction in the roots was reported to be lower in darkness than in light (Glaab and Kaiser, 1993). When calculated from the accumulation of reduced 15N, however, incorporation of 15N-nitrate into insoluble N was slightly higher in the dark than in the light (Rufty et al., 1984; Oji et al., 1989). These seemingly contradictory results can easily be explained by the observations from this study: Nocturnal down-regulation of nitrate reductase refers to the soluble enzyme in the cytoplasm whereas the increase in reduced 15N could well be due to the action of the plasma membrane-bound nitrate reductase in the apoplast. Most strikingly, it has been reported that in situ activity of cytosolic nitrate reductase accounted for only 33% of the real root nitrate reduction (measured with 15N); the missing 67% could be due to PM-NR (Gojon et al., 1986).
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Acknowledgments |
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The authors would like to thank Dr WR Ullrich for helpful discussion and critical reading of the manuscript and G Marx for valuable technical assistance. This research was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 199).
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Notes |
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3 To whom correspondence should be addressed. Fax: +49 6151 164808. E-mail: stoehr{at}bio.tu\|[hyphen]\|darmstadt.de
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Abbreviations |
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cNR, cytosolic nitrate reductase; PM-NR, plasma membrane-bound nitrate reductase; NIR, nitrite reductase; GS, glutamine synthetase..
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References |
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